Identification and functional characterization of arbuscular mycorrhizal fungi effector candidates

丛枝菌根真菌（AMF）与大多数陆地植物的根呈专性共生关系。这种共生关系在植物门的整个进化发展过程中一直保持形态不变。一种AMF可以侵染不同的植物，一种植物可以被多种AMF侵染。因此，植物感染和定殖的机制似乎是古老的和保守的AMF。最近的研究病原体和共生植物-微生物相互作用表明，在感染过程中的相似性。虽然在深入研究的致病系统中，效应蛋白被确定为在这一过程中发挥关键作用，但在共生系统中，类似作用的证据仅限于少数几个例子。病原体效应蛋白抑制植物免疫系统，或者它们可以被植物细胞识别，这导致诱导效应子触发的免疫。我们推测，效应子在AM共生建立的保守过程中发挥着同等的作用。从两个AMF，Rhizophagus irregularis和Rhizophagus clarus的基因组中，我们已经确定了200多个同源基因编码潜在的效应蛋白。基于相似性水平，我们将候选者分为保守的（>90%）和保守性较低的候选者。在这个项目中，我们专注于最保守的潜在效应子的功能表征，因为它们是最有可能在共生建立的保守过程中发挥作用的。在第一步中，我们将选择分泌的候选物，显示核定位，并抑制异源系统中的过敏反应。只有经过验证的候选物将进一步表征其对AM共生的影响，使用百脉根毛状根表达系统。AM共生过程中植物基因的转录分析将用于识别效应作用的靶点。在一个单独的试验中，我们将研究在卵菌植物病原体，Phytophthora palmivora.This研究将提供一个更好的了解共生相互作用的分子过程中的异源表达的致病系统中的效应蛋白的影响。特别感兴趣的将是病原体和共生系统之间的效应作用的相似性和差异。病原菌中的效应基因往往处于多样性选择之下，探索作用于共生体效应基因的进化力量将是一个有趣的课题。

Arbuscular mycorrhiza fungi (AMF) live in obligate symbiosis with roots of the majority of land plants. This symbiosis persisted morphologically unchanged throughout the evolutionary development of the plant phylum. One AMF species can infect different plant species, and one plant species can be mycorrhized by various AMF. Therefore, the mechanisms of plant infection and colonization seem to be ancient and conserved within AMF. Recent studies of pathogenic and symbiotic plant-microbe interactions showed similarities during infection. While in the intensely studied pathogenic systems effector proteins were identified as playing a key role in this process, in symbiotic systems evidence for a similar role is limited to a few examples. Pathogen effector proteins either suppress the plant immune system, or they can be recognized by the plant cell, which results in the induction of effector-triggered immunity. We hypothesize that effectors play an equivalent role during the conserved process of AM symbiosis establishment. From the genomes of two AMF, Rhizophagus irregularis and Rhizophagus clarus, we have identified more than 200 homologous genes encoding potential effector proteins. Based on similarity level we divided the candidates into conserved (>90%) and less-conserved ones. In this project we focus on the functional characterization the most conserved potential effectors, since they are the most likely to play a role during the conserved process of symbiosis establishment. In a first step we will select candidates that are secreted, show nuclear localization, and suppress hypersensitive responses in heterologous systems. Only validated candidates will be further characterized with respect to their effect on AM symbiosis using the Lotus japonicus hairy root expression system. Transcript analysis of plant genes during AM symbiosis will be used to identify targets of effector action. In a separate assay we will investigate the impact of effector proteins in a pathogenic system by heterologous expression in the oomycete plant pathogen, Phytophthora palmivora.This study will provide a better understanding of the molecular processes underlying symbiotic interactions. Of special interest will be the similarities and differences of effector action between pathogenic and symbiotic systems. Effector genes in pathogens tend to be under diversifying selection, and it will be interesting to explore the evolutionary forces acting on symbiont effector genes.