柑橘褐斑病菌NADPH氧化酶复合体介导的基因调控网络及其生物学功能解析

Alternaria alternata is a notorious fungus that causes great losses in economically important crops, including citrus, apple, pear, rice, strawberry, tomato, broccoli, cauliflower, carrot, and potato. This fungus is capable of detoxifying the ROS generated by host during infection. Pioneering work has made significant contribution on understanding the molecular mechanisms of ROS detoxification in A. alternata. NADPH oxidase complex (NOX), however, is one of the most attractive proteins/complexes that function not only in ROS detoxification, but also in biological activities such as ROS producing, osmotic tolerance and conidiation. The mutant of NoxA, a subunit of NOX complex, showed similar impairment on ROS and osmotic tolerance compared with mutants of Ap1, Skn7 and Hog1, implying the regulation beyond. However, the mechanism how NOX mediates the crosstalk between signal pathways is still elusive. In this proposed work, we intend to construct the null mutants of three NOX subunits, and extensively investigate their roles on interacting with the keys proteins in the related signal pathways by using comprehensive approaches at both transcription and protein levels. The prospective results will answer the following questions: (1) whether and how does NOX interact with AP1, SKN7 and HOG1 to orchestrate the ROS detoxification and osmotic tolerance; (2) whether the two MFS genes are functional downstream the NOX; (3) what are those proteins directly interacted with NOX. The fulfillment of this work will expand the knowledge on NOX regulating networks in eukaryotes, and also shed light on the development of novel strategies for disease control.

交链格孢菌是常见的植物病原真菌，对我国柑橘产业威胁重大。已有研究证明交链格孢对侵染过程中产生的ROS解毒能力与其致病性密切相关。前期研究发现病菌自身产生ROS系统，NADPH氧化酶复合体(NOX)，也参与包括ROS解毒在内的多个生物学过程。在抗氧化和抗渗透等方面，NOX一个亚基(NoxA)突变表现与Ap1、Skn7和Hog1等突变类似的表型，但对NoxA如何调控这些基因以及相应的调控层次尚不清楚。本研究拟通过构建NOX亚基的单、双和三基因突变体，结合目的信号通路中已有的关键基因的突变体，通过表型分析、qRT-PCR、基因表达谱和蛋白互作研究，明确NOX复合体与相关通路中关键基因的调控关系，并通过免疫共沉淀方法鉴定其他与NOX复合体直接互作的蛋白，最终阐明NOX介导的交链格孢菌在抗氧化、抗渗透以及产孢等信号途径中的关联调控机理。预期研究结果可为研究和开发防治链格孢菌病害的新途径提供科学依据。

由Alternaria alternata pathotype tangerine引起的褐斑病是柑橘上的重要病害，已有研究证明病菌对ROS解毒能力与其致病性密切相关，而且明确了Nox复合体蛋白NoxA,NoxB和NoxR在ROS解毒等方面的功能，但对复合体其他蛋白的功能及其互作关系并不清楚，对NoxA调控抗氧化的层次尚不清楚。本研究酵母双杂交证明NoxR能与RacA、BemA和Cdc24互作，BemA可与Cdc24和Cdc42互作，并且Cdc42序列中29位的K是与BemA互作所必需的，RacA中61位的W是与NoxR互作所必需的。构建△Cdc42和△RacA和CPCdc42和CPRacA，以及丧失与BemA互作的回补突变体CPgCdc42，丧失与NoxR互作的回补突变体CPgRacA。分析表明Cdc42参与菌丝营养生长，产孢，致病性以及ROS的积累，但与ROS的清除无关；RacA不参与菌丝生长，但参与产孢和致病性，以及ROS的积累和清除。Cdc42与BemA互作是ROS产生必需的，而不是菌丝营养生长，产孢和致病性所必需的；RacA与NoxR互作是生长和致病所需要的。BemA、Cdc42和RacA均定为于细胞质，丧失与BemA和NoxR的互作不影响Cdc42和RacA的定位。研究还发现Cdc42和RacA的双敲突变体以及Cdc24的缺失突变体均致死。缺失突变体△BemA活性氧产生和抗氧化能力均显著下降，表型和△Cdc42不尽相同，说明Cdc42还参与其他的途径。而△RacA的表型与此前报道的NoxR缺失突变体的表型基本一致，说明RacA大部分功能是通过NoxR介导的。.研究表明硫氧还蛋白和谷胱甘肽系统显著受过氧化氢的上调表达，硫氧还蛋白过氧化物酶（Tsa1），硫氧还蛋白还原酶（Trr1）和谷胱甘肽还原酶（Glr1）基因缺失突变体的抗氧化和致病力均显著下降或丧失，说明这些基因均是柑橘褐斑病菌毒力发挥所必需的基因。qRT-PCR 分析发现抗氧化相关的途径AP1、Skn7和Hog1受NoxA的调控，而Tsa1、Gpx3、Tsa1和Trr1在转录不仅受NoxA的调控，还AP1、Skn7和Hog1的调控。此外，研究还发现了甲硫氨酸合成调控因子（MetR）参与甲硫氨酸和半胱氨酸的合成，而半胱氨酸是谷胱甘肽的前体。综上研究推导出Nox复合体介导的抗氧化调控网络。