禾谷镰刀菌cAMP-PKA信号途径下游基因功能鉴定及对DON产生的调控机制

Wheat scab or head blight caused by Fusarium graminearum is one of the most important diseases of wheat and barley worldwide. It not only causes significant yield losses but also results in the contamination of infected kernels with mycotoxins, such as deoxynivalenol (DON) that are harmful to human and animals. Although the functions of cAMP-PKA pathway in F. graminearum have been studied, genetic mechanisms responsible for the regulation of DON synthesis by this pathway are not clear. To date, there are no studies on downstream targets and transcription factors (TFs) of the cAMP-PKA pathway in this important pathogen. In this study, we will further analyze the regulatory mechanisms of cAMP-PKA pathway on DON production and toxisome formation. Genes responsible for suppressing growth, DON production and infection defects of the cpk1 cpk2 double mutant will be identified and characterized for suppressor mutations. We also propose identify and characterize downstream TFs of the cAMP-PKA pathway and determine their functional relationships. Some of them may be co-regulated by the cAMP-PKA singaling and MAP kinase pathways and are important for the cross-talking between these two important pathways. In additional, we will analyze the regulatory mechanisms of downstream TFs on DON production. Results from the proposed study will significantly improve our understanding for the roles of cAMP-PKA pathway in plant infection and DON production in F. graminearum. In the long run, this study will provide useful information for developing novel fungicide targets and scab disease control strategies.

小麦赤霉病严重危害我国和世界小麦生产，发病麦粒中的真菌毒素DON严重危害人畜健康。前期研究发现，cAMP-PKA信号途径可参与调控禾谷镰刀菌致病性并影响DON产生，但该信号途径调控DON产生的分子机制尚不清楚，对其下游转录因子及作用机制也缺乏了解。为此，项目拟深入分析cAMP-PKA信号途径对DON毒素合成的调控机制；通过对该信号通路上PKA两个催化亚基基因双敲除突变体不同表型角变子重测序及结果分析、验证，确定cAMP-PKA信号通路下游相关转录因子；对这些转录因子进行功能分析，并明确它们之间的相互作用关系以及与MAPK信号通路之间的作用关系，确定受两个信号通路共同调控的下游转录因子；同时明确这些下游转录因子在DON毒素产生中的作用。研究结果将会进一步了解cAMP-PKA信号通路在赤霉菌致病和产毒方面的作用机制，为小麦赤霉病的防控和新型杀菌剂的研发提供有效的理论依据。

小麦赤霉病是世界性真菌病害，其主要致病菌-禾谷镰刀菌不仅引起作物产量下降，还可产生引起人畜中毒的真菌毒素DON。虽然已发现禾谷镰刀菌中cAMP-PKA信号通路对DON毒素产生具有重要作用，但具体的作用机制尚不清楚。本项目通过对fac1、cpk1突变体菌株TRI基因定位、产毒小体形成以及菌丝膨大结构处细胞核分裂情况的观察，发现DON毒素的合成应与膨大结构处细胞核分裂密切相关，进一步从细胞学角度明确了DON毒素合成的调控机制；项目以cAMP-PKA信号通路上蛋白激酶A两个催化亚基双敲突变体所产生的角变子入手，通过确定角变子序列位点发生突变的基因，明确了位点突变基因与蛋白激酶A的作用关系，确定了cAMP-PKA信号通路下游相关转录因子FgSFL1并对其功能作用进行了解析，发现该基因的缺失对菌落生长没有影响，但突变体产孢量明显下降，子囊壳变小、形成延迟、数量减少、鞭须形成时间晚，侵染小麦穗和玉米须的致病力均显著下降。通过对预测的三个磷酸化位点功能分析进一步明确了FgSfl1位于cAMP-PKA信号通路下游并受PKA激酶的调控；同时项目还获得了蛋白激酶A调节亚基基因PKR缺失突变体并对其也进行了系统的功能鉴定，结果发现PKR的缺失可导致突变体菌丝核分裂数增多，分生孢子糖原积累变少，自噬现象明显，侵染小麦穗和玉米须的能力显著下降，有性阶段子囊壳无法形成。研究过程中还发现该突变体同样易发生角变，经对角变子表型鉴定、测序等一系列验证后发现CPK1，SNT1基因均存在较高的突变率。这两个基因不同的突变位点，会导致相应角变子的生长速率和菌落形态有不同程度的恢复。进一步研究还发现CPK1突变可抑制PKR缺失对PKA催化亚基基因的上调表达作用。