Download A Conserved Molecular Framework for Compound Leaf Development

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Supporting Online Material for
A Conserved Molecular Framework for Compound Leaf Development
Thomas Blein, Amada Pulido, Aurélie Vialette-Guiraud, Krisztina Nikovics, Halima
Morin, Angela Hay, Ida Elisabeth Johansen, Miltos Tsiantis, Patrick Laufs*
*To whom correspondence should be addressed. E-mail: [email protected]
Published 19 December 2008, Science 322, 1835 (2008)
DOI: 10.1126/science.1166168
This PDF file includes:
Materials and Methods
Figs. S1 to S10
Tables S1 to S7
References
Supporting Online Materiel to
A conserved molecular framework for compound leaf development
Thomas Blein1, Amada Pulido1, Aurélie Vialette-Guiraud1*, Krisztina Nikovics1†, Halima
Morin1,2, Angela Hay3, Ida Elisabeth Johansen4, Miltos Tsiantis3, Patrick Laufs1§
Material and Methods, 10 Supplementary Figures and 7 Supplementary Tables
Material and Methods
Plant material
A. caerulea seeds were bought from a gardener shop (Jardiland, Maurepas, France). S.
lycorpersicum M82 wild type were received from S. Biemelt, the la mutant (accession
LA0335) from the Tomato Genetics Ressource Center and the gob mutants from D. Zamir. P.
sativum (cv Térèse and line NGB5839) were obtained from C. Rameau and the uni mutant
(line JI2171) from J. Hofer. S. tuberosum cv Pompadour were a gift from B. Letarnec.
RNA extraction and RT-PCR
RNA was extract from partially dissected apices or young leaves of the different species using
TRIsol reagent (Invitrogen) according to the manufacturer recommendations. Contaminating
DNA was removed by DNaseI treatment (Invitrogen) and the RNA was reverse transcribed
(SuperScriptTM II reverse transcriptase, Invitrogen). Gene specific primers used for semiquantitative RT-PCR are listed in Supplementary Table 4. PCR products were observed on
ethidium bromide-stained gels or following southern blotting (PsNAM1/2).
Cloning of NAM/CUC genes.
Aquilegia caerulea
Blast analysis identified several Aquilegia formosa X pubescens ESTs with high similarities
with the AtCUC2 and AtCUC3 proteins (TC10566 and TC15387, respectively). Partial
AcNAM and AcCUC3 mRNAs were amplified from reversed transcribed shoot apex RNA
from Aquilegia caerulea using respectively AcNAM1Fw and AcNAM1Rv, and AcCUC3Fw
and AcCUC3Rv primers (Primer sequence available in Supplementary Table 5).
Pisum sativum
Fragments of the NAC domain were amplified from reversed transcribed RNA from small
axillary shoots of pea (cv Térèse) using degenerated primers (NACdeg1Fw1 and
NACdegRv1; NACdegFw2 and NACdegRv2; degCUC3Fw1 and degCUC3Rv1;
degCUC3Fw2 and degCUC3Rv2). The PCR products were cloned into pGEM-T Easy
(Promega) and the insert of several independent clones was sequenced. The resulting
sequences were positioned on a phylogenetic tree containing several NAC proteins including
AtCUC1, AtCUC2 and AtCUC3. The sequences of the NAM/CUC orthologs were extended
by RACE-PCR using the The GeneRacerTM kit (Invitrogen) and nested gene specific primers.
Solanum lycopersicum
A fragment of the SlNAM was amplified from genomic DNA of tomato using nam2 and nam5
primers, cloned into pGEM-T Easy (Promega) and sequenced. The full length SlNAM was
isolated from RNA extracted from vegetative apices by RACE-PCR using the GeneRacerTM
kit (Invitrogen) and nested gene specific primers.
Solanum tubersosum
RNA from S. tuberosum (cv solara) was obtained from C. Navarro. A StNAM fragment was
obtained by RT-PCR using the tomato SlNAM-based primers SlNAM5’ and SlNAM3’. Blast
analysis identified a Solanum chacoense EST (DN983806) showing high similarity to
AtCUC3 and used to define two specific primers cuc3uj2 and revcuc3uj3. These primers were
used to amplify a fragment of StCUC3 which was then extended by RACE PCR using the
GeneRacerTM kit (Invitrogen) and nested gene specific primers.
Cardamine hirsuta
Fragments of ChCUC2 and ChCUC3 were amplified from reversed transcribed shoot apical
meristem Cardamine hirsuta RNA using the Arabidopsis-based primers RLT-FwdAT5G53950 and CUC2 3' and insitu CUC3 L Fwd insitu CUC3 L Rv+T7, respectively. A
fragment of ChCUC1 was amplified using the degenerated primers NACdegFw2 and
miR164bsdegRv1. These fragments were cloned into pGEM-T and sequenced. The sequences
of ChCUC1, 2 and 3 were extended by RACE-PCR.
mRNA in situ hybridisations
They we performed as described (1) using the probes listed in the Supplementary Table 6
Cardamine transgenic lines and dexamethasone induction.
A hairpin cassette was created against the specific domain of ChCUC3 (see Supplementary
Table 7) in pKANNIBAL(2) and transferred into the binary vector pGreen0029(3) to create
the ChCUC3 RNAi vector. The ChCUC3 RNAi plasmid and the 2x35S:MIR164b plasmid(4)
were transferred into Agrobacterium tumefaciens GV3101 and transformed into C. hirsuta as
described(5). Ten-day-old KNOTTED1-GR C. hirsuta transgenic lines were induced with 106
M Dexamethasone (SIGMA) as previously described(6) for 2 weeks for the phenotypic
analysis and for 2 days for the molecular analysis. The STM-GUS line was described in(7).
Virus Induced Gene Silencing
Gene specific fragments of the NAM/CUC genes (see Supplementary Table 7) and of the PDS
genes (8-10) were introduced into TRV (for A. caerulea and S. lycopersicum) or PEBV (for P.
sativum) RNA2. The TRV vectors were kindly provided by E. Kramer. VIGS was done as
described for A. caerulea(9) and P. sativum (line NGB5839)(8). For S. lycopersicum, we
vacuum infiltrated M82 10 days old plants as described for A. caerulea(9).
