Degree Type

Dissertation

Date of Award

2010

Degree Name

Doctor of Philosophy

Department

Agronomy

First Advisor

Madan K. Bhattacharyya

Abstract

Fusarium virguliforme (Fv) is the causal organism of the sudden death syndrome (SDS) in soybean (Glycine max [L.] Merr). The estimated average annual soybean yield loss from this disease is valued at about 300 million dollars. F. virguliforme is a soil borne fungus, which attacks soybean roots and causes root rot as well as chlorosis and necrosis in foliar tissues. The pathogen has never been isolated from the diseased foliar tissues. It was therefore hypothesized that a toxin(s) is involved in the development of the disease.

Cell–free F. virguliforme culture filtrate containing one or more toxins produces foliar SDS symptoms in cut soybean seedlings. Cell–free F. virguliforme culture filtrates were separated by conducting gel filtration and native PAGE and a 13.5 kDa, proteinaceous toxin FvTox1 was purified. Anti–FvTox1 monoclonal antibodies were raised against FvTox1 in mice and used in screening an expression cDNA library. A 516 bp long FvTox1 cDNA was isolated from screening the cDNA library. FvTox1 is a single gene containing two small introns (61 and 50 bp). FvTox1 was expressed in an insect cell line with the aid of baculovirus. Expressed FvTox1 was able to produce chlorosis and necrosis in leaf discs of SDS–susceptible but not SDS–resistant soybean cultivars.

Two single chain variable fragment (scFv) antibody genes, Anti–FvTox1–1andAnti–FvTox1–2, were cloned from the hybridoma cell line expressing the anti–FvTox1 7E8 monoclonal antibody. Escherichia coli expressed Anti–FvTox1–1 and Anti–FvTox1–2 bound to FvTox1 on nylon membranes. Anti–FvTox1 scFv antibody genes with KDEL sequence at their either 5’ or 3’ ends were cloned into a binary vector and four constructs were generated. The Agrobacterium rhizogenes strains carrying these binary constructs were used to generate transgenic roots by infecting soybean cotyledons. Protein preparation from transformed hairy roots with all four constructs except the one carrying Anti–FvTox1–1 with the KDEL sequence fused to its 5’–end were functional. The Anti–FvTox1–1 gene with the KDEL sequence at its 3’end was used to generate stable transgenic soybean plants. The progenies of a transgenic plant carrying the Anti–FvTox1–1 gene were investigated for foliar SDS development. Progenies carrying a copy of the transgene showed enhanced tolerance to the pathogen infection and FvTox1 as compared to the non–transgenic William 82 control plants that went through the same transformation procedures but carry no transgenes. These results suggested that FvTox1 is the pathogenicity factor that induces foliar SDS in soybean and expression of a plant anti–FvTox1 antibody could be a suitable strategy in enhancing foliar SDS resistance.

In order to understand the possible pathogenesis mechanism immunohistochemistry was conducted using the anti–FvTox1 7E8 monoclonal antibody. FvTox1 accumulates in the chloroplasts of the F. virguliforme–infected soybean plants suggesting that the site of FvTox1 action is in chloroplasts. The toxin may interfere the photosynthesis function to initiate foliar SDS symptoms as suggested by an earlier study.

DOI

https://doi.org/10.31274/etd-180810-1152

Copyright Owner

Hargeet Kaur Brar

Language

en

Date Available

2012-04-30

File Format

application/pdf

File Size

151 pages

Share

COinS