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. 2016 Oct 4;28(10):2586–2602. doi: 10.1105/tpc.16.00071

Figure 2.

Figure 2.

HV-Independent Systemic Movement of SatBaMV.

(A) Physical map of pKF4 for generating satBaMV transgenic plants and satBaMV RNA accumulation in transgenic N. benthamiana lines 2-6 and 9-2 by RNA gel blot. r, root; s, stem; L1, 1st leaf; L2, 2nd leaf; L3, 3rd leaf; f, flower. rRNA of ethidium bromide served as loading control. Four independent biological samples, each involving four plants, generated similar results.

(B) and (C) Illustrations of grafting experiments with 40-d-old wild-type and satBaMV-transgenic N. benthamiana (sat). SatBaMV RNA and P20 protein accumulation were examined by RNA gel blot and immunoblot analysis, respectively. rRNA and Coomassie blue staining were used for loading controls. Four independent biological samples, each involving four plants, generated similar results.

(B) Leaf 7 (L7) and leaf 8 (L8) were detached before grafting. L6, L9, and L10 near the graft union were harvested at 12 DAG and L11 at 15 DAG.

(C) Leaves L7 to L11 were detached immediately after grafting; L6 and L12 were harvested at 15 DAG.

(D) RT-PCR and tissue blot detection of satBaMV RNA in grafting scions after dark treatment. The dark treatment of scions started at 12 DAG for 3 d. RNA and protein extracted from L6, L12-L14, and SA were sampled at 15 DAG and examined by RT-PCR and immunoblot analysis, respectively. Plants without dark treatment (w/o dark treatment) were used as controls. rRNA and Coomassie blue staining were used for loading controls. The tissue blots from left to right were prepared from grafting stem tissues between L6 and L12, followed by hybridization with satBaMV-specific probe. Four independent biological samples, each involving four plants, generated similar results.

(E) Detection of transgene RNA in transgenic stocks and wild-type scions after grafting. Wild-type plants were grafted onto transgenic N. benthamiana expressing GFP or BaMV CP or transgenic N. tabacum expressing satCMV. RT-PCR analysis of mRNA level at 15 DAG. Four independent biological samples, each involving four plants, generated similar results.