Silencing of PDS is a good marker for VIGS because it gives a clear phenotype and is
routinely used in VIGS experiments including when developmental processes are analysed
(eg.8, 10-14). PDS is active in green tissues. NAM and CUC3 on the other hand are expressed
in regions of the growing apex. The peak of PDS silencing seen as completely white leaves
and of NAM/CUC seen as altered developmental patterns will therefore appear in different
parts of the plant. So when silencing peaks, the bleaching phenotype will appear in the leaves
whereas the peak in phenotype of NAM/CUC silencing will become visible only later when
the young organs that were affected by the silencing expand to a mature organ. Therefore, the
developmental effect of NAM/CUC3 silencing and the bleaching of the leaf are not always
strictly associated.
1.
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2.
C. Helliwell, P. Waterhouse, Methods 30, 289 (2003).
3.
R. P. Hellens, E. A. Edwards, N. R. Leyland, S. Bean, P. M. Mullineaux, Plant Mol
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6.
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8.
G. D. Constantin et al., Plant J 40, 622 (2004).
9.
E. M. Kramer et al., Plant Cell 19, 750 (2007).
10.
Y. Liu, M. Schiff, S. P. Dinesh-Kumar, Plant J 31, 777 (2002).
11.
T. M. Burch-Smith, M. Schiff, Y. Liu, S. P. Dinesh-Kumar, Plant Physiol 142, 21
(2006).
12.
Y. Dong, T. M. Burch-Smith, Y. Liu, P. Mamillapalli, S. P. Dinesh-Kumar, Plant
Physiol 145, 1161 (2007).
13.
B. Gould, E. M. Kramer, Plant Methods 3, 6 (2007).
14.
F. Ratcliff, A. M. Martin-Hernandez, D. C. Baulcombe, Plant J 25, 237 (2001).
SOM Blein et al.,
Figure S1
Aquilegia caerulea
Pisum sativum
Cardamine hirsute
Solanum lycopersicum
Solanum tuberosum
Figure S1. Characteristics of species analysed in this study.
We have selected 5 broadly distributed and representative Eudicot species that form
compound leaves : a stem Eudicot (Aquilegia caerulea), two Rosids (the Eurosid I, Pisum
sativum and the Eurosid II, Cardamine hirsuta) and two close members of the Asterids (the
Euasterids Solanum lycopersicum and Solanum tuberosum) .This selection of species covers
the two main genetic pathways involved in compound leaf formation, the KNOXI pathway in
C. hirsuta and S. lycopersicum (1, 2), and the LEAFY pathway (UNIFOLIATA,UNI) in P.
sativum(3, 4), whereas no information regarding compound leaf formation is available for A.
caerulea and S. tuberosum. Leaflet primordia arise either basipetally (S. lycopersicum, S.
tuberosum and C. hirsuta) or acropetally (P. sativum). These species also show diverse levels
of margin dissection as A. caerulea, S. lycopersicum and C. hirsuta have lobes and/or
serrations. P. sativum shows leaflet specialisation with tendrils forming at the distal part of the
leaf. The Phylogenetic tree is taken from (5).
1.
2.
3.
4.
5.
D. Hareven, T. Gutfinger, A. Parnis, Y. Eshed, E. Lifschitz, Cell 84, 735 (1996).
A. Hay, M. Tsiantis, Nat Genet 38, 942 (2006).
C. E. Champagne et al., Plant Cell 19, 3369 (2007).
J. Hofer et al., Curr Biol 7, 581 (1997).
THE_ANGIOSPERM_PHYLOGENY_GROUP, Bot. J. Lin. Soc. 141, 399 (2003).
SOM Blein et al.,
Figure S2
A
AtNAC1
StCUC3
PsCUC3
AcCUC3
ChCUC3
94
AtCUC3
91
100
ChCUC1
AtCUC1
AcNAM
90
PsNAM2
100
57
100
PsNAM1
StNAM
SlNAM
ChCUC2
0.1
100
AtCUC2
B
ChCUC2
AtCUC2
StNAM
SlNAM
PsNAM1
PsNAM2
AcNAM
ChCUC1
AtCUC1
YLPPGFRFHPTDEELITHYLLRKVLDGCFSSRAIAEVDLNKCEPWQLPGRAKMGEKEWYFFSLRDRKYPTGLRTNRATEA
YLPPGFRFHPTDEELITHYLLRKVLDGCFSSRAIAEVDLNKCEPWQLPGRAKMGEKEWYFFSLRDRKYPTGLRTNRATEA
HLPPGFRFHPTDEELITYYLLKKVLDCNFTARAIAEVDLNKCEPWELPGKAKMGEKEWYFFSLRDRKYPTGLRTNRATEA
HLPPGFRFHPTDEELITYYLLKKVLDCNFTARAIAEVDLNKCEPWELPGKAKMGEKEWYFFSLRDRKYPTGLRTNRATEA
HLPPGFRFHPTDEELITFYLLKKVLDNTFTARAIAEVDLNKCEPWELPEKAKMGEKEWYFFSLRDRKYPTGLRTNRATEA
HLPPGFRFHPTDEELITFYLLKKVLDNTFTARAIAEVDLNKCEPWELPEKAKMGEKEWYFFSLRDRKYPTGLRTNRATEA
HLPPGFRFHPTDEELITFYLIKKVLDSTFTCRAIAEVDLNKCEPWELPEKAKMGEKEWYFFSLRDRKYPTGLRTNRATEA
LMPPGFRFHPTDEELITYYLLKKVLDSNFSCAAISQVNLNKSEPWELPEKAKMGEKEWYFFTLRDRKYPTGLRTNRATEA
LMPPGFRFHPTDEELITYYLLKKVLDSNFSCAAISQVDLNKSEPWELPEKAKMGEKEWYFFTLRDRKYPTGLRTNRATEA
