Figure 8.

Rhabdoviruses are restricted by host autophagy in a conserved way. (A) Y2H assay showing the interaction between NbBeclin1 and various glycoproteins. Yeast strain Y2HGold cells co-transformed with the indicated plasmids were cultured separately on the SD-Trp-Leu-His-Ade and SD-Trp-Leu selection medium. (B) BiFC analysis of the interaction between NbBeclin1 and various glycoproteins. NbBeclin1-YN was coexpressed with glycoproteins-YC or YC in N. benthamiana leaves. YFP signals were visualized by confocal microscopy. Bars: 20 μm. (C) silencing of NbSnRK1 or NbBeclin1 promotes SYNV infection in N. benthamiana. GFP fluorescence and viral symptoms in plants pre-inoculated with TRV1 together with TRV2-GUS (control), TRV2-NbSnRK1, or TRV2-NbBeclin1 for 7 days and then infected by SYNV-GFP. Plants were photographed under UV light and regular light at 10 dpi. (D) qRT-PCR analysis of SYNV viral RNA accumulation in GUS-, NbSnRK1-, and NbBeclin1-silenced plants. Total RNAs were extracted from the SYNV-GFP infected leaves at 10 dpi. Values represent the mean relative to the GUS-silenced plants (n = 3 biological replicates) and were normalized with NbPP2A as an internal reference. Student’s t test was used for analyses (*P<.05, **P<.01). (E) Western blot analysis of the GFP protein accumulation in GUS-, NbSnRK1-, and NbBeclin1-silenced plants. Total proteins were extracted from the RSMV-GFP infected leaves at 15 dpi, and were detected by anti-GFP antibody. The relative protein band intensity was normalized against that of Actin.