Fig. 8

Specific and efficient knocking-down of four members of group III E2 genes did not significantly affect flg22-induced ROS production and plant development. a Phenotypes of the N. benthamiana plants in which NbUBC12 alone, three E2 genes NbUBC10, 28 and 31, or four E2 genes NbUBC10, 12, 28 and 31 were specifically silenced. The optimized VIGS construct TRV-UBC12/10/28/31* was used to specifically silence NbUBC10, 12, 28 and 31. The non-silenced TRV-infected plant and group III-silenced plant were included as control. Photographs were taken 4 weeks after the approximately 3-week-old seedlings were infiltrated with TRV vector-based VIGS constructs. b The NbUBC10, 12, 28 and 31 genes were specifically and efficiently silenced in N. benthamiana by VIGS. The transcript level of group III E2 genes and a closely-related E2 gene, NbUBC27 (outside the group III) in various VIGS-treated N. benthamiana plants was examined as described. 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 the expression of corresponding gene in plants treated with TRV-UBC12, TRV-UBC10/28/31, and TRV-UBC12/10/28/31*, respectively compared to that in non-silenced TRV control plants. The experiments were repeated three times with similar results. c Knocking down NbUBC10, 12, 28 and 31 genes did not affect significantly flg22-induced ROS production. Leaf disks of the VIGS-treated N. benthamiana plants were incubated with 1.0 μM flg22 to induce ROS production. Error bars indicate standard deviation. Experiments were repeated three times with similar results