ChCUC2
AtCUC2
StNAM
SlNAM
PsNAM1
PsNAM2
AcNAM
ChCUC1
AtCUC1
GYWKATGKDREIYSSKTCALIGMKKTLVFYKGRAPKGEKSNWVMHEYRLEGKFSYHFISRSSKDEWVISRVFQKT
GYWKATGKDREIFSSKTCALVGMKKTLVFYKGRAPKGEKSNWVMHEYRLEGKFSYHFISRSSKDEWVISRVFQKT
GYWKATGKDREIFSSKTCALVGMKKTLVFYRGRAPKGEKSNWVMHEYRLDDKFAYHYISRSSKDEWVISRVFQKS
GYWKATGKDREIFSSKTCALVGMKKTLVFYRGRAPKGEKSNWVMHEYRLDGKFAYHYISRSSKDEWVISRVFQKS
GYWKATGKDREIYSSKTYSLVGMKKTLVFYRGRAPKGEKSNWVMHEYRLEGKFAYHFLSRNSKDEWVISRVFQKS
GYWKATGKDREIYSSKTYSLVGMKKTLVFYRGRAPKGEKSNWVMHEYRLEGKFAYHFLSRNSKDEWVISRVFQKS
GYWKATGKDREIYSSRTSSLVGMKKTLVFYRGRAPKGEKSNWVMHEYRLEGKLSYHYLSRSSKDEWVISRVFQKS
GYWKATGKDREIKSSKTKSLLGMKKTLVFYKGRAPKGEKSSWVMHEYRLDGKFSYHYISSSAKDEWVLCKVCLKS
GYWKATGKDREIKSSKTKSLLGMKKTLVFYKGRAPKGEKSCWVMHEYRLDGKFSYHYISSSAKDEWVLCKVCLKS
80
80
80
80
80
80
80
80
80
154
154
154
154
154
154
154
154
154
C
StCUC3
PsCUC3
AcCUC3
ChCUC3
AtCUC3
GLPPGFRFHPTDEELITFYLASKVFNATFSAIQIPQVDLNRCEPWELPEVAKMGEREWYFFSLRDRKYPTGLRTNRATGA
GLPPGFRFHPTDEELITFYLASKVFKNTFFNVKFAEVDLNRCEPWELPDMAKMGEREWYLFSLRDRKYPTGLRTNRATGA
GLPPGFRFHPTDEELITFYLASKVFNGRFCGVEIAEIDLNRCEPWELPDIAKMGERVWYFFSQRDRKYPTGLRTNRATEA
GLPPGFRFHPTDEELISFYLASKVFDGGLCGIHITEVDLNRCEPWELPEMAKMGEKEWYFYSLRDRKYPTGLRTNRATTA
GLPPGFRFHPTDEELITFYLASKIFHGGLSGIHISEVDLNRCEPWELPEMAKMGEREWYFYSLRDRKYPTGLRTNRATTA
StCUC3
PsCUC3
AcCUC3
ChCUC3
AtCUC3
GYWKATGKDREVYSATNGALLGMKKTLVFYKGRAPRGEKTKWVMHEYRLDGDFSY-R-Y-SCKEEWVICRILHKV
GYWKATGKDKEVYSNSTRALLGMKKTLVFYKGRAPRGEKTKWVMHEYRLHTHLSP---S-TCKEEWVICRIFHKS
GYWKATGKDKEVYSASDDSLLGMKKTLVFYKGRAPRGVKTKWVMHEYRLEGDFSS-F-SHTFKEEWVLCRILQKT
GYWKATGKDKEVFGSGGGQLVGMKKTLVFYKGRAPRGLKTKWVMHEYRLETDLS-HR--HSCKEEWVICRVFNKT
GYWKATGKDKEVFSGGGGQLVGMKKTLVFYKGRAPRGLKTKWVMHEYRLENDHS-HR--HTCKEEWVICRVFNKT
151
150
152
151
151
80
80
80
80
80
SOM Blein et al.,
Figure S2
D
ChCUC2
AtCUC2
StNAM
SlNAM
PsNAM1
PsNAM2
AcNAM
ChCUC1
AtCUC1
miR164
5’-AGCACGUGUCCUGUUUCUCCA-3’
5’-AGCACGUGUCCUGUUUCUCCA-3’
5’-AGCACGUGCCCUGUUUCUCCA-3’
5’-AGCACGUGCCCUGUUUCUCCA-3’
5’-AGCACGUGUCCUGUUUCUCCA-3’
5’-AGCACGUGUCCUGUUUCUCCA-3’
5’-AGCACGUGCCCUGUUUCUCCA-3’
5’-AGCACGUGUCCUGUUUCUCCA-3’
5’-AGCACGUGUCCUGUUUCUCCA-3’
x..
3’-ACGUGCACGGGACGAAGAGGU-5’
Figure S2. Phylogenetic analysis of the NAM/CUC genes.
A. The amino acids of the NAC domains of the NAM/CUC proteins were aligned using the
multiple sequence alignment software ClustalX (v1.83)(1). Maximum likelihood (heuristic
search tree) from this multiple sequence alignment were generated with PhyML(2) using
optimal amino acide substitution model JTT + G select using ModelGenerator (v0.84)(3). The
NAC1 protein from A. thaliana was used as outgroup. Bootstrap confidence values were
obtained by 100 replicates and only bootstrap values above 50% are indicated.
B, C. Alignments of the NAC domain of the NAM and CUC3 proteins are represented in B
and C, respectively.
D. Alignment of the miR164 binding site in the NAM genes showing the complementarity
with miR164
The phylogenetic analysis on the conserved DNA-binding NAC domain showed that the
NAM/CUC genes group either into the NAM clade (AcNAM, SlNAM, StNAM, PsNAM1,
PsNAM2, ChCUC1, ChCUC2) or the CUC3 clade (AcCUC3, StCUC3, PsCUC3 and
ChCUC3). Distinction between members of the two clades extends outside the NAC domain
as all members of the NAM clade contained a putative binding site for miR164, which was
absent in the members of the CUC3 clade. The predicted PsNAM1 and PsNAM2 proteins
showed 95.2% similarity suggesting that they result from a recent duplication. In contrast, the
predicted ChCUC1 and ChCUC2 proteins showed only 52.3% similarity and each showed a
higher sequence similarity to the corresponding A. thaliana protein than to the other C.
hirsuta gene (86.2% and 84.2% for the CUC1 and pairs, respectively). This suggested that
they result from a gene duplication event that took place before the separation between C.
hirsuta and A. thaliana 13-19 MYA(4).
1.
2.
3.
4.
J. D. Thompson, T. J. Gibson, F. Plewniak, F. Jeanmougin, D. G. Higgins, Nucleic
Acids Res 25, 4876 (1997).
S. Guindon, O. Gascuel, Syst Biol 52, 696 (2003).
T. M. Keane, C. J. Creevey, M. M. Pentony, T. J. Naughton, J. O. McLnerney, BMC
Evol Biol 6, 29 (2006).
