Fig. 1: MAPKKKα plays an antiviral role during BBSV infection.
a Schematic representation of BBSV-sfGFP11. 11th β-strand of super-folder GFP (sfGFP11) is fused to the N-terminus of CP in BBSV infectious clone. b Knockdown of MAPKKKα promotes BBSV infection. Agrobacterium containing hairpin MAPKKKα or the control empty vector (EV) was infiltrated into half of N. benthamiana leaves, respectively. Twenty-four hours later, a mixture of Agrobacterium harboring BBSV-sfGFP11 and sfGFP1-10 constructs was infiltrated into the pre-infiltrated leaves. GFP fluorescence was visualized by confocal microscopy at 5 dpi. Scale bars = 100 µm. c The expression level of BBSV CP in the infiltrated leaves was examined by western blot analysis using an anti-CP antibody. Actin served as the loading control. For panels b and c, the experiments were repeated three times with similar results. d RT-qPCR to confirm the downregulation of MAPKKKα. Values represent ± SD of the mean from three biological replicates. An asterisk indicates the significant difference (*P = 0.0104) based on one-sided unpaired Student’s t test. e Locus of the single guide RNA target in the genomic fragment of NbMAPKKKα. Exons are represented by gray boxes, UTRs by white boxes, and introns by black lines. In T2 transformants, deletions were detected in two NbMAPKKKα-KO lines. f Western blot analysis of BBSV CP levels in the infiltrated leaves of wild-type (WT) or NbMAPKKKα-KO plants using an anti-CP antibody. RbcL served as the loading control. The experiment was repeated three times with similar results. g Overexpression of MAPKKKα inhibits BBSV infection. Agrobacterium containing MAPKKKα or MAPKKKαK236M or the control empty vector (EV) was infiltrated into different N. benthamiana leaves. Agrobacterium harboring BBSV infectious clone was infiltrated 24 h later. The accumulation levels of BBSV or phosphorylated NbSIPK and NbWIPK were detected by western blot analysis with an anti-CP or anti-phospho-p44/42 MAPKs (anti-pTEpY) antibody. Actin protein served as the loading control. Each lane represents a sample from individual experiments. h. MAPK activation in BBSV-infected N. benthamiana. Eight-leaf-stage N. benthamiana plants were infiltrated with Agrobacterium carrying empty vector (EV) or BBSV infectious clone. Infiltrated leaves were collected at indicated time points and subjected to western blot analysis with an anti-pTEpY or anti-BBSV CP antibody. Actin protein served as the loading control. i Quantification of bands from blot in panel h, with pSIPK and pWIPK abundance normalized to the intensity of EV control at 0 h. Values represent ± SD of the mean from three biological replicates. An asterisk indicates the significant difference (*P = 0.0498, 0.0284, 0.0134, respectively) based on one-sided paired Student’s t test. j Western blot analysis of BBSV CP accumulation in the TRV-GFP control, TRV-NbMEK2, TRV-NbSIPK, and TRV-NbWIPK inoculated N. benthamiana plants. Actin protein served as the loading control. k RT-qPCR to confirm the downregulation of NbMEK2, NbSIPK, and NbWIPK. Values represent ± SD of the mean from three biological replicates. An asterisk indicates the significant difference (**P = 0.0043, 0.0068, 0.0085, respectively) based on one-sided unpaired Student’s t test. For panels h and j, the experiments were repeated three times with similar results.
