Table_6_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，侵染多种具有重要经济价值的农作物。该真菌通过根部侵入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。本研究对比了异源二倍体长孢轮枝菌（Verticillium longisporum）在不同培养基，或是从宿主植物油菜（Brassica napus）中提取的木质部液中培养时的分泌蛋白质组（exoproteome），并通过无标记液相色谱-串联质谱（label free liquid chromatography-tandem mass spectrometry）筛选鉴定真菌分泌蛋白。长孢轮枝菌存在两种主要分泌模式：一类响应模式由各类非植物相关环境诱导产生；另一类则包含针对植物相关培养基的分泌蛋白响应，涵盖富含果胶的模拟木质部培养基与纯木质部液，二者表现出相似但又各具独特特征的分泌谱。这些分泌蛋白质组共包含由221个分泌且富集程度相似的真菌蛋白构成的共享核心集。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；而木质部液则触发了一组规模更小但独具特色的真菌分泌蛋白质组，包含32种富集蛋白。该组蛋白包含带有已知致病因子结构域的蛋白、金属肽酶以及碳水化合物活性酶，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Nlp2、Nlp3，蜡板蛋白（cerato-platanin）Cp1、Cp2，金属肽酶Mep1、Mep2，以及碳水化合物活性酶Gla1、Amy1与Cbd1。研究人员在单倍体亲本菌株大丽花轮枝菌（V. dahliae）中分析了这些蛋白的致病贡献：对多数对应基因进行敲除后，菌株在非植物培养条件下的生长，或是入侵并定殖番茄植株的过程中均未出现明显表型变化，但MEP1、NLP2与NLP3的敲除菌株在植物侵染过程中致病力显著受损。总体而言，本研究的分泌蛋白质组分析方法揭示，该真菌可在不同环境中诱导特异性的分泌响应：其对非植物相关培养基存在一般性响应模式，而在接触植物材料时则可对分泌蛋白质组进行精细调控。尤为重要的是，木质部液特异性的分泌蛋白质组筛选确定了Nlp2与Nlp3作为大丽花轮枝菌成功定殖所必需的单一效应因子。