Assembly and purification of AvrSr35-induced Sr35 resistosome and determination of its structure by cryo-EM

Meng-Xi Liu; Yan-Bo Zhao; Ze-Kai Li; Tao-Tao Chen; Songying Ouyang

doi:10.1016/j.xpro.2022.101796

Assembly and purification of AvrSr35-induced Sr35 resistosome and determination of its structure by cryo-EM

STAR Protoc. 2022 Oct 31;3(4):101796. doi: 10.1016/j.xpro.2022.101796. eCollection 2022 Dec 16.

Authors

Meng-Xi Liu¹, Yan-Bo Zhao¹, Ze-Kai Li¹, Tao-Tao Chen¹, Songying Ouyang²

Affiliations

¹ Provincial University Key Laboratory of Cellular Stress Response and Metabolic Regulation, the Key Laboratory of Innate Immune Biology of Fujian Province, Biomedical Research Center of South China, Key Laboratory of OptoElectronic Science and Technology for Medicine of the Ministry of Education, College of Life Sciences, Fujian Normal University, Fuzhou 350117, China.
² Provincial University Key Laboratory of Cellular Stress Response and Metabolic Regulation, the Key Laboratory of Innate Immune Biology of Fujian Province, Biomedical Research Center of South China, Key Laboratory of OptoElectronic Science and Technology for Medicine of the Ministry of Education, College of Life Sciences, Fujian Normal University, Fuzhou 350117, China. Electronic address: ouyangsy@fjnu.edu.cn.

Abstract

Sr35, a coiled-coil nucleotide-binding leucine-rich repeat receptor (CC-NLR) from the wheat species Triticum monococcum can directly recognize the pathogen avirulence factor AvrSr35 and confers immunity against wheat stem rust caused by Puccinia graminis f.sp. tritici race Ug99. Assembly of a stable Sr35 resistosome induced by AvrSr35 in vitro is usually limited by protein expression and low assembly efficiency. Here, we describe the expression and purification of AvrSr35 and Sr35, in vitro assembly of Sr35 resistosome for structure determination by cryo-EM. For complete details on the use and execution of this protocol, please refer to Zhao et al. (2022).

Keywords: Cell Biology; Cryo-EM; Molecular Biology; Plant sciences; Protein Biochemistry; Protein expression and purification; Structural Biology.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Basidiomycota* / genetics
Cryoelectron Microscopy
Disease Resistance* / genetics
Genes, Plant
Plant Diseases / genetics