M. Koch, B. Haubold, T. Mitchell-Olds, Am J Bot 88, 534 (2001).
SOM Blein et al.,
AcNAM
lp
m lp
S. tuberosum
AcNAM
lp
D
B
G
SlNAM
I
SlNAM
s
H
fm
AcH4
F
AcH4
stp
J
SlNAM
st
st
fm
st
o
s
StNAM
m
L
R
fm
s
s
fm
SlNAM
M
fm
PsNAM1/2
m
StCUC3
s
S
Q
s
P
ChCUC3
m
ChCUC2
m
fm
PsCUC3
m
lp
ChCUC1
m
lp
N
s
O
StCUC3
m
lp
StNAM
s
E
lp
m
m
s
stp
s
AcCUC3
lp
AcNAM
m
lp
K
P. sativum
C
C. hirsuta
S. lycopersicum
A. caerulea
A
FigureS3
T
PsNAM1/2
sp cmp
VPsNAM1/2 X
sp
PsNAM1/2
o
cw
lp
U
PsCUC3
W
PsH4
Y
PsH4
sp cmp
sp
o
cw
SOM Blein et al.,
FigureS3
Figure S3. NAM/CUC and Histone H4 expression patterns in meristem and reproductive
structures of selected Eudicots: A. caerulea (A-F), S. lycopersicum (G-J), S. tuberosum (KN), C. hirsuta (O-Q) and P. sativum (R-Y).
In A. caerula, AcNAM and AcCUC3 are expressed at the boundaries between leaf primordia (lp)
and the meristem (m) during the formation of the rosette leaves (A, B) and after bolting (C). In
the floral meristem (fm), AcNAM marks the boundary of the numerous stamen primordia (stp, D).
In the vegetative and floral meristem, Histone H4 expression, a marker of dividing cells is downregulated in the domain where AcNAM is expressed (arrows in E and F, compare AcNAM and
Histone H4 expression in C-E and D-F respectively)
SlNAM marks the boundary of the organ primordia in the apical (G) and floral meristem (H, I) of
S. lycopersicum. SlNAM is also expressed at the boundaries between outgrowing ovules (o) and at
the boundary between inner and outer locules of the stamens (st in J).
StNAM and StCUC3 expression in apical and floral meristems of S. tuberosum (K-N).
ChCUC1, ChCUC2 and ChCUC3 show similar expression patterns at the boundary of the
primordia and the meristem in C. hirsuta (O-Q)
PsNAM1/2 and PsCUC3 are expressed at the boundary between organ primordia and meristem of
P. sativum (R, S). Their expression precedes the outgrowth of the primordium in the meristem
and remains detectable in the axillary region of older leaves. In the flower, PsNAM1/2 and
PsCUC3 are expressed in the boundary region of the floral organs (T, U). Pea flowers contain
particular structures called common primordia (cmp) that will be divided into two to generate
petal and a stamen primordia. PsNAM1/2 and PsCUC3 expression can be observed in a fine
domain corresponding to the boundary between the petal and stamen primordia (arrows in T and
U). Within the apex, histone H4 expression is reduced in the region where PsNAM1/2 is
expressed (compare V and W, arrow in W). PsNAM1/2 is also expressed at the inner boundary of
outgrowing teguments within ovules (X), a domain from which Histone H4 expression is
excluded (arrows in Y, compare two successive sections X and Y).
cmp: common primordium; cw: carpel wall; fm: floral meristem; m: meristem; lp: leaf
primordium; o: ovule; s: sepal; sp: sepal primordium; stp: stamen primordium
Bars: 0.1mm
S. tuberosum S. lycopersicum
SOM Blein et al.,
FigureS4
A
SlNAM
ltp1
*
ltp2
1
B
*
2
StNAM
3
4
ltp1
ltp2
*
1
2
SlNAM
C
3
4
5
S. lycopersicum
ltp1
1
m
2
m
3
4
7
8
3
4
ltp1’
5
6
P. sativum
D PsNAM1/2
ltp4
ltp3
ltp2
ltp1
1
2
Figure S4. Detailed analysis of NAM genes expression in serial sections of S. lycopersicum
(A,C), S. tuberosum (B) and P. sativum (D).
A. SlNAM expression in an older S. lycopersicum leaf. Note that SlNAM is not expressed in the
centre of the region between leaflet primordia 1 and 2 (ltp1, ltp2) but is asymmetrically associated
with the distal part of the leaflet primordium (arrow in 2). No SlNAM expression is observed on the
proximal side of the leaflet primordia 1 and 2 (region marked by an asterisk).
B. StNAM expression in an intermediary S. tuberosum leaf. No expression of StNAM is observed on
the proximal side of the young leaflet primordium (ltp2, region marked by an asterisk).
C. SlNAM expression in an young S. lycopersicum leaf. Two leaflet primordia are present (ltp1 and
ltp1’). Expression of SlNAM is observed below these two leaflet primordia (arrows) but no sign of
further leaflet primordium is observed, indicating that SlNAM expression precedes the outgrowth of
the leaflet primordia.
D. PsNAM1/2 expression in a young P. sativum leaf. Polarised PsNAM1/2 expression is observed in
the distal part of primordia 1, 2 and 3 (arrows). Note that strong PsNAM1/2 expression is observed
in distal part of leaflet primordium 4 that has just started to grow out.
Leaflet primordia are numbered from the oldest to the youngest. Note that in S. lycopersicum and S.
tuberosum leaflet primordia form basipetally whereas they form acropetally in P. sativum.
Bars: 0.1mm
SOM Blein et al.,
FigureS5
B
H2O NS
TRV2
AcPDS
NS
S
TRV2
AcPDS
AcCUC3
NS
S
TRV2
AcPDS
AcNAM
AcCUC3
NS
S
2x35S:GFP
_
TRV2
AcPDS
AcNAM
NS
S
2x35S:MIR164B
TRV1 +
cDNA
ChCUC3 RNAi
A
DNA H2O
AcNAM
ChCUC1
AcCUC3
ChCUC2
TRV1
Ch CUC3
TRV2
ChEF1
AcActine
C
PEBV1 +
D
PEBV2
PEBV2 PEBV2 PsPDS
PEBV2 PsPDS PsPDS PsNAM
PsPDS PsNAM PsCUC3 PsCUC3 H2O
_
PsNAM
TRV1 +
TRV2
TRV2 SlPDS
SlPDS SlNAM H2O DNA
SlNAM
PsCUC3
TRV1
PEBV1
TRV2
PEBV2
PsEF1
SlGAPDH
Figure S5. Semi-quantitive RT-PCR experiments revealing the reduction of the NAM/CUC genes
expression following transient or stable gene silencing.
A. Transient TRV-based VIGS in A. caerulea. TRV1 refers to the RNA1 of the TRV virus and TRV2
refers to the RNA2 in which fragments of the AcPDS, AcNAM and/or AcCUC3 genes were inserted.
B. Stable silencing in stable C. hirsuta.
C. Transient PEBV-based VIGS in P. sativum. PEBV1 refers to the RNA1 of the PEBV virus and
PEBV2 refers to the RNA2 in which fragments of the PsPDS, PsNAM and/or PsCUC3 genes were
inserted.
