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Supplementary Material Legends
Table S1. T-DNA insertion sites of target transgenes are defined by NCBI accession (acc.)
numbers for left border or right border T-DNA - A. thaliana chromosomal DNA fusion
sequences. Positions of nucleotides of A. thaliana genomic BAC sequences adjacent to left
border and right border T-DNA sequences are indicated in brackets after the BAC sequence
accession numbers (Forsbach et al., 2003). Left border genomic DNA-T-DNA fusion
sequences were available for 20 of the 26 target transgenes, right border fusion sequences for
23 of the 26. For target transgenes Kchr1-2, Kchr1-9, and Kchr2-2 only the left border genomic
DNA-T-DNA fusion site and for Kchr1-4, Kchr1-5, Kchr3-1, Kchr3-3, Kchr3-6 and Kchr5-3 only the right
border genomic DNA-T-DNA fusion site was known (Suppl. Info. 1). In these cases, it was
assumed that the T-DNA insertion had happened without DNA sequence deletion and
sequence feature analysis for the “unknown” side was started at the nucleotide directly
adjacent to the known genomic DNA-T-DNA fusion site. The newly analysed right border
flanking sequence of target transgene Kchr1-10 was found to correspond to a region of the
bottom arm of chromosome 1 distant from the left border flanking sequence indicating some
sequence rearrangement. All target transgenes (K) contained a single copy of the p35S-GUS
reporter gene (1xr), with exception of line Kchr1-3 which contained two p35S-GUS reporters
(2xr) arranged as a tandem repeat. Different repeat elements were optionally integrated
upstream of the p35S: “1xr” and “2xr” contained no repeat elements, “1xr pAL” 2 copies of a
180 bp centromeric repeat (Martinez-Zapater et al., 1986), “1xr 163” a 450 bp centromeric
repeat from chromosome 4 clone #163 (Thompson et al., 1996), “1xr 164” a 900 bp
centromeric repeat from chromosome 4 clone #164 (Thompson et al., 1996), “1xr 501” 1
copy of a 500 bp HindIII tandem repeat (Simoens et al., 1988), “1xr 502” 2 copies of a 500 bp
HindIII tandem repeat (Simoens et al., 1988) and “1xr rDNA” a fragment of the A. thaliana
rDNA intergenic spacer (Wanzenböck et al., 1997). For detailed information, please see also
Schubert et al., 2004.
Figure S1. Correlation analysis of NPTII real time PCR data (a) No correlation could be
found between the obtained medians of NPTII RNA levels and the variation of measured
values for each of the tested genotypes. Thus, the increased variation values seen in the
generally lower measurements from silenced (red diamonds) compared to non-silenced (blue
diamonds) target transgenes are not due to increased measurement errors. Plotted are the
medians of NPTII RNA levels adjusted to ACTIN2 versus the dispersion of the five measured
values measured for each genotype (Spearman rank order correlation coefficient -0.198;
P=0.158). (b) No clear correlation was found between the efficiency of TGS as measured by
relative NPTII levels and the non-silenced NPTII RNA levels for the different target
transgenes. Thus, efficiency of TGS is not determined by the activity of a target transgene in
the non-silenced state. Plotted are the relative NPTII levels after initiation of silencing in
percent versus the median NPTII RNA levels of the different target transgenes in the nonsilenced state (Spearman rank order correlation coefficient -0.493; P=0.011; Pearson product
moment correlation coefficient -0.274; P=0.175). It should be noted that in none of the cases
tested on kanamycin containing medium, differences in resistance could be detected between
F1 seedlings hemizygous for target transgene and silencer transgene in comparison to F1
seedlings hemizygous for only the target transgene (data not shown), even if plants of the
same genotypes showed substantial reduction of NPTII RNA levels in rosette leaves of
mature plants in the presence of the silencer. This observation hints at a gradual establishment
of pNOS-NPTII reporter gene inactivation from the seedling to the mature rosette stage F1
plants in response to the RNA silencing signal.
Figure S2. Additional DNA methylation analysis by Southern blot in F1 plants (a) Map
indicating cleavage sites of restriction enzymes and target region of probe used in methylation
analysis by Southern blots. DNA was derived from individual plants that were siblings of the
plants represented in Fig. 4b. A basic fragment containing the pNOS and part of the NPTII
gene was released by combined Bsu15I and PstI cleavage. (b) Representative results of
Southern analysis with methylation-sensitive restriction enzymes. After basic cleavage, either
no further restriction enzyme (-) or methylation-sensitive restriction enzymes Psp1406I (P,
CG methylation-sensitive) or NheI (Nh, non-CG methylation-sensitive) were added. Tested
were F1 plants hemizygous for the target transgene alone (K/-;-/-) or hemizygous for target
and silencer transgenes (K/-;H/-). Hybridisation signals at the same position as the ones in (-)
lanes indicate methylated, signals at lower positions non-methylated DNA. (c) Cummulative
quantitative analysis of Southern experiments. Quantification of scans of autoradiograms was
performed using TINA software. At least 2 individual plants were analysed per genotype.
