Download Biological function of RNA interference (RNAi) pathways in the moss

Biological function of RNA interference (RNAi)
pathways in the moss Physcomitrella patens
(Hedw.) Bruch & Schimp.
Inaugural-Dissertation
zur Erlangung der Doktorwürde
der Fakultät für Biologie
der Albert-Ludwigs-Universität
Freiburg im Breisgau
von
Basel Khraiwesh
aus
Jinin Camp - Palästina
Freiburg im Breisgau, 2009
Dekan:
Prof. Dr. Ad Aertsen
Promotionsvorsitzender: Prof. Dr. Eberhard Schäfer
Betreuer:
Prof. Dr. Ralf Reski, PD Dr. Wolfgang Frank
Referent:
Prof. Dr. Ralf Reski, PD Dr. Wolfgang Frank
Koreferent:
Prof. Dr. Wolfgang R. Hess
Tag der Verkündigung des Ergebnisses: 24. April 2009
This work has been created in the
Department of Plant Biotechnology
Institute of Biology II
Faculty of Biology
Albert-Ludwigs University of Freiburg
under the guidance of Prof. Dr. Ralf Reski and PD Dr. Wolfgang Frank
To my marvelous mother and dear family
To my wife and my lovely boys,
For your support, understanding and
always being there for me…
Index
Index
List of contents
Publications and manuscripts related to this work
I
II
1 Chapter Ι: Introduction and Overview……………………………….. 1
1.1
Background………………………………………………………………………… 1
1.1.1
1.1.2
RNA Interference: function and technology…………………………………………… 1
Small RNAs and gene silencing………………………………………………………… 2
1.1.2.1
1.1.2.2
1.1.2.3
1.1.2.4
1.1.2.5
1.1.2.6
1.1.3
1.1.4
1.2
MicroRNAs (miRNAs)…………………………………………………………………3
Trans-acting short interfering RNAs (ta-siRNA)……………………………………5
Repeat-associated RNAs (ra-siRNA)………………………………………………. 6
Natural antisense transcript-derived small interfering RNAs (nat-siRNA)……… 6
Piwi-associated RNAs (piRNAs)……………………………………………………. 7
Secondary transitive siRNA…………………………………………………………. 7
Dicer proteins……………………………………………………………………………... 9
Physcomitrella patens as a model system…………………………………………… 11
Results and Discussion………………………………………………………… 14
1.2.1
DICER-LIKE genes in Physcomitrella patens………………………………………...14
1.2.1.1
Generation and molecular analysis of ΔPpDCL1b knockout mutants…………. 16
1.2.1.1.1
Knockout of PpDCL1b causes developmental disorders……………………..17
1.2.1.1.2
MiRNA biogenesis is not affected and miRNA-directed cleavage of mRNAtargets is abolished in ΔPpDCL1b mutant lines………………………………..17
1.2.1.1.3 Generation of transitive siRNA in ΔPpDCL1b mutant lines…………………...18
Analysis of DNA methylation in ΔPpDCL1b mutants and wild type………….19
1.2.1.1.4
1.2.1.1.5
Analysis of the ta-siRNA pathway in ΔPpDCL1b mutants…………………….20
1.2.1.1.6 Analysis of ΔPpDCL1b mutants and wild type lines expressing
amiR-GNT1…………………………………………………………………………21
1.2.1.1.6.1 Specific methylation of a miRNA1026 target gene in response to the
phytohormone abscisic acid (ABA)………………………………………….. 21
1.2.1.1.7
Expression profiling of transcription factor genes in ΔPpDCL1b mutant
lines…………………………………………………………………………………22
1.2.2
1.3
1.4
2
Highly specific gene silencing by artificial miRNAs in Physcomitrella patens……. 24
Conclusion…………………………………………………………………………27
References………………………………………………………………………… 29
Chapter II: Manuscript 1……………………………………………..34
Transcriptional control of gene expression by microRNAs………………35
3
Chapter III: Publication 1…………………………………………..121
Specific gene silencing by artificial microRNAs in Physcomitrella
patens: An alternative to targeted gene knockout……………………….122
4
4.1
4.2
4.3
4.4
4.5
4.6
4.7
Chapter IV: Appendices……………………………………………. 136
Flow cytometric measurements (FCM)……………………………………...136
Physcomitrella patens DCL1b (PpDCL1b) mRNA……………………….. 137
DNA vectors……………………………………………………………………...140
Genes downregulated in ΔPpDCL1b mutants…………………………….. 141
Genes upregulated in ΔPpDCL1b mutants………………………………… 146
Acknowledgments……………………………………………………………... 152
Erklärung………………………………………………………………………....153
I
Publications
Publications and manuscripts related to this Work:
Manuscript #1
-
Khraiwesh, B., M. A. Arif, G. I. Seumel, S. Ossowski, D. Weigel, R. Reski, W. Frank.
(2009): Transcriptional control of gene expression by microRNAs. Submitted.
Publication #1
-
Khraiwesh, B., S. Ossowski, D. Weigel, R. Reski, W. Frank (2008): Specific gene
silencing by artificial microRNAs in Physcomitrella patens: An alternative to targeted
gene knockouts. Plant Physiology, 148: 684–693.
This work has been presented at the following conferences:
Talks (presented by W. Frank)
−
Frank, W., Khraiwesh, B., Seumel, G. I., Baar, K. M., Reski, R. (2007): Specific
epigenetic control of microRNA target genes to compensate for RNAi dysfunctions in a
Physcomitrella patens DICER-LIKE mutant. Botanical Congress, September 3-7,
2007, University of Hamburg, Germany.
−
Khraiwesh, B., Seumel, G. I., Baar, K. M., Reski, R., Frank, W. (2007): Specific
epigenetic control of microRNA target genes to compensate for RNAi dysfunctions in a
Physcomitrella patens DICER-LIKE mutant. The Annual International Conference
for Moss Experimental Research, August 2-5, 2007, Korea University, Seoul, Korea.
Posters
−
Khraiwesh, B., Ossowski, S., Weigel, D., Reski, R., Frank, W. (2008): Specific gene
silencing by artificial microRNAs in Physcomitrella patens: An alternative to targeted
gene knockouts. Annual Meeting of the RNA Society, July 28-August 3, 2008, Free
University Berlin, Germany.
−
Khraiwesh, B., Seumel, G. I., Baar, K. M., Reski, R., Frank, W. (2007): Knockout of a
DICER-LIKE gene causes silencing of microRNA targets in Physcomitrella patens. 5th
Colmar Symposium: The New RNA Frontiers, November 8-9, 2007, Colmar,
France.
−
Khraiwesh, B., Seumel, G. I., Baar, K. M., Reski, R., Frank, W. (2007): Knockout of a
DICER-LIKE gene causes silencing of microRNA targets in Physcomitrella patens. The
Annual International Conference for Moss Experimental Research, August 25, 2007, Korea University, Seoul, Korea.
II
Chapter I
1
1.1
Background
Chapter Ι: Introduction and Overview
Background
1.1.1 RNA Interference: function and technology
RNA interference (RNAi) is a mechanism regulating gene transcript levels by either
transcriptional gene silencing (TGS) or by posttranscriptional gene silencing (PTGS), which
acts in genome maintenance and the regulation of development (Hannon, 2002; Agrawal et
al., 2003). Since the discovery of RNAi in Caenorhabditis elegans (Lee et al., 1993; Fire et
al., 1998) extensive studies have been performed focusing on the different aspects of RNAi.
In particular, the elucidation of the essential components of RNAi pathways has advanced
extensively (Tomari and Zamore, 2005). RNAi has been discovered in a wide range of
organisms from plants and fungi to insects and mammals suggesting that it arose early in the
evolution of multicellular organisms (Sharp, 2001; Hannon, 2002).
The RNAi pathway is typically initiated by ribonuclease III-like nuclease enzymes, called
Dicer, that cleave double stranded RNA molecules (dsRNAs; typically >200 nt) into small
fragments bearing a 3’ overhang of two nucleotides. One of these two strands is coupled to a
second endonuclease enzyme called Argonaute (AGO) and then integrated into a large
complex (RNA-induced silencing complex, RISC). Subsequently, it has been shown that
RISC contains at least one member of the AGO protein family, which is likely to act as an
endonuclease and cuts the mRNA. In Drosophila and humans, AGO2 has been identified as
being responsible for this cleavage and the catalytic component of the RISC complex. It was
proposed that small interfering RNA (siRNA) guide the cleavage of mRNA. SiRNAs are key
to the RNAi process and they have complementary nucleotide sequences to the targeted RNA
strand. In certain systems, in particular plants, worms and fungi, an RNA dependent RNA
polymerase (RdRP) plays an important role in generating siRNA (Cogoni and Macino, 1999).
Another outcome are epigenetic changes such as histone modification and DNA methylation
(Matzke and Matzke, 2004; Schramke and Allshire, 2004) (Figure1).
In medical research, RNAi is on the way to becoming an important tool to treat HIV,
hepatitis C, and cancer (Hannon and Rossi, 2004) and in plants RNAi technology has been
used to improve their nutritional value (Tang and Galili, 2004). For science in general it is
already a tool of large scale reverse genetic approaches and aids in unravelling gene
functions in many species.
1
Chapter I
Background
Figure 1: Overview of RNA interference (adapted from Matzke and Matzke,
2004). The Dicer enzymes produce siRNA from dsRNA and mature miRNA from hairpin-like
miRNA precursor transcripts. MiRNA or siRNA is bound to an AGO enzyme and an effector complex
is formed, either a RISC or RITS (RNA-induced transcriptional silencing) complex. RITS affects the
rate of transcription by histone and DNA modifications whereas RISC cleaves mRNA or inhibits its
translation.
1.1.2 Small RNAs and gene silencing
Small non-coding RNAs (20-24 nucleotides in size) have been increasingly investigated and
they are important regulators of PTGS in eukaryotes (Hamilton and Baulcombe, 1999; Mello
and Conte, 2004; Baulcombe, 2005). They were first discovered in the nematode
Caenorhabditis elegans (Lee et al., 1993) and are responsible for phenomena described as
RNAi, co-suppression, gene silencing or quelling (Napoli et al., 1990; de Carvalho et al.,
1992; Romano and Macino, 1992). Shortly after these reports were published, it was shown
that PTGS in plants is correlated to small RNAs (Hamilton and Baulcombe, 1999). These
small RNAs regulate various biological processes, often by interfering with mRNA
translation. Based on their biogenesis and function small RNAs are classified as repeatedassociated small interfering RNAs (ra-siRNAs), trans-acting siRNAs (ta-siRNAs), naturalantisense transcript-derived siRNAs (nat-siRNAs) and microRNAs (miRNAs) (Vazquez,
2006) (Table 1).
2
Chapter I
Background
Table 1: Classes of small RNAs identified in eukaryotes (Chapman and Carrington,
2007)
Class
Description
Biogenesis and genomic origin
Function
miRNA
MicroRNA
Processing of foldback miRNA gene
transcripts by members of the Dicer and
RNaseIII-like families
Posttranscriptional regulation of
transcripts from a wide range of
genes
Primary
siRNA
Small interfering
RNA
Processing of dsRNA or foldback RNA by
members of the Dicer family
Secondary
siRNA
Small interfering
RNA
RdRP
activity
at
silenced
loci
(Caenorhabditis elegans) processing of
RdRP derived long dsRNA or long
foldback RNA by members of the Dicer
family (Arabidopsis thaliana)
miRNA-dependent cleavage and RdRP
dependent conversion of TAS gene
transcripts to dsRNA, followed by Dicer
processing
Dicer processing of dsRNA arising from
sense and antisense transcript pairs
Binding to complementary target
RNA; guide for initiation of
RdRP
dependent
secondary
siRNA synthesis
Posttranscriptional regulation of
transcripts;
formation
and
maintenance of heterochromatin
tasiRNA
Trans-acting
siRNA
natsiRNA
Natural antisense
transcriptderived
siRNA
Piwi-interacting
RNA
piRNA
A biogenesis mechanism is emerging
which is Argonaute dependent but Dicerindependent
Posttranscriptional regulation of
transcripts
Posttranscriptional regulation of
genes involved in pathogen
defense and stress responses in
plants
Suppression of transposons and
retroelements in the germ lines
of flies and mammals
1.1.2.1 MicroRNAs (miRNAs)
MiRNAs are ~21nt small RNAs which are encoded by endogenous MIR genes. Their primary
transcripts form precursor RNAs exhibiting a partially double-stranded stem-loop structure
which are processed by DICER-LIKE proteins to release mature miRNAs (Bartel, 2004). In
animals, the primary miRNAs (pri-miRNAs) are cleaved in the nucleus by an enzyme named
Drosha to form the pre-miRNAs (Lee et al., 2003). The pre-miRNAs are then transported
into the cytoplasm where they are processed into the mature double stranded miRNAs,
through cleavage by a second enzyme, Dicer (Bartel, 2004). The first enzyme, Drosha,
required for processing of pri-miRNAs in animals, does not exist in plants, so the precursor
miRNA is directly cleaved within the nucleus to generate the mature miRNA (Baulcombe,
2004) (Figure 2a).
Computational analysis of miRNAs and their potential target mRNAs revealed that many of
the miRNA targets belong to the group of transcription factors (Palatnik et al., 2003; Wang
et al., 2004). In addition to the control of targets at the post-transcriptional level miRNAs
regulate gene expression by epigenetic changes such as DNA and histone methylation (Bao et
al., 2004; Lippman and Martienssen, 2004). Overexpression or knockdown of miRNA genes
can lead to abnormalities during development (Palatnik et al., 2003; Chen, 2004). For
example, plants expressing MIR159, which targets members of MYB transcription factors,
exhibit delayed flowering time and male sterility (Achard et al., 2004; Schwab et al., 2005).
Plants expressing MIR160, which targets members of the ARF transcription factor family,
exhibits agravitropic roots with disorganized root caps and increased lateral rooting (Wang
3
Chapter I
Background
et al., 2005). Plants expressing MIR166, which targets members of HD-ZIP transcription
factors, are arrested in seedling development, and show fasciated apical meristems and femal
sterility (Williams et al., 2005).
(g) Piwi-interacting RNA (piRNA)
Figure 2: Small RNA pathways (modified after Vazquez, 2006; Chapman and
Carrington, 2007). (a) The miRNA (b) trans-acting siRNA precursors are non-coding RNAs (c)
nat-siRNA precursors derive from cis-antisense overlapping coding transcripts. All three precursors
are transcribed by RNA polymerase Pol II. (e) Two miRNAs can guide AGO1-mediated cleavage of
TAS precursors. The resultant 5’ fragment (TAS1a, 1b, 1c and 2) or 3’ fragment (TAS3) is used by
RDR6 and SGS3 as a template for the production of a long dsRNA , which is then cleaved in a phased
fashion every 21-nt by DCL4. (f) ta-siRNAs are then 2’-O-methylated at their 3’ ends by HEN1 and
guide a slicer-competent AGO protein (AGO7 for TAS3 siRNAs or an unidentified AGO protein for
other ta-siRNAs) to their targets for cleavage. (d) A self-amplifying loop believed to depend on RNA
Pol IVa is involved in maintaining ra-siRNA-guided methylation of certain DNA repeats. (g) Piwiinteracting RNA (piRNA) biogenesis. Black bars represent genes, with their transcription initiation
sites indicated by arrows. Thin black strands represent transcripts of genes encoding small RNAs, and
thin blue strands represent target mRNAs. Boxes with broken lines indicate parts of the ta-siRNA and
nat-siRNA pathways for which the cellular location is not well established. In small RNA duplexes, the
red strands correspond to the guide strand and the black strands correspond to the passenger strand
to show that, in the case of siRNAs, the guide strand can originate from either the original sense
strand or the newly RDR-synthesized complementary strand.
Target sites in plant miRNAs normally share perfect or nearly perfect complementarity with
their target sequence and are often in coding regions (Schwab et al., 2005), whereas in
4
Chapter I
Background
animals, target sites are often only partially complementary to their miRNAs and are mostly
located in the 3'UTR of target genes (Filipowicz, 2005). Currently, hundreds of miRNAs have
been
identified
in
plant
species
and
deposited
in
the
miRBase
database
(http://microrna.sanger.ac.uk/sequences/index.shtml) (Table 2).
Table 2: Plant species and number of miRNAs deposited in miRBase database
(Version 12.0, 2008)
Species
Group
Number of miRNAs
Arabidopsis thaliana
Eudicots
184
Medicago truncatula
Eudicots
30
Populus trichocarpa
Eudicots
215
Oryza sativa
Monocots
243
Zea mays
Monocots
96
Mosses
220
Physcomitrella patens
1.1.2.2 Trans-acting short interfering RNAs (ta-siRNA)
MiRNAs are required for the biogenesis of ta-siRNAs, and both miRNAs and ta-siRNAs
regulate mRNA stability and translation (Baulcombe, 2004). Ta-siRNAs arise in plants from
specific TAS loci (Figure 2b). TAS transcripts are RNA polymeraseII-dependent and function
as highly specialized precursors that feed into an RdRP-dependent siRNA biogenesis
pathway. They are targets for cleavage by miRNA-guided mechanisms and yield siRNAs that
are in a 21-nt register with the cleavage site (Allen et al., 2005; Rajagopalan et al., 2006;
Chapman and Carrington, 2007).
Arabidopsis contains different characterized TAS gene families. TAS1a-c and TAS2 ta-siRNA
biogenesis is initiated by miR173-guided cleavage on the 5′ side of the ta-siRNA generating
region, while TAS3 ta-siRNAs form by miR390-guided cleavage on the 3′ side. MiR390 also
interacts in a non cleavage mode with a second site near the 5′ end (Axtell et al., 2006;
Montgomery et al., 2008). The resultant 5’ fragment (TAS1a-c and TAS2) or 3’ fragment
(TAS3) is used by RDR6 and SGS3 as a template for the production of a long dsRNA, which
is then cleaved in a phased fashion every 21-nt by DCL4. This processing involves an
interaction between DCL4 with DRB4 for TAS3. Ta-siRNAs are later incorporated into the
RISC-like complex and guide cleavage of the complementary mRNAs. However, in
Physcomitrella patens, miR173 is absent and therefore miR390 is responsible for the
generation of TAS precursors (Axtell et al., 2006). TAS3 ta-siRNAs, but not those from
TAS1a-c or TAS2, are dependent on a specialized AGO7 (also called ZIP) (Montgomery et al.,
2008). The mechanisms for recognition and routing of transcripts through the ta-siRNA or
RDR6/DCL4-dependent pathway are not well understood. Axtell et al. (2006) proposed a
5
Chapter I
Background
two-hit trigger mechanism, in which transcripts with two or more small RNA target sites are
preferentially routed into the RDR6/DCL4 pathway.
1.1.2.3 Repeat-associated RNAs (ra-siRNA)
The mechanism of small interfering RNAs (siRNA) and ra-siRNA production is quite similar.
They originate from transgenes, viruses and transposons and may require RdRP for dsRNA
formation (Waterhouse et al., 2001; Aravin et al., 2003). Unlike miRNAs, the diced siRNA
products derived from the long complementary precursors are not uniform in sequence, but
correspond to different regions of their precursor. Because these small RNAs are often single
stranded, in fact this means that siRNAs work through the same mechanism, but they are not
evolutionary conserved (Bartel and Bartel, 2003; Allen et al., 2004; Jones-Rhoades et al.,
2006; Axtell et al., 2007). In plants, the cleavage of siRNA occurs by different DICER-LIKE
enzymes than the miRNA processing (Xie et al., 2004). They were first described in plants,
where it was shown that the silencing of three transgenes involved a small antisense RNA
complementary to each targeted mRNA (Hamilton and Baulcombe, 1999; Hamilton et al.,
2002; Bonnet et al., 2006). In plants, siRNA have different functions that can be divided into
two broad categories: those that are involved in formation and maintenance of
heterochromatin and those that derive from and defend against viruses or sense transgene
transcripts (Baulcombe, 2004; Bonnet et al., 2006).
Ra-siRNAs (24-nt) are derived from repetitive elements and control the maintenance of DNA
and histone modifications (Hamilton et al., 2002; Xie et al., 2004) (Figure 2d). Previous
studies on Arabidopsis reported that the transcripts of the two canonical repeats, the
retrotransposable element AtSN1 and the 5S ribosomal DNA are converted by RDR2 into
long dsRNA as a template for DCL3, which processes 24-nt siRNAs that are O-methylated by
HEN1 (Xie et al., 2004; Yang et al., 2006). 24-nt ra-siRNAs guide methylation of AtSN1 and
5S rDNA repeat loci by the action of the AGO4 and they have been implicated in chromatin
modifications (Zilberman et al., 2003). The ra-siRNA pathway is a positive feedback loop
because methylation of these loci is essential for ra-siRNA accumulation (Xie et al., 2004).
1.1.2.4 Natural antisense transcript-derived small interfering RNAs (natsiRNA)
Borsani et al. (2005) identified a new class of small RNAs derived from a natural-antisense
overlapping transcript pair. The transcripts of P5CDH, a stress-related gene, and SRO5, a
gene of unknown function, partially overlap (Figure 2c). In the proposed nat-siRNA
pathway, a 760-nt double-stranded region resulting from pairing of cis-antisense transcripts
is thought to be processed by DCL2 to generate a unique 24-nt nat-siRNA that guides
cleavage of P5CDH transcript through an unidentified AGO protein. In principle, RDR6 and
6
Chapter I
Background
SGS3 could synthesize a strand complementary to cleaved fragments of P5CDH mRNA
leading to a dsRNA that is processed like TAS duplexes in a phased fashion by DCL1. Each
resulting 21-nt nat-siRNA reinforces cleavage of P5CDH mRNAs. NRPD1a could be involved
in a reinforcing amplification loop using the dsRNA generated by RDR6 and SGS3 as a
template. Accumulation of the 24-nt nat-siRNA is correlated with salt stress-induced
transcription of the SRO5 gene and is abolished in dcl2, rdr6, sgs3 and nrpd1a mutants. The
finding that 4–20% of the genes in many eukaryotes show cis-antisense overlapping
organization raises the possibility that the nat-siRNA could be a major mechanism for gene
expression regulation (Borsani et al., 2005).
1.1.2.5 Piwi-associated RNAs (piRNAs)
Recent studies have revealed a new class of 24-30-nt RNAs that are generated by a Dicerindependent mechanism and that interact with members of the Piwi subfamily of AGO
proteins (Chapman and Carrington, 2007; Klattenhoff and Theurkauf, 2008). The proposed
piRNA-biogenesis model involves initial targeting of transcripts from transposons and
retroelements by a Piwi-like protein that is programmed with a small RNA (Figure 2g).
PiRNAs are important for spermatogenesis in mammals and insects. Plants appear to lack
these piRNAs (Faehnle and Joshua-Tor, 2007). In Drosophila, the previously identified rasiRNAs have been shown to bind the Piwi family members Piwi and Aubergine (Aub) and
thus represent a subset of Drosophila piRNAs. Piwi-associated ra-siRNAs (now referred to
as piRNAs) are 24−29nt long than AGO-bound siRNAs/miRNAs and are derived
predominately from repetitive genomic loci like transposons or satellite repeats (Aravin et
al., 2003; Chapman and Carrington, 2007).
1.1.2.6 Secondary transitive siRNA
Previous studies on A. thaliana and C. elegans reported the amplification of silencing related
RNA via transitivity, and explain how strong, persistent silencing can be initiated with small
amounts of initiator dsRNA (Axtell et al., 2006; Pak and Fire, 2007; Sijen et al., 2007). The
emergence of transitivity has an important role for RNAi in controlling gene expression and
for understanding the effects of silencing RNAs on cell function and organism development.
The initiator of transitivity is a dsRNA which is produced by RdRP activity and then
processed by DICER-LIKE family into secondary siRNA or a related type of RNA referred to
as miRNA, these 21-25 nucleotide single stranded RNAs are the primary silencing RNAs in
the transitive process (Baulcombe, 2007; Moissiard et al., 2007). Upon binding to target
transcripts, siRNAs can not only trigger their cleavage and subsequent destruction, but also
serve as primers for RdRP. These extend the local RNA double strands and generate
templates for production of secondary siRNAs by Dicer action. These secondary siRNAs are
7
Chapter I
Background
unrelated in sequence to the initial trigger (Moissiard et al., 2007; Mlotshwa et al., 2008). In
plants, the transitivity occurs in both directions of the initial dsRNA trigger whereas in
animals spreading of the initial signal occurs only upstream of the trigger (Figure 3).
Figure 3: Models for amplification of silencing signals in C. elegans and A.
thaliana (adapted from Chapman and Carrington, 2007). (a) Processing of trigger
dsRNA in C. elegans by Dicer 1 (DCR-1) releases primary siRNAs.The primary siRNA associates with
an AGO protein, such as RDE-1, and the complexes bind to complementary target RNA. The bound
complexes might then recruit RdRP, which uses the target transcript as template for synthesis of the
secondary siRNA. Abundant secondary siRNAs are formed by independent initiation events (rather
than by Dicer-mediated processing), are complementary to the target RNA and accumulate in phased
pools. (b) Processing of trigger dsRNA in A. thaliana by one or more Dicer-like enzymes releases
primary siRNAs, which associate with an AGO protein, such as AGO1, and guide cleavage of the target
RNA. This event is proposed to recruit an RdRP, such as RDR6, which uses the target transcript as
template for synthesis of a long dsRNA. This dsRNA precursor is processed by DCL enzymes to release
abundant secondary siRNAs.
8
Chapter I
Background
1.1.3 Dicer proteins
Dicer and DICER-LIKE (DCL) proteins are RNAaseIII-type enzymes that cleave RNA
molecules with dsRNA features into small fragments bearing a 3’ overhang of two
nucleotides during PTGS (Elbashir et al., 2001). Dicer is a large multidomain protein
conserved in most eukaryotes, consisting of DExD-helicase, helicase-C, Duf283, PAZ (PiwiArgonaute-Zwille), dual RNAase III , and double stranded RNA-binding (dsRB) domains
(Bartel and Bartel, 2003) (Figure 4).
Figure 4: Crystal structure of Dicer (adapted from Macrae et al., 2006). The linear
arrangement of domains typically found in DCL or DCR proteins is depicted above the figure. (a)
Front and side view ribbon representations of Dicer showing the N-terminal platform domain (blue),
the PAZ domain (orange), the connector helix (red), the RNase IIIa domain (yellow), the RNase III b
domain (green) and the RNase-bridging domain (gray). Disordered loops are drawn as dotted lines.
(b) Close-up view of the Dicer catalytic sites; conserved acidic residues (sticks); erbium metal ions
(purple); and erbium anomalous difference electron density map, contoured at 20s (blue wire mesh).
The DExD and helicase-C domains are found towards the N-terminal and C-terminal
regions, respectively. There are always two RNase III domains (termed A and B) in a Dicer
protein, and the Duf283 is a domain of unknown function but which is strongly conserved
among Dicer proteins. The role of the dsRB domain in human Dicer is generally thought to
mediate unspecific reactions with dsRNA, with the PAZ, RNase III A and RNase III B
domains being crucial for the recognition and spatial cleavage of dsRNAs into siRNA or
miRNA (Zhang et al., 2004). In organisms with only one Dicer, this enzyme, with its
associated proteins, is presumably the only generator of siRNAs and miRNAs. In organisms
with two or more Dicers, there is probably a division of labour. Plants have at least four main
types of DCLs (Margis et al., 2006). In A. thaliana, the four different DICER-LIKE proteins
(DCL1-DCL4) exhibit predominant functions in particular small RNAs pathways, even
9
Chapter I
Background
though functional redundancies among these proteins were identified (Henderson et al.,
2006). DCL1 produces mature miRNAs which direct cleavage of transcripts containing
sequence elements in reverse complementary orientation (Kurihara and Watanabe, 2004).
DCL2 mediates the generation of siRNA from RNA of exogenous sources (Xie et al., 2004).
DCL3 is required for the formation of heterochromatin-associated endogenous siRNA (Xie et
al., 2004) and DCL4 is needed for the formation of ta-siRNA involved in systemic cell-to-cell
transmission of silencing signals (Xie et al., 2005). Also all DCLs have been shown to be
involved in generation of viral-derived RNAs in coordinated hierarchical actions (Moissiard
and Voinnet, 2006). Human, mice and nematodes each contain one Dicer gene, involved in
miRNA biogenesis and the generation of siRNAs. Insects and fungi possess two Dicer genes,
the two Dicers have related but different roles, one processes miRNAs and the other is
necessary for siRNA-mediated RNAi (Margis et al., 2006). Knockout and knockdown
experiments indicate that Dicer is essential for vertebrate development (Jaskiewicz and
Filipowicz, 2008). Disruption of Dicer in mice arrests embryogenesis (Bernstein et al.,
2003). Dicer function was also found to be essential for Zebrafish development and many
processes in C. elegans. In Drosophila, Dicer involved in miRNA biogenesis is likewise an
essential gene (Jaskiewicz and Filipowicz, 2008). In mammals, Dicer is important for
protection against influenza A virus infection (Matskevich and Moelling, 2007). Dicer is also
required for the maintenance of epigenetic silencing in human to protect against cancer
(Ting et al., 2008).
Loss-of-function mutants of DCL1 in Arabidopsis revealed its role in a number of
developmental processes including embryogenesis and flower morphogenesis (Golden et al.,
2002; Park et al., 2002). The pleiotropic effects observed in these mutants were ascribed to
the lack of miRNA which was caused by the loss of miRNA biogenesis. However, other
mechanisms controlled by Dicer and related to RNAi, such as DNA methylation, chromatin
structure and centromeric silencing, may also contribute to developmental or cellular
defects.
Although recombinant Dicer is active as a dsRNA endonuclease in vitro, in cells it generally
functions in association with other proteins as a component of multiprotein complexes. In
animals, Dicer proteins were shown to be associated with other proteins forming complexes
which act as RISC or RISC loading complexes (RLC) (Tomari et al., 2004). Thus, miRNA
processing and target-RNA cleavage could be coupled. In Drosophila, it was shown that Dcr2, which produces siRNA, also acts in the RISC assembly together with its partner R2D2 by
loading one of the two siRNA strands into RISC (Tomari et al., 2004). The C. elegans
homologue of this protein, RDE-4 was also found to interact with Dicer (Tabara et al., 2002).
Similarly, human Dicer may function in loading siRNA into RISC, as siRNA does not cause
PTGS in human cells lacking Dicer (Doi et al., 2003). However, this is dependent on
10
Chapter I
Background
particular cell types as siRNA triggers gene silencing in Dicer knockout embryonic stem cells
(Kanellopoulou et al., 2005). Moreover, human Dicer, TRBP and AGO2 were present in a
protein complex which is able to perform siRNA or miRNA directed target RNA cleavage
(Gregory et al., 2005). In plants, members of the HYL1/DRB family proteins were identified
as DCL-interacting dsRBD (dsRNA-binding domain) partners and implicated in small RNA
pathways in Arabidopsis (Hiraguri et al., 2005). Another group of well characterized Dicer
partners in animals is represented by PPD or AGO proteins. Members of the PPB protein
family contain two signature domains: a PAZ domain in the center and a PIWI domain at the
carboxyl terminus (Carmell et al., 2002; Tolia and Joshua-Tor, 2007). Several other proteins
have been found to interact with Dicer. RNA-helicase-related protein, which is required for
RNAi, was found to interact with RDE-4 and Dicer in C. elegans (Tabara et al., 2002).
FMRP, an mRNA-binding protein involved in the pathogenesis of fragile X syndrome, has
been shown to interact with Dicer and AGO-1 in mammalian cells (Jin et al., 2004).
Biochemical analysis of fission yeast Dicer (Dcr1) revealed its physical and functional
association with RNA-directed RNA polymerase complex (RDRC) in transcriptional
silencing (Shiekhattar, 2007). Identification of so many Dicer-interacting proteins indicates
that Dicer participates in many cellular processes (Jaskiewicz and Filipowicz, 2008).
1.1.4 Physcomitrella patens as a model system
The moss Physcomitrella patens is a member of the bryophytes. Physcomitrella patens
occupies an important phylogenetic position for the elucidation of the development of higher
plants (Figure 5), including other model organisms, such as Arabidopsis, and plants of
commercial importance, such as poplar, corn, soybean, sorghum, and rice. In terms of
evolutionary distance, Physcomitrella is to the flowering plants what fish is to humans.
Figure 5: Land plant
evolution
(adapted
from Rensing et al.,
2008). Bryophytes comprise
three separate lineages which,
together with the vascular
plants (including the flowering
plants),
make
up
the
embryophytes (land plants).
These four lineages, remnants
of the initial radiation of land
plants in the Silurian, began to
diverge from each other about
450 million years ago.
11
Chapter I
Background
The gametophytic phase is divided into the protonema and the gametophore stages, which
produce the sporophyte upon particular conditions. Typical for mosses is the heteromorphicheterophasic alteration of generations, which is responsible for the predominant haploid
phase of the gametophyte (Figure 6), and a diploid phase that produces haploid spores.
Physcomitrella patens is a monoecious, self-fertile species, i.e. one plant carries both the
male (antheridia) and the female (archegonia) sex organs.
Figure 6: Life cycle of Physcomitrella patens (adapted from http://www.plantbiotech.net). A haploid spore germinates and grows into the filamentous protonema cells.
Starting with a three-faced apical cell bud formation is initiated which gives rise to the leafy adult
gametophyte. In monoecious moss species both sex organs (antheridia and archegonia) are present on
one and the same plant. Fertilization of the egg cell takes place in the presence of water. From the
fertilized egg the sporophyte grows out of the archegonia. Within the spore capsule the cells undergo
meiosis and new spores are formed.
These features, among others, make Physcomitrella advantagous for scientific use. Because
protonema grows quickly and simultaneously, it can be cultivated in a bioreactor as a
genetically stable cell suspension. Another advantage is that Physcomitrella can be easily
manipulated using molecular biology methods (Reski, 1998a).
A unique feature is the high efficiency of homologous recombination, therefore targeted
disruption and manipulation of single genes can be performed easily (strepp et al., 1998;
Schaefer and Zryd, 1997). The rate of homologous recombination in Physcomitrella is found
to be several orders of magnitudes higher than in any other characterized plant species
12
Chapter I
Background
(Reski, 1998b). The high rate of homologous recombination together with the predominant
haploid phase make Physcomitrella a highly suitable system to initiate forward and reverse
genetics approaches, enabling the study of gene functions related to almost all aspects of
plant biology. A considerable collection of mutants (Egener et al., 2002) and around 210.000
expressed sequence tag (EST) sequences are available (Rensing et al., 2002). Analyses have
shown that around 95% of Physcomitrella’s transcriptome is covered by these data.
The moss Physcomitrella patens genome comprises about 511 Mbp which are dispersed on
27 chromosomes. The sequence contains approximately 30,000 protein coding genes. Most
predicted genes are supported by multiple types of evidence, and 84% of the predicted
proteins appear to be complete. About 20% of the analyzed genes show alternative splicing, a
frequency similar to that of A. thaliana and O. sativa (Rensing et al., 2008). Despite its low
evolutionary position at the basis of land plants Physcomitrella shares more features with
the seed plant A. thaliana, than Arabidopsis as dicotyledonous plant with the
monocotyledon O. sativa (Reski, 1998b). Recently, a small RNA database has been
established in Physcomitrella (Arazi et al., 2005; Axtell et al., 2006; Axtell et al., 2007;
Fattash et al., 2007) and they are highly conserved in plants. Recent reports have shown that
the RNAi machinery is present and working correctly in Physcomitrella. However, in
contrast to A. thaliana and other plant species the biological function of the RNAi pathways
in Physcomitrella were not studied.
The objectives of this study are:
1. To study the biological function of RNAi pathways in the moss Physcomitrella patens,
focussing on the function of the key protein of RNAi, Dicer, by the generation of targeted
knockout plants and analyzing the pathways of small RNAs and miRNA target genes in Dicer
mutants.
2. To study gene silencing using artificial miRNAs (amiRNAs) in the moss Physcomitrella
patens as an alternative tool to targeted gene knockouts.
13
Chapter I
1.2
Results and Discussion
Results and Discussion
1.2.1 DICER-LIKE genes in Physcomitrella patens
To identify genes encoding DCL proteins BLAST searches of a Physcomitrella EST database
(Rensing et al., 2002) were performed using the four Arabidopsis DCL proteins as query.
The corresponding cDNA clones of the identified ESTs were sequenced. Analysis of these
partial cDNA sequences suggested the existence of four DCL genes in Physcomitrella (Table
3).
Table 3: Identification of DICER-LIKE genes in Physcomitrella patens. Closest A.
thaliana homologue obtained by reverse BLAST searches using the deduced amino acid sequences of
the four P. patens genes in the GenPept/nr database. The PpDCL1b sequence used to generate
ΔPpDCL1b mutant lines are underlined.
A.th. homologues
(Acc. No.)
P.p.
DCL cDNA
(Acc. No.)
PpDCL1a (EF670436)
PpDCL1b (DQ675601)
PpDCL3 (EF670437)
AtDCL1
(Q9SP32)
AtDCL2
(NP_566199)
AtDCL3
(NP_189978)
AtDCL4
(P84634)
69% identity
81% similarity
65% identity
78% similarity
32% identity
48% similarity
PpDCL4 (EF670438)
35% identity
53% similarity
These DCL genes have recently been deduced from the Physcomitrella patens genome
sequence independently (Axtell et al., 2007). Two of the Physcomitrella patens DCL proteins
(PpDCL1a and PpDCL1b) group together with AtDCL1 (Figure 7), the only A. thaliana DCL
involved in miRNA processing (Kurihara and Watanabe, 2004). Prediction of protein
domains in the Pfam database (Bateman et al., 2004) revealed the existence of all functional
domains in the two Physcomitrella patens DCL1 proteins present in the AtDCL1 protein
(Figure S2 and S3, Manuscript 1). However, compared to AtDCL1 the PpDCL1b protein
lacks approximately 240 amino acids at the N terminus. The other two PpDCL proteins are
homologs of AtDCL3 and AtDCL4, whereas an AtDCL2 homolog is lacking.
Using the homologous recombination system, M. Asif Arif generated and analyzed (in his
ongoing Ph.D. work) two targeted PpDCL1a knockout mutants (ΔPpDCL1a) (Figure S2,
Manuscript 1). The ΔPpDCL1a mutant lines show severe developmental abnormalities.
14
Chapter I
Results and Discussion
Most drastically the ΔPpDCL1a mutant lines are not able to develop leafy gametophores and
are developmentally arrested at the protonema stage (Figure 1A and 1B, Manuscript 1). The
results show that PpDCL1a is the functional equivalent of the A. thaliana DCL1 protein
required for the biogenesis of miRNAs and ta-siRNAs. Compared to wild type the expression
levels of miRNA and ta-siRNA target genes were upregulated in ΔPpDCL1a mutant lines
(Figure 1C-E, Manuscript 1). However, in Physcomitrella patens miRNAs might be
processed by additional DCLs as the detection particular miRNAs albeit at significantly
reduced expression levels (Figure 1, Manuscript 1).
The additional presence of a second AtDCL1 homolog in Physcomitrella patens suggested
potential differences in endogenous RNAi pathways in comparison to the seed plant A.
thaliana.
Figure 7: Neighbour-joining tree showing the phylogenetic relationships
between DICER-LIKE proteins. DICER-LIKE proteins from animals and plants are indicated
by vertical lines. The four groups of DICER-LIKE proteins in plants are marked by coloured boxes.
Species abbreviations are At (Arabidopsis thaliana), Ce (Caenorhabditis elegans), Cr
(Chlamydomonas reinhardtii), Dm (Drosophila melanogaster), Hs (Homo sapiens), Mm (Mus
musculus), Mt (Medicago truncatula), Nc (Neurospora crassa), Os (Oryza sativa), Pp
(Physcomitrella patens), Pt (Populus trichocarpa), Sp (Schizosaccharomyces pombe). Pp DCL
proteins are highlighted in bold. * The sequence of DCL from Chlamydomonas reinhardtii can be
retrieved at: http://genome.jgi-psf.org/chlre2. (Figure S1, Manuscript 1).
15
Chapter I
Results and Discussion
1.2.1.1 Generation and molecular analysis of ΔPpDCL1b knockout
mutants
To generate PpDCL1b knockout lines a PpDCL1b gene disruption construct was prepared by
inserting an nptII selection marker cassette into a 560 bp fragment of the PpDCL1b cDNA
which encompasses the coding region of the second RNAseIII domain present in the DCL1b
protein (Seumel, 2004; Figure S3, Manuscript 1). The resulting construct was used for
transfection of Physcomitrella protoplasts. After selection of regenerating plants they were
analyzed by PCR to identify mutant lines which had integrated the disruption construct at
the DCL1b genomic locus. Out of a total of 520 analyzed transgenic lines 8 lines (1.54%)
unable to produce PpDCLb1 transcripts were identified. Four lines were used for further
studies (ΔPpDCL1b 1-4). The full-length cDNA sequence of PpDCL1b was obtained (Figure 8,
Appendix 4.2), the cDNA was termed DCL1b encoding a protein of 1695 amino acids.
Furthermore, the haploidy of all ΔPpDCL1b mutant lines was verified by flow cytometry to
exclude the possible generation of diploid lines by protoplast fusion during the
transformation process (Appendix 4.1).
Figure 8: Cloning and sequencing of the PpDCL1b cDNA. The PpDCL1b cDNA is
indicated by a black line. The colored arrows above depict cDNA fragments obtained by different
cloning steps. The numbers in the arrows refer to the corresponding nucleotide positions in the DCL1
cDNA. First, a cDNA clone comprising the 3’ end of PpDCL1b was sequenced. Subsequently, three 5’
RACE-PCRs using the BD Smart RACE cDNA Amplification Kit (Clontech) and one RT-PCR was
performed. The primers for the RT-PCR were derived from available Physcomitrella genomic trace
files. All PCR and 5’ RACE primers were selected to give rise to overlapping PCR fragments of already
known sequence stretches to confirm that the amplicons were derived from the same cDNA. The
following primers were used:
5‘RACE-PCR 1: 5’- GAACTCCCAACGATGGTCGAGACGC-3’
5’RACE-PCR 2: 5’- CCAGCT CATCGTGATCAGTAAAGTCGGG -3’
5‘RACE-PCR 3: 5’-TCCCAGCGCCCGTGTCTAGAAATGCAAC -3’
RT-PCR: 5’-GAGAGGCGGTCTGTGTCGAGGTCTAG -3’ and 5’-TTGTAGCCACCAGCAACGTCACCCGT
-3’
16
Chapter I
Results and Discussion
1.2.1.1.1 Knockout of PpDCL1b causes developmental disorders
The ΔPpDCL1b mutant lines showed developmental disorders throughout all stages of
protoplast regeneration including abnormalities in cell division, growth polarity, cell size,
cell shape and growth of tissues (Figure 2A, Manuscript 1). Moreover, these mutants
developed only a small number of gametophores, which in addition were malformed (Figure
9). The observed developmental effects are consistent with previous studies of Dicer mutants
in animals and plants. The pleiotropic effects observed in these mutants were ascribed to the
lack of miRNA which was caused by the loss of miRNA biogenesis.
Figure 9: Phenotypic analysis of the ΔPpDCL1b mutants. Electron micrographs of
gametophores from wild type plants and ΔPpDCL1b mutant 1 (Figure 2B, Manuscript 1).
1.2.1.1.2 MiRNA biogenesis is not affected and miRNA-directed cleavage
of mRNA-targets is abolished in ΔPpDCL1b mutant lines
The isolated PpDCL1b gene from Physcomitrella shows 65% identitiy and 78% similarity to
to the DCL1 gene from Arabidopsis (Table 3), encoding the essential enzyme required for the
generation of miRNAs from pre-miRNA precursors. If the high sequence conservation causes
considerable overlap in function one would expect the absence of miRNAs in the ΔPpDCL1b
mutant lines. Interestingly, the accumulation of miRNAs in ΔPpDCL1b mutant lines
compared to the wild-type was present in almost equal amounts (Figure 2C, Manuscript 1),
indicating that PpDCL1b is not required for processing of miRNA precursors in
Physcomitrella. In contrast, miRNA-triggered cleavage of miRNA target genes, encoding
different transcription factors, was abolished in the ΔPpDCL1b mutant lines (Figure 3A,
Manuscript 1). The abolished miRNA-directed cleavage of target mRNAs in the ΔPpDCL1b
mutant lines suggests a direct involvement of PpDCL1b in this step of miRNA action. The
requirement of DCL proteins for target cleavage was not shown in plants. It is unlikely that
PpDCL1b directly cleaves mRNA targets as this function is commonly associated with AGO
proteins present in the RISC (Liu et al., 2003). Studies in animals have shown Dicer in
17
Chapter I
Results and Discussion
association with protein complexes (Tabara et al., 2002; Liu et al., 2003; Lee et al., 2004;
Pham et al., 2004). Some of these complexes, like the Dcr-2/R2D2 heterodimer from
Drosophila act in loading siRNA into RISC (Liu et al., 2003). It is possible that
Physcomitrella patens DCL1b functions in RISC loading like Dcr-1 and Dcr-2 from
Drosophila. Until now, only the Arabidopsis protein HYL1 was shown to interact with
Arabidopsis DCL1 in vitro (Hiraguri et al., 2005). However, even though HYL1 shows high
similarity to the Drosophila R2D2, a function in RISC loading is unlikely as dsRNA triggered
gene silencing is not affected in hyl1 mutants. Analysis of miRNA expression indicated that
HYL1 plays a role in miRNA biogenesis as miRNA levels were reduced in the hyl1 mutant
(Han et al., 2004).
1.2.1.1.3 Generation of transitive siRNA in ΔPpDCL1b mutant lines
In Physcomitrella wild type, 5’RACE-PCRs performed from the miRNA targets yielded
additional fragments besides the expected cleavage products, indicating additional cleavage
of the mRNAs at sites other than the miRNA binding site (Figure 3A, Manuscript 1). The
mRNA cleavage products may serve as templates for synthesizing cRNA by RdRP (Vaistij et
al., 2002) leading to the formation of dsRNA. Subsequently, the dsRNA may be processed
into secondary siRNAs resulting in spreading of the initial miRNA signal (Figure 3B,
Manuscript 1). In plants, this mechanism, known as transitivity, is initiated by dsRNA
triggers (e. g. viruses and transgene transcripts) and transcripts that are targeted by more
than one small RNA (Moissiard et al., 2007). In seed plants, the generation of transitive
siRNAs from miRNA cleavage products is the exception. To prove the occurrence of
transitivity in Physcomitrella, sense and antisense oligonucleotides derived from PpARF and
PpC3HDZIP1 mRNA regions upstream and downstream of the miRNA binding sites were
used. Sense and antisense siRNAs were only detected in wild type whereas siRNAs derived
from miRNA targets were lacking in the ΔPpDCL1b mutants (Figure 10). In Physcomitrella
the generation of siRNAs depends on PpDCL1b function and is specific for miRNA-directed
cleavage of target RNAs.
Figure 10: Detection of
transitive siRNAs derived
from miRNA target genes.
Detection of sense and antisense
siRNAs derived from PpARF and
PpC3HDZIP1 with oligonucleotides
derived from regions upstream and
downstream the miRNA binding
sites.
Hybridisation
with
an
antisense probe for U6snRNA served as control to indicate equal loading (Figure 3C, Manuscript 1).
18
Chapter I
Results and Discussion
1.2.1.1.4 Analysis of DNA methylation in ΔPpDCL1b mutants and wild
type
When miRNA targets are not cleaved, the respective mRNAs are likely to accumulate to
higher levels in the ΔPpDCL1b mutant lines. Conversely, in ΔPpDCL1b mutants all miRNA
targets analyzed had reduced transcript levels when compared to wild type (Figure 11),
although these mRNAs were not cleaved in these mutants. It is tempting to speculate that
other RNAi components may sense the defective target cleavage as some of them were shown
to direct heterochromatin formation and gene silencing. One probable explanation for these
unexpected findings is a yet undiscovered epigenetic control of genes encoding miRNA
targets in Physcomitrella. Since methylation of cytosine residues is the most prominent
mechanism for transcriptional silencing in eukaryotes (Bender, 2004), this possibility was
tested by methylation-specific PCR from the miRNA target genes and the control gene
(PpGNT1) which is not regulated by a miRNA.
Figure 11: Expression levels of miRNA target
genes in ΔPpDCL1b mutants and wild type. RNA
blots analysis of miRNA target genes PpARF, PpC3HDZIP1,
PpHB10 and PpSBP3 and two control genes, PpGNT1 and
PpEF1α. (Figure 4B, Manuscript 1).
In some cases, endogenous siRNAs have an influence
on
epigenetic
control,
DNA
methylation
and
chromatin structure at target loci and are associated with RNA-directed DNA methylation
(RdDM) and chromatin remodeling (Hamilton et al., 2002; Zilberman et al., 2003; Xie et al.,
2004). In plants, dsRNAs which contain sequences that are homologous promoter regions
can trigger promoter methylation and transcriptional gene silencing (Melquist and Bender,
2003; Matzke and Birchler, 2005). A function of miRNA 165/166 in directing DNA
methylation was shown in the regulation of the homeodomain-leucine zipper (HD-ZIP)
transcription factor genes PHABULOSA (PHB) and PHAVOLUTA (PHV) in Arabidopsis
(Bao et al., 2004).
Promoter regions of the miRNA target genes and the control gene were unmethylated in wild
type, whereas in the ΔPpDCL1b mutants the promoters of the genes encoding miRNA targets
were methylated (Figure 4D, Manuscript 1). In the latter, methylation occurred specifically
at CpG residues (Figure S7, Manuscript 1). In contrast, the promoter of the control gene
PpGNT1 remained unaffected in the mutants. Taken together, this reveals a specific
epigenetic control of genes encoding miRNA targets upon PpDCL1b dysfunction and
subsequent impeded miRNA-directed mRNA cleavage.
19
Chapter I
Results and Discussion
In Physcomitrella the pC3HDZIP1 and PpHB10 harbor an intron within their miRNA
binding site (Figure S8, Manuscript 1). Therefore, it is unlikely that DNA methlyation is
initiated by the formation of an miRNA:DNA hybrid. The miRNA:mRNA duplex may be
required to control the DNA methlyation. In Arabidopsis, the composition of a nucleolar
complex involved in the siRNA-directed silencing of endogenous repeat regions has been
recently identified (Bao et al., 2004). This complex combines several proteins which have
been linked to RdDM including RDR2, DCL3 and AGO4. In the yeast Schizosaccharomyces
pombe, the RITS complex containing AGO1, a chromodomain protein (Chp1) and other
proteins, was shown to bind siRNAs to direct DNA heterochromatin formation (Verdel et al.,
2004). In Physcomitrella, the detection of the miRNA:mRNA duplexes in the ΔPpDCL1b
mutant lines (Figure 4G, Manuscript 1) suggests that the miRNAs are not incorporated
into the RISC but may form a free duplex. Subsequently, this duplex guides the RITS
complex to the corresponding genomic region resulting in the initiation of DNA methylation.
