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V. Chaturvedi, S. Chaturvedi; Fusarium Solani Sporulation and Biofilm-Deficient Mutants Constructed by Agrobacterium - Mediated Transformation. Invest. Ophthalmol. Vis. Sci. 2008;49(13):827.
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There is virtually no information available about the virulence traits of Fusarium solani, an important causal agent of fungal keratitis. We report a genome-wide random mutagenesis by Agrobacterium tumefaciens -mediated transformation for the discovery of fungal virulence genes.
F. solani, MYC23-06, a corneal ulcer isolate, was transformed with A. tumefaciens EHA105 strain harboring T-DNA plasmid pTAS10, and the transformants were selected by hygromycin resistance. The insertion of marker gene (Hyg) in the mutants was confirmed by PCR and Southern hybridization. Stable transformants were further tested for phenotypic changes in pigmentation, sporulation and biofilm-formation.
Acetosyringone-activated A. tumefacines (1 X 106) and young conidia of F. solani (1 X 106) yielded 20-30 transformants per 100 µl of the suspension after 72-hour co-cultures on induction medium. A total of 580 transformants were pooled onto 24-well tissue culture plates. A random selection of 11 transformants, probed for Hyg insertion, revealed single tagging of genes in 60% of the mutants. Phenotypic screens showed that 13 mutants produced scant to no spores and greatly diminished biofilms.
Insertion mutagenesis of F. solani yielded sporulation and biofilm-deficient mutants, which will serve as the building blocks for characterization of virulence factors in fungal keratitis.
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