Mechanisms of basidiomycete yeast function in complex leaf microbial communities

定植植物的微生物进化出复杂的微生物相互作用，其中真菌和卵菌是植物相关细菌多样性和丰度的主要决定因素。由于真菌和卵菌为争夺植物生态位而竞争，因此了解这些有机体群体如何在叶片叶圈中相互作用是关键。叶球中的一个关键枢纽微生物是卵菌Albugo laibchii。在我们的实验中，我们鉴定了担子菌酵母菌Moeszimyces Bullatus ex Albugo on Arabitopsis(MBA)作为A.laibchii的拮抗剂。转录学指导下的基因缺失确定了MBA的糖苷水解酶家族25(GH25)是抑制A.laibchii所必需的。酶活性测定表明，GH25蛋白具有溶菌酶活性，对拟南芥的侵染有较强的抑制作用。GH25的系统发育分析揭示了担子菌门的两个主要分支。然而，一些担子菌不包含GH25的同源基因，包括囊状担子菌，这是我们确定的核心类群，在群落稳定的顶峰时，拟南星藻叶围中出现的次数最多。Cystofilasidium在群落环境中抑制A.laibchii，暗示了一种不依赖于GH25的拮抗机制。我们将研究GH25介导的微生物拮抗作用及其在群落组装中的作用。作为补充，我们将探索不同担子菌酵母菌中GH25独立的机制。我们将测试GH25作为不同卵菌抑制剂的功能保守性，以及它对选定的植物伴生真菌和细菌的影响。这些一对一的相互作用将引导我们在社区背景下调查MBA GH25突变和过表达株的影响。我们假设缺乏GH25会破坏群落的稳定，导致样本之间的差异增加。为了研究GH25依赖和独立抑制莱巴赫杆菌对微生物群落的影响，我们将在MBA GH25缺失突变体中表达来自广泛担子菌的MBA同源基因。同时，我们将通过一系列的胞菌分离株来推断GH25独立抑制莱巴奇菌的机制。我们将分析在存在/不存在A.laibchii的情况下MBA和Cystofilobasiums在细菌群落中作用的差异和共同点。我们假设，在不同的条件下，GH25的存在/不存在会影响酵母的适合度，从而产生我们将通过对Cystofilobasidium分离株的全基因组分析来推断的基因组特征。为了汇编所有数据并测试我们关于群落组装的假设，我们将开发计算管道来预测微生物宿主的范围和生活方式，确定公共亚结构，以及它们如何受到关键功能基因的存在/不存在的影响，如GH25。因此，我们提出的项目是一种组合方法，通过直接微生物相互作用的功能分子分析来阐明微生物群落的行为和稳定性。

Plant-colonizing microbes evolved complex microbial interactions, in which fungi and oomycetes are major determinants of diversity and abundance of plant-associated bacteria. Since fungi and oomycetes compete for the plant niche, it is key to understand how these organism groups interact in the leaf phyllosphere. A key hub microbe in the leaf phyllosphere is the oomycete Albugo laibachii. In our experiments we identified the basidiomycete yeast Moesziomyces bullatus ex Albugo on Arabidopsis (MbA) as an antagonist of A. laibachii. Gene deletion guided by transcriptomics identified a Glucoside hydrolase-family 25 (GH25) of MbA being required for inhibition of A. laibachii. Enzyme assays showed a lysozyme activity of the GH25 protein, which effectively inhibited A. laibachii infection when applied on Arabidopsis. Phylogenetic analyses of GH25 revealed two main clades throughout the Basidiomycota. However, several basidiomycetes do not contain orthologs of GH25, including the Cystofilobasidiales, which we identified as core taxa with a maximum occurrence in the A. thaliana phyllosphere at the peak of community stability. Cystofilobasidium inhibits A. laibachii in a community context, suggesting a GH25-independent mechanism of antagonism. We will study GH25 mediated microbial antagonism and its role in community assembly. Complementary, we will explore GH25-independent mechanisms in diverse basidiomycete yeasts. We will test functional conservation of GH25 as an inhibitor of different oomycetes, as well as its effects on selected plant associated fungi and bacteria. These one-to-one interactions will guide us to investigate the effects of MbA GH25 mutant and overexpression lines in a community context. We hypothesize that absence of GH25 de-stabilizes the community, resulting in increased sample-to-sample variations.To study the effects of GH25 dependent and independent repression of A. laibachii on microbial communities, we will express MbA orthologs from a broad range of basidiomycetes in the MbA GH25 deletion mutant. In parallel, we will infer the mechanism of GH25 independent suppression of A. laibachii by a range of Cystofilobasidium isolates. We will analyze differences and communalities of MbA versus Cystofilobasidum function on bacterial communities in presence/absence of A. laibachii. We hypothesize that under varying conditions, presence/absence of GH25 affects the yeast’s fitness, resulting in genomic signatures we will infer by whole genome analyses of Cystofilobasidium isolates. To compile all data and test our hypotheses on community assembly, we will develop computational pipelines to predict microbial host ranges and lifestyles, to identify communal sub-structures, and how they are affected by presence/absence of key functional genes such as the GH25. Thus, our proposed project is a combinatorial approach to elucidate microbial community behavior and stability with a functional molecular analysis of direct microbial interactions.