D. Transient TRV-based VIGS in S. lycopersicum. TRV1 refers to the RNA1 of the TRV virus and
TRV2 refers to the RNA2 in which fragments of the SlPDS and SlNAM genes were inserted.
H20: control PCR with no template, DNA: control PCR with genomic DNA as template, NS: cDNA
extracted from non silenced tissues, S: cDNA extracted from silenced tissues.
SOM Blein et al.,
Figure S6
No VIGS
A
C
AcPDS
B
AcPDS AcCUC3
*
*
% of plants
A. caerulea
*
A
E
D
60%
AcPDS
50%
AcPDS
AcNAM
40%
AcPDS
AcCUC3
30%
AcPDS
AcNAM
AcCUC3
20%
10%
AcPDS AcNAM
0%
smoothed
simple
simple entire
leaflets dissected leaf
leaf
1
F
2
No VIGS
G
PsPDS
D
E
H
4
PsPDS
PsNAM
I
J
F
G
6
5
4
3
20%
***
PsPDS
PsNAM
PsPDS
PSCUC3
PsPDS
PsNAM
PsCUC3
PsPDS PsCUC3
10%
PsPDS PsNAM PsCUC3
*
*
3.5
3
*
*
*
2.5
2
1.5
1
0.5
Q
SlPDS
SlNAM
R
SlPDS
SlNAM
S
number of leaflets
9
II
8
7
50%
6
5
4
3
2
1
0
I
T
SlPDS
SlPDS SlNAM
primary secondary intercalary
leaflets
40%
30%
20%
10%
0%
***
SlPDS
***
P
0
% of leaflet fusion
No VIGS
1
***
0%
**
0
Int
PsPDS PsNAM
4
1
O
PsPDS
N
PsPDS
PsNAM
distance leaflet-tendril
7
***
8
*
9
30%
***
% of leaflet and tendril fusion
10
M
PsPDS
Distance leaflet-tendril
L
***
K
2
S. lycopersicum
PsPDS
PsNAM
PsPDS
PsNAM
PsCUC3
number of leaflets+tendrils
P. sativum
C
3
SOM Blein et al.,
Figure S6
Figure S6. Leaf phenotype induced by NAM/CUC gene silencing in compound leaf species.
A-E: Silencing of AcNAM and AcCUC3 in Aquilegia caerulea
A. Control leaf that was not infiltrated. Note the three leaflets (*) which are highly dissected into
three structures that are themselves lobed. The two lateral leaflets have been cut.
B, C. Leaves silenced for the AcPDS gene and for both the AcPDS and AcCUC3 genes. Note that the
architecture is unchanged compared to a leaf that was not infiltrated.
D. Successive leaves formed on a plant silenced for the AcPDS and AcNAM genes. Note the
progressive smoothening of the leaflet margins from leaf 1 to 3. At the final stage (leaf 4), simple
leaves with highly dissected margins are formed.
E. Scoring of the leaf phenotypes in the different conditions. “smoothed margins” refers to leaves
such as leaf 3 in panel D, “simple dissected leaves” refers to leaves like leaf 4 in panel D and “simple
entire leaf” refers to leaves similar to leaf 4 in panel D but with smoothed margins.
F-N: Silencing of PsNAM/PsCUC genes in Pisum sativum
F. Control leaf that was not infiltrated.
G. Leaf silenced for the PsPDS gene. The overall architecture of the leaf is unchanged although the
leaf is smaller compared to a leaf that was not infiltrated.
H. Leaf silenced for the PsPDS PsNAM genes. The pairs of leaflets are not properly spaced along the
rachis and leaflets are fused to the rachis (arrows).
I. Leaf silenced for the PsPDS, PsNAM and PsCUC3 genes. Note the fusion of the tendrils (arrows)
detailed in the inset (Bar=1mm, for the detail).
J. Leaves silenced for the PsPDS PsNAM genes with no leaflet that terminates with a pair of tendrils
and a terminal tendril.
K. Quantification of the leaflet and tendril number.
L. Quantification of leaflet and tendril fusion.
M. Leaf silenced for the PsPDS PsNAM genes. The reduction of the leaflet and tendril number is
reflected in a longer distance between the last pair of leaflets and the first couple of tendrils.
N. Quantification of the distance between the last pair of leaflets and the first couple of tendrils.
n=64 for PsPDS, n=14 for PsPDS PsNAM, n=3 for PsPDS PsCUC3, n=20 for PsPDS PsNAM
PsCUC3 leaves (rank 17-19) of the main stem were analysed in K, L and N
O-T: Silencing of SlNAM in Solanum lycopersicum
O. Control leaf that was not infiltrated. “I” and “II” point respectively to a primary and secondary
leaflets, “int” points to an intercalary leaflet.
P. Leaf silenced for the SlPDS gene. Note that although the size of the leaf is reduced, the overall
architecture is unchanged compared to a leaf that was not infiltrated.
Q,R. Leaves silenced for the SlPDS SlNAM. Note the absence of secondary leaflets and the reduction
of the intercalary leaflet number. The leaflet margins are smoothed (arrowheads) and fusion between
leaflets or with the rachis occur (arrow).
S. Quantification of leaflet number.
T. Quantification of leaflet fusion.
n=33 for SlPDS, n=63 for SlPDS SlNAM (rank 5-7) of the main stem were analysed in K, L and N
Error bars=SE. Significance of the differences with the control values (PDS control) were assessed by
a Student’s t test (*=P<0.05;**=P<0.01; ***=P<0.001)
Bars=1cm when another value is not specified.
SOM Blein et al.,
FigureS7
gob2 e3883-m1
G378A G95D
A
95
272 273
gob1 e1976-m1
C593T Q167Stop
gob3 n5126-m1
28 nt deleted (1001-1030)
556 557
1153
Intron 1, 144nt
100nt
Intron 2, 782nt
B
D
C
gob3
WT
gob3
E
WT
gob3
G
F
gob3
H
gob3
WT
I
J
WT
WT
gob3
gob3
gob3
gob3
WT
gob3
Figure S7. Molecular and Phenotypic characterisation of the tomato goblet mutants
A. LeNAM structure and mutations. The cDNA, the ORF (thick lines) and the introns are shown.
The effects of the 3 goblet mutations and the corresponding lines are represented (the modification
of the nucleotide and of the amino acid are indicated)
B, C. Wild-type and gob3 seedlings showing the fused cotyledons and the absence of leaf
development in the gob3 line.
