WOPR转录因子调控菰黑粉菌致病力的作用机制研究

Dimorphic switch and spore formation are critical to plant panthogens pathogenicity. Previous studies showed that WOPR family of transcriptional regulator regulated pathogenicity through activating or depressing dimorphic switch and spore formation. Unfortunately, most studies focused on ascomycete and pathogenic fungi. As the endophyte of Zizania latifolia, which was the second largest planted aquicolous vegetable in China, appropriate pathogenicity of Ustilago esculenta was essential to normal gall formation. But results related to WOPR regulating pathogenicity in Ustilago esculenta was not reported in until now. We have cloned two homologs of WOPR transcriptional regulator, and signal pathways inflencing the dimorphic switch and teliospore formation were identified. In this study, we will employ inducable promotor, gene knock-out or over-expression, artificial inoculation to investigate the mechanisms involved in WOPR regulating pathogenicity in Ustilago esculenta. And the directly binding genes and binding site will be indentified. Our expected findings will uncover the underlying mechanisms involved in WOPR regulating pathogenicity, and advance our understanding of WOPR function in basidiomycete and endophyte.

形态转换和孢子形成是关系植物病原真菌致病力的关键。研究显示，WOPR转录因子通过调控形态转换和孢子形成来调节真菌致病力。但相关研究多集中于子囊菌和病原真菌。作为我国第二大水生蔬菜茭白的内生真菌-菰黑粉菌，其致病力的强弱对于形成正常茭白至关重要，但关于WOPR转录因子调控菰黑粉菌致病力的相关研究未见报道。项目组前期克隆了两个菰黑粉菌WOPR转录因子同系物，明确了参与形态转换和影响冬孢子形成的信号途径及关键基因。本研究结合诱导型启动子、基因敲除或过表达、人工接种等试验方法，及ChIP-Seq、转录组测序及组织切片等技术，明确WOPR转录因子调控菰黑粉菌形态转换及冬孢子形成的作用途径、直接作用基因及作用位点，深入分析WOPR转录因子调控菰黑粉菌致病力的可能机制。所得结果不仅可以阐明WOPR转录因子调控菰黑粉菌致病力的内在机制，更有助于明晰WOPR转录因子在担子菌及内生真菌中的作用。

WOPR转录因子在植物病原真菌中被报道参与调控形态转换和孢子形成来调节真菌致病力。本项目在菰黑粉菌中鉴定并克隆到到两个WOPR转录因子UeItd1和UePac2。通过对UeItd1和UePac2基因敲除突变体的表型分析，发现UeItd1基因参与调控菰黑粉菌侵染后期的冬孢子形成，但是并不参与调控二态型转换、菌丝生长和侵染能力；而UePac2基因敲除突变体表型相比于野生型没有差异。进一步通过RNA-Seq技术分析UeItd1的调控网络，鉴定并克隆到一个p53-like家族的转录因子基因UeTsf1，其基因敲除突变体与UeItd1基因敲除突变体表型一致；结合基因敲除表达、转录激活、酵母单杂交等实验证明UeItd1处于UeTsf1上游，并通过结合其基因启动子区域来调控其表达。最后通过qRT-PCR进一步解析了UeTsf1负调控菰黑粉菌与细胞周期和冬孢子形成紧密相关的b交配型位点的基因（bE和bW）和UeRbf1。本项目的研究明确了一条由WOPR转录因子UeItd1通过调控UeTsf1来影响b交配型位点的基因和UeRbf1的表达，从而参与调控冬孢子形成的基因通路。为研究菰黑粉菌以及其它黑粉菌冬孢子的形成提供理论基础。此外，这还帮助更加深入了解茭白田间灰茭的形成机理，为田间的灰茭防治提供新的思路。