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

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.

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. Movement of small RNAs from plants to pathogens has been explored using HIGS. Although we tried HIGS methods several times in Arabidopsis to study functions of ATR1, ATR13 and ATR5 for Hyaloperonospora arabidopsidis (Hpa) in our laboratory, it proved unsuccessful (Tör Group, unpublished results). However, we discovered that application of sRNA can be effectively used to elucidate gene function in Hpa. We will Investigate the properties of sRNA-mediated silencing, optimize, and test in other oomycetes including Peronospora viciae, Phytophthora parasitica and P. capsici. We will Generate gene-specific sRNAs for highly regulated genes in Hpa 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