Fig. 4: NbMPK3/6 phosphorylates the Ser59 residue of P7 and enhances viral infection. | Nature Communications

Fig. 4: NbMPK3/6 phosphorylates the Ser59 residue of P7 and enhances viral infection.

From: A viral protein activates the MAPK pathway to promote viral infection by downregulating callose deposition in plants

Fig. 4

a In vivo phosphorylation of P7-Flag. P7-Flag was immunoprecipitated with anti-Flag from the total soluble proteins extracted from the leaves of WT, OE-NbMPK3, OE-NbMPK6 and mpk3−/−mpk6RNAi N. benthamiana plants transiently overexpressing P7-Flag and harvested at 48 hpi. N. benthamiana plants were infiltrated with Agrobacterium containing empty vector (EV) as the mock control. Phosphorylation levels of P7 were analysed by western blot with pIMAGO analysis. Accumulation of P7 was analysed by western blot with an anti-Flag antibody. The level of MAPK phosphorylation was detected by western blot analysis with an anti-pTEpY antibody. The molecular weight of P7-Flag is approximately 25 kDa. Each lane represents a sample from an individual experiment. Each treatment included three biological replicates. b In vitro phosphorylation assays showing phosphorylation of P7 by NbMPK3/6. P7-GST protein was incubated with preactivated NbMPK3/6-GST or NbMPK3m/NbMPK6m-GST in 1× kinase reaction buffer. The NbMPK3m-GST and NbMPK6m-GST in the TEY mutant (T218A/Y220A) served as negative control. The phosphorylation level of P7 was analysed by western blot with pIMAGO analysis. Coomassie blue (CBB) staining was used to assess protein loading. The molecular weight of P7-GST is 35 kDa. c Phosphorylation assays of P7S59A-Flag in vivo. P7S59A-Flag was immunoprecipitated with an anti-Flag antibody from the total soluble proteins extracted from the leaves of transiently over-expressing P7S59A-Flag in WT, OE-NbMPK3, OE-NbMPK6 and mpk3−/−mpk6RNAi N. benthamiana harvested at 48 hpi. N. benthamiana plants were infiltrated with Agrobacterium containing empty vector (EV) as the mock control. Phosphorylation levels of P7 S59A were analysed by western blot with pIMAGO analysis. Accumulation of P7S59A was analysed by western blot with an anti-Flag antibody. The level of MAPK phosphorylation was detected by western blot analysis with an anti-pTEpY antibody. The molecular weight of P7S59A-Flag is approximately 25 kDa. Each lane represents a sample from an individual experiment. Each treatment included three biological replicates. d Phosphorylation assays of P7S59A by NbMPK3/6 in vitro. The P7S59A-GST protein was incubated with preactivated NbMPK3/6-GST or NbMPK3m/NbMPK6m-GST in 1× kinase reaction buffer. The NbMPK3m-GST and NbMPK6m-GST served as negative control. The phosphorylation level of P7S59A was analysed by western blot with pIMAGO analysis. CBB staining was used to assess protein loading. The molecular weight of P7 S59A-GST is 35 kDa. e Symptoms of N. benthamiana plants infiltrated with ToCV, ToCVS59D and ToCVS59A at 30 dpi (scale bar = 3 cm); a magnified view is shown. N. benthamiana plants were infiltrated with Agrobacterium containing empty vector (EV) as the mock control. f Western blot analysis of ToCV CP accumulation in ToCV-, ToCVS59D- and ToCVS59A-inoculated N. benthamiana plants. Actin served as the loading control. Each lane represents a sample from an individual experiment. Each treatment included three biological replicates. g Symptoms of WT or mpk3−/−mpk6RNAi plants infiltrated with Agrobacteria containing ToCV, ToCVS59D or ToCVS59A at 30 dpi; a magnified view is shown (scale bar = 3 cm). N. benthamiana plants were infiltrated with Agrobacterium containing empty vector (EV) as the mock control. h Western blot analysis of ToCV CP accumulation in ToCV-, ToCVS59D- and ToCVS59A-inoculated WT or mpk3−/−mpk6RNAi plants. Actin served as the loading control. Each lane represents a sample from an individual experiment. Each treatment included three biological replicates. i Symptoms of WT, OE-NbMPK3 and OE-NbMPK6 plants infiltrated with Agrobacteria containing ToCV, ToCVS59D and ToCVS59A at 30 dpi; a magnified view is shown (scale bar = 3 cm). N. benthamiana plants were infiltrated with Agrobacterium containing empty vector (EV) as the mock control. j Western blot analysis of ToCV CP accumulation in ToCV-, ToCVS59D- and ToCVS59A-inoculated WT OE-NbMPK3 or OE-NbMPK6 plants. Actin served as the loading control. Each lane represents a sample from an individual experiment. Each treatment included three biological replicates. For (b, d) the experiments were repeated three times with similar results. Source data are provided as a Source Data file.

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