Fig. 1: The MAPK cascade positively regulates ToCV infection.
a MAPK activation in ToCV-infected N. benthamiana. Four-leaf-stage N. benthamiana plants were infiltrated with Agrobacterium containing empty vector (EV) (Mock) or a ToCV infectious cDNA clone. Systemic leaves were collected at the indicated time points. The accumulation levels of ToCV and that of MAPK phosphorylation were detected by western blot analysis with an anti-CP or anti-phospho-p44/42 MAPKs (anti-pTEpY) antibody. Actin served as the loading control. Each lane represents a sample from an individual experiment. Each treatment included three biological replicates. The molecular weights of pMPK6, pMPK3, CP and Actin are 44, 42, 30 and 43 kDa, respectively. b Western blot analysis of ToCV CP accumulation and MAPK phosphorylation in N. benthamiana plants inoculated with TRV-00 control, TRV-MPK3, TRV-MPK6, TRV-MKK2 and TRV-MAPKKKα. Actin served as the loading control. Each lane represents a sample from an individual experiment. Each treatment included three biological replicates. c Accumulation of ToCV RNA1 and RNA2 in N. benthamiana plants inoculated with TRV-00 control, TRV-MPK3, TRV-MPK6, TRV-MKK2 and TRV-MAPKKKα, as determined by qRT-PCR using P22 and CP gene-specific primers. Data are presented as means ± SDs and were analysed using Student’s t-test (one-sided, P = 0.0022, 0.0023, 0.0004, 0.0021, 0.0038, 0.0016, 0.0015, 0.0024, respectively). Each treatment included three biological replicates. **P < 0.01, ***P < 0.001. d Overexpression of MAPKKKα enhances ToCV infection. Agrobacterium harbouring ToCV infectious cDNA was infiltrated into different N. benthamiana leaves for 27 days. Then, Agrobacterium containing MAPKKKα-HA, MAPKKKαK236M-HA or empty vector (EV) was infiltrated into ToCV-infected leaves. After 3 days, accumulated levels of ToCV or MAPK phosphorylation were detected by western blot analysis with an anti-CP or anti-pTEpY antibody. MAPKKKα-HA and MAPKKKαK236M-HA protein levels were determined by western blot analysis with an anti-HA antibody. Actin served as the loading control. Each lane represents a sample from an individual experiment. Each treatment included three biological replicates. The molecular weights of MAPKKKα-HA and MAPKKKαK236M-HA are 75 kDa. e Accumulation of ToCV RNAs in N. benthamiana plants coinfected with ToCV and EV, ToCV and MAPKKKαK236M-HA or ToCV and MAPKKKα-HA determined by qRT-PCR using P22 and CP gene-specific primers. Data are presented as means ± SDs and were analysed using Student’s t-test (one-sided, P = 0.1472, 0.0036, 0.4097, 0.0001, respectively). Each treatment included three biological replicates. **P < 0.01, ***P < 0.001, ns, not significant. Source data are provided as a Source Data file.
