Fig. 5: Nb14-3-3a-mediated antiviral defense depends on the MAPKKKα.

a Overexpression of 14-3-3a inhibits BBSV infection. Agrobacterium containing indicated construct above the panel was infiltrated into N. benthamiana leaves followed by mechanical inoculation with 300 ng BBSV virions 24 h later. Inoculated leaves were collected at 3 dpi and analyzed by western blot analysis with an anti-CP or anti-Flag antibody. Actin served as the loading control. b Knockdown of Nb14-3-3a increases the accumulation of BBSV. The opposite halves of N. benthamiana leaves were agroinfiltrated with 14-3-3a-RNAi construct and the control empty vector (EV). Agrobacterium carrying BBSV infection clone was infiltrated into the previously infiltrated regions 24 h later. Leaf samples from infiltrated regions were harvested at 3 dpi and subjected to western blot with an anti-CP or anti-14-3-3a antibody. CBB-stained RbcL served as the loading control. For panels a and b, the experiments were repeated three times with similar results. c Phenotypes of BBSV-infected WT, 14-3-3a-OE, and 14-3-3a-KD plants. N. benthamiana leaves were inoculated with 100 ng BBSV virions. Photographs were taken at 14 dpi and a representative result is shown. d The percentages of BBSV-infected wild-type (WT), 14-3-3a-OE, and 14-3-3a-KD plants with different disease symptom grades. Error bars indicate ± SD of the mean three independent experiments (n = 3) with at least six plants in each experiment. e Western blot analysis of BBSV CP levels in the indicated plants using an anti-CP or anti-14-3-3a antibody. Actin served as the loading control. f Western blot analysis of BBSV CP levels in the infiltrated leaves of WT or NbMAPKKKα-KO plants using an anti-CP or anti-Flag antibody. Actin served as the loading control. For panels e and f, the experiments were repeated three times with similar results.