Fig. 3
The group III ubiquitin E2 s are required for ROS production and the induction of NbRbohB. a Phenotype of the N. benthamiana plants in which the group III E2 genes were silenced. The non-silenced TRV empty vector (TRV)-infected plant was used as control. Photographs were taken 4 weeks after the approximately 3-week old seedlings were infiltrated with TRV or TRV-III VIGS constructs. b The group III ubiquitin E2 genes were efficiently and specifically silenced in N. benthamiana by VIGS. The transcript level of group III E2 genes and closely-related E2 genes NbUBC13 and NbUBC27 (outside the group III) in non-silenced TRV control (TRV) and group III E2 genes-silenced (TRV-III) N. benthamiana plants was examined by semi-quantitative PCR. NbEF1α was used as an internal reference for determining the amount of cDNA template to be used. Numbers under the gel bands denote the percentage of reduction (%) in expression of the corresponding gene in TRV-III-infected plants compared to that in control plants. Experiments were repeated three times with similar results. c Silencing the group III E2 genes resulted in reduced ROS production induced by flg22 in a chemiluminescence assay. Leaf discs of the group III E2 genes-silenced (TRV-III) and non-silenced TRV control (TRV) N. benthamiana plants were incubated with 1.0 μM flg22 to induce ROS production. The experiment was repeated three times with similar results. Error bars indicate standard deviation. d Knocking down the group III ubiquitin E2 genes down-regulates the induction of gene NbRbohB by flg22. The expression of NbRbohB in 2.0 μM flg22-treated leaves of group III E2 genes-silenced and non-silenced TRV control N. benthamiana plants was analyzed by real time PCR at the indicated time points after flg22 treatment. The X-axis marks different time points (hour) after flg22 treatment. The experiment was performed with three technical repeats in each of the three biological replicates. Error bars indicate standard deviation. Asterisks mark significant reduction of the expression of NbRbohB in group III E2 genes-silenced plants compared with that in non-silenced control plants based on one-way ANOVA (P < 0.01)