Supplementary Figure captions
Figure S1: Symptoms of TSWV infection on grafted plant.
Symptoms of TSWV infection on leaves of wt scions grafted on 30.4 R4 rootstocks (a), or on wt rootstock (b)
inoculated two weeks after grafting; (c) leaf from healthy scion.
Figure S2: 5’ transcript sequences obtained with 5’RACE in 30.4
Different classes of transcripts amplified with the 5’RACE from 30.4 R3 and 110.1 tomato plants. All the
three classes are with a 5’ CAP and are present in both the lines. Colors represent: violet=35S sequence;
light blue=polylinker junction; yellow=coding sequence of TSWV nucleocapsid protein. The starting ATG
codon is in bold, the number of clones sequenced belonging to each category are reported in the two lines.
Figure S3: Summary of the N protein 3’ mRNA sequences produced in the 30.4 tomato line.
For both transcript classes (N628 and N728) all the clones recovered are reported based on the length; for
same variants more than one clone has been sequenced. The 3’ tails of the N transcripts in the 110.1
transgene expressing line have been sequenced as control; the new putative stop codon is indicated with
an asterisk.
Figure S4: Identification of fragments in previously reported southern blot analysis on 30.4 tomato
Southern blotting of XbaI-digested total genomic DNA from 30.4 transgenic R2 and R0 plants, and wild-type
control, hybridized with a NptII and N probes (Nervo et al. 2003). The size of each probed fragment is
indicated by red arrows, and has been predicted by sequencing data relative to the transgenic locus model
proposed.
Figure S5: Scheme of 30.4 tomato progeny production.
Number of TSWV- inoculated plants for each individual progeny is indicated. Till the R5 progeny all seeds
have been collected from resistant TSWV-challenged plants. R6 seeds were only obtained from a noninoculated R5 #7 plant.
Figure S6: PstI digestion patterns of tomato genomic DNAs following electrophoresis on agarose gel .
PstI-treated genomic DNA of 30.4 R5s and R4r plants, subjected to electrophoresis, are showing similar
degree of digestion following ethidium bromide staining. DNAs were blotted on a nylon membrane and
processed by Southern blot analysis using Digoxigenin N and NptII probes, following the procedure
described by Nervo et al. 2003. Southern blot results are shown in Figure 4a.
Figure S7: Analysis of transgenic transcripts in resistant and susceptible 30.4 progeny.
Tomato line 110.1 is included to show the full-length N transcript: a) Northern blotting –TSWV-N probe.
Red arrow: full-length N transcript; blue arrow: aberrant 30.4 N transcripts. b) Ethidium bromide-stained
gels .
Figure S8: Sequences of the 3’ terminal obtained from 3’RACE in 30.4 R2 plants show presence of
termination consensus signals.
The termination sequences in N728 and N628 transcript classes are obtained from the longest variant
sequenced for each class in the 3’RACE (Figure S3). The sequence of the CaMV terminator (originally
present on the transgenic construct and sequenced from the control line 110.1) is shown as comparison.
The canonical NUE signal is highlighted in red, the consensus UUGUAA and the UG motifs typical of the FUE
sequences are highlighted in yellow and in light blue, respectively.
Figure S9: Termination signals can be found flanking the 30.4 transgenic insertion in tomato chromosome
4.
Search for the top 5 most common NUEs described in Arabidopsis from Loke and coworker (2005) shows
NUE sequences enrichment in the region surrounding the transgenic locus; schematic drawing is produced
using Geneious™ software. The integrated transgenic cassettes are shown in red.
Figure S10: Model of evolution of silencing in the 30.4 transgenic locus.
In the R2 plants the production of aberrant mRNA from the N573 cassette induces the silencing
machinery to degrade the transcript and generate both 21-22 and 24 classes of siRNA directing
translational repression of N transcripts and de-novo methylation of the transgenes, respectively.
In the R4 plants the methylation spreads on the flanking region and is maintained independently by the
siRNAs (probably through MET1).
In the R5 and R6 susceptible plants the methylation spans over all the four transgenic inserts precluding
PolII from binding in the locus. As consequence of the lack of transgenic transcript as degradation target,
the production of siRNA collapses and the plant becomes susceptible to TSWV. DNA methylation is
represented above the gene cassettes with red and blue dots, respectively for CG and not CG context.
Figure S11: Estimation of bisulfite conversion rate.
Conversion rate of cytosines on PHAVULOTA gene sequence in Arabidopsis and on tomato chloroplast DNA.
Each line represents a single clone, and each dot a cytosine. Estimation of bisulfite conversion rate is 98%
and 96%, respectively. KISMETH (Gruntman et al. 2008) was used to obtain the graphs.