Methylation levels in percent were calculated by devision of the intensities of hybridisation
signals resistant to the respective restriction enzymes by the intensities of (-) control signals.
CG, non-CG and average methylation levels are listed in comparison to the corresponding
relative NPTII levels. Good correlation is found between CG (correlation coefficient -0.739;
P=0,000) and non-CG methylation (correlation coefficient -0,567; P=0,014) and relative
NPTII levels. (d) Bar diagram illustrating the generally high correlation of relative NPTII
(grey bars) and average DNA methylation levels in presence of the silencer (black bars) for 18
target transgenes. Please note that Kchr2-3 is an exception to this rule.
Figure S3. Additional DNA methylation analysis by Southern blot in F3 plants (a) Additional
results of DNA methylation analysis by Southern blot for F3 plants doubly homozygous for
target and silencer transgenes in comparison to plants homozygous for respective target
transgenes only. DNA was derived from individual plants that were siblings of the plants
represented in Fig. 4b. Please refer to legend of Fig. 4 for experimental details.(b)
Comparison of DNA methylation levels in F3 versus F1 plants in presence of the silencer.
Quantitative analysis of Southern experiments was performed as described in the legend of
Suppl. Info. 3. Inhibition of restriction enzyme Psp1406I (P) was considered indicative for
CG methylation, inhibition of restriction enzymes NheI (Nh) and Alw26I (A) for non-CG
methylation
Figure S4. DNA methylation analysis by bisulfite sequencing (a) Comparison of DNA
methylation levels determined by bisulfite sequencing for selected target transgenes. The
quantitative analysis of bisulfite sequencing results [%] was confined to the region of the
pNOS sequence in target transgenes that is covered by the pNOS dsRNA arising from the
silencer transgene. Total “bisulfite” DNA methylation levels were calculated by deviding the
number of methylated cytosines by the total number of cytosines/100 in the analysed
sequences. Accordingly, separate DNA methylation levels for cytosines in CG, CNG and
CHH (H stands for A, C or T) context were calculated by deviding the number of methylated
cytosines in the particular context by the respective total number of cytosines/100 in the same
context in the analysed sequences. (b)-(h) pNOS top strand DNA methylation patterns
determined for F1 plants of genotypes: (b) Kchr1-10 / - ; - / - (c) Kchr1-10 / - ; H/ - (d) Kchr2-3 / - ; / - (e) Kchr2-3 / - ; H/ - (f) Kchr4-4 / - ; H/ - (g) Kchr5-2 / - ; - /- and (h) Kchr5-2 / - ; H/ - . Each row
of symbols indicates one sequenced clone. Circles indicate cytosines in CG context, squares
in CNG context and triangles in CHH context. Symbols filled in black represent methylated
cytosines, open symbols non-methylated cytosines. Positions for which DNA sequencing
results were ambiguous are filled in grey. A grey bar below the sequence indicates the region
covered by the pNOS dsRNA arising from the silencer. The sequence context in which
cytosine methylation occurs is of interest, as different DNA methyltransferases, MET1,
CMT3 and DRM1/DRM2, respectively, are thought to be responsible for methylation in the
different contexts. pNOS-NPTII methylation occurred predominantly in CNG context (76%
for Kchr1-10, 88% for Kchr4-4, 18% for Kchr5-2 and 49% for Kchr2-3), followed by CG (78% for
Kchr1-10, 68% for Kchr4-4, 11% for Kchr5-2 and 37% for Kchr2-3) and CHH (69% for Kchr1-10, 65%
for Kchr4-4, 4.6% for Kchr5-2 and 29% for Kchr2-3) in all cases.
Figure S5. Quantification of silencer-derived pNOS-siRNAs by Northern blot. SiRNAs
homologous to the pNOS sequence were detected by Nothern blot analysis of RNA fractions
enriched for small RNA molecules according to Aufsatz et al., 2002a. A 23 nt DNA
oligonucleotide running with approximately the same electrophoretic mobility as 21 nt RNA
molecules was employed as size standard. No pNOS siRNAs were detected in plants
containing target transgenes only. In F1 plants hemizygous for target and silencer transgenes,
comparable pNOS siRNA amounts were detected for all lines tested irrespective of the
corresponding relative NPTII levels. Thus, differences in silencing efficiency at different
target transgenes are not based on varying pNOS siRNA amounts.
