氧固醇结合蛋白调控稻瘟菌致病力及营养生长的分子基础

Oxysterol-binding proteins (OSBP) are a class of proteins that widely exist in eukaryotic organisms ranging from yeast to humans. OSBPs bind small lipid molecules, including steroids, phosphatidylinositol-4-phosphate, and phosphatidylserine, through the conserved ligand-binding domain and have important roles in a range of cellular processes including lipid metabolism, vesicle and non-vesicle trafficking, and signal transduction. Up to date, the role of OSBPs in mediating plant-microbe interaction has not been revealed, although these proteins have been reported to mediate polar growth during the budding process in Saccharomyces cerevisiae, and colony radial growth and sporulation in Aspergillus nidulans. We have identified 6 OSBP genes from the rice blast fungus, and found that they have overlapping but diversified functions in modulation of fungal virulence and vegetative growth: OSBP1 and OSBP2 are collectively key for the fungus to cause disease in planta, whereas OSBP1 and OSBP3 are essential in combination to colony radial growth on the plate. Based on these observations, we propose to further characterize the biological phenotypes of double mutants deficient in both OSBP1 and OSBP2 genes, and OSBP1 and OSBP3 genes, thereby identifying the exact pathological and cellular aspects where the OSBPs are involved. We will use an approach integrating multidisciplinary methods and technology, including plant pathology, biochemistry, cellular biology, transcriptomic and metabolomic analyses, to unravel the molecular basis of OSBP-mediated biology. Findings from this study will provide new insight on the mechanism of regulation of virulence and polar growth in Maganaporthe oryzae, and shed new light on the development of novel strategies for the control of the rice blast disease in future.

氧固醇结合蛋（OSBPs）广泛存在于由酵母到人类的许多真核生物中，它们通过保守的结构域与类固醇等脂类分子结合，参与调控细胞的脂类代谢、囊泡及非囊泡运输以及信号传导等过程。真菌中OSBP可介导酵母出芽生殖与曲霉产孢及菌落的辐射生长。稻瘟菌含有6个OSBP基因，我们发现其中三个基因为菌丝生长及致病过程所必需。本研究将立足这一新发现，通过进一步分析突变体表型，确定稻瘟菌OSBP蛋白影响生长及侵染的具体环节；通过转录组与代谢组分析，明确OSBP调控基因表达与代谢的关键途径；通过研究OSBP的亚细胞定位，分离鉴定其结合底物及互作蛋白，解析它们行使功能的分子基础。本研究将为揭示稻瘟菌侵染致病及生长发育机制提供新的视角，为未来研发稻瘟病防控新策略提供重要依据。

氧固醇结合蛋白基因家族在真核生物中广泛存在，它们参与调控囊泡运输、信号转导及自噬、胞吞胞吐等多种细胞过程。我们前期发现稻瘟菌MoOSBP基因家族成员中MoOSBP1、MoOSBP2、MoOSBP3是侵染致病和菌丝生长过程必需的基因。本研究通过检查MoOSBP1/2和MoOSBP1/3两个双敲除菌株侵染和生长过程的表型，发现上述三个MoOSBP基因参与调控稻瘟菌侵染植物的多个关键环节，包括孢子萌发后的程序性细胞死亡、附着胞形态建成、膨压形成、侵入钉穿透以及侵染菌丝扩展等。此外，MoOSBP双敲除菌株的胞质效应子递送及抑制寄主活性氧积累的能力相应受损。在稻瘟菌菌丝生长方面，我们发现极性胞吐作用在MoOSBP1/3双敲除菌株中受损，这可能导致其生长严重缺陷的重要原因之一；同时发现双敲除菌株的胞吞及自噬过程严重缺陷并伴随出现胞内甾醇分布异常。亚细胞定位研究发现三个MoOSBP蛋白主要分布于细胞质，但MoOSBP1和MoOSBP2也会在质膜上形成点状定位，蛋白互作研究发现MoOSBP3可能与MoSsz1、MoLCTL存在互作但其互作的意义有待进一步研究。稻瘟菌MoOSBP蛋白不含分泌信号肽，但本研究首次发现培养条件下及侵染大麦叶片时它们均可被分泌到胞外，同时原核表达的MoOSBP可以触发拟南芥先天免疫反应，暗示该类蛋白可能扮演了病原相关分子模式的角色。上述结果为深入揭示OSBP在稻瘟菌生长、致病及植物互作过程中的作用奠定了基础。