Figure 4.

Determination of the NSm²¹ Recognition Sites in Sw-5b.

(A) Expression of FLAG-tagged Sw-5b (i) and chimeric NB-ARC^Heinz-LRR^Sw-5b (iii) induces cell death when coexpressed with NSm²¹ in N. benthamiana plants. Sw-5b^Heinz (ii) and chimeric NB-ARC^Sw-5b-LRR^Heinz (iv) failed to induce cell death when coexpressed with NSm²¹. Expression of individual proteins was confirmed by immunoblot assay using FLAG and NSm antibodies.

(B) Alignment of the LRR sequence of Sw-5b and Sw-5b^Heinz. Boxed regions indicate 15 different polymorphic sites/region between Sw-5b and Sw-5b^Heinz.

(C) Each polymorphic site in Sw-5b LRR was replaced with the cognate site found in the Heinz paralog in full-length Sw-5b and Sw-5b-NB-ARC-LRR and expressed alone or coexpressed with NSm or NSm²¹ in N. benthamiana plants. Normal cell death, reduced cell death, or no cell death response was recorded 5 d after expression. The polymorphic sites 3 and 4 are shown in blue; the polymorphic sites 5 and 6 are shown in green; the polymorphic sites 1, 2, and 7 to 15 are shown in black.

(D) Sw-5b FLAG-LRR, FLAG-LRR^M3, FLAG-LRR^M4, FLAG-LRR^M5, or FLAG-LRR^M6 immunoprecipitate NSm²¹-YFP when coexpressed in N. benthamiana leaves. The amount of NSm²¹-YFP, FLAG-LRR and mutants precipitated were quantified using ImageQuant software. The relative fold of mutant in each lane was compared with immunoprecipitated wild type. Similar results were obtained from more than three biological repeats. IP, immunoprecipitation with specific antibody; IB, immunoblot with specific antibody.

(E) Mapping of four polymorphic sites 3, 4, 5, and 6 on the three-dimensional homology structure model of Sw-5b-NB-ARC-LRR. I954 at polymorphic site 3, and F977 and E978 at polymorphic site 4 are in a blue sphere. M996 (highlighted with sphere), L998, Y1000, D1002, and L1004 at polymorphic sites 3 are shown in green. V1047 and G1048 at polymorphic site 4 are shown in a green sphere.