Figure S6. Correlation of silencing efficiency with features of target integration sites
(a) The table summarize the data for initial reporter gene activity (NPTII RNA levels in nonsilenced hemizygous F1 plants) and silencing efficiency (relative NPTII levels in F1 plants in
presence of the silencer) at the different target transgenes with the presence of varying repeat
sequence elements within the T-DNAs and of different sequence features identified by in
silico database analysis of transgene-flanking region. A window of 10 kb (5 kb to the left and
right of the insertion site) was used to screen for genes, repeats, matrix attachment regions
(MARs), global DNA methylation clusters and matches to endogenous siRNAs. Repeat
elements integrated in different T-DNA types are defined in Suppl. Info. 1.
RG-index: Genomic sequences flanking transgene insertion sites to both sides were grouped
according to MAtDB and RepeatMasker annotations into genes (G), pseudogenes (PG) and
repetitive sequences (R). RG-index values were calculated by adding values 1 for a gene or
pseudogene and 0 for a repetitive sequence.
RG-Index left border: Genomic sequence bordering the transgene to the left border side of
the T-DNA (flanking the NOS-promotor) only were analysed as above. G indicates a gene,
PG a pseudogene and R a repetitive sequence.
MAR-sites: Matrix attachment regions were determined by the MAR-wiz1.5 program
including the core and the new MAR rules (Singh et al., 1997; http://futuresoft.org/MARWiz).
DNA-methylation cluster classes: According to Methylome database, absence of methylation
within the 10 kb was assigned to class 1, single site sparse methylation to class 2, single site
clustered methylation to class 3, multiple site clustered methylation to class 4 and extended
methylation to class 5.
#siRNA from MPSSplus: Matches of siRNA (20bp signature) in flanking sequences were
detected in the MPSSplus database.
siRNA cluster class: Accordingly, classes were assigned as class 0 for no siRNA match, class
1 for 1-5 non-clustered signatures, class 2 for 5-15 non-clustered signatures, class 3 for >10
clustered signatures and class 4 for >40 clustered signatures.
(b) No correlation could be found between the presence of particular repeat elements in target
T-DNA and non-silenced NPTII RNA levels (top panel, grey diamonds) or the relative NPTII
levels in F1 plants in presence of the silencer (bottom panel, red diamonds).
(c) No correlation could be found between the presence of matrix attachment regions (MARs)
within 5 kb adjacent to the target transgene insertion site and the non-silenced NPTII RNA
levels (left panel, grey diamonds; Spearman rank order correlation coefficient -0.024,
P=0.907; Pearson product moment correlation coefficient -0.048, P=0.816) or the relative
NPTII levels in F1 plants in presence of the silencer (right panel, red diamonds; Spearman
rank order correlation coefficient -0.046, P=0.823; Pearson product moment correlation
coefficient -0.073, P=0.722).
(d) Promotion of RNA-directed transcriptional gene silencing by repetitive sequences
flanking target transgenes.Genomic sequences flanking target transgenes at both sides were
grouped according to MAtDB (http://mips.gsf.de/proj/thal/db) and RepeatMasker
(http://repeatmasker.org/) database annotations into genes, pseudogenes and repetitive
sequences. Repeat / gene index values were calculated by adding value 1 for presence of a
gene or pseudogene and 0 for a repetitive sequence. Relative NPTII levels in F1 plants in
presence of the silencer were plotted on a logarithmic scale against the repeat / gene index.
Kruskal-Wallis one way analysis of variance identified a clear difference between target
transgenes of different repeat / gene index value with regard to their silenced relative NPTII
levels (H=11,026 with 2 degrees of freedom; P=0.004). Pairwise multiple comparison by
Dunn´s method demonstrated a correlation (Q=3.038, P<0.05; Spearman rank order
correlation coefficient 0.594, P=0.0014) between silenced relative NPTII levels and repeat /
gene index values of target transgenes. Focussing on T-DNA left border flanking regions only
even improves this correlation (Fig. 6).
(e) Analysis of correlation between DNA methylation or siRNA matches in target transgene
flanking regions and relative NPTII levels in F1 plants in presence of the silencer. Left panel:
No clear correlation is found between DNA methylation of target transgene flanking regions
as measuered by the DNA methylation index defined in Suppl. Info. 7a and relative NPTII
levels (Spearman rank order correlation coefficient -0.024, P=0.907; Pearson product
moment correlation coefficient -0.048, P=0.816). Right panel: No clear correlation is found
between the number of siRNA matches in target transgene flanking regions and relative
NPTII levels (Spearman rank order correlation coefficient -0.289, P=0.15; Pearson product
moment correlation coefficient -0.244, P=0.229).