D, E. Longitudinal sections through a wild-type and a gob3 seedling revealing the absence of
meristem in the gob3 line.
F. Regenerated gob3 plant
G. Leaves from regenerated wild-type and gob3 plants showing the simplified leaf structure and the
smoothed margins of the gob3 line
H. Inflorescences from regenerated wild-type and gob3 showing the modified structure of the gob3
plant
I. Flowers from regenerated wild-type and gob3 showing the increased sepal fusion of the gob3 plant
J. Fruits of a wild-type and a regenerated gob3 plant. Note the outer floral organs fused to the fruit
(arrowheads) and the absence of seeds in the gob3 plant
Bars=1cm
SOM Blein et al.,
FigureS8
A Wild type
2x35S:MIR164B
R4
R8
R4
Wild type
C1
2x35S:MIR164B
C2
C1
R8
ChCUC3 RNAi
C2
C1
C2
2x35S:MIR164B
6
ChCUC3 RNAi
2x35S:MIR164B
ChCUC3 RNAi
4
3
2
***
50%
40%
30%
20%
0%
R1
R2
R3
R4
R5
R6
leaf rank
R7
R8
R9
R10
10
Rosette leaves 1-10
60%
7
WT
6
2x35S:GFP
5
2x35S:MIR164B
4
3
ChCUC3 RNAi
2
2x35S:MIR164B
ChCUC3 RNAi
1
0
C1
C2
leaf rank
C3
50%
40%
***
8
***
9
% of leaflet fusion
number of leaflets
60%
10%
1
D
C2
70%
7
5
C1
80%
2x35S:GFP
8
R8
2x35S:MIR164B
ChCUC3 RNAi
WT
9
0
R4
***
10
R4
30%
20%
***
number of leaflets
C
R8
2x35S:MIR164B
ChCUC3 RNAi
% of leaflet fusion
B
ChCUC3 RNAi
10%
0%
Cauline leaves 1-3
Figure S8. Leaf phenotype induced by ChCUC1, ChCUC2 and ChCUC3 silencing in Cardamine
hirsuta
A. Rosette leaf 4 (R4) and 8 (R8). Arrows point to examples of fusion between leaflets or between a
leaflet and the rachis.
B. First (C1) and second (C2) cauline leaf. Arrowheads indicate smoothed leaflet margins. Arrows
point to examples of fusion between leaflets or between a leaflet and the rachis.
C. Quantification of the rosette leaf parameters.
D. Quantification of the cauline leaf parameters
10 plants analysed. Bars=SE. Student’s t test ***= P<0.001
Bars=1cm
SOM Blein et al.,
A
la
B
FigureS9
SlNAM
C
uni
D
PsNAM1/2
E
PsCUC3
Figure S9. The NAM/CUC genes act downstream of genes required for leaflet formation
in S. lycopersicum and P. sativum.
The la mutant forms a simple leaf, which shows some lobes but no leaflets (A). No
expression of SlNAM is detected in a young developing leaf of the la mutant (B) in contrast
to wild type. SlNAM is still expressed in the meristem (arrow).
The uni pea mutant forms a simple leaf (C). The expression of the PsNAM1/2 and PsCUC3
genes is not detected in the developing uni leaf (D,E) in contrast to wild type. PsNAM1/2
and PsCUC3 expression is still observed in the meristem and in the leaf axils (arrows).
SOM Blein et al.,
FigureS10
A
B
Distal
Cell
proliferation
Leaf axis
NAM
CUC
ltp
ltp
ltp
Leaflet
formation
Proximal
KNOX1
LFY-like
Leaf Margin
Figure S10. Role for the NAM/CUC genes in leaf dissection and promotion of leaflet formation
A. View of a tomato apex showing the developing leaflet primordia (ltp).
B. Schematic representation of the role of the NAM/CUC genes during the formation of the leaflet
highlighted in red in A. The NAM/CUC genes are expressed in discrete strips along the margin of the
leaf primordium (shown here in yellow). This leads in local repression of cell proliferation, and thus
to the formation of a groove. At the same time, the NAM/CUC genes promote cell proliferation in the
cells proximal to their expression domain, thus promoting leaflet formation. This long distance effect
of the NAM/CUC genes may rely on movement of the NAM/CUC proteins themselves, or on the
production of a mobile signal. NAM/CUC genes are part of a feed-forward regulatory loop with
KNOX1 and LFY-like genes, activating their expression and being activated by them.
Supplementary Table 1. Quantification of VIGS effects in Aquilegia caerulea
survivorsa
VIGS positiveb
leaf phenotyped
nb of
infiltrated
plants
nb of plants
%
nb of plants
%c
nb of plants
%e
TRV1 + TRV2 AcPDS
96
88
92%
46
52%
0
0%
TRV1 + TRV2 AcPDS AcNAM
107
97
91%
58
60%
33
57%
TRV1 + TRV2 AcPDS AcCUC3
108
106
98%
65
61%
0
0%
TRV1 + TRV2 AcPDS AcNAM AcCUC3
131
121
92%
49
40%
27
55%
Constructs
a
determined 4 weeks after inoculation
determined 4 weeks after inoculation as plants with white sectors
c
of survivors
d
determined 8 weeks after inoculation as plants that show reduced leaf complexity
e
of plants showing sectors
b
Supplementary Table 2. Quantification of VIGS effects in Solanum lycopersicum
survivorsa
VIGS positiveb
leaf phenotyped
nb of
infiltrated
plants
nb of plants
%
nb of plants
%c
nb of plants
%e
TRV1 + TRV2 SlPDS
38
37
97%
37
100%
0
0%
TRV1 + TRV2 SlPDS SlNAM
50
49
98%
49
100%
38
78%
Constructs
a
determined 4 weeks after inoculation
determined 4 weeks after inoculation as plants with white sectors
c
of survivors
d
determined 8 weeks after inoculation as plants that show reduced leaf complexity
e
of plants showing sectors
b
Supplementary Table 3. Quantification of VIGS effects in Pisum sativum
survivorsa
VIGS positiveb
leaf phenotyped
nb of
infiltrated
plants
nb of plants
%
nb of plants
%c
nb of plants
%e
PEBV1 + PEBV2 PsPDS
93
93
100%
79
85%
0
0%
PEBV1 + PEBV2 PsPDS PsNAM
95
94
99%
81
86%
45
56%
PEBV1 + PEBV2 PsPDS PsCUC3
95
95
100%
80
84%
4
5%
PEBV1 + PEBV2 PsPDS PsNAM PsCUC3
92
92
100%
77
84%
17
22%
constructs
a
determined 6 weeks after inoculation
determined 6 weeks after inoculation as plants with white sectors
c
of survivors
d
determined 6 weeks after inoculation as plants that show reduced leaf complexity
e
of plants showing sectors
b
The experiments were repeated twice and the numbers indicated in the tables are the sum of the independant experiments.
