FIG 6.

Complete Wheat streak mosaic virus (WSMV) NIa-Pro is required for superinfection exclusion. (A) Schematic diagram of Triticum mosaic virus (TriMV) genome (a) with expanded views of WSMV NIa-Pro cistron (b) or NIa-Pro with a series of deletions covering the entire NIa-Pro cistron (c to f). Deleted amino acids in the NIa-Pro cistron are indicated with black rectangles. (B and C) Presented fluorescent images are local foci (B) at 5 days post-challenge inoculation (dpci) and systemic infection (C) at 9 dpci by the challenge virus (WSMV-GFP) on wheat infected with TriMV and TriMV with WSMV NIa-Pro or NIa-Pro deletions. WSMV, wheat infected with WSMV; buffer and buffer-NC, buffer-inoculated wheat subsequently challenge inoculated or not challenge inoculated, respectively. Bars, 500 μm. (D and E) Western blotting of total proteins from local foci at 5 dpci (D) and systemic infection at 9 dpci with challenge virus (E). The blots are probed with GFP monoclonal antibody and TriMV polyclonal antibodies as indicated. Below are SDS-PAGE gels stained with Coomassie blue showing the amount of the large subunit of wheat RubisCO protein as a Western blot loading control. Asterisks indicate wheat proteins reacting nonspecifically with TriMV antiserum. (F and G) RT-PCR products of WSMV cistrons using TriMV-specific primers to demonstrate the stability of inserts in the challenge virus-inoculated (F) and upper noninoculated (G) wheat leaves at 5 and 9 dpci, respectively. (D to G) Lanes 1 to 6, wheat infected with TriMV (lane 1) or TriMV with WSMV NIa (lane 2), NIa-ProΔ3-60aa (lane 3), NIa-ProΔ61-120aa (lane 4), NIa-ProΔ121-180aa (lane 5), or NIa-ProΔ181-229aa (lane 6); lane 7, WSMV-infected wheat; lane 8, buffer-inoculated wheat with challenge inoculation; lane 9, buffer-inoculated wheat with no challenge inoculation; lane +, pTriMV as a positive control; lane M, 1.0-kbp DNA ladder.