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Amplification and Cloning Strategy for Viral Segments in Transgenic Potato Plants

This figure illustrates the methodology used for amplifying viral segments to create 600-bp tandem fusions. Panel (a) details the six primers utilized for PCR, producing three 200-bp segments, essential for generating overlapping sequences for subsequent fusion. Panel (b) explains the amplification of the 600-bp fusion segments with specific primers for cloning into the Gateway system. Panel (c) describes the introduction of the fused segments into a plant transformation vector, emphasizing the Gateway recombination process. Primer sequences are provided in Table S1.

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Amplification and Cloning Strategy for Viral Segments in Transgenic Potato Plants

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  1. Supplementary Figure

  2. PVY CP PLRV CP PVA CI Fig. S1 PVY CP-F (a) PVY CP -R PLRV CP-F PLRV CP-R attB2 PVA CI-F (b) PLRV CP PVY CP PVA CI PVA CI-R attB1 600bp P35S 600bp Intron T35S Kan (c) LB RB pK7GWIWG2(I) Intron R Intron F P35SR T35SF

  3. Fig. S1 Strategy used for amplifying viral segments of 200 bp and joining the segments to generate 600-bp tandem fusions. (a) Six primers were used for the PCR to generate the three 200-bp segments, as well as the 600 bp tandem fusions. The internal primers contained 5’ proximal sequences of the flanking virus sequence complementary to the 3’ proximal sequences of another primer for synthesis on the opposite strand. This was used to produce the overlapping sequences necessary to facilitate joining of the 200-bp PCR products to form various fusions. (b) The 600-bp tandem fusion was amplified using primers for the various attachment regions required for cloning by using the Gateway system. Different primers were required to attach the attB1 and attB2 for constructing inserts with the IN orientation versus the OUT orientation. (c) After cloning into the Gateway entry vector pDONR207, the cassette of tandem, fused viral segments was introduced into the plant transformation vector pKGWIWG2(I) twice, in opposite orientations, via the gateway recombination system. The sequences of the various primers required are given in Table S1

  4. Fig. S2 PLRV-inoculated 600-102-6-IN34 Mock-inoculated Non-transformed VS

  5. Fig. S2 Potato plants of cv. Vales Sovereign (VS) inoculated with PLRV using aphids. Transgenic potato plants (line pK7GWIWG2(I)/600-102-6-IN34 and non-transformed potato plants were inoculated with PLRV using 10 aphids per plant or mock-inoculated, respectively. Plants were assessed for infection 30 days after inoculation

  6. Fig. S3 PVA-inoculated 600-102-6-IN34 Mock-inoculated Non-transformed VS

  7. Fig. S3 Potato plants of cv. Vales Sovereign (VS) inoculated with PVA using aphids. Transgenic potato plants (line pK7GWIWG2(I)/600-102-6-IN34 and non-transformed potato plants were inoculated with PVA using 10 aphids per plant or mock-inoculated, respectively. Plants were assessed for infection 30 days after inoculation

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