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. 2021 Jun 7;19(11):2164–2176. doi: 10.1111/pbi.13641

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© 2021 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

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Analysis of single, double and triple mutations of L33P, K319E and R927A in the SD and LRR domains of Sw‐5b for their ability to induce HR to WT NSm or NSm^C118Y or NSm^T120N mutants in N. benthamiana leaves. (a) (YFP)‐Sw‐5b^L33P/R927A, (YFP)‐Sw‐5b^K319E/R927A, (YFP)‐Sw‐5b^{L33P/K319E/R927A}, (YFP)‐Sw‐5b^L33P, (YFP)‐ (YFP)‐Sw‐5b^K319E, (YFP)‐Sw‐5b^L33P/K319E, (YFP)‐Sw‐5b^{L33P/K319E/D642E}, (YFP)‐Sw‐5b^{L33P/K319E/R927Q} and (YFP)‐Sw‐5b^{L33P/K319E/R927L} were co‐expressed with and without NSm, NSm^C118Y and NSm^T120N, respectively, in N. benthamiana leaves via agro‐infiltration. HR phenotypes were monitored at 3–10 dpi and photographed at 7 dpi. (b) Western blotting analysis of YFP‐tagged Sw‐5b, Sw‐5b^L33P/R927A, Sw‐5b^K319E/R927A, Sw‐5b^{L33P/K319E/R927A}, Sw‐5b^L33P, Sw‐5b^K319E, Sw‐5b^L33P/K319E, (YFP)‐Sw‐5b^{L33P/K319E/D642E}, (YFP)‐Sw‐5b^{L33P/K319E/R927Q} and (YFP)‐Sw‐5b^{L33P/K319E/R927L} co‐expressed with NSm, NSm^C118Y and NSm^T120N in N. benthamiana leaves using YFP‐specific and NSm‐specific antibodies. Plant leaves agro‐infiltrated with empty vector (EV) were used as a negative control. The rubisco large subunit was stained with Ponceau S to indicate sample loading. Protein size is indicated on the left.