玉米钙依赖蛋白激酶CDPKs参与诱导系统性抗性ISR防御禾谷镰孢菌茎腐病的功能和机制研究

Gibbrella stem rot, caused by Fusarium graminearum, one of the most devasting fungal pathogens of cereal crops, such as corn, can cause not only the significant yield loss, but also the reduced quality in maize production worldwidely. The effetive way nowdays to control the gibbrella stem rot is to exhume and identify the novel genes that can be utilized as reistance resources for crop improvement. Induced systemic resistance (ISR) triggered by the non-pathogenic microorganisms in the rhizosphere has become an effective stragegy of plants for broad-spectrum resistance to pathogens. However, there is no report so far about the function and mechanisms of ISR in the maize resistance to F. graminearum. Previous studies by other groups have suggested that Calcium-Dependent Protein Kinases (CDPKs) might play important roles in the plant systemic resistance to diverse pathogens. Our recent studies showed that differential CDPKs could modulate the distinct pathways of Effector-Triggered Immunity (ETI) in Arabidopsis. We also found recently that in maize the calcium-signaling inhibitor could suppress the ISR activated by a beneficial microorganism Trichodema virens, and that the expression of different CDPKs is likely associated with different types of systemic resistance, e.g., JA- and SA-dependent resistance. The findings together imply that CDPKs are very likely involved in the ISR to the stem rot caused by F. graminearum. To better understant the function of CDPKs in the activation of ISR to F. graminearum, as well as to explore the mechanism behind this, we propose first to screen and clone the candidate CDPK genes responsible for the ISR using functional genomics approaches. A series of biochemical experiments will be conductedto identify the interacting proteins of CDPKs, followed by the investigation of how those individual CDPKs and signaling components contribute to the ISR activated by T. virens to stem rot. We will also plan to investigate ISR phenotypes by mutating and overexpressing the individual CDPKs, while the spatial and temporal changes of endogenous phytohormones and resistant gene expression will be measured in the mutants vs wild types upon infection with F. graminearum. The findings obtained from this proposal will not only help us to understand the function and molecular and biochemical mechanisms of CDPKs -mediated maize ISR resistance to F. graminearum stem rot, but also provide an effective alternative to control maize stem rot, and will eventually be helpful to molecular breeding for disease resistance in other cereal crops.

禾谷镰孢菌茎腐病是导致玉米严重减产和品质下降的世界性病害，发掘鉴定新型基因抗源是控制该病的有效途径。根际非病原微生物诱导的系统性抗性ISR是一种有效的新型广谱抗性策略，但迄今未见有关玉米ISR抗茎腐病的功能和机制的报道。前人研究表明钙依赖蛋白激酶在植物系统抗性中发挥重要作用；我们近期研究发现: 拟南芥CDPKs家族不同成员参与调控先天性免疫的不同分支途径；钙离子抑制剂可降低玉米ISR水平；CDPK的表达因诱导抗性方式而异，提示CDPKs参与ISR抗茎腐病的可能性。为深入了解CDPK在ISR中的功能和机制，项目将筛选并克隆候选CDPK基因，鉴定其互作组分，考查CDPK基因突变和过表达对茎腐病ISR的影响，解析不同CDPK基因及其信号途径对ISR的贡献。研究不仅能帮助弄清CDPK在介导ISR抗茎腐病中的功能和分子生化机制，还能为有效控制玉米茎腐病乃至其它禾谷类作物的抗病分子育种提供理论依据。

禾谷镰孢菌茎腐病（GSR）是导致玉米严重减产和品质下降的世界性病害，发掘鉴定新型基因抗源是控制该病的有效途径。根际非病原微生物诱导的系统性抗性ISR是一种有效的新型广谱抗性策略，但有关玉米ISR抗茎腐病的功能和机制的报道尚不清楚。前人研究表明钙离子信号在植物系统抗性中发挥重要作用，提示钙信号蛋白基因如CDPKs可能参与ISR茎腐病抗性。本项目的主要研究内容为筛选、鉴定和克隆候选CDPK基因，了解CDPK在ISR中的功能和机制，解析钙离子信号途径对ISR的贡献。项目利用绿木霉Trichodema virens孢子粉和解淀粉芽孢杆菌Bacillus amyloliquefaciens SQR9处理玉米B73根部后，对地上部用钙离子抑制剂氯化镧处理，然后在茎部接种禾谷镰孢菌。结果表明，两种有益菌均可激活对GSR的ISR反应，且显著增强活性氧和胼胝体的积累。但氯化镧处理明显抑制SQR9的促进作用，提示钙离子信号途径可能参与有益菌激活的ISR。为了进一步明确这种作用，我们利用qRT-PCR检测了SQR9处理后不同部位（根、茎和叶）、不同时间点的CDPK基因亚家族成员和其它激素信号途径，如JA和SA途径基因的表达模式，明确了CDPK参与茎腐病ISR抗性的贡献，并解析了激素信号途径在钙离子信号途径介导的ISR反应中的作用。另外，利用RNA-seq我们在全基因组水平检测了SQR9对玉米全基因组mRNA表达的影响，发现SQR9处理后，根和茎中的基因表达模式表现出相反的趋势；结合qRT-PCR挖掘出了ISR标记基因100381655- Glutamate decarboxylase 1、107457600- Putative WRKY DNA-binding domain superfamily protein、100282375-adhesive/proline-rich protein和PR1基因。在此基础上，我们系统阐述了有益菌在诱导ISR中的功能、作用机制和相关进展，提出了利用有益菌结合多种措施来激活ISR、实现对玉米镰刀菌茎腐病的有效防控，为有效控制玉米茎腐病乃至其它禾谷类作物的抗病分子育种提供理论依据。