效应物在杨盘二孢半活养方式及亲和寄主致病性中的作用

Effectors secreted by plant pathogens have been referred as targets of selection pressures in pathosystem.The divergent evolution in effectors between pathogens promote differential pathogenicity and disease occurrence. It is unclear whether some effectors of hemibiotrophic fungi on trees are determinants for woody pathogens with the similar functions. The fungus, Marssonina brunnea, a pathogen causing a severe leave spot disease on poplars in China and other areas of the world, has been found to evolve two genotypes in specific pathogenicity, M.brunnea f.sp. monogermtubi and M.brunnea f.sp.multigermtubi, which has own compatible and incompatible hosts among poplar sections in nature, respectively. This phenomenon of this pathosystems is probably stemmed from the divergence evolution on effectors in M.brunnea species. To test the hypothesis, we are going to establish pathosytems for the two genotypes with their compatible and incompatible hosts in laboratory. Within the pathosystem, their effectors are about to be investigated on their transcriptomic characterizations during infection with next generation sequencing technology, genomic and bioinformatic tools. The fungal effectors and their target molecules of hosts responsible to the pathogenicity and host specificity will be determined. The functions of the putative effectors will furthermore be confirmed through yeast two hybrid system and in Planta expression system.The results from this study could have an insight into the interactive nature of the effectors in the pathology of poplar black blot, moreover, could facilitate to clarify the roles of effectors from hemibiotrophic fungi on trees in pathogenicity and host specificity in the course of evolutionary interaction pathosystems between pathogens and tree hosts.

效应物是病害互作体系中承受选择压力的病原分子靶标，其趋异性进化是致病性分化及病害发生的基础。目前对树木半活体营养型病原菌效应物的这些作用了解甚少。杨树黑斑病病原杨盘二孢菌存在单芽管和多芽管专化型，且分别高感不同派组的杨树树种，而派外杨树树种免疫或高抗，导致这专化型和寄主互作亲和关系的原因可能是二者靶标效应物组成和表达存在差异的结果。为验证该推论，我们通过建立杨盘二孢不同专化型与其亲和非亲和寄主互作的病害实验体系，设计对比病理学试验；利用新一代测序和组学技术、基因克隆表达等手段；结合对效应物及其功能认识的最新进展，运用生物信息学工具等，系统揭示杨树黑斑病互作体系之间转录组中靶标效应物基因的表达特征，分离鉴别致病差异性效应物及其寄主作用靶标基因分子，经酵母双杂交和in planta体系表达验证功能。研究结果有助于阐述杨树黑斑病这些基本现象的本质，还有助于了解和进一步研究树木半活养菌效应物在病原与树木寄主互作进化中的致病作用和机制。

效应物是病害互作体系中承受选择压力的病原分子靶标，其趋异性进化是致病性分化及病害发生的基础。杨树黑斑病病原杨盘二孢菌存在单芽管和多芽管专化型，且分别高感不同派组的杨树树种，而导致这专化型和寄主互作亲和关系的原因可能是二者靶标效应物组成和表达存在差异的结果。本项目立项的主要目的之一是揭示杨盘二孢（杨生褐盘二孢，Marssonina brunnea）侵染过程，系统阐明杨树黑斑病互作体系之间转录组中靶标效应物基因的表达特征，分离鉴别致病差异性效应物及其寄主作用靶标基因分子，促进杨树褐斑病侵染机制科学地深入研究。项目首次完整地展现了杨盘二孢两个专化型侵染发育全过程，并发现了该菌在寄主细胞膜和壁间发育的侵染囊泡状吸器（infection vihiecles），详实地证实了杨盘二孢的半活养寄生方式。项目目的之二是通过建立杨盘二孢不同专化型与其亲和非亲和寄主互作的病害实验体系，设计对比病理学试验，建立杨盘二孢专化型互作体系，以期用于长期深入探索研究杨树半活养致病菌与寄主互作的机制。通过对杨盘二孢遗传分化以及接种及交叉接种试验，建立和筛选出以多芽管专化型菌株Aig39（M. brunnea f. sp. multigermtubi Aig39）与黑杨品系黑金（Populus deltoids cv. ‘Zj - 2’）、单芽管专化型菌株Leu45（M. brunnea f. sp. monogermtubi Leu45）与白杨品系白金（Populus alba × Populus alba var. pyramidalis）的互作体系。项目目的之三是从基因组水平的转录组尤其是效应物表达特点，探索分泌性蛋白毒力因子的表达在杨盘二孢多芽管和单芽管专化型特异侵染专化性（亲和性）寄主分化中的作用。通过对侵染过程杨盘二孢附着胞形成初期入侵阶段、囊泡吸器活养阶段至次生菌丝形成死养阶段的组织转录组，进行详细比对和分析，首次明确地了解到两专化型病原菌侵染的基因表达调控机制和其感病寄主响应的基因表达调控机制存在显著性差异，转录组分析结果和蛋白组表达结果均积极地支持分泌性蛋白毒力因子尤其是效应物（子）在两互作体系的分化中可能起到了重要作用。项目结果可以极大地促进树木半活养病原菌与寄主互作机制的基础研究。