Document Type

Thesis - Open Access

Award Date

2016

Degree Name

Master of Science (MS)

Department

Biology and Microbiology

First Advisor

Yang Yen

Keywords

fgsiR34, Fusarium graminearum, Fusarium head blight, pathogenicity, small RNAs, trichothecenes

Abstract

Fusarium graminearum is an ascomycetous fungal pathogen that causes Fusarium head blight (FHB) disease in wheat and other cereal grains. Mycotoxin produced by the fungus, predominantly deoxynivalenol (DON), is considered as an important virulence factor for the spread of disease. Our previous study of a Dicer-like 2 knockdown mutant has led to our hypothesis that a silencing RNA, fgsiR34, might play a key role in regulating DON biosynthesis and some other virulent factors. To test this hypothesis, we generated an fgsiR34 over-expressing mutant (ΔfgsiR34+) using Inverse Repeat Transgene method and studied the pathogenicity of the mutant in wheat. Though no phenotypic alterations, such as spore production and growth rate on solid media, were found in the mutant in comparison with the wildtype strain, altered expressions of Tri genes and other pathogenic genes were observed. Tri4, Tri5, Tri6, Tri10, and Tri14 were all significantly downregulated, while the cell wall degrading enzymes (CWDEs) were upregulated in ΔfgsiR34+ strain. Wheat spikelets inoculated with ΔfgsiR34+ showed a significant downregulation of both Tri5 and Tri6. The disease progression and F. graminearum biomass were significantly reduced in ΔfgsiR34+-inoculated FHBsusceptible NIL compared to the wildtype-inoculated ones. To understand the mechanism of the pathogenic role by fgsiR34, we analyzed the methylation pattern of the seed region of fgsiR34 at nearly 1000 bp upstream of Tri5 to elucidate if fgsiR34 induces methylation to suppress expression of Tri genes. After bisulfite treatment and methylation-specific PCR we found that the seed region of fgsiR34 in both the wildtype and the ΔfgsiR34+ strains was not methylated. In summary, our results suggested that fgsiR34 negatively regulates Tri genes biosynthesis pathway, while positively regulates CWDEs. All these results imply a significant, complex role of fgsiR34 in regulating Tri genes biosynthesis. It seems that methylation is not involved in repressing the expression of Tri genes. Interestingly, our RT-qPCR assay of non-coding transcript within the seed region revealed that the fgsiR34 seed region was transcribed with increased transcript abundance in the ΔfgsiR34+ mutant over the wildtype, suggesting a role of the non-coding transcript in regulating expression of Tri genes. More research is, therefore, needed to elucidate the mechanisms of FHB pathogenicity by fgsiR34.

Library of Congress Subject Headings

Wheat -- Disease and pest resistance -- Genetic aspects.

Fusarium diseases of plants.

RNA interference.

Gene silencing.

Description

Includes bibliographical references (pages 82-121)

Format

application/pdf

Number of Pages

150

Publisher

South Dakota State University

Rights

Copyright © 2016 Subha Dahal

Available for download on Sunday, December 09, 2018

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