Supplementary Table 4. Primers used for semi quantitive RT-PCR
primer 1 name
primer 1 sequence (5'->3')
primer 2 name
primer 2 sequence (5'->3')
Reference
Aquilegia caerulea
species
AcActine
gene
AcActine-1
GAT GGA TCC TCC AAT CCA GAC ACT GTA
AcActine-2
GTA TTG TGT TGG ACT CTG GTG ATG GTG T
Kramer et al. (2007) Plant Cell, 19, 750-766
Aquilegia caerulea
AcCUC3
Ac CUC3 Fw
TCC ATC ACA TAG CTT CTT CTT
Ac CUC3 Rv
AGC ATC CTG AGG TCC ATA TTG
This work
Aquilegia caerulea
AcNAM1
Ac NAM1 Fw
TCA ATG GAG ATC ATT AGT TTT
Ac NAM1 Rv
TAC CCT TCA CAT AGC TAG CAA
This work
Cardamine hirsuta
ChCUC1
Ch CUC1 Fw3
CAA CAG AAG CTG GTT ACT GGA AA
Ch CUC1 Stop
TCA GAG AGC AAA CGG CCA GTA ACT
This work
Cardamine hirsuta
ChCUC2
Ch CUC2 Start
ATG GAC ATT CCA TAC TAC CAC TA
Ch CUC2 Rv1
TAG AGA TCA CCC ATT CAT CCT T
This work
Cardamine hirsuta
ChCUC3
Ch CUC3 Start
ATG ATG CTT GCG GTA GAA GAT GT
Ch CUC3 Rv2
CCA AAG ACC TCC TTA TCT TTT
This work
Cardamine hirsuta
EF1
EF1af
ATG CCC CAG GAC ATC GTG ATT TCA T
EF1ar
TTG GCG GCA CCC TTA GCT GGA TCA
This work
Pea Early Browning Virus
PEBV1
PEBV1-Rv
GAA AGA ACC CAA TCA AT
PEBV1-Fw
TGG CAT AAA TAT AAT ACT TGT
This work
Pea Early Browning Virus
PEBV2
PEBV2-Rv
ATT TTG GGC ACA TAT CAA AC
PEBV2-Fw
GCT GCG GTT AGA AAT AAG AA
This work
PsCUC3-Start
ATG ATG TTA GCA ATG GAA GA
PsCUC3-Stop
TTA AAC AAG CTG AGC CTG T
This work
AGG GAG ACA ACA TGA TTG AG
RTPsEF1aRv
TAA CCA TTT CCA ATC TGT CC
This work
Pisum sativum
PsCUC3
Pisum sativum
PsEF1
Pisum sativum
PsNAM1/2
PsNAM-Start
ATG GAG GGC TTC TAC CA
PsNAM2-Stop
TTA ATA ATT CCA CAT GCA AT
This work
Pisum sativum
UNI
RTPsUNIFwd
GGT ATG CAA AGA AAG CTG
RTPsUNIRv
ATT GGT GGA AGG GAG GTT TT
Bai and DeMason (2006) Plant Cell Physiol, 47, 935-48
Lae/e R
TTC AAG TAG AGC ATT TCC CTG TCA
LaCS L
GGG ACC CCT TCA GTC CAG TA
Ori et al. (2007) Nat. Genet. 39, 787-91
RTPsEF1aFwd
Solanum lycopersicum
LA
Solanum lycopersicum
SlGAPBH
SlGAPDH-F
TGG AAT CAG GAA CCC TGA AG
SlGAPDH-R
GAT CGA CAA CGG AGA CAT CA
Jasinski et al. (2007) Planta, 226, 1255-63
Solanum lycopersicum
SlLFY
SlLFY-Fw1
TGG AGG AAT AAG CGA GAG A
SlLFY-Rv1
GGA CAT ACC AAA TGG CTA GTC
This work
Solanum lycopersicum
SlNAM
Sl NAM5'
ATG GAG ATT TAT CAT CAG ATG CA
SlNAM3'
TCA GTA GCT CCA CAT ACA GTC AA
This work
Solanum lycopersicum
SlPHAN
SlPHAN-F
GCT GAA GAG GAT GCT TTG
SlPHAN-R
TGC TCG TCT TCA GTG AGT GAT C
Jasinski et al. (2007) Planta, 226, 1255-63
Solanum lycopersicum
SlT6/TKn2
SlT6/Tkn2-F
TAA GCA GGA GTT CAT GAA GAA G
SlT6/Tkn2-R
TAC ATG CAC ACA AGT AAT ATG C
Jasinski et al. (2007) Planta, 226, 1255-63
Solanum lycopersicum
TKn1
Tkn1-F
AGA GAT CTC GTT GAG TAG TTG
Tkn1-R
GTT AGA ACA TTG AGG ATG AGC
Jasinski et al. (2007) Planta, 226, 1255-63
Tobacco Rattle Virus
TRV1
TRV1-1
CTT GAA GAA GAA GAC TTT CGA AGT CTC
TRV1-2
GTA AAA TCA TTG ATA ACA ACA CAG ACA AAC
Hileman et al. (2005) Plant J, 44, 334-341
Tobacco Rattle Virus
TRV2
TRV2-1
GGT CAA GGT ACG TAG TAG AG
TRV2-2
CGA GAA TGT CAA TCT CGT AGG
Hileman et al. (2005) Plant J, 44, 334-341
Supplementary Table 5. Primers used for the isolation of the NAM/CUC genes
primer name
primer sequence (5'->3')
AcCUC3Fw1
TCC ATC ACA TAG CTT CTT CTT
AcCUC3Rv1
AGC ATC CTG AGG TCC ATA TTG
AcNAMFw1
TCA ATG GAG ATC ATT AGT TTT
AcNAMRv1
TAC CCT TCA CAT AGC TAG CAA
CUC2 3'
TCA GTA GTT CCA AAT ACA GTC
cuc3uj2
AGC TTA TCA CTT TTT ACT TAG CCT C
degCUC3Fw1
GGT TYC AYC CNA CTG AYG ARG AGC T
degCUC3Fw2
GAT CTY AAY MGN TGT GAR CCN TGG GA
degCUC3Rv1
CTG CAN ATN ACC CAT TCY TCC TT
degCUC3Rv2
TCR AGN CGR TAY TCR TGC AKR ACC CA
insitu CUC3 L Fwd
ATG ATG CTT GCG GTG GAA GA
insitu CUC3 L Rv+T7
TGT AAT ACG ACT CAC TAT AGG GCC TGA TAC TAT TTC GCA AGA C
