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of ATR1, ATR13 and ATR5 for Hyaloperonospora arabidopsidis (Hpa) in our laboratory, it proved unsuccessful (Tor Group, unpublished results).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
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