1.2.1.1.5 Analysis of the ta-siRNA pathway in ΔPpDCL1b mutants
To challenge the findings obtained from the transitivity and miRNA-dependent DNA
methylation the ta-siRNA pathway was analysed. In Physcomitrella, all four ta-siRNA
precursors (TAS1-4 RNAs) analyzed to date are cleaved within two distinct miRNA390
binding sites resulting in the production of ta-siRNAs (Axtell et al., 2006; Talmor-Neiman et
al., 2006). The mRNA encoding an EREBP/AP2 transcription factor is targeted by one of the
ta-siRNAs derived from the TAS4 precursor (Talmor-Neiman et al., 2006). The abolished
miRNA390-directed cleavage of TAS4 precursor resulted the lack of ta-siRNAs in ΔPpDCL1b
mutant lines (Figure 5A and B, Manuscript 1) revealing that PpDCL1b is required to
initiate the ta-siRNA pathway. According to the findings obtained from miRNA target genes,
the lack of ta-siRNAs in the ΔPpDCL1b mutants should abolish cleavage of the EREBP/AP2
mRNA and subsequently transitive siRNAs derived from it should be missing. In agreement
with findings for miRNA target genes the level of target TAS4-RNA was down-regulated in
the ΔPpDCL1b mutants (Figure 12), and the TAS4 genomic locus was methylated in the
ΔPpDCL1b mutants but not in wild type (Figure 5D, Manuscript 1). Indeed, the mRNA
level of EREBP/AP2 was increased in the ΔPpDCL1b mutants (Figure 12) and the cognate
genomic locus was unmethylated in wild type but methylated in the ΔPpDCL1b mutants
(Figure 5D, Manuscript 1).
Figure 12: Analysis of expression levels of
PpTAS4 and PpEREBP/AP2 in ΔPpDCL1b
mutants and wild type. RNA blots analysis of
PpTAS4 and PpEREBP/AP2. Ethidium bromide staining
shown as loading control below. (Figure 5C,
Manuscript 1).
20
Chapter I
Results and Discussion
1.2.1.1.6 Analysis of ΔPpDCL1b mutants and wild type lines expressing
amiR-GNT1
To check whether the mechanism of epigenetic silencing occurred in Physcomitrella wild
type, an amiRNA targeting the control gene PpGNT1 in Physcomitrella wild type as well as in
the ΔPpDCL1b mutants was used. PpGNT1-amiRNA was expressed from the Arabidopsis
thaliana miR319a precursor fused to a constitutive promoter. Transgenic Physcomitrella
lines harboring the overexpression construct showed precise processing of the PpGNT1amiRNA (Figure 6B, Manuscript 1). However, normalization of the PpGNT1-amiRNA
hybridization signal to the U6snRNA control revealed amiRNA expression levels which
differed between the individual lines (Figure 6B, Manuscript 1). In agreement with the
results obtained from miRNA target genes, the cleavage product of PpGNT1 in the
ΔPpDCL1b mutant background was not detect (Figure 6C, Manuscript 1), and an efficient
knock-down of the PpGNT1 gene in the plants expressing the PpGNT1-amiRNA and the
transcript level of PpGNT1 even lower in the ΔPpDCL1b mutant background (Figure 6D,
Manuscript 1). The PpGNT1 promoter was methylated in the ΔPpDCL1b mutant
background (Figure 6E and Figure S9, Manuscript 1). Also DNA methylation at the
PpGNT1 promoter in the wild type background which showed a strong expression of the
PpGNT1-amiRNA was detected whereas the PpGNT1 promoter was unmethylated in the wild
type background expressing the PpGNT1-amiRNA at a low level (Figure 6E and Figure S9,
Manuscript 1). This finding suggests that the ratio of the miRNA and its target mRNA is
crucial for the induction of DNA methylation at the target locus. At low concentrations the
miRNA might be effectively loaded into a cleavage competent RISC to direct target cleavage.
If the miRNA concentration reaches a certain threshold the RISC loading capacity for the
miRNA might be limited and excessive miRNAs form a duplex with their targets, the excess
miRNA might be loaded immediately into an effector complex such as RITS triggering
duplex formation that directs DNA methylation (Figure 7, Manuscript 1).
1.2.1.1.6.1 Specific methylation of a miRNA1026 target gene in response
to the phytohormone abscisic acid (ABA)
The results obtained from the analysis of PpGNT1-amiRNA expressing lines indicated that in
Physcomitrella patens miRNAs control the expression of target RNAs at the posttranscriptional and transcriptional level (Figure 7, Manuscript 1). Expression profiling
experiments using a Physcomitrella microarray (unpublished data) revealed an ABAmediated repression of a gene encoding a basic helix-loop-helix transcription factor
(PpbHLH) in wild type and was down-regulated in ΔPpDCL1b mutants (Figure 13). This
21
Chapter I
Results and Discussion
gene has been predicted to be targeted by the Physcomitrella miRNA1026 (Axtell et al.,
2007).
Figure 13: Expression profile of PpbHLH.
Expression level of PpbHLH down-regulated in response
to 10 µM ABA and in ΔPpDCL1b mutants.
RNA gel blots confirmed the down-regulation of
PpbHLH in response to ABA and corresponding
ABA-induced increase of miRNA1026 expression
levels (Figure 6G and H, Manuscript 1). In
agreement with hypothesis that miRNAs control the expression of their targets at the posttranscriptional and transcriptional level, the PpbHLH promoter as well as intragenic regions
were found to be methylated in the plants treated with ABA (Figure 6J, Manuscript 1).
From these results, epigenetic silencing of miRNA target loci contributes to the control of
target gene expression in Physcomitrella was concluded. Although this phenomenon in
ΔPpDCL1b mutants was initially discovered, subsequent analyses of the miR1026/PpbHLH
regulon confirmed that this type of miRNA-dependent control operates also in wild type.
1.2.1.1.7 Expression profiling of transcription factor genes in ΔPpDCL1b
mutant lines
Approximately 6% of the protein coding genes are considered to encode transcription factors
in Arabidopsis (Riechmann et al., 2000). In addition, more than 50 % of the predicted
miRNA target genes belong to the class of transcription factor encoding mRNAs (Rhoades et
al., 2002). In Physcomitrella patens, the comparison of the expression pattern of
transcription factor encoding genes between wild type and ΔPpDCL1b mutants will identify
putative candidate genes, which are regulated by RNAi. It is likely that more genes which are
miss-regulated in the ΔPpDCL1b mutants and direct miRNA and ta-siRNA targets were able
to be identified.
RNA from wild type and two ΔPpDCL1b mutants was hybridized on a custom Combimatrix
12K oligonucleotide microarray representing 1,427 Physcomitrella patens assembled
transcript sequences encoding more than 400 Transcription Associated Proteins (TAPs).
Corresponding gene models assigned for 1,200 assembled transcripts (Richardt, 2009). In
ΔPpDCL1b mutants, all previously analyzed miRNA targets (PpARF, PpC3HDZIP1, PpHB10
and PpSBP3) were downregulated and the ta-siRNA target gene (PpEREBP/AP2) was
upregulated when compared to wild type. I hypothesized that the downregulated genes of
transcription factors in Physcomitrella ΔPpDCL1b mutants could be putative miRNA target
genes and the upregulated ones could be ta-siRNA target genes. Clustering of expression
22
Chapter I
Results and Discussion
profiles showed different gene expression between ΔPpDCL1b mutant lines and wild-type
plants (Figure 14).
Figure 14: Expression profiling of genes in ΔPpDCL1b mutant lines and Wild
type. Differential gene expression in ΔPpDCL1b mutant lines and wild-type plants, 213 genes which
were downregulated and 273 genes upregulated in ΔPpDCL1b mutant lines (Appendix 4 and 5).
MiRNA and ta-siRNA target genes supposed to be downregulated and upregulated in ΔPpDCL1b
mutant lines, respectively.
46 miRNA target genes are present on the Combimatrix 12K oligonucleotide microarray.
Normalization and statistical analysis identified 20 miRNA target genes differentially
expressed within Physcomitrella ΔPpDCL1b mutant lines; the analysis revealed 13 miRNA
target genes downregulated (Table 4) and 7 miRNA target genes upregulated in ΔPpDCL1b
mutant lines. By analyzing all upregulated genes in ΔPpDCL1b mutants, the ta-siRNA target
genes were predicted using the RNA hybrid program (I. Fattash, personal communication).
The parameters used in this program are adopted from Schwab et al. (2005). In agreement
with findings for ta-siRNA target genes, the analysis revealed 19 ta-siRNA target genes
upregulated in ΔPpDCL1b mutant lines (Table 5).
23
Chapter I
Results and Discussion
Table 4: MiRNA target genes downregulated in ΔPpDCL1b mutant lines
MiRNAs
Target accession
(Gene model)
miR1026ab
miR1026ab
miR166
miR166
miR166
miR166
miR414
miR414
miR477a
miR538abc
miR538abc
miR902f
miR904
Phypa1_132150 †
Phypa1_209063 ‡
Phypa1_116038 ‡
Phypa1_182184 ‡
Phypa1_184087 ‡
Phypa1_192868 ‡
Phypa1_167487 ‡
Phypa1_145753 ‡
Phypa1_130477 ‡
Phypa1_109598 ‡
Phypa1_94754 ‡
Phypa1_199042 †
‡
Phypa1_141045
Sequence ID
(EST)
PP015054317R
PP_12500_C1
PP015020123R
PP020016117R
PP_SD_92_C1
BJ580674
PP_9369_C1
PP_4238_C1
PP_323_C1
PP020062195R
PP_SD_0_C1
PP030015063R
PP015071162R
Target description (Annotation)
12-oxophytodienoate reductase (OPR1)
basic helix-loop-helix (bHLH) family protein
class III HD-Zip protein HB12
class III HD-Zip protein HB11
class III HD-Zip protein HB10
class III HD-Zip protein HB14
Helix-loop-helix DNA-binding
translation initiation factor 3 subunit 3 / eIF-3
Photosystem subunit V, chloroplast precursor
MADS-domain protein PPM2
agamous-like MADS box protein AGL1
polyubiquitin (UBQ4), identical to GI:17677
AGO1-1 (Nicotiana benthamiana)
Fold
change
-1.5
-3.2
-1.5
-2.0
-2.0
-2.0
-1.6
-1.6
-1.6
-2.0
-2.0
-1.5
-1.4
Table 5: Ta-siRNA target genes upregulated in ΔPpDCL1b mutant lines
TasiRNAs
Target accession
(Gene model)
†
PpTAS2
Phypa1_160018
PpTAS1
PpTAS1
PpTAS2
PpTAS1
PpTAS1
PpTAS2
PpTAS1
PpTAS1
PpTAS3
PpTAS1
PpTAS1
PpTAS1
PpTAS1
PpTAS1
PpTAS1
PpTAS1
PpTAS3
PpTAS3
Phypa1_188484 †
Phypa1_170836 †
Phypa1_53217 †
Phypa1_184404 †
Phypa1_123311 †
Phypa1_61310 †
Phypa1_168363 †
Phypa1_175333 †
Phypa1_203982 †
†
Phypa1_13874
Phypa1_216494 †
Phypa1_165365 †
Phypa1_142162 †
Phypa1_15899†
Phypa1_138749 †
Phypa1_167719 †
Phypa1_79139 ‡
Phypa1_161831 †
‡
Sequence ID
(EST)
PP_10130_C2
PP_10320_C1
PP_13554_C1
PP_12145_C1
PP_13985_C1
PP_15546_C1
PP_15997_C1
PP_17900_C1
PP_18393_C1
PP_10621_C1
PP_584_C1
PP_12254_C1
PP_8332_C1
PP_8343_C1
PP004007192R
PP004043210R
PP004103024R
PP015028003R
PP020043294R
Target description (Annotation)
Q8H9A2 Dehydratiion responsive element
binding protein 1 like protein
Putative nuclear DNA-binding protein G2p
Q9LKG4 Putative DNA binding protein.
Homolog of hypothetical protein sativa
Arabidopsis thaliana genomic DNA,
Q9LW84 Gb|AAF26996.1.
Q9SI75 F23N19.11 Hypothetical protein
Homolog of (AJ131113) VP1/ABI3-like protein
not annotated Physcomitrella patens
Q9FPV8 Putative methionine aminopeptidase
scarecrow-like transcription factor 3 (SCL3)
Homolog of lateral suppressor protein
Homolog of AP2 domain,
Putative 2-isopropylmalate synthase
Q9FJ91 Dbj|BAA78737.1 AT5g52010
Q9SGT9 T6H22.8.2 protein.
O99018 Chloroplast protease precursor.
Homolog of zinc finger B-box type family
Mitochondrial transcription termination factor
Fold
change
1.8
1.8
1.3
1.8
1.4
1.7
3.0
1.4
2.0
1.3
2.1
1.7
7.8
3.0
2.0
4.1
1.7
3.6
3.3
Target validated, † Target predicted
1.2.2 Highly specific gene
Physcomitrella patens
silencing
by
artificial
miRNAs
in
Artificial miRNA (amiRNA) are single-stranded 21-nt small RNAs, which have been used to
downregulate single or multiple protein coding genes by guiding their cleavage based on
sequence complementarity. Their sequences are designed according to known determinants
of target selection for natural miRNAs (Schwab et al., 2005; Schwab et al., 2006). Previous
reports have shown that DNA sequences encoding Arabidopsis pre-miRNAs can be
expressed from the constitutive CaMV35S promoter in transgenic plants to produce mature
24
Chapter I
Results and Discussion
miRNAs. Moreover, alterations of several nucleotides within a miRNAs 21-nt sequence do
not affect its biogenesis and maturation (Vaucheret et al., 2004). These findings raise the
possibility of modifying miRNA sequences according to the determinant miRNA target
selection, such that the 21-nt specifically silence their intended target gene(s). In humans
miR30 precursor has been modified to generate an amiRNA to downregulate gene
expression by translation inhibition (Boden et al., 2004; Dickins et al., 2005). Arabidopsis
miRNA precursors have been modified to silence endogenous and exogenous target genes in
the dicotyledonous plants Arabidopsis, tomato and tobacco (Parizotto et al., 2004; Alvarez et
al., 2006; Niu et al., 2006; Schwab et al., 2006; Qu et al., 2007). Gene silencing in
monocotyledon species by amiRNAs has been reported (Warthmann et al., 2008). Previous
results have shown that artificial ta-siRNAs (ata-siRNAs) confer consistent and effective
gene silencing in Arabidopsis by engineering the TAS1c (ta-siRNAs1c) locus to silence the
FAD2 gene (de la Luz Gutierrez-Nava et al., 2008). So amiRNAs and ata-siRNAs make an
effective tool for specific gene silencing in plants.
In the moss Physcomitrella patens analysis of gene function can be carried out by the
generation of targeted gene knockout lines. However, the development of an amiRNA
expression system will be a valuable alternative to speed up such analyses. As a proof of
concept two amiRNAs, targeting the gene PpFtsZ2-1, which is indispensable for chloroplast
division
(Strepp
et
al.,
1998),
and
the
gene
PpGNT1
encoding
an
N-
acetylglucosaminyltransferase (Koprivova et al., 2003) were designed.
Both amiRNAs were expressed from the Arabidopsis thaliana miR319a precursor fused to a
constitutive promoter (Figure 1A, Publication 1). Based on the conservation of the miR319
family among land plants and similar secondary structures of miR319 precursor transcripts
from Arabidopsis and Physcomitrella (Figure 1B, Publication 1), the PpFtsZ2-1-amiRNA
and PpGNT1-amiRNA were correctly processed from the Arabidopsis miR319a precursor.
Transgenic Physcomitrella lines harboring the overexpression constructs showed precise
processing of the amiRNAs and an efficient knockdown of the cognate target mRNAs (Figure
1D and 2A, Publication 1). Furthermore, chloroplast division was impeded in PpFtsZ2-1amiRNA lines which phenocopied PpFtsZ2-1 knockout mutants (Figure 15). The formation of
macrochloroplasts in the PpFtsZ2-1-amiRNA lines was observed in all tissues and cells
analyzed indicating an efficient production of mature amiRNAs from constitutively
expressed precursor transcripts. To investigate the possibility of transitivity, sense and
antisense oligonucleotides derived from a PpFtsZ2-1 and PpGNT1 mRNA regions
downstream the amiRNA recognition site were used for RNA gel blot analysis.
Sense and antisense siRNAs were only detected in PpFtsZ2-1-amiRNA and PpGNT1amiRNA lines, but not in wild type (Figure 2B and C, Publication 1). Additionally, these
25
Chapter I
Results and Discussion
siRNAs do not seem to have a major effect on sequence-related mRNAs, confirming
specificity of the amiRNA approach.
Figure 15: Impeded plastid divison and formation of macrochloroplasts in
PpFtsZ2-1-amiRNA overexpressors. A, Light microscopy from protonema and leaves of wild
type (WT) and one PpFtsZ2-1-amiRNA overexpression line (Size bars: 100 µm). B, Confocal laser
scanning microscopy from protonema and leaves of wild type (WT) and one PpFtsZ2-1-amiRNA
overexpression line (Size bars: 50 µm). Red: chlorophyll autofluorescence in plastids. (Figure 3,
Publication 1).
26
Chapter I
1.3
Conclusion
Conclusion
These findings reveal the existence of novel RNAi pathways in Physcomitrella patens. As
opposed to Arabidopsis thaliana, the miRNA-directed posttranscriptional control of target
mRNAs in Physcomitrella patens is amplified by transitive siRNAs. Furthermore, we
identified a pathway that depends on miRNA:targetRNA duplexes and triggers the silencing
of genes encoding miRNA targets. Consequently, ΔPpDCL1b mutants deficient in miRNA
target cleavage are not viable in some plants and animals. In contrast, Physcomitrella
ΔPpDCL1b mutant lines are viable, although severely affected in several cellular features and
in development. From that a model for gene-specific sensing of the levels of specific miRNAs
and their target-RNAs (by miRNA:mRNA or miRNA:TAS-RNA duplex formation) was
proposed, effective (or ineffective) target cleavage, and subsequent epigenetic control of
target-RNA accumulation (Figure 16).
In summary, the conclusions are:
1- PpDCL1a is the functional equivalent of Arabidopsis AtDCL1 (miRNA biogenesis).
2- PpDCL1b is essential for miRNA target cleavage (including the ta-siRNA pathway).
3- In Physcomitrella, amplification of miRNA and ta-siRNA signals by transitive siRNAs is a
common mechanism.
4- The accumulation of miRNAs and their cognate RNA targets in the ΔPpDCL1b mutants
causes a specific hypermethylation of the corresponding genomic loci.
5- MiRNAs induce a specific epigenetic silenicng of miRNA target genes which depends on
the miRNA:target ratio and is mediated by the formation of stable miRNA:RNA duplexes.
6- The expression of amiRNAs in Physcomitrella leads to an efficient silencing of their target
mRNAs comparable to the effects of targeted gene knockouts.
7- The amplification of the initial amiRNA signal by secondary transitive siRNAs, these
siRNAs do not have a major effect on highly conserved gene families, confirming specificity
of the amiRNA approach in Physcomitrella.
27
Chapter I
Conclusion
Figure 16: Model for the post-transcriptional and epigenetic control of miRNA
target genes in Physcomitrella patens. (A) Pathways leading to miRNA target cleavage.
The maturation of miRNAs from stem-loop precursors is catalyzed by PpDCL1a. PpDCL1b is required
for loading miRNAs into cleavage competent RISC. After loading of miRNAs into RISC (consisting of
PpDCL1b, AGO and unknown proteins) transient miRNA:target-RNA duplexes form based on
sequence complementarity. Subsequently, target-RNAs are cleaved. From the cleavage products
dsRNA is produced by the action of RdRP. Subsequently, the dsRNA is processed to generate
transitive siRNAs (from mRNA cleavage products) or ta-siRNAs (from TAS-RNAs).Transitive siRNAs
lead to an amplification of the miRNA trigger; ta-siRNAs are directed to their mRNA targets guiding
their cleavage. (B) Epigenetic control of miRNA target genes. In the ΔPpDCL1b mutant lines miRNAs
are not loaded into cleavage competent RISC and target cleavage is abolished. Also in Physcomitrella
patens wild type miRNAs can accumulate which cannot be loaded efficiently into RISC. In both cases
miRNAs may be loaded into alternative complexes such as the RITS complex and targeted to cognate
target-RNAs. These miRNA:RNA duplexes bound by RITS enter the nucleus and initiate DNA
methylation at complementary genomic loci. The RITS complex expands into adjacent regions (e. g.
promoters) and completes CpG methylation of the entire genomic locus. In consequence, genomic loci
are silenced and accumulation of mRNAs is inhibite
28
Chapter I
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33
Chapter II
Transcriptional control of gene expression by microRNAs
2 Chapter II: Manuscript 1
Transcriptional control of gene expression by microRNAs
Own contribution:
Carried out all molecular and phenotypic analyes of the ΔPpDCL1b mutants, generated and
analyzed the amiR-GNT1 overexpression lines, and performed the complete analysis of the
miR1026 and its PpbHLH target. Writing parts of the manuscript and preparing the figures.
The work was supervised by W. Frank
34
Transcriptional control of gene expression by microRNAs
Basel Khraiwesh1, M. Asif Arif1, Gotelinde I. Seumel1, Stephan Ossowski2, Detlef
Weigel2, Ralf Reski1,3,4, Wolfgang Frank1,3*
1
Plant Biotechnology, Faculty of Biology, University of Freiburg, Schänzlestraße 1, D-
79104 Freiburg, Germany
2
Department of Molecular Biology, Max Planck Institute for Developmental Biology, D-
72076 Tübingen, Germany
3
Freiburg Initiative for Systems Biology (FRISYS), Faculty of Biology, University of
Freiburg, Schänzlestr. 1, D-79104 Freiburg, Germany
4
Centre for Biological Signalling Studies (bioss), University of Freiburg, Schänzlestr. 1,
D-79104 Freiburg, Germany
* Corresponding author
Phone:
+49 (0)761-203-2820
Fax:
+49 (0)761-203-6945
Email:
[email protected]
1
Summary
MicroRNAs (miRNAs) control gene expression in animals and plants. They share with
another class of small RNAs, siRNAs, the ability to post-transcriptionally affect target
mRNAs. In contrast to siRNAs, however, the role of miRNAs in transcriptional
regulation has been less clear. Here we reveal dual transcriptional and posttranscriptional activities of miRNAs in Physcomitrella patens. In plants lacking activity
of one DICER-LIKE gene (PpDCL1b), miRNA target genes are silenced. The specific
function of PpDCL1b in miRNA-mediated target cleavage suggests that changes in the
ratio of the miRNA and its targets cause miRNA:target-RNA duplex formation, which in
turn triggers DNA methylation. We propose that miRNA-mediated transcriptional
silencing, which also occurs in wild type plants, provides a mechanism critical for
homeostasis of miRNA-dependent gene expression.
Introduction
Small RNAs (sRNAs) are important regulators of post-transcriptional and transcriptional
gene expression (Meister and Tuschl, 2004). In plants, microRNAs (miRNAs), which
are produced from hairpin-like precursor transcripts, are also required for the
biogenesis of trans-acting small interfering RNAs (ta-siRNAs). Both miRNAs and tasiRNAs regulate mRNA stability and translation, siRNAs, which originate from perfectly
double-stranded RNA (dsRNA) precursors post-transcriptionally silence transposons,
viruses and transgenes and are important for the establishment and maintenance of
cytosine DNA methylation (Baulcombe, 2004). Even though the role of plant siRNAs in
the methylation of cognate genomic loci is well understood (Matzke et al., 2007),
evidence for a similar function of miRNAs in directing DNA methylation is limited. The
biogenesis of sRNAs from dsRNA is catalyzed by Dicer proteins and the size of the
Dicer gene family varies between organisms, reflecting different degrees of
specialization of Dicer proteins. For example, in D. melanogaster Dcr1 produces
2
miRNAs from hairpin precursors, whereas Dcr2 generates siRNAs from dsRNA
molecules (Tomari and Zamore, 2005). By contrast, in C. elegans the single Dicer
protein DCR-1 is directed by accessory proteins such as PIR-1, ER-1 and RRF-3 to
produce sRNAs from different dsRNA triggers (Duchaine et al., 2006). Besides their
function in dicing dsRNA, animal Dicers are associated with accessory proteins in
complexes which act as RISC (RNA-induced silencing complex) or RISC loading
complexes (Doi et al., 2003; Pham et al., 2004). Thus, Dicer proteins are also essential
components in the executive phase of RNAi, indicating that miRNA/siRNA processing
and target RNA cleavage are coupled. Dcr-2 from Drosophila, which produces siRNA,
acts together with its partner R2D2 to load one of the two siRNA strands into RISC(Liu
et al., 2003; Tomari et al., 2004). Similarly, human Dicer associated with Ago2, TRBP
and RHA acts in RISC assembly (MacRae et al., 2008) which is further supported by
the observation that siRNAs cannot cause post-transcriptional gene silencing in human
cells lacking Dicer (Doi et al., 2003).
In the plant A. thaliana, the four DCL proteins (AtDCL1-4) act in specific sRNA
pathways, with some functional redundancies of the four isoforms (Gasciolli et al.,
2005; Henderson et al., 2006). The maturation of miRNAs from imperfect RNA
foldbacks relies on AtDCL1 activity. In consequence, A. thaliana dcl1 mutants have
significantly reduced miRNA levels and a corresponding increase in target mRNA
levels, which causes a multitude of developmental defects (Golden et al., 2002; Park et
al., 2002). AtDCL2 mediates the generation of siRNAs from exogenous RNA sources
(Xie et al., 2004), AtDCL3 is required for the formation of heterochromatin-associated
endogenous siRNAs (Herr et al., 2005; Xie et al., 2004) and AtDCL4 is needed for the
formation of ta-siRNAs involved in systemic cell-to-cell transmission of silencing signals
(Dunoyer et al., 2005; Xie et al., 2005).
The genome of the moss Physcomitrella patens encodes four DCL proteins
(Axtell et al., 2007). PpDCL1a and PpDCL1b are very similar to AtDCL1 (Figure S1).
3
PpDCL3 and PpDCL4 proteins are orthologs of AtDCL3 and AtDCL4, whereas an
AtDCL2 ortholog is lacking. The primary PpDCL1a transcript harbors a miRNA
precursor within one intron, which is reminiscent of AtDCL1, and suggests a conserved
autoregulatory control of mRNA maturation (Axtell et al., 2007). Together with the
slightly greater sequence similarity, this led us to hypothesize that PpDCL1a as the
functional equivalent of AtDCL1 is required for miRNA biogenesis, while the additional
presence of PpDCL1b suggested also potential differences in sRNA pathways between
P. patens and A. thaliana. Here, we present an analysis of P. patens ΔPpDCL1a and
ΔPpDCL1b knockout mutants, which supports differences in sRNA pathways such as
the formation of transitive siRNAs. Moreover, we propose a mechanism for the miRNAmediated transcriptional silencing of miRNA target genes that relies on miRNA
abundance, formation of miRNA:target-RNA duplexes and DNA methylation.
Results
Requirement of PpDCL1a for miRNA biogenesis
Taking advantage of the efficient homologous recombination system in P. patens
(Strepp et al., 1998) we generated two PpDCL1a knockout mutants (ΔPpDCL1a)
(Figure S2). Complete loss of PpDCL1a function resulted in retarded growth and
developmental disorders including abnormalities in cell size, and cell shape. The
mutants were arrested at the filamentous protonema stage and did not form
gametophores (Figure 1A and 1B).
To test whether miRNA biogenesis is affected in the ΔPpDCL1a mutants, we
analyzed the accumulation of miR156, 160, 166, and 390 (Arazi et al., 2005; Fattash et
al., 2007). Due to the limited amount of plant material of the ΔPpDCL1a mutant lines,
miRNA expression was investigated by RT-PCR (Varkonyi-Gasic et al., 2007). PCR
products were sequenced to rule out unspecific amplification. Compared to wild type,
4
miR156, 160 and 166 had drastically reduced levels, while miR390 was undetectable in
ΔPpDCL1a mutants (Figure 1C). In P. patens, all known trans-acting siRNA (ta-siRNA)
precursors (PpTAS1-4 RNAs) are initially cleaved at two distinct miR390 target sites.
Subsequently, dsRNAs are generated from the cleavage products and processed in a
phased manner to generate ta-siRNAs (Axtell et al., 2006; Talmor-Neiman et al., 2006).
Two ta-siRNAs (pptA079444 processed from PpTAS1, and pptA013298 processed
from PpTAS3) (Axtell et al., 2006) were detected in wild type, but were absent in the
ΔPpDCL1a mutants, indicating that the lack of miR390 abolishes ta-siRNA production
(Figure 1D). To test whether reduced levels of miRNAs result in elevated transcript
levels of miRNA targets, as observed in A. thaliana dcl1 mutants, we analyzed the
expression of the miRNA targets PpSPB3 for miR156 (Arazi et al., 2005),
PpC3HDZIP1 and PpHB10 for miR166 (Axtell et al., 2007; Floyd and Bowman, 2004),
PpARF for miR160 (Fattash et al., 2007), and PpTAS1 for miR390(Axtell et al., 2006).
RT-PCR analysis revealed increased transcript levels of all analyzed miRNA targets in
the ΔPpDCL1a mutants (Figure 1E). From these results we conclude that PpDCL1a is
the major P. patens DCL protein required for the processing of miRNAs and thus the
functional equivalent of A. thaliana DCL1.
Requirement of PpDCL1b for miRNA guided target cleavage
The presence of PpDCL1b as a second P. patens AtDCL1 homolog raised the question
whether PpDCL1b acts redundantly with PpDCL1a, given that some miRNAs were not
completely abolished in ΔPpDCL1a mutants. We generated four targeted PpDCL1b
knockout mutants (ΔPpDCL1b) (Figure S3). The PpDCL1b mutants were strongly
affected in cell division, growth polarity, cell size, cell shape and tissue differentiation
(Figure 2A and Figure S4A). The developmentally arrested mutants produced only a
small number of malformed gametophores (Figure 2B and Figure S4B). Thus, while
5
both ΔPpDCL1a and ΔPpDCL1b mutants suffer from severe developmental defects,
the exact mutant phenotypes differed.
Analysis of six different miRNAs, miR156, 160, 166, 390, 535, and 538(Arazi et
al., 2005; Fattash et al., 2007), revealed that their levels were unchanged in ΔPpDCL1b
mutants (Figure 2C-2E). Thus, PpDCL1b is not essential for miRNA maturation from
precursor RNAs. The severe developmental defects of the mutants prompted us
nevertheless to examine miRNA targets PpSPB3, PpC3HDZIP1, PpHB10, and PpARF.
We verified cleavage of these miRNA targets in P. patens wild type based on 5’ RACE,
cDNA cloning and sequencing (Figure 3A). An unrelated mRNA (PpGNT1) (Koprivova
et al., 2003), which is not miRNA regulated, was examined as control (Figure 3A).
Although the ΔPpDCL1b mutants produced apparently normal levels of miRNAs, the
miRNA target transcripts were not cleaved (Figure 3A), indicating a surprising
requirement of PpDCL1b for miRNA-guided mRNA cleavage. We propose that
PpDCL1b may act in loading miRNAs into an RNA-cleavage competent RISC, in
analogy to what has been reported for animal Dicer proteins (Doi et al., 2003; Liu et al.,
2003; MacRae et al., 2008; Pham et al., 2004; Tomari et al., 2004).
Generation of transitive siRNA triggered by miRNA-guided transcript cleavage
In P. patens wild type we had not only detected 5’ RACE products resulting from
miRNA-guided cleavage of the target mRNA, but also a variety of shorter and longer
products (Figure 3A). In analogy with other plant systems, where targeting of a
transcript with dsRNA-derived siRNAs or multiple miRNAs (Axtell et al., 2006; Howell et
al., 2007; Vaistij et al., 2002) causes the production of secondary siRNAs, the miRNA
cleavage products may serve as templates for synthesizing cRNA by RNA-dependent
RNA polymerase (RdRP). Subsequently, the resulting dsRNA may be processed into
secondary siRNAs resulting in spreading of the initial trigger signal (Figure 3B). In
6
flowering plants, this phenomenon, known as transitivity, is, however, rarely observed
after targeting of an mRNA with a single miRNA (Axtell et al., 2006; Howell et al.,
2007).
To investigate the possibility of transitivity in P. patens, we asked whether one
could synthesize cDNA from both the sense and antisense strand of miRNA target
mRNAs (PpARF and PpC3HDZIP1). Indeed, this was the case in wild type, indicating
the presence of dsRNA. Such dsRNA molecules were lacking in ΔPpDCL1b mutants
(Figure 3C). To determine whether transitive siRNAs were generated from these
dsRNAs, we performed small RNA blots using probes for both upstream and
downstream sequences relative to the miRNA targeting site. Such small RNAs
corresponding to sense and antisense strands of PpARF and PpC3HDZIP1 mRNAs
were detected in wild type, but not in ΔPpDCL1b mutants (Figure 3D). Thus, in P.
patens transitive siRNAs arise from regions upstream as well as downstream of the
miRNA targeting motif after miRNA-directed cleavage of mRNAs. These siRNAs most
likely cause cleavage of the cognate mRNAs at additional sites, which explains why we
observed additional mRNA fragments in the 5’ RACE analyses. We did not detect such
siRNAs in the ΔPpDCL1b mutants, nor for the control mRNA (PpGNT1) in wild type
(Figure 3C and 3D), indicating that the generation of transitive siRNAs depends on
PpDCL1b and is specific for mRNAs subject to miRNA-directed cleavage.
DNA methylation of miRNA target loci in ΔPpDCL1b mutants
A. thaliana ago1 and dcl1 mutants are defective in miRNA-directed target cleavage or
miRNA biogenesis, respectively. Consequently, transcript levels of miRNA targets are
elevated in both mutants (Ronemus et al., 2006). Conversely, all miRNA targets
analyzed had reduced transcript levels in ΔPpDCL1b mutants (Figure 4A and 4B),
even though they were not cleaved. As explanation for these unexpected findings we
7
considered the possibility that miRNA target loci are under epigenetic control in P.
patens. Since methylation of cytosine residues is the most prominent mechanism for
transcriptional silencing in plants and other eukaryotes (Bender, 2004), we evaluated
this scenario by methylation-specific PCR of four miRNA target loci, along with an
unrelated locus, PpGNT1 (Figure 4C-4F and Figure S5 and Figure S6). Promoters of
all five genes were unmethylated in wild type, but in ΔPpDCL1b mutants the four
miRNA target promoters were methylated (Figure 4D). These findings were confirmed
by sequencing the PCR products of the PpARF promoter from wild type and
ΔPpDCL1b mutants. In the latter, methylation occurred specifically at CpG residues
(Figure S7). Taken together, we conclude that disruption of PpDCL1b causes specific
epigenetic changes in genes encoding miRNA targets, and that this is accompanied by
a loss of miRNA-directed mRNA cleavage.
It is well-known that siRNA pathways govern DNA methylation in A. thaliana,
e.g. at repeat-associated loci (Herr et al., 2005). However, only one study has
suggested a function of miRNAs in the silencing of cognate target genes, at the PHB
and PHV loci, which are targeted by miR165/166. Normally methylated DNA
sequences downstream of the miRNA complementary motif became hypomethylated in
plants with dominant alleles of PHB and PHV, while the promoters remained
unmethylated (Bao et al., 2004). The dominant alleles carry mutations in the miRNA
targeting motif, such that the encoded mRNAs are no longer susceptible to miRNAguided cleavage.
Like PHB and PHV, the P. patens HD-ZIP homologs PpC3HDZIP1 and PpHB10
harbor an intron within their miRNA binding site (Figure S8), whereas the miRNA
targeting motif in PpARF is not disrupted by an intron. Similar to the promoters,
PpC3HDZIP1 and PpARF sequences flanking the miRNA targeting motif as well as the
8
intron disrupting the miR166 binding site of PpC3HDZIP1 were CpG methylated in
ΔPpDCL1b mutants, but not in P. patens wild type (Figure 4E and 4F and Figure S7).
One scenario that can account for the findings presented so far is that
PpDCL1b normally acts in loading miRNAs into an RNA-cleavage competent RISC. In
the absence of PpDCL1b, miRNAs might be loaded instead into an RNA-induced
transcriptional silencing complex (RITS) directing DNA methylation of miRNA target
loci. As the sequence of the miR166 binding site is disrupted by introns in two genes
(PpC3HDZIP1 and PpHB10), it is unlikely that their methylation in ΔPpDCL1b mutants
is initiated by the formation of miRNA:DNA hybrids. Instead, the miRNA-loaded RITS
complex might interact with the target mRNA, resulting in the formation of a stable
miRNA:mRNA duplex. Subsequently, this duplex could guide the RITS complex to the
corresponding genomic region, resulting in the initiation and spreading of DNA
methylation.
If stable miRNA:mRNA duplexes are present in ΔPpDCL1b mutants, it should
be possible to synthesize cDNA without added exogenous primers, which can
subsequently be detected by conventional PCR. In support of such a scenario we
obtained RT-PCR products for all miRNA targets examined, but not for a control locus
in ΔPpDCL1b mutants. No such products were obtained with RNA from wild type plants
(Figure 4G). In addition, in the ΔPpDCL1b mutants no PCR products were obtained
when using PCR primers located downstream of the miRNA targeting site. As a further
control, we heated the RNA samples prior to cDNA synthesis. This should lead to
denaturation of a miRNA:mRNA complex and hence eliminate priming; indeed, this
procedure prevented the amplification of PCR products in the ΔPpDCL1b mutants
(Figure 4H). These results are compatible with base-paired miRNA:mRNA duplexes
being present specifically in RNA samples from ΔPpDCL1b mutants.
9
To further scrutinize our hypotheses of transitivity and miRNA-dependent DNA
methylation, we analyzed the ta-siRNA pathway in ΔPpDCL1b mutants. After miR390mediated cleavage of TAS precursors, the RNA cleavage products are converted into
dsRNA and further processed into ta-siRNAs (Axtell et al., 2006; Talmor-Neiman et al.,
2006). The mRNA encoding an EREBP/AP2 transcription factor is targeted by one of
the ta-siRNAs derived from the TAS4 precursor (Talmor-Neiman et al., 2006). Hence,
the production of ta-siRNAs presents an intermediate step in the miRNA-dependent
control of mRNAs. TAS4 RNA cleavage products resulting from miRNA390-directed
cleavage were detected by 5’ RACE-PCR in P. patens wild type, but not in the
ΔPpDCL1b mutants (Figure 5A), even though miR390 was present in equal amounts in
ΔPpDCL1b mutants and wild type (Figure 2C). Furthermore, ta-siRNAs of both sense
and antisense orientation were present in wild type, but were undetectable in
ΔPpDCL1b mutants (Figure 5B), confirming that PpDCL1b is required to initiate the tasiRNA pathway.
In agreement with our findings for other miRNA targets, TAS4 transcript levels
were reduced in ΔPpDCL1b mutants (Figure 5C). Likewise, the TAS4 genomic locus
was methylated only in ΔPpDCL1b mutants (Figure 5D). If, similar to miRNAs, tasiRNA-mediated cleavage of target mRNAs also initiates the generation of transitive
secondary siRNAs, the lack of ta-siRNAs in ΔPpDCL1b mutants should abolish both
cleavage of EREBP/AP2 mRNA and transitive siRNAs. Consistent with this scenario,
the EREBP/AP2 mRNA was cleaved in wild type, but not in ΔPpDCL1b mutants (Figure
5A), and only wild type produced EREBP/AP2 mRNA-derived siRNAs in sense and
antisense orientation (Figure 5B). This observation indicates that the siRNA-dependent
amplification of target RNA degradation that was initially triggered by ta-siRNA/miRNAguided cleavage is a common mechanism in P. patens. In addition, we expected that,
in contrast to direct miRNA targets, EREBP/AP2 mRNA levels should be elevated in
10
ΔPpDCL1b mutants, as stable ta-siRNA:mRNA duplexes that could guide DNA
methylation at the corresponding genomic locus should be absent. Indeed, the
EREBP/AP2 RNA levels were increased in ΔPpDCL1b mutants (Figure 5C), and the
genomic locus was methylated neither in wild type nor in ΔPpDCL1b mutants (Figure
5D).
Dependence of DNA methylation at miRNA target loci on miRNA expression
levels
We propose that the formation of stable miRNA:target RNA duplexes leads to
methylation of the corresponding genomic regions in ΔPpDCL1b mutants. Is this
mechanism of epigenetic silencing also relevant in wild type P. patens? To investigate
this question, we generated wild type and ΔPpDCL1b mutant plants expressing
different levels of an artificial miRNA targeting our control gene PpGNT1.
Artificial
miRNAs
(amiRNAs)
can
be
generated
by
exchanging
the
miRNA/miRNA* sequence of endogenous miRNA precursor genes, while maintaining
the general pattern of matches and mismatches in the foldback. We engineered an
amiRNA against PpGNT1 into the A. thaliana miR319a precursor (Khraiwesh et al.,
2008) and expressed the hybrid construct in P. patens wild type and ΔPpDCL1b
mutants (Figure 6A). RNA blots confirmed precise maturation of amiR-GNT1 in
transformed lines, independent of expression level (Figure 6B). The expected PpGNT1
mRNA cleavage products were present in wild type, but not in ΔPpDCL1b mutants
(Figure 6C). Consequently, compared to P. patens wild type the PpGNT1 transcript
levels were reduced in amiR-GNT1 plants (Figure 6D). Despite abolished amiRNAdirected cleavage of PpGNT1 mRNA, transcript levels were even lower in the
ΔPpDCL1b mutant background (Figure 6D).
11
Consistent with our model of miRNA-dependent epigenetic silencing, the
PpGNT1 promoter was methylated in the ΔPpDCL1b mutant background (Figure 6E
and Figure S9). Importantly, the PpGNT1 promoter was also methylated in wild type
lines, with strong expression of the amiR-GNT1, while it was unmethylated in lines with
low levels of amiR-GNT1 (Figure 6E and Figure S9). Thus, specific methylation of
miRNA target loci is not limited to ΔPpDCL1b mutants. Based on the observation that
methylation in wild type is miRNA-dosage dependent, we hypothesized that the ratio of
the miRNA and its target mRNA is crucial for the induction of DNA methylation at the
target locus. If the miRNA concentration exceeds a certain threshold, the miRNA may
either interact directly with its target and the duplex might then be recruited into a DNA
methylation silencing complex, or the excess miRNA might be loaded immediately into
an effector complex such as RITS triggering duplex formation that directs DNA
methylation. We obtained supporting evidence for the expected amiR-GNT1:PpGNT1mRNA duplexes by cDNA synthesis without exogenous primers and subsequent PCR
in ΔPpDCL1b mutants. Importantly, we could amplify such products also in a wild type
line with high levels of amiRNA expression, but not in a wild type line expressing only
moderate amounts of amiR-GNT1 (Figure 6F).
Hormone-dependent DNA methylation of a miR1026 target locus
The analysis of amiRNA-GNT1 lines had shown that miRNA-directed epigenetic
silencing occurs also wild type, but that it is dependent on miRNA levels. We therefore
sought to identify endogenous miRNAs that might be induced to high levels in
response to specific stimuli, which in turn should be reflected by downregulation of
target mRNAs. In separate experiments we had found that treatment with the hormone
abscisic acid (ABA) strongly represses expression of a basic helix-loop-helix (bHLH)
transcription factor gene, PpbHLH, which has been predicted to be targeted by
12
miR1026 (Axtell et al., 2007). ABA is a well known signaling molecule in abiotic stress
signaling pathways in plants including mosses (Frank et al., 2005b). RNA gel blots
confirmed downregulation of PpbHLH in response to ABA (Figure 6G). This effect
correlated well with an ABA-induced increase of miR1026 levels (Figure 6H),
suggesting direct regulation of PpbHLH by miR1026. We confirmed miR1026-mediated
cleavage of PpbHLH transcript by 5’ RACE (Figure 6I).
To evaluate transcriptional effects of miR1026, we analyzed DNA methylation of
the PpbHLH gene, including the promoter and transcribed sequences. Upon ABA
treatment, PpbHLH became methylated at specific CpG sites (Figure 6J and Figure
S10), consistent with the methylation patterns we had found before in ΔPpDCL1b
mutants. The promoter of an unrelated gene, PpGNT1, was unmethylated regardless
of ABA treatment (Figure 6J).
Our model posits that DNA methylation will be initiated if the miRNA target ratio
exceeds a certain threshold. That DNA methylation of the PpbHLH locus is not
quantitative, as deduced from the observation that unmethylation-specific primers
allowed albeit inefficient PCR amplification in ABA-treated samples, may reflect cell
type-specific differences in miRNA or target expression levels. Finally, we tried to
obtain evidence for stable miR1026:PpbHLH-mRNA duplexes by unprimed RT-PCR.
Consistent with the DNA methylation status, such duplexes were only found in the
ABA-treated samples (Figure 6K).
We conclude that in P. patens, epigenetic silencing of miRNA target loci
contributes to the control of target gene expression. Although we initially discovered
this
phenomenon
in
ΔPpDCL1b
mutants,
subsequent
analyses
of
the
miR1026/PpbHLH regulon confirmed that this type of miRNA-dependent control
operates also in wild type.
13
Discussion
Our studies suggest that PpDCL1a is the functional ortholog of AtDCL1 required for
miRNA and ta-siRNA biogenesis. Even though PpDCL1b shares a similar level of
sequence identity with AtDCL1, we propose that it has a distinct function, since its
inactivation does not affect miRNA biogenesis, but abolishes miRNA-directed target
cleavage. It is unlikely that PpDCL1b directly cleaves target RNAs, as AGO proteins in
RISC are the catalytic enzymes in sRNA-dependent target cleavage (MacRae et al.,
2008). Biochemical analysis of AGO1 complexes immunoprecipitated from Arabidopsis
dcl1-7, dcl2-1 and dcl3-1 mutants provided evidence for distinct functional properties.
An AGO1 complex extracted from dcl1-7 mutants was not able to cleave RNA targets
due to the lack of ~21 nt small RNA accumulation in this mutant. In contrast, cleavage
of RNA targets was not affected in AGO1 complexes from dcl2-1 and dcl3-1 mutants
(Qi et al., 2005). Furthermore, purification of Arabidopsis AGO1 revealed a ~160 kDa
complex, most likely only consisting of AGO1 and associated sRNA (Baumberger and
Baulcombe, 2005). Thus, there is so far no evidence to support a function of plant DCL
proteins in sRNA-mediated target cleavage. In contrast, studies in animals have shown
that Dicer proteins are part of the RNA loading complex (RLC), which loads sRNAs into
RISC. Human RLC comprises the proteins Ago2, Dicer and TRBP and the purified
protein components assemble spontaneously in vitro without requirement of any
cofactors. The reconstituted RLC is fully functional and once Ago2 is loaded with a
miRNA it tends to dissociate from the rest of the complex (MacRae et al., 2008).
Similarly, Dcr-2 from D. melanogaster, which produces siRNA, acts in the RISC
assembly together with its partner R2D2 by loading one of the two siRNA strands into
RISC (Liu et al., 2003; Tomari et al., 2004). The C. elegans homolog of this protein,
RDE-4, was also found to interact with Dicer (Tabara et al., 2002). Given this particular
function of animal Dicer proteins, we hypothesize that P. patens PpDCL1b may exhibit
14
an equivalent function in loading miRNAs into RISC, making it indispensable for
miRNA-directed target cleavage.
Arabidopsis dcl1 and ago1 mutants, which are affected in miRNA biogenesis or
miRNA-directed target cleavage, respectively, exhibit elevated transcript levels of
miRNA targets (Ronemus et al., 2006). Likewise, miRNA target transcripts are
increased in ΔPpDCL1a mutants due to the lack of miRNAs. In contrast, levels of
miRNA target mRNAs are drastically reduced in ΔPpDCL1b mutants, in spite of
abolished target RNA cleavage. We have shown that cytosine residues within the
corresponding genomic loci are methylated in ΔPpDCL1b mutants, suggesting
epigenetic control at the transcriptional level. Small RNAs initiate transcriptional
silencing of homologous sequences by methylation of cytosine residues at CpG,
CpNpG, and CpHpH sequence motifs or by histone modifications (Bender, 2004; Cao
and Jacobsen, 2002). In all genomic regions analyzed in the ΔPpDCL1b mutants, we
only detected methylation at CpG dinucleotides, but cannot exclude that cytosine
methylation may also occur at different sequence contexts in other regions. Moreover,
we detected CpG methylation in large regions of the genomic loci encoding miRNA
targets including introns, exons and promoter regions pointing to methylation that is
able to spread over considerably long distances.
Although spreading of siRNA-directed DNA methylation into adjacent non
repeated sequences is not common in A. thaliana, siRNA-mediated spreading of DNA
methylation has been observed for the SUPPRESSOR OF drm1 drm2 cmt3 (SDC)
locus, where methylation spreads beyond siRNA generating repeat regions present in
the SDC promoter (Henderson and Jacobsen, 2008). In Arabidopsis, cytosine
methylation can also spread in the PHV and PHB genes, which are targets of
miR165/166 (Bao et al., 2004). In both genes, the miR165/166 complementary motif is
disrupted by an intron and the coding sequence was found to be heavily methylated
15
downstream of the miRNA complementary site in differentiated, but not undifferentiated
cells of wild type plants. Furthermore, methylation was reduced in phv-1d and phb-1d
mutants, which have an altered miRNA recognition motif or a mutation in the intron
splice donor sequence, suggesting that miR165/166 needs to bind to nascent PHV and
PHB transcripts to trigger gene silencing (Bao et al., 2004).
Similarly, the P. patens HD-Zip genes PpC3HDZip1 and PpHB10 are targeted
by miR166 and the miR166 binding sites are only reconstituted after splicing of an
intron from the primary transcripts. These loci are hypermethylated in ∆PpDCL1b
mutants, but not in wild type, suggesting that the initiation of CpG methylation upon
defective target cleavage cannot be mediated by miR166, but involves binding of
miR166 to the cognate target mRNAs. We have obtained evidence for the presence of
stable duplexes of a miRNA and its target RNA in ∆PpDCL1b mutants. We propose
that such duplexes guide a DNA modification complex.
In Arabidopsis, RNA-directed DNA methylation (RdRM) by siRNAs requires
RDR2, DCL3 and RNA PolIVa, which are all involved in siRNA biogenesis (Herr et al.,
2005; Kanno et al., 2005; Onodera et al., 2005; Pontier et al., 2005; Xie et al., 2004),
whereas AGO4, DRM2, DRD1 and RNA PolIVb are indispensable for DNA methylation
(Cao and Jacobsen, 2002; Kanno et al., 2005; Zilberman et al., 2004). In fission yeast,
RNA-directed heterochromatic gene silencing at centromeres relies on two different
complexes, the RITS complex comprising Ago1, Chp1 and TAS3, and the argonaute
siRNA chaperone complex (ARC) comprising Ago1, Arb1 and Arb2. However, these
complexes are required to direct histone H3 Lys9 methylation, but do not direct
cytosine methylation. Nevertheless, it has been proposed that their action involves the
recognition of nascent transcripts by RITS-bound siRNAs to promote recruitment of
chromatin-modifying enzymes that implement silencing (Buker et al., 2007).
We also detected the specific silencing of miRNA target genes in P. patens wild
type, where the expression of amiR-GNT1 caused methylation of the PpGNT1 genomic
16
locus. Moreover, we found that methylation of the locus is dependent on amiR-GNT1
abundance and only obtained evidence for amiR-GNT1:PpGNT1-mRNA duplexes in
lines with high amiRNA levels, supporting the hypothesis that miRNA:target-RNA
duplexes are required for DNA methylation. Finally, we have been able to show that the
genomic region of the miR1026 target PpbHLH becomes methylated in response to
ABA, which upregulates miR1026 expression. Also in this case, DNA methylation was
miR1026 dosage-dependent and appeared to correlate with the formation of stable
miR1026:PpbHLH-mRNA duplexes. As ABA acts as a mediator of abiotic stress
signaling, we assume that the miR1026-regulated silencing of PpbHLH is part of stress
adaptation in P. patens.
