Global phosphoproteome analysis identifies PeVYV P4-induced host protein phosphorylation changes that facilitate viral proliferation

Figures

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  Fig. 1. Phosphoproteomic profiling of P4-overexpressing transgenic (P4-OE) and wild-type (control) N. tabacum plants. A Phosphorylation levels of wild-type (Mock) and P4-OE (P4−1 and P4−2) transgenic lines of N. tabacum by Western blot using anti-9611 S antibody. B Principal Component Analysis (PCA) of phosphoproteomic data. C SPearson correlation coefficient of of phosphoproteomic data. D Bar graph of up-regulated and down-regulated differentially phosphorylated proteins

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  Fig. 2. Phosphorylation sites and motif analysis of differentially phosphorylated proteins. A Statistics of phosphorylation sites of phosphorylated proteins. B Phosphate site analysis of differentially phosphorylated proteins. C MOTIF enrichment analysis of differentially phosphorylated proteins. D Analysis of phosphorylated MOD of differentially phosphorylated proteins. E Analysis of phosphorylation protein phosphorylation sites motif

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  Fig. 3. Prediction of secondary structure of differentially phosphorylated proteins and analysis of subcellular localization. A Netsurf P predicted secondary structure and a pie chart of phosphorylated proteins. B Subcellular localization analysis of differentially phosphorylated proteins

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  Fig. 4. Hierarchical clustering analysis, COG, GO, and KEGG enrichment analysis of significantly regulated phosphorylated proteins. A Hierarchical cluster analysis of phosphopeptides in P4-OE Nicotiana tabacum to screen the top 40 phosphopeptides with significant differences. B COG analysis of differentially expressed phosphorylated proteins. C GO enrichment analysis of differentially expressed phosphorylated proteins. All phosphorylated proteins were clustered in the Biological Process (BP), cellular component (CC), and Molecular Function (MF) GO categories based on GO Slim terms. D KEGG pathway enrichment analysis of differentially expressed phosphorylated proteins. Circles represent the number of enriched genes, with larger circles indicating more enriched genes

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  Fig. 5. The subcellular localization and effects on PeVYV infectionof SATF and its phosphorylation site mutant. A Subcellular localization of Agrobacterium-mediated transient expressed SATF-GFP, MCM2-GFP, SAC2-GFP, and SATFS360A-GFP. MCM2S183A-GFP, SAC2S791A-GFP in H2B-RFP transgenic N. benthamiana leaves. Confocal images were captured 48 h after infiltration. Scale bar = 50 μm. B-D Accumulation of PeVYV in N. benthamiana plants expressing the target protein was detected by RT-qPCR at 10 dpi after infiltration, with 35 S: GFP as the control. E Western blot analysis was performed using Phospho-(Ser/Thr) Akt Substrate Antibody #9611 at 10 dpi after infiltration, and GFP antibody (Sangon Biotech, Order NO D110008) detection was used to determine the target protein level. Coomassie brilliant blue (CBB) was used to show the loading amount. Bars represent means ± SD (n = 3) (*ρ < 0.05, **ρ < 0.01 and *** ρ < 0.005, Student’s t test)

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  Fig. 6. Effect of MAPK kinase on PeVYV infection in plants. A Gene expression of MAPK3, MAPK6, MEK2, and MAPKKKα in TRV-MAPK3, TRV-MAPK6, TRV-MEK2, and TRV-MAPKKKα in gene knocking-down N.benthamiana plants. B Quantification of PeVYV virus accumulation in gene knocking-down N.benthamiana plants infected with PeVYV. C Western blot analysis of phosphorylation levels of plants in (B) at 10 d using Phospho-(Ser/Thr) Akt Substrate Antibody #9611. D Western blot analysis of phosphorylation levels of plants in (B) at 20 usingPhospho-(Ser/Thr) Akt Substrate Antibody #9611. Bars represent means ± SD (n = 3) (*ρ < 0.05, **ρ < 0.01 and *** ρ < 0.005, Student’s t test)