Location: Sunflower and Plant Biology Research
Project Number: 3060-21220-031-20-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Jul 1, 2017
End Date: Dec 31, 2020
RNA-based fungicides hold great potential for the development of strategies to limit disease losses from Sclerotinia sclerotiorum (Lib.) de Bary, which infects nearly 400 plant species and causes Sclerotinia stem rot also known as white mold, a disease with annual losses exceeding $200 million in the United States. However, little is known about the molecular mechanisms by which RNA silencing genes responsible for debilitated phenotypes of fungi. Emerging evidence suggests that alterations in small RNA production induced by interactions between fungal RNA-mediated silencing which is also involved in antiviral defense produce changes in fungal gene expression. The reductions in pathogenicity or virulence induced by viral infection can be informative of ways to design RNA-based fungicides. Our proposed work will elucidate the roles of S. sclerotiorum RNA silencing-related genes in alteration of fungal gene expression. Such knowledge in the long term will enable us to devise new methods for controlling not only S. sclerotiorum specifically, but other similar necrotrophic fungi including the mildews and rusts that are commonly found on most plants. It has been shown that in fungi, RNA silencing functions primarily as an antiviral defense mechanism. In addition, fungi export small RNAs to block the function of plant RNA silencing proteins as shown in Botrytis cinerea. Recently, it has been illustrated that pathogenic fungi also process RNAs from host plants or external application and that plants export microRNAs to inhibit fungal virulence gene expression, clearly demonstrating RNA silencing and small RNAs can be manipulated to increase plant disease protection. It is possible that these findings apply to all the host-pathogen interactions, including white mold Sclerotinia. Hence, the objectives are to determine the roles of RNA silencing genes in (1) changes in phenotypes of disruption mutants; (2) changes in virulence/pathogenicity and determine whether there is reduced ability of the fungal mutant to produce small RNAs to block plant RNAi; (3) changes in susceptibility to viral infections; (4) changes in small RNA accumulation/profile; in order to design RNA-based fungicides.
The above objectives will be accomplished by (1) Generate disruption mutants of S. sclerotiorum genes encoding homologues of AGO, DCL and RdRp proteins; (2) Evaluate the effects of the disruption mutations for alterations in growth, sclerotial development, sensitivity to hypovirulent mycoviruses, pathogenicity on canola, dry bean, pea, lentil, chickpea, soybean and sunflower; (3) Detect and quantify host and virus small RNA profile during viral infection in wild-type S. sclerotiorum and disruption mutants; (4) Examine fungal uptake of RNAs externally that confer plant protection. Three replications of the biological samples of wildtype and two selected disruption mutants challenged with a mycovirus or non-infection will be collected for sequencing. The selection will be based on the levels of hypovirulence (reduced virulence). Around twenty-seven libraries will be generated for smRNA. Libraries will be pooled and sequenced in one lane. Multiple RNA fungicides will be designed based on prediction of data generated from the disruption mutants.