Document Type

Dissertation - Open Access

Award Date

2020

Degree Name

Doctor of Philosophy (PhD)

Department

Biology and Microbiology

First Advisor

Shin-Yi Lee Marzano

Keywords

Fungi, Mycovirus, RNA interference, RNA-editing, Sclerotinia sclerotiorum, small RNA

Abstract

In eukaryotes, small RNAs (sRNAs) are key regulators of RNA silencing which is also known as RNA interference (RNAi). RNAi is an essential mechanism known in plants, animals and fungi that regulates various biological activities, including defense against foreign nucleic acids and viruses. Fungal pathogens, such as Sclerotinia sclerotiorum, severely limit crop production in all parts of the world. Sclerotinia sclerotiorum cause white mold infection, affecting all dicotyledonous plants including many economically important field crops, vegetables, and floriculture. There does not exist known host resistance, and only limited chemical control can be achieved. Given the growing cost and environmental impacts of using fungicides to control these pathogens, an alternative method that exploits RNA silencing in fungi is warranted. The objectives of my research were: (i) to understand RNA-editing mechanism in virus derived small interferring RNAs (VsiRNAs), (ii) to discover mycoviruses from metatranscriptomic study of arbuscular mycorrhizal fungus Rhizophagus spp., and (iii) to characterize the antiviral roles of RNAi genes in S. sclerotiorum. This dissertation includes three chapters from my PhD research. In chapter 1, I have demonstrated that RNA editing mechanism is common in fungi, including S. sclerotiorum, Botrytis cinerea, and Fusarium graminearum, as well as in higher metazoans. My analysis showed that the virus-infected wild-type and RNAi mutant strains of S. sclerotiorum accumulate virusderived small RNAs with distinct patterns of internal and terminal modifications. Chapter 2 deals with the discovery of mycoviruses infecting arbuscular mycorrhizal fungus and their evolution with respect to their counterparts infecting pathogenic fungi. Finally, chapter 3 focuses on the characterization of RNAi genes in S. sclerotiorum. While RNA silencing in fungi is primarily involved in antiviral defense against foreign nucleic acids, pathogenic fungi also utilize RNA silencing mechanism to silence host defense genes in plants through the delivery of small RNA molecules as virulence effectors. Beginning with the discovery of RNA editing events, this study investigates the roles of RNAi genes in fungal pathogen S. sclerotiorum using various approaches in bioinformatics. The present study dissects the RNA silencing pathway in S. sclerotiorum by disrupting its key silencing genes using the split-marker recombination method in order to probe the contributions of these genes, specifically argonautes, to fungal virulence and viral defense mechanisms. Following gene disruption, mutants were studied for changes in phenotype, pathogenicity, viral susceptibility, and small RNA processing compared to the wild-type strain, DK3. Among the argonaute mutants, the Δagl-2 mutant had significantly slower growth and virulence prior to and following virus infection. Additional analyses indicated that the virus-infected wild-type strain accumulated virusderived small RNAS (vsiRNAs) with distinct patterns of internal and terminal nucleotide mismatches. Δdcl-1 mutant produced less vsiRNA compared to Δdcl-2 mutant and the wild type strain, suggesting the two dicers are not in the state of complete redundancy. An emerging area of interest is the use of external dsRNA-based pest control which will require detailed characterization of the RNA silencing pathways in S. sclerotiorum. In an attempt to investigate the utility of dsRNA-based pest control strategy for white mold, we studied the roles of argonaute enzymes, agl-2 and agl-4, in small RNA metabolism in S. sclerotiorum. Our study has shown that RNA silencing deficient S. sclerotiorum show increased susceptibility to virus infection. Additionally, we also profiled different classes of small RNAs, including vsiRNAs, from different gene mutants to study viral susceptibility, internal modification, stability, and small RNA processing compared to wild-type strain, DK3, of S. sclerotiorum. Results from this study show that fungal pathogen S. sclerotiorum host robust RNA silencing mechanisms to defend against foreign nucleic acid and viruses and to facilitate fungal infection of host plants through trans-kingdom RNAi. These findings expand our overall understanding of S. sclerotiorum and has important implications for any current or future uses of dsRNA and mycoviruses as disease control agents.

Format

application/pdf

Number of Pages

91

Publisher

South Dakota State University

Rights

In Copyright - Educational Use Permitted
http://rightsstatements.org/vocab/InC-EDU/1.0/

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