In plants, epigenetic changes as a response to stress conditions have been
previously shown to include DNA methylation, histone modifications and chromatin
remodeling (Boyko and Kovalchuk, 2008; Dyachenko et al., 2006; Henderson and
Dean, 2004). Our analysis of the miR1026:PpbHLH regulon suggests that miRNAs
may act in the epigenetic control of stress-responsive genes in plants.
Taken together, we propose that silencing of genomic loci can be triggered by
stable duplexes of a miRNA and its target RNA, which can be either an mRNA or a
primary TAS transcript. The epigenetic control of genes encoding miRNA target RNAs
discovered in P. patens presents a new mechanism that affects the homeostasis of
miRNA-regulated RNAs (Figure 7). The specific equilibrium of a cleavage-competent
RISC and a DNA-modifying RITS loaded with the same miRNA may determine the
relative contribution of both pathways to miRNA-mediated downregulation of gene
expression. In addition, siRNA-mediated transitivity as a major factor in amplifying the
original miRNA- and ta-siRNA-directed cleavage signal appears to be more prevalent
than in the flowering plant A. thaliana. It seems not unlikely that similar modifications
and specializations of RNAi pathways will be common, which indicates that care needs
17
to be exercised when interpolating the results from single model organisms, in either
plants or animals.
Experimental Procedures
Plant material
Culture of P. patens, protoplast transformation, and molecular analyses of transgenic
plants were performed according to standard procedures (Frank et al., 2005a). Abscisic
acid treatment was carried out by application of 10 µM (±)-cis-trans ABA to P. patens
liquid cultures.
Generation of ΔPpDCL1a and ΔPpDCL1b mutant lines
An nptII selection marker cassette was cloned into single restriction sites present in
PpDCL1a and PpDCL1b, respectively. The gene disruption constructs were transfected
into P. patens protoplasts and G418-resistant lines were analyzed by PCR to confirm
precise integration events at the corresponding genomic loci. Loss of PpDCL1a and
PpDCL1b transcript, respectively, was confirmed by RT-PCR.
P. patens lines expressing amiR-GNT1
The generation of an amiRNA targeting PpGNT1 was described previously (Khraiwesh
et al., 2008). The amiRNA expression construct was transfected into P. patens wild
type and ΔPpDCL1b mutant lines.
RT-PCR of small RNAs
RT-PCR analyses of miRNAs and ta-siRNAs was carried out as described (VarkonyiGasic et al., 2007). Oligonucleotides used for the cDNA synthesis and subsequent
PCR reactions are listed in Table S1.
18
DNA methylation analysis
DNA sequences were analyzed with the MethPrimer program (Li and Dahiya, 2002) to
deduce methylation-specific (MSP) and unmethylation-specific primers (USP) (Figure
S6) for PCR analysis of bisulfite-treated DNA. Two µg of genomic DNA were used for
sodium bisulfite treatment with the EpiTect Bisulfite Kit (Qiagen).
Detection of miRNA:mRNA duplexes by RT-PCR
cDNA was synthesized from 4 µg total RNA with Superscript III (Invitrogen) without the
addition of primers, with the exception of a primer specific for the PpEF1α transcript to
monitor the efficiency of cDNA synthesis. RT-PCRs were carried out with gene-specific
primers located upstream of miRNA binding sites (Table S1). Control experiments were
performed by heating RNA samples to 95°C for 5 min prior to cDNA synthesis. PpEF1α
control primers were added after cooling of the samples.
Supplemental Data
Supplemental Data include Figure S1-S10, Table S1 and Supplemental Experimental
Procedures and can be found with this article online
Acknowledgements
This work was supported by the Landesstiftung Baden-Württemberg (P-LS-RNS/40 to
D.W., W.F. and R.R.), the German Federal Ministry of Education and Research
(FRISYS: 0313921 to W.F. and R.R.), the Excellence Initiative of the German Federal
and State Governments (EXC 294 to R.R.), the European Community FP6 IP
SIROCCO (contract LSHG-CT-2006-037900; D.W.), and the German Academic
Exchange Service (M.A.A.). We thank G. Gierga for assisting us in the small RNA blot
19
technique and T. Laux, W.R. Hess, R. Baumeister, and P. Beyer for comments on the
manuscript.
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Figure Legends
Figure 1. Analysis of ΔPpDCL1a mutants
(A) Protonema filaments of identical density from wild type (WT) and two ΔPpDCL1a
mutants grown for 28 days on solid medium. (B) Protonema filaments of plants grown
in liquid cultures. (C) RT-PCR expression analysis of miR156, 160, 166, and 390. (D)
RT-PCR expression analysis of ta-siRNAs pptA013298 (processed from PpTAS3) and
pptA079444 (processed from PpTAS1). (E) RT-PCR expression analysis of miRNA
target genes in wild type and ΔPpDCL1a mutants. Error bars indicate standard errors
with n=3.
Figure 2. Analysis of ΔPpDCL1b mutants (1-4)
(A) Regeneration of protoplasts from wild type (WT) and ΔPpDCL1b mutant 1 over
indicated time points. (B) Scanning electron micrographs of gametophores. See
Supplementary Fig. 5 for phenotypes of other ΔPpDCL1b mutants. (C) Small RNA blots
with 30 µg total RNA from protonema, probed for miR156, miR390, miR535, and
miR538. An antisense probe for U6snRNA served as loading control. (D) Small RNA
25
blot with 80 µg total RNA from protonema treated with 5 µM auxin (NAA) for 8 hours,
probed for miR160. (E) Small RNA blot with 80 µg total RNA from gametophores,
probed for miR166. Ethidium bromide staining shown as loading control at the bottom
for d and e. Size bars correspond to 100 µm in a, except for the 18 d and 8 week old
plants, 500 µm.
Figure 3. RNA cleavage products, antisense transcripts, and transitive siRNAs of
miRNA target genes
(A) 5’ RACE products of miRNA targets and a control transcript, PpGNT1, from wild
type and ΔPpDCL1b mutants. Arrows indicate PCR fragments of the expected size for
cleavage products. Numbers above miRNA:target alignments indicate sequenced
RACE products with the corresponding 5’ end. (B) Scheme for the generation of
transitive siRNAs. Double stranded RNA is synthesized from cleaved miRNA targets by
an RNA-dependent RNA polymerase (RdRP), processed into transitive siRNAs, which
subsequently mediate cleavage of the miRNA target mRNA upstream and downstream
of the miRNA recognition motif. Black line: mRNA; grey box: miRNA binding site;
curved line: miRNA; arrows indicate oligonucleotide primers for RT-PCR, with grey
indicating primers for cDNA synthesis from antisense strand, and black for sense
strand. (C) RT-PCR products derived from antisense or sense-specific cDNAs from
wild type and two ΔPpDCL1b mutants (KO1, KO2). (D) Detection of sense and
antisense transitive siRNAs derived from PpARF and PpC3HDZIP1 RNAs, using
hybridization probes targeting regions upstream and downstream of the miRNA binding
sites. U6snRNA was used as control.
Figure 4. Expression of miRNA target genes, DNA methylation, and detection of
miRNA:mRNA duplexes
26
(A) RT-PCR expression analysis of miRNA target genes and the control gene PpGNT1
in wild type and ΔPpDCL1b mutants (KO1-KO4). Bars indicate standard error (n=3). (B)
RNA blot analysis of miRNA target genes PpARF and PpC3HDZIP1 and two control
genes, PpGNT1 and PpEF1α. (C) Specificity analysis of bisulfite PCR, using primers
specific for unmodified sequences. PCR was performed with untreated and bisulfitetreated genomic DNA of wild type and two ΔPpDCL1b mutants (KO1, KO2). (D-F) PCR
reactions
with
bisulfite-treated
genomic
DNA
using
methylation
(MSP)
and
unmethylation specific primers (USP). (D) Bisulfite PCR for promoters of miRNA target
genes and the PpGNT1 control. (E) Bisulfite PCR analysis of PpARF sequences
surrounding the miR160 targeting motif. (F) Bisulfite PCR analysis of PpC3HDZIP1
sequences upstream of, the intron disrupting, and sequences downstream of the
miR166 targeting motif. Arrows in d-f mark primer bands. (G) PCR products of miRNA
target genes using cDNA synthesized from wild type and two ΔPpDCL1b mutants (1
and 2) without addition of exogenous primers. For the PpGNT1 control, no PCR
products were detected in either wild type or ΔPpDCL1b mutants (not shown). A
PpEF1α primer specific for cDNA synthesis from the sense transcript was added as an
internal control to all reactions, to monitor the efficiency of cDNA synthesis. (H) The
same experiment performed with RNA samples that had been heated for 5 min to 95°C
prior to cDNA synthesis. The control PpEF1α primer was added after cooling of the
RNA samples.
Figure 5. The ta-siRNA pathway in wild type and ΔPpDCL1b mutants
(A) 5’ RACE-PCRs from wild type and ΔPpDCL1b mutants (1-4) for the miR390 target
PpTAS4 and the ta-siRNA target PpEREBP/AP2. Arrows indicate products of the size
expected for cleavage products. The number of sequenced RACE-PCR products with
the corresponding 5’ end is indicated above the alignment. (B) ta-siRNAs derived from
27
PpTAS4 and transitive siRNAs derived from PpEREBP/AP2. U6snRNA served as
control. (C) RNA blots for PpTAS4 and PpEREBP/AP2 transcripts. Ethidium bromide
staining shown as loading control below. (D) Bisulfite PCR with methylation specific
(MSP) and unmethylation specific primers (USP) for PpTAS4 and PpEREBP/AP2. The
arrow marks primer dimers.
Figure 6. Lines expressing amiR-GNT1 and analysis of miR1026 target PpbHLH
(A) PCR-based identification of two transgenic lines each harboring the PpGNT1amiRNA expression construct in wild type (lines #1, #2), ΔPpDCL1b mutant 1 (lines #3,
#4), and ΔPpDCL1b mutant 2 (lines #5, #6) backgrounds. PpEF1α served as control.
(B) Detection of amiR-GNT1 on a small RNA blot loaded with 50 µg of total RNA.
U6snRNA served as control. (C) Cleavage mapping of PpGNT1 in amiR-GNT1 lines by
5’ RACE-PCR. The number of sequenced RACE-PCR products with the corresponding
5’ end is indicated above the alignment. (D) RNA blot of wild type and amiR-GNT1
lines, probed for PpGNT1. Hybridization signals were normalized to rRNA. Levels
relative to wild type are indicated (E) Bisulfite PCR on genomic DNA from amiR-GNT1
lines using methylation (MSP) and unmethylation specific primers (USP) derived from
the PpGNT1 promoter. (F) RT-PCR to detect amiR-GNT1:PpGNT1-mRNA duplexes,
using cDNA synthesized without the addition of exogenous primers. PCR was carried
out with a primer pair upstream of the amiR-GNT1 target motif. Amplification controls
were as in Figure 4G and 4H. Arrows mark primer dimers. (G) RNA blots with 20 µg
total RNA from untreated (Untr.) and ABA-treated wild type plants using probes for
PpbHLH, the loading control PpEF1α, and PpCOR47, a known ABA-induced gene.
PpbHLH levels were normalized to PpEF1α. Relative PpbHLH mRNA levels compared
to wild type are given. (H) Small RNA blot with 50 µg total RNA from untreated (Untr.)
and ABA-treated wild type. MiR1026 levels were normalized to the U6snRNA control.
28
Numbers indicate miR1026 levels relative to wild type. (I) 5’ RACE-PCR for PpbHLH
using RNA from untreated (Untr.) and wild type treated for 4 h with ABA. Arrows
indicate PCR fragments of the expected size for cleavage products. Numbers above
miRNA:target
alignments
indicate
sequenced
RACE-PCR
products
with
the
corresponding 5’ end. (J) Bisulfite PCR reactions on DNA from untreated (Untr.) and
ABA-treated wild type using methylation (MSP) and unmethylation specific primers
(USP) targeting PpbHLH genomic sequences. PpGNT1 promoter served as control.
Arrows mark primer dimers. (K) RT-PCR to detect miR1026:PpbHLH-mRNA duplexes,
using cDNA synthesized without the addition of exogenous primers. PCR was carried
out with a primer pair upstream of the miR1026 binding site. Amplification controls were
as in Figure 4G and 4H. Arrows mark primer dimers.
Figure
7.
MiRNA
expression
levels
determine
post-transcriptional
and
transcriptional silencing of miRNA target genes in P. patens
At low miRNA:target-RNA ratios, miRNA targets are regulated primarily at the posttranscriptional level. The maturation of miRNAs from stem-loop precursors is catalyzed
by PpDCL1a. PpDCL1b is required for loading miRNAs into cleavage competent RISC.
After loading of miRNAs into RISC, transient miRNA:target-RNA duplexes form based
on sequence complementarity resulting in target RNA cleavage. In P. patens, the
amplification of the miRNA signal by the generation of transitive siRNAs appears to be
widespread. Elevated miRNA expression levels cause an increase in the miRNA:target
RNA ratio. In addition to the loading of miRNA into RISC (dotted arrow), miRNAs form
stable duplexes with their cognate target RNAs. MiRNAs are either loaded into a RITS
complex and subsequently interact with their target to form a duplex, or these duplexes
are formed at first and then loaded into RITS. The miRNA:RNA duplexes bound by
RITS initiate DNA methylation at complementary genomic loci. The RITS complex is
29
able to act on adjacent regions (e. g., promoters) to complete CpG methylation of the
entire genomic locus.
30
Figure 1
A
DPpDCL1a
mutant 1
WT
0.5 cm
B
0.5 cm
DPpDCL1a
mutant 1
WT
100 µM
DPpDCL1a
mutant 2
0.5 cm
DPpDCL1a
mutant 2
100 µM
C
100 µM
D
DPpDCL1a
mutants
WT 1 2
DPpDCL1a
mutants
WT
1
2
miR156
miR160
miR166
miR390
ta-siRNA pptA013298
ta-siRNA pptA079444
Relative transcript levels
E
7
6
WT
5
DPpDCL1a mutant 1
4
DPpDCL1a mutant 2
3
2
1
0
PpSBP3 PpARF
PpC3 PpHB10 PpTAS1
HDZIP1
Figure 2
A
DPpDCL1b
mutant 1
WT
B
DPpDCL1b
mutant 1
WT
100 µm
4d
200 µm
C
WT
DPpDCL1b mutants
3
1
2
4
miR156
miR390
6d
miR535
miR538
8d
U6snRNA
D
miR160
EtBr
18 d
E
miR166
EtBr
8 weeks
Figure 3
A
DPpDCL1b
mutants
WT 1 2 3 4
6/9
5’ UGGCAUGCAGGGGGCCAGGCA 3’
3’ ACCGUAUGUCCCUCGGUCCGU 5’
PpARF
miR160
7/9
U
GG AUGAAGCCUGGUCCGG 3’
CC UACUUCGGACCAGGCU 5’
CC U
CU
PpC3HDZIP1 5’
miR166
3’
7/9
U
GG AUGAAGCCUGGUCCGG 3’
CC UACUUCGGACCAGGCU 5’
CC U
CU
PpHB10
miR166
5’
3’
PpSBP3
miR156
U 8/9
5’ GUGCUC CUCUCUUCUGUCA 3’
3’ CACGAG GAGAGAAGACAGU 5’
U
PpGNT1 (control)
B
5’
3’
5’
3’ 5’
5’
3’
3’ 5’
RdRP
3’
RdRP
transitive
siRNAs
5’
3’
5’
3’
5’
5’
C
PpARF
KO KO
WT 1 2
antisense
cDNA
PpC3HDZIP1
KO KO
WT 1 2
antisense
cDNA
PpGNT1
KO KO
WT 1 2
antisense
cDNA
D
3’
3’
5’
3’
3’
5’
PpEF1a
PpARF
PpEF1a
KO KO
KO KO
KO KO
WT 1 2 WT 1 2 WT 1 2
control
sense
cDNA
control
PpC3HDPpEF1a
ZIP1
KO KO
KO KO
KO KO
WT 1 2 WT 1 2 WT 1 2
PpEF1a
control
sense
cDNA
control
PpEF1a
PpGNT1 PpEF1a
KO KO
KO KO
KO KO
WT 1 2 WT 1 2 WT 1 2
control
sense
cDNA
control
Upstream
Downstream
Sense
Sense
Antisense
Antisense
KO KO
KO KO
KO KO
KO KO
WT 1 2
WT 1 2 WT 1 2
WT 1 2
PpARF
PpC3HDZIP1
U6 sRNA
Figure 4
A
Relative transcript levels
14
DPpDCL1b
3
WT 1 2
4
WT KO1 KO2 KO3 KO4
PpARF
12
PpC3HDZIP1
10
DNA
Untreated
+ Bisulfite
DNA
KO KO
KO KO
2 WT 1
2
WT 1
PpARF
PpGNT1
8
PpC3HDZIP1
PpEF1a
6
PpHB10
4
PpSBP3
2
PpGNT1
0
D
C
B
16
PpGNT1
PpC3
HDZIP1
PpHB10
PpSBP3
PpARF
E
MSP
KO KO
WT 1 2
USP
KO KO
WT 1
2
Exon
MSP
KO KO
WT 1 2
PpARF
Intron
USP
KO KO
WT 1 2
MSP
KO KO
WT 1 2
USP
KO KO
WT 1 2
upstream
of miR160
binding site
PpC3HDZIP1
PpHB10
downstream
of miR160
binding site
PpSBP3
PpGNT1
F
Intron upstream
of miR166
binding site
MSP
KO KO
WT 1 2
Intron located in
miR166
binding site
USP
MSP
KO KO
KO KO
WT 1 2 WT 1 2
Exon downstream
of miR166
binding site
USP
MSP
USP
KO KO
KO KO
KO KO
WT 1 2 WT 1 2
WT 1 2
G
DPpDCL1b
mutants
WT 1 2
PpARF
PpC3HDZIP1
PpHB10
PpSBP3
PpEF1a
H
PpEF1a
KO KO
WT 1 2
PpC3HDPpARF
ZIP1
KO KO
KO KO
WT 1 2 WT 1 2
PpHB10
PpSBP3
KO KO
KO KO
WT 1 2 WT 1 2
Figure 5
A
DPpDCL1b
mutants
WT 1 2 3 4
PpTAS4
miR390-5’
5/6
5’ GGCGUUAUCCCUCUUGAGCUG 3’
3’ CCGCGAUAGGGAGGACUCGAA 5’
U
A
A 8/9
5’ G UGU UAUC CUCCUGAGCUA 3’
3’ C GCG-AUAG GAGGACUCGAA 5’
C
G
C
G 8/8
PpEREBP/AP2 5’ GAAGCA UCAUCACACCCUA 3’
ta-siRNA6(+)3’ CUUCGU AGUAGUGUGGGAU 5’
A
G
PpTAS4
miR390-3’
C
B
Antisense
KO KO
WT 1 2
DPpDCL1b
WT 1 2 3 4
Sense
KO KO
WT 1 2
PpTAS4
PpTAS4
PpEREBP/AP2
PpEREBP/AP2
rRNA
U6 sRNA
D
PpEREBP/AP2
PpTAS4
MSP
KOKO
WT 1 2
Coding Sequence
USP
MSP
KOKO
KOKO
WT 1 2 WT 1 2
USP
KOKO
WT 1 2
Promoter
MSP
USP
KOKO
KOKO
WT 1 2
WT 1 2
A
C
B
amiRNA
WT lines
#1 #2
#3 #4
#5 #6
WT background
DPpDCL1b
+ amiRNA
KO1 KO2
WT #3 #5
DPpDCL1b
WT + KO1 + KO2 +
amiR amiR amiR
WT #1 #2 #3 #4 #5 #6
WT +amiRNA#1
Figure 6
amiRGNT1
DPpDCL1b KO1
DPpDCL1b KO2
PpEF1a
PpGNT1
amiRNA
U6 snRNA
E
D
WT +
amiRNA
#1
#2
WT
DPpDCL1b
KO1 +
KO2 +
amiRNA
amiRNA
#3
#4
#5
#6
PpGNT1
1.0
F
MSP
WT + DPpDCL1b
amiRNA KO1 KO2
#1 #2 #3 #5
USP
WT + DPpDCL1b
amiRNA KO1 KO2
#1 #2 #3 #5
6/8
C
5’ AA CGUCCUGAUUAUUUGGAG 3’
3’ UU GCAGGACUAAUAAACCUU 5’
C
DPpDCL1b
WT +
amiRNA KO1 KO2
#3 #5
#2
#1
DPpDCL1b
WT +
amiRNA KO1 KO2
#3 #5
#1
#2
PpGNT1
0.22 0.11 0.022 0.017 0.017 0.017
PpEF1a
rRNA
Duplex RT-PCR
G
I
Untr.
Untr.
H
ABA
1h 2h 4h 6h 8h
ABA
4h
ABA
1h 2h 4h 6h 8h
PpbHLH
miR1026
1.0 2.9 2.5 3.3
1.0 0.5 0.3 0.3 0.4 0.4
U6snRNA
ABA
4h 6h 8h
4h
ABA
6h 8h
Untr.
Untr.
Untr.
ABA
4h 6h 8h
K
USP
Untr.
PpCOR47
MSP
Untr.
PpbHLH
5’ CC
C 3’
8/10
UCCUCUCAAGUCUUUCUC
AGGAGAGUUCAGAAAGAG
3’ AC
U 5’
miR1026
3.0 3.0
PpEF1a
J
Heating control
4h
ABA
6h 8h
PpbHLH
PpbHLH
intragenic
PpbHLH
Promoter
PpGNT1
Promoter
PpEF1a
Duplex RT-PCR
Heating control
Figure 7
Cytosol
RISC
Low miRNA:
target-RNA ratio
RISC
AAAAA
PpDCL1b
target-RNA
cleavage
target-RNA
(mRNA or TAS-RNA)
+
AAAAA
Nucleus
AAAAA
mRNA or
TAS-RNA
cleavage
products
RdRP
pre-miRNA
double-stranded
mRNA
cleavage
products
double-stranded
TAS-RNA
cleavage
products
transitive
siRNAs
PpDCL1a
ta-siRNAs
Amplification of
the miRNA signal
Targeting and cleavage
of ta-siRNA targets
miRNA
transitive
siRNAs
AAAAA
High miRNA:
target-RNA ratio
RITS
target-RNA gene
(mRNA or TAS-RNA)
CH3
CH3
AAAAA
CH3
RITS
RITS
AAAAA
AAAAA
elevated
miRNA level
AAAAA
Formation of
miRNA:target-RNA
duplexes
Epigenetic Silencing
Formation of
miRNA:target-RNA
duplexes
Supplemental Data
Transcriptional control of gene expression by microRNAs
Basel Khraiwesh1, M. Asif Arif1, Gotelinde I. Seumel1, Stephan Ossowski2, Detlef
Weigel2, Ralf Reski1,3,4, Wolfgang Frank1,3*
1
Plant Biotechnology, Faculty of Biology, University of Freiburg, Schänzlestraße 1, D-
79104 Freiburg, Germany
2
Department of Molecular Biology, Max Planck Institute for Developmental Biology, D-
72076 Tübingen, Germany
3
Freiburg Initiative for Systems Biology (FRISYS), Faculty of Biology, University of
Freiburg, Schänzlestr. 1, D-79104 Freiburg, Germany
4
Centre for Biological Signalling Studies (bioss), University of Freiburg, Schänzlestr. 1,
D-79104 Freiburg, Germany
Supplementary Figures
Figure S1. Neighbor-joining tree of DICER-LIKE proteins
Figure S2. Generation of ΔPpDCL1a mutants
Figure S3. Generation of ΔPpDCL1b mutants
Figure S4. Phenotypic analysis of ΔPpDCL1b mutants
Figure S5. Gene models of PpARF, PpC3HDZIP1, PpHB10, PpSBP3 and PpGNT1
Figure S6. Primer design for bisulfite PCR analyses
Figure S7. DNA methylation analysis of promoter and intragenic regions of the PpARF
gene in P. patens wild type and two ΔPpDCL1b mutants
Figure S8. The miR166 binding sites of PpC3HDZIP1 and PpHB10 are disrupted by
introns
Figure S9. DNA methylation analysis of the PpGNT1 promoter region in lines
expressing the amiR-GNT1
Figure S10. DNA methylation analysis of promoter and intragenic regions of PpbHLH
in untreated and ABA-treated P. patens wild type
Supplementary Tables
Table S1. Primers used in this study
Supplemental Experimental Procedures
Figure S1
MmDCL1_NP_683750
HsDCL1_NP_803187
DmDCL1_AAF56056
Animals
CeDCL1_NP_498761
DmDCL2_NM_079054
SpDCL1_Q09884
NcDCL1_XP_961898
PpDCL1b_DQ675601
PpDCL1a_EF670436
PtDCL1_Pt02g14226280
MtDCL1_AC150443
DCL1
AtDCL1_NP_171612
OsDCL1_NP_912466
PtDCL2a_Pt06g11470720
PtDCL2_Pt08g4686890
AtDCL2_NP_566199
DCL2
OsDCL2_XP_463595
Plants
AtDCL3_NP_189978
PtDCL3_Pt10g16358340
OsDCL6_Os09g14610
OsDCL5_Os03g38740
DCL3
OsDCL3_NP_922059
PpDCL3_EF670437
PtDCL4_Pt18g3481550
AtDCL4_NP_197532
OsDCL4_Os04g43050
DCL4
PpDCL4_EF670438
CrDCL*
Figure S1. Neighbor-joining tree of DICER-LIKE proteins
DICER-LIKE proteins from animals and plants are indicated by vertical lines. The four groups of DICER-LIKE
proteins in plants are marked by coloured boxes. Species abbreviations are At (Arabidopsis thaliana), Ce
(Caenorhabditis elegans), Cr (Chlamydomonas reinhardtii), Dm (Drosophila melanogaster), Hs (Homo
sapiens), Mm (Mus musculus), Mt (Medicago truncatula), Nc (Neurospora crassa), Os (Oryza sativa), Pp
(Physcomitrella patens), Pt (Populus trichocarpa), Sp (Schizosaccharomyces pombe). P. patens DCL proteins
are highlighted in bold. * The sequence of DCL from Chlamydomonas reinhardtii can be retrieved at:
http://genome.jgi-psf.org/chlre2.
Phylogenetic tree construction: The multiple sequence alignment was performed using the PROBCONS
program. The phylogenetic tree was constructed by a neighbor-joining method using a WAG matrix model for
amino acid substitution.
Figure S2
A
DEAD
HEL
DUF
B
PAZ
nptII
nosP
RNAse III
dsrm
nosT
EcoRV
PpDCL1a
gDNA
DCL1 cDNA
PpDCL1a nosP
F2
PpDCL1a
nptII
nosT PpDCL1a
nptII
nosT PpDCL1a PpDCL1a
F1
F3
PpDCL1a nosP
R2
C
*
*
D
WT
R1
WT
Genomic
PCR-Screen
E
DPpDCL1a
mutants
1
2
PpDCL1a
PpEF1a
R3
DPpDCL1a
mutants
1
2 WT
5’ integration
3’ integration
Figure S2. Generation of DPpDCL1a mutants
(A) Predicted domain structure of the P. patens DCL1a protein. DEAD: DEAD box helicase, HEL: helicase C,
DUF: domain of unknown function, PAZ: PAZ domain, RNAseIII: ribonuclease III domain, dsrm: doublestranded RNA binding motif. (B) Scheme illustrating the generation of the DPpDCL1a mutants. The nptII
cassette was cloned into a single EcoRV site of a PpDCL1a genomic DNA fragment (gDNA). Middle: Resulting
PpDCL1a knockout construct. Bottom: Expected genomic structure of the PpDCL1a locus after integration of
the PpDCL1a knockout construct by homologous recombination. Primers used for molecular analyses of the
transgenic lines are indicated by arrows. White box: nptII cassette; grey boxes: PpDCL1a gDNA fragment;
black boxes: genomic PpDCL1a locus. (C) PCR analysis of transgenic lines using genomic DNA performed
with primers F1 and R1. Transgenic lines which failed to give rise to a PCR product are marked by asterisks.
WT: wild type control; the remaining samples were derived from transgenic lines. (D) Analysis of PpDCL1a
mRNA expression. Top: RT-PCR studies from two DPpDCL1a mutants and wild type (WT) with primers F1 and
R1. Bottom: RT-PCR performed with PpEF1a control primers. (E) PCR analysis of DPpDCL1a mutants using
genomic DNA to confirm 5’ and 3’ integration of the PpDCL1a knockout construct. PCR products obtained from
PCR reactions with primers F2 and R2 (5’ integration), F3 and R3 (3’ integration). Sequences of primers used
for the molecular analyses are listed in Experimental Procedures and Table S1.
Figure S3
A
HEL DUF
DEAD
B
RNAse III
PAZ
nosP
4550
nptII
dsrm
nosT
Eco72I
5108
PpDCL1b
cDNA
DCL1 cDNA
PpDCL1b nosP
F3
nptII
nosT PpDCL1b PpDCL1b
F4
PpDCL1b nosP
R4
R3
D
nosT PpDCL1b
F2 F1
PpDCL1b
C
nptII
WT
*
R1 R2
** *
E
DPpDCL1b mutants
1
2
3
4
PpDCL1b
PpEF1a
F
DPpDCL1b mutants
1
WT
Genomic
PCR-Screen
2
3
4
WT
PpDCL1b
PpEF1a
DPpDCL1b mutants
1
2
3
4
5’ integration
3’ integration
Control PpEF1a
Figure S3. Generation of DPpDCL1b mutants
(A) Predicted domain structure of the P. patens DCL1b protein. DEAD: DEAD box helicase, HEL: helicase C,
DUF: domain of unknown function, PAZ: PAZ domain, RNAseIII: ribonuclease III domain, dsrm: doublestranded RNA binding motif. (B) Scheme illustrating the generation of the DPpDCL1b mutants. The nptII
cassette was cloned into a single Eco72I site of a PpDCL1b cDNA fragment. Numbers indicate nucleotide
positions in the PpDCL1b cDNA. Middle: Resulting PpDCL1b knockout construct. Bottom: Expected genomic
structure of the PpDCL1b locus after integration of the PpDCL1b knockout construct by homologous
recombination. Primers used for molecular analyses of the transgenic lines are indicated by arrows. White box:
nptII cassette; grey boxes: PpDCL1b cDNA fragment; black boxes: genomic PpDCL1b locus. (C) PCR analysis
of transgenic lines using genomic DNA performed with primers F1 and R1. Transgenic lines which failed to give
rise to a PCR product are marked by asterisks. WT: wild type control; the remaining samples were derived from
transgenic lines. (D) Analysis of PpDCL1b mRNA expression. Top: RT-PCR studies from four DPpDCL1b
mutants and wild type (WT) with primers F2 and R2. Bottom: RT-PCR performed with PpEF1a control primers.
(E) Top: RT-PCR analysis of PpDCL1b expression in DPpDCL1b mutants and wild type using primers F3 and
R3 located upstream of the knockout construct integration site. Bottom: RT-PCR performed with PpEF1a
control primers. (F) PCR analysis of DPpDCL1b mutants using genomic DNA to confirm 5’ and 3’ integration of
the PpDCL1b knockout construct. PCR products obtained from PCR reactions with primers F2 and R4 (5’
integration), F4 and R2 (3’ integration) and PpEF1a control primers to confirm integrity of the used genomic
DNA. Sequences of primers used for the molecular analyses are listed in Experimental Procedures and Table
S1.
Figure S4
A
DPpDCL1b
mutant 1
WT
DPpDCL1b
mutant 2
DPpDCL1b
mutant 3
DPpDCL1b
mutant 4
4d
6d
8d
18 d
8 weeks
B
200 µm
100 µm
100 µm
90 µm
80 µm
Figure S4. Phenotypic analysis of DPpDCL1b mutants
(A) Regeneration of protoplasts from wild type plants (WT) and DPpDCL1b mutants was monitored
at the indicated time points. Size bars 4 d, 6 d, and 8 d: 100 µm; 18 d, 8 weeks: 500 µm.(B) Electron
micrographs of gametophores from wild type and DPpDCL1b mutants.
Figure S5
Start codon
1 - 999
PpARF
1
1315-1317
Intron 1
Intron 2
1893-2105
2731-2860
1 - 1000
2000
I2
I4
3041-3247
3673-3984
I6
I8
I 12
I 14
I 16
6208-6446
7039-7300
7807-8039
Stop codon
8181-8183
8183
miRNA BS
3662-3672
3985-3894
I1
I3
2504-2839
3351-3581
Start codon
1393-1395
4000
I5
6000
I7
I9
8000
I 11
4193-4402 4796-4929 5243-5363 5919-6130
I 13
I 15
6588-6726
7407-7658
Stop codon
miRNA BS
2072-2092
4033-4035
4590
Promoter Region
1000
2000
Start codon
1 - 1000
3000
3280-3282
Promoter Region
3282
2000
Start codon
1 - 1000
4000
Stop codon
miRNA BS
2687-2706
1300-1302
1000
PpGNT1 1
I 10
4489-4641 5007-5135 5553-5735
4000
Promoter Region
1 - 999
PpSBP3 1
3000
2329-2331
2000
PpHB10 1
3959-3961
4194
Start codon
1
Stop codon
Intron 3
3749-3907
Promoter Region
1000
PpC3
HDZIP1
miRNA BS
3115-3135
3000
Stop codon
1300-1302
2713-2715
Promoter Region
2715
1000
2000
Figure S5. Gene models of PpARF, PpC3HDZIP1, PpHB10, PpSBP3 and PpGNT1
For PpARF and PpC3HDZIP1, complete gene models including intron sequences were predicted by comparing
available cDNA sequences with the P. patens genomic trace files. For PpHB10, PpSBP3 and PpGNT1, promoter
regions were analyzed following the same strategy. For PpHB10 the 5’ untranslated region was included based on the
available full-length cDNA sequence. In the case of PpSBP3 and PpGNT1, cDNA sequences encompassing the open
reading frame were available. Here, regions lying 300 nucleotides upstream of the start codon were used for promoter
analysis. I 1 – I 16: Intron 1 to Intron 16; miRNA BS: miRNA binding site. The genomic nucleotide sequences were
deposited in Genbank with the following accession numbers: BK006047 (PpHB10), BK006048 (PpC3HDZIP1),
BK006049 (PpGNT1), BK006050 (PpSBP3) and BK006051 (PpARF).
Figure S6
Promoter region of PpC3HDZIP1
1 CCTGCCTGCGCTGTCCTATCCTCCTCCTCTTCCTACTTCCCCTCACCTCCTCCGCCTCTG
::||::||++:|||::|||::|::|::|:||::||:||::::|:|::|::|:++::|:||
1 TTTGTTTGCGTTGTTTTATTTTTTTTTTTTTTTTATTTTTTTTTATTTTTTTCGTTTTTG
61 CGCTCTGTGCACTGTCCCTTCCATGTCGTGCCAGGCTCTGCGGAGGGTGCGGCCAGGCAG
++:|:||||:|:|||:::||::||||++||::|||:|:||++|||||||++|::|||:||
61 CGTTTTGTGTATTGTTTTTTTTATGTCGTGTTAGGTTTTGCGGAGGGTGCGGTTAGGTAG
WTfwd >>>>>>>>>>>>>>>>>>>>>
121 GCAGCTGTGATGGCGGTGTTGTACTGCCGCATGATTCTGGACCAACCGGGCCAGGGCGGG
|:||:||||||||++||||||||:||:++:||||||:||||::||:++||::||||++||
121 GTAGTTGTGATGGCGGTGTTGTATTGTCGTATGATTTTGGATTAATCGGGTTAGGGCGGG
MSPfwd >>>>>>>>>>>>>>>>>>>>>>>>>
USPfwd >>>>>>>>>>>>>>>>>>>>>>>>
181 CGACTGTTACTGCAGACTGTTACTGACTCCTGAGGGCGCAGCAGGCTGGTGAGAGGGACG
++|:|||||:||:|||:|||||:|||:|::||||||++:||:|||:||||||||||||++
181 CGATTGTTATTGTAGATTGTTATTGATTTTTGAGGGCGTAGTAGGTTGGTGAGAGGGACG
<<<<
241 GCCGCGGGTGGGGCGGGGCGAAGAAGAGAAAAAGGTGTGTAGGCGCGGGAGGCGTGCTTT
|:++++|||||||++|||++|||||||||||||||||||||||++++|||||++||:|||
241 GTCGCGGGTGGGGCGGGGCGAAGAAGAGAAAAAGGTGTGTAGGCGCGGGAGGCGTGTTTT
<<<<<<<<
<<<<<<<<<<
<<<<<<<<<<<<<<<<< WTrev
301 GTATGCGCACTACATGCCTTGAGCCTGGTGGTTGTTCATGACACTGTTCATCGCAGTATT
|||||++:|:||:|||::|||||::|||||||||||:||||:|:||||:||++:||||||
301 GTATGCGTATTATATGTTTTGAGTTTGGTGGTTGTTTATGATATTGTTTATCGTAGTATT
<<<<<<<<<<<<<<<<< MSPrev
<<<<<<<<<<<<<<<< USPrev
361 CTACAACGACATCGTCCCCAGCTGGTAGTAGTATTGCAGTTGTATAAGTTGTCGCTGCAG
:||:||++|:||++|::::||:||||||||||||||:|||||||||||||||++:||:||
361 TTATAACGATATCGTTTTTAGTTGGTAGTAGTATTGTAGTTGTATAAGTTGTCGTTGTAG
421 CCAGGCGCCGCCAGTCAGCATCTTCTCGTTAGTGTTCAGTTAGTAGTTGAAGCGAGGGAG
::|||++:++::|||:||:||:||:|++||||||||:|||||||||||||||++||||||
421 TTAGGCGTCGTTAGTTAGTATTTTTTCGTTAGTGTTTAGTTAGTAGTTGAAGCGAGGGAG
481 TATATTCCGCCTTCGATTTTTTGTTTCTCAGGGAGTCACAGCGCTGCGAATCAGAAGCCT
||||||:++::||++|||||||||||:|:|||||||:|:||++:||++|||:|||||::|
481 TATATTTCGTTTTCGATTTTTTGTTTTTTAGGGAGTTATAGCGTTGCGAATTAGAAGTTT
541 GTGAGAGCTTTGGGAACTGGTTTTCGTGTTTTAGAAAGCGAGGCCAACGAGAGAGCGAGA
|||||||:||||||||:|||||||++||||||||||||++|||::||++||||||++|||
541 GTGAGAGTTTTGGGAATTGGTTTTCGTGTTTTAGAAAGCGAGGTTAACGAGAGAGCGAGA
601 TCGAGAGAGAGAGAGAGCGCGAGCGACAGCATGTCACGCATGAGAGGAGAGAAGAACAGA
|++||||||||||||||++++||++|:||:||||:|++:|||||||||||||||||:|||
601 TCGAGAGAGAGAGAGAGCGCGAGCGATAGTATGTTACGTATGAGAGGAGAGAAGAATAGA
661 GGACGGAGCAGGGCTGGCCTATTGGTGTTACAGGAAGGGGGTTGCAGGAATTTGTAGGCG
|||++|||:||||:|||::|||||||||||:|||||||||||||:|||||||||||||++
661 GGACGGAGTAGGGTTGGTTTATTGGTGTTATAGGAAGGGGGTTGTAGGAATTTGTAGGCG
721 TGGCCGTCACTGTTTGGTTTTTGAAAGCTAGTGCTGCGACAAGAGATGCGGGTGGTCCTA
|||:++|:|:|||||||||||||||||:|||||:||++|:||||||||++||||||::||
721 TGGTCGTTATTGTTTGGTTTTTGAAAGTTAGTGTTGCGATAAGAGATGCGGGTGGTTTTA
781 GCTTGAGTACTTGTGCTAGGCGTCTGAGGCGTGAAGTTTCGGCTAGCTGATTGCAAATTC
|:|||||||:|||||:||||++|:|||||++||||||||++|:|||:||||||:|||||:
781 GTTTGAGTATTTGTGTTAGGCGTTTGAGGCGTGAAGTTTCGGTTAGTTGATTGTAAATTT
841 AGTAAGATTGGAGAGGGCAATGGCTGACGGTCCGCATCCATTCGTACAAGAATGCCTTCT
|||||||||||||||||:|||||:|||++||:++:||::|||++||:|||||||::||:|
841 AGTAAGATTGGAGAGGGTAATGGTTGACGGTTCGTATTTATTCGTATAAGAATGTTTTTT
901 TCTTGAAAAGCTGGTTGATCCTCGTCGTTGTAATCCGACGGTGCGGCTACGGAGCTAAAG
|:||||||||:||||||||::|++|++|||||||:++|++|||++|:||++|||:|||||
901 TTTTGAAAAGTTGGTTGATTTTCGTCGTTGTAATTCGACGGTGCGGTTACGGAGTTAAAG
961 TTCAAACGCTTAGTCTCTTCTTTTCTGGTGTGAAGTAGGT
||:|||++:|||||:|:||:||||:|||||||||||||||
961 TTTAAACGTTTAGTTTTTTTTTTTTTGGTGTGAAGTAGGT
Used primers:
Forward MSP: 5’-ATTGTCGTATGATTTTGGATTAATC-3’
Reverse MSP: 5’-ACATATAATACGCATACAAAACACG-3’
Forward USP: 5’-GTTGTATGATTTTGGATTAATTGG-3’
Reverse USP: 5’-CATATAATACACATACAAAACACACC-3’
