Journal: Viruses

Article Title: The C4 Protein of TbLCYnV Promotes SnRK1 β2 Degradation Via the Autophagy Pathway to Enhance Viral Infection in N. benthamiana

doi: 10.3390/v16020234

Figure Lengend Snippet: Demonstrated interaction of TbLCYnV C4 with NbSnRK1 β2 in vitro and in vivo. ( A ) Interaction between TbLCYnV C4 and NbSnRK1 β2 by Y2H assay. Yeast strains of Gold co–transformed with the indicated plasmids were subjected to 10–fold serial dilutions and grown on an SD-Trp/-Leu/-His/-Ade medium. The strain–carried combinations of AD-T7/BD-P53 were used as a positive control, and those with AD-T7/BD-lam or with empty vectors BD and AD were used as negative controls. ( B ) BiFC assay showing the interaction between TbLCYnV C4 with NbSnRK1 β2 in vivo. The H2B-RFP transgenic N. benthamiana leaves co-expressing 2YN-C4 and 2YC-NbSnRK1 β2, 2YN-C4 and 2YC, or 2YN and 2YC-NbSnRK1 β2 were examined and imaged under confocal microscope. Expression of H2B-RFP was used a nuclear marker. Columns from top to bottom represent YFP, red fluorescence (RFP), and YFP/RFP merged field (merge). Scale bars represent 50 μm. ( C ) Interaction between C4 and NbSnRK1 β2 by Co-IP assay in vivo. N. benthamiana leaves were co-infiltrated TbLCYnV C4 with NbSnRK1 β2-GFP or GFP. Total proteins were extracted from the infiltrated leaf samples at 2 dpi. The samples were carried out with anti-GFP mAb-magnetic beads. The input and the co–immunoprecipitated proteins were analyzed by Western blot using anti-GFP or anti-mCherry antibody.

Article Snippet: Immunoblotting was performed with primary antibodies: Mouse anti-GFP-Tag mAb antibody (ABclonal, Wuhan, China), Mouse anti-mCherry-Tag mAb antibody (ABclonal, Wuhan, China), and then HRP* Goat anti-Mouse IgG antibody (ImmunoWay, Suzhou, China).

Techniques: In Vitro, In Vivo, Y2H Assay, Transformation Assay, Positive Control, Bimolecular Fluorescence Complementation Assay, Transgenic Assay, Expressing, Microscopy, Marker, Fluorescence, Co-Immunoprecipitation Assay, Magnetic Beads, Immunoprecipitation, Western Blot

Journal: Viruses

Article Title: The C4 Protein of TbLCYnV Promotes SnRK1 β2 Degradation Via the Autophagy Pathway to Enhance Viral Infection in N. benthamiana

doi: 10.3390/v16020234

Figure Lengend Snippet: TbLCYnV C4 promotes the degradation of NbSnRK1 β2 via the autophagy pathway. ( A ) Western blot analysis and semi-qRT-PCR assay of NbSnRK1 β2 protein and mRNA levels. N. bent hamiana leaves co-expressed NbSnRK1 β2-GFP with C4-mCherry or mCherry, respectively. Samples were analyzed by immunoblot using anti-GFP or anti-mCherry antibody. The amount of NbSnRK1 β2-GFP expression was calculated by ImageJ 1.8.0, and the data of NbSnRK1 β2-GFP co-infiltrated with mCherry were set as 1.00. Ponceau Red Stained Rubisco was used as a loading control. Semi-qRT-PCR assay was used to detect the transcription levels of NbSnRK1 β2 in plants. The expression of Actin gene was used as an internal control. ( B ) The expression of NbSnRK1 β2-GFP was calculated by GraphPad Prism 8. The data are shown as means and SD of three biological replicates. ** indicates a significant difference between the two treatments at the p < 0.01 by Student’s t -test. ( C ) Protease inhibitor treatment of NbSnRK1 β2 by Western blot. The N. benthamiana leaves transiently expressing NbSnRK1 β2-GFP were treated with 100 μM CHX together with 100 μM E64d, 5 mM 3-MA, 100 μM MG132, or DMSO, respectively. Samples were analyzed by immunoblot using anti-GFP antibody. The relative accumulation level of NbSnRK1 β2 in DMSO treatments were set as 1.00. Ponceau Red Stained Rubisco was used as a loading control. ( D ) The expression levels of NbSnRK1 β2-GFP by Western blot. The N. benthamiana leaves transiently co-expressing C4-mCherry or mCherry with NbSnRK1 β2-GFP were treated with 100 μM E64d or DMSO, respectively. The amount of NbSnRK1 β2-GFP expression was calculated using ImageJ 1.8.0. The relative accumulation level of NbSnRK1 β2 in the co-infiltrated with NbSnRK1 β2 and mCherry plants with DMSO treatment was set at 1.00. Ponceau Red Stained Rubisco was used as a loading control. ( E ) Semi-in-vivo protein stability assay of NbSnRK1 β2 by Western blot. NbSnRK1 β2 was transiently co-expressed with C4 (D22A)-mCherry or C4-mCherry in N. benthamiana plants, and E64d treatment was performed 48 h later. The data of NbSnRK1 β2-GFP co-infiltrated with C4-mCherry were set as 1.00. Ponceau Red Stained Rubisco was used as a loading control.

Article Snippet: Immunoblotting was performed with primary antibodies: Mouse anti-GFP-Tag mAb antibody (ABclonal, Wuhan, China), Mouse anti-mCherry-Tag mAb antibody (ABclonal, Wuhan, China), and then HRP* Goat anti-Mouse IgG antibody (ImmunoWay, Suzhou, China).

Techniques: Western Blot, Quantitative RT-PCR, Expressing, Staining, Control, Protease Inhibitor, In Vivo, Stability Assay