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Additional file 2: Figure S1. Analysis of pWX56 infected plants. A. RT-PCR analysis of target gene insertion of pWX56 plant 40 days post VPI, lanes: MDMV-VIGS (primers WX317/WX176: 938 bp); MDMV-ZmChlI (primers WX291/WX321: 382 bp); MDMV-ZmIspH (primers WX291/WX325:636 bp), MDMV-ZmPDS (primers WX327/WX315:413 bp); pWX56-infected plant (lanes 2–5); pWX6-infected plant (lane 6, with primers WX317/WX315: 176 bp); pWX56 DNA control (primers WX317/WX315: 965 bp) (lane 8). B. Chlorophyll content measurement (μmol per m2) of newest fully emerged leaf of each plant: healthy (HC), pWX6, and pWX56. C. Representative images of GFP and photobleaching after pWX56 rub-inoculation passages (see Table S5). 0P = plant rub-inoculated from VPI tissue, 1P-5P = plants rub-inoculated with 14 dpi pooled tissue from prior inoculation, all shown 14 dpi. Images were taken with a Leica DFC460C camera using fluorescence imaging with NIGHTSEA Green-only bandpass filter at 3-s exposure and bottom panel images are taken with the same camera without fluorescence at 1 s exposure. D. pWX56-infected whole plant silencing 90 days post inoculation. Figure S2. Representative gels showing GFP and VIGS insertion stability analysis by RT-PCR. A. pWX27-inoculated plants tested with primers WX111/112 (Table S1) spanning NIb/CP insertion site. B. pWX68-inoculated plants tested with primers WX315/317 (Table S1) spanning P1/HCPro insertion site. C. pWX56 [GFP] tested with primers WX358/367 (Table S1) spanning NIb/CP insertion site. D. pWX56 [VIGS] tested with primers WX315/317 (Table S1) spanning P1/HCPro insertion site. For each construct, assays from 20 rub-inoculated plants are shown in first lanes, followed by control assays from either five pWX6-inoculated control plants and five mock-inoculated control plants (A-B) or five pWX27-inoculated, five pWX6-inoculated, and five mock-inoculated control plants (C-D). Samples of the newest fully emerged leaf from top of each test plant was collected and fresh tissue used for one-step RT-PCR at 7, 14, and 21 days post inoculation as indicated, with gradient of bands shown spliced from three gels in each rightmost panel. Expected full-length amplicon sizes for test construct (top) vs. pWX6 no-insert control (bottom) with arrows to the left of each leftmost panel. Figure S3. Analysis of pWX56 infected plants transmitted by aphids R. padi. A. Photobleaching phenotype of pWX56 infected plants cv. Early Sunglow by aphid R. padi 28 days post aphid transmission. HC: healthy plant; WT: wild type pWX6 infected plant; L1-L5 leaves of pWX56 infected plant from newest (L1) to oldest (L5) leaves, photographed at 28 days post transmission. B. Aphid transmission of pWX56 inoculum using the newest fully emerged young leaves from top of the plants were collected, GFP and VIGS were assayed at 17 days post inoculation by PCR with primers spanning the insertion site. pWX6: wild type MDMV OH5 transmitted plants; controls: pWX56 plasmid DNA and PCR master mix without template. Expected amplified PCR fragment sizes: pWX56 GFP full-length insert vs WT: WX358/WX367 (1115 bp vs. 335 bp); pWX56 VIGS WX317/WX315 (965 bp vs. 176 bp). |