Forward WT: 5’-ACTGCCGCATGATTCTGGACC-3’
Reverse WT: 5’-GCATGTAGTGCGCATACAAAG-3’
Promoter region of PpHB10
1 AGGAGGTGGAGGAGGTGGAGGGTTCCAAGGTGAGGGAGCAAGCTGTCATACCGGTAGGAG
||||||||||||||||||||||||::||||||||||||:|||:|||:|||:++|||||||
1 AGGAGGTGGAGGAGGTGGAGGGTTTTAAGGTGAGGGAGTAAGTTGTTATATCGGTAGGAG
61 TCCGTAGAGGGAAATAGAGAGGAAGCAAGTCAGGAAGTGTTGGTGAAGGGGGAGAGAAAG
|:++|||||||||||||||||||||:||||:|||||||||||||||||||||||||||||
61 TTCGTAGAGGGAAATAGAGAGGAAGTAAGTTAGGAAGTGTTGGTGAAGGGGGAGAGAAAG
121 AGAGCGAGAGGAGGAGGAGGAGTAGTAGAGGTGGTCGTGTCGATGATGGAAGAGATGATG
||||++|||||||||||||||||||||||||||||++|||++||||||||||||||||||
121 AGAGCGAGAGGAGGAGGAGGAGTAGTAGAGGTGGTCGTGTCGATGATGGAAGAGATGATG
181 GTGTAGTTTTTGGTTGTATGTAGTAGTAGCCATGAAGGAGGGGTTGTTTTTACGGGTAAT
|||||||||||||||||||||||||||||::|||||||||||||||||||||++||||||
181 GTGTAGTTTTTGGTTGTATGTAGTAGTAGTTATGAAGGAGGGGTTGTTTTTACGGGTAAT
241 GGTTGTTGTTCGGAAGGTATGTACAAATGGAGAGGGCTATGTCGGGGATCAGCTGGAGTG
||||||||||++|||||||||||:||||||||||||:|||||++|||||:||:|||||||
241 GGTTGTTGTTCGGAAGGTATGTATAAATGGAGAGGGTTATGTCGGGGATTAGTTGGAGTG
301 ATGATTGATTGAGTGGAGAGGGAGTGGCGGTAGATATGGGGATGGAGTGGAATGGGGTTC
|||||||||||||||||||||||||||++||||||||||||||||||||||||||||||+
301 ATGATTGATTGAGTGGAGAGGGAGTGGCGGTAGATATGGGGATGGAGTGGAATGGGGTTC
361 GTATGTCATCATTAGAATCCAAGAGTGGAGAGTAGTTTACCTGGAGCAGCAGCGTTGTGC
+|||||:||:||||||||::|||||||||||||||||||::|||||:||:||++|||||:
361 GTATGTTATTATTAGAATTTAAGAGTGGAGAGTAGTTTATTTGGAGTAGTAGCGTTGTGT
421 TCTTGCGCATCCTGGCGATGGACATTTGTGTTTGAGTAGTAGAGGTGGAGGCGTTGCTGT
|:|||++:||::|||++|||||:||||||||||||||||||||||||||||++|||:|||
421 TTTTGCGTATTTTGGCGATGGATATTTGTGTTTGAGTAGTAGAGGTGGAGGCGTTGTTGT
481 TGTTGTTGTCGTGGTTGTTGTGTGGTAGTGGTAGTAGTGTGACTCTGTAGTGGCTATGGT
|||||||||++|||||||||||||||||||||||||||||||:|:||||||||:||||||
481 TGTTGTTGTCGTGGTTGTTGTGTGGTAGTGGTAGTAGTGTGATTTTGTAGTGGTTATGGT
541 GGGTCTATTGCTGATGGTTTTTGTGTTGCGTCAGGCCGGCGTCACGGTCGTGTAGCATCG
||||:|||||:|||||||||||||||||++|:|||:++|++|:|++||++|||||:||++
541 GGGTTTATTGTTGATGGTTTTTGTGTTGCGTTAGGTCGGCGTTACGGTCGTGTAGTATCG
601 AGGGCGACGAAAGGTGAATGAACAAAGGGTGTGATTGTGTATAGGCATCCACATATTCTC
||||++|++|||||||||||||:||||||||||||||||||||||:||::|:|||||:|+
601 AGGGCGACGAAAGGTGAATGAATAAAGGGTGTGATTGTGTATAGGTATTTATATATTTTC
WTfwd >>>>>>>>>>>>>>>>>>>>>>>>
661 GGCTGTGGAAGTTGGGAACAGGGATGCCTTGTGTGCGATTCAACTCGTGGTATAGAAGAA
+|:|||||||||||||||:|||||||::|||||||++|||:||:|++|||||||||||||
661 GGTTGTGGAAGTTGGGAATAGGGATGTTTTGTGTGCGATTTAATTCGTGGTATAGAAGAA
MSPfwd >>>>>>>>>>>>>>>>>>>>>>>>
USPfwd >>>>>>>>>>>>>>>>>>>>>>>>>
721 GAAGAAGAGGAGCTTGAAGGTTGTCAAGAAAAGGGTAGGGTGTTGCTGCAGCAGCAGTAG
||||||||||||:|||||||||||:||||||||||||||||||||:||:||:||:|||||
721 GAAGAAGAGGAGTTTGAAGGTTGTTAAGAAAAGGGTAGGGTGTTGTTGTAGTAGTAGTAG
<<<<<<<<<<<<<<<<<<<<<<<<< WTrev
781 CAGCAGGAGCATCAGTAGCAGCTTGAGAGGACGAGGACCTAGGAGGAACAGAAGCTCTTG
:||:|||||:||:|||||:||:|||||||||++||||::|||||||||:|||||:|:|||
781 TAGTAGGAGTATTAGTAGTAGTTTGAGAGGACGAGGATTTAGGAGGAATAGAAGTTTTTG
<<<<<<<<<<<<<<<<<<<<<<<<< MSPrev
<<<<<<<<<<<<<<<<<<<<<<<<<< USPrev
841 CGTGGTCTGTGAGGAGAATTCTTTGTTAGGGGTTGGAAGCTTCTAGGTTGGGCACGTAGT
++||||:|||||||||||||:||||||||||||||||||:||:|||||||||:|++||||
841 CGTGGTTTGTGAGGAGAATTTTTTGTTAGGGGTTGGAAGTTTTTAGGTTGGGTACGTAGT
901 AGTGCGTTCTTTGTGTCTTGTCAACTGGGGTTTCAGTCGTATGAGTTGAACACGGGCTGT
||||++||:|||||||:||||:||:||||||||:|||++|||||||||||:|++||:|||
901 AGTGCGTTTTTTGTGTTTTGTTAATTGGGGTTTTAGTCGTATGAGTTGAATACGGGTTGT
961 CGTCACCAACCAGCAATTCGCAACCGGGCCTGCTCACGA
++|:|::||::||:||||++:||:++||::||:|:|++|
961 CGTTATTAATTAGTAATTCGTAATCGGGTTTGTTTACGA
Used primers:
Forward MSP: 5’-GATGTTTTGTGTGCGATTTAATTC-3’
Reverse MSP: 5’-ACTTCTATTCCTCCTAAATCCTCGT-3’
Forward USP: 5’-ATGTTTTGTGTGTGATTTAATTTGT-3’
Reverse USP: 5’-ACTTCTATTCCTCCTAAATCCTCATC-3’
Forward WT: 5’-GATGCCTTGTGTGCGATTCAACTC-3’
Reverse WT: 5’-GCTTCTGTTCCTCCTAGGTCCTCGT-3’
Promoter region of PpARF
1 AGGAGTGGTTTGTGATGCGAAGCTGGGAGGGTGACAGAAAGGACATCAGTGGATCTATGC
|||||||||||||||||++|||:|||||||||||:||||||||:||:|||||||:||||:
1 AGGAGTGGTTTGTGATGCGAAGTTGGGAGGGTGATAGAAAGGATATTAGTGGATTTATGT
61 TCTTATTAGTCCTAGTATGGATTAGTATTCATTGATTATAGAGGCTGCGCGGGAGAAAAT
|:||||||||::|||||||||||||||||:||||||||||||||:||++++|||||||||
61 TTTTATTAGTTTTAGTATGGATTAGTATTTATTGATTATAGAGGTTGCGCGGGAGAAAAT
121 GGAGAGACTAAGAAGATGAATTCTTCGTAGTTGTGACGAGATGGAAGGTTATTCAATTTA
|||||||:||||||||||||||:||++|||||||||++|||||||||||||||:||||||
121 GGAGAGATTAAGAAGATGAATTTTTCGTAGTTGTGACGAGATGGAAGGTTATTTAATTTA
181 TATTAGGGTACAATGGAAGGAATGCACTTAATTTTTGAAAGTTTTTCGCACGCCAGGATG
||||||||||:|||||||||||||:|:|||||||||||||||||||++:|++::||||||
181 TATTAGGGTATAATGGAAGGAATGTATTTAATTTTTGAAAGTTTTTCGTACGTTAGGATG
241 GAACTCTTGATAATTGCGTATTATCTACGTATTGTTGAGTTTTCAATTTTCCCATACTGT
|||:|:||||||||||++||||||:||++||||||||||||||:||||||:::|||:|||
241 GAATTTTTGATAATTGCGTATTATTTACGTATTGTTGAGTTTTTAATTTTTTTATATTGT
301 CTGTCTGGATTTGCTTCTCATGATACAGGAGTTGTCTGTGAATCTCATTGGATATTTCCG
:|||:||||||||:||:|:||||||:|||||||||:|||||||:|:|||||||||||:++
301 TTGTTTGGATTTGTTTTTTATGATATAGGAGTTGTTTGTGAATTTTATTGGATATTTTCG
361 GATGGTTATTAACCGGGTCCAGTTGATCGTCCAAGCTCCCTTGGCATTTGTGAGGGGTTC
||||||||||||:++|||::|||||||++|::|||:|:::||||:||||||||||||||:
361 GATGGTTATTAATCGGGTTTAGTTGATCGTTTAAGTTTTTTTGGTATTTGTGAGGGGTTT
421 TACTAGTTGTGTGAACTCAGCACACGTAAATTTATAGATTTCCTCTCAAGGCTCAAAGTA
||:||||||||||||:|:||:|:|++|||||||||||||||::|:|:||||:|:||||||
421 TATTAGTTGTGTGAATTTAGTATACGTAAATTTATAGATTTTTTTTTAAGGTTTAAAGTA
WTfwd >>>>>>>>>>>>>>>>>>>>>>
481 CCAGACTTTTTATGCTAGGACAATCTTTGGATATGTGCCAGCGTATCTTGTGATCGTGGT
::|||:||||||||:|||||:|||:||||||||||||::||++|||:|||||||++||||
481 TTAGATTTTTTATGTTAGGATAATTTTTGGATATGTGTTAGCGTATTTTGTGATCGTGGT
MSPfwd >>>>>>>>>>>>>>>>>>>>>>>>>
USPfwd >>>>>>>>>>>>>>>>>>>>>>>>>
541 TCTTAAGGGTCGAGTGCTTAGCTCCTCATCCTCATGCTTAGGTCTGGAAATATGTAAAAG
|:||||||||++||||:||||:|::|:||::|:|||:||||||:||||||||||||||||
541 TTTTAAGGGTCGAGTGTTTAGTTTTTTATTTTTATGTTTAGGTTTGGAAATATGTAAAAG
<<<<<<<<<<<<<<<<<<<<< WTrev
601 GGGACGTAATGACAACACGAAGCTTATAAAAACTCAAAGCTATATGATCATAGGGCTTTC
||||++||||||:||:|++|||:|||||||||:|:||||:||||||||:||||||:|||:
601 GGGACGTAATGATAATACGAAGTTTATAAAAATTTAAAGTTATATGATTATAGGGTTTTT
<<<<<<<<<<<<<<<<<<<<<<<<<< MSPrev
<<<<<<<<<<<<<<<<<<<<<<<<<< USPrev
661 ACGATGAGCGAGATATTTTCTCTCAAGCCTGTGAAGCATTTTGAACGTCTTTATTCTAGG
|++|||||++|||||||||:|:|:|||::|||||||:||||||||++|:||||||:||||
661 ACGATGAGCGAGATATTTTTTTTTAAGTTTGTGAAGTATTTTGAACGTTTTTATTTTAGG
721 AAGACGAGTTTGATGTTTATTGGTATTGAGTTTCGCTCTTTCAGAAGTATTTTCAGAAGT
||||++|||||||||||||||||||||||||||++:|:|||:|||||||||||:||||||
721 AAGACGAGTTTGATGTTTATTGGTATTGAGTTTCGTTTTTTTAGAAGTATTTTTAGAAGT
781 TAGCAACGATTTCCTATGTTAGGTTCTGTTATTGGTTTTTTGCGTATGATTCGTGTCCTT
|||:||++||||::|||||||||||:||||||||||||||||++|||||||++|||::||
781 TAGTAACGATTTTTTATGTTAGGTTTTGTTATTGGTTTTTTGCGTATGATTCGTGTTTTT
841 CTGGTTGTAACCAAGCTGTACAAAAAAACGTGCAATTGATATCATTTGGTGGCGATTAGA
:|||||||||::|||:||||:|||||||++||:|||||||||:|||||||||++||||||
841 TTGGTTGTAATTAAGTTGTATAAAAAAACGTGTAATTGATATTATTTGGTGGCGATTAGA
901 CATTTTGGTGTCATTGACAAGTTCCAATGTACACTTCTCTTTAAGGTTTTTATTTAATTC
:||||||||||:|||||:|||||::||||||:|:||:|:||||||||||||||||||||:
901 TATTTTGGTGTTATTGATAAGTTTTAATGTATATTTTTTTTTAAGGTTTTTATTTAATTT
961 CTAAGTATTGATATTTTATTTATTTATTTGTTGTGGTCA
:||||||||||||||||||||||||||||||||||||:|
961 TTAAGTATTGATATTTTATTTATTTATTTGTTGTGGTTA
Used primers:
Forward MSP: 5’-GGATAATTTTTGGATATGTGTTAGC-3’
Reverse MSP: 5’-AACTTTAAATTTTTATAAACTTCGTA-3’
Forward USP: 5’-ATAATTTTTGGATATGTGTTAGTGT-3’
Reverse USP: 5’-AACTTTAAATTTTTATAAACTTCATA-3’
Forward WT: 5’-GGACAATCTTTGGATATGTGCC-3’
Reverse WT: 5’-AAGCTTCGTGTTGTCATTACG-3’
Promoter region of PpSBP3
1 ATAAAAGTCGTAAGGATCTCACTGGGTCCCTCTCACATTTCTCCCTGAAAAATGACGACG
||||||||++|||||||:|:|:|||||:::|:|:|:||||:|:::||||||||||++|++
1 ATAAAAGTCGTAAGGATTTTATTGGGTTTTTTTTATATTTTTTTTTGAAAAATGACGACG
61 TCGTTTTCATGACGGTGATTCTCGGTTGTCCATTTGTGGCCTTGACGGAAATGTGTGGGC
|++||||:||||++||||||:|++|||||::||||||||::||||++||||||||||||+
61 TCGTTTTTATGACGGTGATTTTCGGTTGTTTATTTGTGGTTTTGACGGAAATGTGTGGGC
121 GATCTTTGATGGCCACTCTTTTTGTTTTGTTGCCAATCCTCCTCCTATATTTAGTGACTG
+||:||||||||::|:|:||||||||||||||::|||::|::|::||||||||||||:||
121 GATTTTTGATGGTTATTTTTTTTGTTTTGTTGTTAATTTTTTTTTTATATTTAGTGATTG
181 GAGGATCTTTGCTGTTGCTGATTTCCTGGCTTATCCTGGGCGCTGCTATAAGTTAGGCTT
||||||:||||:|||||:||||||::|||:||||::||||++:||:|||||||||||:||
181 GAGGATTTTTGTTGTTGTTGATTTTTTGGTTTATTTTGGGCGTTGTTATAAGTTAGGTTT
241 TTCTTCATCCATTTTGAGGTGTCACAATATATTTATGGTCGTCGTAATTGTTTTTAATTT
||:||:||::||||||||||||:|:||||||||||||||++|++||||||||||||||||
241 TTTTTTATTTATTTTGAGGTGTTATAATATATTTATGGTCGTCGTAATTGTTTTTAATTT
301 TACCTCCGTCGGGGTCTGCGCCACCATATGCTTGATAAATTGCAGATTTCAAAGCAGAAC
||::|:++|++||||:||++::|::|||||:|||||||||||:||||||:||||:||||+
301 TATTTTCGTCGGGGTTTGCGTTATTATATGTTTGATAAATTGTAGATTTTAAAGTAGAAC
361 GTTTCGGTGATGCATGGTCACTTGTGCAGGTTTCTAGTTACCTGGTTGGTTATTTCTTTT
+|||++||||||:|||||:|:|||||:||||||:||||||::|||||||||||||:||||
361 GTTTCGGTGATGTATGGTTATTTGTGTAGGTTTTTAGTTATTTGGTTGGTTATTTTTTTT
421 TTGTTTATTTCTCGAGTTTGCGGGTAGTGGTGGAGTTATGGATGCTTAGAACGCTGCAAA
||||||||||:|++||||||++||||||||||||||||||||||:||||||++:||:|||
421 TTGTTTATTTTTCGAGTTTGCGGGTAGTGGTGGAGTTATGGATGTTTAGAACGTTGTAAA
481 TAGGCCAGTTTGGTGTTGGTGATGAGGATTGCGCTCCTTCCAGTCACGATTGTGTGCCTG
||||::|||||||||||||||||||||||||++:|::||::|||:|++||||||||::||
481 TAGGTTAGTTTGGTGTTGGTGATGAGGATTGCGTTTTTTTTAGTTACGATTGTGTGTTTG
541 CATTCTGTGGAGTCTGTAATCCGCAGTTCAGTTTTTGTGTTTTAGCAAATTAGCGCATGC
:|||:||||||||:||||||:++:||||:||||||||||||||||:|||||||++:|||:
541 TATTTTGTGGAGTTTGTAATTCGTAGTTTAGTTTTTGTGTTTTAGTAAATTAGCGTATGT
601 TTCGCAGTCTTACGTGCTTATGACGTTCCTATGGACGTCCTTCTATCGTTGCCCGAATTT
||++:|||:|||++||:||||||++||::||||||++|::||:|||++|||::++|||||
601 TTCGTAGTTTTACGTGTTTATGACGTTTTTATGGACGTTTTTTTATCGTTGTTCGAATTT
661 TCTGTGCTTCTTTCAAAGTCGCTGGCAATTGCAGACCTGGAAATTGGGTATTGTTTCCTC
|:||||:||:|||:|||||++:|||:|||||:|||::|||||||||||||||||||::|:
661 TTTGTGTTTTTTTTAAAGTCGTTGGTAATTGTAGATTTGGAAATTGGGTATTGTTTTTTT
721 AGTTGCTTACTCTAAGTGCGAATACTACTTAGACGTGCTGTTGAGGGTAAACTTGCTTCT
|||||:|||:|:||||||++||||:||:|||||++||:|||||||||||||:|||:||:|
721 AGTTGTTTATTTTAAGTGCGAATATTATTTAGACGTGTTGTTGAGGGTAAATTTGTTTTT
WTfwd >>>>>>>>
781 GAGGCTCTCCACAGTTTTAGAAGTTTGATTAATAAGATATAGAGGCTTTTCTCTGATCAC
||||:|:|::|:|||||||||||||||||||||||||||||||||:||||:|:||||:|:
781 GAGGTTTTTTATAGTTTTAGAAGTTTGATTAATAAGATATAGAGGTTTTTTTTTGATTAT
MSPfwd >>>>>>>>
USPfwd >>>>>>>>
>>>>>>>>>>>>>>>>>>
841 TTCAAATGGATGGTGATCGTGTTCTTTGATACTGCTGAAGCTTGGCGAGTTTTTTTGGTT
||:||||||||||||||++||||:|||||||:||:|||||:||||++|||||||||||||
841 TTTAAATGGATGGTGATCGTGTTTTTTGATATTGTTGAAGTTTGGCGAGTTTTTTTGGTT
>>>>>>>>>>>>>>>>>>
>>>>>>>>>>>>>>>>>>
<<<<<<<<<<<
901 CAAATCTCCGAAGCCTATGGACCATTCAGCAGCCTGAGCTTCCAATTTGGCCGTCAGTGT
:||||:|:++|||::||||||::|||:||:||::||||:||::|||||||:++|:|||||
901 TAAATTTTCGAAGTTTATGGATTATTTAGTAGTTTGAGTTTTTAATTTGGTCGTTAGTGT
<<<<<<<<<<<
<<<<<<<<<<<
<<<<<<<<<<<<<<< WTrev
961 CGTATGTTACTCCTATGTTGAAGCTTGTGGGCTGGATCGC
++|||||||:|::||||||||||:|||||||:|||||++:
961 CGTATGTTATTTTTATGTTGAAGTTTGTGGGTTGGATCGT
<<<<<<<<<<<<<< MSPrev
<<<<<<<<<<<<<< USPrev
Used primers:
Forward MSP: 5’-TTGATTATTTTAAATGGATGGTGATC-3’
Reverse MSP: 5’-AAAAATAACATACGACACTAACGAC-3’
Forward USP: 5’-TTGATTATTTTAAATGGATGGTGATT-3’
Reverse USP: 5’-AAAAATAACATACAACACTAACAAC-3’
Forward WT: 5’-CTGATCACTTCAAATGGATGGTGATC-3’
Reverse WT: 5’-TAGGAGTAACATACGACACTGACGGC-3’
Promoter region of PpGNT1
1 CTGTTGATGTATCCTAGATATTGTTGCATAGTTCTTGTCTAGTTTATTAAAATAAGAATA
:|||||||||||::||||||||||||:||||||:||||:|||||||||||||||||||||
1 TTGTTGATGTATTTTAGATATTGTTGTATAGTTTTTGTTTAGTTTATTAAAATAAGAATA
61 ATAATAATAAATGTTTATATATTTAATATTAAAATAACCAATGTACAAAATATGTTAGAC
|||||||||||||||||||||||||||||||||||||::||||||:|||||||||||||:
61 ATAATAATAAATGTTTATATATTTAATATTAAAATAATTAATGTATAAAATATGTTAGAT
121 ATTTTTGTATCAAATTCAAAAATATATTAAAAAAAGTACACAACATAGGTTACAATGGAT
||||||||||:|||||:|||||||||||||||||||||:|:||:||||||||:|||||||
121 ATTTTTGTATTAAATTTAAAAATATATTAAAAAAAGTATATAATATAGGTTATAATGGAT
181 CATAAATCATTAATTATTCTTGATATTATGTTAAAAAAGTTGAGAAACATCTACAATTAG
:||||||:||||||||||:||||||||||||||||||||||||||||:||:||:||||||
181 TATAAATTATTAATTATTTTTGATATTATGTTAAAAAAGTTGAGAAATATTTATAATTAG
241 TTAGAAACTTTCATATTGTTTAAAATCATTTTGTTATAAAAACAATACCATTTAATAAAG
|||||||:|||:||||||||||||||:|||||||||||||||:||||::|||||||||||
241 TTAGAAATTTTTATATTGTTTAAAATTATTTTGTTATAAAAATAATATTATTTAATAAAG
301 ATGAATCTTATTAATAGGTAATTCTGTTGATATATTTCCTTGACACAGCAATATGGATAG
||||||:||||||||||||||||:|||||||||||||::||||:|:||:|||||||||||
301 ATGAATTTTATTAATAGGTAATTTTGTTGATATATTTTTTTGATATAGTAATATGGATAG
361 GAATCATAGTCTTAGATATAGTAGTTTTAAGGTGATTAATGTCAAAAGAACATAACTAGC
||||:|||||:|||||||||||||||||||||||||||||||:|||||||:||||:|||:
361 GAATTATAGTTTTAGATATAGTAGTTTTAAGGTGATTAATGTTAAAAGAATATAATTAGT
421 AAAAGAATTAAAGCATAGTCCACCAAACATATATTTTGAATAGCAAGATAATATAAATTA
|||||||||||||:|||||::|::|||:|||||||||||||||:||||||||||||||||
421 AAAAGAATTAAAGTATAGTTTATTAAATATATATTTTGAATAGTAAGATAATATAAATTA
481 CTTTAAAACAGAATATAATATAATATTAAATTTACTTTTATATTATTTTTAGATTAATGA
:|||||||:|||||||||||||||||||||||||:|||||||||||||||||||||||||
481 TTTTAAAATAGAATATAATATAATATTAAATTTATTTTTATATTATTTTTAGATTAATGA
541 AACTTCACAATAATACATGAAAGAAATTTTTGTGACTTTGGCACCTTTTATTAGCAATGT
||:||:|:|||||||:|||||||||||||||||||:|||||:|::|||||||||:|||||
541 AATTTTATAATAATATATGAAAGAAATTTTTGTGATTTTGGTATTTTTTATTAGTAATGT
601 ATTACTCTTACTATGTAAAAGTATCAAATTTAACAAAAATTGAAAAAATATACATCCACT
||||:|:|||:|||||||||||||:||||||||:||||||||||||||||||:||::|:|
601 ATTATTTTTATTATGTAAAAGTATTAAATTTAATAAAAATTGAAAAAATATATATTTATT
661 TATACTATCAATTAATTAATTAAAATTTTATTTATTTTTTAATTTTTTGTTGACTTAAAA
||||:|||:||||||||||||||||||||||||||||||||||||||||||||:||||||
661 TATATTATTAATTAATTAATTAAAATTTTATTTATTTTTTAATTTTTTGTTGATTTAAAA
721 TATATCTATATATATATATATATATATATATATGTATATTCACATTCTTGAACAAGAATT
|||||:||||||||||||||||||||||||||||||||||:|:|||:|||||:|||||||
721 TATATTTATATATATATATATATATATATATATGTATATTTATATTTTTGAATAAGAATT
WTfwd >>>>>>>>>>>>>>>>>>>>>>>
781 TTGGATTCAAGGAGGTGAATGCTTTGCACAAAAAAAAGTTTTATCTCTAAATTCTTAGAC
|||||||:|||||||||||||:||||:|:|||||||||||||||:|:||||||:|||||:
781 TTGGATTTAAGGAGGTGAATGTTTTGTATAAAAAAAAGTTTTATTTTTAAATTTTTAGAT
MSPfwd >>>>>>>>>>>>>>>>>>>>>
USPfwd >>>>>>>>>>>>>>>>>>>>
841 AACGTCATTCAAAATAAGTTTTAAAACAGCGACTAGTCATAAAATACGTATTTACACACT
||++|:|||:||||||||||||||||:||++|:||||:||||||||++||||||:|:|:|
841 AACGTTATTTAAAATAAGTTTTAAAATAGCGATTAGTTATAAAATACGTATTTATATATT
>>>>
>>>>>
901 TGTATATGATGTACCATAGACGGTAATCGTACATATTTGCCGACACCCTGCAATTAATAG
|||||||||||||::|||||++|||||++||:|||||||:++|:|:::||:|||||||||
901 TGTATATGATGTATTATAGACGGTAATCGTATATATTTGTCGATATTTTGTAATTAATAG
<<<<<<<<<<<<<<<<<<<<<<< WTrev
961 AGTTCGAATATCCCCGCCGCGTTCAAGTCGCCTCGTGCAA
||||++|||||:::++:++++||:||||++::|++||:||
961 AGTTCGAATATTTTCGTCGCGTTTAAGTCGTTTCGTGTAA
<<<<<<<<<<<<<<<<<<< MSPrev
<<<<<<<<<<<<<<<<<<<<< USPrev
Used primers:
Forward MSP: 5’-TTTATTTTTAAATTTTTAGATAACG-3’
Reverse MSP: 5’-AACGACTTAAACGCGACGA-3’
Forward USP: 5’-TTATTTTTAAATTTTTAGATAATGT-3’
Reverse USP: 5’-AAACAACTTAAACACAACAAA-3’
Forward WT: 5’-GTTTTATCTCTAAATTCTTAGAC-3’
Reverse WT: 5’-GGCGACTTGAACGCGGCGGGGAT-3’
Intron 4 within the miRNA166 binding site of PpC3HDZIP1
1 GGTATGAAGGTATGGATGCCATGCCTTCCTACGGCACGTTCTACAGTGTATTGTGGAGTA
||||||||||||||||||::|||::||::||++|:|++||:||:||||||||||||||||
1 GGTATGAAGGTATGGATGTTATGTTTTTTTACGGTACGTTTTATAGTGTATTGTGGAGTA
MSPfwd >>>>>>>>>>>>>>>>>>>>>>
USPfwd >>>>>>>>>>>>>>>>>>>>>>>
61 GCGAGCCTCACCTGTAACTCTTGATCTATAGATTCCATTATCAGAGATATGATCGCACGA
|++||::|:|::|||||:|:|||||:||||||||::|||||:|||||||||||++:|++|
61 GCGAGTTTTATTTGTAATTTTTGATTTATAGATTTTATTATTAGAGATATGATCGTACGA
<<<<<<<
<<<<<<<
121 AATAACTCTTTGTTCCAACCTTTTGTAAAATAAGTATTAGCGGAGTCATGGTACTGGAGC
|||||:|:||||||::||::||||||||||||||||||||++||||:||||||:|||||:
121 AATAATTTTTTGTTTTAATTTTTTGTAAAATAAGTATTAGCGGAGTTATGGTATTGGAGT
<<<<<<<<<<<<<<< MSPrev
<<<<<<<<<<<<<<<<<< USPrev
181 AAAGTCAAACAAATTAATTTGACTCAAAACACGACTTCGAATTAATTTAGGAGCTAACAA
|||||:|||:||||||||||||:|:||||:|++|:||++||||||||||||||:|||:||
181 AAAGTTAAATAAATTAATTTGATTTAAAATACGATTTCGAATTAATTTAGGAGTTAATAA
241 GGTAATGATATTGATTCTTTAATTCAAATTAAAGTGGTTGATTGCAAATGCCATTGCTGA
||||||||||||||||:|||||||:|||||||||||||||||||:|||||::||||:|||
241 GGTAATGATATTGATTTTTTAATTTAAATTAAAGTGGTTGATTGTAAATGTTATTGTTGA
301 TACGTCACTAGT
||++|:|:||||
301 TACGTTATTAGT
Used primers:
Forward MSP: 5’-GTTATGTTTTTTTACGGTACGT-3’
Reverse MSP: 5’-AAACAAAAAATTATTTCGTACG-3’
Forward USP: 5’-TGTTATGTTTTTTTATGGTATGT-3’
Reverse USP: 5’-TTAAAACAAAAAATTATTTCATACA-3’
Intron 1 upstream of the miRNA166 binding site of PpC3HDZIP1
1 GGTATGTATCCGTGTCCTTCGCCAGATTCTAGGAGAGGAAAATTGTTTGGGCATAAACCT
|||||||||:++|||::||++::|||||:||||||||||||||||||||||:|||||::|
1 GGTATGTATTCGTGTTTTTCGTTAGATTTTAGGAGAGGAAAATTGTTTGGGTATAAATTT
61 CGTATAGAAGTTCGTTCGATCTTGAAATCTTTCTCAAACGGAGATTCTGGATGACATGCA
++||||||||||++||++||:|||||||:|||:|:|||++||||||:|||||||:|||:|
61 CGTATAGAAGTTCGTTCGATTTTGAAATTTTTTTTAAACGGAGATTTTGGATGATATGTA
MSPfwd >>>>>>>>>>>>>>>>>>>>>>>>>
USPfwd >>>>>>>>>>>>>>>>>>>>>>>>>
121 CGTGAAGTTGATTCACTATATTATGTCGCCTCGAATTTCACTTCGTATTGAGCCATCTCA
++|||||||||||:|:||||||||||++::|++|||||:|:||++|||||||::||:|:|
121 CGTGAAGTTGATTTATTATATTATGTCGTTTCGAATTTTATTTCGTATTGAGTTATTTTA
181 TGTTTTATTCACTTCTGTCTTGAGATTGTCATACGTGCCGCTCCGTGATGTGCGGATAGG
|||||||||:|:||:|||:||||||||||:|||++||:++:|:++|||||||++||||||
181 TGTTTTATTTATTTTTGTTTTGAGATTGTTATACGTGTCGTTTCGTGATGTGCGGATAGG
<<<<<<<<
<<<<<<<<<<
241 TGGACAGGTGCACAACATGATAATCCATGTTGTGGTCGAGGGGTAGGGGGGTGGTACACG
||||:|||||:|:||:||||||||::||||||||||++||||||||||||||||||:|++
241 TGGATAGGTGTATAATATGATAATTTATGTTGTGGTCGAGGGGTAGGGGGGTGGTATACG
<<<<<<<<<<<<<<<<< MSPrev
<<<<<<<<<<<<<<<< USPrev
301 ACACAATTAATTGAAATGAGTGGAGAGTGTATTGCAG
|:|:||||||||||||||||||||||||||||||:||
301 ATATAATTAATTGAAATGAGTGGAGAGTGTATTGTAG
Used primers:
Forward MSP: 5’-TTCGATTTTGAAATTTTTTTTAAAC-3’
Reverse MSP: 5’-TATTATACACCTATCCACCTATCCG-3’
Forward USP: 5’-TGATTTTGAAATTTTTTTTAAATGG-3’
Reverse USP: 5’-ATTATACACCTATCCACCTATCCACA-3’
Exon 14 downstream of the miRNA166 binding site of PpC3HDZIP1
1 GGACGGAATCGGGCTGAATCGTACACTTGATCTGGCTTCCACACTTGAAGATCACGAGGC
|||++||||++||:|||||++||:|:|||||:|||:||::|:|:||||||||:|++|||:
1 GGACGGAATCGGGTTGAATCGTATATTTGATTTGGTTTTTATATTTGAAGATTACGAGGT
MSPfwd >>>>>>>>>>>>>>>>>>>>>>>>>
USPfwd >>>>>>>>>>>>>>>>>>>>>>>>>
61 AGGATTGAATGGAGAGAGCAAGTCTAATGGCAGCTCTAGCCAAGTGCGATCCGTTCTGAC
||||||||||||||||||:||||:||||||:||:|:|||::|||||++||:++||:|||:
61 AGGATTGAATGGAGAGAGTAAGTTTAATGGTAGTTTTAGTTAAGTGCGATTCGTTTTGAT
121 AATAGCTTTTCAGTTTGCGTATGAAGTTCATACACGCGAAACATGCGCAGTGATGGCCCG
|||||:||||:||||||++|||||||||:|||:|++++|||:|||++:||||||||::++
121 AATAGTTTTTTAGTTTGCGTATGAAGTTTATATACGCGAAATATGCGTAGTGATGGTTCG
<<<<<<<<<<<<<<<
<<<<<<<<<<<<<<<<<
181 CCAGTATGTTCGCACAGTGGTTGCATCCGTGCAGCGGGTTGCCATGGCTTTGGCACCGTC
::||||||||++:|:||||||||:||:++||:||++|||||::||||:|||||:|:++|:
181 TTAGTATGTTCGTATAGTGGTTGTATTCGTGTAGCGGGTTGTTATGGTTTTGGTATCGTT
<<<<<<<<<< MSPrev
<<<<<<<< USPrev
241 CCGTGGTCAGCCCCGTCCAGCACTGGGCAACTCAGATGCCATCAGTTTGGCGCGTCACAT
:++||||:||:::++|::||:|:||||:||:|:|||||::||:|||||||++++|:|:||
241 TCGTGGTTAGTTTCGTTTAGTATTGGGTAATTTAGATGTTATTAGTTTGGCGCGTTATAT
301 CCTGAGCAGCTACAG
::||||:||:||:||
301 TTTGAGTAGTTATAG
Used primers:
Forward MSP: 5’-TGGTTTTTATATTTGAAGATTACGA-3’
Reverse MSP: 5’-AACATACTAACGAACCATCACTACG-3’
Forward USP: 5’-TGGTTTTTATATTTGAAGATTATGA-3’
Reverse USP: 5’-CATACTAACAAACCATCACTACACA-3’
Exon 1 upstream of the miRNA160 binding site of PpARF
1 GGTATCGATCTGGAGCCCGTTGCAAACTCAATGGTGTATTTTATAGGGCAAAAGTCTGAT
|||||++||:|||||::++|||:|||:|:|||||||||||||||||||:||||||:||||
1 GGTATCGATTTGGAGTTCGTTGTAAATTTAATGGTGTATTTTATAGGGTAAAAGTTTGAT
61 CTATATGGAATGCATCCTCTCAGAGTTGCAAATCATGGACTGCATGTCACTCTGGGTTAT
:|||||||||||:||::|:|:|||||||:||||:|||||:||:||||:|:|:||||||||
61 TTATATGGAATGTATTTTTTTAGAGTTGTAAATTATGGATTGTATGTTATTTTGGGTTAT
121 TCTCGATCACCTAGCTTTGCTGGAGTTCAAATTGGTGAGTACGAGTATTATGAGTGATCT
|:|++||:|::|||:||||:|||||||:|||||||||||||++|||||||||||||||:|
121 TTTCGATTATTTAGTTTTGTTGGAGTTTAAATTGGTGAGTACGAGTATTATGAGTGATTT
181 CGAGTTTATGGTCCCCTTCTTTCATGATCAAGGGTAATTTATATCAAGGGTGTATATGAG
++||||||||||::::||:|||:|||||:|||||||||||||||:|||||||||||||||
181 CGAGTTTATGGTTTTTTTTTTTTATGATTAAGGGTAATTTATATTAAGGGTGTATATGAG
241 AGATACGCACTTATTGAGTGGACCTTTTCTCATACTGCATTTACACCCCTGTCAGTTGCA
|||||++:|:||||||||||||::||||:|:|||:||:|||||:|::::|||:|||||:|
241 AGATACGTATTTATTGAGTGGATTTTTTTTTATATTGTATTTATATTTTTGTTAGTTGTA
301 GCATCCTGGTTTGGAATGCCGGGTCCAGTCCCTCTATTATCCATGAGTGTAAAATCGGAG
|:||::||||||||||||:++|||::|||:::|:||||||::|||||||||||||++|||
301 GTATTTTGGTTTGGAATGTCGGGTTTAGTTTTTTTATTATTTATGAGTGTAAAATCGGAG
361 AGTCTCGATGACATTGGAGGTCACGAGAAAAAATCTGTAACTGGGTCGGAAGTGGGTGGC
|||:|++||||:|||||||||:|++|||||||||:|||||:|||||++|||||||||||:
361 AGTTTCGATGATATTGGAGGTTACGAGAAAAAATTTGTAATTGGGTCGGAAGTGGGTGGT
421 CTCGATGCTCAGCTGTGGCATGCCTGTGCTGGGGGTATGGTTCAACTGCCTCATGTGGGT
:|++|||:|:||:|||||:|||::||||:|||||||||||||:||:||::|:||||||||
421 TTCGATGTTTAGTTGTGGTATGTTTGTGTTGGGGGTATGGTTTAATTGTTTTATGTGGGT
481 GCTAAGGTTGTCTATTTTCCCCAAGGCCATGGCGAACAAGCTGCTTCAACTCCCGAGTTC
|:|||||||||:||||||::::||||::||||++||:|||:||:||:||:|::++||||:
481 GTTAAGGTTGTTTATTTTTTTTAAGGTTATGGCGAATAAGTTGTTTTAATTTTCGAGTTT
541 CCCCGCACTTTGGTTCCAAATGGAAGTGTTCCCTGCCGAGTTGTGTCAGTTAACTTTCTG
:::++:|:|||||||::|||||||||||||:::||:++||||||||:||||||:|||:||
541 TTTCGTATTTTGGTTTTAAATGGAAGTGTTTTTTGTCGAGTTGTGTTAGTTAATTTTTTG
MSPfwd >>>>>
USPfwd >>>>>
601 GCTGATACAGAAACAGACGAGGTATTTGCTCGTATTTGCCTGCAGCCTGAGATTGGCTCC
|:|||||:|||||:|||++|||||||||:|++||||||::||:||::|||||||||:|::
601 GTTGATATAGAAATAGACGAGGTATTTGTTCGTATTTGTTTGTAGTTTGAGATTGGTTTT
>>>>>>>>>>>>>>>>>>>>
>>>>>>>>>>>>>>>>>>>>
661 TCCGCTCAGGATTTAACAGATGATTCTCTTGCGTCTCCGCCTCTAGAGAAACCAGCTTCA
|:++:|:|||||||||:||||||||:|:|||++|:|:++::|:||||||||::||:||:|
661 TTCGTTTAGGATTTAATAGATGATTTTTTTGCGTTTTCGTTTTTAGAGAAATTAGTTTTA
721 TTTGCCAAAACGCTCACTCAAAGTGATGCAAACAACGGTGGAGGCTTTTCAATACCTCGT
||||::||||++:|:|:|:|||||||||:|||:||++|||||||:||||:||||::|++|
721 TTTGTTAAAACGTTTATTTAAAGTGATGTAAATAACGGTGGAGGTTTTTTAATATTTCGT
<<<<<<<<<<<<<<<<<<<<<<<<<MSPrev
<<<<<<<<<<<<<<<<<<<<<<<<<USPrev
781 TATTGTGCTGAAACTATTTTCCCACCTCTCGATTACTGTATCGATCCTCCTGTTCAAACT
|||||||:|||||:||||||:::|::|:|++||||:|||||++||::|::||||:|||:|
781 TATTGTGTTGAAATTATTTTTTTATTTTTCGATTATTGTATCGATTTTTTTGTTTAAATT
841 GTTCTTGCAAAAGATGTCCATGGAGAGGTGTGGAAATTTCGTCACATTTACAGG
|||:|||:|||||||||::||||||||||||||||||||++|:|:|||||:|||
841 GTTTTTGTAAAAGATGTTTATGGAGAGGTGTGGAAATTTCGTTATATTTATAGG
Used primers:
Forward MSP: 5’-TTTTGGTTGATATAGAAATAGACGA-3’
Reverse MSP: 5’-GAAATATTAAAAAACCTCCACCGTT-3’
Forward USP: 5’-TTTTGGTTGATATAGAAATAGATGA-3’
Reverse USP: 5’-AAAATATTAAAAAACCTCCACCATT-3’
Exon 4 downstream of the miRNA160 binding site of PpARF
1 AGGAATTCCATGGAGACAGTCAGACGCCTCATACTCCTGCATCTGGTAGCCAATGAGGCT
|||||||::|||||||:|||:|||++::|:|||:|::||:||:||||||::|||||||:|
1 AGGAATTTTATGGAGATAGTTAGACGTTTTATATTTTTGTATTTGGTAGTTAATGAGGTT
61 AAAGCTTGATCATAGCTCATAACCCTCTCACAGGACGTAATGGGGGTGACAACATGCTAA
||||:|||||:||||:|:||||:::|:|:|:||||++||||||||||||:||:|||:|||
61 AAAGTTTGATTATAGTTTATAATTTTTTTATAGGACGTAATGGGGGTGATAATATGTTAA
MSPfwd >>>>>>>>>>>>>>>>>>>>>>>>>
USPfwd >>>>>>>>>>>>>>>>>>>>>>>>>>
121 CAGAATTGCACGGTAAAGGAAAACTGTACTAGGCATGTTATATGGGAATTCGGATCGCTT
:|||||||:|++|||||||||||:||||:||||:||||||||||||||||++|||++:||
121 TAGAATTGTACGGTAAAGGAAAATTGTATTAGGTATGTTATATGGGAATTCGGATCGTTT
181 CTTGCAATTAAACACGCTAGCGCCGTTTGGTGCCAATGTTATTCTGGCATTTGTTTTGTT
:|||:|||||||:|++:|||++:++|||||||::|||||||||:|||:||||||||||||
181 TTTGTAATTAAATACGTTAGCGTCGTTTGGTGTTAATGTTATTTTGGTATTTGTTTTGTT
<<<<<<<<<<<<<<<<<<<<<<<< MSPrev
<<<<<<<<<<<<<<<<<<<<<<<<<< USPrev
241 TCCTTTGGAAACAAATTGCTATATTTCAAAGTCCTTTGGAGGAGCTCGC
|::||||||||:||||||:|||||||:|||||::||||||||||:|++:
241 TTTTTTGGAAATAAATTGTTATATTTTAAAGTTTTTTGGAGGAGTTCGT
Used primers:
Forward MSP: 5’-AGTTTATAATTTTTTTATAGGACGT-3’
Reverse MSP: 5’-AAAATAACATTAACACCAAACGAC-3’
Forward USP: 5’-TAGTTTATAATTTTTTTATAGGATGT-3’
Reverse USP: 5’-CCAAAATAACATTAACACCAAACAAC-3’
Intron 2 upstream of the miRNA160 binding site of PpARF
1 AGTTCTCCATGGCGGGTTCTGCAGGTTAGCTTTTTGTTTGTCTAATCAAAGCAATCAATG
||||:|::||||++||||:||:|||||||:|||||||||||:||||:||||:|||:||||
1 AGTTTTTTATGGCGGGTTTTGTAGGTTAGTTTTTTGTTTGTTTAATTAAAGTAATTAATG
MSPfwd >>>>>>>>>>>>>>
USPfwd >>>>>>>>>>>>>>>
61 TGCTAATGAACGTGCTGCCTGCATTGTCTGCAATGACTATGTACTGCGTAGTCAGTGGAA
||:|||||||++||:||::||:|||||:||:|||||:||||||:||++||||:|||||||
61 TGTTAATGAACGTGTTGTTTGTATTGTTTGTAATGATTATGTATTGCGTAGTTAGTGGAA
>>>>>>>>>>>>>
>>>>>>>>>>>>>
121 ATAGGTGGTTGATTATGAGATTTTGGTTGTGCAGGTCACTTGGGATGAGCCGGACCTATT
|||||||||||||||||||||||||||||||:||||:|:||||||||||:++||::||||
121 ATAGGTGGTTGATTATGAGATTTTGGTTGTGTAGGTTATTTGGGATGAGTCGGATTTATT
181 GCAGGGAGTGAATCGTGTAAGCCCATGGCAGTTAGAGCTTGTGGCGACACTTCCTATGCA
|:|||||||||||++||||||:::||||:||||||||:||||||++|:|:||::||||:|
181 GTAGGGAGTGAATCGTGTAAGTTTATGGTAGTTAGAGTTTGTGGCGATATTTTTTATGTA
<<<<<<<<<<<<<<<<<<
<<<<<<<<<<<<<<<<<<
241 GCTGCCCCCTGTCTCTCTTCCCAAAAAGAAACTGCG
|:||:::::|||:|:|:||:::|||||||||:||++
241 GTTGTTTTTTGTTTTTTTTTTTAAAAAGAAATTGCG
<<<<<<< MSPrev
<<<<<<< USPrev
Used primers:
Forward MSP: 5’-TAAAGTAATTAATGTGTTAATGAACGT-3’
Reverse MSP: 5’-AAACAACTACATAAAAAATATCGCC-3’
Forward USP: 5’-TTAAAGTAATTAATGTGTTAATGAATGT-3’
Reverse USP: 5’-AAACAACTACATAAAAAATATCACC-3’
Intron 3 downstream of the miRNA160 binding site of PpARF
1 AGGATCAGGTTTGTATCAACAAATCTCTAGTTCGTTTTGGCATGGAGTTCACAATGTGCT
|||||:||||||||||:||:||||:|:|||||++||||||:||||||||:|:||||||:|
1 AGGATTAGGTTTGTATTAATAAATTTTTAGTTCGTTTTGGTATGGAGTTTATAATGTGTT
MSPfwd >>>>>>>>>>>>>
USPfwd >>>>>>>>>>>>>
61 CTTCAAGCTTCGTTGTATGCTAAACTCTACTGCAATACTGCTACGGCGTGCCTTTTCTTT
:||:|||:||++|||||||:||||:|:||:||:||||:||:||++|++||::||||:|||
61 TTTTAAGTTTCGTTGTATGTTAAATTTTATTGTAATATTGTTACGGCGTGTTTTTTTTTT
>>>>>>>>>>>>>
>>>>>>>>>>>>>
121 TTTCGAAGTATGATATGATGACCTAATGGTCTTTTTGAATACGACAGGAATTCCATGGAG
|||++||||||||||||||||::|||||||:||||||||||++|:|||||||::||||||
121 TTTCGAAGTATGATATGATGATTTAATGGTTTTTTTGAATACGATAGGAATTTTATGGAG
<<<<<<<<<<<<<<<<<<<<<
<<<<<<<<<<<<<<<<<<<<<
181 ACAGTCAGACGCCTCATACTCCTGCATCTGGTAGCCAATGAGGCTAAAGCTTGATC
|:|||:|||++::|:|||:|::||:||:||||||::|||||||:|||||:|||||:
181 ATAGTTAGACGTTTTATATTTTTGTATTTGGTAGTTAATGAGGTTAAAGTTTGATT
<<<< MSPrev
<<<< USPrev
Used primers:
Forward MSP: 5’-TTTATAATGTGTTTTTTAAGTTTCGT-3’
Reverse MSP: 5’-CTATCTCCATAAAATTCCTATCGTA-3’
Forward USP: 5’-TTTATAATGTGTTTTTTAAGTTTTGT-3’
Reverse USP: 5’-CTATCTCCATAAAATTCCTATCATA-3’
Sequence of PpTAS4
1 ACCAAAGTAGATTGAATCAATCCGTGCATCGATTCACAGGAGCACTGACCTGATCTATGC
|::||||||||||||||:|||:++||:||++|||:|:|||||:|:|||::||||:||||+
1 ATTAAAGTAGATTGAATTAATTCGTGTATCGATTTATAGGAGTATTGATTTGATTTATGC
61 GACGGTGCGAGAAAAATCATCCCAGCGTGGTGCTACGCTAGTCACCTAGTCATCAGCATC
+|++|||++||||||||:||:::||++|||||:||++:||||:|::||||:||:||:||+
61 GACGGTGCGAGAAAAATTATTTTAGCGTGGTGTTACGTTAGTTATTTAGTTATTAGTATC
MSPfwd >>>>>>>>>>>>>>>>>>>>>>>
USPfwd >>>>>>>>>>>>>>>>>>>>>>>>
121 GGTGCTACGTAAACTTATGGCAATGGTGCGTTCCTATCAGTGCGTCACTTCAAGGAAGCC
+|||:||++||||:||||||:|||||||++||::|||:||||++|:|:||:|||||||::
121 GGTGTTACGTAAATTTATGGTAATGGTGCGTTTTTATTAGTGCGTTATTTTAAGGAAGTT
<<<<<<<<<<<<<<<<<<<<
<<<<<<<<<<<<<<<<<<<<
181 CTTCCCAAGTCTTCATCCGGCCCGCTACCTTTGCGTAGCTGCTTCACTGGAGGCCTGGGT
:||:::||||:||:||:++|::++:||::||||++|||:||:||:|:||||||::|||||
181 TTTTTTAAGTTTTTATTCGGTTCGTTATTTTTGCGTAGTTGTTTTATTGGAGGTTTGGGT
<<<<< MSPrev
<<<<< USPrev
241 GGAGACTGGCGGACAGCTTTGCGATGTTACGGTTGTAGCCAATTCTTGTTGCACTTAGAT
|||||:|||++||:||:||||++||||||++|||||||::||||:||||||:|:||||||
241 GGAGATTGGCGGATAGTTTTGCGATGTTACGGTTGTAGTTAATTTTTGTTGTATTTAGAT
301 TTCCACTGGGCGTTATCCCTCTTGAGCTGAGAAGACAAGGGCTCCCTCCTAGGGGGCGAA
||::|:||||++||||:::|:|||||:||||||||:|||||:|:::|::|||||||++||
301 TTTTATTGGGCGTTATTTTTTTTGAGTTGAGAAGATAAGGGTTTTTTTTTAGGGGGCGAA
361 AATAGGTGAGCTGGGGTCACCTTGTTAGCGGGGTGTTAAGCATTTGAATGCAACACTCCT
||||||||||:||||||:|::|||||||++||||||||||:|||||||||:||:|:|::|
361 AATAGGTGAGTTGGGGTTATTTTGTTAGCGGGGTGTTAAGTATTTGAATGTAATATTTTT
421 ACGCAAGACCCTAGCTATGGCTCCATAGGGTGTGATGAGTGCTTCATCCGGTGCTCTTCT
|++:||||:::|||:|||||:|::|||||||||||||||||:||:||:++|||:|:||:|
421 ACGTAAGATTTTAGTTATGGTTTTATAGGGTGTGATGAGTGTTTTATTCGGTGTTTTTTT
481 ACTGCCTTGCCCACCTACCCTTGTGATATGGGCCGCGCGTGTCTGCGTGTCTCCTGTATC
|:||::|||:::|::||:::||||||||||||:++++++|||:||++|||:|::|||||+
481 ATTGTTTTGTTTATTTATTTTTGTGATATGGGTCGCGCGTGTTTGCGTGTTTTTTGTATC
541 GGTTGTATATCACTCCTGAGCTACGGGTGTGCAATTCCCATGTCTTTTGGGAATAGGCGT
+|||||||||:|:|::||||:||++||||||:||||:::||||:|||||||||||||++|
541 GGTTGTATATTATTTTTGAGTTACGGGTGTGTAATTTTTATGTTTTTTGGGAATAGGCGT
601 CAAGACTAGAGGTAGTTTTGTTGTCTTAGCCGGCCACAGGCGGCGGTGATAAAACCTGCA
:||||:||||||||||||||||||:||||:++|::|:|||++|++|||||||||::||:|
601 TAAGATTAGAGGTAGTTTTGTTGTTTTAGTCGGTTATAGGCGGCGGTGATAAAATTTGTA
661 GTTGATGTAATGGAGTCACATACTGAATCCACTTGACTGGCTGTGGCTGAAATAAAAACA
||||||||||||||||:|:|||:|||||::|:||||:|||:|||||:|||||||||||:|
661 GTTGATGTAATGGAGTTATATATTGAATTTATTTGATTGGTTGTGGTTGAAATAAAAATA
721 TTTTCCAC
||||::|:
721 TTTTTTAT
Used primers:
Forward MSP: 5’-GGTGCGAGAAAAATTATTTTAGC-3’
Reverse MSP: 5’-AAAAAAACTTCCTTAAAATAACGCA-3’
Forward USP: 5’-GTGTGAGAAAAATTATTTTAGTGT-3’
Reverse USP: 5’-AAAAAAACTTCCTTAAAATAACACA-3’
Coding Sequence of PpEREBP/AP2
1 NATGTCTGGTAGCGGAAGCATAGGCACTTCCGGAGTGGACTCATGGGTTGAGCAGAGCTA
|||||:||||||++||||:|||||:|:||:++|||||||:|:||||||||||:||||:||
1 NATGTTTGGTAGCGGAAGTATAGGTATTTTCGGAGTGGATTTATGGGTTGAGTAGAGTTA
MSPfwd >>>>>>>>>>>>>>>>>>>>>>>>
USPfwd >>>>>>>>>>>>>>>>>>>>>>>>>
61 ACTGACATGCTTTGCGGGGGAATTACTCACTGATTCAGCAGCACTGGCAGATCTGTGTGA
|:|||:|||:||||++|||||||||:|:|:|||||:||:||:|:|||:||||:|||||||
61 ATTGATATGTTTTGCGGGGGAATTATTTATTGATTTAGTAGTATTGGTAGATTTGTGTGA
121 GGCGTTGAAGGTTTTCTACGAGTCGCTGCTTCCCCAGCTAATCCAAAGAGATGCGGATAG
||++|||||||||||:||++|||++:||:||::::||:||||::|||||||||++|||||
121 GGCGTTGAAGGTTTTTTACGAGTCGTTGTTTTTTTAGTTAATTTAAAGAGATGCGGATAG
<<<<<<<<<<<<<<<<<<<<<<<<< MSPrev
<<<<<<<<<<<<<<<<<<<<<<<<< USPrev
181 AGATTCCGCAACATCATGCTGAAGTGGAAGAAAGTGTTGCGAGTGTGGGTTTATAGATTC
|||||:++:||:||:|||:||||||||||||||||||||++||||||||||||||||||+
181 AGATTTCGTAATATTATGTTGAAGTGGAAGAAAGTGTTGCGAGTGTGGGTTTATAGATTC
241 GCGCTTTATTGACGATTGAGGTGAAGCAGAGGGTGGGGTTTTCGGAAAGCTGCAGCGTCA
+++:||||||||++||||||||||||:|||||||||||||||++|||||:||:||++|:|
241 GCGTTTTATTGACGATTGAGGTGAAGTAGAGGGTGGGGTTTTCGGAAAGTTGTAGCGTTA
301 CTCGAGTGCAGAGGGTGTGCCAGGTTGTAGTGGTTTTCGAATTTCAGCGGACGAGGTAAG
:|++||||:||||||||||::||||||||||||||||++|||||:||++||++|||||||
301 TTCGAGTGTAGAGGGTGTGTTAGGTTGTAGTGGTTTTCGAATTTTAGCGGACGAGGTAAG
361 GAAGCCTAGCAGACGAGAACGTGAGTAAGTCAGCGCAAGATGGTAGGCAAAGTGCAGGCG
||||::|||:|||++||||++|||||||||:||++:|||||||||||:||||||:|||++
361 GAAGTTTAGTAGACGAGAACGTGAGTAAGTTAGCGTAAGATGGTAGGTAAAGTGTAGGCG
421 TCCCTAGCTGGTGCCCATGGCAAGCAGTCTACTCATCATGCCATGGTTCGAAGCAGTCAT
|:::|||:|||||:::||||:|||:|||:||:|:||:|||::||||||++|||:|||:||
421 TTTTTAGTTGGTGTTTATGGTAAGTAGTTTATTTATTATGTTATGGTTCGAAGTAGTTAT
481 CACACCCTAGTTCCGGAGATCATGGGTCGCTCGCGACCTGTTTCACAAAAGCTGAAAGCC
:|:|:::|||||:++|||||:||||||++:|++++|::|||||:|:|||||:||||||:+
481 TATATTTTAGTTTCGGAGATTATGGGTCGTTCGCGATTTGTTTTATAAAAGTTGAAAGTC
541 GCCAGCATCAAGAGGGCCAAAAAGGTTCAAGAGGGGAGGTACAGAGGGGTGCGGCAGCGG
+::||:||:|||||||::|||||||||:|||||||||||||:|||||||||++|:||++|
541 GTTAGTATTAAGAGGGTTAAAAAGGTTTAAGAGGGGAGGTATAGAGGGGTGCGGTAGCGG
