芦笋茎枯病菌LysM结构域效应因子的鉴定及其致病调控作用机理研究

Asparagus officinalis L. is a high value healthy vegetable rich in bioactive factors such as saponins, rutin, and organic selenium. At present, China is the largest asparagus planted country around the world. Asparagus stem blight is a high destructive disease of asparagus worldwide. Filamentous fungus Phmopsis asparagi is identified as the causing pathogen. However, its molecular pathogenic mechanism on pathogen and plant interaction is poorly known. Identification of pathogenic related genes is of great importance for understanding of molecular pathogenic mechanism on asparagus stem blight. Recently, our lab has completed the genome and transcriptome sequencing project of this fungus strain Pa1100. Further more, based on the constructed T-DNA insertion mutant library, we already screened one pathogenic related candidate LysM domain-contaning effector preotein named PaLdp1, which significantly up-regulated during plant infection based on our transcriptome sequencing data and also confirmed by real-time fluorescent quantitative PCR (qRT-PCR). Moreover, another two strongly homologous candidate proteins, named PaLdp2-PaLdp3, were found in P. asparagi genome based on BlastP analysis. In this study, further identification and characterization will be conducted on these LysM domain-contaning effectors (PaLdp1-PaLdp3) to analyze their biological functions. These candidate effectors will be firstly identified based on genome sequence data directly, then be confirmed by PCR amplication and southern blot. Gene knockout, complement, and over-expression technologies will be applied for effectors functional analysis on biological characteristics of morphology growth, infection process, abilities of binding chitin, and pathogenicity. Moreover, the key pathogenic effectors’ expression patterns, subcellular localization and accumulation characteristics during plant infection will be analyzed by using scanning electron microscope (SEM), qRT-PCR, and GFP-fusion methods to get more meaningful understanding about the reasons of their defect on pathogenicity. Further more, the deletion or point mutation of key domains such as SignalP and LysM or the conserved sites will be conducted to deeply reveal the important functional parts and the pathogenic mechanism of key LysM effectors in P. asparagi.

芦笋是重要的保健型蔬菜，我国种植面积居世界首位，茎枯病是生产上的毁灭性真菌病害，目前国内外缺乏病菌与芦笋致病互作机制报道。本项目前期对芦笋茎枯病菌Pa1100完成了全基因组和转录组测序，并通过T-DNA突变体库筛选到一个含LysM结构域的致病相关候选效应因子，暂命名PaLDP1，进一步发现基因组中还含有另外两个与PaLDP1高度同源的LysM结构域基因（PaLDP2-PaLDP3）。为深入鉴定这些候选效应因子，拟通过基因敲除、互补和过表达技术探讨它们对病菌形态建成、组织侵染、几丁质结合能力和致病生化调控的生物学功能。同时，借助扫描电镜结合qRT-PCR和GFP定位技术，阐述在病菌－芦笋生物学互作过程中，关键效应因子的时空表达与分泌定位特性及其对致病性的影响。进一步，通过对含LysM结构域效应因子中的致病结构区域进行去除或定点突变，剖析致病关键功能区域，深入阐明其致病调控作用机理。

芦笋茎枯病危害严重制约着芦笋产业健康发展。本项目在前期研究基础上，成功鉴定了三个致病相关效应蛋白 PaLDP1，PaLDP2 和 PaLDP3，并分析了其分子序列、保守结构域和分子进化树，完成分子克隆。进一步，利用分子遗传转化、基因敲除、互补、基因表达定位、致病侵染分析、点突变等技术手段，探讨它们对病菌形态建成、组织侵染、几丁质结合能力和致病生化调控的生物学功能，筛选到芦笋茎枯病菌致病关键候选 LysM 效应蛋白PaLDP3，并深入探讨了其在病菌－芦笋生物学互作过程中可能的作用机制。致病性和致病侵染进程分析表明，候选效应蛋白 PaLDP3 致病力相比野生型对照显著下降，扫描电镜分析表明致病性侵染菌丝生长明显受阻。进一步，对候选效应蛋白的几丁质结合力分析表明，PaLDP3 基因编码的蛋白 PaLdp3 能够特异性与几丁质结合。基因时空表达结果表明，PaLDP3在致病菌侵染初期（5 dpi）表现出显著增强表达，这可能为其更好地参与致病调控起到了一定作用。对PaLDP3基因关键区域（功能位点）点突变结果表明，SignalP和LysM关键区域对基因功能发挥具有重要作用。更有趣的是，通过对寄主芦笋对应的候选模式识别受体蛋白 CEBiP的基因沉默转化子株系的致病试验发现，病原菌PaLDP3 基因敲除突变体的致病力得到了恢复，这说明 PaLDP3 可能是通过与芦笋 CEBiP 蛋白竞争结合几丁质以便抑制由几丁质诱导的寄主抗病免疫反应。项目研究结果对于深入了解病害发生进程和病害科学防控具有重要的现实意义。