FIG 2.

Inhibition of rSYNV-RFP localized infections by ectopic expression of SYNV proteins. (A) Fluorescence microscopy observation of N. benthamiana leaves after agroinfiltration to initiate rSYNV local infections and to express each of the SYNV N, P, sc4, M, and G proteins or an empty vector control. Note that each of the viral proteins was expressed from a dual binary plasmid that also contains a GFP expression cassette to visualize the infiltrated leaf regions. Infiltrated leaves were observed under a stereo fluorescence microscope at 8 dpi and again at 12 dpi to estimate local infections during the 4-day movement period. The top two panels show RFP expression, and the bottom panel shows merged images of RFP and GFP expression in leaf cells surrounding the rSYNV RFP foci. Bar, 200 μm. (B) Western blotting to detect GFP expression levels and the presence of viral proteins using polyclonal antibodies against SYNV virions (α:SYNV), sc4 (α:sc4), and GFP (α:GFP). The stained RuBisCO large subunit was used as a loading control. (C) Calculation of the average numbers of RFP-expressing cells per rSYNV-RFP infection focus at 12 dpi. Bars represent standard deviations. Asterisks denote statistical significance of the M protein inhibition by a Student's t test (P < 0.001; n = 24). (D) Observation of the local movement of PVX-GFP coinfiltrated with SYNV M or empty vector at 60 hpi and 6 dpi with a fluorescence microscope. Bar, 200 μm. (E) Western blot analysis of GFP expression levels in leaf tissues infiltrated with PVX-GFP with or without SYNV M protein at 6 dpi. The stained RuBisCO large subunit was used as a loading control. (F) Calculation of the average areas of PVX-GFP infection foci at 6 dpi. NS: not statistically significant (P > 0.05; n = 20) as analyzed by Student's t test.