601 CCGTGGGGGCGATTTGCGGCGGAGATTAGAGACCCCAATACTAAGGAACGGAAGTGGCTA
:++||||||++|||||++|++|||||||||||::::||||:|||||||++|||||||:||
601 TCGTGGGGGCGATTTGCGGCGGAGATTAGAGATTTTAATATTAAGGAACGGAAGTGGTTA
661 GGCACTTTTGACACTGCTGAGGATGCAGCTCTCGCTTACGACACTGGTAAGAATATCAAC
||:|:||||||:|:||:||||||||:||:|:|++:|||++|:|:||||||||||||:||:
661 GGTATTTTTGATATTGTTGAGGATGTAGTTTTCGTTTACGATATTGGTAAGAATATTAAT
721 TTCTCCATTGCGCATTTGGTTACAAGGTGGCGATGACGATCACGTATCTCTATCCCTGAT
||:|::||||++:|||||||||:|||||||++||||++||:|++|||:|:|||:::||||
721 TTTTTTATTGCGTATTTGGTTATAAGGTGGCGATGACGATTACGTATTTTTATTTTTGAT
781 AGAGCTAACTCTATCCGCCTTCCGTTTCTTGTAGCGGCAAGATCTATGAGAGGACCTAAG
||||:|||:|:|||:++::||:++|||:||||||++|:|||||:||||||||||::||||
781 AGAGTTAATTTTATTCGTTTTTCGTTTTTTGTAGCGGTAAGATTTATGAGAGGATTTAAG
841 GCACGTACCAACTTTGTGTACCCTACGCATGAGACCTGTCTTCTTTCCGCTGCAGCGGCA
|:|++||::||:||||||||:::||++:||||||::|||:||:|||:++:||:||++|:|
841 GTACGTATTAATTTTGTGTATTTTACGTATGAGATTTGTTTTTTTTTCGTTGTAGCGGTA
901 CTGGCGGCGCCAAATGGTAATTCGCAGCATCACCAGGTGGGTCTAATCGCTCAGAAGACC
:|||++|++::|||||||||||++:||:||:|::||||||||:||||++:|:||||||::
901 TTGGCGGCGTTAAATGGTAATTCGTAGTATTATTAGGTGGGTTTAATCGTTTAGAAGATT
961 TTGGGAAGTGCTGCTGCTCTCAGCAGCAGTACCGGCTTATTGCACNNAACCCTNNNNNNN
||||||||||:||:||:|:|:||:||:||||:++|:||||||:|:||||:::||||||||
961 TTGGGAAGTGTTGTTGTTTTTAGTAGTAGTATCGGTTTATTGTATNNAATTTTNNNNNNN
1021 GGGGA
|||||
1021 GGGGA
Used primers:
Forward MSP: 5’-GGTAGCGGAAGTATAGGTATTTTC-3’
Reverse MSP: 5’-TTAACTAAAAAAACAACGACTCGTA-3’
Forward USP: 5’-GTAGTGGAAGTATAGGTATTTTTGG-3’
Reverse USP: 5’-TTAACTAAAAAAACAACAACTCATA-3’
Promoter region of PpEREBP/AP2
1 GCGTGACATCGTAGATATTGAGGATGAAGATTCGTCTGAGAATGGAACTTGCGTGGATAG
|++|||:||++|||||||||||||||||||||++|:|||||||||||:|||++|||||||
1 GCGTGATATCGTAGATATTGAGGATGAAGATTCGTTTGAGAATGGAATTTGCGTGGATAG
61 CAGACATTTTCTGGGCTCGATGACTCCAAGCTCTGAACCTGTGTCTTCGAAATTTACGAT
:|||:|||||:||||:|++||||:|::|||:|:||||::|||||:||++|||||||++||
61 TAGATATTTTTTGGGTTCGATGATTTTAAGTTTTGAATTTGTGTTTTCGAAATTTACGAT
121 CACGCTGGAGTCCACGTACTTCGACACTACTTGGTCCAGTGTGCCACTTATAATTTTAGA
:|++:||||||::|++||:||++|:|:||:|||||::||||||::|:|||||||||||||
121 TACGTTGGAGTTTACGTATTTCGATATTATTTGGTTTAGTGTGTTATTTATAATTTTAGA
181 CTCAAACTGCCACTGCATTAGTGCTTGCGAAGGAGAAGTTACTGTAGAAGCTTTTTTGTA
:|:|||:||::|:||:|||||||:|||++||||||||||||:||||||||:|||||||||
181 TTTAAATTGTTATTGTATTAGTGTTTGCGAAGGAGAAGTTATTGTAGAAGTTTTTTTGTA
241 AAGCATAATCGAATCGCTTGGTGGTGAATCGTTTTCGGAGGAAACTGAGATATCATCGCT
|||:|||||++|||++:||||||||||||++||||++|||||||:||||||||:||++:|
241 AAGTATAATCGAATCGTTTGGTGGTGAATCGTTTTCGGAGGAAATTGAGATATTATCGTT
301 GTCAAAGACCTGCCACAAGGATTTGAAAAAGTGGTGAACTCCAGTAAGGTGGGATATCTC
||:|||||::||::|:||||||||||||||||||||||:|::|||||||||||||||:|:
301 GTTAAAGATTTGTTATAAGGATTTGAAAAAGTGGTGAATTTTAGTAAGGTGGGATATTTT
361 CAAGGATGAAATGCATTTAGCAGAGCTTATTCCAATAACAACTGCACCATCACTACGTAA
:||||||||||||:||||||:||||:|||||::|||||:||:||:|::||:|:||++|||
361 TAAGGATGAAATGTATTTAGTAGAGTTTATTTTAATAATAATTGTATTATTATTACGTAA
421 TGACCAAATAAACTAGTTATAAACAAAATGCTACGAGCATCTTCATAATGCGAAACATAA
|||::|||||||:||||||||||:||||||:||++||:||:||:||||||++|||:||||
421 TGATTAAATAAATTAGTTATAAATAAAATGTTACGAGTATTTTTATAATGCGAAATATAA
481 AGCCTGCTTCAAACACACCTTGAAACAGGTGTGAGTATTCACGTTGCTGGTTCACAAGAC
||::||:||:|||:|:|::||||||:|||||||||||||:|++|||:|||||:|:||||:
481 AGTTTGTTTTAAATATATTTTGAAATAGGTGTGAGTATTTACGTTGTTGGTTTATAAGAT
541 TCCGGAAAAAGTAATAAGTTTCTGGATCGAGTAGTGAAAGAGAATTACCTGAAATGGTTG
|:++|||||||||||||||||:|||||++||||||||||||||||||::|||||||||||
541 TTCGGAAAAAGTAATAAGTTTTTGGATCGAGTAGTGAAAGAGAATTATTTGAAATGGTTG
601 GGATCCTGCTCCGCAATAGCGTGACACAAACAGCTCGGAACTGACAGTTGGACTCCGTTT
||||::||:|:++:|||||++|||:|:|||:||:|++|||:|||:|||||||:|:++|||
601 GGATTTTGTTTCGTAATAGCGTGATATAAATAGTTCGGAATTGATAGTTGGATTTCGTTT
MSPfwd >>>>>>>>>>>>>>>>>>>>>>>>>
USPfwd >>>>>>>>>>>>>>>>>>>>>>>>>>
661 GAATTGGGTCAGACGAAATTGTTGGTCTTCGTCAGAGACGCAATGCCATTGCTCTTGCTT
|||||||||:|||++|||||||||||:||++|:|||||++:||||::||||:|:|||:||
661 GAATTGGGTTAGACGAAATTGTTGGTTTTCGTTAGAGACGTAATGTTATTGTTTTTGTTT
721 CCCCGGACTTACTTTAAAACCTTCTACCTCAACCTTGTTGATCGAGGGACTTTTCGGCAT
:::++||:|||:|||||||::||:||::|:||::||||||||++|||||:||||++|:||
721 TTTCGGATTTATTTTAAAATTTTTTATTTTAATTTTGTTGATCGAGGGATTTTTCGGTAT
<<<<<<<<<<<<<<<<<<
<<<<<<<<<<<<<<<<<<<<
781 CACCAATCTTTCATCCTGCGAAGGGCTGTGGAGAGCGTTGGGAGGAGGCATCTGTTGAGA
:|::|||:|||:||::||++|||||:|||||||||++|||||||||||:||:||||||||
781 TATTAATTTTTTATTTTGCGAAGGGTTGTGGAGAGCGTTGGGAGGAGGTATTTGTTGAGA
<<<<<<< MSPrev
<<<<< USPrev
841 AGCTGGTTGGATCTTTCTGTCTACGTACCTACACCCATACCCAGGCAACCTGTATCCTTC
||:|||||||||:|||:|||:||++||::||:|:::|||:::|||:||::|||||::||:
841 AGTTGGTTGGATTTTTTTGTTTACGTATTTATATTTATATTTAGGTAATTTGTATTTTTT
901 TACCAGTTGCTCCTGTACTGCTGGTGCATTGAACATATTGTTGGAATGTTCATCATAATT
||::|||||:|::||||:||:|||||:||||||:||||||||||||||||:||:||||||
901 TATTAGTTGTTTTTGTATTGTTGGTGTATTGAATATATTGTTGGAATGTTTATTATAATT
961 CAGCTGAGTATTTTTCCAGTTGCAGTTGCGAGCATTGTTT
:||:|||||||||||::|||||:|||||++||:|||||||
961 TAGTTGAGTATTTTTTTAGTTGTAGTTGCGAGTATTGTTT
Used primers:
Forward MSP: 5’-TAATAGCGTGATATAAATAGTTCGG-3’
Reverse MSP: 5’-ATTAATAATACCGAAAAATCCCTCG-3’
Forward USP: 5’-GTAATAGTGTGATATAAATAGTTTGG-3’
Reverse USP: 5’-TAATAATACCAAAAAATCCCTCAAT-3’
Promoter region of PpbHLH
1 GAACAAGGGTTTAAAGCATTGCAGGCAGGTGATTGCATTTGTATTAACCGAGTAGTACAA
|||:||||||||||||:||||:|||:|||||||||:|||||||||||:++|||||||:||
1 GAATAAGGGTTTAAAGTATTGTAGGTAGGTGATTGTATTTGTATTAATCGAGTAGTATAA
61 TTCGAGTTTGTGTGTCATTTCGCAGAATATTGGTGGTTGGGGTTCCATGATATTGTTCAC
||++|||||||||||:||||++:|||||||||||||||||||||::|||||||||||:|:
61 TTCGAGTTTGTGTGTTATTTCGTAGAATATTGGTGGTTGGGGTTTTATGATATTGTTTAT
121 TGCTTTGATGTTTTTATTTGTGTGATTGTGATTTTATCATGATCAAACGCAAACAAAAGT
||:||||||||||||||||||||||||||||||||||:|||||:|||++:|||:||||||
121 TGTTTTGATGTTTTTATTTGTGTGATTGTGATTTTATTATGATTAAACGTAAATAAAAGT
181 ATTCTTCTGTTGCTGCTGTATCACGTTTTACTGTGGGTTGAAGAATGTTGCAGTCTAACA
|||:||:|||||:||:|||||:|++|||||:|||||||||||||||||||:|||:|||:|
181 ATTTTTTTGTTGTTGTTGTATTACGTTTTATTGTGGGTTGAAGAATGTTGTAGTTTAATA
241 ATGTGGTTCTCTAGAAGGACTGTCTAAGGCGACGGAATATTTCAGGCCTCTGTTGGGCTG
||||||||:|:||||||||:|||:|||||++|++||||||||:|||::|:|||||||:||
241 ATGTGGTTTTTTAGAAGGATTGTTTAAGGCGACGGAATATTTTAGGTTTTTGTTGGGTTG
301 TGTTTATTATTTCTTTTTTGTTTCTTCTTCTTCTTCTTCTTCTTCTTCTTTTACCTCATT
||||||||||||:||||||||||:||:||:||:||:||:||:||:||:|||||::|:|||
301 TGTTTATTATTTTTTTTTTGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTATTTTATT
361 CATATATATATATATATATATATATATATATATGTGTATGGATTTGTGAATGATATGAAT
:|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
361 TATATATATATATATATATATATATATATATATGTGTATGGATTTGTGAATGATATGAAT
421 TCACAGTTTTAATCATAACCTTGGATGCTGGGGGATACTCTGAATGAAATAGTTTTCCCC
|:|:|||||||||:||||::|||||||:|||||||||:|:||||||||||||||||::::
421 TTATAGTTTTAATTATAATTTTGGATGTTGGGGGATATTTTGAATGAAATAGTTTTTTTT
481 TACAGCAGTTATTCACGAAGTTGCTTTGAGCAATACCCGATATTACCATGGCTCAAGCTT
||:||:|||||||:|++||||||:||||||:||||::++||||||::||||:|:|||:||
481 TATAGTAGTTATTTACGAAGTTGTTTTGAGTAATATTCGATATTATTATGGTTTAAGTTT
541 ATTGAATCTTTCAAATTCCGCTTCCCTCGCACATGACTAAATCTAACATAATTTCTAAAC
|||||||:|||:|||||:++:||:::|++:|:||||:|||||:|||:|||||||:||||:
541 ATTGAATTTTTTAAATTTCGTTTTTTTCGTATATGATTAAATTTAATATAATTTTTAAAT
MSPfwd >>>>>>>>>>>>>>>>>>>>>>>>>
USPfwd >>>>>>>>>>>>>>>>>>>>>>>>>
601 TGCTGATTTTGTCCCATCGGTGTGTCAGGAAAGACTTCGACTCGTCAGCTGTAACTTCAG
||:|||||||||:::||++||||||:||||||||:||++|:|++|:||:|||||:||:||
601 TGTTGATTTTGTTTTATCGGTGTGTTAGGAAAGATTTCGATTCGTTAGTTGTAATTTTAG
661 TTTCGAAATCCCAGCTTGGACAGAACTTCGTTTTATCTAGACGGAGGTCACCAGGACTGG
|||++||||:::||:|||||:||||:||++||||||:||||++|||||:|::||||:|||
661 TTTCGAAATTTTAGTTTGGATAGAATTTCGTTTTATTTAGACGGAGGTTATTAGGATTGG
<<<<<<<<<<<<<<<<<<<<<
<<<<<<<<<<<<<<<<<<<<<
721 TAACACGTCAAGAATTGGCATCGGCTCTTCAATTAGTACTATTGAACTTTCCGAGGACGT
|||:|++|:|||||||||:||++|:|:||:||||||||:|||||||:|||:++||||++|
721 TAATACGTTAAGAATTGGTATCGGTTTTTTAATTAGTATTATTGAATTTTTCGAGGACGT
<<<< MSPrev
<<<< USPrev
781 GCTATGGCGTCGCAATCCAATTCCGACATGTGGATGGGCGCTGCATCATCAGAAATCAGG
|:|||||++|++:|||::||||:++|:|||||||||||++:||:||:||:||||||:|||
781 GTTATGGCGTCGTAATTTAATTTCGATATGTGGATGGGCGTTGTATTATTAGAAATTAGG
841 TTCAGTTCATGATCACCCCTGCATTTCCTTCCAACAACCATTCTCATTACGAACAGGAGT
||:||||:|||||:|::::||:||||::||::||:||::|||:|:||||++||:||||||
841 TTTAGTTTATGATTATTTTTGTATTTTTTTTTAATAATTATTTTTATTACGAATAGGAGT
901 TCTGCTGGTTTCTGTGATTGATAAAGACTCGGTTCGGAGTTAAATCACTTGGAATAAATC
|:||:||||||:|||||||||||||||:|++|||++|||||||||:|:|||||||||||:
901 TTTGTTGGTTTTTGTGATTGATAAAGATTCGGTTCGGAGTTAAATTATTTGGAATAAATT
961 CATGAATGTGTTTTTTTTATTTTTTATTTTATTTTCCCATAGTTTGCCT
:||||||||||||||||||||||||||||||||||:::||||||||::|
961 TATGAATGTGTTTTTTTTATTTTTTATTTTATTTTTTTATAGTTTGTTT
Used primers:
Forward MSP: 5’- ATTTTTTAAATTTCGTTTTTTTCGT-3’
Reverse MSP: 5’- ATTACCAATCCTAATAACCTCCGTC-3’
Forward USP: 5’- ATTTTTTAAATTTTGTTTTTTTTGT-3’
Reverse USP: 5’- ATTACCAATCCTAATAACCTCCATC-3’
Gene model of PpbHLH (Phypa1_209063)
1 ATTACCAAATCAAGTTGATCCATCGATTGCTAATTTGCAGACTGGAGTGCAGGAAAATGT
||||::||||:||||||||::||++||||:|||||||:|||:|||||||:||||||||||
1 ATTATTAAATTAAGTTGATTTATCGATTGTTAATTTGTAGATTGGAGTGTAGGAAAATGT
61 AGGAACACCAAGTTTCAGCAAGGGCGTGCTGGACGAGGAGTGGTACACGCCCGAGACCTC
|||||:|::||||||:||:|||||++||:||||++||||||||||:|++::++|||::|:
61 AGGAATATTAAGTTTTAGTAAGGGCGTGTTGGACGAGGAGTGGTATACGTTCGAGATTTT
121 CTTAATGGAGCTCTCTTACTCATTACCATATGGGATATCCGATACTCGCACAGGCTTTGG
:|||||||||:|:|:|||:|:||||::|||||||||||:++|||:|++:|:|||:|||||
121 TTTAATGGAGTTTTTTTATTTATTATTATATGGGATATTCGATATTCGTATAGGTTTTGG
181 AATGCTCGAGTCGTCGCTGAATTTTGACAGCAGCAGCAACCTCATGTCTAGCTTCCGCCC
||||:|++|||++|++:||||||||||:||:||:||:||::|:||||:|||:||:++:::
181 AATGTTCGAGTCGTCGTTGAATTTTGATAGTAGTAGTAATTTTATGTTTAGTTTTCGTTT
241 TGCTCCCTCAGCCTTGAGCATGGGCCTTGAGAGCAACCGCAGTCTGGAGGATCTCGTTTG
||:|:::|:||::|||||:|||||::|||||||:||:++:|||:||||||||:|++||||
241 TGTTTTTTTAGTTTTGAGTATGGGTTTTGAGAGTAATCGTAGTTTGGAGGATTTCGTTTG
301 CACTGGTCAGGGCTCGAGCAACGTTGGCCTCCTCTCAAGTCTTTCTCCAGGTCTTGTGGT
:|:||||:||||:|++||:||++||||::|::|:|:||||:|||:|::||||:|||||||
301 TATTGGTTAGGGTTCGAGTAACGTTGGTTTTTTTTTAAGTTTTTTTTTAGGTTTTGTGGT
361 CTTGTCCCATTTTTCAGCAATTGTACACTTGTGATATCCGTTTCTCAACACATACACCGC
:||||:::||||||:||:|||||||:|:|||||||||:++|||:|:||:|:|||:|:++:
361 TTTGTTTTATTTTTTAGTAATTGTATATTTGTGATATTCGTTTTTTAATATATATATCGT
421 AGCATTTTATAAAATTCATCTCAACAATGGATGAGAACCATGGTACCTGCTCAACTTACA
||:|||||||||||||:||:|:||:||||||||||||::||||||::||:|:||:|||:|
421 AGTATTTTATAAAATTTATTTTAATAATGGATGAGAATTATGGTATTTGTTTAATTTATA
481 AGGTCCTCAAGTAGGGAATTGAAGGATATCCTGTTGGTTGTGATTGCAGGCGGTCAACTC
||||::|:|||||||||||||||||||||::|||||||||||||||:|||++||:||:|+
481 AGGTTTTTAAGTAGGGAATTGAAGGATATTTTGTTGGTTGTGATTGTAGGCGGTTAATTC
541 GGCCGGAGCACCGTAATGGAAAGCTTCAGCTCAGGTCTGCCAACAAGCTTCAACCAAGGA
+|:++|||:|:++||||||||||:||:||:|:||||:||::||:|||:||:||::|||||
541 GGTCGGAGTATCGTAATGGAAAGTTTTAGTTTAGGTTTGTTAATAAGTTTTAATTAAGGA
601 ATCATCAACGCTGGTGGAAGCATAACCAACATCACCAGTAGCAACATAAATAACGTCCGC
||:||:||++:|||||||||:||||::||:||:|::|||||:||:||||||||++|:++:
601 ATTATTAACGTTGGTGGAAGTATAATTAATATTATTAGTAGTAATATAAATAACGTTCGT
661 TCTAACTTCCCCCTCATGGCCTCACCTTCGAACTTTTCCGATGCGTACCGCGCTCGATCA
|:|||:||:::::|:||||::|:|::||++||:||||:++|||++||:++++:|++||:|
661 TTTAATTTTTTTTTTATGGTTTTATTTTCGAATTTTTTCGATGCGTATCGCGTTCGATTA
721 GTGAGCGAAGACAAGTCTGGGAAAGTTGTTGGCTCTGGCGGCCCACGGAATGAACTTGTG
|||||++||||:||||:|||||||||||||||:|:|||++|:::|++|||||||:|||||
721 GTGAGCGAAGATAAGTTTGGGAAAGTTGTTGGTTTTGGCGGTTTACGGAATGAATTTGTG
781 CCATATCACAGAAACAAGGGGGCGGAAACTCGGAGCCATGGTCAGGGTCAGCAAACTCTA
::||||:|:|||||:|||||||++||||:|++|||::|||||:|||||:||:|||:|:||
781 TTATATTATAGAAATAAGGGGGCGGAAATTCGGAGTTATGGTTAGGGTTAGTAAATTTTA
841 TTCTTGAAGCGCGCAGCTTCTCGCCGTTGTGCGGGGTCTAGTGGGACTGTCTCTCCAGTG
||:||||||++++:||:||:|++:++|||||++||||:||||||||:|||:|:|::||||
841 TTTTTGAAGCGCGTAGTTTTTCGTCGTTGTGCGGGGTTTAGTGGGATTGTTTTTTTAGTG
901 AGCAAGTCACCCCCACGCGTGGTCACTAGTGCCTCCAACGATTCTTCTGTGGACACGCCG
||:||||:|:::::|++++||||:|:|||||::|::||++|||:||:||||||:|++:++
901 AGTAAGTTATTTTTACGCGTGGTTATTAGTGTTTTTAACGATTTTTTTGTGGATACGTCG
961 GATAAAGACAGCCCTCATCCGCGTAATGCTCACTTGCAGAGCGCATCTGGAAGATTAAAT
||||||||:||:::|:||:++++|||||:|:|:|||:||||++:||:|||||||||||||
961 GATAAAGATAGTTTTTATTCGCGTAATGTTTATTTGTAGAGCGTATTTGGAAGATTAAAT
1021 ATCAACTCCGGATCGGATGATCCCAACGACATGGGATTAGATGGTGACGACTACGATGCC
||:||:|:++|||++||||||:::||++|:|||||||||||||||||++|:||++|||::
1021 ATTAATTTCGGATCGGATGATTTTAACGATATGGGATTAGATGGTGACGATTACGATGTT
1081 AAAGACGACGATGATTTGGATGAGAGTGGTGACGGCTCAGGGGGGCCCTACGAGGTGGAA
|||||++|++||||||||||||||||||||||++|:|:|||||||:::||++||||||||
1081 AAAGACGACGATGATTTGGATGAGAGTGGTGACGGTTTAGGGGGGTTTTACGAGGTGGAA
1141 GAAGGCGCAGGCAATGGAGCAGATCAAAGCATTGGAAAGGGAAACGGCAAAGGGAAACGA
|||||++:|||:|||||||:||||:||||:||||||||||||||++|:|||||||||++|
1141 GAAGGCGTAGGTAATGGAGTAGATTAAAGTATTGGAAAGGGAAACGGTAAAGGGAAACGA
1201 GGACTTCCTGCGAAAAACCTCATGGCTGAGCGCAGGCGCCGCAAAAAACTCAACGATCGC
|||:||::||++|||||::|:||||:||||++:|||++:++:||||||:|:||++||++:
1201 GGATTTTTTGCGAAAAATTTTATGGTTGAGCGTAGGCGTCGTAAAAAATTTAACGATCGT
MSPfwd >>>>>>>>>>>>>>>>>>>>>>
USPfwd >>>>>>>>>>>>>>>>>>>>>>>>>
1261 CTGTACACGCTACGGTCTGTAGTTCCTAAGATTACAAAGGTGCTTCCAAACTCTATCTTT
:||||:|++:||++||:|||||||::||||||||:|||||||:||::|||:|:|||:|||
1261 TTGTATACGTTACGGTTTGTAGTTTTTAAGATTATAAAGGTGTTTTTAAATTTTATTTTT
1321 GAACATGTTGCCCGCCTCGATTGCTGAATTGCACATCATGTATGTTGAGATGTCCACTTA
|||:||||||::++::|++||||:|||||||:|:||:||||||||||||||||::|:|||
1321 GAATATGTTGTTCGTTTCGATTGTTGAATTGTATATTATGTATGTTGAGATGTTTATTTA
1381 CGAATCAGTGGGGTGTGGAGTACAGATGGATAGAGCCTCCATATTGGGGGATGCGATTGA
++|||:||||||||||||||||:||||||||||||::|::|||||||||||||++|||||
1381 CGAATTAGTGGGGTGTGGAGTATAGATGGATAGAGTTTTTATATTGGGGGATGCGATTGA
1441 GTACCTAAAGGAGCTCCTGCAACGCATCAATGAAATCCATAACGAACTGGAAGCAGCAAA
|||::||||||||:|::||:||++:||:||||||||::||||++||:||||||:||:|||
1441 GTATTTAAAGGAGTTTTTGTAACGTATTAATGAAATTTATAACGAATTGGAAGTAGTAAA
<<<<<<<<<<<<<<<<<<<
<<<<<<<<<<<<<<<<<<<<
1501 GCTGGAGCAGTCGCGGTCGATGCCGTCTAGCCCCACTCCACGATCCACCCAAGGTTATCC
|:|||||:|||++++||++|||:++|:|||::::|:|::|++||::|:::||||||||::
1501 GTTGGAGTAGTCGCGGTCGATGTCGTTTAGTTTTATTTTACGATTTATTTAAGGTTATTT
<<<<<< MSPrev
<<<<< USPrev
1561 AGCTACAGTTAAAGAAGAATGCCCCGTCTTGCCGAATCCTGAATCCCAGCCTCCTCGAGT
||:||:|||||||||||||||:::++|:|||:++|||::|||||:::||::|::|++|||
1561 AGTTATAGTTAAAGAAGAATGTTTCGTTTTGTCGAATTTTGAATTTTAGTTTTTTCGAGT
1621 ATGTTGTTTATAATTTCTCACCTTCTTGGAATTGCATCTCAGTACTTATTTCGCAATGCC
||||||||||||||||:|:|::||:|||||||||:||:|:||||:||||||++:||||::
1621 ATGTTGTTTATAATTTTTTATTTTTTTGGAATTGTATTTTAGTATTTATTTCGTAATGTT
1681 AACGACGTTCTGAAATGTCTACACTTTGCACTGTTCTGAAGTTCTGGAATGCTGAACATA
||++|++||:||||||||:||:|:||||:|:||||:|||||||:|||||||:||||:|||
1681 AACGACGTTTTGAAATGTTTATATTTTGTATTGTTTTGAAGTTTTGGAATGTTGAATATA
1741 GTTTACTTTGCACATTGTTTCATAGGTGGAAGTGAGGAAAAGAGAAGGTCAGGCCCTCAA
|||||:||||:|:|||||||:||||||||||||||||||||||||||||:|||:::|:||
1741 GTTTATTTTGTATATTGTTTTATAGGTGGAAGTGAGGAAAAGAGAAGGTTAGGTTTTTAA
1801 CATTCATATGTTCTGTGCCCGCCGGCCTGGACTCCTCCTCTCTACTGTGAAGGCGCTGGA
:|||:|||||||:||||::++:++|::||||:|::|::|:|:||:||||||||++:||||
1801 TATTTATATGTTTTGTGTTCGTCGGTTTGGATTTTTTTTTTTTATTGTGAAGGCGTTGGA
1861 CGCCCTTGGCTTGGATGTACAACAGGCTGTCATCAGCTGCTTCAATGGTTTCGCCCTTGA
++:::||||:|||||||||:||:|||:|||:||:||:||:||:||||||||++:::||||
1861 CGTTTTTGGTTTGGATGTATAATAGGTTGTTATTAGTTGTTTTAATGGTTTCGTTTTTGA
1921 CCTCTTCCGTGCAGAGGTAAGAGTCTTTCGCCTCAAGAATTCGATGTGATTGCAACTAAT
::|:||:++||:||||||||||||:|||++::|:|||||||++|||||||||:||:||||
1921 TTTTTTTCGTGTAGAGGTAAGAGTTTTTCGTTTTAAGAATTCGATGTGATTGTAATTAAT
1981
AGAGTTTGTGCGTTGACATGGGCGGGAAATGTACATCGGTTGTCTTATTTAGCTGTGTTA
||||||||||++||||:|||||++|||||||||:||++|||||:||||||||:|||||||
1981 AGAGTTTGTGCGTTGATATGGGCGGGAAATGTATATCGGTTGTTTTATTTAGTTGTGTTA
2041 GGCCTCAAGCTGAAGATCACCTTGGGTGTGGGTTATTGCTGTGGGCAGGCCAAAGATGTG
||::|:|||:|||||||:|::|||||||||||||||||:||||||:|||::|||||||||
2041 GGTTTTAAGTTGAAGATTATTTTGGGTGTGGGTTATTGTTGTGGGTAGGTTAAAGATGTG
2101 GACGTTGGACCAGAAGAAATAAAGGCCGTTCTGCTGCTCACTGCGGGATGTGATTTGCAC
||++|||||::||||||||||||||:++||:||:||:|:|:||++||||||||||||:|:
2101 GACGTTGGATTAGAAGAAATAAAGGTCGTTTTGTTGTTTATTGCGGGATGTGATTTGTAT
2161 TCTTTGCAGTAGATCCCACAATGCAGACGGACAAGTTGAATGAATTCTCTTCTTTTCTGC
|:||||:|||||||:::|:||||:|||++||:||||||||||||||:|:||:||||:||:
2161 TTTTTGTAGTAGATTTTATAATGTAGACGGATAAGTTGAATGAATTTTTTTTTTTTTTGT
2221 ATGGGAAACAAAACACAAATTGATACGAGTGTGGTTTCAAAGTCTCTCCTCACCAGGCAG
||||||||:||||:|:|||||||||++||||||||||:|||||:|:|::|:|::|||:||
2221 ATGGGAAATAAAATATAAATTGATACGAGTGTGGTTTTAAAGTTTTTTTTTATTAGGTAG
2281 TGTTCTTCAATTTTCACCATACAAGCTAAAAAATTTGACGCTACCTAAATTCAGTGGTTT
||||:||:||||||:|::|||:|||:||||||||||||++:||::||||||:||||||||
2281 TGTTTTTTAATTTTTATTATATAAGTTAAAAAATTTGACGTTATTTAAATTTAGTGGTTT
2341 GAACCTGACATTGTTGTAGGCTGTACTCAGCTTTTTCTGTTTTTGTATAACTTAGTATAC
|||::|||:|||||||||||:||||:|:||:|||||:|||||||||||||:||||||||+
2341 GAATTTGATATTGTTGTAGGTTGTATTTAGTTTTTTTTGTTTTTGTATAATTTAGTATAC
2401 GGTAAAGGCCCACAGAGCAGAGAACGGTGGGACCTTCACGGTACTGCCACTAGACCAAGC
+|||||||:::|:||||:||||||++||||||::||:|++|||:||::|:||||::|||:
2401 GGTAAAGGTTTATAGAGTAGAGAACGGTGGGATTTTTACGGTATTGTTATTAGATTAAGT
2461 CTTGAAAACGTTATGCAGGTGAAATGTTTCTGTACATCATCTTCAAGACTTGTGTGCTGT
:|||||||++|||||:|||||||||||||:||||:||:||:||:||||:|||||||:|||
2461 TTTGAAAACGTTATGTAGGTGAAATGTTTTTGTATATTATTTTTAAGATTTGTGTGTTGT
2521 GTGTGCCTCTATGTGTTTGCCTATGTAACCAAGTTGTCCGTTGCTATCCAACACCTGCCA
|||||::|:||||||||||::|||||||::|||||||:++|||:|||::||:|::||::|
2521 GTGTGTTTTTATGTGTTTGTTTATGTAATTAAGTTGTTCGTTGTTATTTAATATTTGTTA
Used primers:
Forward MSP: 5’-GCGAAAAATTTTATGGTTGAGC-3’
Reverse MSP: 5’-TCCAACTTTACTACTTCCAATTCGT-3’
Forward USP: 5’-TGTGAAAAATTTTATGGTTGAGTGT-3’
Reverse USP: 5’-CCAACTTTACTACTTCCAATTCATT-3’
Figure S6. Primer design for bisulfite PCR analyses
Primer design for promoter, exon and intron regions of miRNA target genes
(PpC3HDZIP1, PpHB10, PpSBP3, PpARF, PpbHLH, PpTAS4), the ta-siRNA target
gene PpEREBP/AP2, and the PpGNT1 control gene as described in the manuscript. In
the gene model sequence of PpbHLH start and stop codons are highlighted in grey and
intron sequences are marked in blue. The upper strand of each sequence depicts the
wild type sequence, the lower strand indicates the expected cytosine to thymine
conversions after the bisulfite treatment. Upright lines mark unaltered nucleotides,
double points mark cytosine to thymine conversions. CpG dinucleotides are marked by
plus signs (+).
Figure S7
A
WT
WT BT
KO1 BT
KO2 BT
GGACAATCTTTGGATATGTGCCAGCGTATCTTGTGATCGTGGTTCTTAAGGGTCGAGTGCTTAGCT
GGATAATTTTTGGATATGTGTTAGTGTATTTTGTGATTGTGGTTTTTAAGGGTTGAGTGTTTAGTT
GGATAATTTTTGGATATGTGTTAGCGTATTTTGTGATTGTGGTTTTTAAGGGTCGAGTGTTTAGTT
GGATAATTTTTGGATATGTGTTAGCGTATTTTGTGATTGTGGTTTTTAAGGGTCGAGTGTTTAGTT
WT
WT BT
KO1 BT
KO2 BT
CCTCATCCTCATGCTTAGGTCTGGAAATATGTAAAAGGGGACGTAATGACAACACGAAGCTTATAA
TTTTATTTTTATGTTTAGGTTTGGAAATATGTAAAAGGGGATGTAATGATAATATGAAGTTTATAA
TTTTATTTTTATGTTTAGGTTTGGAAATATGTAAAAGGGGACGTAATGATAATACGAAGTTTATAA
TTTTATTTTTATGTTTAGGTTTGGAAATATGTAAAAGGGGACGTAATGATAATACGAAGTTTATAA
WT
WT BT
KO1 BT
KO2 BT
AAACTCAAAGCT
AAATTTAAAGTT
AAATTTAAAGTT
AAATTTAAAGTT
B
WT
WT BT
KO1 BT
KO2 BT
TTTCTGGCTGATACAGAAACAGACGAGGTATTTGCTCGTATTTGCCTGCAGCCTGAGATTGGCTCC
TTTTTGGTTGATATAGAAATAGATGAGGTATTTGTTTGTATTTGTTTGTAGTTTGAGATTGGTTTT
TTTTTGGTTGATATAGAAATAGACGAGGTATTTGTTTGTATTTGTTTGTAGTTTGAGATTGGTTTT
TTTTTGGTTGATATAGAAATAGACGAGGTATTTGTTTGTATTTGTTTGTAGTTTGAGATTGGTTTT
WT
WT BT
KO1 BT
KO2 BT
TCCGCTCAGGATTTAACAGATGATTCTCTTGCGTCTCCGCCTCTAGAGAAACCAGCTTCATTTGCC
TTTGTTTAGGATTTAATAGATGATTTTTTTGTGTTTTTGTTTTTAGAGAAATTAGTTTTATTTGTT
TTCGTTTAGGATTTAATAGATGATTTTTTTGCGTTTTTGTTTTTAGAGAAATTAGTTTTATTTGTT
TTCGTTTAGGATTTAATAGATGATTTTTTTGCGTTTTTGTTTTTAGAGAAATTAGTTTTATTTGTT
WT
WT BT
KO1 BT
KO2 BT
AAAACGCTCACTCAAAGTGATGCAAACAACGGTGGAGGCTTTTCAATACCTC
AAAATGTTTATTTAAAGTGATGTAAATAATGGTGGAGGTTTTTTAATATTTT
AAAATGTTTATTTAAAGTGATGTAAATAACGGTGGAGGTTTTTTAATATTTC
AAAATGTTTATTTAAAGTGATGTAAATAACGGTGGAGGTTTTTTAATATTTC
C
WT
WT BT
KO1 BT
KO2 BT
TAGCTCATAACCCTCTCACAGGACGTAATGGGGGTGACAACATGCTAACAGAATTGCACGGTAAAG
TAGTTTATAATTTTTTTATAGGATGTAATGGGGGTGATAATATGTTAATAGAATTGTATGGTAAAG
TAGTTTATAATTTTTTTATAGGACGTAATGGGGGTGATAATATGTTAATAGAATTGTACGGTAAAG
TAGTTTATAATTTTTTTATAGGACGTAATGGGGGTGATAATATGTTAATAGAATTGTACGGTAAAG
WT
WT BT
KO1 BT
KO2 BT
GAAAACTGTACTAGGCATGTTATATGGGAATTCGGATCGCTTCTTGCAATTAAACACGCTAGCGCC
GAAAATTGTATTAGGTATGTTATATGGGAATTTGGATTGTTTTTTGTAATTAAATATGTTAGTGTT
GAAAATTGTATTAGGTATGTTATATGGGAATTTGGATCGTTTTTTGTAATTAAATACGTTAGTGTC
GAAAATTGTATTAGGTATGTTATATGGGAATTTGGATCGTTTTTTGTAATTAAATACGTTAGTGTC
WT
WT BT
KO1 BT
KO2 BT
GTTTGGTGCCAATGTTATTCTGG
GTTTGGTGTTAATGTTATTTTGG
GTTTGGTGTTAATGTTATTTTGG
GTTTGGTGTTAATGTTATTTTGG
D
WT
WT BT
KO1 BT
KO2 BT
TCAAAGCAATCAATGTGCTAATGAACGTGCTGCCTGCATTGTCTGCAATGACTATGTACTGCGTAG
TTAAAGTAATTAATGTGTTAATGAATGTGTTGTTTGTATTGTTTGTAATGATTATGTATTGTGTAG
TTAAAGTAATTAATGTGTTAATGAACGTGTTGTTTGTATTGTTTGTAATGATTATGTATTGCGTAG
TTAAAGTAATTAATGTGTTAATGAACGTGTTGTTTGTATTGTTTGTAATGATTATGTATTGCGTAG
WT
WT BT
KO1 BT
KO2 BT
TCAGTGGAAATAGGTGGTTGATTATGAGATTTTGGTTGTGCAGGTCACTTGGGATGAGCCGGACCT
TTAGTGGAAATAGGTGGTTGATTATGAGATTTTGGTTGTGTAGGTTATTTGGGATGAGTTGGATTT
TTAGTGGAAATAGGTGGTTGATTATGAGATTTTGGTTGTGTAGGTTATTTGGGATGAGTCGGATTT
TTAGTGGAAATAGGTGGTTGATTATGAGATTTTGGTTGTGTAGGTTATTTGGGATGAGTCGGATTT
WT
WT BT
KO1 BT
KO2 BT
ATTGCAGGGAGTGAATCGTGTAAGCCCATGGCAGTTAGAGCTTGTGGCGACACTTCCTATGCAGCT
ATTGTAGGGAGTGAATTGTGTAAGTTTATGGTAGTTAGAGTTTGTGGTGATATTTTTTATGTAGTT
ATTGTAGGGAGTGAATTGTGTAAGTTTATGGTAGTTAGAGTTTGTGGCGATATTTTTTATGTAGTT
ATTGTAGGGAGTGAATTGTGTAAGTTTATGGTAGTTAGAGTTTGTGGCGATATTTTTTATGTAGTT
WT
WT BT
KO1 BT
KO2 BT
GCCC
GTTT
GTTT
GTTT
E
WT
WT BT
KO1 BT
KO2 BT
TTCACAATGTGCTCTTCAAGCTTCGTTGTATGCTAAACTCTACTGCAATACTGCTACGGCGTGCC
TTTATAATGTGTTTTTTAAGTTTTGTTGTATGTTAAATTTTATTGTAATATTGTTATGGTGTGTT
TTTATAATGTGTTTTTTAAGTTTCGTTGTATGTTAAATTTTATTGTAATATTGTTACGGCGTGTT
TTTATAATGTGTTTTTTAAGTTTCGTTGTATGTTAAATTTTATTGTAATATTGTTACGGCGTGTT
WT
WT BT
KO1 BT
KO2 BT
TTTTCTTTTTTCGAAGTATGATATGATGACCTAATGGTCTTTTTGAATACGACAGGAATTCCATG
TTTTTTTTTTTTGAAGTATGATATGATGATTTAATGGTTTTTTTGAATATGATAGGAATTTTATG
TTTTTTTTTTTTGAAGTATGATATGATGATTTAATGGTTTTTTTGAATACGATAGGAATTTTATG
TTTTTTTTTTTTGAAGTATGATATGATGATTTAATGGTTTTTTTGAATACGATAGGAATTTTATG
WT
WT BT
KO1 BT
KO2 BT
GAGACAG
GAGATAG
GAGATAG
GAGATAG
Figure S7. DNA methylation analysis of promoter and intragenic regions of the
PpARF gene in P. patens wild type and two ΔPpDCL1b mutants
Nucleotide sequences of PCR products obtained from methylation-specific PCRs are
aligned. (A) Promoter region. (B) Exon 1 upstream of the miR160 binding site. (C) Exon
4 downstream of the miR160 binding site. (D) Intron 2 upstream of the miR160 binding
site. (E) Intron 3 downstream of the miR160 binding site. WT: Wild type nucleotide
sequence of the analyzed region; CpG residues are highlighted in green. WT BT:
Sequences of PCR products obtained with USP primers from bisulfite-treated DNA
from wild type. KO1+2 BT: Sequences of PCR products obtained with MSP primers
from bisulfite-treated DNA from two ΔPpDCL1b mutants. Cytosine residues of CpG
dinucleotides which are methlyated in the ΔPpDCL1b mutants are indicated in red.
Cytosine residues of CpG dinucleotides which are not methylated are highlighted in
yellow. Cytosine to thymine conversions are highlighted in bold and are underlined.
Five independent clones from each PCR product were sequenced.
Figure S8
A
PpC3HDZIP1 mRNA
PpC3HDZIP1 genomic
PpC3HDZIP1 mRNA
GACGGATTTCCTGGCGAAGGCAACGGGAACCGCAGTGGATTGGATACAGT
||||||||||||||||||||||||||||||||||||||||||||||||||
gacggatttcctggcgaaggcaacgggaaccgcagtggattggatacagt
PpC3HDZIP1 genomic
TACCTGGTATGAAG-----------------------------------||||||||||||||
tacctggtatgaagGTatggatgccatgccttcctacggcacgttctaca
PpC3HDZIP1 mRNA
--------------------------------------------------
PpC3HDZIP1 genomic
gtgtattgtggagtagcgagcctcacctgtaactcttgatctatagattc
PpC3HDZIP1 mRNA
--------------------------------------------------
PpC3HDZIP1 genomic
cattatcagagatatgatcgcacgaaataactctttgttccaaccttttg
PpC3HDZIP1 mRNA
--------------------------------------------------
PpC3HDZIP1 genomic
taaaataagtattagcggagtcatggtactggagcaaagtcaaacaaatt
PpC3HDZIP1 mRNA
--------------------------------------------------
PpC3HDZIP1 genomic
aatttgactcaaaacacgacttcgaattaatttaggagctaacaaggtaa
PpC3HDZIP1 mRNA
--------------------------------------------------
PpC3HDZIP1 genomic
tgatattgattctttaattcaaattaaagtggttgattgcaaatgccatt
PpC3HDZIP1 mRNA
--------------------------CCTGGTCCGGATGCCATTGGCATC
||||||||||||||||||||||||
gctgatacgtcactagtgcaatgcAGcctggtccggatgccattggcatc
PpC3HDZIP1 genomic
PpC3HDZIP1 mRNA
PpC3HDZIP1 genomic
ATTGCTATATCCCATGGTTGCGTGGGCATAGCAGCTCGAGCGTGCGGCCT
||||||||||||||||||||||||||||||||||||||||||||||||||
attgctatatcccatggttgcgtgggcatagcagctcgagcgtgcggcct
B
PpHB10 mRNA
PpHB10 genomic
PpHB10 mRNA
AGCTACCGCTGAGGAGACGCTGACAGAATTCCTGGCTAAAGCCACAGGAA
||||||||||||||||||||||||||||||||||||||||||||||||||
agctaccgctgaggagacgctgacagaattcctggctaaagccacaggaa
PpHB10 genomic
CGGCGGTGGATTGGATTCAGTTACCTGGTATGAAG--------------|||||||||||||||||||||||||||||||||||
cggcggtggattggattcagttacctggtatgaagGTatgtcatctctcc
PpHB10 mRNA
--------------------------------------------------
PpHB10 genomic
gcgatggtgatgagtgattcaccgcatcacctcttaccgtaatctgagtg
PpHB10 mRNA
--------------------------------------------------
PpHB10 genomic
atttgaagtaattgcaatgctctgaaattgaaatctgaagttctgaaagg
PpHB10 mRNA
--------------------------------------------------
PpHB10 genomic
gcggtttggctgaatttgttaactggtgagactattgctgttgcactaat
PpHB10 mRNA
--------------------CCTGGTCCGGATGCCATTGGCATTATTGCT
||||||||||||||||||||||||||||||
agtaagatgtttatttgcAGcctggtccggatgccattggcattattgct
PpHB10 genomic
Figure S8. The miR166 binding sites of PpC3HDZIP1 and PpHB10 are disrupted
by introns
(A) PpC3HDZIP1; (B) PpHB10. The miR166 binding sites are indicated in red and are
underlined. Intron borders (GT and AG) are marked in bold.
Figure S9
WT
WT1+amiRNA
WT2+amiRNA
KO1+amiRNA
KO2+amiRNA
BT
BT
BT
BT
TTTATCTCTAAATTCTTAGACAACGTCATTCAAAATAAGTTTTAAAACAGCGACTAGTCATAAAATA
TTTATTTTTAAATTTTTAGATAATGTTATTTAAAATAAGTTTTAAAATAGTGATTAGTTATAAAATA
TTTATTTTTAAATTTTTAGATAACGTTATTTAAAATAAGTTTTAAAATAGCGATTAGTTATAAAATA
TTTATTTTTAAATTTTTAGATAACGTTATTTAAAATAAGTTTTAAAATAGCGATTAGTTATAAAATA
TTTATTTTTAAATTTTTAGATAACGTTATTTAAAATAAGTTTTAAAATAGCGATTAGTTATAAAATA
WT
WT1+amiRNA
WT2+amiRNA
KO1+amiRNA
KO2+amiRNA
BT
BT
BT
BT
CGTATTTACACACTTGTATATGATGTACCATAGACGGTAACCGTACATATTTGCCGACACCCTGCAA
TGTATTTATATATTTGTATATGATGTATTATAGATGGTAATTGTATATATTTGTTGACATTTTGTAA
CGTATTTATATATTTGTATATGATGTATTATAGACGGTAATTGTATATATTTGTTGACATTTTGTAA
CGTATTTATATATTTGTATATGATGTATTATAGACGGTAATTGTATATATTTGTTGACATTTTGTAA
CGTATTTATATATTTGTATATGATGTATTATAGACGGTAATTGTATATATTTGTCGACATTTTGTAA
WT
WT1+amiRNA
WT2+amiRNA
KO1+amiRNA
KO2+amiRNA
BT
BT
BT
BT
TTAATAGAGTTCGAATATCCCCGCCGCGTTCAAGTCGCCT
TTAATAGAGTTTGAATATTTTTGTTGTGTTTAAGTTGTTT
TTAATAGAGTTCGAATATTTTCGTCGCGTTTAAGTCGTTT
TTAATAGAGTTTGAATATTTTCGTCGCGTTTAAGTCGTTT
TTAATAGAGTTTGAATATTTTCGTCGCGTTTAAGTCGTTT
Figure S9. DNA methylation analysis of the PpGNT1 promoter region in lines
expressing the amiR-GNT1
Nucleotide sequences of PCR products obtained from methylation-specific PCRs are
aligned. WT: Wild type nucleotide sequence of the analyzed region; CpG residues are
highlighted in green. WT1 + amiRNA BT: Sequences of PCR products obtained with
USP primers from bisulfite-treated DNA from wild type line 1 expressing the amiRGNT1 at low levels. WT2 + amiRNA BT: Sequences of PCR products obtained with
MSP primers from bisulfite-treated DNA from wild type line 2 expressing the amiRGNT1 at high levels. KO1 + amiRNA BT and KO2 + amiRNA BT: Sequences of PCR
products obtained with MSP primers from bisulfite-treated DNA from ΔPpDCL1b
mutants expressing the amiR-GNT1. Cytosine residues of CpG dinucleotides which are
methlyated are indicated in red. Cytosine residues of CpG dinucleotides which are not
methylated are highlighted in yellow. Cytosine to thymine conversions are highlighted
in bold and are underlined. Five independent clones from each PCR product were
sequenced.
Figure S10
A
WT
WT/Con. BT
WT/ABA BT
ATCTTTCAAATTCCGCTTCCCTCGCACATGACTAAATCTAACATAATTTCTAAACTGCTGATTTTG
ATTTTTTAAATTTTGTTTTTTTTGTATATGATTAAATTTAATATAATTTTTAAATTGTTGATTTTG
ATTTTTTAAATTTCGTTTTTTTCGTATATGATTAAATTTAATATAATTTTTAAATTGTTGATTTTG
WT
WT/Con. BT
WT/ABA BT
TCCCATCGGTGTGTCAGGAAAGACTTCGACTCGTCAGCTGTAACTTCAGTTTCGAAATCCCAGCTT
TTTTATTGGTGTGTTAGGAAAGATTTTGATTTGTTAGTTGTAATTTTAGTTTTGAAATTTTAGTTT
TTTTATTGGTGTGTTAGGAAAGATTTCGATTCGTTAGTTGTAATTTTAGTTTTGAAATTTTAGTTT
WT
WT/Con. BT
WT/ABA BT
GGACAGAACTTCGTTTTATCTAGACGGAGGTCACCAGGACTGGTAAC
GGATAGAATTTTGTTTTATTTAGATGGAGGTTATTAGGATTGGTAAT
GGATAGAATTTCGTTTTATTTAGACGGAGGTTATTAGGATTGGTAAT
B
WT
WT/Con. BT
WT/ABA BT
GCGAAAAACCTCATGGCTGAGCGCAGGCGCCGCAAAAAACTCAACGATCGCCTGTACACGCTACGG
GTGAAAAATTTTATGGTTGAGTGTAGGTGTTGTAAAAAATTTAATGATTGTTTGTATATGTTATGG
GCGAAAAATTTTATGGTTGAGCGTAGGTGTCGTAAAAAATTTAATGATCGTTTGTATACGTTACGG
WT
WT/Con. BT
WT/ABA BT
TCTGTAGTTCCTAAGATTACAAAGGTGCTTCCAAACTCTATCTTTGAACATGTTGCCCGCCTCGAT
TTTGTAGTTTTTAAGATTATAAAGGTGTTTTTAAATTTTATTTTTGAATATGTTGTTTGTTTTGAT
TTTGTAGTTTTTAAGATTATAAAGGTGTTTTTAAATTTTATTTTTGAATATGTTGTTTGTTTCGAT
WT
WT/Con. BT
WT/ABA BT
TGCTGAATTGCACATCATGTATGTTGAGATGTCCACTTACGAATCAGTGGGGTGTGGAGTACAGAT
TGTTGAATTGTATATTATGTATGTTGAGATGTTTATTTATGAATTAGTGGGGTGTGGAGTATAGAT
TGTTGAATTGTATATTATGTATGTTGAGATGTTTATTTACGAATTAGTGGGGTGTGGAGTATAGAT
WT
WT/Con. BT
WT/ABA BT
GGATAGAGCCTCCATATTGGGGGATGCGATTGAGTACCTAAAGGAGCTCCTGCAACGCATCAATGA
GGATAGAGTTTTTATATTGGGGGATGTGATTGAGTATTTAAAGGAGTTTTTGTAATGTATTAATGA
GGATAGAGTTTTTATATTGGGGGATGTGATTGAGTATTTAAAGGAGTTTTTGTAACGTATTAATGA
WT
WT/Con. BT
WT/ABA BT
AATCCATAACGAACTGGAAGCAGCAAAGCTGGA
AATTTATAATGAATTGGAAGTAGTAAAGTTGGA
AATTTATAACGAATTGGAAGTAGTAAAGTTGGA
Figure S10. DNA methylation analysis of promoter and intragenic regions of
PpbHLH in untreated and ABA-treated P. patens wild type
Nucleotide sequences of PCR products obtained from methylation-specific PCRs are
aligned. (A) Promoter region of PpbHLH. (B) Coding Sequence of PpbHLH (intron
sequences are marked in blue). WT: Wild type nucleotide sequence of the analyzed
region; CpG residues are highlighted in green. WT/Con. BT: Sequences of PCR
products obtained with USP primers from bisulfite-treated DNA from untreated wild
type. WT/ABA BT: Sequences of PCR products obtained with MSP primers from
bisulfite-treated DNA from ABA-treated wild type. Cytosine residues of CpG
dinucleotides which are methlyated in the ABA-treated plants are indicated in red.
Cytosine residues of CpG dinucleotides which are not methylated are highlighted in
yellow. Cytosine to thymine conversions are highlighted in bold and are underlined.
Five independent clones from each PCR product were sequenced.
Table S1. Primers used in this study
Primer sequence (5’ Ö 3’)
Description of experiment
TGGCATACAGGGAGCCAGGCA
Antisense oligonucleotide of miR160.
GGGGAATGAAGCCTGGTCCGA
Antisense oligonucleotide of miR166.
GGCGCTATCCCTCCTGAGCTT
Antisense oligonucleotide of miR390.
GTGCTCACTCTCTTCTGTCA
Antisense oligonucleotide of miR156.
GCGTGCTCTCTCTCGTTGTCA
Antisense oligonucleotide of miR535.
TCCAGACATAGACTCCATGCAA
Antisense oligonucleotide of miR538.
TGTCCTCTCAAGTCTTTCTCA
Antisense oligonucleotide of miR1026.
