Figure 3.

Sw-5b Interacts with NSm²¹ in Vitro and in Planta.

(A) GST pull-down assay of the interaction between the full-length Sw-5b and NSm²¹-YFP. GST-Sw-5b or GST was expressed and purified from E. coli. NSm²¹-YFP was expressed in N. benthamiana plants and harvested at 24 h postagroinfiltration. The purified GST-Sw-5b or GST was mixed with plant extracts of NSm²¹-YFP and pulled down with GST beads. GST-Sw-5b pulled down a significant amount of NSm²¹-YFP compared with the YFP control. GST alone failed to pull down NSm²¹-YFP.

(B) Sw-5b NB-ARC-LRR and NSm²¹-YFP interact in an in vitro GST pull-down assay. Purified GST-Sw-5b-NB-ARC-LRR expressed in E. coli pulled down purified NSm²¹-YFP-6xhis. NSm²¹-YFP-6xhis was not pulled down with GST alone.

(C) Sw-5b NB-ARC-LRR^K568R associates with NSm²¹-YFP in planta. Sw-5b P-loop mutant NB-ARC-LRR^K568R fused to FLAG tag coimmunoprecipitated NSm²¹-YFP when coexpressed in N. benthamiana leaves but not with YFP alone.

(D) Sw-5b LRR associates with NSm²¹-YFP in planta. Sw-5b LRR fused to FLAG tag coimmunoprecipitated with NSm²¹-YFP when coexpressed in N. benthamiana leaves but not with YFP alone.

IB, immunoblot with specific antibody; IP, immunoprecipitation with specific antibody.