Figure 6.

Nonelicitor NSm^21(C118Y) and NSm^21(T120N) Could Enhance Cell Death Induced by Sw-5b NB-ARC-LRR^R927A.

(A) Cell death assay of NSm^21(C118Y) and NSm^21(T120N) with Sw-5b FLAG-NB-ARC-LRR^R927A in the leaves of 12-week-old N. benthamiana plants. The 8th leaf on a 12-week-old N. benthamiana plant was coinfiltrated with Sw-5b FLAG-NB-ARC-LRR^R927A and NSm²¹-YFP, FLAG-NB-ARC-LRR^R927A and NSm^21(C118Y)-YFP or FLAG-NB-ARC-LRR^R927A and NSm^21(T120N)-YFP. Areas infiltrated with FLAG-NB-ARC-LRR^R927A alone or with both FLAG-NB-ARC-LRR^R927A and the empty expression vector were used as controls. The infiltrated leaf was photographed at 5 d postinoculation. The infiltrated area for each treatment is marked with dotted line.

(B) Time course of ion leakage caused by expression of Sw-5b FLAG-NB-ARC-LRR^R927A (R927A), FLAG-NB-ARC-LRR^R927A + empty vector (R927A+EV), FLAG-NB-ARC-LRR^R927A + NSm^21(WT)-YFP [R927A+NSm^21(WT)], FLAG-NB-ARC-LRR^R927A + NSm^21(C118Y)-YFP [R927A+NSm^21(C118Y)], and FLAG-NB-ARC-LRR^R927A + NSm^21(T120N)-YFP [R927A+NSm^21(T120N)] in the leaves of 12-week-old N. benthamiana plants are shown.

(C) Immunoblot detection of FLAG-NB-ARC-LRR^R927A and the wild-type and mutant NSm²¹ proteins using a FLAG-specific and an YFP-specific antibody, respectively. The sizes of proteins in kilodaltons are shown to the left of the blot. Ponceau S staining was used as a protein loading control.

(D) FLAG-NB-ARC-LRR ^{K568RandR927A} was coexpressed with NSm^21(WT)-YFP, NSm^21(C118Y)-YFP, or NSm^21(T120N)-YFP in N. benthamiana leaves followed by co-IP assays using a FLAG-specific and a YFP-specific antibody. Leaf tissues expressing NSm^21(WT)-YFP, NSm^21(C118Y)-YFP, or NSm^21(T120N)-YFP were used as a negative control. IP, immunoprecipitation with specific antibody; IB, immunoblot with specific antibody.