miR164bsdegRv1
TGG AGA ARC AGG RCA CGW GCT C
NACdegFw1
TYG AYC TSA ACA AGT GYG AGC CWT GG
NACdegFw2
ATG GGR GAG AAR GAG TGG TAC TTY T
NACdegRv1
TCR AGN CGR TAY TCR TCC ATR ACC CA
NACdegRv2
TAR AAR ACD AGN GTY TTC TTC AT
nam2
TAA CTC CAC ATG CAA TCA AGT TCA G
nam5
GAG CAC GTG TCC TGT TTC TCC A
revcuc3uj3
CGG TAA GAG AAA TCA CCG TCA AGA CG
RLT-Fwd-AT5G53950
CAG CCG TAG CAC CAA CAC AA
SlNAM3'
TCA GTA GCT CCA CAT ACA GTC AA
SlNAM5'
ATG GAG ATT TAT CAT CAG ATG CA
Supplementary Table 6. Probes used for in situ hybridisation
gene
length
Aquilegia caerulea
species
AcNAM
1141
position primer 1 sequence (5'->3')
1-1140
AcNAMFw1
TCA ATG GAG ATC ATT AGT TTT
primer 2 sequence (5'->3')
in situ Ac NAM1 T7
TGT AAT ACG ACT CAC TAT AGG GCT ACC CTT CAC ATA GCT AGC AA
Aquilegia caerulea
AcCUC3
1131
1-1130
AcCUC3Fw1
TCC ATC ACA TAG CTT CTT CTT
insitu CUC3 L Rv+T7
TGT AAT ACG ACT CAC TAT AGG GCC TGA TAC TAT TTC GCA AGA C
Cardamine hirsuta
ChCUC1
844
343-1186 ChCUC1fd2
CCC AAC GGG ACT GAG AAC GAA CA
insitu ChCUC1 Rv T7
TGT AAT ACG ACT CAC TAT AGG GCG AAG GAT AGA AGT CAA TAT TTT AAA A
Cardamine hirsuta
ChCUC2
1156
104-1259 insituCUC2LFwd
ATG GAC ATT CCG TAT TAC CA
insitu ChCUC2 Rv T7
TGT AAT ACG ACT CAC TAT AGG GCA ACA AAC CCT AAC GGG ACA CAC
Cardamine hirsuta
ChCUC3
1008
75-1082
insituCUC3LFwd
ATG ATG CTT GCG GTG GAA GA
insitu CUC3 L Rv T7
TGT AAT ACG ACT CAC TAT AGG GCC TAC AGC TGG AAT CCT AAA
Pisum sativum
PsCUC3
1242
1-1241
PsCUC3Fd3
CAT TTT CAT TCA TCA TCA TCT A
T7-PsCUC3Rv3
TGT AAT ACG ACT CAC TAT AGG GCT ATT CTA CAC ATG TAG AGT TA
Pisum sativum
PsNAM1/2
1180
15-1194
PsNAM5'UTR1
TAA CGG CTC TAC TCT CTC TCT CAT TT
T7PsNAM1Rv
TGT AAT ACG ACT CAC TAT AGG GCT TCC ACA TGC AAT CAA GCT
Solanum lycopersicum
SlNAM
1208
95-1302
SlNAM5'
ATG GAG ATT TAT CAT CAG ATG CA
insitucucrev4
TGT AAT ACG ACT CAC TAT AGG GCA GCA TGC ATT AAA TTA CGC TGA ACG
Solanum tuberosum
StCUC3
1166
8-1173
StCUC35'
CAT ATC TTG AAA ATC TCT CAC TTT T
in situ St CUC3 T7
TGT AAT ACG ACT CAC TAT AGG GCG GCT AAA GGG TAG CTC TTT ATA TT
Solanum tuberosum
StNAM
1063
1-1062
SlNAM5'
ATG GAG ATT TAT CAT CAG ATG CA
insitucucrev4
TGT AAT ACG ACT CAC TAT AGG GCA GCA TGC ATT AAA TTA CGC TGA ACG
For each probe, its length and position are inidicated as well as the primers used to amplify the DNA template used for the in vitro transcription. The sequence of the T7 promoter is underlined
Supplementary Table 7. Sequence used for the transient and stable NAM/CUC gene silencing
gene
length
position primer 1 sequence (5'->3')
primer 2 sequence (5'->3')
Aquilegia caerulea
species
AcCUC3
587
579-1165 AcCUC3BamHI
GGA TCC ACT ATT CCA TGC CCA CAA C
AcCUC3SacI
GAG CTC AGC ATC CTG AGG TCC ATA TTG
Aquilegia caerulea
AcNAM1
598
543-1140 AcNAM1SacI
GAG CTC TAG TGG TAC AGG GAG TAA
AcNAM1XhoI
CTC GAG TAC CCT TCA CAT AGC TAG CAA
Cardamine hirsuta
ChCUC3
469
616-1084 ChCUC3XbaI
TCT AGA TAG CCA AAT CAG CTG CCT C
ChCUC3HindIII
AAG CTT GCC TAC AGC TGG AAT CCT AAA
Cardamine hirsuta
ChCUC3
469
616-1084 ChCUC3EcoRI
GAA TTC ATA GCC AAA TCA GCT GCC TC
ChCUC3KpnI
GGT ACC TAC AGC TGG AAT CCT AAA
Pisum sativum
PsCUC3
508
688-1195 PsCUC3BamHI
GGA TCC CCC CAA AAA GCT GTC TAC
PsCUC3SacI
GAG CTC CAA GAA TCA ATT CCC ATA GGA GC
Pisum sativum
PsNAM1/2
525
613-1137 PsNAM1SacI
GAG CTC ACC GCA AGT GGT TCA AAA AAG
PsNAM1XhoI
CTC GAG TCA ACC ACC CCT CTT TGC TC
SlNAM
511
752-1262 SlNAMSacI
GAG CTC CGC CGC AGC TAT CGT AAT C
SlNAMXhoI
CTC GAG CAA TAC CAC TAA AGC TGC CAC ACG
Solanum lycopersicum
For each gene, the length and position of the fragment used to induce the silencing are indicated as well as the primers used to amplify it. The sequence of the restrictions sites are underlined