致病相关分泌蛋白SS1G_08128在核盘菌致病过程中的作用及其机理研究

The necrotrophic fungal phytopathogen Sclerotinia sclerotiorum could cause sclerotinia diseases in rapeseed crop, causing serious economic losses each year. The pathogenicity-associated secretory proteins play significant roles during the infection of S. sclerotiorum on the host plants. The rapeseed sclerotinia diseases would be controlled effectively and safely by inhibiting the activity of S. sclerotiorum pathogenicity-associated secretory proteins. However, until now, the genes screen and the functional mechanisms of the pathogenicity-associated secretory proteins involved in interference of plant defense or inducing necrosis are still largely unknown. In our previous study, the S. sclerotiorum transcriptome during the early stage of infection had been analyzed and identified a small molecular secretory protein SS1G_08128. Targeted deletion of SS1G_08128 gene resulted in a significant reduction in virulence, indicating that SS1G_08128 is pathogenicity associated secretory proteins. In addition, expression of SS1G_08128 in N. benthamiana by the using of Agrobacterium- mediated transient expression developed necrosis, indicating that SS1G_08128 is a pathogenicity-associated secretory proteins by inducing necrosis. In this study, the correlation between the SS1G_08128 and S. sclerotiorum pathogenicity will be defined further by gene complementation and over expression assaying. Further more, the function mechanisms of SS1G_08128 in S. sclerotiorum infection will be further characterized by the gene expression analysis, protein location and the yeast two-hybrid system. The results of this study will be useful in elucidating S. sclerotiorum pathogenesis and also provide the new theoretical basis for the exploration of inhibition strategy on the activity of pathogenicity-associated secretory proteins, cultivation of the sclerotinia diseases-resistant crops and the development of new type fungicide.

由核盘菌引起的油菜菌核病可造成油菜严重减产，而致病相关分泌蛋白在核盘菌的侵染过程中起着重要作用，研究致病相关分泌蛋白的功能将会为油菜菌核病的防控提供理论依据。但迄今为止，对抑制植物抗性或诱导植物死亡的核盘菌致病相关分泌蛋白的功能机理的研究还相对较少。在前期研究中，通过对核盘菌侵染早期转录组的分析，筛选到一个小分子分泌蛋白SS1G_08128，该基因敲除转化子的致病力显著降低，并且通过农杆菌对其进行瞬时表达后可引起烟草叶片的坏死，研究结果表明SS1G_08128可能通过诱导植物的坏死从而参与核盘菌的致病。本研究拟从该基因着手，通过基因互补和超表达技术进一步明确该基因与致病力的关系，并通过基因表达分析、蛋白定位以及筛选互作蛋白等方法深入研究该基因的功能及其调控核盘菌致病的作用机制，研究结果将进一步阐明核盘菌的致病机理，为其致病相关分泌蛋白阻断策略的研发和抗病作物的培育提供理论指导。

致病相关分泌蛋白在核盘菌致病过程中起着重要作用，研究其作用机理将会为菌核病的防治提供理论基础。目前对核盘菌致病相关分泌蛋白功能的研究相对较少。在前期研究中，通过对核盘菌侵染早期RAN-Seq 分析，筛选到了两个候选基因（SS1G_00736 和 SS1G_08128），通过分割标记对这两个基因敲除后，敲除转化子的致病力降低。在此基础上，本研究以这两个基因为研究对象，通过基因互补明确这两个基因在致病过程中的作用。同时通过基因表达动态分析、蛋白分泌性分析、蛋白对植物的影响的等深入研究这两个基因的功能及其调控核盘菌致病作用机理。研究发现，这两个基因在核盘菌侵染早期能被大量诱导表达。对SS1G_00736 基因深入分析发现，与灰霉中的BcGAR2具有99%的同源性，属于D-半乳糖醛酸代谢途径中的一员。因此在核盘菌中将其命名为SsGAR2。对核盘菌中D-半乳糖醛酸代谢途径中的其它基因SsGAR1,SsLGD1和SsLGA1也进行了研究。结果证明除SsGAR1外,SsGAR2，SsLGD1和SsLGA1基因敲除转化子致病力显著下降。当核盘菌侵染大豆和菜豆早期，SsGAR2，SsLGD1和SsLGA1基因都被诱导表达。以D-半乳糖醛酸和果胶为唯一碳源的培养上生长时SsGAR2，SsLGD1和SsLGA1基因敲除转化子生长显著受抑制。研究还发现D-半乳糖醛酸代谢途径相关基因与核盘菌菌丝对细胞壁抑制剂抗逆性正向调控，对渗透压力抗逆性负向调控。本研究表明，D-半乳糖醛酸代谢途径相关基因在核盘菌致病力、抗逆性和与植物互作过程中起着重要作用，为深入揭示核盘菌的致病机理奠定了基础，也为菌核病的绿色安全防治提供了线索。