AAGCGTCCTGATTATTTGGAA
Antisense oligonucleotide of amiR-GNT1.
GGGGCCATGCTAATCTTCTCTG
Antisense oligonucleotide of U6snRNA.
GGGTGTACAAGAGCTCTATAGTGCCACCG
5’ RACE primer to detect the cleavage product of
PpC3HDZIP1.
GCCACCGTTTCCTGTCGGGAGAGTTCC
5’ RACE nested primer to detect the cleavage
product of PpC3HDZIP1.
AACCGCCGCCATCACACGGCCGGATC
5’ RACE primer to detect the cleavage product of
PpHB10.
CACACGGCCGGATCAGGTAACCACTTG
5’ RACE nested primer to detect the cleavage
product of PpHB10.
CGCAGATCGGTGAACCCGCGGTGCTCAC
5’ RACE primer to detect the cleavage product of
PpSBP3.
CCCGCGGTGCTCACCAACTGAGACCGGA
5’ RACE nested primer to detect the cleavage
product of PpSBP3.
ATGAGGGCTGTCTTTATCCGGCGTGT
5’ RACE primer to detect the cleavage product of
PpbHLH.
ACTTTGGAGCAAGTTCTTCCCAGGTGGA
5’ RACE primer to detect the cleavage product of
PpGNT1 in PpGNT1-amiRNA expressing lines.
CGGTGAGAAATACACGCTTTTGACCCT
5’ RACE primer of the control gene PpGNT1.
Primer sequence (5’ Ö 3’)
Description of experiment
GATGCTTACCATCCCCAGCAACGGA
5’ RACE primer to detect the cleavage product of
PpARF.
PpARF reverse primer to detect sense and
antisense transcript downstream of the miR160
binding site by RT-PCR.
Primer used for the synthesis of the PpARF sense
transcript derived cDNA.
CAAGATCATCAAGTCTTCCATCCT
PpARF forward primer to detect sense and
antisense transcript downstream of the miR160
binding site by RT-PCR.
Primer used for the synthesis of the PpARF
antisense transcript derived cDNA.
CAAAGAGTGTCCAATCCTGGC
PpC3HDZIP1 forward primer to detect sense and
antisense transcript upstream of the miR166
binding site by RT-PCR.
Primer used for the synthesis of the PpC3HDZIP1
antisense transcript derived cDNA.
PpC3HDZIP1 forward primer to detect the miRNA:
mRNA duplexes by RT-PCR.
TTGAAGCCACACCAGCCTGAC
PpC3HDZIP1 reverse primer to detect sense and
antisense transcript upstream of the miR166
binding site by RT-PCR.
Primer used for the synthesis of the PpC3HDZIP1
sense transcript derived cDNA.
PpC3HDZIP1 reverse primer to detect the miRNA:
mRNA duplexes by RT-PCR.
CAAAGATGTGGCGGAGAAAT
PpGNT1 forward primer to detect sense and
antisense transcript by RT-PCR.
Primer used for the synthesis of the PpGNT1
antisense transcript derived cDNA.
PpGNT1 forward primer for expression analysis by
RT-PCR.
PpGNT1 forward primer to detect the miRNA:
mRNA duplexes by RT-PCR.
Forward primer used for the amplification of the
PpGNT1 hybridisation probe from cDNA.
Primer sequence (5’ Ö 3’)
Description of experiment
ATAACCTGGCGACCTTTCCT
PpGNT1 reverse primer to detect sense and
antisense transcript by RT-PCR.
Primer used for the synthesis of the PpGNT1
sense transcript derived cDNA.
PpGNT1 reverse primer for expression analysis by
RT-PCR.
PpGNT1 reverse primer to detect the miRNA:
mRNA duplexes by RT-PCR.
Reverse primer used for the amplification of the
PpGNT1 hybridisation probe from cDNA.
CCCCTGTCAGTTGCAGCATCC
PpARF forward primer to detect the miRNA:
mRNA duplexes by RT-PCR.
CTAGAGGCGGCGACGCAAGAG
PpARF reverse primer to detect the miRNA:
mRNA duplexes by RT-PCR.
GGAAAGAAGCAACAAGGTTGG
PpHB10 forward primer to detect the miRNA:
mRNA duplexes by RT-PCR.
ATCCCGCAGGACTGGAATCGC
PpHB10 reverse primer to detect the miRNA:
mRNA duplexes by RT-PCR.
GTGCAGGGTTGTGATGCCGAC
PpSBP3 forward primer to detect the miRNA:
mRNA duplexes by RT-PCR.
ATGCAAGAAACTGGACTGCTTC
PpSBP3 reverse primer to detect the miRNA:
mRNA duplexes by RT-PCR.
TTGATCCATCGATTGCTAATTT
PpbHLH forward primer to detect the miRNA:
mRNA duplexes by RT-PCR.
AGCCCTGACCAGTGCAAAC
PpbHLH reverse primer to detect the miRNA:
mRNA duplexes by RT-PCR.
AGCGTGGTATCACAATTGAC
PpEF1α forward primer for expression analysis by
RT-PCR and PCRs from genomic DNA.
Forward primer used for the amplification of the
PpEF1α hybridisation probe from cDNA.
Primer sequence (5’ Ö 3’)
Description of experiment
GATCGCTCGATCATGTTATC
PpEF1α reverse primer for for expression analysis
by RT-PCR and PCRs from genomic DNA.
Primer used for the synthesis of PpEF1α sense
transcript derived cDNA.
Reverse primer used for the amplification of the
PpEF1α hybridisation probe from cDNA.
CAGGCTTTCGCGTAATTCCCGTTG
Forward primer used for the amplification of the
PpC3HDZIP1 hybridisation probe from cDNA.
AGTGCCTCCAACTTCGGGCCTAAC
Reverse primer used for the amplification of the
PpC3HDZIP1 hybridisation probe from cDNA.
TTCTGCTGTCACTGGTGGACTT
PpC3HDZIP1
forward
primer
for
expression
primer
for
expression
analysis by RT-PCR.
AGAGTTCCAAGAACCTCCATGC
PpC3HDZIP1
reverse
analysis by RT-PCR.
GATTCTGCTGTCACTGGTGGTC
PpHB10 forward primer for expression analysis by
RT-PCR.
GTCTTGTAACCAACGTGGACGA
PpHB10 reverse primer for expression analysis by
RT-PCR.
GGCTATCACTTCCTGGATGGAC
PpSBP3 forward primer for expression analysis by
RT-PCR.
ACAAGGAAGTTGCAGATGGTGA
PpSBP3 reverse primer for expression analysis by
RT-PCR.
TGGTTCTCGGTTCAAGATGAAA
PpARF forward primer for expression analysis by
RT-PCR.
CAACTTGTTGGACTGCTGAGGA
PpARF reverse primer for expression analysis by
RT-PCR.
GGTGAACGTTTTGAGGTTGTG
PpARF forward primer for the amplification of the
PpARF hybridisation probe.
CAAAGGAAACAAAACAAATGCC
PpARF reverse primer for the amplification of the
PpARF hybridisation probe.
GGACGTTGGACCAGAAGAAA
PpbHLH forward primer for the amplification of the
PpbHLH hybridisation probe.
Primer sequence (5’ Ö 3’)
Description of experiment
CGCTTTATTCAGCCTCCTCA
PpbHLH reverse primer for the amplification of the
PpbHLH hybridisation probe.
GGTTGGTCATGGGTTGCG
PpCOR47 forward primer for the amplification of
the PpCOR47 hybridisation probe.
GAGGTCAACTGTCTCGCC
PpCOR47 reverse primer for the amplification of
the PpCOR47 hybridisation probe.
CAGCCACAGCCAGTCAAGTGGATTCAGT
5’ RACE primer to detect the cleavage product of
PpTAS4.
ATGTGACTCCATTACATCAACTGCAGGT
5’ RACE nested primer to detect the cleavage
product of PpTAS4.
GACCCACCTGGTGATGCTGCGAATTACC
5’ RACE primer to detect the cleavage product of
PpEREBP/AP2.
ATTTGGCGCCGCCAGTGCCGCTGCAGCG
5’ RACE nested primer to detect the cleavage
product of PpTAP2.
GCACTTAGATTTCCACTGGGCG
PpTAS4 forward primer for expression analysis by
RT-PCR.
Forward primer used for the amplification of the
PpTAS4 hybridisation probe from cDNA.
AAGACATGGGAATTGCACACCC
PpTAS4 reverse primer for expression analysis by
RT-PCR.
Reverse primer used for the amplification of the
PpTAS4 hybridisation probe from cDNA.
TTTGCGATGTTACGGTTGTAGC
PpTAS1 forward primer for expression analysis by
RT-PCR.
ACAGCCAGTCAAGTGGATTCAG
PpTAS1 reverse primer for expression analysis by
RT-PCR.
CCGCAACATCATGCTGAAGTGG
PpEREBP/AP2 forward primer for expression
analysis by RT-PCR.
Forward primer used for the amplification of the
PpEREBP/AP2 hybridisation probe from cDNA.
GATGCTGGCGGCTTTCAGCTTT
PpEREBP/AP2 reverse primer for expression
analysis by RT-PCR.
Reverse primer used for the amplification of the
PpEREBP/AP2 hybridisation probe from cDNA.
Primer sequence (5’ Ö 3’)
Description of experiment
GAAGGAAGCAACGAGGCTGGTGGCGTGAAT
Oligonucleotide to detect PpC3HDZIP1 sense
GCTAAGCTGACAGCC
siRNA upstream of the miR166 binding site.
GTTCTTGGAACTCTCCCGACAGGAAACGGTG
Oligonucleotide to detect PpC3HDZIP1 sense
GCACTATAGAGCTCTT
siRNA downstream of the miR166 binding site.
GGCTGTCAGCTTAGCATTCACGCTCACCAGC
Oligonucleotide to detect PpC3HDZIP1 antisense
CTCGTTGCTTCCTTC
siRNA upstream of the miR166 binding site.
AAGAGCTCTATAGTGCCACCGTTTCCTGTCG
Oligonucleotide to detect PpC3HDZIP1 antisense
GGAGAGTTCCAAGAAC
siRNA downstream of the miR166 binding site.
TACACGATTCACTCCCTGCAATAGGTCCGGC
Oligonucleotide to detect PpARF sense siRNA
TCACTCCAAGTGAC
upstream of the miR160 binding site.
CTCCAGCTGTGAGCTTCCAGAAGCAGACATG
Oligonucleotide to detect PpARF sense siRNA
AAGGCTAAGGGCTG
downstream of the miR160 binding site.
GTCACTTGGGATGAGCCGGACCTATTGCAGG
Oligonucleotide to detect PpARF antisense siRNA
GAGTGAATCGTGTA
upstream of the miR160 binding site.
CAGCCCTTAGCCTTCATGTCTGCTTCTGGAA
Oligonucleotide to detect PpARF antisense siRNA
GCTCACAGCTGGAG
downstream of the miR160 binding site.
TGAAGCACTCATCACACCCTATGGAGCCATA
Oligonucleotide to detect PpTAS4 sense ta-
GCTAGGGTCTTGCG
siRNA.
TGGCTCCATAGGGTGTGATGATGTCTTCATC
Oligonucleotide to detect PpTAS4 antisense ta-
CGGTGCTCTTCTACTGCCTT
siRNA.
GGCAAAGTGCAGGCGTCCCTAGCTGGTGCC
Oligonucleotide to detect PpEREBP/AP2 sense
CATGGCAAGCAGTCT
siRNA.
AGACTGCTTGCCATGGGCACCAGCTAGGGA
Oligonucleotide
CGCCTGCACTTTGCC
antisense siRNA.
GTCGTATCCAGTGCAGGGTCCGAGGTATTCG
Oligonucleotide
CACTGGATACGACGTGCTC
synthesis.
GTCGTATCCAGTGCAGGGTCCGAGGTATTCG
Oligonucleotide
CACTGGATACGACTGGCAT
synthesis.
GTCGTATCCAGTGCAGGGTCCGAGGTATTCG
Oligonucleotide
CACTGGATACGACGGGGAA
synthesis.
GTCGTATCCAGTGCAGGGTCCGAGGTATTCG
Oligonucleotide
CACTGGATACGACGGCGCT
synthesis.
to
detect
PpEREBP/AP2
for
miR156-specific
cDNA
for
miR160-specific
cDNA
for
miR166-specific
cDNA
for
miR390-specific
cDNA
Primer sequence (5’ Ö 3’)
Description of experiment
GTCGTATCCAGTGCAGGGTCCGAGGTATTCG
Oligonucleotide
CACTGGATACGACGCAGAG
specific cDNA synthesis.
GTCGTATCCAGTGCAGGGTCCGAGGTATTCG
Oligonucleotide
CACTGGATACGACTTGCCC
specific cDNA synthesis.
GCGGCGGTGACAGAAGAGAGT
Forward primer for miR156 RT-PCR.
CCTCCCGTGCCTGGCTCCCTGT
Forward primer for miR160 RT-PCR.
GCGGCGGTCGGACCAGGCTTCA
Forward primer for miR166 RT-PCR.
GCGGCGGAAGCTCAGGAGGGAT
Forward primer for miR390 RT-PCR.
GCGGCGGGTGATTGCACTGCAG
Forward primer for ta-siRNA (pptA013298) RT-
for
for
ta-siRNA
ta-siRNA
(pptA013298)-
(pptA079444)-
PCR.
GCGGCGGATCACAAGGGTAGGT
Forward primer for ta-siRNA (pptA079444) RTPCR.
GTGCAGGGTCCGAGGTAT
Universal reverse primer for miRNA and ta-siRNA
RT-PCR.
Supplemental Experimental Procedures
Isolation of PpDCL full-length cDNAs
Partial cDNA sequences of P. patens DCL genes were initially identified by tblastn
searches in P. patens EST sequences (Rensing et al., 2002) using A. thaliana DCL1-4
protein sequences (accession numbers P84634, Q9SP32, NP_189978, NP_566199) as
queries. Corresponding cDNA clones were sequenced and used to obtain the full-length
sequences. The cloning of full-length cDNA sequences was performed by 5’RACEPCRs and RT-PCRs using primers derived from available P. patens genomic sequence
data. To confirm that the amplicons were derived from the same cDNA all PCR and 5’
RACE primers were selected to produce overlapping PCR fragments of already known
sequence stretches.
Generation and molecular analysis of PpDCL1a and PpDCL1b knockout lines
For the generation of PpDCL1a and PpDCL1b knockout constructs we amplified a
PpDCL1a genomic region with the primers 5’-CCAGTTGCGCATAAAGTTGA-3’ and 5’TCCAAGGCATCCAGAGAGTC-3’ and a PpDCL1b cDNA region using the primers 5’GCATTCCTGTGGAGTTTGATG-3’ and 5’-ACCTTCCACACTTGGTGTGTG-3’. An nptII
selection marker cassette was cloned into a single Eco72I restriction site present in the
PpDCL1b cDNA fragment and into a single EcoRV restriction site of the PpDCL1a
genomic fragment. The complete knockout cassettes were released from the vector prior
to transformation. Primers used to identify ΔPpDCL1a transgenic lines were: 5’TTATGTGGATTCAGTGCGCTTC-3’ and 5’-CCATCGACTTAGCCAAACCAGT-3’. To
confirm a precise 5’ and 3’ integration of the PpDCL1a knockout construct we used the
primers
5’-TTTGCAGTTGACTGACCTCAAGA-3’
GCGGCTGAGTGGCTCCTTCA-3’
CCAAGGATCCCGGAAGAGGA-3’
(5’
and
integration)
and
and
5’-AAATTATCGCGCGCGGTGTC-3’
5’5’(3’
integration). To confirm the loss of PpDCL1a transcript by RT-PCR the primers 5’TTGGTCCGTTGGAATACACA-3’ and 5’- AATCTTTGTGCGCCTCTCAC-3’ were used.
Primers
used
for
the
screening
GCATTCCTGTGGAGTTTGATG-3’
of
and
transgenic
ΔPpDCL1b
lines
were:
5’-ACCTTCCACACTTGGTGTGTG-3’.
5’The
same primers were used to confirm the loss of PpDCL1b transcript by RT-PCR. A
second primer pair upstream of the integration site was used for RT-PCR: 5’AGGATTGTTACTGCGGTGCA-3’ and 5’-AAGCTCTGCACGCTCATAGC-3’. To confirm
a precise 5’ and 3’ integration of the PpDCL1b knockout construct we used the primers
5'-TGCTACTCACTTCATGAACTG-'3
integration)
and
and
5'-ACGTGACTCCCTTAATTCTCC-'3
5'-CCCGCAATTATACATTTAATACG-'3
and
(5’
5'-
GCACCATGGCTGCAACAAAG-'3 (3’ integration). RT-PCR control primers for the
PpEF1α control gene are listed in Table S1.
P. patens lines expressing an artificial miRNA (amiRNA) targeting PpGNT1
The amiR-GNT1 sequence was introduced into the A. thaliana miRNA319a precursor
by overlapping PCR as described (Khraiwesh et al., 2008). The resulting construct was
used for transfections of P. patens wild type and ΔPpDCL1b mutant lines. To identify
transgenic lines harboring the amiR-GNT1 expression construct PCR was performed
using
the
primers
5’-TGATATCTCCACTGACGAAAGGG-3’
and
5’-
GGATCCCCCCATGGCGATGCCTTAAAT-3’.
Detection of RNA cleavage products
Synthesis of 5’ RACE-ready cDNAs was carried out according to Zhu et al. (Zhu et al.,
2001) with the BD Smart RACE cDNA Amplification Kit (Clontech). PCR reactions were
performed using the UPM Primer-Mix in combination with gene-specific primers derived
from target RNAs (Table S1). Cleavage products were excised from the gel, cloned and
sequenced.
Small RNA Blots
Total RNA was separated in a 12% denaturing polyacrylamide gel containing 8.3 M urea
in TBE buffer. The RNA was electroblotted onto nylon membranes for 1 h at 400 mA.
Radiolabeled probes were generated by end-labeling of DNA oligonucleotides
complementary to miRNA, siRNA and ta-siRNA sequences and the U6snRNA control
(Table S1) with γ32P-ATP using T4 polynucleotide kinase. Blot hybridization was carried
out in 0.05 M sodium phosphate (pH 7.2), 1 mM EDTA, 6 x SSC, 1 x Denhardt’s, 5%
SDS. Blots were washed 2-3 times with 2 x SSC, 0.2% SDS and one time with 1 x SSC,
0.1% SDS. Blots were hybridized and washed at temperatures 5°C below the Tm of the
oligonucleotide. The sequences of the oligonucleotides used for the detection of small
RNAs are listed in Table S1.
Detection of small RNAs by RT-PCR
The RT-PCR analyses of the miR156, 160, 166, 390, and the ta-siRNAs pptA079444
(processed from the PpTAS1 gene) and pptA013298 (processed from the PpTAS3
gene) were carried out as described (Varkonyi-Gasic et al., 2007). The sequences of
oligonucleotides used for the cDNA synthesis and subsequent PCR reactions are listed
in Table S1.
Expression analysis by RT-PCR and RNA gel blots
RT-PCRs were performed for PpEF1α, PpGNT1, PpC3HDZIP1, PpHB10, PpSBP3,
PpARF, and PpTAS1 from three independent biological replicates with gene-specific
primers (Table S1). PCR products were quantified with the Quantity One Software (BioRad). The relative amounts of the transcripts were normalized to the constitutive control
PpEF1α. 20 µg of total RNA isolated from wild type, ΔPpDCL1b mutants and transgenic
lines expressing the amiR-GNT1, respectively, were separated in denaturing agarose
gels and blotted onto nylon membranes. Hybridization probes for PpARF, PpC3HDZIP1,
PpGNT1, PpEF1α, PpTAS4 PpEREBP/AP2, PpbHLH, and PpCOR47 were amplified
from wild type cDNA (primers listed in Table S1). The ABA-responsive gene PpCOR47
(Frank et al., 2005) was used to control the efficiency of the ABA treatments.
DNA methylation analysis
The cDNA sequences of PpC3HDZIP1 (DQ385516), PpHB10 (AB032182), PpARF
(AR452951), PpSBP3 (AJ968318) and PpGNT1 (AJ429143) were used for BLASTN
searches to identify corresponding genomic sequences from the P. patens wholegenome-shotgun (WGS) traces (accessible via www.ncbi.nlm.nih.gov/Traces/trace.cgi).
The identified genomic sequences were clustered and assembled using the Paracel
Transcript Assembler to determine the genomic exon/intron structure (Figure S5). The
parameters for clustering threshold, overlap length and overlap identity were 100 nt, 80
nt and 90%, respectively. Primers to analyse the PpTAS4 genomic locus were derived
from the reported PpTAS4 sequence (Talmor-Neiman et al., 2006). Primers for the
analysis of the PpEREBP/AP2 and PpbHLH gene were derived from the corresponding
gene model of the available P. patens genomic sequence (http://genome.jgipsf.org/Phypa1_1/Phypa1_1.home.html;
gene
model
accession
numbers
Phypa1_129196 [PpEREBP/AP2] and Phypa1_209063 [PpbHLH]). The derived
promoter, exon and intron regions were analyzed with the MethPrimer program (Li and
Dahiya, 2002) to deduce methylation-specific (MSP) and unmethylation-specific primers
(USP) (Figure S6) for PCR analysis of bisulfite-treated DNA.
Detection of sense and antisense transcripts
cDNA from wild type plants and ΔPpDCL1b mutants was synthesized from 4 µg total
RNA with Superscript III (Invitrogen) using primers specific for sense and antisense
transcripts, respectively (Table S1). To monitor the efficiency of cDNA synthesis, primers
specific for the PpEF1α sense transcript were added to each cDNA synthesis reaction.
RT-PCRs were carried out with gene-specific primers (Table S1).
Supplemental Experimental Procedures References
Frank, W., Ratnadewi, D., and Reski, R. (2005). Physcomitrella patens is highly tolerant
against drought, salt and osmotic stress. Planta 220, 384-394.
Khraiwesh, B., Ossowski, S., Weigel, D., Reski, R., and Frank, W. (2008). Specific gene
silencing by artificial microRNAs in Physcomitrella patens: An alternative to targeted
gene knockouts. Plant Physiol.
Li, L. C., and Dahiya, R. (2002). MethPrimer: designing primers for methylation PCRs.
Bioinformatics 18, 1427-1431.
Rensing, S. A., Rombauts, S., Van de Peer, Y., and Reski, R. (2002). Moss
transcriptome and beyond. Trends Plant Sci 7, 535-538.
Talmor-Neiman, M., Stav, R., Klipcan, L., Buxdorf, K., Baulcombe, D. C., and Arazi, T.
(2006). Identification of trans-acting siRNAs in moss and an RNA-dependent RNA
polymerase required for their biogenesis. Plant J 48, 511-521.
Varkonyi-Gasic, E., Wu, R., Wood, M., Walton, E. F., and Hellens, R. P. (2007).
Protocol: a highly sensitive RT-PCR method for detection and quantification of
microRNAs. Plant Methods 3, 12.
Zhu, Y. Y., Machleder, E. M., Chenchik, A., Li, R., and Siebert, P. D. (2001). Reverse
transcriptase template switching: a SMART approach for full-length cDNA library
construction. Biotechniques 30, 892-897.
Chapter III
3
Artificial microRNAs in Physcomitrella patens
Chapter III: Publication 1
Specific gene silencing by artificial microRNAs in Physcomitrella
patens: An alternative to targeted gene knockout
Plant Physiology, October 2008, Vol. 148, pp. 684–693
Received August 13, 2008
Accepted August 22, 2008
Own contribution:
Carried out all experimental work reported in the publication and contributed to prepare the
manuscript (drafting the manuscript and preparing the figures). The work was supervised by
W. Frank.
121
Breakthrough Technologies
Specific Gene Silencing by Artificial MicroRNAs in
Physcomitrella patens: An Alternative to Targeted
Gene Knockouts1[C][W][OA]
Basel Khraiwesh, Stephan Ossowski, Detlef Weigel, Ralf Reski, and Wolfgang Frank*
Plant Biotechnology, Faculty of Biology (B.K., R.R., W.F.), Freiburg Initiative for Systems Biology (R.R., W.F.),
and Centre for Biological Signaling Studies (R.R.), University of Freiburg, 79104 Freiburg, Germany; and
Department of Molecular Biology, Max Planck Institute for Developmental Biology, 72076 Tuebingen,
Germany (S.O., D.W.)
MicroRNAs (miRNAs) are approximately 21-nucleotide-long RNAs processed from nuclear-encoded transcripts, which
include a characteristic hairpin-like structure. MiRNAs control the expression of target transcripts by binding to reverse
complementary sequences directing cleavage or translational inhibition of the target RNA. Artificial miRNAs (amiRNAs) can
be generated by exchanging the miRNA/miRNA* sequence within miRNA precursor genes, while maintaining the pattern of
matches and mismatches in the foldback. Thus, for functional gene analysis, amiRNAs can be designed to target any gene of
interest. The moss Physcomitrella patens exhibits the unique feature of a highly efficient homologous recombination mechanism,
which allows for the generation of targeted gene knockout lines. However, the completion of the Physcomitrella genome
necessitates the development of alternative techniques to speed up reverse genetics analyses and to allow for more flexible
inactivation of genes. To prove the adaptability of amiRNA expression in Physcomitrella, we designed two amiRNAs, targeting
the gene PpFtsZ2-1, which is indispensable for chloroplast division, and the gene PpGNT1 encoding an N-acetylglucosaminyltransferase. Both amiRNAs were expressed from the Arabidopsis (Arabidopsis thaliana) miR319a precursor fused to a
constitutive promoter. Transgenic Physcomitrella lines harboring the overexpression constructs showed precise processing of
the amiRNAs and an efficient knock down of the cognate target mRNAs. Furthermore, chloroplast division was impeded in
PpFtsZ2-1-amiRNA lines that phenocopied PpFtsZ2-1 knockout mutants. We also provide evidence for the amplification of the
initial amiRNA signal by secondary transitive small interfering RNAs, although these small interfering RNAs do not seem to
have a major effect on sequence-related mRNAs, confirming specificity of the amiRNA approach.
During the last decade, small nonprotein-coding
RNAs (20–24 nucleotides [nt] in size) have been demonstrated to be involved in RNA-mediated phenomena
such as RNA interference (RNAi), cosuppression, gene
silencing, and quelling (Matzke et al., 1989; Napoli et al.,
1990; de Carvalho et al., 1992; Romano and Macino, 1992;
Lee et al., 1993; Hamilton and Baulcombe, 1999). Major
1
This work was supported by the Landesstiftung BadenWürttemberg (grant no. P–LS–RNS/40 to W.F., R.R., and D.W.), the
Federal Ministry of Education and Research (Freiburg Initiative for
Systems Biology grant no. 0313921 to R.R. and W.F.), the Excellence
Initiative of the German Federal State Governments (Biological
Signaling Studies grant no. EXC294 to R.R.), and European Community FP6 IP SIROCCO (contract no. LSHG–CT–2006–037900 to
D.W.).
* Corresponding author; e-mail wolfgang.frank@biologie.
uni-freiburg.de.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy
described in the Instructions for Authors (www.plantphysiol.org) is:
Wolfgang Frank ([email protected]).
[C]
Some figures in this article are displayed in color online but in
black and white in the print edition.
[W]
The online version of this article contains Web-only data.
[OA]
Open Access articles can be viewed online without a subscription.
www.plantphysiol.org/cgi/doi/10.1104/pp.108.128025
684
classes of small RNAs include microRNAs (miRNAs)
and small interfering RNAs (siRNAs), which differ with
respect to their biogenesis (Bartel, 2004; Chapman and
Carrington, 2007). MiRNAs are approximately 21-nt
RNAs that are encoded by endogenous MIR genes.
Their primary transcripts form precursor RNAs exhibiting a partially double-stranded stem-loop structure
that is processed by DICER-LIKE proteins to release
mature miRNAs (Bartel, 2004). MiRNAs are recruited
to the RNA-induced silencing complex (RISC), where
they become activated by unwinding of the double
strand and subsequently bind to complementary mRNA
sequences resulting in either direct cleavage of the
mRNA or repression of their translation by RISC
(Bartel, 2004; Kurihara and Watanabe, 2004; Brodersen
et al., 2008). Recently, miRNAs have been identified as
important regulators of gene expression in both plants
and animals (Jones-Rhoades et al., 2006), and particular
miRNA families were shown to be highly conserved in
evolution (Jones-Rhoades et al., 2006; Axtell et al., 2007;
Fahlgren et al., 2007; Fattash et al., 2007; Axtell and
Bowman, 2008). In contrast, precursors of siRNAs
form perfectly complementary double-stranded RNA
(dsRNA) molecules (Myers et al., 2003). They originate
from transgenes, viruses, and transposons and may
require RNA-dependent RNA polymerases for dsRNA
formation (Waterhouse et al., 2001; Aravin et al., 2003).
Plant Physiology, October 2008, Vol. 148, pp. 684–693, www.plantphysiol.org Ó 2008 American Society of Plant Biologists
Artificial MicroRNAs in Physcomitrella patens
Unlike miRNAs, the diced siRNA products derived from
the long complementary precursors are not uniform in
sequence, but correspond to different regions of their
precursor. Whereas miRNAs mainly mediate posttranscriptional control of endogenous transcripts, siRNAs
have been implicated in transcriptional silencing of
transposable elements as well as posttranscriptional
control of endogenous and exogenous RNAs, for example, viral transcripts (Waterhouse et al., 2001; Aravin
et al., 2003; Myers et al., 2003; Ossowski et al., 2008).
Previous reports demonstrated that the alteration of
several nucleotides within the miRNA sequence does
not affect its biogenesis as long as the positions of
matches and mismatches within the precursor stem
loop remain unaffected (Vaucheret et al., 2004). This
raises the possibility of modifying miRNA sequences
and creating artificial miRNAs (amiRNA) directed
against any gene of interest resulting in posttranscriptional silencing of the corresponding transcript
(Zeng et al., 2002; Parizotto et al., 2004; Alvarez et al.,
2006; Niu et al., 2006; Schwab et al., 2006; Warthmann
et al., 2008). In addition, genome-wide expression
analyses in Arabidopsis (Arabidopsis thaliana) have
shown that plant amiRNAs exhibit high specificity
similar to natural miRNAs (Schwab et al., 2005, 2006),
such that their sequences can easily be optimized to
knock down the expression of a single gene or several
highly conserved genes without affecting the expression of other genes.
The moss Physcomitrella patens has become a recognized model system to study diverse processes in
plant biology, which was mainly based on the unique
ability to efficiently integrate DNA into its nuclear
genome by means of homologous recombination enabling the generation of targeted gene knockout lines
(Schaefer, 2002). Furthermore, based on the predominant haploid phase of Physcomitrella’s life cycle, the
frequency of phenotypic deviations caused by the
disruption of a single gene is higher compared to
seed plants (Egener et al., 2002). Nevertheless, the
generation of targeted knockout mutants in Physcomitrella has limitations. For example, despite the haploid
genome, homologs might still compensate for each
other and one cannot recover knockouts of genes with
essential functions. Furthermore, the targeted knockout of a single gene requires several cloning steps,
repetitive selection of transgenic lines, and detailed
molecular analysis of putative knockout candidates.
The recently published Physcomitrella genome (Rensing
et al., 2008) now opens the way for medium- to largescale analysis of gene functions in a postgenomic era
(Quatrano et al., 2007) requiring the development of
new techniques. The posttranscriptional silencing of
genes by amiRNAs may serve as an appropriate tool
to speed up such analyses because they can be designed to target several genes (as long they contain at
least one conserved sequence stretch), amiRNAs can
be expressed from inducible promoters, and amiRNA
constructs can easily be generated using a standardized cloning procedure (Schwab et al., 2006).
Plant Physiol. Vol. 148, 2008
Alternative approaches to analyze gene functions
in Physcomitrella were recently reported, which were
based on the expression of classical inverted repeat
sequences resulting in the formation of dsRNA molecules, which give rise to siRNAs and consequently
silence the target transcript (Bezanilla et al., 2003, 2005;
Vidali et al., 2007). One drawback to applying classical
RNAi constructs is the production of a diverse set
of siRNAs from the complete dsRNA, which may affect off-target transcripts. Furthermore, in some cases,
gene silencing triggered by the expression of inverted
repeat sequences was found to be unstable in Physcomitrella (Bezanilla et al., 2005). Additional differences in the action of siRNAs and amiRNAs may
result from their varying mobility within the plant.
Recent studies have shown that transgene-derived or
viral-induced siRNAs are able to move from cell to
cell, whereas miRNAs are not mobile and act cell
autonomously (Tretter et al., 2008).
Independent studies on small RNAs in Physcomitrella
revealed the existence of a diverse miRNA repertoire,
including highly conserved miRNA families (Arazi
et al., 2005; Axtell et al., 2006, 2007; Talmor-Neiman
et al., 2006; Fattash et al., 2007). Furthermore, their
corresponding precursor transcripts share the characteristic hairpin-like structure known from seed plants.
Thus, the design and expression of amiRNAs for the
specific knock down of genes in Physcomitrella should
be feasible. To test amiRNA function in Physcomitrella,
we targeted the gene PpFtsZ2-1, which is required
for chloroplast division (Strepp et al., 1998), and the
PpGNT1 gene encoding an N-acetylglucosaminyltransferase (Koprivova et al., 2003). PpFtsZ2-1 null mutants
form macrochloroplasts, presenting an obvious phenotype, which enables direct evaluation of the efficiency of
the intended amiRNA approach.
RESULTS
Expression and Detection of PpFtsZ2-1-amiRNA and
PpGNT1-amiRNA in Physcomitrella
The use of amiRNAs for efficient gene silencing has
been reported in various seed plants (Alvarez et al.,
2006; Niu et al., 2006; Schwab et al., 2006; Warthmann
et al., 2008), but it has not been tested in nonseed
plants such as the bryophyte P. patens. This is an issue
because functional studies of essential members of the
RNAi machinery in Physcomitrella, such as Dicer and
Argonaute proteins, are still missing (Axtell et al.,
2007). Furthermore, the complement of essential RNAirelated proteins in Physcomitrella differs from that in
seed plants (Axtell et al., 2007; Rensing et al., 2008).
We designed two amiRNAs that were predicted to
target the genes PpFtsZ2-1 and PpGNT1, respectively,
using the amiRNA designer interface WMD (Schwab
et al., 2006; Ossowski et al., 2008). The designed
amiRNAs contain a uridine residue at position 1 and
an adenine residue at position 10, both of which are
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Khraiwesh et al.
overrepresented among natural plant miRNAs and
increase the efficiency of miRNA-mediated target cleavage (Schwab et al., 2005). Furthermore, we also preferred that the amiRNAs exhibit 5# instability relative
to the miRNA*, which positively affects separation of
both strands during RISC loading (Fig. 1A; Mallory
et al., 2004; Schwab et al., 2005). In previous studies, the
Arabidopsis miR319a precursor was used to introduce
specific nucleotide changes within the miRNA/miRNA*
stem-loop region. Based on the conservation of the
miR319 family among land plants (Jones-Rhoades et al.,
2006; Fattash et al., 2007; Axtell and Bowman, 2008;
Warthmann et al., 2008) and similar secondary structures of miR319 precursor transcripts from Arabidopsis
and Physcomitrella (Fig. 1B; Supplemental Fig. S1),
we hypothesized that the PpFtsZ2-1-amiRNA and
PpGNT1-amiRNA will be correctly processed from the
Arabidopsis miR319a precursor. The PpFtsZ2-1-amiRNA
and PpGNT1-amiRNA and the corresponding miRNA*
sequences were introduced into the miR319a precursor by overlapping PCR using primers harboring the
respective amiRNA and miRNA* sequences, cloned
into the plant expression vector pPCV downstream
of a double cauliflower mosaic virus 35S promoter
(Fig. 1A) and used for transfection of Physcomitrella
protoplasts. After selection of regenerating plants,
genomic DNA of individual lines was analyzed by
PCR with primers flanking the amiRNA sequence
present in the expression constructs to identify transgenic lines that had integrated the PpFtsZ2-1-amiRNA
and PpGNT1-amiRNA constructs, respectively. Eight
of 12 regenerated lines derived from the transformation with the PpFtsZ2-1-amiRNA construct and seven
of 12 regenerated lines derived from the transformation with the PpGNT1-amiRNA construct produced
the expected PCR amplicon. Thus, we cannot exclude that some lines survived the antibiotic selection
without the integration of the DNA constructs. From
the lines harboring an overexpression construct, three
PpFtsZ2-1-amiRNA lines and two PpGNT1-amiRNA
lines were selected for further analysis (Fig. 1C). As the
generated amiRNA overexpression constructs do not
contain homologous sequences of the Physcomitrella genome, the constructs are expected to integrate into the
Physcomitrella genome by an illegitimate recombination event. To prove the correct maturation of the
PpFtsZ2-1-amiRNA and PpGNT1-amiRNA from the
Arabidopsis miR319a precursor and its accumulation
in the transgenic lines, we performed small RNA gelblot analyses with antisense probes for both amiRNAs.
Accumulation of the mature PpFtsZ2-1-amiRNA and
PpGNT1-amiRNA was detected in all lines analyzed,
demonstrating that the amiRNAs are efficiently processed from the Arabidopsis miR319a precursor in
Physcomitrella (Fig. 1D). However, normalization of
the PpFtsZ2-1-amiRNA and PpGNT1-amiRNA hybridization signals to the U6snRNA controls revealed
amiRNA expression levels that differed up to 8-fold
for the PpFtsZ2-1-amiRNA and up to 5-fold for the
PpGNT1-amiRNA between the individual lines (Fig. 1D).
686
AmiRNA-Directed Cleavage of PpFtsZ2-1 and
PpGNT1 mRNAs
The expression of the PpFtsZ2-1-amiRNA and
PpGNT1-amiRNA should cause cleavage of the cognate mRNAs within the region complementary to the
amiRNA sequences. To prove this, we performed 5#
RACE-PCRs to detect specific PpFtsZ2-1 and PpGNT1
mRNA cleavage products. Using 5# RACE-ready
cDNA prepared from one PpFtsZ2-1-amiRNA, one
PpGNT1-amiRNA overexpression line, and wild type,
cleavage products of the expected size were only
amplified from the amiRNA lines. Conversely, in
wild type, the 5# RACE-PCRs yielded exclusively
fragments derived from the full-length transcripts
(Fig. 1E). The PCR products corresponding to the
expected size of the PpFtsZ2-1 and PpGNT1 mRNA
cleavage products in the amiRNA lines were cloned
and sequenced to determine the precise mRNA cleavage sites. In 12 of 18 clones analyzed, PpFtsZ2-1 mRNA
cleavage occurred between nucleotide positions 11
and 12 with respect to the PpFtsZ2-1-amiRNA sequence, whereas the remaining six clones resulted
from cleavage of the PpFtsZ2-1 mRNA between nucleotides 12 and 13 (Fig. 1E). Normally, in plants,
cleavage within a target transcript that is mediated by
a 21-nt miRNA occurs between positions 10 and 11
with respect to the miRNA sequence (Llave et al.,
2002), suggesting that the actual amiRNAs produced
from the PpFtsZ2-1-amiRNA construct were shifted by
1 or 2 nt. However, the sequencing of six independent
clones of PpGNT1 mRNA cleavage products revealed
that cleavage occurred between positions 10 and 11
with respect to the PpGNT1-amiRNA sequence (Fig.
1E), indicating precise processing of the PpGNT1amiRNA from the precursor construct.
Target sites in plant mRNAs normally share high
sequence complementarity to the respective miRNA
(Schwab et al., 2005). To prove the specificity of the
expressed PpFtsZ2-1-amiRNA, we analyzed whether
the mRNA of PpFtsZ2-2, the closest homolog of
PpFtsZ2-1, is targeted by the PpFtsZ2-1-amiRNA.
Compared to the PpFtsZ2-1-amiRNA recognition site
in PpFtsZ2-1, the corresponding region within the
PpFtsZ2-2 sequence contains two mismatches at positions 12 and 16. 5# RACE-PCRs were performed using a
PpFtsZ2-2 gene-specific primer. PCR products indicating amiRNA-guided cleavage products were not obtained. Instead, the 5# RACE-PCR yielded exclusively
fragments corresponding to the PpFtsZ2-2 full-length
transcript (Fig. 1E). Thus, the PpFtsZ2-1-amiRNA exhibits high specificity, comparable to natural miRNAs.
AmiRNAs Efficiently Down-Regulate PpFtsZ2-1 and
PpGNT1 mRNA Levels
As we detected amiRNA-directed cleavage of the
PpFtsZ2-1 and PpGNT1 target mRNAs, we next analyzed the target transcript levels by RNA gel blots.
Compared to wild type, we detected strongly reduced
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Artificial MicroRNAs in Physcomitrella patens
Figure 1. Analysis of Physcomitrella lines expressing PpFtsZ2-1-amiRNA and PpGNT1-amiRNA. A, Scheme illustrating the
PpFtsZ2-1-amiRNA and PpGNT1-amiRNA overexpression constructs. The modified ath-miRNA319a precursor DNA fragments
were cloned into the SmaI and BamHI sites of the pPCV plant expression vector containing a double 35S promoter, nos
terminator, and hpt selection marker cassette. Primers that were used for molecular analyses of the transgenic lines are indicated
Plant Physiol. Vol. 148, 2008
687
Khraiwesh et al.
steady-state levels of PpFtsZ2-1 and PpGNT1 mRNAs in
the respective amiRNA overexpression lines (Fig. 2A).
However, PpFtsZ2-1 transcript levels were reduced
to 1% to 2% in PpFtsZ2-1-amiRNA lines, whereas
PpGNT1 mRNA levels dropped to 10% to 20% in
PpGNT1-amiRNA lines when compared to wild-type
plants. Furthermore, the efficiency of posttranscriptional silencing of PpFtsZ2-1 was similar in all three
amiRNA overexpression lines, even though they differed with respect to the PpFtsZ2-1-amiRNA accumulation (Fig. 1D), whereas the reduction of PpGNT1
transcript levels correlated with the PpGNT1-amiRNA
expression levels. From these results, we conclude that
amiRNAs confer efficient down-regulation of their
target mRNAs in Physcomitrella. As a control, we also
analyzed the steady-state levels of the sequencerelated PpFtsZ2-2 mRNA in PpFtsZ2-1-amiRNA overexpression lines. In agreement with the absence of
amiRNA-induced mRNA cleavage products, PpFtsZ2-2
transcript levels were similar in wild-type and the three
PpFtsZ2-1-amiRNA lines (Fig. 2A).
The 5# RACE-PCR experiments performed with
one of the PpFtsZ2-1-amiRNA lines yielded additional
fragments that differed substantially in size from the
expected cleavage products (Fig. 1E). After amiRNAmediated cleavage of the mRNA, the cleavage products may serve as templates for synthesizing cRNA
by RNA-dependent RNA polymerase (Vaistij et al.,
2002) leading to the formation of dsRNA. Subsequently, the dsRNA may be processed into secondary
siRNAs, resulting in spreading of the initial amiRNA
signal (Fig. 2B). This mechanism, known as transitivity, usually is initiated by dsRNA triggers. In plants,
the transitivity occurs in both directions of the initial
dsRNA trigger (Moissiard et al., 2007), whereas in
animals, spreading of the initial signal occurs only
upstream of the trigger (Pak and Fire, 2007). However,
the onset of transitivity is a rare event after miRNAmediated target cleavage (Howell et al., 2007; Moissiard
et al., 2007) and is normally not observed after
amiRNA-mediated target cleavage (Schwab et al.,
2006). To investigate the possibility of transitivity, we
used sense and antisense oligonucleotides derived
from PpFtsZ2-1 and PpGNT1 mRNA regions downstream of the amiRNA recognition site for RNA gelblot analysis. Sense and antisense siRNAs were only
detected in PpFtsZ2-1-amiRNA and PpGNT1-amiRNA
lines, respectively, but not in wild type (Fig. 2C). We
conclude that amiRNAs allow for efficient downregulation of mRNAs in Physcomitrella and the generation of transitive siRNAs from mRNA cleavage products
may amplify the initial amiRNA trigger. However,
the transitive effects are apparently not sufficient to
have a major impact on sequence-related genes, as the
PpFtsZ2-2 steady-state RNA levels were unaffected in
PpFtsZ2-1-amiRNA overexpression lines (Fig. 2A).
PpFtsZ2-1-amiRNA Overexpressors Phenocopy PpFtsZ2-1
Null Mutants
In this study, we have chosen two genes to evaluate
the use of an amiRNA expression system in Physcomitrella. The targeted deletion of PpGNT1 that is involved in the N-glycosylation of proteins did not cause
any phenotypic deviations (Koprivova et al., 2003). In
agreement with this previous study, the two characterized PpGNT1-amiRNA lines were indistinguishable from Physcomitrella wild-type plants. In contrast,
PpFtsZ2-1 null mutants, which were generated by targeted gene disruption and lack expression of PpFtsZ2-1
mRNA, are impeded in chloroplast division leading
to the formation of macrochloroplasts (Strepp et al.,
1998). In our study, the expression of PpFtsZ2-1amiRNA led to strongly reduced PpFtsZ2-1 mRNA
levels. To compare knockout and amiRNA lines, we
investigated the phenotypes of the three PpFtsZ2-1amiRNA lines. In all lines, the accumulation of the
amiRNA targeting PpFtsZ2-1 resulted in impaired
chloroplast division and the formation of macrochloroplasts that phenocopied the PpFtsZ2-1 null mutants
(Fig. 3; Supplemental Fig. S2). The formation of macrochloroplasts in the PpFtsZ2-1-amiRNA lines was observed in all tissues and cells analyzed indicating
an efficient production of mature amiRNAs from constitutively expressed precursor transcripts. Furthermore, we did not observe any particular phenotypic
Figure 1. (Continued .)
by arrows. B, Secondary structures of foldbacks of the P. patens miR319d precursor (ppt-MIR319d) and Arabidopsis miR319a
precursor (ath-MIR319a). The mature miRNA is highlighted in green with uppercase letters. C, PCR screen to identify transgenic
lines harboring the PpFtsZ2-1-amiRNA and PpGNT1-amiRNA expression constructs. WT, Wild type; amiRNA lines, 1, 2, and 3
for PpFtsZ2-1-amiRNA; 1 and 2 for PpGNT1-amiRNA; PpEF1a, control PCRs. D, Expression analysis of PpFtsZ2-1-amiRNA and
PpGNT1-amiRNA in Physcomitrella wild type (WT), and lines harboring the PpFtsZ2-1-amiRNA or PpGNT1-amiRNA
expression constructs. Fifty micrograms of each RNA was blotted and hybridized with a PpFtsZ2-1-amiRNA and PpGNT1amiRNA antisense probe, respectively. Hybridization with an antisense probe for U6snRNA served as control. PpFtsZ2-1amiRNA and PpGNT1-amiRNA expression levels were normalized to the U6snRNA control hybridization. Numbers indicate the
relative PpFtsZ2-1-amiRNA and PpGNT1-amiRNA expression levels. E, Top, 5# RACE-PCRs for the genes PpFtsZ2-1 and
PpFtsZ2-2 from wild type (WT) and line 1 expressing the PpFtsZ2-1-amiRNA; bottom, 5# RACE-PCR for the gene PpGNT from
wild type (WT) and line 1 expressing the PpGNT1-amiRNA. The arrows mark PCR fragments corresponding to the expected size
of the cleavage products that were isolated, cloned, and sequenced. The right images show the sequence complementarity of
PpFtsZ2-1, PpFtsZ2-2, and PpGNT1 to the amiRNA sequences. The determined cleavage sites within the PpFtsZ2-1 and PpGNT1
mRNAs are marked by vertical arrows and numbers above indicate the number of sequenced products cleaved at this site.
[See online article for color version of this figure.]
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Artificial MicroRNAs in Physcomitrella patens
Figure 2. Expression analysis of PpFtsZ2-1, PpFtsZ2-2,
and PpGNT1, and detection of transitive siRNAs. A,
Left, RNA gel blots (20 mg each) from wild type (WT)
and PpFtsZ2-1-amiRNA overexpression lines (1–3)
hybridized with PpFtsZ2-1 and PpFtsZ2-2 probes;
right, RNA gel blots (20 mg each) from wild type (WT)
and PpGNT1-amiRNA overexpression lines (1 and 2)
hybridized with a PpGNT1 probe. The ethidium
bromide-stained gels below indicate equal loading.
The hybridization signals were normalized to the rRNA
bands, and the PpFtsZ2-1, PpFtsZ2-2, and PpGNT1
expression levels in wild type were set to 1. Numbers
indicate the relative PpFtsZ2-1, PpFtsZ2-2, and
PpGNT1 mRNA levels. B, Scheme illustrating the
generation of transitive siRNAs from amiRNA target
cleavage products requiring an RNA-dependent RNA
polymerase (RdRP) to generate dsRNA, which is
subsequently processed into siRNAs. Black line,
mRNA; gray box, amiRNA binding site; curved line,
amiRNA. C, Detection of sense and antisense transitive
siRNAs produced from PpFtsZ2-1 (left) and PpGNT1
(right) mRNA cleavage products by RNA gel blots
hybridized with oligonucleotides derived from regions
downstream of the amiRNA binding sites. Hybridization with an antisense probe for U6snRNA served
as control.
differences among the transgenic lines expressing the
PpFtsZ2-1-amiRNA, which is consistent with the similar degree of PpFtsZ2-1 mRNA reduction. Our results
demonstrate that the expression of amiRNAs in Physcomitrella leads to efficient silencing of their target
mRNAs comparable to the effects of targeted gene
knockouts.
DISCUSSION
The successful use of amiRNAs for the specific
down-regulation of genes was shown for the dicotyledonous plants Arabidopsis, tomato (Solanum lycopersicum), and tobacco (Nicotiana tabacum), and for the
monocot rice (Oryza sativa; Parizotto et al., 2004;
Alvarez et al., 2006; Niu et al., 2006; Schwab et al.,
2006; Qu et al., 2007; Ossowski et al., 2008; Warthmann
et al., 2008). In most cases, the amiRNA was expressed
from endogenous miRNA precursors. However, high
expression rates of amiRNAs were achieved in tobacco
and tomato using the Arabidopsis miR164b precursor
sequence indicating correct processing of conserved
Plant Physiol. Vol. 148, 2008
pre-miRNAs within seed plants (Alvarez et al., 2006).
In our study, we tested the application of amiRNAs for
the specific silencing of genes in the bryophyte Physcomitrella making use of an amiRNA expression system, where the Arabidopsis miR319a precursor serves
as the backbone for amiRNA expression and subsequent maturation and was developed to control gene
expression in Arabidopsis (Schwab et al., 2006). The
miR319 family belongs to the highly conserved amiRNA
families, even over large evolutionary distances, and
was also found in Physcomitrella (Arazi et al., 2005;
Jones-Rhoades et al., 2006; Fattash et al., 2007). Notably, miR319 stands out in that there is also considerable sequence conservation in the foldback, not only
in the miRNA itself. Our comparison of the Arabidopsis miR319a precursor and the Physcomitrella miR319
precursor sequences confirmed nucleotide sequence
conservation outside the miRNA/miRNA* region,
implying similar foldback structures of the Arabidopsis and Physcomitrella miR319 pre-miRNAs. Indeed,
we detected the correct processing of a mature 21-nt
PpFtsZ2-1-amiRNA and PpGNT1-amiRNA, respectively, from the Arabidopsis miR319a precursor in
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Khraiwesh et al.
