Characterization of the Largest Effector Gene Cluster of Ustilago maydis

In the genome of the biotrophic plant pathogen Ustilago maydis, many of the genes coding for secreted protein effectors modulating virulence are arranged in gene clusters. The vast majority of these genes encode novel proteins whose expression is coupled to plant colonization. The largest of these gene clusters, cluster 19A, encodes 24 secreted effectors. Deletion of the entire cluster results in severe attenuation of virulence. Here we present the functional analysis of this genomic region. We show that a 19A deletion mutant behaves like an endophyte, i.e. is still able to colonize plants and complete the infection cycle. However, tumors, the most conspicuous symptoms of maize smut disease, are only rarely formed and fungal biomass in infected tissue is significantly reduced. The generation and analysis of strains carrying sub-deletions identified several genes significantly contributing to tumor formation after seedling infection. Another of the effectors could be linked specifically to anthocyanin induction in the infected tissue. As the individual contributions of these genes to tumor formation were small, we studied the response of maize plants to the whole cluster mutant as well as to several individual mutants by array analysis. This revealed distinct plant responses, demonstrating that the respective effectors have discrete plant targets. We propose that the analysis of plant responses to effector mutant strains that lack a strong virulence phenotype may be a general way to visualize differences in effector function.在活体营养型植物病原菌玉米黑粉菌（Ustilago maydis）的基因组中，诸多编码调控致病力的分泌蛋白效应子（secreted protein effectors）的基因呈基因簇（gene clusters）排布。此类基因的绝大多数编码新型蛋白质，其表达与植物定殖过程紧密耦合。其中最大的基因簇19A可编码24种分泌型效应子。完整敲除该基因簇会导致病原菌致病力严重减弱。本研究针对该基因组区域开展功能解析。我们证实，19A缺失突变体的行为模式与内生真菌（endophyte）类似：其仍可定殖宿主植物并完成完整的侵染循环，但作为玉米黑粉病最典型的症状，肿瘤仅极少形成，且侵染组织内的真菌生物量显著降低。通过构建并分析携带亚缺失（sub-deletions）的菌株，我们鉴定出多个在幼苗侵染阶段对肿瘤形成具有显著贡献的基因。另有一类效应子可特异性介导侵染组织的花青素（anthocyanin）积累诱导。鉴于这些基因对肿瘤形成的单独贡献较弱，我们通过基因芯片分析（array analysis）探究了玉米植株对整个簇缺失突变体以及数种单个效应子缺失突变体的转录响应。结果揭示了差异化的植物响应模式，证明各对应效应子拥有独立的植物靶标。我们提出，针对无强致病力表型的效应子缺失突变株开展植物响应分析，或可成为可视化区分效应子功能差异的通用研究策略。