大丽轮枝菌异分支酸水解酶VdIch1在植物中的转运及致病机制解析

Biosynthesis of salicylic acid in plant mainly through chorismate-isochorimate pathway, and majority of fungi contains the isochorismatases. It was predicted that this enzyme will target the direct precursor isochorimate for suppress the biosynthesis of salicylic acid, but was not reported. We previously found that an isochorismatase VdIch1 in V. dahliae was involved in pathogenicity of V. dahliae. When the gene was knockout, the pathogenicity was significantly reduced and the salicylic acid level in plant infected by knockout mutant was increased, and we also found it secreted protein. Collectively, it was proposed that VdIch1 wound have function in plant. Here, we will fuse HA-tag with VdIch1 and overexpress it in V. dahliae, and use immune colloidal gold technique to determine the fusion protein whether go into the plant cell; fuse the VdIch1 with red fluorescent protein and overexpress it in V. dahliae and observe VdIch1 whether spread into neighbouring cells. We also want to transfer VdIch1 in tobacco and test the resistance of the transgenic plant to pathogens and reveal the function of VdIch1 in plant, and use RNA-seq to determine the signal pathway of Vdich1 in transgenic tobacco. The study will help us to understand the translocation function of isochorismatase VdIch1 and its role in plant.

植物水杨酸（SA）主要通过分支酸-异分支酸途径合成，大多病原真菌含有异分支酸水解酶基因，推测该基因作用于SA合成的直接前体（异分支酸）抑制植物SA合成，但未有报道证实。我们发现大丽轮枝菌异分支酸水解酶基因VdIch1敲除后，大丽轮枝菌的致病力下降、被侵染棉花根系的SA含量上升，表明VdIch1基因参与了致病过程。Western杂交证明VdIch1蛋白可以分泌到真菌胞外，推测其可能在植物细胞内行使功能。本项目拟将VdIch1分别与HA-tag和RFP融合后在大丽轮枝菌过量表达，明确VdIch1蛋白在侵染过程中的定位，确认它能否转运到寄主细胞内并扩散到邻近细胞；将VdIch1在烟草过量表达，测定烟草SA含量及其抗病性，研究VdIch1对植物SA及其抗病性的影响，并通过转基因烟草转录组解析VdIch1作用的信号通路。本项目将在明确VdIch1基因致病功能的基础上，进一步阐述其转运能力及致病机制。

大丽轮枝菌侵染引起的棉花黄萎病目前是棉花的不治之症，其和寄主互作的机制研究较少。本研究主要研究了一个对致病性非常关键的异分支酸水解酶基因VdIch1在侵染.过程中的作用机理。证明了VdIch1可进入寄主细胞，并可在寄主细胞内扩散；发现VdIch1在侵染过程中主要定位于吸器，通过吸器分泌出去；明确了VdIch1可增强植物感病性；实验表明，VdIch1主要通过降低植物水杨酸含量促进病原菌侵染。通过对大丽轮枝菌异分支酸水解酶VdIch1在侵染过程中的作用解析，发现了病原菌是如何克服寄主的先天免疫，为进一步制定针对黄萎病的防治措施奠定基础。