pvy cp-f atgccaactgtgatgaat plrv cp+pvy cp-r actcggcccgaaggtga cgcatttctatatacgctt
DESCRIPTION
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 ACTCGGCCCGAAGGTGA CGCATTTCTATATACGCTT - PowerPoint PPT PresentationTRANSCRIPT
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
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600-3 OUT C1 Non-transgenic
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50 KDa
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600-3 OUT E1 Non-transgenic
600-3-OUT-B2
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600-3 OUT B2 Non-transgenic
siRNA
siRNA
siRNA
Western
Western
Western
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1000-1-2 OUT-B1
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1000-1-2-OUT-A1
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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).