小分泌蛋白在盾壳霉-核盘菌互作中的作用及应用研究

Coniothyrium minitans is an important sclerotial mycoparasite, and could be used to control crop diseases caused by fungi in the genus of Sclerotinia. Nowadays, commercial biological control agents with conidia of C. minitans as major active ingredient are available. C. minitans has a narrow host range; it could only parasitize fungi in the genus of Sclerotinia, but cannot parasitize Botrytis cinerea, a fungal plant pathogen which shares the same family with Sclerotinia spp., this fact suggests that there exists a compatible interaction between C. minitans and its hosts. Previously, we compared the gene expression pattern of C. minitans when it contacted its host S. sclerotiorum and its non-host B. cinerea with RNA_Seq analysis, and we found that the gene expression patterns between C. minitans/S. sclerotiorum and C. minitans/B. cinerea were significantly different. This experiment results further suggested that the compatible interaction exists really between C. minitans and S. sclerotiorum. Furthermore, we also screened some small secretory proteins of C. minitans whose genes were highly expressed during the early stage of parasitization, and these small proteins are similar to these of effectors of crop pathogens, such as small and rich in cysteine residue. We suspect that these small proteins may function as effectors of plant pathogens. In this proposal, four small proteins will be chosen for further study. First, we will further detect the expression patterns of genes encoding the four small proteins when C. minitans contacts its host S. sclerotiorum, the function of these four proteins on the parasitism of C. minitans, subcellular location of these proteins in S. sclerotiorum cells. We also will examine the effect of these small proteins of C. minitans on the growth, sclerotial formation and pathogenicity of S. sclerotiorum when transgenically introduced into S. sclerotiorum. If these proteins could attenuate the growth and pathogenicity of S. sclerotiorum, we will express these genes on rapeseed plants transiently and insistently to probe the possibility as resistance genes to improve the resistance of plants against S. sclerotiorum. Our study is likely to enhance our understanding the molecular interaction between C. minitans and S. sclerotiorum, and it may also supply new resistance genes for genetic modification of crop resistance against Sclerotinia diseases.

盾壳霉是一种重要的核盘菌重寄生真菌，可以用于作物菌核病的生物防治，目前在国内外均有以盾壳霉分生孢子为主要成分的生防菌剂。盾壳霉能寄生核盘菌属真菌，但不能寄生与核盘菌具较近亲缘关系的灰葡萄孢，预示盾壳霉与核盘菌存在亲和性互作。我们前期研究表明，盾壳霉与核盘菌和灰葡萄孢接触后，在基因表达上有显著的差异，表明这种亲和性互作客观存在；同时也从盾壳霉与核盘菌互作中筛选到一些具有分泌特征的小分子蛋白。本项目拟以盾壳霉-核盘菌互作体系为模型，研究4个在盾壳霉侵染核盘菌早期诱导表达的、具有信号肽、无跨膜结构的分泌蛋白对盾壳霉寄生特性的影响，分析它们直接进入核盘菌的可能性及对核盘菌生长和致病作用的干扰，以解析小分泌蛋白在盾壳霉-核盘菌亲和互作中的作用，并探索在油菜中异源表达盾壳霉分泌蛋白增强作物抗菌核病的可能性。研究结果将有助于我们理解盾壳霉寄生核盘菌的分子机制，并为培育抗菌核病材料积累抗性基因资源。

盾壳霉是一种重要的核盘菌重寄生真菌，可以用于作物菌核病的生物防治，目前在国内外均有以盾壳霉分生孢子为主要成分的生防菌剂。盾壳霉能寄生核盘菌属真菌，但不能寄生与核盘菌具较近亲缘关系的灰葡萄孢，预示盾壳霉与核盘菌存在亲和性互作。我们前期研究表明，盾壳霉与核盘菌和灰葡萄孢接触后，在基因表达上有显著的差异，表明这种亲和性互作客观存在；同时也从盾壳霉与核盘菌互作中筛选到一些具有分泌特征的小分子蛋白。依托本项目我们对盾壳霉的基因组进行了重新注释，并在转录组水平分析了盾壳霉寄生核盘菌和核盘菌对盾壳霉寄生的反应。研究了四个盾壳霉分泌蛋白基因CMZS1_03102、CMZS1_07825、CMZS1_06645和CMZS1_08750 4对盾壳霉、核盘菌、灰葡萄孢、烟草和拟南芥的影响，发现它们的敲除对盾壳霉的生长和寄生无显著影响；超量表达（有和没有信号肽）对核盘菌生长和菌落形态有较大影响，而对灰葡萄孢影响较小，并且超量表达不能使灰葡萄孢获得被盾壳霉寄生的能力；超量表达可以在发病前期提高植株植物对菌核病的抗病能力，但后期不显著；其中CMZS1_03102可能在核盘菌和烟草细胞外其作用，而CMZS1_06645可能会进入细胞内。我们希望能在油菜上进一步评价基因在抗病中应用的潜能。同时我们也对盾壳霉线粒体蛋白基因CmAim24、铁载体铁转运类似蛋白基因CmSIT1和转录因子基因CmMR1的功能开展了研究，鉴定了它们在盾壳霉寄生、拮抗和抗逆中的作用；初步研究了核盘菌坏死和乙烯诱导蛋白基因SSNEP2在核盘菌与盾壳霉互作中的作用。研究结果将有助于我们理解盾壳霉选择性寄生核盘菌的分子机制，并为培育抗菌核病材料积累抗性基因资源。