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

剖析大麦黑粉病真菌UhAvr1无毒效应子的旅程和功能

• 批准号: RGPIN-2014-04976
• 负责人: Bakkeren, Guus
• 金额: $ 2.55万
• 依托单位: University of British Columbia
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2014
• 资助国家: 加拿大
• 起止时间: 2014-01-01 至 2015-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=566865
• 关键词: Dissecting; journey; functions; barley; smut

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.

效应生物学，研究微生物病原体的小分泌蛋白，是人类/动物和植物科学中新兴的研究领域。研究效应子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，包括防御和抗性反应。因此，效应子告诉我们病原体是如何引起疾病的，但也可以用作探针来阐明基本的生物过程。真菌有大量的效应子，基因组计划揭示，但他们的交付模式，宿主细胞的目标和功能仍然在很大程度上未知。在对几种感染谷物的担子菌病原体（大麦黑穗病菌和相关的小麦叶锈菌）的基因组序列的产生和分析做出贡献之后，我们现在可以专注于许多预测效应子的功能。我们最近在美国发现。大麦是一种在用抗性基因Ruh 1感染大麦品种时触发防御的效应子。这种无毒效应子UhAVR 1仅在与宿主接触时表达，从真菌分泌，并且其与Ruh 1的相互作用或其作用导致局部宿主细胞死亡和对感染的免疫。我们将Ruh 1基因定位在大麦1号染色体短臂上一个约200 kb的区域。本建议旨在阐明三个基本问题：1。UhAVR 1分泌和宿主细胞摄取的机制是什么？2. UhAVR 1在大麦细胞中的（毒力）功能是什么，它靶向什么？3. UhAVR 1如何实现其无毒功能/与Ruh 1的相互作用？在第一部分中，我们将通过在U. Hordei，并通过共聚焦显微镜测定它们对与荧光蛋白部分连接UhAVR 1的递送的影响。使用针对UhAVR 1的抗体，我们还将通过电子显微镜进行免疫定位实验，并在细胞室分级分离后检测UhAVR 1。其他参与效应子传递的基因将通过用solopathogenic单倍体菌株的标记插入物进行随机诱变来揭示，这依赖于无毒基因将提供的强选择。也就是说，输送中断将导致Ruh 1植物产生孢子。在2中，比较了当用野生型感染与UhAvr 1缺失的感染进行攻击时大麦中基因表达的变化。将使用以UhAVR 1为诱饵的酵母双杂交筛选来鉴定潜在的大麦靶蛋白。在生物化学方法中，UhAVR 1将与固体支持物结合，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进行分析。可用的真菌和大麦基因组序列允许快速鉴定候选基因，并且将使用适应的基因沉默系统来研究功能。在图3中，大麦基因表达与图2中进行比较，但现在是在抗性宿主中。Ruh 1将通过将选择的大麦大插入基因组DNA克隆与UhAvr 1一起引入大麦胚芽鞘或原生质体中来鉴定;当Ruh 1存在于克隆上时，可以测定防御触发。阐明该模型系统中的效应子传递机制、宿主靶标识别和防御激发将有助于研究困难但经济上更重要的谷物锈病致病系统。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，以获得作物持久的抗病性，减少对有害农药的依赖。保守的真菌组分对效应子传递至关重要，是设计特定控制措施（如RNA沉默）的主要目标。