title: A comprehensive mutational analysis of the Arabidopsis resistance protein RPW8.2 reveals key amino acids for defense activation and protein targeting.
abstract: The Arabidopsis thaliana resistance to powdery mildew8.2 (RPW8.2) protein is specifically targeted to the extrahaustorial membrane (EHM) encasing the haustorium, or fungal feeding structure, where RPW8.2 activates broad-spectrum resistance against powdery mildew pathogens. How RPW8.2 activates defenses at a precise subcellular locale is not known. Here, we report a comprehensive mutational analysis in which more than 100 RPW8.2 mutants were functionally evaluated for their defense and trafficking properties. We show that three amino acid residues (i.e., threonine-64, valine-68, and aspartic acid-116) are critical for RPW8.2-mediated cell death and resistance to powdery mildew (Golovinomyces cichoracearum UCSC1). Also, we reveal that two arginine (R)- or lysine (K)-enriched short motifs (i.e., R/K-R/K-x-R/K) make up the likely core EHM-targeting signals, which, together with the N-terminal transmembrane domain, define a minimal sequence of 60 amino acids that is necessary and sufficient for EHM localization. In addition, some RPW8.2 mutants localize to the nucleus and/or to a potentially novel membrane that wraps around plastids or plastid-derived stromules. Results from this study not only reveal critical amino acid elements in RPW8.2 that enable haustorium-targeted trafficking and defense, but also provide evidence for the existence of a specific, EHM-oriented membrane trafficking pathway in leaf epidermal cells invaded by powdery mildew.
authors.affiliation: Rice Research Institute, Sichuan Agricultural University, Chengdu 611130, China.
authors.forename: Wenming
authors.initials: W
authors.lastname: Wang
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhang
authors.forename: Yingqiang
authors.initials: Y
authors.lastname: Wen
authors.forename: Robert
authors.initials: R
authors.lastname: Berkey
authors.forename: Xianfeng
authors.initials: X
authors.lastname: Ma
authors.forename: Zhiyong
authors.initials: Z
authors.lastname: Pan
authors.forename: Dipti
authors.initials: D
authors.lastname: Bendigeri
authors.forename: Harlan
authors.initials: H
authors.lastname: King
authors.forename: Qiong
authors.initials: Q
authors.lastname: Zhang
authors.forename: Shunyuan
authors.initials: S
authors.lastname: Xiao
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000596:Amino Acids / Q000276:immunology*; D017360:Arabidopsis / Q000235:genetics / Q000276:immunology* / Q000382:microbiology; D029681:Arabidopsis Proteins / Q000235:genetics / Q000276:immunology*; D001203:Ascomycota; D004252:DNA Mutational Analysis; D018744:DNA, Plant / Q000235:genetics; D060467:Disease Resistance; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010935:Plant Diseases / Q000276:immunology / Q000382:microbiology; D057865:Plant Immunity; D018515:Plant Leaves / Q000166:cytology / Q000382:microbiology; D030821:Plants, Genetically Modified / Q000235:genetics / Q000276:immunology / Q000382:microbiology
chemical_list: D000596:Amino Acids; D029681:Arabidopsis Proteins; D018744:DNA, Plant; C418463:RPW8.2 protein, Arabidopsis