效应蛋白FOL1及其互作蛋白在西瓜-镰刀菌相互作用中的功能分析

The phytopathogenic fungus Fusarium oxysporum f. sp. Niveum causes Fusarium wilt of watermelon, one of the most destructive watermelon diseases. Wild Germplasm PI296341 is an excellent resource carrying resistance to all Fusarium oxysporum f. sp. Niveum races. However, incorporation of the resistance trait into commercial types has proved to be difficult, thus it is very significant to isolate resistance related genes and to understand the molecular mechanisms underlying the interactions between watermelon and F. oxysporum. Increasing studies demonstrated that many plant pathogens secrete effector proteins that play a crucial role in establishment of a successful infection. These effector proteins interact with host factors to suppress or elicit host defense responses, resulting in plant susceptibility or resistance. Therefore, isolation and characterization of fungal effectors and their host targets will benefit the elucidation of molecular basis of host-fungi interactions. In previous work we showed that a secreted lipase FOL1 play an important role of FOL1 during plant infection. To identify potential FOL1 physically interacting targets in watermelon, we screened PI296341 yeast cDNA expression libraries using the FOL1 protein as a bait. Based on these studies, our objectives were: (1) to further investigate the function of FOL1 through inoculation of wild-type and gene deletion strains to both resistant and susceptible watermelon germplasms and analysis of FOL1 expression patterns during host infection; (2) to preliminarily analyze the identified FOL1 interacting proteins by bioinformatics, gene expression, and so on, and further investigate one or two FOL1 interacting proteins as potential targets for disease resistance. This study will lay the foundation for control of Fusarium wilt and deepen the understanding of signaling pathways regulating disease resistance in watermelon- F. oxysporum interactions.

枯萎病是西瓜生产中最主要的致命性病害。野生西瓜种质PI296341是公认的抗枯萎病优异资源。如何获取其中的抗病基因并明确抗病机理显得尤为重要。越来越多的研究证实病原菌分泌效应蛋白与寄主蛋白互作后启动或抑制抗病反应。本项目组前期研究表明西瓜专化型尖孢镰刀菌的一个分泌性酯酶（FOL1）在侵染过程中具有重要作用，并采用酵母双杂交的方法在已构建的PI296341酵母表达文库中完成初步筛选。在此基础上，本研究计划：１、根据抗、感枯萎病种质材料接种真菌野生型和缺失突变体后的抗病反应以及FOL1在侵染前后的表达情况，进一步明确FOL1的功能。2、通过对初步获得的蛋白进行生物信息学、基因表达等分析，选取1-2抗病相关基因进行进一步的功能研究。根据上述结果推断"FOL1-靶蛋白"在西瓜-镰刀菌互作中的功能。上述研究将为防治枯萎病及深入了解西瓜-枯萎病抗病信号传导网络途径提供良好的基础和借鉴意义。

枯萎病是西瓜生产中最主要的致命性病害，该病是由西瓜专化型尖孢镰刀菌（Fon）侵染西瓜造成的。Fon存在三个生理小种0、1、2，其中以Fon1引发的枯萎病占主导地位。目前，西瓜种质PI296341-FR是公认的抗枯萎病三个生理小种的优异资源。因此，寻找Fon效应蛋白和其相互作用的西瓜蛋白对理解西瓜专化型尖孢镰刀菌-西瓜间的相互作用和防治枯萎病具有重要意义。.项目组从Fon1中分离获得了一个分泌性酯酶（命名为FOL1），发现FOL1基因存在Fon的三个生理小种中，过表达该蛋白可以激发烟草的过敏性反应。通过基因组比对后克隆获得该基因的上下游序列，构建Fon1的FOL1缺失菌株ΔFOL1和互补菌株ΔFOL1+FOL1，接种西瓜后发现致病力明显下降，说明FOL1是一个致病因子，在Fon侵染西瓜过程中抑制西瓜抗病基因的表达。.另一方面，构建了一个满足实验需要的PI296341-FR酵母表达文库，以FOL1为诱饵筛选获得了相互作用的西瓜蛋白10个。通过生物信息学分析后发现，其中protein phosphatase 2c 、calcium lipid binding protein和zinc-binding protein可能涉及到西瓜抗病反应。克隆获得了calcium lipid binding protein和zinc-binding protein全长并发现calcium lipid binding protein与FOL1蛋白间存在相互作用，命名为FOLR1，发现该基因在根部受Fon1诱导表达。上述研究将为采用防治枯萎病提供理论依据及为西瓜抗病提供基因支持。