PKA磷酸化对橡胶树胶孢炭疽菌CgCAP20蛋白功能的影响

Perilipin is the most abundant lipid-associated protein on the surfaces of lipid droplets, and regulates lipid metabolisam, appressorial tugor pressure and virulence in plant pathogenic fungi. As we known, perilipin is phosphorylated by protein kinase A and serves important functions in the regulation of lipolysis in the adipose tissues of mammals and insects. But the regulation mechanism of perilipin homolog in fungi are still unknown. In our early study, we cloned the CgCAP20 gene in C.gloeosporioides, the pathogen of Colletotrichum leaf fall disease of Hevea brasilience. Sequence and preliminary functional analysis showed that CAP20 protein was perilipin homolog, and important to fully functional appressorial formation and fungal virulence. This suggests that there is probably PKA-CAP20 regulated cellular lipid metabolismin in fungi. In this study, we intend to use the real-time fluorescent quantitative PCR and bimolecular fluorescence complementation (BiFC) technology to demonstrate whether or not the CAP20 is regulated by protein kinase A in mRNA transcription and protein expression level in C.gloeosporioides. If so, we will further analysis the function of three cAMP-dependent protein kinase A phosphorylation sites of CAP20 respectively in protein-protein interaction by site-directed mutagenesis method based on overlap extension PCR and BiFC. In the mean time, we construct the phosphorylation sites mutation respectively. And functional analysis of PKA phosphorylation sites by observing phenotype of these mutants comparatively, and testing the relationship between the phenotype varieties will be finished. And we also will determine whether the relationship between PKA phosphorylation sites and CgCAP20 subcellular localization by fusing GFP protein to CgCAP20 mutation. The research results will help us further to understand the regulation of lipolysis in fungi and clarify the accumulation of turgor pressure in appressoria and pathogenesis of pathogenic fungus.

脂滴包被蛋白与病原真菌附着胞穿透寄主能力和致病性关系密切。已知哺乳动物的脂滴包被蛋白受PKA调控，对细胞内甘油三酯的水解与合成过程起着双重调控作用，但至今未见病原真菌脂滴包被蛋白的调控机制研究报道。根据已有基础，推测病原真菌中也有PKA调控脂滴包被蛋白的机制。本项目在证实CgCAP20蛋白是橡胶树胶孢炭疽菌脂滴包被蛋白并影响附着胞穿透寄主能力和致病力的基础上，在转录水平、蛋白水平和细胞学水平上，采用实时定量荧光PCR技术、双分子荧光互补技术和定点突变等技术，验证CgCAP20与PKA的调控关系，研究胶孢炭疽菌CgCAP20三个PKA磷酸化位点对该蛋白调控的影响，和对该蛋白的定位、以及该蛋白对病菌生长发育、附着胞膨压和致病力等功能的作用。本项目的完成有助于阐明植物病原真菌脂滴包被蛋白的调控机制，以期为理解植物病原真菌的致病机理及制定植物病害防控策略提供科学依据。

脂滴包被蛋白与病原真菌附着胞穿透寄主能力和致病性关系密切。但病原真菌脂滴包被蛋白的调控机制未见报道。本研究在前期获得橡胶树炭疽菌脂滴包被蛋白CgCap20基因和获得基因缺失突变体的基础上，重点围绕CgCap20蛋白的互作蛋白及调控机制开展工作，主要研究结果有：.（1）进一步完善了CgCap20基因的功能研究，证实该蛋白与病原菌附着胞细胞壁孔径大小、膨压大小和致病性存在关系；.（2）构建了橡胶树炭疽菌cDNA酵母文库，采用酵母双杂技术从酵母文库中筛选获得16个互作蛋白，主要有环腺苷酸依赖性蛋白激酶催化亚基（PKAC1）、蛋白激酶、乙酸激酶、疏水蛋白、组氨酸酸性磷酸酶、铁还原酶样跨膜成分、翻译起始因子、bys1 结构域蛋白、磷酸盐：H+转运体、过敏反应诱导蛋白、以及6个假定蛋白；.（3）重点分析了PKA亚基基因PKAC1与CgCap20间的表达和互作关系，证实了在炭疽菌中CgCap20基因的表达受到PKA亚基基因的正向影响，以及PKAC1与CgCap20发生互作；同时还证实了乙酸激酶CsAck和CgCap20可互作；.（4）证实了CgCap20蛋白为磷酸化蛋白。初步分析了PKA磷酸化位点对CgCap20基因功能的影响，结果显示，未发生磷酸化位点突变的互补转化子，能一定程度恢复基因缺失突变体表型，但三个磷酸化位点突变体未能恢复表型，初步证实磷酸化位点对分生孢子瘦弱、分生孢子长度是否均一以及细胞壁抗逆性等方面具有重要作用；.（5）对互作蛋白疏水蛋白CsHydr功能进行分析，结果显示，与野生型相比，基因缺失突变体分生孢子大小显著缩小，菌丝表面疏水性降低，菌落生长略大；突变体在含氯化钠和刚果红培养基中生长比野生型快，都有一定的抗逆性，致病力分析显示基因缺失致病能力显著降低。推测脂滴包被蛋白和疏水蛋白互作可能参与细胞壁组分调控，从而影响病原菌致病力。.该研究获得了脂滴包被蛋白的互作蛋白，证实了植物病原真菌炭疽菌脂滴包被蛋白受到PKA蛋白调控，此外它还可能受到乙酸激酶、组氨酸酸性磷酸酶等其他蛋白调控。初步证实其磷酸位点对功能具有重要作用。同时还分析了CgCap20的互作蛋白疏水蛋白的功能。下一步将重点阐明这些互作蛋白与脂滴包被蛋白互作的生物学意义。该研究结果为阐明植物病原真菌脂滴包被蛋白的调控机制和了解其如何参与调控病原真菌致病机制奠定基础。