Figure 3. Impeded plastid division and
formation of macrochloroplasts in
PpFtsZ2-1-amiRNA overexpressors. A,
Light microscopy from protonema and
leaves of wild type (WT) and one
PpFtsZ2-1-amiRNA overexpression line
(size bars, 100 mm). B, Confocal laserscanning microscopy from protonema
and leaves of wild type (WT) and one
PpFtsZ2-1-amiRNA overexpression line
(size bars, 50 mm). Red, Chlorophyll autofluorescence in plastids. See Supplemental
Figure S2 for phenotypes of the other
two PpFtsZ2-1-amiRNA lines.
transgenic Physcomitrella lines, indicating that the reconstructed miR319a pre-miRNA contains the essential recognition and processing information to enter
the Physcomitrella miRNA biogenesis pathway. The
PpFtsZ2-1 mRNA cleavage products were, however,
offset by 1 and 2 nt relative to the expected products
(Llave et al., 2002), suggesting that the PpFtsZ2-1amiRNA was shifted by 1 or 2 nt, respectively, relative
to the intended amiRNA. A similar effect has been
observed for some Arabidopsis amiRNAs (Schwab
et al., 2006). Because the originally designed amiRNAs
were perfectly complementary, the shifted amiRNAs
should still adhere to the targeting rules for miRNAs.
The observation of shifted cleavage products suggested
that release of the PpFtsZ2-1-amiRNA/miRNA* duplex from the precursor was not always precise,
consistent with observations on endogenous miRNAs
(Rajagopalan et al., 2006). Nevertheless, the Arabidopsis miR319a precursor can be used routinely for the
expression of amiRNAs in Physcomitrella as the apparent shift by 1 nt during the maturation of the amiRNA
may result in a mismatch at the 3# end of the miRNA,
which is not affecting target mRNA cleavage (Schwab
690
et al., 2005). Furthermore, cleavage of the PpFtsZ2-1
mRNA within the amiRNA recognition site indicates
correct amiRNA/amiRNA* duplex recognition and
amiRNA loading into the RISC complex.
Previous studies have shown that the transcript
levels of amiRNA targets are in most cases anticorrelated with corresponding amiRNA levels (Schwab
et al., 2006). Among PpFtsZ2-1-amiRNA and PpGNT1amiRNA lines analyzed, the amiRNA expression levels
varied 8-fold and 5-fold, respectively. Nevertheless, the
amiRNA expression caused a similar reduction of
PpFtsZ2-1 and PpGNT1 mRNA levels to 1% to 2% and
10% to 20%, respectively, compared to transcript levels
in wild type. This suggests that the amount of amiRNAs
is not limiting in any of the lines. Instead, it is
likely that the competition of natural miRNAs and
amiRNAs in RISC loading determines the efficiency
of posttranscriptional silencing of the PpFtsZ2-1 and
PpGNT1 transcripts.
The formation of macrochloroplasts in the PpFtsZ2-1amiRNA lines indicated impeded plastid division
and resembled the phenotype of PpFtsZ2-1 knockout
lines, which completely lack a functional transcript
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Artificial MicroRNAs in Physcomitrella patens
(Strepp et al., 1998). We therefore conclude that the
remaining PpFtsZ2-1 transcripts in the amiRNA lines
are not able to generate sufficient PpFtsZ2-1 protein to
support proper plastid division. In addition, amiRNA
expression in the transgenic lines seems to be stable
over long time periods as we did not observe any
phenotypic reversion to wild-type plastids in the
PpFtsZ2-1-amiRNA overexpression lines after 1 year of
subculture. We anticipate that the described amiRNA
expression system will result in similar silencing
efficiencies of any target gene and thus can be routinely used as an alternative to the generation of
knockout mutants in Physcomitrella.
The efficient silencing of PpFtsZ2-1 and PpGNT1 by
amiRNAs might be enhanced by the generation of
transitive siRNAs, as we detected such siRNAs from
the 3# cleavage products of the PpFtsZ2-1 and PpGNT1
mRNAs. Usually, transitive siRNAs are produced
from exogenous RNA sequences such as viruses or
sense transgene transcripts (Baulcombe, 2004), but the
formation of transitive siRNAs from miRNA-guided
cleavage products appears to be the exception (Howell
et al., 2007; Moissiard et al., 2007). Furthermore, transitivity was suggested not to be a major factor contributing to amiRNA efficacy in previous studies,
although this was inferred only indirectly from the
lack of effects on sequence-related transcripts (Schwab
et al., 2006; Warthmann et al., 2008). Although we
cannot exclude that siRNAs, which are produced from
amiRNA-mediated mRNA cleavage products, can affect other genes not targeted by the original amiRNA,
the PpFtsZ2-1 homolog PpFtsZ2-2, which shares high
identity in sequence stretches of the coding region
(Supplemental Fig. S3), seemed unaffected in PpFtsZ2-1amiRNA lines. We detected neither cleavage products
by 5# RACE-PCR, indicating siRNA-mediated cleavage of PpFtsZ2-2 transcripts, nor reduced PpFtsZ2-2
steady-state mRNA levels, pointing to a posttranscriptional silencing of this gene. Thus, even though
transitivity might be more common in Physcomitrella,
the specificity of posttranscriptional silencing is apparently sufficient to silence single members of highly
conserved gene families. Moreover, it might be preferable to design amiRNAs lacking perfect sequence
complementarity at the 3# end, as this reduces transitivity (Moissiard et al., 2007).
CONCLUSION
Compared to the conventional targeted gene knockout approach in Physcomitrella, the expression of
amiRNA provides several advantages. (1) The generation and molecular analysis of amiRNA overexpression lines is sped up as each regenerated transgenic
line harboring an amiRNA expression construct and
should produce the desired mature amiRNA. (2)
Instead of the generation of multigene knockout lines,
which is experimentally difficult, but feasible (Hohe
et al., 2004), amiRNAs are likely to be particularly
Plant Physiol. Vol. 148, 2008
useful for targeting groups of closely related genes
(Alvarez et al., 2006; Schwab et al., 2006). (3) AmiRNAs
can be expressed from inducible or tissue-specific
promoters (Schwab et al., 2006) enabling the analysis
of genes with essential functions that cannot be analyzed by targeted gene disruption. Provided that other
amiRNAs have a similar effect on the knock down of
their cognate target genes in Physcomitrella as observed
in this study, they can be considered as an efficient
alternative tool to the targeted gene knockout approach
for reverse genetics studies in Physcomitrella.
MATERIALS AND METHODS
Plant Material and Growth Conditions
Physcomitrella patens plants were cultured in modified liquid Knop medium containing 250 mg L21 KH2PO4, 250 mg L21 KCl, 250 mg L21
MgSO4·7H2O, 1,000 mg L21 Ca(NO3)2, and 12.5 mg L21 FeSO4·7H2O (pH 5.8)
or on solid Knop plates. Erlenmeyer flasks containing 400 mL of suspension
culture were agitated on a rotary shaker at 120 rpm at 25°C under a 16-h-light/
8-h-dark regime (Philips TLD 25; 50 mM m22 s21). Liquid cultures were
mechanically disrupted every week to maintain the plants in the protonema
stage. Gametophore development was induced by transferring protonema
tissue to solidified Knop medium.
Transformation of Physcomitrella Protoplasts
Polyethylene glycol-mediated transformation of Physcomitrella protoplasts
was performed according to standard procedures (Frank et al., 2005). Briefly,
transformation was carried out using 25 mg of linearized plasmid DNA.
Transformed protoplasts were cultivated for 24 h under standard conditions
in the dark and were then transferred to light. After 10 d, the protoplasts were
transferred to solid Knop medium. Three days later, regenerating plants were
transferred to Knop medium supplemented with hygromycin (Promega). The
selection lasted 2 weeks and was followed by a 2-week release period on Knop
medium without antibiotic followed by another round of selection and
release. Plants surviving the second round of selection were screened by
PCR to confirm integration of the DNA construct.
Generation of Physcomitrella Lines Expressing
AmiRNAs Targeting PpFtsZ2-1 and PpGNT1
AmiRNAs targeting PpFtsZ2-1 (accession no. AJ001586; amiRNA,
5#-TTCGTAATTAACGTGTCCGCG-3#) and PpGNT1 (accession no. AJ429143;
amiRNA, 5#-TTCCAAATAATCAGGACGCTT-3#) were designed using the
amiRNA designer interface WMD (Schwab et al., 2006; Ossowski et al., 2008).
The PpFtsZ2-1-amiRNA and PpGNT1-amiRNA sequences were introduced into
the Arabidopsis (Arabidopsis thaliana) miR319a precursor by overlapping PCR using the following primers. PpFtsZ2-1-amiRNA, miRNA-sense, 5#-GATTCGTAATTAACGTGTCCGCGTCTCTCTTTTGTATTCC-3#; miRNA-antisense, 5#-GACGCGGACACGTTAATTACGAATCAAAGAGAATCAATGA-3#; miRNA*sense, 5#-GACGAGGACACGTTATTTACGATTCACAGGTCGTGATATG-3#;
miRNA*-antisense, 5#-GAATCGTAAATAACGTGTCCTCGTCTACATATATATTCCT-3#; primer A, 5#-CCCGGGTGCAGCCCCAAACACACGCTC-3#; primer
B, 5#-GGATCCCCCCATGGCGATGCCTTAAAT-3#. PpGNT1-amiRNA, miRNAsense, 5#-GATTCCAAATAATCAGGACGCTTTCTCTCTTTTGTATTCC-3#;
miRNA-antisense, 5#-GAAAGCGTCCTGATTATTTGGAATCAAAGAGAATCAATGA-3#; miRNA*-sense, 5#-GAAAACGTCCTGATTTTTTGGATTCACAGGTCGTGATATG-3#; miRNA*-antisense, 5#-GAATCCAAAAAATCAGGACGTTTTCTACATATATATTCCT-3#; same primers A and B as described above. The
plasmid pRS300 harboring the Arabidopsis miR319a precursor was used as
PCR template (Schwab et al., 2006). The resulting precursor fragments were
cloned into the pJET1.2 cloning vector (Fermentas) and sequenced. The modified
ath-miRNA319a precursor DNA fragments were cloned into SmaI and BamHI
sites of the plant expression vector pPCV (Koncz et al., 1989) containing the
cauliflower mosaic virus 35S promoter, nos terminator, and hpt selection marker
cassette. Transgenic lines were analyzed by PCR to identify lines that had
691
Khraiwesh et al.
integrated the amiRNA overexpression constructs using the primers 5#-TGATATCTCCACTGACGAAAGGG-3# and 5#-GGATCCCCCCATGGCGATGCCTTAAAT-3#. PCR primers for the amplification of the Physcomitrella control gene
EF1a were 5#-AGCGTGGTATCACAATTGAC-3# and 5#-GATCGCTCGATCATGTTATC-3#. The one-step isolation of genomic DNA was performed according to the method of Schween et al. (2002).
Sequence data from this article can be found in the GenBank/EMBL data
libraries under accession numbers AJ001586 (PpFtsZ2-1), XM_001766723
(PpFtsZ2-2), and AJ429143 (PpGNT1).
Supplemental Data
The following materials are available in the online version of this article.
Small RNA Blots
Total RNA was isolated from protonema using TRIzol reagent (Invitrogen)
and separated in a 12% denaturing polyacrylamide gel containing 8.3 M urea
in Tris-borate/EDTA buffer. The RNA was electroblotted onto nylon membranes for 1 h at 400 mA. Radiolabeled probes were generated by end labeling
of DNA oligonucleotides with [g-32P]ATP using T4 polynucleotide kinase.
The following probes were used. Antisense probe for PpFtsZ2-1-amiRNA,
5#-CGCGGACACGTTAATTACGAA-3#; antisense probe for PpGNT1-amiRNA,
5#-AAGCGTCCTGATTATTTGGAA-3#; detection of sense transitive PpFtsZ2-1
siRNAs, 5#-CCCCAGTGACGGAAGCGTTCAATCTTGCAGACGACATCCTTCGGC-3#; detection of antisense transitive PpFtsZ2-1 siRNAs, 5#-GCCGAAGGATGTCGTCTGCAAGATTGAACGCTTCCGTCACTGGGG-3#; detection of
sense transitive PpGNT1 siRNAs, 5#-GTGAATTTCCTGCAGCATTTAGATGAAAATCCTCCCAAGACAAGG-3#; detection of antisense transitive
PpGNT1 siRNAs, 5#-CCTTGTCTTGGGAGGATTTTCATCTAAATGCTGCAGGAAATTCAC-3#; detection of the U6snRNA control, 5#-GGGGCCATGCTAATCTTCTCTG-3#. Blot hybridization was carried out in 0.05 M sodium
phosphate (pH 7.2), 1 mM EDTA, 63 SSC, 13 Denhardt’s, 5% SDS. Blots were
washed three times with 23 SSC, 0.2% SDS, and one time with 13 SSC, 0.1% SDS.
Blots were hybridized and washed at temperatures 5°C below the melting
temperature of the oligonucleotide.
Detection of mRNA Cleavage Products
Synthesis of 5# RACE-ready cDNAs was carried out according to Zhu et al.
(2001) using the BD Smart RACE cDNA amplification kit (CLONTECH).
Subsequent PCR reactions were performed using the UPM Primer-Mix
supplied with the kit in combination with gene-specific primers derived
from the target gene PpFtsZ2-1 (5#-GACTATCCCTGTGGCTCGCTCAATACCC-3#), a PpFtsZ2-1 nested primer (5#-CCAATAGAGGAGATTGGATTGCGCTCA-3#), a gene-specific primer derived from the gene PpFtsZ2-2
(5#-CCAATACGCGACTTGCATACTGCATAC-3#), and a gene-specific primer
derived from the gene PpGNT1 (5#-ACTTTGGAGCAAGTTCTTCCCAGGTGGA-3#). Amplification products corresponding to the size of the expected
cleavage products were excised from the gel, cloned and sequenced.
Total RNA Gel Blots
Twenty micrograms of total RNA were mixed with an equal volume of
RNA denaturing buffer and incubated for 10 min at 65°C. The RNA gel was
blotted to a Hybond-N+ nylon membrane (GE Healthcare) using a Turbo
blotter (Schleicher & Schuell) with 203 SSC. RNA was fixed by UV crosslinking. Hybridization was carried out with an [a-32P]dCTP-labeled DNA
probe derived from PpFtsZ2-1 amplified with primers 5#-AGACACGTCATTAAAGGT-3# and 5#-TAAGTGTGCAAGAAGATA-3#, a probe derived from
PpFtsZ2-2 amplified with primers 5#-AAGGTAGTACAAATGGGATGGC-3#
and 5#-TCATTAAGTCTGCCACTCCAC-3#, and a probe derived from
PpGNT1 amplified with primers 5#-GCACTCTCGATCGGATTCTC-3# and
5#-TCGGGAGAGATTTCCATGTC-3#. DNA labeling was carried out with the
Rediprime II random prime labeling kit (GE Healthcare). Prehybridization
was carried out at 67°C for 4 h, subsequent hybridization at 67°C overnight.
Blots were washed three times with 0.53 SSC, 0.1% SDS, and one time with
13 SSC, 0.1% SDS at 67°C. Signals were detected using the Molecular Imager
FX (Bio-Rad).
Microscopy
For microscopic analyses, we used the Axioplan 2 epifluorescence microscope equipped with an AttoArc HBO 100-W bulb and the stereomicroscope
Stemi 2000-C (Carl Zeiss). Image acquisition was achieved using the Canon
digital camera EOS D300 (Canon), and confocal laser-scanning microscopy
(TCS 4D; Leica).
692
Supplemental Figure S1. Analysis of Physcomitrella and Arabidopsis
miR319 precursors.
Supplemental Figure S2. Impeded plastid division and formation of
macrochloroplasts in PpFtsZ2-1-amiRNA overexpressors.
Supplemental Figure S3. Nucleotide sequence alignment of PpFtsZ2-1 and
PpFtsZ2-2 coding regions.
ACKNOWLEDGMENTS
We thank Andras Viczian for providing the pPCV expression vector, Enas
Qudeimat for assisting with confocal laser-scanning microscopy, and Björn
Voß and Isam Fattash for advice on miR319 precursor sequence analysis.
Received August 13, 2008; accepted August 22, 2008; published August
27, 2008.
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693
Supplemental Figure S1
A
5’
3’
ppt-MIR319a
5’
3’
ppt-MIR319b
5’
3’
ppt-MIR319c
5’
3’
ppt-MIR319d
5’
3’
ath-MIR319a
B
miRNA*
miRNA
Supplemental Figure S1. Analysis of Arabidopsis and Physcomitrella miRNA319 precursors. A, Secondary
structures of foldbacks of Physcomitrella patens miR319a-d precursors (ppt-MIR319a, b, c, d) and Arabidopsis
thaliana miR319a precursor (ath-MIR319a). The mature miRNA is highlighted in green with uppercase letters.B,
Multiple sequence alignment of Physcomitrella patens miR319a-d precursors (ppt-MIR319a, b, c, d) and the
Arabidopsis thaliana miR319a precursor (ath-MIR319a). Black boxes with underlined sequences indicate
miRNA/miRNA* sequences.
Supplemental Figure S2
A
PpFtsZ2-1-amiRNA lines
WT
1
3
2
Protonema
-
-
-
100µm
-
Leaves
-
-
-
-
B
Protonema
-
--
Leaves
-
--
50µm
Supplemental Figure S2. Impeded plastid divison and formation of macrochloroplasts in PpFtsZ2-1-amiRNA overexpressors.
A, Light microscopy from protonema and leaves of wild type (WT) and three PpFtsZ2-1-amiRNA overexpression lines (1-3)
(Size bars: 100 µm). B, Confocal laser scanning microscopy from protonema and leaves of wild type (WT) and three PpFtsZ2-1amiRNA overexpression lines (1-3) (Size bars: 50 µm). Red: chlorophyll autofluorescence in plastids.
Supplemental Figure S3
FtsZ2-1
FtsZ2-2
FtsZ2-1
FtsZ2-2
FtsZ2-1
FtsZ2-2
FtsZ2-1
FtsZ2-2
FtsZ2-1
FtsZ2-2
FtsZ2-1
FtsZ2-2
FtsZ2-1
FtsZ2-2
FtsZ2-1
FtsZ2-2
FtsZ2-1
FtsZ2FtsZ2-1
FtsZ2-2
FtsZ2-1
FtsZ2-2
FtsZ2-1
FtsZ2-2
FtsZ2-1
FtsZ2-2
FtsZ2-1
FtsZ2-2
FtsZ2-1
FtsZ2-2
1 ATGGCGTTGTTTAGTGGGTGCTCGGGATGGGCGGGGCTCAAGGTGTCATC
|||||| |||| | | ||||||| | || ||| |||| ||||
||
1 ATGGCGCTGTTAGGCAGTCGCTCGGGCTTGGTGGGCCTCAGGGTGAGCTC
50
50
51 GCGAGTGGGTGGGGAGGCTTGCAGAA-----CCCCCCCCGTT-GTTCACT 94
||||||||| ||||||
| ||||
||| | ||
| || ||
51 GCGAGTGGGCGGGGAGAGCAGTAGAATAGTGCCCGCGACGAGAGATCGCT 100
95 GCAGCATGCATTCTAGGTCAAGCGTTCGAGCTCTACGCCGAATCGACCGA 144
| || |||| | ||| | ||| ||| || | || || | || |
101 TCTGCGTGCACTTGAGGCCGAGCACTCGGGCGCATCGTCGTCTGGATAGG 150
145 GCTTTGAGTAATGGGGGTCTTTGCAATTTTGGAGAGAGGGACTTGTTGGC 194
|| | | |||| | ||||||||| |
| ||||||||||
||||
151 ACTGTAGGGAATGAGAGTCTTTGCACTCCCCGGGAGAGGGACT---TGGC 197
195 TTTGGAAGCAAAATC---GCCTTTGCGATGTGAACCCCCCTCGA------ 235
| ||| | ||||
||
| |||| || | | ||
198 TGCGGAGCCTAAATTCTTGCACACGGGATGGGAGTCTTCTTCTTCTTCTT 247
236 ------------GCGTGATGCGGAATCCTGTCATGGCATTTGAAGGAAGC 273
||| ||
||| || |||| |||||||| ||| |
248 CTTCTTCTTCTTGCGAGACTGGGATACCCGTCACGGCATTTGGAGGTAAT 297
274 GGAGACGACACTGGAAGTTATAACGAAGCGAAAATTAAAGTAATAGGGGT 323
||||||||
|| |||| || || |||||||||||||| ||||| ||
298 GGAGACGAATATGAGAGTTCCAATGAGGCGAAAATTAAAGTGATAGGCGT 347
324 CGGAGGTGGGGGTTCCAACGCCGTAAACCGAATGCTTGAGAGCGAGATGC 373
|| || ||||||||||||||||| |||||||||||||||||||| ||||
348 GGGGGGCGGGGGTTCCAACGCCGTCAACCGAATGCTTGAGAGCGAAATGC 397
374 AAGGGGTAGAATTTTGGATCGTGAATACTGATGCGCAGGCTATGGCCTTG 423
|||| || ||||| ||||| |||||||| ||||| ||||| ||||| |||
398 AAGGTGTGGAATTCTGGATTGTGAATACGGATGCTCAGGCAATGGCGTTG 447
424 TCCCCTGTTCCGGCTCAGAATCGTCTGCAGATTGGGCAAAAATTGACGAG 473
|| || ||||||||||||||||||||||||||||| || ||||||||| |
448 TCTCCGGTTCCGGCTCAGAATCGTCTGCAGATTGGTCAGAAATTGACGCG 497
474 AGGTCTGGGGGCGGGCGGGAATCCAGAAATAGGGTGTAGTGCTGCGGAAG 523
||||||||||||||| || ||||| ||||||||||||||||| |||||||
498 AGGTCTGGGGGCGGGTGGTAATCCGGAAATAGGGTGTAGTGCCGCGGAAG 547
524 AGAGCAAAGCTATGGTGGAAGAAGCCCTACGCGGAGCTGACATGGTTTTC 573
|||||||||||||||||||||||||| |||||||||||||||||||||||
548 AGAGCAAAGCTATGGTGGAAGAAGCCTTACGCGGAGCTGACATGGTTTTC 597
574 GTAACGGCGGGTATGGGTGGCGGCACTGGCAGCGGTGCAGCACCAATAAT 623
|| || ||||| |||||||| ||||||||||||||||| |||||||| ||
598 GTTACAGCGGGCATGGGTGGTGGCACTGGCAGCGGTGCTGCACCAATCAT 647
624 TGCGGGTGTGGCGAAGCAGTTGGGAATTCTTACTGTAGGAATAGTTACTA 673
||| ||||| |||||||| |||||||||||||| || |||||||| ||||
648 TGCTGGTGTAGCGAAGCAATTGGGAATTCTTACCGTGGGAATAGTAACTA 697
674 CTCCTTTCGCCTTTGAAGGGCGGAGACGAGCTGTCCAAGCCCACGAGGGT 723
| ||||| ||||||||||||||||||||| | || ||||| ||||| ||
698 CGCCTTTTGCCTTTGAAGGGCGGAGACGATCCGTTCAAGCTCACGAAGGC 747
FtsZ2-1
FtsZ2-2
FtsZ2-1
FtsZ2-2
FtsZ2-1
FtsZ2-2
FtsZ2-1
FtsZ2-2
FtsZ2-1
FtsZ2-2
FtsZ2-1
FtsZ2-2
FtsZ2-1
FtsZ2-2
FtsZ2-1
FtsZ2-2
FtsZ2-1
FtsZ2-2
FtsZ2-1
FtsZ2-2
FtsZ2-1
FtsZ2-2
FtsZ2-1
FtsZ2-2
FtsZ2-1
FtsZ2-2
FtsZ2-1
FtsZ2-2
724 ATTGCAGCTCTCAAAAATAACGTGGACACGTTAATTACGATTCCAAACAA
|| || |||||||||||||| || ||||| ||||||||||| ||||||||
748 ATCGCGGCTCTCAAAAATAATGTTGACACTTTAATTACGATACCAAACAA
773
824 CAAACTTTTGACTGCAGTTGCGCAGTCTACCCCAGTGACGGAAGCGTTCA
||| ||||||||||||||||||||||||||||| ||||||||||| ||||
798 CAAGCTTTTGACTGCAGTTGCGCAGTCTACCCCCGTGACGGAAGCATTCA
823
824 ATCTTGCAGACGACATCCTTCGGCAGGGAGTGCGGGGTATTTCAGATATT
||||||| || |||||||||||||||||||||||||||||||||||||||
848 ATCTTGCCGATGACATCCTTCGGCAGGGAGTGCGGGGTATTTCAGATATT
873
874 ATCACGGTCCCTGGGCTGGTTAACGTAGATTTTGCCGACGTGCGGGCGAT
||||| || ||||| || |||||||| || ||||| || |||||||||||
898 ATCACTGTTCCTGGTCTCGTTAACGTGGACTTTGCGGATGTGCGGGCGAT
923
924 CATGGCTAATGCAGGATCATCTTTGATGGGCATAGGGACCGCCACAGGTA
|||||| ||||||||||||||||||||||| || || ||||| ||||| |
948 CATGGCCAATGCAGGATCATCTTTGATGGGAATTGGAACCGCTACAGGGA
973
797
847
897
947
997
974 AGTCAAGAGCTAGAGAAGCAGCATTGAGCGCAATCCAATCTCCTCTATTG 1023
|||||| ||||||||| ||||||||||| || || || ||||| | |||
998 AGTCAAAAGCTAGAGAGGCAGCATTGAGTGCCATTCAGTCTCCATTGTTG 1047
1024 GATGTGGGTATTGAGCGAGCCACAGGGATAGTCTGGAATATCACTGGGGG 1073
||||||||||||||||||||||||||||| || |||||||| ||||||||
1048 GATGTGGGTATTGAGCGAGCCACAGGGATCGTTTGGAATATTACTGGGGG 1097
1074 AAGCGACATGACTCTCTTTGAGGTAAATGCTGCAGCAGAGGTGATTTATG 1123
|||||||||||| |||||||| || ||||||||||||||||| || ||||
1098 AAGCGACATGACCCTCTTTGAAGTCAATGCTGCAGCAGAGGTAATCTATG 1147
1124 ATTTGGTCGATCCCAACGCAAATCTTATTTTTGGAGCCGTAGTAGACGAA 1173
||||||| ||||| ||||||||||||||||| ||||||||||||||||||
1148 ATTTGGTGGATCCTAACGCAAATCTTATTTTCGGAGCCGTAGTAGACGAA 1197
1174 GCACTTCATGGCCAAGTTAGTATAACTTTGATAGCAACAGGATTTAGTTC 1223
|||||||||| |||| |||| ||||| || ||||||||||| ||||||||
1198 GCACTTCATGACCAAATTAGCATAACCTTAATAGCAACAGGGTTTAGTTC 1247
1224 TCAAGATGAACCTGATGCGCGTAGTATGCAAAATGTGAGTCGTATTTTGG 1273
||||||||| |||||||| || |||||||| ||| ||||| | ||||
1248 TCAAGATGATCCTGATGCACGGAGTATGCAGTATGCAAGTCGCGTATTGG 1297
1274 ATGGACAAGCTGGTCGATCACCGACAGGTTTATCTCAAGGCAGCAATGGC 1323
| || ||||||||||||||| ||| | | ||| | ||| ||||| ||
1298 AGGGTCAAGCTGGTCGATCATCGATGGCCTCATCCCGAGGTGGCAATAGC 1347
1324 TCTGCGATCAATATACCAAGTTTCTTAAGGAAGCGAGGCCAGACACGTCA 1373
||| |||| || ||||||| ||||||| | |||||||| || |
1348 TCTACGATTAACATACCAAATTTCTTACGAAAGCGAGGGCAAAGG----- 1392
1374 TTAA 1377
||
1393 –TAG 1395
Supplemental Figure S3. Nucleotide sequence alignment of PpFtsZ2-1 and PpFtsZ2-2 coding regions. The highly
conserved central region with 89% sequence identity is highlighted in yellow. The PpFtsZ2-1-amiRNA target site is
highlighted in green.
Chapter IV
Appendices
4 Chapter IV: Appendices
4.1
Flow cytometric measurements (FCM)
For ploidy level determination 10-20 mL of protonema liquid culture were used. The plant
material was harvested four to seven days after the last sub-culturing, resuspended with 2
mL of DAPI buffer, and chopped up with a razor blade in a Petri dish. The solution was
filtered through a sieve of 30 μm pore size prior to measuring the fluorescence intensity with
a PAS cell analyser (Partec, Münster) using a 100 W high-pressure mercury lamp for
detection. The ploidy level was derived from the resulting histograms. For Physcomitrella, a
prominent peak at a fluorescence intensity of about 200 indicates a haploid genotype,
whereas signals at fluorescence intensities of about 400 represent a diploid plant.
Figure 1: Flow cytometric analysis. Flow cytometric histograms of protonema from WT and
ΔPpDCL1b mutants (1-4) grown in parallel in Knop medium. The abscissa represents the channel
numbers corresponding to the relative fluorescence intensities of analyzed particles (linear mode),
while the ordinate indicates the number of events counted.
136
Chapter IV
4.2
Appendices
Physcomitrella patens DCL1b (PpDCL1b) mRNA
LOCUS
DEFINITION
ACCESSION
VERSION
KEYWORDS
SOURCE
ORGANISM
DQ675601
6052 bp
mRNA
linear
PLN 03-JUL-2008
Physcomitrella patens Dicer-like 1b protein (DCL1b) mRNA,
complete cds.
DQ675601
DQ675601.1 GI: 110520366
Physcomitrella patens
Physcomitrella patens
Eukaryota; Viridiplantae; Streptophyta; Embryophyta;
Bryophyta; Moss Superclass V; Bryopsida; Funariidae;
Funariales; Funariaceae; Physcomitrella
REFERENCE
1 (bases 1 to 6052)
AUTHORS
Khraiwesh, B., Seumel, G.I., Reski, R. and Frank, W.
TITLE
Direct genetic evidence for the involvement of a Dicer-like
gene in microRNA mediated target cleavage in plants.
JOURNAL
Unpublished
REFERENCE
2 (bases 1 to 6052)
AUTHORS
Khraiwesh, B., Seumel, G.I., Reski,R. and Frank,W.
TITLE
Direct Submission
JOURNAL
Submitted (06-JUN-2006) Plant Biotechnology, University of
Freiburg, Schaenzlestrasse 1, Freiburg 79104, Germany
FEATURES
Location/Qualifiers
Source
1..6052
/organism="Physcomitrella patens"
/mol_type="mRNA"
/db_xref="taxon: 3218"
Gene
1..6052
/gene="DCL1b"
CDS
607..5694
/gene="DCL1b"
/codon_start=1
/product="Dicer-like 1b protein"
/protein_id="ABG74922.1"
/db_xref="GI:110520367"
/translation="MRRARSVRLENGLNGKDEGEEKARTYQLEVLAQAKV
KITVAFLDTGAGKTLIAILLMKHKHQVLREYDKRMLALFLVPKVPLVYQQADVIRNGTKFSVGHYCGEMGSRFWD
ARGWQREFDTKDVFVMTAQILLNILRHSIVKMEAIHLLILDECHHAVKKHPYSLVMSEFYLMTPKDKRPCVFGMI
ASPVNLKGVSNQEDCAIEIRNLESKLDSIVCTIRDRKELEKHVPLPSETMILYDKPALLFSLRKERKQMEATVEK
AANASVRRSKWKCMGARDAGAKEELQLVYSVAERTESDGAASLSQKLRAITYALDELGQWCAYKVSLGYLTSLHN
DERVNHQLDVKFQKLYLKKVCTLLRCSLREGAAGWEVPAEIGESEGDKAQDPMDVEEGSFLTLVSVGEHLDEILG
AAVADGKVTPKVQSLIKVLIGYQHTDDFRAIIFVERVWVGVTLCRSLQSCPSLKFVKCASLIGHNNNQDMPTRQM
QETISKFRDGRVTLLVATSVAAEGLDIRQCNVVIRFDLAKTVLAYIQSRGRARKPGSDYILMLERGNLQHEAFFR
NAKNSEETLRKEAIERTDLGEKRENAILASIDIGEGEIYQVPATGAVVSMNSAVGLIHFYCSQLPSDRYSLLRPE
FIMNKIEDQRGAIRYSCRLQLPCHAPFEAVEGPECNSMRGAQQSCVLEGLQKMHEMGAFTDMLLSNKGSREEAAK
LEGSEEGESLPGTSRHREYYPEGIADILKGDRIVAEKDSDTKEGSKVLVFMYTVKCENVGFSRDSLLTETSDFTL
LVGQQLHDQVLTMTINLFVANPTLLITMSWKKRDLDCSNSQLTELKSFHVRLMSIVLDVNVEPATTRWDPAKAYL
FAPVLHKDASDPKDLVDWVVMRRTIETDSWSNPLQRASPDVNLGTDERALGGDRREYGFGKLRCSLAFGQGAHPT
YGARGAKAQFDVVKATGLLPTSDMVEETTVQEVPPEGKLLIVDGFVEVEVLVGRIVTAVHSGKRLYVDSVRFDMT
ADSSFPQKDGYLGPLEYTSYADYYKQKYGVELVCKKQPLLRGRGVSHCKNLLSPRFETSGDSLDALDKTYYVMLP
PELCLIHPLPGSLVRGAQRLPSVMRRVESMLLAIQLKHQIDYPIAASKVALTAASGQETSYERAELLSDAYLEWV
VSHRLFLKFPSKHEGQLTRMRQKIVSNSVLYQHALEKGLQSYIQADRFAPSRWAAPGVPPAFDEDLRDGDDSDKE
SKPEVEREVVEIVGEEGEIVKELNTESENMEDGEIEGDSGSYRVLSSKTLADVVEVFMGMYYVEGGGEAATHFMN
WVGIPVEDDVETDLATGGCQVPETVMRSIDFSSLQKNVGHEFRERSLLVEAITHASRPSLGVPCYQRLEFVGDAV
LDHLITRYLFFKYTNLPPGRLTDLRAAAVNNENFARVAVKHSYHLHLRHGSTALETQIRNFVNDIHSELDKPGVN
SFGLGDFKAPKVLGDIFESIAGALFLDARLDTHQVWKVFEPLLQPMVSPETLPIHPVRGLQERCQQEAEGLEYKV
SRAESVATVEVYVDGVQIGSTQSAQKKMAQKLGARNALVKLKDKEVIKVKAEAENGDLNAGKSSKNGHTNFTRKT
INDLCLKRQWPMPQYKCVLESGPAHAKKFTLSVRVLTTTDGWTEECVGEPMASVKKAKDSAALVLLATLRRSYPL
RNNIIDC"
U
U
U
U
137
Chapter IV
Appendices
ORIGIN
1
61
121
181
241
301
361
421
481
541
601
661
721
781
841
901
961
1021
1081
1141
1201
1261
1321
1381
1441
1501
1561
1621
1681
1741
1801
1861
1921
1981
2041
2101
2161
2221
2281
2341
2401
2461
2521
2581
2641
2701
2761
2821
2881
2941
3001
3061
3121
3181
3241
3301
3361
3421
3481
aacgcgggga
tgaattgtca
gtttgatgga
gaggctcgtg
atgaagggta
tctgtgtcga
gcagggaggg
agaagagggg
tgacgcgcga
ctcgggagcg
tttgaaatga
ggtgaggaga
acggttgcat
cataagcacc
aaagtaccgc
ggtcactact
tttgatacca
agcattgtaa
aagaaacatc
cgaccgtgtg
gaagattgtg
atcagggatc
tacgataagc
gtagaaaagg
gatgcgggtg
gatggcgcag
ggtcaatggt
agggttaatc
cttctgcgat
gagtctgagg
ctcgtatcag
gtgactccga
ttccgagcta
cagagttgcc
caagacatgc
acgttgctgg
gtcatccgtt
cggaagcctg
ttttttcgga
gatctgggtg
atttaccagg
cacttctact
atgaacaaaa
tgccatgctc
cagtcctgtg
ctattatcta
gagtctcttc
ctgaagggcg
ctcgtattca
accgagacat
atgacaataa
agggatttgg
atgagcattg
gcgtatctct
tgggtcgtta
tcacctgatg
gggtttggaa
gctcgtggcg
gacatggtgg
ggtggagaag
agtaaggaac
gcaggtgggg
atcgagggaa
gaggtggtgg
ggtctaggtc
gaggaagcga
gcggcatttc
cagaccttgg
atttgttgcg
gaagggcgcg
aggcgcggac
ttctagacac
aggtgttgcg
tcgtctacca
gcggagagat
aagatgtttt
aaatggaagc
cctattcttt
tctttgggat
caatagagat
ggaaagagct
cggccttgct
ctgctaatgc
ctaaagagga
ctagtctttc
gtgcttacaa
atcagttaga
gcagtctacg
gcgataaagc
tgggtgaaca
aggtgcagtc
ttatatttgt
cttcattgaa
cgacacggca
tggctacaag
ttgatcttgc
gttcagatta
atgcaaaaaa
aaaaacggga
tgccagccac
gctctcagct
ttgaggatca
cgtttgaagc
tgcttgaagg
ataaaggaag
ctggcacatc
atcggatagt
tgtacacggt
cagactttac
atctttttgt
attgctctaa
ttttagacgt
ttgctccagt
tgagaaggac
tgaacttggg
aactgcgatg
ctaaagctca
aggagacaac
tggctctttt
gattcaatta
agcggagggg
gaggagccgg
tggtgtgaga
agagagaggc
aggtcggctt
catgaattgg
attggcagag
tcgtctcgtg
aagcgtgaga
ttatcagctt
gggcgctggg
ggagtatgac
gcaagcagat
gggatcaaga
tgtaatgacc
cattcatcta
ggtgatgtct
gatagcatcg
tcgaaattta
cgaaaagcac
tttctcgttg
aagtgtcaga
actgcaactt
tcaaaagctt
ggtctcgctg
cgtgaagttt
tgaaggtgct
acaagatcca
tttggatgag
tttaattaag
ggagcgagtc
gtttgtgaaa
gatgcaggag
cgtggccgca
taaaaccgtg
tattttaatg
tagtgaggag
gaatgcgatt
tggggcagtc
tcccagtgac
aagaggtgct
tgtggaaggc
cttgcaaaaa
tagggaagaa
ccgtcatcga
ggctgagaaa
gaagtgtgaa
cttacttgtc
cgcaaacccg
ttcccagttg
aaatgtcgag
tctgcataag
gatcgagact
gactgacgaa
tagtctggcc
atttgatgtt
tgtgcaggaa
tctagcacta
gagcgccgcg
tgaaatgcgc
gagagcgacg
ggattcgtca
ctaaagagag
tcgaatatgc
gagattatcg
gggattgcga
atcgggagag
ctggagaatg
gaagtgctgg
aagaccctaa
aagcgtatgc
gtgattcgca
ttttgggacg
gcacagattc
cttattctcg
gaattctatc
cctgtgaacc
gaaagcaagt
gtgcctttgc
cggaaagaga
cgcagcaaat
gtgtacagtg
agagccatta
ggatatctga
caaaagttgt
gcagggtggg
atggatgtgg
attcttgggg
gttttaatag
tgggtgggtg
tgtgccagtc
actatttcca
gagggattag
ttagcctaca
cttgagagag
actttacgga
ctggcctcca
gtgagcatga
aggtattctc
ataagatact
ccagaatgta
atgcacgaaa
gctgctaagt
gaatattatc
gattcggata
aatgttggct
ggccaacagc
actttactga
actgagctca
ccggcaacaa
gatgcctccg
gattcatgga
cgtgctcttg
tttgggcagg
gtgaaagcca
gtacctcccg
tccctctcga
aattgattga
aagcaagggg
ggagagttgg
ggaggagcag
ggcggagatg
ctttgatgat
agattaccac
ccggcgatca
agagtgggag
ggctgaatgg
ctcaggcgaa
ttgcgattct
tcgctctgtt
acggcacaaa
cccgagggtg
ttttgaacat
atgagtgcca
ttatgacacc
tcaaaggggt
tggactcgat
cgtcagagac
gaaaacagat
ggaaatgcat
tcgcggagag
cctatgcact
caagtcttca
acttgaagaa
aggtacctgc
aagaaggaag
ccgctgtagc
gttatcagca
ttacgctttg
tgatagggca
agtttcgaga
atattcgcca
tccagtctcg
gaaatctgca
aggaggctat
ttgacattgg
actcagctgt
tcttgcgtcc
cgtgcagact
attctatgcg
tgggggcatt
tggagggtag
cagaggggat
caaaggaagg
tctcgagaga
ttcatgacca
tcacgatgag
agagttttca
ctcgttggga
atcctaaaga
gtaatcccct
gtggggatcg
gagcgcatcc
caggtctact
agggtaagct
ggagcggagg
attacgagtg
tgtacatgac
cgaggctgga
ggagaggcgg
acgggagaca
aggcgcgatg
ggttccatag
aggatggagg
cggtcgcgtg
gaaggatgag
ggtgaagatt
gttgatgaag
cctcgtccct
gtttagtgtt
gcagcgagaa
ccttaggcat
ccatgccgtg
taaagataag
atcaaaccag
agtgtgtaca
aatgattctg
ggaggccact
gggcgctcgg
aacggaaagc
tgatgaatta
taatgatgaa
ggtttgtact
tgaaattgga
cttcctgact
agatggaaaa
tacggatgat
caggtctttg
caacaataac
tggacgggtg
atgtaatgtg
tggtcgtgct
acatgaggcg
tgaaagaact
ggaaggggag
aggtcttatt
tgagttcatt
gcagttgcct
aggagcgcag
cacggacatg
tgaagaggga
tgcagatatt
cagcaaggtg
cagccttttg
ggtgttaacc
ctggaaaaag
tgtgaggctt
tcccgccaag
cttggtggac
ccagcgcgca
tagagagtac
aacgtatggt
tcctacctca
gttgatagtg
138
Chapter IV
3541
3601
3661
3721
3781
3841
3901
3961
4021
4081
4141
4201
4261
4321
4381
4441
4501
4561
4621
4681
4741
4801
4861
4921
4981
5041
5101
5161
5221
5281
5341
5401
5461
5521
5581
5641
5701
5761
5821
5881
5941
6001
gatggttttg
aagaggcttt
aaggatggat
tacggtgtcg
tgcaaaaatt
aagacgtatt
ttggtgagag
gccatacaac
gctgcgtctg
ctcgaatggg
cttacacgta
aaaggtcttc
ggagtgcctc
aaacctgaag
gaactaaata
tatcgagtgc
tatgtggagg
gagtttgatg
atgcggagca
agtttattgg
caaaggctgg
tttaaatata
aacgaaaatt
tcaaccgctt
aagcctggag
attttcgaat
tggaaggttt
ccagtacgag
tctcgtgcag
acgcaaagtg
ttgaaggata
ggaaaatcga
cttaagagac
gctaagaagt
tgtgttgggg
ttggctactt
ccaaattgat
atatccgtgc
tagccagcgc
catgatatgt
gttcaaagaa
tgtctttcat
Appendices
ttgaagttga
atgtggattc
accttggtcc
agttggtttg
tattgtcgcc
atgtgatgct
gcgcacaaag
taaagcacca
gtcaagagac
ttgttagtca
tgagacagaa
agagttacat
ctgcattcga
ttgaaagaga
cagaaagtga
tttcgagtaa
ggggggggga
acgtggagac
tagacttttc
tagaggccat
agtttgtggg
ctaatttgcc
tcgcacgtgt
tagaaactca
tgaactcttt
ccattgcagg
ttgagccttt
ggttgcagga
agagtgttgc
ctcagaagaa
aggaggtgat
gcaagaacgg
agtggccgat
ttacgctctc
agcctatggc
tgagacgatc
cagaaaacat
cccgtcaaca
cagttcgttc
tttgtctttt
agtagtgagt
ttttcagttt
agtattggtg
ggtgcgcttt
actggaatac
caagaaacag
acgttttgag
gccacctgag
attgccatcg
aatcgattac
attcagctat
tcgattgttc
aattgtcagc
tcaggccgac
tgaggacttg
agtagtggag
aaatatggaa
aaccttggca
ggctgctact
agacttagcc
atcattacaa
cacgcacgcg
ggatgccgtg
cccaggtagg
tgctgtgaag
gattcgcaat
tggactaggg
cgctctattc
gttgcagccc
gcgttgtcaa
gaccgtggag
aatggcccaa
caaagtgaaa
tcacactaac
gccacagtac
tgtacgggtt
gagtgtgaag
atatcctttg
acagaactac
taaatttgtg
aaggagctgc
tggatcaaat
ttttgtaccg
ttcagagaaa
ggaaggattg
gacatgacag
acatcgtatg
cctctgttga
acctctggcg
ctttgcctta
gtcatgagac
cctattgctg
gagcgtgcag
ctgaagttcc
aattccgttc
cgctttgcac
agagatggcg
attgtcggtg
gacggtgaaa
gacgtggtag
cacttcatga
acaggtggct
aaaaacgttg
tctcgaccat
ttggaccatc
ttgaccgatt
cactcgtatc
ttcgtgaatg
gattttaagg
ctggacgctc
atggtgtccc
caagaagctg
gtgtatgtag
aaattaggtg
gctgaggcag
ttcactcgca
aaatgcgttc
ctgaccacca
aaagctaagg
cgtaataata
ataccggcct
aatgcacaaa
aggccagctc
tgtgagagac
taagacagct
aaaaaaaaaa
ttactgcggt
ccgacagctc
cggattatta
ggggtcgtgg
actctctgga
tacatcctct
gtgtagagag
cttcgaaggt
agcttttaag
ctagtaaaca
tgtatcaaca
cgtcccggtg
atgattcgga
aggaaggtga
ttgaaggtga
aggtattcat
actgggtagg
gccaagttcc
gccatgaatt
cgttgggagt
tgattacacg
tgcgagctgc
atcttcattt
atatacactc
cccctaaagt
gtcttgacac
cagagacatt
aaggtctgga
acggtgtaca
ctcgtaatgc
agaatggtga
aaacaattaa
tggagagcgg
ctgatggatg
actctgcagc
ttatagactg
gtgggtgctt
tcacaaggtt
agccctcgtt
agagcacagg
gcgtctctcc
aaaaaaaaaa
gcattctggg
ttttcctcaa
caaacaaaag
ggtttctcat
tgccttggat
tccgggatcc
catgttgctt
agcgttgacg
cgacgcgtac
tgaggggcag
tgccctagag
ggccgcaccg
taaggagtcg
aattgttaag
ttccggttcc
gggaatgtat
cattcctgtg
tgaaaccgtt
tcgtgaacga
tccttgctac
ttatctattc
tgcagtgaat
gcggcatggt
ggagttagac
gctgggtgat
acaccaagtg
gccgatccat
gtacaaagtg
gataggttct
gttggtcaaa
cttgaacgct
cgacctttgt
accagcgcat
gaccgaagaa
tcttgtactt
ctaaaatgac
caggttcaac
tggatagcac
ttataccttt
tcagtatacc
ctcttaattt
aa
139
Chapter IV
4.3
Appendices
DNA vectors
Origin of DNA vectors that were used for cloning and transformations
Name
pCR4-TOPO
Backbone
pCR4-TOPO
Insert
PpDCL1b cDNA region; nptII
Reference
Invitrogen,
Karlsruhe
PpFtsZ2-1-amiRNA and PpGNT1pPCV
(Figure 2)
----
amiRNA sequences with A.
Koncz, C. et al.,
thaliana miRNA319a Precursor
1989
Figure 2: pPCV plant overexpression vector containing a double 35S promoter,
nos terminator and hpt selection marker cassette.
140
Chapter IV
4.4
Appendices
Genes downregulated in ΔPpDCL1b mutants
Cosmoss Annotation
Fold change
-1.46
-1.49
-1.59
-1.82
-1.59
-1.80
-3.15
-2.21
-1.60
-1.66
-1.43
-1.34
-1.50
PP_10382_C1
PP_10385_C1
PP_10479_C1
PP_10658_C1
PP_1073_C1
PP_10745_C1
PP_10817_C1
PP_11302_C1
PP_11331_C1
PP_11112_C1
PP_11390_C1
PP_1146_C3
PP_11513_C1
PP_11795_C1
PP020018263R
**: Homolog of OSJNBa0003O19.20|putative MYC transcription factor
***: Q7XN04 OSJNBb0038F03.7 protein.
**: Homolog of Similar to phytochrome and flowering time 1 protein
***: Q8RYB8 Aldehyde dehydrogenase Aldh21A1.
**: Homolog of Roc1-related|o_sativa|chr_8|P0020B10|4196
**: Homolog of (AB032182) homeobox protein PpHB10 [Physcomitrella patens]
**: Homolog of aldehyde dehydrogenase, putative|o_sativa|chr_11|OSJNBa0052O08|5272
**: Homolog of GRAS family transcription factor, putative|o_sativa|chr_1|P0406G08|2927
**: Homolog of Helicase conserved C-terminal domain,
***: Q84WK0 At4g33880.
**: Homolog of (68417.m02544 vernalization 2 protein VRN2)
***: Q8SA80 Disease-resistent-related protein.
**: Homolog of 68416.m05958 protein kinase family protein contains eukaryotic protein
kinase domain
***: PSAG_ARATH Photosystem I reaction center subunit V, chloroplast precursor
***: Q6Z5T6 Putative intensifier.
***: Q8S1X3 Putative SUVH4.
**: Homolog of AP2 domain, putative|o_sativa|chr_6|P0021C04|3631
**: Homolog of AT3g02790/F13E7_27|o_sativa|chr_6|P0621D05|1946
**: Homolog of (AB084898) mitochondrial aldehyde dehydrogenase [Sorghum bicolor]
not annotated Physcomitrella patens
***: Q8X1E7 Histidine kinase.
***: Q9M551 Polyubiquitin.
***: Q948P1 Peroxisomal ascorbate peroxidase.
***: Q41067 Polyubiquitin.
***: Q9LV44 Similarity to signal peptidase.
***: Q9M077 Putative serine/threonine protein kinase.
***: Q93V58 Putative serine threonine-protein kinase.
**: Homolog of 68418.m05603 YEATS family protein contains Pfam domain
PP_12005_C1
PP_12101_C1
PP_12167_C1
PP_12301_C1
PP_12301_C1
PP_12365_C2
PP_12367_C1
PP_125_C1
PP_12576_C1
PP_12599_C1
PP_12681_C1
PP_12858_C1
PP_12940_C1
PP_13101_C1
PP_13149_C1
PP_13160_C1
PP_13170_C1
PP_13256_C1
PP_13508_C1
**: Homolog of Neutral/alkaline nonlysosomal ceramidase|o_sativa|chr_1|P0501G01|2708
***: Q9ARE4 ZF-HD homeobox protein.
**: Homolog of (AY034888) aldehyde dehydrogenase Aldh21A1 [Tortula ruralis]
contains: (COIL:coil)
contains: (COIL:coil)
***: Q9FH40 Similarity to unknown protein (TAF14b) (Hypothetical protein At5g45600).
***: Q8W314 Putative dehydratase/deaminase.
**: Homolog of (68415.m03211 plectin-related contains
**: Homolog of SNF2 family N-terminal domain
**: Homolog of expressed protein|o_sativa|chr_4|OSJNBa0013K16|5362
***: Q7XPK1 OSJNBa0087O24.9 protein.
**: Homolog of 68417.m05252 expressed protein contains Pfam profile PF04784
***: Q7Y1Z3 Putative small nuclear ribonucleoprotein Prp4p.
***: Q43303 Histone H3 (Fragment).
***: Q9ZPN6 Transcription factor MYC7E (Fragment).
***: RL71_ARATH 60S ribosomal protein L7-1.
***: Q9SII9 Putative ubiquitin protein.
**: Homolog of expressed protein|o_sativa|chr_2|P0506A08|3865
contains: Protein kinase-like(InterPro:IPR011009,SUPERFAMILY:SSF56112)
Sequence ID
EST
BJ165956
BJ172212
BJ200093
BJ200754
BJ579811
BJ580674
BJ583348
BJ583460
BJ587267
BJ589432
BJ601044
PP_10059_C1
PP_10115_C2
-3.18
-1.58
-1.90
-1.50
-1.70
-1.45
-1.39
-2.11
-1.86
-2.22
-1.66
-1.60
-1.87
-1.35
-1.50
-1.58
-1.65
-4.19
-2.40
-2.06
-1.35
-1.71
-1.25
-1.70
-2.34
-2.51
-1.74
-1.42
-1.43
-1.79
-1.38
-1.42
-1.85
-1.58
141
Chapter IV
PP_13734_C1
PP_13750_C1
PP_13846_C1
PP_1387_C1
PP_14366_C1
PP_14581_C1
PP_14609_C1
PP_14674_C1
PP_1468_C1
PP_15181_C1
PP_1665_C2
PP_17120_C1
PP_1761_C1
PP_18023_C1
PP_18168_C1
PP_11285_C4
PP_18237_C1
PP_18520_C1
PP_2007_C1
PP_2015_C1
PP_2094_C3
PP_2103_C2
PP_2104_C1
PP_214_C9
PP_224_C1
PP_2272_C1
PP_2294_C1
PP_2113_C3
PP_2320_C1
PP_2360_C1
PP_233_C4
PP_2537_C1
PP_2633_C1
PP_2646_C2
PP_2738_C1
PP_276_C1
PP_2920_C1
PP_2921_C1
PP_285_C2
PP_2980_C1
PP_2817_C1
PP_6215_C1
PP_3091_C1
PP_323_C1
PP_3684_C1
PP_3846_C1
PP_3876_C1
PP_3950_C1
PP_3864_C1
PP_4087_C4
Appendices
**: Homolog of 68415.m02009 ARID/BRIGHT DNA-binding domain-containing protein
contains
**: Homolog of (AB015183) transcription factor Vp1 [Mesembryanthemum crystallinum]
***: Q8LB52 Scarecrow-like protein.
***: Q7X9V3 Nuclear shuttle interacting protein.
**: Homolog of (Transcription factor E2F/dimerisation partner TDP)
**: Homolog of (AY077758) WRKY transcription factor 1 [Physcomitrella patens]
***: Q39183 Serine/threonine protein kinase (Protein kinase (EC 2.7.1.37) 5)
(AT5g47750/MCA23_7).
