Figure 6.

The effect of virus-induced gene silencing (VIGS) of NbSOBIR1 and NbBAK1 on the necrotizing activity of Sclerotinia sclerotiorum necrosis-inducing effectors. (A) Nicotiana benthamiana SOBIR1 and BAK1 expression after VIGS treatment as determined by qRT-PCR analysis. Expression is relative to actin as an endogenous control. Means and standard errors of three biological replicates are shown. (B) N. benthamiana plants were subjected to VIGS using the TRV-based vectors (TRV : NbBAK1 and TRV : NbSOBIR1) and leaves infiltrated with Agrobacterium tumefaciens strains carrying effector constructs three weeks after TRV infiltration. TRV : GFP was used as a VIGS negative control. Bcl2-Associated protein X (BAX) was used as a BAK1/SOBIR1-independent positive control (P) for necrosis induction and A. tumefaciens carrying an empty vector as a negative control (N). Leaves in each row were infiltrated with the four constructs at the same locations as shown for the first leaf. Arrows show lesions that were smaller than the control plants. (C) The effect of NbSOBIR1 and NbBAK1 silencing on the necrotizing activity of recombinant SsNE2. N. benthamiana plants were subjected to VIGS using TRV : NbBAK1 and TRV : NbSOBIR1. TRV : GFP was used as a VIGS negative control. Leaves were infiltrated with recombinant SsNE2 expressed in Escherichia coli three weeks after TRV infiltration. Bcl2-Associated protein X (BAX) was used as a BAK1/SOBIR1-independent positive control (P) for necrosis induction and A. tumefaciens carrying an empty vector as a negative control (N).