落叶松-杨栅锈菌Kss1/Fus3类和Slt2类MAPK基因的功能分析

Poplar foliar rust caused by Melampsora larici-populina Kleb. (MLP) is an important forest disease worldwide. During the last decades, breeders have generated a collection of hybrid poplars with complete resistances to M. larici-populina. Nowadays, almost all poplar cultivars are susceptible to the rust fungus. In order to define new strategies to control the disease, it’s crucial to identify the molecular mechanism that allows M. larici-populina to establish infection in the host. Very little is known about the process because it belongs to the obligate biotroph fungi. It can generally be assumed that Kss1/Fus3-type MAPKs are multifunctional pathogenicity factors required for virulence of biologically and taxonomically diverse phytopathogenic fungi based on more than 30 published studies. Functional characterization of the SLT2 homolog in several plant pathogenic fungi has indicated that this MAPK is well conserved among fungal pathogens and plays important roles during plant infection. However, so far less is known about their roles in the virulence of M. larici-populina. In this study, we aim to identify Kss1/Fus3-type and Slt2-type MAPKs from M. larici-populina, analyze the transcript level using qRT-PCR assays, and finally perform the functional characterization by the complementation assays and the approach of host induced-gene silencing, which will accelerate the research about the molecular mechanism and lay the foundation for improvement of the rust resistant breeding.

由落叶松-杨栅锈菌侵染引起的杨树叶锈病是一种严重危害杨树生产的世界性重大真菌病害，其控制主要依赖于抗病品种的选育和应用，但病菌新生理小种的出现导致品种抗病性降低或丧失。解析落叶松-杨栅锈菌致病的分子机理可为杨树叶锈病的持久控制提供新策略和新技术。然而，活体专性寄生特性使得对该病菌致病的分子机理研究甚少。Kss1/Fus3类和Slt2类MAPK基因在许多植物病原真菌的侵染与致病过程中具有重要作用，但在落叶松-杨栅锈菌中的功能目前尚不清楚。本研究拟克隆落叶松-杨栅锈菌Kss1/Fus3类和Slt2类MAPK基因；利用实时荧光定量PCR技术分析基因的表达特征；采用模式真菌突变体互补技术分析基因的功能，并进一步通过寄主诱导的基因沉默技术分析基因在病菌侵染与致病过程中的作用，以便在揭示落叶松-杨栅锈菌致病的分子机理的同时，为实施杨树抗叶锈病的遗传改良提供理论依据和技术支撑。

专性寄生真菌落叶松-杨栅锈菌在世界范围内引起杨树叶锈病，常引起树苗叶子大量提前脱落，严重降低其生长量从而造成重大经济损失。解析落叶松-杨栅锈菌致病分子机理可为杨树抗叶锈的新材料的创制提供重要的理论依据和技术支撑。MAPK基因在许多植物病原真菌的生长发育和致病过程中具有重要作用。然而，MAPK基因在落叶松-杨栅锈菌中的功能目前尚不明确。本项目采用荧光染色技术、光学显微镜和电子显微镜技术，系统研究了落叶松-杨栅锈菌在感病杨树叶片上的发育过程；利用同源克隆获得落叶松-杨栅锈菌MAPK基因MlpHog1、PKA催化亚基MlpcPKA1、腺苷酸环化酶互作蛋白MlpCAP1及丝裂原活化蛋白激酶激酶MlpMKK1/2的ORF片段，并对其序列进行生物信息学分析；借助模式物种异源互补技术推断MlpHog1基因在落叶松-杨栅锈菌侵染菌丝生长和响应环境胁迫方面具有一定的作用，及MlpcPKA1基因在该锈菌营养生长和侵染菌丝生长方面具有一定的作用；确定偶联Alexa 488荧光素的小麦凝集素荧光染色方法可作为研究落叶松-杨栅锈菌与寄主杨树互作的一种组织病理学方法。本项目的开展为落叶松-杨栅锈菌致病分子机理的研究奠定理论基础，同时为杨树叶锈病的有效防治提供帮助。