Table_1_Verticillium longisporum Elicits Media-Dependent Secretome Responses With Capacity to Distinguish Between Plant-Related Environments.xlsx

Verticillia cause a vascular wilt disease affecting a broad range of economically valuable crops. The fungus enters its host plants through the roots and colonizes the vascular system. It requires extracellular proteins for a successful plant colonization. The exoproteomes of the allodiploid Verticillium longisporum upon cultivation in different media or xylem sap extracted from its host plant Brassica napus were compared. Secreted fungal proteins were identified by label free liquid chromatography-tandem mass spectrometry screening. V. longisporum induced two main secretion patterns. One response pattern was elicited in various non-plant related environments. The second pattern includes the exoprotein responses to the plant-related media, pectin-rich simulated xylem medium and pure xylem sap, which exhibited similar but additional distinct features. These exoproteomes include a shared core set of 221 secreted and similarly enriched fungal proteins. The pectin-rich medium significantly induced the secretion of 143 proteins including a number of pectin degrading enzymes, whereas xylem sap triggered a smaller but unique fungal exoproteome pattern with 32 enriched proteins. The latter pattern included proteins with domains of known pathogenicity factors, metallopeptidases and carbohydrate-active enzymes. The most abundant proteins of these different groups are the necrosis and ethylene inducing-like proteins Nlp2 and Nlp3, the cerato-platanin proteins Cp1 and Cp2, the metallopeptidases Mep1 and Mep2 and the carbohydrate-active enzymes Gla1, Amy1 and Cbd1. Their pathogenicity contribution was analyzed in the haploid parental strain V. dahliae. Deletion of the majority of the corresponding genes caused no phenotypic changes during ex planta growth or invasion and colonization of tomato plants. However, we discovered that the MEP1, NLP2, and NLP3 deletion strains were compromised in plant infections. Overall, our exoproteome approach revealed that the fungus induces specific secretion responses in different environments. The fungus has a general response to non-plant related media whereas it is able to fine-tune its exoproteome in the presence of plant material. Importantly, the xylem sap-specific exoproteome pinpointed Nlp2 and Nlp3 as single effectors required for successful V. dahliae colonization.

轮枝菌属（Verticillia）可引发维管束萎蔫病，危害多种具有重要经济价值的作物。该真菌通过根部侵染宿主植物，并定殖于维管束系统，成功完成植物定殖依赖于胞外蛋白的参与。本研究对异源二倍体长孢轮枝菌（Verticillium longisporum）在不同培养基，以及从宿主植物甘蓝型油菜（Brassica napus）中提取的木质部汁液中培养时的外蛋白质组（exoproteome）进行了比较分析。采用无标记液相色谱-串联质谱（label free liquid chromatography-tandem mass spectrometry）技术筛选鉴定真菌分泌蛋白。结果显示，长孢轮枝菌存在两种主要的分泌模式：一种响应模式由各类非植物相关环境诱导产生；第二种模式则对应植物相关培养基（富含果胶的模拟木质部培养基与纯木质部汁液）引发的外蛋白响应，该模式表现出相似但又具有独特差异的特征。
这些外蛋白质组共包含221个分泌型且富集程度相似的真菌核心蛋白。富含果胶的培养基可显著诱导143种蛋白的分泌，其中包含多种果胶降解酶；而木质部汁液则触发了一组规模更小但特异性更强的真菌外蛋白质组，包含32种富集蛋白。该特异性组包含具有已知致病因子结构域的蛋白、金属肽酶（metallopeptidases）以及碳水化合物活性酶（carbohydrate-active enzymes）。这些不同类群中丰度最高的蛋白分别为坏死与乙烯诱导样蛋白（necrosis and ethylene inducing-like proteins）Nlp2与Nlp3、拟盘多毛孢素蛋白Cp1与Cp2、金属肽酶Mep1与Mep2，以及碳水化合物活性酶Gla1、Amy1与Cbd1。
研究以单倍体亲本菌株大丽轮枝菌（V. dahliae）为对象，分析了这些蛋白的致病贡献。对多数对应基因的敲除并未导致菌株在非植物培养条件下的生长，或是侵染定殖番茄植株的表型变化。但研究发现，MEP1、NLP2与NLP3基因的敲除菌株在植物侵染过程中致病力受损。
综上，本研究通过外蛋白质组学分析发现，该真菌在不同环境下会诱导特异性的分泌响应：对非植物相关环境产生通用型响应，而在接触植物材料时则可精细调控其外蛋白质组。尤为重要的是，木质部汁液特异性的外蛋白质组分析证实，Nlp2与Nlp3是大丽轮枝菌成功定殖宿主所需的单一效应因子（effector）。