StPKA-c调控玉米大斑病菌侵染能力的分子机制

The function of StPKA-c，a gene encoding the catalytic subunit of cAMP-dependent protein kinase A in Setosphaeria turcica was investigated through RNA interference technology in our preliminary study. The StPKA-c-silenced transformants showed delayed appressorial development, weakened activity and downregulted transcription level of the xylnase and decreased penetration capacity. The results indicated that StPKA-c might regulate the appressorial development and the expression of extracellular hydrolases to control the infection ability of S. turcica. However, its regulatory mechanisms are still unknown. In this reaearch, we will ulteriorly confirm the regulatory functions of StPKA-c on the appressorial development and the expression of extracellular hydrolases through creating StPKA-c null mutants and revertants labeled with Green Fluorescence Protein (GFP). The candidate downstream transcription factors (TFs) contianing the phosphorylation site R-[KR]-x-S of PKA will be firstly acquired from the Genome database of of S. turcica with the bioinformatics analysis. Then all candidate TFs will be detected using Yeast two-hybrid system to reveal their interactions with StPKA-c in vitro and the interaction will finally be verified in vivo by Bimolecular Fluorescence Complementation. Furthermore,the regulatory functions of exact downstream effectors on the invasive ablility will be analyzed with Gene-knockout and over-expression technologies. This research will clarify the molecular mechanisms of StPKA-c during it regulates the infection of S. turcica and provide some new targets for the discovery of efficient and durable prevention and control strategies of S. turcica and the research of new fungicieds.

本课题组发现玉米大斑病菌StPKA-c基因沉默导致病菌附着胞形成与发育过程延迟，胞外水解酶活性及表达水平显著下降，推测StPKA-c通过调控病菌的附着胞发育及胞外水解酶活性影响其侵染能力，但其调控机制有待深入探索。申请人拟创制StPKA-c敲除及回复突变体，进一步证实StPKA-c对病菌附着胞发育、胞外水解酶活性的调控作用；利用生物信息学方法分析玉米大斑病菌基因组数据库，获取具有PKA磷酸化位点序列R-[KR]-x-S的转录因子作为候选效应蛋白，继而运用酵母双杂交技术检测其与StPKA-c的体外互作关系，以双分子荧光互补技术确立其与StPKA-c在菌体中的互作关系；将与StPKA-c互作关系明确的转录因子基因分别敲除、过表达，分析突变体侵染力，从分子水平解析StPKA-c调控病菌侵染能力的机制。研究结果将为寻找高效、持久的病害防控策略及发掘杀菌剂设计的新靶点奠定理论基础。

玉米大斑病是由大斑刚毛座腔菌（Setosphaeria turcica）引起的严重威胁玉米生产安全的重要真菌性病害之一。本课题组前期研究发现，cAMP信号途径（cAMP-dependent signal pathway）对玉米大班病菌的营养菌丝生长及致病性具有重要调控作用，并克隆了cAMP最主要的下游效应分子PKA催化亚基（PKA-C）基因StpkaC1，利用RNAi技术证明该基因调控病菌的营养生长、产孢及侵染过程，但其调控机制尚待明确。本课题组按照项目计划书的规划，对玉米大斑病菌PKA催化亚基的功能及其作用机制进行了系统研究。结果表明：1）玉米大斑病菌基因组中含有StpkaC1和StpkaC2 2个PKA-C基因，分属不同类型的PKA-C；利用qPCR技术分析发现StpkaC2基因相比于StpkaC1基因在芽管形成及附着胞的成熟阶段表达更活跃，StpkaC1和StpkaC2在侵入丝生长阶段表达均明显上调。2）通过基因敲除技术研究了StpkaC1和StpkaC2的基因功能。StpkaC2基因负调控大斑病菌菌丝体生长，StpkaC1对菌丝体生长无明显影响；StpkaC1和StpkaC2均为产孢必需基因。StpkaC1和StpkaC2通过共同正向调控病菌黑色素生物合成及膨压积累而调控病菌附着胞及侵染菌丝形成。在菌丝形成附着胞阶段，StpkaC2缺失可引起MAPK编码基因STK1、STK3及CaMK编码基因CaMK1、CaMK2表达上调，CaMK3、CaMK4基因下调。3）对WT、△StpkaC2菌株进行转录组测序分析，发现差异表达基因2103个，上调基因917，下调基因1186；对差异表达基因进行功能注释并筛选出产孢相关基因32个，黑色素生物合成相关基因12个，菌丝生长相关基因68个。4）获得了该病菌EFG1同源基因StEFG1，利用酵母双杂交技术证实了该转录因子与StpkaC2存在互作关系。本项目的研究成果为深入揭示丝状真菌附着胞发育调控的分子机制奠定了基础。