A Pathogen Type III Effector with a Novel E3 Ubiquitin Ligase Architecture

Type III effectors are virulence factors of Gram-negative bacterial pathogens delivered directly into host cells by the type III secretion nanomachine where they manipulate host cell processes such as the innate immunity and gene expression. Here, we show that the novel type III effector XopL from the model plant pathogen Xanthomonas campestris pv. vesicatoria exhibits E3 ubiquitin ligase activity in vitro and in planta, induces plant cell death and subverts plant immunity. E3 ligase activity is associated with the C-terminal region of XopL, which specifically interacts with plant E2 ubiquitin conjugating enzymes and mediates formation of predominantly K11-linked polyubiquitin chains. The crystal structure of the XopL C-terminal domain revealed a single domain with a novel fold, termed XL-box, not present in any previously characterized E3 ligase. Mutation of amino acids in the central cavity of the XL-box disrupts E3 ligase activity and prevents XopL-induced plant cell death. The lack of cysteine residues in the XL-box suggests the absence of thioester-linked ubiquitin-E3 ligase intermediates and a non-catalytic mechanism for XopL-mediated ubiquitination. The crystal structure of the N-terminal region of XopL confirmed the presence of a leucine-rich repeat (LRR) domain, which may serve as a protein-protein interaction module for ubiquitination target recognition. While the E3 ligase activity is required to provoke plant cell death, suppression of PAMP responses solely depends on the N-terminal LRR domain. Taken together, the unique structural fold of the E3 ubiquitin ligase domain within the Xanthomonas XopL is unprecedented and highlights the variation in bacterial pathogen effectors mimicking this eukaryote-specific activity.

III型效应蛋白（Type III effectors）是革兰氏阴性病原菌的毒力因子，通过III型分泌纳米机器（type III secretion nanomachine）直接递送至宿主细胞内，进而操控宿主细胞的多种生理过程，例如先天免疫与基因表达。本研究表明，来自模式植物病原菌野油菜黄单胞菌辣椒致病变种（Xanthomonas campestris pv. vesicatoria）的新型III型效应蛋白XopL，在体外及植物体内均具有E3泛素连接酶（E3 ubiquitin ligase）活性，可诱导植物细胞死亡并抑制植物免疫。E3泛素连接酶活性定位于XopL的C端区域，该区域可特异性结合植物E2泛素结合酶（E2 ubiquitin conjugating enzymes），并介导以K11连接的多泛素链为主的多泛素链形成。XopL C端结构域的晶体结构显示其为单结构域，具有此前未在任何已表征的E3泛素连接酶中发现的新型折叠，被命名为XL盒（XL-box）。XL盒中央空腔内的氨基酸突变会破坏E3泛素连接酶活性，并阻断XopL诱导的植物细胞死亡。XL盒中缺乏半胱氨酸残基，这表明其不存在硫酯键连接的泛素-E3连接酶中间体，且XopL介导的泛素化采用非催化机制。XopL N端区域的晶体结构证实其含有富亮氨酸重复（LRR）结构域，该结构域可作为蛋白质相互作用模块参与泛素化靶标的识别。尽管E3泛素连接酶活性是诱导植物细胞死亡所必需的，但抑制PAMP响应仅依赖于N端的LRR结构域。综上，黄单胞菌XopL所含的E3泛素连接酶结构域具有独特的折叠模式，这在以往研究中尚无先例，凸显了模拟真核生物特有活性的病原菌效应蛋白的多样性。