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-RPVA CI+PLRV CP-R

PLRV CP PVA CIPVY CP

PLRV CP+PVY CP -R

①

②

③

④

⑤

⑥

①②③

④⑤

⑥

PLRV CP

PVA CI

PVY CP

aFig. S1

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-RPVA CI+PLRV CP-R

PLRV CPPVY CP

PLRV CP+PVY CP -R

①

②

③

④

⑤

’⑥

①②③

④⑤

PVA CI TRV REP PMTV REP

’⑥

bFig. S1 con’t.

TemplatePrimer no.

600 bp 1000 bp

PVY-O 1, 2 1, 2

PLRV 3, 4 3, 4

PVA 5, 6 5, 6’

TRV - 7, 8

PMTV - 9, 10

PCR primers for generating 200 bp fragments including overlapping sequences

cFig. S1 con’t.

PVY CP PLRV CP PVY CP PLRV CP

Overlapping PCR for generation of 600 bp or 1000 bp fusion of fragments

Step TemplatePrimer

no.PCR products

1st 1, 4

2nd

1, 6

1, 6’

3rd 7, 10

4th 1, 10

PVY CP PLRV CP PVY CP PLRV CP PVA CI PVA CI

TRV REP PMTV REPTRV REP PMTV REP

TRV REP PMTV REP

PVY CP PLRV CP PVA CI TRV REP PMTV REP

PVY CP PLRV CP PVA CI

Fig. S1 con’t.d

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.

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

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

600-3 OUT C1 Non-transgenic

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

600-3 OUT E1 Non-transgenic

600-3-OUT-B2

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

600-3 OUT B2 Non-transgenic

siRNA

siRNA

siRNA

Western

Western

Western

a

c

b

1000-1-2 OUT-B1

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

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

1550 KDa

40 KDa 30 KDa

50 KDa

40 KDa 30 KDa

Non-transgenic

Non-transgenic 1000-1-2 OUT A1

1000-1-2 OUT B

siRNA

Western

siRNA

Western

Fig. S2 con’t.

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).