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. 2016 Nov 14;90(23):10886–10905. doi: 10.1128/JVI.01697-16

FIG 5.

FIG 5

Wheat streak mosaic virus NIa-Pro and CP but not RNA sequence is required for superinfection exclusion. (A) Schematic diagram of the genomic organization of Triticum mosaic virus (TriMV) with expanded views of WSMV NIa-Pro and CP cistrons with a series of frameshifts or nucleotide substitution mutations (indicated with vertical red lines) for a continuous reading frame but with a different amino acid sequence. NIa-Pro-NS and CP-NS cistrons of WSMV with a 9-amino-acid NIb/CP cleavage peptide of TriMV were inserted between the P1 and HC-Pro cistrons. (B and C) Fluorescent images shown are local foci at 5 days post-challenge inoculation (dpci) (B) and systemic infection at 9 dpci (C) with WSMV-GFP (challenge virus) on wheat leaves systemically infected with TriMV or TriMV expressing WSMV NIa-Pro-NS, NIa-Pro, CP-NS, or CP cistrons. Buffer, buffer-inoculated wheat subsequently challenge inoculated. (D and E) Western blot assays of total proteins from inoculated (D) and upper noninoculated (E) leaves at 5 and 9 dpci, respectively. The blots were probed with GFP monoclonal antibody and TriMV polyclonal antibodies as indicated. Coomassie blue-stained SDS-PAGE gels show the amount of the large subunit of wheat RubisCO protein as loading controls for the amount of protein loaded per well in Western blot assays. Full-length and truncated TriMV CP are indicated with arrows and arrowheads, respectively. Asterisks indicate a wheat protein reacting nonspecifically with TriMV antiserum. (F and G) Presented ethidium bromide-stained agarose gels are RT-PCR products of WSMV cistrons using TriMV-specific primers from inoculated (F) and upper noninoculated (G) wheat leaves at 5 and 9 dpci, respectively. (D to G) Lanes 1 to 5, wheat infected with TriMV (lane 1) or TriMV with WSMV NIa-Pro-NS (lane 2), NIa-Pro (lane 3), CP-NS (lane 4), and CP (lane 5); lane 6, buffer-inoculated wheat subsequently challenge inoculated; lane 7, buffer-inoculated wheat with no challenge inoculation; lane +, pTriMV as a positive control; lanes M, 1.0-kbp DNA ladder.