FIG 4.
Suppression of viral transcription by the M protein in an SYNV minireplicon (MR) system. (A) Schematic diagrams of the SYNV genome and a negative-sense MR cassette. The (−)MR contains 3′ leader (le) and 5′ trailer (tr) sequences flanking GFP and RFP reporter genes substituted for the SYNV N and P genes. (B) Visualization of GFP and RFP reporter proteins in N. benthamiana leaves agroinfiltrated with Agrobacterium cultures harboring plasmids for expression of the SYNV MR and the N, P, and L core proteins. Additional bacterial cultures containing the empty vector (Vec), pGD-M, pGD-MUAA, and pGD-sc4 plasmids, as indicated on the top of each panel, were also included in the mixtures to test their effects on reporter expression. Infiltrated leaves were photographed at 8 dpi with a fluorescence microscope. Bar, 200 μm. (C) Average numbers of cells per field expressing GFP and RFP at 8 dpi in each SYNV MR treatment. Bars represent standard deviations. Asterisk denotes Student's t test significance of transcription inhibition by the M protein (P < 0.001; NS, not significant; n = 12). (D) Western blots confirming GFP and RFP expression in each group. Expression of the N and P core proteins or of the M or sc4 protein, was detected with polyclonal antibodies against SYNV virion or against the sc4 protein, respectively. The Coomassie blue-stained RuBisCO large subunit provides a loading control. (E) Northern blot hybridization showing inhibitory effects of the M protein on SYNV MR RNA synthesis. Total RNA extracted from N. benthamiana leaf tissues shown in panel B was blotted with a negative-sense GFP probe to detect the MR agRNAs and GFP mRNAs. RNA from leaf tissues infiltrated with (−)MR in the absence of the N, P, and L proteins was used as a negative control. In vitro T7 transcripts corresponding in size to the GFP and MR agRNA (agMR) were used as size markers. The 25S rRNA was stained with ethidium bromide to show equal RNA loading. (F and G) Lack of inhibitory effects of the SYNV M protein on GFP reporter gene expression from a PVX replicon. N. benthamiana leaves were agroinfiltrated with mixtures of Agrobacterium strains harboring the PVX-GFPΔp25 plasmid together with pGD-M or an empty vector. Single-cell GFP foci were imaged with a fluorescence microscope at 3 dpi, and average numbers of cells per field were calculated and statistical significance was analyzed by Student's t test. NS, not statistically significant (P > 0.05; n = 12).