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PVY CP-F ATGCCAACTGTGATGAAT PLRV CP+PVY CP-R ACTCGGCCCGAAGGTGA CGCATTTCTATATACGCTT PVY CP+PLRV CP-F AAGCGTATATAGAAATGCG TCACCTTCGGGCCGAGT PVA CI+PLRV CP-R TCTCAACTTGTCCAAGCTTC AATTTGGAACTTGTTGACGT PLRV CP+PVA CI-F ACGTCAACAAGTTCCAAATT GAAGCTTGGACAAGTTGAGA PVA CI-R TTCTCTCGAAATAGCTAATACT PVY CP-F PVY CP+PLRV CP-F PLRV CP+PVA CI-F PVA CI-R PVA CI+PLRV CP-R PLRV CP PVA CIPVY CP PLRV CP+PVY CP -R PLRV CP PVA CI PVY CP a Fig. S1

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PVY CP-F ATGCCAACTGTGATGAAT PLRV CP+PVY CP-R ACTCGGCCCGAAGGTGA CGCATTTCTATATACGCTT PVY CP+PLRV CP-F AAGCGTATATAGAAATGCG TCACCTTCGGGCCGAGT PVA CI+PLRV CP-R TCTCAACTTGTCCAAGCTTC AATTTGGAACTTGTTGACGT PLRV CP+PVA CI-F ACGTCAACAAGTTCCAAATT GAAGCTTGGACAAGTTGAGA TRV REP+PVA CI-R AGCACCAGGCTTCAAAGAAACCTTCTCTCGCCATAGCTAA PVA CI+TRV REP-F AGCTATGGCGAGAGAAGGTTTCTTTGAAGCCTGGTGCT PMTV REP+TRV REP-R TCTTGCAGACAAATCATCCTTAAGTACTCCCGACCTCT TRV REP+PMTV REP-F AGAGGTCGGGAGTACTTAAGGATGATTTGTCTGCAAGA PMTV REP-R TCACCACGGATGTACCATA PVY CP-F PVY CP+PLRV CP-F PLRV CP+PVA CI-F PVA CI-R PVA CI+PLRV CP-R PLRV CP PVY CP PLRV CP+PVY CP -R PVA CITRV REPPMTV REP b Fig. S1 cont.

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Template Primer no. 600 bp1000 bp PVY-O1, 2 PLRV3, 4 PVA5, 6 TRV-7, 8 PMTV-9, 10 PCR primers for generating 200 bp fragments including overlapping sequences c Fig. S1 cont.

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PVY CPPLRV CP PVY CP PLRV CP Overlapping PCR for generation of 600 bp or 1000 bp fusion of fragments StepTemplate Primer no. PCR products 1st1, 4 2nd 1, 6 3rd7, 10 4th1, 10 PVY CPPLRV CP PVY CP PLRV CPPVA CI TRV REPPMTV REP TRV REPPMTV REP TRV REPPMTV REP PVY CPPLRV CPPVA CI TRV REP PMTV REP PVY CP PLRV CPPVA CI Fig. S1 cont. d

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Fig. S1. Strategy for amplifying viral segments of 200 bp and joining the segments to generate 600 bp and 1000 bp tandem segments. (a) The six primers used for the PCR to generate the three 200 bp segments and the 600 bp tandem fusions. The internal primers contained 5 proximal sequences of a different virus complementary to the 3 proximal sequences of another primer for synthesis on the opposite strand. This is to produce the overlapping sequences necessary to facilitate joining of the 200 bp PCR products to form various fusions. (b) The ten primers used for the PCR to generate the five 200 bp segments and the 1000 bp tandem fusions, with internal primers containing overlapping ends as for (a). (c) Summary of the primers used to generate each 200 bp segment for generating the 600 bp tandem fusions or the 1000 bp tandem fusions. (d) Summary of the steps and PCR reactions involving the numbered primers to generate intermediate tandem fusions and final 600 bp and 1000 bp tandem fusions.

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Fig. S2 600-3-OUT- C1 600-3-OUT-E1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1234512345 6 7 8 9 10 11 12 13 14 15 600-3 OUT C1Non-transgenic 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1234512345 6 7 8 9 10 11 12 13 14 15 50 KDa 40 KDa 30 KDa 50 KDa 40 KDa 30 KDa 600-3 OUT E1Non-transgenic 600-3-OUT-B2 1234512345 6 7 8 9 10 11 12 13 14 15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 50 KDa 40 KDa 30 KDa 600-3 OUT B2Non-transgenic siRNA Western a c b

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1000-1-2 OUT-B1 1234512345 6 7 8 9 10 11 12 13 14 15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1000-1-2-OUT-A1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 2 3 4 5`` 6 7 8 9 10 11 12 13 14 15 50 KDa 40 KDa 30 KDa 50 KDa 40 KDa 30 KDa Non-transgenic 1000-1-2 OUT A1 1000-1-2 OUT B siRNA Western siRNA Western Fig. S2 cont. e d Fig. S2. Analysis of transgenic lines for expression of siRNAs against PVY and resistance to PVY-O as determined by visual symptoms and western blots for the presence of PVY-O CP. The gels above each blot show the loading controls for small RNAs. The numbers above the gel lanes refer to individual transgenic plants identified in the plant photos. The sizes and positions of protein molecular weight markers is shown on the left of each western blot. T2-generation transgenic plants were from lines 600-3-OUT-B2 (a), 600-3-OUT-C1 (b), 600-3-OUT-E1 (c), 1001-1- 2-OUT-A1 (d), and 1000-1-2-OUT-B1 (e).

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