Dissecting the journey and functions of barley smut fungus Ustilago hordei avirulence effector UhAvr1

效应生物学，研究微生物病原体的小分泌蛋白，是人类/动物和植物科学的一个新兴研究领域。研究效应物已经揭示了这些蛋白质与许多基本宿主过程和信号通路相互作用和操纵的方式。这包括发育、蛋白质降解和基因调控，其中包括防御和抵抗反应。因此，效应物告诉我们病原体是如何引起疾病的，但也可以作为探针来阐明基本的生物过程。基因组计划揭示了真菌有大量的效应器，但它们的传递方式、宿主细胞靶点和功能在很大程度上仍然未知。在对几种侵染谷物的担子菌病原菌（大麦黑穗病菌黑穗病菌和相关的小麦叶锈病菌小麦锈病菌）的基因组序列进行生成和分析之后，我们现在可以专注于许多预测效应菌的功能。我们最近在美国发现了一种效应物，当感染具有抗性基因Ruh1的大麦品种时，它会触发防御。这种无毒效应物UhAVR1仅在与宿主接触时表达，由真菌分泌，其与Ruh1的相互作用或其作用导致宿主局部细胞死亡并对感染产生免疫。我们将Ruh1基因定位在大麦1号染色体短臂上约200 kb的区域。这一建议旨在阐明三个基本问题：1。UhAVR1分泌和宿主细胞摄取的机制是什么？2. UhAVR1在大麦细胞中的（毒力）功能是什么？它的目标是什么？3. UhAVR1如何发挥其无毒功能/与Ruh1相互作用？在1中，我们将通过在U. hordei中删除已知基因来研究分泌途径中的已知基因，并通过共聚焦显微镜检测它们对与荧光蛋白片段相关的UhAVR1传递的影响。我们还将使用针对UhAVR1的抗体，通过电子显微镜进行免疫定位实验，并在细胞隔室分离后检测UhAVR1。其他参与效应传递的基因将通过随机诱变发现，随机诱变的插入单致病性单倍体菌株，依靠强选择的无毒基因将提供。也就是说，交货中断将导致Ruh1植物上的孢子产生。在2中，比较了野生型感染与UhAvr1缺失时大麦基因表达的变化。以UhAVR1为诱饵的酵母2杂交筛选将用于鉴定潜在的大麦靶蛋白。在生化方法中，UhAVR1将结合到固体载体上，以分离与其相互作用的蛋白质或复合物，并使用蛋白质组学技术对其进行分析。现有的真菌和大麦基因组序列允许快速鉴定候选基因和功能，将使用适应性基因沉默系统进行研究。在3中，大麦基因表达与2中比较，但现在在抗性宿主中。Ruh1将通过在大麦胚芽组织或原生质体中与UhAvr1一起引入大麦大插入基因组DNA克隆来鉴定；当Ruh1出现在克隆体上时，可以分析防御触发。在这个模型系统中阐明效应物的传递机制、宿主靶标的识别和防御机制将有助于研究困难的、但在经济上更重要的谷物锈病病理系统。已鉴定的抗性基因可用于育种计划，宿主效应靶基因可通过基因组编辑进行修饰，从而使作物获得持久的抗病能力，减少对有害农药的依赖。保守的真菌成分对效应递送至关重要，是设计特定控制措施（如RNA沉默）的主要目标。

Effector biology, the study of small secreted proteins from microbial pathogens, is a burgeoning research field in both human/animal and plant sciences. Studying effectors has already revealed insight into ways these proteins interact with and manipulate many basic host processes and signaling pathways. This includes development, protein degradation and gene regulation which include defense and resistance responses. Therefore, effectors teach us how pathogens cause disease but can also be used as probes to elucidate basic biological processes. Fungi have large suites of effectors, revealed by genome projects, but their mode of delivery, host cell targets and functions remain largely unknown. Having contributed to the generation and analyses of genome sequences of several cereal-infecting Basidiomycete pathogens (barley smut fungus Ustilago hordei and the related wheat leaf rust fungus Puccinia triticina), we can now focus on functions of the many predicted effectors. We recently identified in U. hordei an effector that triggers defense when infecting a barley cultivar with resistance gene Ruh1. This avirulence effector UhAVR1 is only expressed upon contact with the host, is secreted from the fungus, and its interaction with Ruh1 or its action leads to local host cell death and immunity to the infection. We genetically located Ruh1 on the short arm of barley chromosome 1 to a ~200-kb region. This proposal aims to shed light on 3 basic questions: 1. What are the mechanisms of UhAVR1 secretion and host cell uptake? 2. What is the (virulence) function of UhAVR1 in barley cells, what does it target? 3. How does UhAVR1 carry out its avirulence function / interaction with Ruh1? In 1, we will investigate known genes in the secretory pathways by deleting them in U. hordei and assay by confocal microscopy their effect on delivery of UhAVR1 linked to a fluorescent protein moiety. Using antibodies against UhAVR1, we will also conduct immunolocalisation experimenets by electron microscopy and detect UhAVR1 after cell compartment fractionations. Other genes involved in effector delivery will be revealed by random mutagenesis with tagged inserts of a solopathogenic haploid strain, relying on the strong selection the avirulence gene will provide. That is, interruption of delivery will result in spore production on Ruh1 plants. In 2, changes in gene expression in barley, when challenged with a wildtype infection versus one where UhAvr1 is deleted, is compared. A yeast 2-hybrid screen with UhAVR1 as bait will be used to identify potential barley target proteins. In a biochemical approach, UhAVR1 will be bound to solid supports to isolate proteins or complexes it interacts with, which will be analysed using proteomic techniques. Available fungal and barley genome sequences allow rapid identification of candidate genes and functions will be investigated using an adapted gene silencing system. In 3, barley gene expression is compared as in 2 but now in a resistant host. Ruh1 will be identified by introducing select barley large-insert genomic DNA clones together with UhAvr1 in barley coleoptiles or protoplasts; when Ruh1 is present on a clone, defense triggering can be assayed. Elucidating mechanisms of effector delivery, identification of their host target(s) and defense elicitation in this model system will aid studying difficult, yet economically more important cereal rust pathosystems. Identified resistance genes can be used in breeding programs and host effector target genes can be modified by genome editing to obtain durable disease resistance in crop plants and less reliance on harmful pesticides. Conserved fungal components essential for effector delivery are prime targets for designing specific control measures such as RNA silencing.