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

卵菌包括几百种微生物，包括独特的生物营养、坏死性和半生物营养植物病原体群。它们表面上与丝状真菌相似，但在几个方面与丝状真菌不同：据报道卵菌的细胞壁主要由B-1-3葡聚糖和纤维素组成，很少或没有几丁质，卵菌的菌丝是共胞的（多核，没有隔分裂），它们的营养细胞核处于二倍体状态。卵霉菌植物病原菌引起的病害包括幼苗枯萎病、枯枯病、根腐病、叶枯病和霜霉病。总的来说，卵菌估计每年造成数十亿美元的损失，因为它们具有很高的进化潜力，可以使宿主跳跃，对杀菌剂产生抗性，以及抑制或逃避宿主抗性基因。一些经济上最重要的卵菌病原菌有：疫霉（番茄和马铃薯晚疫病）、橡树猝死病、辣椒病（黄瓜和辣椒的茎和果实腐病）、樟病（鳄梨和菠萝的枯萎病）、葡萄霉病（葡萄霜霉病）、halstedii病（向日葵霜霉病）、大霉病（枯萎病和根腐病）、莴苣霜霉病（莴苣白疱锈病）和白念珠菌病（十字花科植物的白疱锈病）。拟南芥透明operonospora拟南芥（拟南芥）生物营养性卵菌在拟南芥野生种群中作为霜霉病病原体共同进化，并作为研究基因对基因理论的分子基础和植物与卵菌相互作用其他方面的实验模型已有30多年的历史。专性卵菌不适合遗传转化，因此阻碍了遗传分析。包括我们在内的几个研究小组已经采用了几种不同的方法来测定植物中效应基因的功能，包括：a)使用GUS基因对植物细胞进行共轰击试验，以表明无毒活性；b)使用细菌分泌系统递送效应基因；c)在植物启动子的控制下，创建表达效应基因的稳定转化植物。然而，所有这些方法都将效应基因从病原体中剥离出来，其中一个基因的表达水平可能与本地背景中不一样。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运作的分泌效应蛋白。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，在专性卵菌中使用反向遗传学。本项目中描述的这种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。我们的目标是利用高通量基因筛选来鉴定和研究与孢子萌发、感染、菌丝发育、产孢、营养吸收和宿主免疫抑制有关的基因。我们将研究srna介导的沉默特性，优化并在其他卵菌中进行测试。我们将在孢子萌发、菌丝发育和产孢过程中生成高度调控基因的基因特异性sRNAs。然后，我们将应用基因特异性sRNAs来鉴定沉默后表现出表型的基因。利用这一技术，我们还将在自然条件下研究一些众所周知的效应基因。这将导致病原体基因的鉴定和表征，从而成为疾病控制的目标。从这项工作中获得的结果可以很容易地转移到其他专性霜霉病的葡萄藤，生菜，或芸苔。

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.