PVY CP-F ATGCCAACTGTGATGAAT PLRV CP+PVY CP-R ACTCGGCCCGAAGGTGA CGCATTTCTATATACGCTT

1. PVY CP PLRV CP PVA CI Fig. S1 a ③ ⑤ ① PVY CP+PLRV CP-F PLRV CP+PVA CI-F PVY CP-F PLRV CP PVY CP PVA CI PVA CI+PLRV CP-R PLRV CP+PVY CP -R PVA CI-R ⑥ ④ ② PVY CP-F ATGCCAACTGTGATGAAT PLRV CP+PVY CP-R ACTCGGCCCGAAGGTGACGCATTTCTATATACGCTT PVY CP+PLRV CP-F AAGCGTATATAGAAATGCGTCACCTTCGGGCCGAGT PVA CI+PLRV CP-R TCTCAACTTGTCCAAGCTTCAATTTGGAACTTGTTGACGT PLRV CP+PVA CI-F ACGTCAACAAGTTCCAAATT GAAGCTTGGACAAGTTGAGA PVA CI-R TTCTCTCGAAATAGCTAATACT ① ② ③ ④ ⑤ ⑥

2. Fig. S1 con’t. b ③ ⑤  ①  PVY CP+PLRV CP-F PLRV CP+PVA CI-F PVY CP-F PLRV CP PVY CP PVA CI TRV REP PMTV REP PVA CI-R PVA CI+PLRV CP-R PLRV CP+PVY CP -R ② ④ ⑥’   PVY CP-F ATGCCAACTGTGATGAAT PLRV CP+PVY CP-R ACTCGGCCCGAAGGTGACGCATTTCTATATACGCTT PVY CP+PLRV CP-F AAGCGTATATAGAAATGCGTCACCTTCGGGCCGAGT PVA CI+PLRV CP-R TCTCAACTTGTCCAAGCTTCAATTTGGAACTTGTTGACGT 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 ① ② ③ ④ ⑤ ⑥’    

3. Fig. S1 con’t. c PCR primers for generating 200 bp fragments including overlapping sequences

4. PVY CP PVY CP PLRV CP PLRV CP PVY CP PVY CP PLRV CP PLRV CP PVA CI PVA CI PVY CP PLRV CP PVA CI TRV REP TRV REP TRV REP PMTV REP PMTV REP PMTV REP Fig. S1 con’t. d Overlapping PCR for generation of 600 bp or 1000 bp fusion of fragments PVY CP PLRV CP PVA CI TRV REP PMTV REP

5. 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.

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

7. Fig. S2 con’t. 1000-1-2 OUT A1 Non-transgenic 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 6 7 8 9 10 11 12 13 14 15 1 2 3 4 5`` 1000-1-2-OUT-A1 siRNA d 50 KDa Western 40 KDa 30 KDa 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Non-transgenic 1000-1-2 OUT B 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 siRNA 1000-1-2 OUT-B1 e 50 KDa Western 40 KDa 30 KDa 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).