Data_Sheet_1_Thioredoxin VdTrx1, an unconventional secreted protein, is a virulence factor in Verticillium dahliae.DOCX

Understanding how plant pathogenic fungi adapt to their hosts is of critical importance to securing optimal crop productivity. In response to pathogenic attack, plants produce reactive oxygen species (ROS) as part of a multipronged defense response. Pathogens, in turn, have evolved ROS scavenging mechanisms to undermine host defense. Thioredoxins (Trx) are highly conserved oxidoreductase enzymes with a dithiol-disulfide active site, and function as antioxidants to protect cells against free radicals, such as ROS. However, the roles of thioredoxins in Verticillium dahliae, an important vascular pathogen, are not clear. Through proteomics analyses, we identified a putative thioredoxin (VdTrx1) lacking a signal peptide. VdTrx1 was present in the exoproteome of V. dahliae cultured in the presence of host tissues, a finding that suggested that it plays a role in host-pathogen interactions. We constructed a VdTrx1 deletion mutant ΔVdTrx1 that exhibited significantly higher sensitivity to ROS stress, H2O2, and tert-butyl hydroperoxide (t-BOOH). In vivo assays by live-cell imaging and in vitro assays by western blotting revealed that while VdTrx1 lacking the signal peptide can be localized within V. dahliae cells, VdTrx1 can also be secreted unconventionally depending on VdVps36, a member of the ESCRT-II protein complex. The ΔVdTrx1 strain was unable to scavenge host-generated extracellular ROS fully during host invasion. Deletion of VdTrx1 resulted in higher intracellular ROS levels of V. dahliae mycelium, displayed impaired conidial production, and showed significantly reduced virulence on Gossypium hirsutum, and model plants, Arabidopsis thaliana and Nicotiana benthamiana. Thus, we conclude that VdTrx1 acts as a virulence factor in V. dahliae.

解析植物病原真菌如何适应宿主，对于保障作物最优产量至关重要。当受到病原攻击时，植物会产生活性氧（reactive oxygen species, ROS），作为其多重防御反应的一部分。而病原物则进化出ROS清除机制，以破坏宿主的防御系统。硫氧还蛋白（Thioredoxins, Trx）是一类高度保守的氧化还原酶，具有二硫醇-二硫键活性位点，可作为抗氧化剂保护细胞免受ROS等自由基的损伤。然而，硫氧还蛋白在重要的维管束病原真菌大丽轮枝菌（Verticillium dahliae）中的功能尚不清楚。通过蛋白质组学分析，我们鉴定出一个缺乏信号肽的假定硫氧还蛋白VdTrx1。VdTrx1存在于宿主组织诱导培养的大丽轮枝菌的外分泌蛋白质组中，这一发现提示其在宿主-病原互作中发挥作用。我们构建了VdTrx1缺失突变体ΔVdTrx1，该突变体对ROS胁迫、过氧化氢（H2O2）以及叔丁基过氧化氢（tert-butyl hydroperoxide, t-BOOH）的敏感性显著升高。活细胞成像的体内实验与蛋白质免疫印迹（western blotting）的体外实验均显示，尽管缺乏信号肽的VdTrx1可定位于大丽轮枝菌细胞内，但VdTrx1也可依赖ESCRT-II蛋白复合物成员VdVps36进行非经典分泌。ΔVdTrx1菌株在宿主侵染过程中无法完全清除宿主产生的胞外ROS。敲除VdTrx1会导致大丽轮枝菌菌丝的胞内ROS水平升高，分生孢子产生能力受损，并且在陆地棉（Gossypium hirsutum）以及模式植物拟南芥（Arabidopsis thaliana）和本氏烟草（Nicotiana benthamiana）上的致病力显著降低。综上，我们认为VdTrx1是大丽轮枝菌的一个致病因子。