"**: Homolog of (68417.m04184 acid phosphatase class B family protein similar to acid
phosphatase
not annotated Physcomitrella patens
***: O82527 Polyubiquitin (Fragment).
***: RL71_ARATH 60S ribosomal protein L7-1.
**: Homolog of 68417.m02122 myb family transcription factor contains Pfam profile
contains: Nascent polypeptide-associated complex
NAC(InterPro:IPR002715,PFAM:PF01849)
**: Homolog of (68415.m03211 plectin-related contains
***: SU91_HUMAN Histone-lysine N-methyltransferase, H3 lysine-9 specific 1
-2.23
-1.91
-1.37
-1.51
-1.74
-1.75
-1.56
-1.58
-1.92
-1.46
-2.11
-1.55
-1.89
-2.11
-1.63
-1.80
-1.73
-1.56
-1.95
-1.38
-1.71
-1.43
**: Homolog of (AB112672) auxin response factor 2 [Cucumis sativus]
***: ARP_ARATH Apurinic endonuclease-redox protein (DNA-(apurinic or apyrimidinic site)
"**: Homolog of ((AP004849) putative CCR4-NOT transcription complex
***: Q7XU22 OSJNBb0034G17.2 protein (Transcription factor DREB).
***: Q8LKS8 Early drought induced protein.
***: Q8RXD3 ABI3-interacting protein 2.
***: Q8GRK2 Somatic embryogenesis receptor kinase 1.
**: Homolog of Similar to chloroplast DNA-binding protein
PD3|o_sativa|chr_2|P0017C12|3841
***: AHM7_ARATH Potential copper-transporting ATPase 3 (EC 3.6.3.4).
**: Homolog of 68417.m02632 lil3 protein identical to Lil3 protein [Arabidopsis thaliana]
***: O81077 Putative cytochrome P450.
**: Homolog of expressed protein|o_sativa|chr_6|OSJNBa0019F11|6897
***: Q9FIX3 Gb|AAD30619.1.
***: Q852K5 Putative zinc finger protein (Putative zinc finger transcription factor ZFP38).
***: AHM1_ARATH Potential cadmium/zinc-transporting ATPase HMA1 (EC 3.6.3.3) (EC
3.6.3.5).
***: Q943L1 Putative Ubiquitin carrier protein UBC7.
***: Q9ZS93 T4B21.6 protein.
contains: Transcription factor, MADS-box
contains: GCN5-related N-acetyltransferase
***: O23310 CCAAT-binding transcription factor subunit A(CBF-A)
**: Homolog of (AB106274) SCARECROW-like protein [Lilium longiflorum]
**: Homolog of GRAS family transcription factor, putative|o_sativa|chr_1|P0406G08|2927
***: T2AG_ARATH Transcription initiation factor IIA gamma chain (TFIIA-gamma).
**: Homolog of AT3g02790/F13E7_27|o_sativa|chr_6|P0621D05|1946
***: Q8H1G0 Putative flowering protein CONSTANS (GATA-type zinc finger protein).
contains: Protein of unknown function DUF296(InterPro:IPR005175,PFAM:PF03479)
***: Q8S9V3 Putative zinc finger protein.
***: Q9SNA4 Receptor-like protein kinase homolog.
***: PSAG_ARATH Photosystem I reaction center subunit V, chloroplast precursor (PSI-G).
"**: Homolog of (X58577) DNA-binding protein; bZIP type [Petroselinum crispum]
**: Homolog of aldehyde dehydrogenase,
**: Homolog of (68414.m05059 Ras-related GTP-binding protein
**: Homolog of (AB028078) homeobox protein PpHB7 [Physcomitrella patens]
**: Homolog of (68416.m02515 basic helix-loop-helix bHLH) family protein
"**: Homolog of (68416.m01203 aspartate/glutamate/uridylate kinase family protein
-1.74
-2.08
-1.34
-40.91
-1.59
-2.07
-1.65
-1.64
-1.96
-2.16
-1.67
-1.66
-1.82
-1.50
-1.45
-1.56
-1.31
-1.37
-1.56
-2.49
-1.53
-1.59
-1.70
-1.85
-1.36
-1.58
-2.01
-1.50
142
Chapter IV
PP_4109_C1
PP_4163_C1
PP_4175_C1
PP_4238_C1
PP_4183_C1
PP_4368_C1
PP_438_C1
PP_4383_C1
PP_4394_C1
PP_4227_C2
PP_4501_C1
PP_4570_C3
PP_4710_C1
PP_4819_C1
PP_4820_C1
PP_4988_C1
PP_5004_C1
PP_5046_C1
PP_5138_C1
PP_5262_C1
PP_5396_C1
PP_5526_C1
PP_5624_C1
PP_5836_C2
PP_5905_C1
PP_6171_C1
PP_6223_C1
PP_6242_C1
PP_6285_C1
PP_6114_C1
PP_6587_C1
PP_646_C2
PP_6731_C1
PP_6766_C1
PP_683_C1
PP_6888_C1
PP_6969_C1
PP_6973_C1
PP_6875_C1
PP_7128_C1
PP_7321_C2
PP_11287_C4
PP_7371_C1
PP_7586_C1
PP_7694_C1
PP_7708_C1
PP_775_C1
PP_7994_C1
PP_8047_C1
PP_8107_C1
Appendices
**: Homolog of (M62985) protein kinase [Zea mays]
**: Homolog of (AF029984) COP1 homolog [Lycopersicon esculentum]
**: Homolog of (68414.m07887 basic helix-loop-helix bHLH) family protein
**: Homolog of (68414.m01246 eukaryotic translation initiation factor 3 subunit 3 / eIF-3
**: Homolog of (AY077758) WRKY transcription factor 1 [Physcomitrella patens]
**: Homolog of (68418.m04281 histidine kinase AHK2) identical to histidine kinase
**: Homolog of 68418.m05840 myb family transcription factor contains Pfam profile
**: Homolog of (68415.m02898 basic helix-loop-helix bHLH) family protein
**: Homolog of Protein kinase domain, putative|o_sativa|chr_1|OJ1529_G03|4777
**: Homolog of (transcription initiation factor iib general transcription factor tfiib).
**: Homolog of OSJNBa0003O19.1|putative AT-Hook DNA-binding protein
"**: Homolog of (68418.m08464 F-box family protein similar to unknown protein
(dbj|BAA78736.1)
**: Homolog of 68416.m05166 Dof-type zinc finger domain-containing protein [Arabidopsis
thaliana]
**: Homolog of Similar to histidine kinase-like protein|o_sativa|chr_6|P0709F06|1935
**: Homolog of (AF378125) GAI-like protein 1 [Vitis vinifera]
**: Homolog of (68415.m05233 basic helix-loop-helix bHLH) family protein
"**: Homolog of (68418.m01242 sensory transduction histidine kinase-related
"**: Homolog of (68415.m04408 zinc finger (C3HC4-type RING finger) family protein
**: Homolog of (AF439278) ethylene-responsive transciptional coactivator-like protein
[Retama raetam]
**: Homolog of 68415.m04505 PHD finger transcription factor, putative
**: Homolog of (AF311224) C2H2 zinc-finger protein [Zea mays]
**: Homolog of 68418.m04803 Dof-type zinc finger domain-containing protein [Arabidopsis
thaliana]
**: Homolog of TAZ zinc finger, putative|o_sativa|chr_1|P0696G06|5647
**: Homolog of 68416.m05063 myb family transcription factor
**: Homolog of OSJNBa0053C23.4|putative serine/threonine protein kinase
***: Q84XK6 Peroxisomal targeting signal type 2 receptor.
contains: Helix-turn-helix, Fistype(InterPro:IPR002197,GO:0003700,GO:0006355,PRINTS:PR01590)
not annotated Physcomitrella patens
**: Homolog of (Y10685) G/HBF-1 [Glycine max]
**: Homolog of F-box domain, putative|o_sativa|chr_4|OSJNBa0043A12|8129
***: O82064 Putative beta-subunit of K+ channels.
**: Homolog of Dof domain, zinc finger, putative|o_sativa|chr_1|P0453A06|2679
not annotated Physcomitrella patens
***: Q8W2B8 Serine acetyltransferase (Hypothetical protein At4g35640).
***: YPT6_CHLRE Ras-related protein YPTC6.
contains: Ubiquitin-conjugating enzymes
**: Homolog of (68415.m03211 plectin-related contains
"***: Q9C8A0 Serine/arginine-rich protein, putative; 48931-50251 (TAF7) (At1g55300)."
contains: U2 snRNP auxilliary factor
**: Homolog of (AF467900) hypothetical transcription factor [Prunus persica]
***: Q9FJC9 26S proteasome regulatory particle chain RPT6-like protein
(AT5g53540/MNC6_8).
***: RL5_ARATH 60S ribosomal protein L5.
contains: Tubby(InterPro:IPR000007,PFAM:PF01167)
***: DR1D_ARATH Dehydration responsive element binding protein
***: Q39031 Protein kinase.
***: Q7X976 Putative AT-Hook DNA-binding protein.
***: Q9SQ79 Helix-loop-helix protein 1A.
**: Homolog of (AY077758) WRKY transcription factor 1 [Physcomitrella patens]
**: Homolog of 68417.m01380 KOW domain-containing transcription factor family protein
chromatin
**: Homolog of Similar to TINY-related|o_sativa|chr_2|OJ1711_D06|4260
-1.86
-1.68
-2.79
-1.61
-1.50
-2.10
-1.42
-1.56
-3.02
-1.39
-1.43
-1.63
-1.53
-2.13
-4.01
-1.80
-1.70
-3.59
-3.39
-1.92
-1.73
-1.31
-1.62
-27.29
-1.50
-1.29
-3.32
-2.62
-1.94
-1.91
-3.86
-2.29
-1.89
-2.19
-1.97
-1.65
-9.85
-1.39
-1.60
-2.40
-1.71
-1.55
-2.04
-1.54
-2.02
-1.43
-1.79
-1.50
-1.45
-2.55
143
Chapter IV
PP_8413_C1
PP_8463_C1
PP_8293_C1
PP_8547_C1
PP_86_C1
PP020016117R
PP_723_C1
PP_9108_C3
PP_12500_C1
PP_9253_C1
PP_9264_C1
PP_9369_C1
PP_9394_C1
PP_9399_C1
PP_9419_C1
PP_9627_C1
PP_9785_C1
PP_9953_C1
PP_9960_C1
PP_6995_C2
PP020064243R
PP_SD_251_C1
PP_SD_60_C1
PP_SD_88_C1
PP_SD_92_C1
PP_SD_245_C1
PP_5766_C1
PP_SD_0_C1
PP001001061F
PP001008059F
PP001009093F
PP001019006F
PP001030028F
PP001030033F
PP001068061R
PP001085009R
BJ165389
PP004021140R
PP004058038R
PP004067310R
PP004086076R
PP004087236R
BI488008
PP004095105R
PP013015004R
PP015004308R
PP015011331R
PP015020123R
PP015029288R
Appendices
contains: Zn-finger, RING(InterPro:IPR001841,PFAM:PF00097)
***: Q86AZ8 Similar to Anabaena sp. (Strain PCC 7120). Hypothetical WD-repeat protein
alr2800.
contains: (COIL:coil)
**: Homolog of 68414.m01463 hypothetical protein
**: Homolog of (AY566696) unknown [Xerophyta humilis]
**: Homolog of (AB032182) homeobox protein PpHB10 [Physcomitrella patens]
***: Q8LST6 Mitochondrial aldehyde dehydrogenase.
***: FTH2_ARATH Cell division protein ftsH homolog 2, chloroplast precursor
**: Homolog of (68414.m01494 basic helix-loop-helix bHLH) family protein / F-box family
protein
***: Q9C1Q7 Putative two-component histidine kinase Fos-1.
"***: Q9SRM4 Putative nucleic acid binding protein (At3g11200/F11B9.12)
***: Q9FJ00 Gb|AAF24948.1.
***: O94094 Histidine kinase FIK.
***: Q8X1E7 Histidine kinase.
**: Homolog of 68417.m04480 WRKY family transcription factor contains Pfam profile
***: Q7WZ30 MmoS.
***: Q8LPA5 MADS-box protein PpMADS1.
***: Q852U6 At1g49850.
contains: Nascent polypeptide-associated complex NAC
(InterPro:IPR002715,PFAM:PF01849)
***: Q9ZNX9 Sigma-like factor precursor (RNA polymerase sigma subunit SigE).
***: Q9LWW0 Oryza sativa (japonica cultivar-group) genomic DNA, chromosome 6,
clone:P0425F02.
**: Homolog of (AB115546) phototropin 2 [Adiantum capillus-veneris]
****: Physcomitrella patens mRNA for homeobox protein PpHB9, complete cds.
not annotated Physcomitrella patens
****: Physcomitrella patens PpHB10 mRNA for homeobox protein PpHB10, complete cds.
**: Homolog of (68418.m05453 disease resistance protein TIR-NBS-LRR class)
**: Homolog of (68418.m02952 zinc finger B-box type) family protein similar to
CONSTANS-like protein
****: Physcomitrella patens subsp. patens mRNA for MADS-box protein PpMADS1,
complete cds.
**: Homolog of (X98744) chloroplast DNA-binding protein PD3 [Pisum sativum]
**: PP_CL_6374.Singlet Homolog of (AB046872) PpSIG2 [Physcomitrella patens]
**: Homolog of OSJNBa0010C11.3|putative transcription regulatory protein
**: PP_CL_8395.Singlet Homolog of (68416.m00477 RNA recognition motif (RRM)
**: Homolog of (AC005315) putative non-LTR retroelement reverse transcriptase
"**: Homolog of 68414.m08061 paired amphipathic helix repeat-containing protein
**: PP_CL_18208.Singlet Homolog of 68415.m05104 expressed protein
**: PP_CL_6389.Singlet Homolog of (AY324646) katanin [Gossypium hirsutum]
***: Q41067 Polyubiquitin.
****: Physcomitrella patens subsp. patens mRNA for MADS-box protein PpMADS1,
complete cds.
***: O49977 Ubiquitin (Fragment).
**: PP_CL_7066.Singlet Homolog of (AB026657) unnamed protein product
***: Q41067 Polyubiquitin.
****: Physcomitrella patens subsp. patens mRNA for MADS-box protein PpMADS1,
complete cds.
***: O82527 Polyubiquitin (Fragment).
***: O82527 Polyubiquitin (Fragment).
**: Homolog of OSJNBb0070O09.2|unknown protein
***: Q8LNW1 Putative transcription factor.
**: Homolog of (68414.m08326 GCN5-related N-acetyltransferase GNAT) family protein
**: Homolog of (AB032182) homeobox protein PpHB10 [Physcomitrella patens]
"**: PP_CL_5054.Singlet Homolog of (68418.m08510 TAZ zinc finger family protein
-1.47
-1.76
-2.52
-1.84
-1.83
-1.93
-1.51
-1.70
-3.17
-1.41
-1.33
-1.59
-1.81
-1.67
-2.93
-3.06
-1.73
-1.59
-2.92
-1.77
-1.40
-1.33
-1.87
-3.49
-2.00
-1.66
-1.47
-1.96
-2.13
-1.52
-1.35
-1.54
-1.96
-1.47
-2.18
-1.60
-1.41
-1.48
-1.31
-1.60
-1.58
-1.52
-1.59
-1.59
-1.51
-1.70
-1.43
-1.47
-1.99
144
Chapter IV
PP015033075R
PP015037157R
PP015044155R
PP015054317R
PP015071162R
PP020027384R
PP020032226R
PP020034124R
PP020036015R
PP020036307R
PP020044335R
PP020051260R
PP020062195R
PP020065285R
PP020069226R
PP030015063R
AY123146
Appendices
**: Homolog of (AB121445) histidine kinase 3 [Zea mays]
**: Homolog of Helicase conserved C-terminal domain,
putative|o_sativa|chr_8|OJ1034_C08|7210
**: PP_CL_1670.Singlet Homolog of (AJ419328) putative MADS-domain transcription
factor
**: Homolog of Helix-loop-helix DNA-binding domain,
putative|o_sativa|chr_1|OSJNBa0093F16|4658
***: AGO1_ARATH Argonaute protein.
****: Physcomitrella patens subsp. patens PPLFY1 mRNA for FLORICAULA/LEAFY
homolog
**: PP_CL_18682.Singlet Homolog of (AJ011828) NDX1 homeobox protein [Lotus
corniculatus
**: PP_CL_4373.Singlet Homolog of (AB115546) phototropin 2 [Adiantum capillus-veneris]
**: PP_CL_9604.Singlet Homolog of OJ1365_D05.10|putative response regulator protein
***: Q9FT60 Histidine kinase-like protein.
"**: Homolog of 68416.m03380 oligopeptide transporter OPT family protein
***: Q9SXL4 Histidine kinase 1.
**: Homolog of (AJ419328) putative MADS-domain transcription factor [Physcomitrella
patens]
**: PP_CL_10589.Singlet Homolog of (AL670011) related to regulatory protein SET1
***: Q9LRI1 Homeobox protein PpHB10.
***: O82527 Polyubiquitin (Fragment).
PP_SD_72.Singlet not annotated Physcomitrella patens
-4.00
-1.73
-1.50
-1.53
-1.40
-1.40
-1.59
-1.65
-2.19
-1.44
-1.94
-2.12
-1.82
-1.49
-1.60
-1.55
-1.76
145
Chapter IV
4.5
Appendices
Genes upregulated in ΔPpDCL1b mutants
Sequence ID
EST
AW561368
BJ160934
BJ163321
BJ181458
BJ187175
BJ189887
BJ609923
BJ610672
BU051751
PP_10130_C2
PP_10143_C1
PP_10308_C1
PP_1034_C1
PP_10379_C1
PP_10320_C1
PP_10567_C1
PP_10875_C1
PP_10880_C1
PP_10919_C1
PP_11080_C1
PP_1112_C1
PP_11139_C1
PP_12499_C1
PP_13554_C1
PP_11359_C1
PP032001093R
PP_18394_C1
PP_1179_C1
PP_1012_C1
PP_12120_C1
PP_12140_C1
PP_12145_C1
PP001005057F
PP_12587_C1
PP_12713_C2
PP_12802_C1
PP_1303_C1
PP_13105_C1
PP_13136_C1
PP_13592_C1
PP_13985_C1
PP_14045_C1
PP_14113_C1
PP_1440_C1
PP_14547_C1
Cosmoss Annotation
Fold change
"**: Homolog of (68417.m01135 F-box family protein (FBL8) FBL24) contains
**: Homolog of (AF004165) 2-isopropylmalate synthase [Lycopersicon pennellii]
***: Q8S0L3 Ankyrin-kinase-like protein.
***: Q9AV93 Response regulator 8.
***: Q84VL6 Putative polyubiquitin (Fragment).
**: PP_CL_6916.Singlet Homolog of ((AP003273) histone H1-like protein (Oryza sativa )
***: EFTM_ARATH Elongation factor Tu, mitochondrial precursor.
**: Homolog of expressed protein|o_sativa|chr_4|OSJNBa0013K16|5362
***: Q7X864 OSJNBa0093F12.4 protein.
***: Q8H9A2 Dehydratiion responsive element binding protein 1 like protein.
***: Q8YMN1 All4902 protein.
**: Homolog of OSJNBb0015I11.23|putative ubiquitin
protein|o_sativa|chr_10|OSJNBb0015I11|31
***: Q9SEK4 Putative succinic semialdehyde dehydrogenase
"**: Homolog of (68417.m00335 AAA-type ATPase family protein
***: Q96327 Putative nuclear DNA-binding protein G2p (Nuclear DNA-binding protein)
not annotated Physcomitrella patens
***: Q94H06 Putative zinc finger protein.
***: Q8GYN7 Putative SCARECROW gene regulator.
***: Q8S8P6 Putative salt-inducible protein.
***: Q9SA69 F10O3.17.
***: Q9ZVG0 Putative ATP-dependent DNA helicase RECG.
**: Homolog of 68414.m03428 expressed protein
***: PRS7_ARATH 26S protease regulatory subunit 7 (26S proteasome subunit 7)
***: Q9LKG4 Putative DNA binding protein.
**: Homolog of 68416.m01881 DNA-binding protein-related
**: PP_CL_11223.Singlet Homolog of 68414.m05053 expressed protein
**: Homolog of RNA polymerase I specific transcription initiation factor
**: Homolog of Similar to probable zinc finger protein [imported] - Arabidopsis thaliana
**: Homolog of 68418.m05158 kelch repeat-containing F-box family protein contains Pfam
profiles
***: Q7X6J0 RNA binding protein Rp120.
**: Homolog of (AF470350) WD40 [Tortula ruralis]
**: Homolog of hypothetical protein|o_sativa|chr_1|P0470A12|2880
***: O82527 Polyubiquitin (Fragment).
**: Homolog of AP2-related transcription factor,
putative|o_sativa|chr_4|OSJNBa0079A21|8302
***: Q9GZS3 Homo sapiens cDNA: FLJ21101 fis, clone CAS04682 (G protein beta
subunit)
***: Q7XXN2 Putative serine/threonine-protein kinase ctr1.
contains: Dihydrodipicolinate synthetase(InterPro:IPR002220,PFAM:PF00701)
***: Q84QC2 Putative AP2 domain transcription factor.
***: Q9SGP0 F3M18.14.
***: Q94DZ5 Putative helicase-like transcription factor.
***: Q9FHJ4 Arabidopsis thaliana genomic DNA, chromosome 5, P1 clone: MFC19.
***: O65567 Puative protein.
***: IF35_ARATH Eukaryotic translation initiation factor 3 subunit 5 (eIF-3 epsilon) (eIF3
p32 subunit)
contains: RNA-binding region RNP-1 (RNA recognition motif)
**: Homolog of (68414.m00475 zinc finger (C3HC4-type RING finger) family protein
1.32
2.24
1.94
1.97
1.81
1.30
1.43
1.70
1.55
1.76
1.87
1.62
1.53
1.95
1.75
1.57
2.11
1.67
1.69
1.39
1.41
1.38
1.43
1.30
2.03
1.58
2.11
2.07
2.43
1.73
1.41
1.78
1.65
2.46
1.56
2.19
1.86
1.72
1.96
2.17
1.39
1.81
1.47
1.69
1.32
146
Chapter IV
Appendices
PP_14811_C1
**: Homolog of (AF098674) lateral suppressor protein [Lycopersicon esculentum]
1.40
PP_15007_C1
PP_15083_C1
PP_15177_C2
***: Q9ZV05 Expressed protein.
**: Homolog of zinc finger protein, putative|o_sativa|chr_6|P0550B04|1978
***: Q9SJR0 Putative AP2 domain transcription factor (Putative AP2/EREBP transcription
factor).
***: Q9FHA7 Emb|CAB62312.1 (Putative bHLH transcription factor).
***: Q7XJM3 Putative mitochondrial translation elongation factor G.
***: O81763 Protein kinase-like protein.
**: Homolog of 68416.m05464 phototropic-responsive protein
**: Homolog of 68417.m00399 elongation factor Tu, putative / EF-Tu, putative
***: Q9LW84 Gb|AAF26996.1.
**: Homolog of Similar to DNA helicase-like|o_sativa|chr_2|P0724B10|6621
***: MAT1_MOUSE CDK-activating kinase assembly factor MAT1 (RING finger protein
MAT1)
***: Q9SJW0 Transfactor-like protein.
***: Q8VZG7 AT5g07350/T2I1_60.
**: Homolog of expressed protein|o_sativa|chr_4|OSJNBa0013K16|5362
contains: Zn-binding protein, LIM(InterPro:IPR001781,PFAM:PF00412)
***: EFTM_ARATH Elongation factor Tu, mitochondrial precursor.
***: Q9SI75 F23N19.11 (Hypothetical protein At1g62750).
***: Q851S7 Pescadillo-like protein.
***: Q9LVF7 Gb|AAD14441.1.
"**: Homolog of (68414.m09356 coatomer protein complex, subunit beta 2 (beta prime),
***: Q8S3E7 Putative bHLH transcription factor.
***: Q9LSQ8 Arabidopsis thaliana genomic DNA, chromosome 5, BAC clone: F24B18.
***: EFGM_ARATH Probable elongation factor G, mitochondrial precursor (mEF-G).
contains: Pathogenesis-related transcriptional factor and ERF
***: O82527 Polyubiquitin (Fragment).
**: Homolog of auxin response factor 1 [imported] - Arabidopsis
thaliana|o_sativa|chr_2|P0506A08|3865
***: Q9LS31 Homeobox protein Pphb7 short form.
**: Homolog of OSJNBa0003O19.20|putative MYC transcription factor
contains: Zn-finger, Dof type(InterPro:IPR003851,GO:0003677,PFAM:PF02701)
***: Q8W3M3 AP2 domain containing protein (Putative AP2/EREBP transcription factor).
***: Q9SAK5 T8K14.15 protein.
**: Homolog of (AJ131113) VP1/ABI3-like protein [Chamaecyparis nootkatensis]
***: Q7XSB1 OJ991113_30.18 protein.
***: Q9SSF9 F25A4.28 protein.
contains: (COIL:coil)
not annotated Physcomitrella patens
**: Homolog of RNA polymerase I specific transcription initiation factor
***: O65567 Puative protein.
"**: Homolog of (68417.m02830 nucleoside phosphatase family protein / GDA1/CD39
family protein
**: Homolog of Kelch motif, putative|o_sativa|chr_6|OJ1378_E04|3445
not annotated Physcomitrella patens
***: Q8SB10 Putative crp1 protein.
**: Homolog of EREBP-type transcription factor, putative|o_sativa
***: Q9ZVU6 T5A14.12 protein.
***: Q8GZ22 Putative ankyrin (At2g03430).
***: Q84TU4 Arm repeat-containing protein.
contains: (SUPERFAMILY:SSF54171)
***: Q94ID6 ERF domain protein12 (Ethylene responsive element binding factor, putative).
***: Q9M8Z0 T6K12.4 protein.
***: Q9FWR5 F14P1.8 protein.
1.60
1.69
10.73
PP_15255_C1
PP_15299_C1
PP_15344_C1
PP_15382_C1
PP_15384_C1
PP_15546_C1
PP_15582_C1
PP_15610_C1
PP_15633_C1
PP_15636_C1
PP_15695_C1
PP_1585_C2
PP_15995_C1
PP_15997_C1
PP_16050_C1
PP_1618_C1
PP_16284_C1
PP_1662_C1
PP_1663_C1
PP_16437_C1
PP_1496_C1
BJ181914
PP_16865_C1
PP_1691_C1
PP_16923_C1
PP_1738_C3
PP_17440_C1
PP_17575_C1
PP_17900_C1
PP_17924_C1
PP_1804_C1
PP_18357_C1
PP_18393_C1
PP_18403_C1
PP_1844_C1
PP_18489_C1
PP_18663_C1
PP_18676_C1
PP_1999_C1
PP_2158_C1
PP_2271_C5
PP_2334_C1
PP_2362_C1
PP_7120_C2
PP_2324_C1
PP_2520_C1
PP_2344_C1
1.41
2.15
1.84
1.59
1.50
1.65
1.42
1.44
2.38
2.24
24.51
1.53
1.96
2.72
4.28
1.44
3.16
1.84
1.98
2.48
3.69
1.61
2.62
1.91
1.52
2.99
20.48
1.34
1.43
1.89
1.76
1.54
2.06
2.84
1.42
1.67
1.72
2.63
1.70
2.02
1.40
1.52
1.50
2.14
1.58
2.89
1.35
147
Chapter IV
Appendices
PP_2372_C1
***: Q9SAI2 F23A5.13 protein (Putative CCR4-associated factor).
1.39
BQ041789
PP_10621_C1
PP_3086_C1
PP_3132_C1
PP_319_C1
PP_3479_C1
PP_3728_C1
PP_3738_C1
***: Q9LMP8 F7H2.20 protein (At1g15870/F7H2_19).
***: Q9FPV8 Putative methionine aminopeptidase.
***: Q949D4 Putative AP2-related transcription factor.
"**: Homolog of (68415.m02229 expressed protein contains Pfam profiles
***: Q9LQ28 F14M2.12 protein (Putative AP2/EREBP transcription factor).
"**: Homolog of 68416.m01095 KOW domain-containing transcription factor family protein
**: Homolog of 68417.m00223 WRKY family transcription factor
"**: Homolog of 68418.m07197 protein kinase family protein similar to protein kinase
[Glycine max]
**: Homolog of DRE-binding protein 1A|o_sativa|chr_8|OJ1323_A06|7300
"**: Homolog of (AC016529) putative AP2 domain transcription factor
**: Homolog of OSJNBb0033N16.2|putative RNA
**: Homolog of (68414.m05749 basic helix-loop-helix bHLH) family protein contains Pfam
profile
**: Homolog of myb-like DNA-binding domain, SHAQKYF class
"**: Homolog of (68414.m00292 GCN5-related N-acetyltransferase (GNAT) family protein
not annotated Physcomitrella patens
**: Homolog of (AB067689) MADS-box protein PpMADS2 [Physcomitrella patens]
1.55
1.33
3.09
1.49
1.55
1.51
1.79
1.67
PP_4015_C1
PP_4112_C6
PP_4166_C1
PP_4300_C1
PP_4459_C1
PP_4595_C1
PP_4414_C1
PP_4697_C1
PP_4719_C1
PP_4825_C1
PP_4986_C1
PP_5002_C1
PP_5288_C1
PP_5296_C1
PP_543_C1
PP_4563_C1
PP_5681_C1
PP_5719_C1
PP_573_C2
PP_5803_C2
PP_584_C1
PP_560_C1
PP_5870_C1
PP_5912_C2
PP_12254_C1
PP_6275_C1
PP_63_C1
PP_6340_C1
PP_6354_C2
PP_6368_C4
PP_6455_C1
PP_648_C2
PP_18379_C1
PP_6564_C1
PP_6651_C1
PP_6682_C1
PP_6732_C1
PP_7252_C1
PP_729_C1
PP_753_C1
PP_7668_C1
PP_10278_C1
5.29
6.24
1.31
1.51
1.77
1.35
2.27
2.22
**: Homolog of 68414.m01735 expressed protein
**: Homolog of OSJNBa0053C23.4|putative serine/threonine protein kinase
"**: Homolog of (68415.m03129 transducin family protein / WD-40 repeat family protein
**: Homolog of (68415.m04914 eukaryotic translation initiation factor 3 subunit 5 / eIF-3
epsilon
**: Homolog of (AY346455) histone deacetylase [Solanum chacoense]
**: Homolog of ((AP002092) unnamed protein product [Oryza sativa japonica cultivar
group)]
**: Homolog of 68416.m05511 expressed protein
**: Homolog of (AF184886) LIM domain protein WLIM2 [Nicotiana tabacum]
**: Homolog of (AB111943) hypothetical protein [Nicotiana benthamiana]
**: Homolog of expressed protein|o_sativa|chr_5|OSJNBb0015A05|5000
***: Q852S5 Nucleoside diphosphate kinase.
**: Homolog of (AB042267) response regulator 5 [Zea mays]
**: Homolog of (68414.m05651 scarecrow-like transcription factor 3 (SCL3)
"**: Homolog of (68418.m07708 no apical meristem (NAM) family protein
**: Homolog of 68418.m01679 expressed protein
**: Homolog of (AF506028) CTV.22 [Poncirus trifoliata]
**: Homolog of (AF098674) lateral suppressor protein [Lycopersicon esculentum]
contains: Pathogenesis-related transcriptional factor and ERF
***: Q7Y1U0 Kinesin-like calmodulin binding protein.
***: Q9SR03 Ankyrin-like protein.
***: Q9SAU3 CAO.
***: Q762A0 BRI1-KD interacting protein 114 (Fragment).
***: Q9SZM7 Protein kinase like protein.
**: Homolog of (L76926) putative zinc finger protein [Arabidopsis thaliana]
not annotated Physcomitrella patens
"**: Homolog of 68414.m01006 protein kinase
***: O22826 Putative splicing factor (At2g43770).
***: Q7XMI6 OSJNBb0006N15.13 protein.
not annotated Physcomitrella patens
**: Homolog of ((AP005190) putative p53 binding protein [Oryza sativa japonica cultivargroup)]
**: Homolog of GRAS family transcription factor, putative|o_sativa|chr_1|P0466H10|5982
***: Q94D32 P0712E02.24 protein (P0700A11.5 protein).
***: PCNA_TOBAC Proliferating cell nuclear antigen (PCNA).
***: Q9FP06 P0038C05.18 protein.
22.24
1.53
1.45
1.54
1.67
1.46
2.02
1.37
1.51
1.70
1.54
2.33
2.15
1.75
2.76
1.37
1.70
1.43
1.81
1.52
1.58
1.33
2.29
1.79
1.78
1.64
1.64
1.37
1.66
1.65
1.66
1.88
1.40
2.75
148
Chapter IV
PP_793_C4
PP_7999_C1
PP_8_C1
PP_8013_C1
PP_14769_C1
PP_8021_C1
PP_8274_C1
PP_8314_C1
PP_8332_C1
PP_8337_C1
PP_8348_C1
PP_8372_C1
PP_8392_C1
PP_8343_C1
PP_8584_C1
PP_8642_C1
PP_8479_C1
PP_8784_C1
PP_8794_C1
PP_8838_C1
PP_8990_C3
PP_900_C1
PP_8906_C1
PP_9142_C1
PP_9252_C1
PP_765_C1
PP_9420_C1
PP_9498_C1
PP_9599_C1
PP_9374_C1
PP_9607_C1
PP_9750_C1
PP_976_C1
PP_17043_C1
PP_9949_C1
PP_SD_12_C1
PP_SD_17_C1
PP_SD_252_C1
PP_SD_46_C1
PP_SD_67_C1
PP_SD_90_C1
PP_10747_C1
PP001063096R
PP001072036R
PP001077051R
PP001090095R
PP002015081R
PP002023001R
PP004003286R
PP004006023R
Appendices
**: Homolog of (68418.m08455 basic helix-loop-helix bHLH) family protein
**: Homolog of 68415.m03352 DC1 domain-containing protein contains Pfam profile
contains: Protein synthesis factor, GTP-binding
***: RM21_ARATH 50S ribosomal protein L21, mitochondrial precursor.
**: Homolog of GRAS family transcription factor, putative|o_sativa|chr_1|P0406G08|2927
**: Homolog of putative AP2 domain transcription
factor|o_sativa|chr_4|OSJNBb0034G17|5454
not annotated Physcomitrella patens
**: Homolog of Similar to Lil3 protein|o_sativa|chr_2|P0018H03|4896
**: Homolog of AP2 domain, putative|o_sativa|chr_6|P0638H11|5506
***: Q7XNE0 OSJNBa0088A01.11 protein.
***: Q9ZV05 Expressed protein.
"***: Q9CAN3 Transcription factor SCARECROW, putative; 52594-50618."
***: Q8RYF8 P0592G05.19 protein.
"***: Q9C550 2-isopropylmalate synthase
***: Q39216 RNA polymerase subunit (Isoform B).
**: Homolog of (AY192369) ethylene response factor 3 [Lycopersicon esculentum]
contains: Basic helix-loop-helix dimerisation region bHLH
contains: Protein kinase-like(InterPro:IPR011009,SUPERFAMILY:SSF56112)
contains: Pathogenesis-related transcriptional factor
***: Q7XU78 OSJNBa0029H02.4 protein.
**: Homolog of (GDA1/CD39 nucleoside phosphatase) family, putative
***: O49591 Putative zinc finger protein.
***: Q9M551 Polyubiquitin.
"**: Homolog of 68418.m03534 bZIP transcription factor family protein
***: Q84QD7 Avr9/Cf-9 rapidly elicited protein 276.
***: O80582 Expressed protein (At2g44130/F6E13.26).
***: Q7XU22 OSJNBb0034G17.2 protein (Transcription factor DREB).
**: Homolog of 68416.m01360 PHD finger family protein contains Pfam domain
**: Homolog of similar to CH6 and COP9 complex subunit
6|o_sativa|chr_8|OJ1118_A06|3036
**: Homolog of SelR domain|o_sativa|chr_3|OSJNBa0048D11|3659
***: O65639 Glycine-rich protein.
***: Q8LBL6 Cell division protein FtsH-like protein.
***: Q6ZHJ5 Pentatricopeptide (PPR) repeat-containing protein-like.
**: Homolog of ((AP004068) GCN5-related N-acetyltransferase protein-like (Oryza sativa
japonica )
**: Homolog of Myb-like DNA-binding domain, putative|o_sativa|chr_1|P0038F12|2733
****: Physcomitrella patens mRNA for RNA polymerase alpha subunit, complete cds.
****: Physcomitrella patens WRKY transcription factor 1 (WRKY1) gene, complete cds.
**: Homolog of 68418.m05899 protein kinase, putative similar to protein kinase G11A
[Oryza sativa]
****: Physcomitrella patens mRNA for homeobox protein PpHB7, complete cds.
****: Physcomitrella patens MADS-domain protein PPM1 (ppm1) mRNA, complete cds.
not annotated Physcomitrella patens
***: O81763 Protein kinase-like protein.
**: PP_CL_18202.Singlet Homolog of (AC026238) Hypothetical protein [Arabidopsis
thaliana]
**: PP_CL_15170.Singlet Homolog of 68418.m06538 myb family transcription factor
***: O49459 Predicted protein.
**: PP_CL_15183.Singlet Homolog of Similar to Z97341 apetala2 domain TINY like protein
**: PP_CL_5597.Singlet Homolog of (AC006072) putative tubby protein [Arabidopsis
thaliana]
***: O24460 Calmodulin-like domain protein kinase.
"**: PP_CL_18294.Singlet Homolog of (68414.m07536 gibberellin regulatory protein
RGL1)
****: Physcomitrella patens phytochrome (phy2) gene, complete cds.
1.56
1.37
1.62
1.51
1.32
8.04
1.66
1.61
7.71
1.79
1.68
2.32
2.63
2.61
2.05
3.41
1.63
1.67
1.87
2.11
1.59
2.44
1.42
1.55
1.49
2.39
3.06
1.63
1.35
1.91
1.33
1.28
1.74
1.79
1.71
2.09
1.55
1.49
3.51
1.37
2.32
1.54
1.89
1.32
1.89
1.59
1.56
4.72
2.15
1.44
149
Chapter IV
PP004007192R
PP004009367R
PP004012159R
PP004015085R
PP004020107R
PP004024038R
PP004029122R
PP004030378R
PP004032225R
PP004040295R
PP004043210R
PP004046136R
PP004054012R
PP004054209R
PP004057142R
PP004062128R
PP004075103R
BQ827548
PP004034373R
PP004082282R
PP004083344R
PP004088245R
PP004092140R
PP004094219R
PP004095066R
PP004095253R
PP004096225R
PP004097116R
PP004103024R
PP004105269R
PP006002067R
PP010001010R
PP010002038R
PP010008086R
PP011003080R
PP011005059R
PP011006015R
PP015001075R
PP015006184R
PP015015236R
PP015024237R
PP015028003R
PP015028194R
PP015030306R
PP015033189R
PP015040077R
PP015041065R
PP015041271R
PP015042038R
PP015044222R
Appendices
***: Q9FJ91 Dbj|BAA78737.1 (AT5g52010/MSG15_9).
**: Homolog of (AF004165) 2-isopropylmalate synthase [Lycopersicon pennellii]
**: Homolog of 68414.m06061 mechanosensitive ion channel domain
**: Homolog of OSJNBa0010C11.3|putative transcription regulatory protein
**: PP_CL_11223.Singlet Homolog of 68414.m05053 expressed protein
***: Q8VYE7 Putative calcium-dependent protein kinase.
***: RPOA_PSINU DNA-directed RNA polymerase alpha chain (EC 2.7.7.6) (PEP)
**: Homolog of Similar to DRE binding factor 1|o_sativa|chr_6|P0516A04|3745
**: Homolog of (AB164647) vascular plant one zinc finger protein [Physcomitrella patens]
**: PP_CL_18294.Singlet Homolog of (AY269087) GAI-like protein [Lycopersicon
esculentum]
***: Q9SGT9 T6H22.8.2 protein.
**: Homolog of VIP2 protein|o_sativa|chr_2|OJ1311_D08|4248
**: PP_CL_15491.Singlet Homolog of (68418.m00567 zinc finger (C3HC4-type RING
finger)
PP_SD_13.Singlet not annotated Physcomitrella patens
***: Q94C56 Putative FtsH protease (Fragment).
**: Homolog of 68418.m06038 phototropic-responsive NPH3 family protein
***: Q7XPK1 OSJNBa0087O24.9 protein.
***: Q8H386 Casein kinase II alpha subunit.
****: Physcomitrella patens phytochrome (phy2) gene, complete cds.
**: Homolog of (AJ579910) NIN-like protein 1 [Lotus corniculatus var. japonicus]
***: Q40164 Ubiquitin.
***: Q9M1K4 Leucine zipper-containing protein AT103.
**: PP_CL_18318.Singlet Homolog of (AB028621) unnamed protein product [Arabidopsis
thaliana]
***: Q8LST4 Mitochondrial aldehyde dehydrogenase.
**: Homolog of NF-X1 type zinc finger, putative|o_sativa|chr_1|P0041E11|2738
**: Homolog of (AF328842) homeodomain protein HB2 [Picea abies]
****: PP_SD_29.Singlet Physcomitrella patens mRNA for putative P-type II calcium
**: Homolog of ((AP005243) VP1/ABI3 family regulatory protein-like [Oryza sativa]
***: O99018 Chloroplast protease precursor.
**: Homolog of 68416.m01747 scarecrow transcription factor family protein
***: Q9M378 TATA box binding protein (TBP) associated factor (TAF)-like protein.
***: Q8GV68 Phytochrome.
**: Homolog of (AC068602) F14D16.2 [Arabidopsis thaliana]
**: Homolog of (AL163912) putative protein [Arabidopsis thaliana]
"**: PP_CL_17423.Singlet Homolog of (68418.m02981 transducin family protein
**: Homolog of ATP-dependent metalloprotease FtsH,
putative|o_sativa|chr_5|OJ1362D02|3955
**: Homolog of (AY514604) gibberelin response modulator dwarf 8 Zea mays
2.04
1.77
1.79
1.64
1.92
2.59
1.56
1.59
1.57
2.13
4.14
1.43
1.39
1.92
2.33
1.34
1.52
1.47
1.58
2.23
1.67
1.43
2.09
1.33
1.48
1.44
2.55
1.60
1.72
1.60
2.78
1.60
1.64
1.94
1.52
2.28
3.63
****: PP_SD_46.Singlet Physcomitrella patens gene for homeobox protein
**: PP_CL_2250.Singlet Homolog of Cyclin, N-terminal domain, putative
****: PP_SD_46.Singlet Physcomitrella patens mRNA for homeobox protein PpHB7,
complete cds.
**: PP_CL_3101.Singlet Homolog of (68414.m03658 DNA-directed RNA polymerase
family protein
**: Homolog of (68414.m07827 zinc finger B-box type) family protein
**: Homolog of 2-isopropylmalate synthase|o_sativa|chr_12|OSJNBb0034E23|7059
"**: PP_CL_3039.Singlet Homolog of (68417.m04350 translation initiation factor 3 IF-3)
**: Homolog of (68414.m08377 CCAAT-box-binding transcription factor-related
**: Homolog of (AF098674) lateral suppressor protein [Lycopersicon esculentum]
**: Homolog of Similar to RSSG8|o_sativa|chr_12|OJ1396_C02|7247
**: Homolog of Helix-loop-helix DNA-binding domain,
putative|o_sativa|chr_11|P0410D09|7510
**: PP_CL_6025.Singlet Homolog of expressed protein|o_sativa|chr_1|B1064G04|2860
**: PP_CL_18339.Singlet Homolog of (AF328786) EIL3 [Lycopersicon esculentum]
3.50
2.35
3.32
1.58
3.56
2.35
1.66
2.41
1.54
1.75
2.00
1.72
1.32
150
Chapter IV
PP015050353R
PP015058275R
PP015060167R
PP020009267R
PP020016269R
PP020019294R
PP020024305R
PP020026165R
PP020029315R
PP020031042R
PP020031185R
PP020032141R
PP020039216R
PP020041086R
PP020043294R
PP020053142R
PP020054111R
PP020054231R
PP020058260R
PP020060244R
PP020063226R
PP020063256R
PP020070127R
PP030007003R
PP030013070R
PP032009070R
Appendices
***: Q9S786 Calcium-dependent protein kinase.
***: Q9FGR7 Similarity to salt-inducible protein.
***: Q8W3N8 26S proteasome regulatory particle triple-A ATPase subunit4b (Fragment).
***: Q9SI82 F23N19.4.
**: Homolog of OSJNBb0033N16.3|putative protein kinase|o sativa|chr
3|OSJNBb0033N16|761
**: PP_CL_15.Singlet Homolog of (AF534891) type-B response regulator [Catharanthus
roseus]
***: O04235 Transcription factor.
contains: Response regulator receiver
"**: Homolog of (68418.m00739 transcription factor jumonji (jmjC) domain-containing
protein
***: Q9LPC6 F22M8.8 protein.
**: Homolog of expressed protein|o_sativa|chr_5|P0683B12|6231
"**: Homolog of (68416.m05316 bacterial transferase hexapeptide repeat-containing
protein
**: Homolog of A67797 unnamed protein product-related|o sativa|chr
8|OSJNBa0054L03|4675
***: Q9LTD4 Similarity to unknown protein.
***: Q6K7E2 Mitochondrial transcription termination factor-like.
***: Q9S729 GlsA.
"**: PP_CL_10402.Singlet Homolog of (68414.m01147 NF-X1 type zinc finger family
protein
***: Q9FP06 P0038C05.18 protein.
***: Q9FLM7 Gb|AAC33480.1 (MYB transcription factor).
**: PP_CL_10622.Singlet Homolog of (AB107691) AG-motif binding protein-3 [Nicotiana
tabacum]
***: Q9LV30 Emb|CAB40755.1.
**: Homolog of Protein kinase domain, putative|o_sativa|chr_1|P0695A04|2742
PP_SD_242.Singlet not annotated Physcomitrella patens sporophyte
***: Q8S9J9 At1g14000/F7A19_9.
***: O82527 Polyubiquitin (Fragment).
***: Q8GV68 Phytochrome.
1.50
1.66
1.39
1.42
1.63
1.34
1.72
1.90
1.56
2.58
1.64
1.54
1.41
1.96
3.27
1.78
1.85
1.50
1.51
1.77
1.48
1.46
1.81
1.93
1.34
2.22
151
Acknowledgment
4.6
Acknowledgments
First of all I would like to thank Prof. Dr. Ralf Reski for giving me the opportunity of
doing my PhD in his research group and for his support and encouragement along the way. I
am also indebted to him for his guidance
I would like to thank PD Dr. Wolfgang Frank for his invaluable supervision, great efforts
in guidance, encouragement throughout the research work.
Great appreciation is also due to my family for their encouragement and support.
Special thanks to my wife Enas for valuable help, encouragement and support.
I would like to say thanks to:
Dr. Volker Speth and Dr. Claudia Gack for sample preparation and valuable help at the
scanning electron micrographs
Andras Viczian for providing the pPCV expression vector
Björn Voß for advice on miR319 precursor sequence analysis
Gregor Gierga for assisting in the small RNA blots technique
Richard Haas for practical support in the lab
Anne Katrin Prowse for proofreading my thesis.
Special thanks are expressed to my dearest colleagues; Fattash, I., Arif, M. A., Rödel, P.,
Tomek, M.
Finally, I would like to say thanks to all members from the Reski group for the wonderful
working ambience.
Thank you all…
152
Erklärung
4.7
Erklärung
Hiermit erkläre ich, dass ich die vorliegende Arbeit ohne unzulässige Hilfe Dritter und ohne
Benutzung anderer als der angegebenen Hilfsmittel angefertigt habe. Die aus anderen
Quellen direkt oder indirekt übernommenen Daten und Konzepte sind unter Angabe der
Quellen gekennzeichnet. Insbesondere habe ich hierfür nicht die entgeltliche Hilfe von
Vermittlungs,
beziehungsweise
Beratungsdiensten
(Promotionsberater
oder
anderer
Personen) in Anspruch genommen. Niemand hat von mir unmittelbar oder mittelbar
geldwerte Leistungen für Arbeiten erhalten, die im Zusammenhang mit dem Inhalt der
vorgelegten Dissertation stehen.
Die Arbeit wurde bisher weder im In noch im Ausland in gleicher oder ähnlicher Form einer
anderen Prüfungsbehörde vorgelegt.
Die Bestimmungen der Promotionsordnung der Fakultät für Biologie der Universität
Freiburg sind mir bekannt; insbesondere weiß ich, dass ich vor Vollzug der Promotion zur
Führung des Doktortitels nicht berechtigt bin.
Basel Khraiwesh
März, 2009
Freiburg,
153