Journal: Journal of Virology

Article Title: The Matrix Protein of a Plant Rhabdovirus Mediates Superinfection Exclusion by Inhibiting Viral Transcription

doi: 10.1128/JVI.00680-19

Figure Lengend Snippet: Mutual exclusion of SYNV variants tagged with GFP and RFP in N. benthamiana. (A) Schematic diagrams showing rSYNV antigenomes engineered to express GFP (rSYNV-GFP) and RFP (rSYNV-RFP). (B) Confocal microscopy z-stack images showing spatial separation of GFP and RFP fluorescent foci in upper systemically infected leaves of N. benthamiana plants taken at 12 days postinoculation (dpi) with leaf extract containing mixtures of rSYNV-GFP and rSYNV-RFP. The bottom panels show higher magnifications of the boxed areas. Bar, 100 μm. (C) Confocal microscopy z-stack images of contiguous GFP and RFP foci in N. benthamiana leaf tissue at 15 dpi with agrobacterial mixtures harboring rSYNV-RFP and rSYNV-GFP derivatives. Right panels show magnified views of the boxed sectors on the left to highlight the exclusion boundaries of rSYNV-GFP and rSYNV-RFP in adjacent cells of two foci. Bar, 50 μm. (D) GFP and RFP fluorescence in upper leaves of N. benthamiana plants after an initial sap inoculation with one SYNV variant or mock inoculation, followed 12 days later with a challenge inoculation with the other SYNV variant indicated on the left of the panels. Images were captured with a stereo fluorescence microscope with GFP or RFP channels at 20 days after primary inoculation and again 20 days after challenge inoculation. 1st, primary inoculum; 2nd, challenge inoculum. Bar, 2 mm. (E) Confocal microscopy of N. benthamiana leaves after initial sap inoculation with rSYNV-RFP, followed by a PVX-GFP challenge inoculation 6 days later. The images show overlapping GFP and RFP fluorescence in upper infected leaves taken at 6 days after PVX infection. Bar, 100 μm.

Article Snippet: Fluorescence in N. benthamiana leaves was captured with a Lumar 31 filter set for RFP detection (excitation, 565/30 nm; emission, 620/60 nm) and a Lumar 38 filter set for GFP detection (excitation, 470/40; emission, 525/50).

Techniques: Confocal Microscopy, Infection, Fluorescence, Variant Assay, Microscopy

Journal: Journal of Virology

Article Title: The Matrix Protein of a Plant Rhabdovirus Mediates Superinfection Exclusion by Inhibiting Viral Transcription

doi: 10.1128/JVI.00680-19

Figure Lengend Snippet: 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.

Article Snippet: Fluorescence in N. benthamiana leaves was captured with a Lumar 31 filter set for RFP detection (excitation, 565/30 nm; emission, 620/60 nm) and a Lumar 38 filter set for GFP detection (excitation, 470/40; emission, 525/50).

Techniques: Inhibition, Expressing, Fluorescence, Microscopy, Plasmid Preparation, Western Blot, Staining, Infection

Journal: Journal of Virology

Article Title: The Matrix Protein of a Plant Rhabdovirus Mediates Superinfection Exclusion by Inhibiting Viral Transcription

doi: 10.1128/JVI.00680-19

Figure Lengend Snippet: Identification of the SYNV M protein as a viral superinfection exclusion (SIE) determinant. N. benthamiana leaves were coinfected via agroinoculation with the pair rSYNV-GFP and rSYNV-RFP, rSYNV-GFP and rSYNV-RFP-ΔM, rSYNV-GFP and rSYNV-RFP-ΔG, or rSYNV-GFP and rSYNV-RFP-MUAA. Contiguous GFP and RFP foci in inoculated leaf tissues at 15 dpi were imaged by confocal microscopy to show fluorescence exclusion between foci. The white arrows indicate boundary cells expressing both GFP and RFP. Bar, 100 μm.

Article Snippet: Fluorescence in N. benthamiana leaves was captured with a Lumar 31 filter set for RFP detection (excitation, 565/30 nm; emission, 620/60 nm) and a Lumar 38 filter set for GFP detection (excitation, 470/40; emission, 525/50).

