Document Type

Dissertation - Open Access

Award Date

2020

Degree Name

Doctor of Philosophy (PhD)

Department / School

Biology and Microbiology

First Advisor

Yang Yen

Keywords

Fhb1, Fusarium graminearum Virus 1, Fusarium Head Blight, Fusarium pathogenicity, Host-Pathogen interactions, WFhb1-1

Abstract

Fusarium head blight (FHB) is a severe disease of wheat (Triticum aestivum L.). It not only reduces the quantity of the harvested grains but also decreases the grain quality due to mycotoxins contamination, especially Deoxynivalenol (DON). Qfhb1 (or simply called Fhb1) is the most important quantitative trait locus (QTL) for FHB resistance. Our lab has previously identified wheat gene WFhb1-1 (aka. Wfhb1_c1) as a candidate for FHB resistance gene. Here we report that WFhb1-1 has been cloned. The gene (GenBank # KU304333.1) consists of a single exon, encoding a putative membrane protein of 127 amino acids. WFhb1-1 protein produced in Pichia pastoris inhibits the growth of both F. graminearum and P. pastoris in culture. Western Blotting with anti- WFhb1-1 antibody revealed a significant decrease (p < 0.01) in WFhb1-1 accumulation 12 hours post Fusarium inoculation in Qfhb1-non carrier wheat but not in Qfhb1-carrier wheat. Overexpressing WFhb1-1 in Qfhb1-non carrier wheat led to a significant decrease (p < 0.01) in Fusarium-damaged rachis rate, Fusarium-diseased kernel rate and DON content in harvested kernels, while silencing WFhb1-1 in Qfhb1-carrier wheat resulted in a significant increase (p < 0.01) in FHB severity. Therefore, WFhb1-1 is an important FHB resistance gene with a potential antifungal function and probably a key functional component of Qfhb1 in wheat. A model regarding how WFhb1-1 functions in FHB resistance/susceptibility is hypothesized and discussed. All the published reports on Fhb1 candidate genes has been also reviewed, discussed, and a multi-gene model of Qfhb1 associated regulation mechanism against FHB has been proposed. DON toxin production during the infection of F. graminearum in small grain crops is one of the most harmful virulence factors associated with economic losses. F. graminearum strain, Fg-4-2, having mycovirus FgV1-SD4 showed significantly reduced virulence against wheat compared to the wildtype not having the virus. Additionally, no DON accumulation was detected in the harvested wheat seeds infected by Fg-4-2, whereas ~18 ppm DON was detected in seeds infected by Fg-4-1, (F. graminearum not having FgV1-SD4). In this study, we propose a pathway of regulation of DON biosynthesis by FgV1 infection. We also made an infectious clone of the mycovirus, FgV1-SD4, and tested if this strain of FgV1 can infect F. graminearum and F. oxysporum by electroporation method of transfection. Although, we could not confirm the transfection of F. oxysporum and F. graminearum, differences in culture morphology and pathogenicity were observed between the transfected and the wildtype strains. If this mycovirus strain and the protocol of transfection can be optimized to infect broad range of F. graminearum and F. oxysporum strains, it can open a possibility of using this FgV1 strain as a potential biocontrol agent against F. graminearum infections to reduce the DON accumulation level in small grain crops due to this fungal disease. A protocol for the overexpression of WFhb1-1 gene using BSMV virus-mediated over-expression (VOX) is also developed. Using a three plasmid BSMV expression system, up to ~30-fold overexpression of WFhb1-1 gene could be achieved in generation T0 of wheat plants, whereas we could detect ~2.5-fold overexpression of the gene in T1. generation transmitted through seeds into the new generation. As far as we know, this is the first report of transmission BSMV-mediated gene overexpression through seeds into the new generation. This has a huge potential for further exploration in the use of BSMV in overexpression of genes in many generations, and optimization of the process for gene editing using CRISPR/Cas9 technique.

Library of Congress Subject Headings

Wheat -- Diseases and pests.
Fusarium diseases of plants.
Plant-pathogen relationships.
Mycotoxins.

Format

application/pdf

Number of Pages

175

Publisher

South Dakota State University

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Rights Statement

In Copyright