Deciphering pathogenicity and development in obligate downy mildew pathogen using small RNA approach.

使用小 RNA 方法破译专性霜霉病病原体的致病性和发育。

• 批准号: BB/V014609/1
• 负责人: Mahmut Tör
• 金额: $ 79.79万
• 依托单位: University of Worcester
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FV014609%2F1
• 关键词: Deciphering; pathogenicity; development; obligate; downy

The oomycetes comprise several hundred microbial species including unique groups of biotrophic, necrotrophic and hemibiotrophic plant pathogens. They have superficial similarity to filamentous fungi but are distinct from them in several areas: the cell walls of oomycetes have been reported to be primarily B-1-3 glucans and cellulose with little or no chitin, oomycetes' hyphae are coenocytic (multinucleate with no division by septa) and their vegetative nuclei are in a diploid state. The diseases caused by oomycete plant pathogens include seedling blights, damping-off, root rots, foliar blights and downy mildews. Collectively, oomycetes estimated to cause 10's of billions in losses annually, due to their high evolutionary potential that enables host jumps, resistance to fungicides, and suppression or evasion of host resistance genes. Some of the most economically important oomycete pathogens are Phytophthora infestans (tomato and potato late blight), P. ramorum (sudden oak death), P. capsici (stem and fruit rot of cucumber and pepper), P. cinnamomi (dieback in avocado, pineapple), Plasmopora viticola (grapevine downy mildew), P. halstedii (sunflower downy mildew), Pythium ultimum (damping off and root rot), Bremia lactuca (lettuce downy mildew), and Albugo candida (white blister rust of crucifers).The biotrophic oomycete Hyaloperonospora arabidopsidis has co-evolved as a downy mildew pathogen in wild populations of Arabidopsis thaliana and has been used for more than 30 years as an experimental model for investigating the molecular basis of the gene-for-gene theory and other aspects of plant-oomycete interactions.Obligate oomycetes are not amenable to genetic transformation, thus hindering genetic analysis. Several groups including ours have relied on alternative approaches to assay effector function in planta including: a) co-bombardment assays into plant cells using the GUS gene to indicate avirulence activity, b) delivering effectors using bacteria secretion system, and c) creation of stably transformed plants expressing effector genes under control of plant promoters. However, all of these methods stripped the effector gene away from the pathogen where the expression level of a gene may not be comparable to that in the native background. Moreover, single-gene assays do not accurately capture gene function in the native milieu. Finally, these approaches are only applicable to secreted effector proteins that operate inside host cells. Our approach breaks the current barriers and employs reverse genetics in obligate oomycetes by applying sRNA directly to spores to trigger gene silencing.This innovative approach described in this project focuses on the to use of a small RNA (sRNA) approach to increase our understanding of plant - biotrophic oomycete microbe interactions. We aim to use high-throughput genetic screen to identify and study genes specifically involved in spore germination, infection, mycelial development, sporulation, nutrient uptake, and host immune suppression. We will investigate the properties of sRNA-mediated silencing, optimize, and test in other oomycetes. We will Generate gene-specific sRNAs for highly regulated genes in spores, during germination, mycelial development and sporulation. We will then apply gene specific sRNAs to identify genes showing a phenotype upon silencing. Using this technique, we will also investigate some of the well-known effector genes under native conditions. These would lead to identification and characterization of pathogen genes that could be targeted for disease control. Results obtained from this work can easily be transferred to other obligate downy mildews of grapevine, lettuce, or brassica.

卵菌包括数百种微生物，包括独特的生物营养型、坏死型和半生物营养型植物病原菌。它们表面上与丝状真菌相似，但在几个方面有所不同：据报道，卵菌的细胞壁主要是B-1-3葡聚糖和纤维素，很少或没有几丁质，卵菌的菌丝是共核的(多核的，不被隔膜分裂)，营养核处于二倍体状态。由卵菌类植物病原体引起的病害包括苗枯病、立枯病、根腐病、叶枯病和霜霉病。据估计，卵菌每年造成S数十亿美元的损失，因为它们具有很高的进化潜力，可以使寄主跳跃，对杀菌剂产生抗性，以及抑制或逃避寄主抗性基因。一些经济上最重要的卵菌病原菌是致病疫霉(番茄和马铃薯晚疫病)、枝疫霉菌(橡树猝死)、辣椒疫霉(黄瓜和辣椒的茎和果实腐烂)、肉桂疫霉(鳄梨、菠萝)、葡萄霜霉病(葡萄霜霉病)、哈斯特迪疫霉(向日葵霜霉病)、腐霉(倒伏和根腐病)、乳杆菌(生菜霜霉病)、作为霜霉病病原菌在拟南芥野生种群中的共同进化，30多年来一直被用作研究基因换基因理论的分子基础和植物与卵菌相互作用的实验模型。包括我们在内的几个小组依赖于另一种方法来测试植物的效应器功能，包括：a)使用GUS基因向植物细胞进行共轰击测试以表明无毒活性，b)使用细菌分泌系统运送效应器，以及c)在植物启动子的控制下建立稳定表达效应器基因的植物。然而，所有这些方法都将效应基因从病原体中剥离出来，在这种情况下，基因的表达水平可能无法与自然背景中的基因相比较。此外，单基因分析不能准确地捕捉到原生环境中的基因功能。最后，这些方法只适用于在宿主细胞内工作的分泌效应蛋白。我们的方法打破了目前的障碍，通过将sRNA直接应用于孢子来触发基因沉默，在专性卵菌中采用了反向遗传学。本项目中描述的这一创新方法侧重于使用小RNA(SRNA)方法来增加我们对植物-生物营养卵菌微生物相互作用的理解。我们的目标是使用高通量遗传筛选来鉴定和研究与孢子萌发、感染、菌丝发育、产孢量、营养吸收和宿主免疫抑制相关的基因。我们将研究sRNA介导的沉默的特性，优化并在其他卵菌中进行测试。我们将在孢子萌发、菌丝发育和孢子形成过程中为高度调控的基因产生基因特异的sRNA。然后，我们将应用基因特异性sRNAs来识别沉默时表现出表型的基因。利用这项技术，我们还将研究一些已知的自然条件下的效应基因。这些将导致识别和表征可作为疾病控制目标的病原体基因。从这项工作中获得的结果可以很容易地转移到葡萄藤、生菜或油菜的其他专性霜霉病上。