Techniques: Confocal Microscopy, Fluorescence, Expressing

Journal: Journal of Virology

Article Title: The Matrix Protein of a Plant Rhabdovirus Mediates Superinfection Exclusion by Inhibiting Viral Transcription

doi: 10.1128/JVI.00680-19

Figure Lengend Snippet: 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).

Article Snippet: Fluorescence in N. benthamiana leaves was captured with a Lumar 31 filter set for RFP detection (excitation, 565/30 nm; emission, 620/60 nm) and a Lumar 38 filter set for GFP detection (excitation, 470/40; emission, 525/50).

Techniques: Expressing, Plasmid Preparation, Fluorescence, Microscopy, Inhibition, Western Blot, Staining, Northern Blot, Hybridization, Negative Control, In Vitro

Journal: Journal of Virology

Article Title: The Matrix Protein of a Plant Rhabdovirus Mediates Superinfection Exclusion by Inhibiting Viral Transcription

doi: 10.1128/JVI.00680-19

Figure Lengend Snippet: Subcellular localization, interactions with the N protein, and the ability to suppress MR transcription by the SYNV M nuclear localization mutants. (A) Confocal micrographs showing RFP-histone 2B reporter protein (RFP-H2B) transgenic N. benthamiana leaf epidermal cells expressing the CFP-M, CFP-MRARA, and CFP-MAAAA fusions. Bar, 20 μm. (B) Bimolecular fluorescence complementation (BiFC) assays to determine interactions of the N protein with the wild-type M or the MRARA or MAAAA mutant protein. Leaf epidermal cells of the N. benthamiana RFP-H2B transgenic plants were agroinfiltrated to express the YFP N-terminal fusion to N (YN-N) and the YFP C-terminal fusions to M derivatives (YC-M). Confocal micrographs of YFP (BiFC), RFP-H2B (nucleus), and the merged channel are shown. Bar, 20 μm. (C) Fluorescence microscopy visualization of SYNV (−)MR reporter gene expression at 8 dpi in N. benthamiana leaves coinfiltrated to express either an empty vector control (Vec) or the M, MRARA, or MAAAA protein. Bar, 200 μm. (D) Average numbers of cells per field expressing GFP and RFP at 8 dpi in each SYNV MR treatment shown in panel C. Bars represent standard deviations. ***, P < 0.001; NS, not significant (by Student's t test; n = 20). (E) Western blots showing expression of the M, MRARA, or MAAAA protein with the N and P proteins in each group. The Coomassie blue-stained RuBisCO large subunit provides a loading control.

Article Snippet: Fluorescence in N. benthamiana leaves was captured with a Lumar 31 filter set for RFP detection (excitation, 565/30 nm; emission, 620/60 nm) and a Lumar 38 filter set for GFP detection (excitation, 470/40; emission, 525/50).

Techniques: Transgenic Assay, Expressing, Fluorescence, Mutagenesis, Microscopy, Plasmid Preparation, Western Blot, Staining

Journal: Journal of Virology

Article Title: The Matrix Protein of a Plant Rhabdovirus Mediates Superinfection Exclusion by Inhibiting Viral Transcription

doi: 10.1128/JVI.00680-19

Figure Lengend Snippet: SIE activities of rSYNV and rSYNV M nuclear localization mutants. N. benthamiana leaves were coinfected via agroinoculation with the virus pairs rSYNV-GFP and rSYNV-RFP, rSYNV-GFP and rSYNV-RFP- MRARA, or rSYNV-GFP and rSYNV-RFP- MAAAA. Contiguous GFP and RFP foci in inoculated leaf tissues at 15 dpi were imaged by confocal microscopy to show fluorescence exclusion between the foci. The white arrows indicate boundary cells expressing both GFP and RFP. Bar, 100 μm.

Article Snippet: Fluorescence in N. benthamiana leaves was captured with a Lumar 31 filter set for RFP detection (excitation, 565/30 nm; emission, 620/60 nm) and a Lumar 38 filter set for GFP detection (excitation, 470/40; emission, 525/50).

Techniques: Confocal Microscopy, Fluorescence, Expressing