拮抗木霉菌外切葡聚糖酶与玉米根系互作诱导抗叶斑病分子机理

The systemic induced resistance to maize foliar disease by antagonistic Trichoderma is a major mechanism for biocontrol of maize foliar disease. Previous study showed that cellulase（exo-glucanase）and cellobiose as ISR effectors (elicitors) produced in the maize root colonization by T. harzianum is able to trigger maize leaf immunity response to foliar diseases, however, nowadays it is still unclear that what receptors in maize root cortex are responsible for the interaction with those T.harzianum effectors. To uncover the nature of both kinds of receptors and their way to trigger host plant defense response expression against infection of three kinds of foliar pathogen (Exserohilum turcicum, Bipolaris maydis, Curvularia lunata), we are going to identify the structure, subcellular distribution and degradation pathway of receptors (PRR) which is responsible for the recognition to exo-glucanase and cellubiose produced along the colonization of T.harzianum on root system. Furthermore the molecular interaction sites and biochemical characteristics between the interface of receptor-effectors as well as the changes of receptor conformation to fit binding effectors are investigaged. Meanwhile the signal long distance transduction pathway for triggering defense response is investigated from root to leaf. Then the differential expression modes of defense response initiated by the recognition of either single receptor or both receptors to effectors are characterized along time course in different maize organs. Taken together, a molecular mechanism is understood of the far distance-induced maize resistance against foliar diseases by the interaction of T.harzianum and host roots so as to innovate plant basic immunity theory supported by the synergistic action of MAMPs/DAMPs in the presence of antagonistic Trichoderma, and provide novel insight for performing molecular maize resistant breeding at micro-ecological level.

木霉菌系统诱导玉米免疫反应是其生物防治玉米叶斑病重要途径。前期工作表明，哈茨木霉菌定殖玉米根系过程中分泌的外切葡聚糖酶以及释放的纤维二糖作为诱导抗性效应因子（激发子）激发玉米对叶斑病的免疫反应，但不清楚效应因子如何与根系发生互作、诱导宿主防御反应系统表达。本研究以大斑病菌、小斑病菌和弯孢叶斑病菌为靶标，鉴定哈茨木霉菌定殖玉米根系产生的外切葡聚糖酶和纤维二糖在根系皮层细胞上的受体结构及其亚细胞分布和代谢途径；研究效应因子与受体互作位点分子和生化特征，效应因子对受体构象的影响；示踪宿主防御反应信号（JA/ET、Ca2+、ROS等）从根系到叶片长距离转导通路；分析两种受体单一表达与协同表达时空规律及对宿主防御反应信号长距离转导的影响，揭示木霉菌-根系互作远程诱导玉米抗叶斑病分子机理。本研究将创新木霉菌诱导下的MAMPs/DAMPs协同作用的植物免疫学理论，为玉米微生态抗病分子育种奠定基础。

研究哈茨木霉菌（Trichoderma harzianum）激发子与玉米根系分子互作机制是国内外的研究的难点。木霉菌分泌的纤维素酶（外切葡聚糖酶）和纤维二糖可分别起到MAMPs和DAMPs分子的作用，但互作根系的受体或靶标及诱导的寄主防御反应信号转导方式缺少深入研究。本研究以玉米弯孢叶斑病和小斑病为靶标病害，主要通过酵母双杂交和免疫共沉淀等方法筛选根系与哈茨木霉菌外切葡聚糖酶互作用的受体或靶标基因，以及哈茨木霉菌外切葡聚糖酶系统诱导防御反应信号转导的方式。首先克隆了2种木霉菌外切葡聚糖酶基因thph1和thph2，获得两基因的单敲除及双敲除突变株，并对T30（野生株）、∆thph1、∆thph2进行GFP荧光标记。发现木霉菌T30的thph1、thph2基因可促进其在玉米根系内皮层和维管束中的定殖，Thph1、Thph2均能诱导玉米防御反应基因JA/ET信号（Opr7、Pr4、Aoc1、Erf1）系统、SA信号（PR1）和抗氧化酶（SOD，PAL）表达。为了筛选与Thph1和Thph2互作的蛋白，通过酵母双杂的方法对玉米cDNA库中进行筛选，分别得到可能与Thph1和Thph2可能存在互作关系的ZmATG3（BT085821.1）、ZmGLP（NP_001141258.1），并且通过Co-IP方法验证了Thph2和玉米根系ZmGLP之间的互作关系，发现Thph2上调玉米根系根系中GLP表达，诱导玉米叶片水杨酸信号的系统表达和对玉米小斑病的抗性。成功构建了转ZmGLP的工程玉米植株，进一步验证Thph1与玉米根系GLP的互作关系。