Preliminary Studies On Agrobacterium Tumefaciens-mediated Transformation Of Fusarium Graminearum

Fusarium graminearim is the causal agent of Fusarium head blight disease of barley and wheat. Understanding the molecular basis of the pathogenicity of F. graminearim is beneficial for control of Fusarium head blight disease and breeding of resistant host cultivars. To this end, we construct a T-DNA insertion population of F. graminearum via Agrobacterium tumefaciens-mediated transformation (ATMT), screen for pathogenicity mutants and study the functions of the corresponding genes in these mutants. In this study, an ATMT system of F. graminearim was established and optimized, and the main results are as follows.1. Some factors, which could affect ATMT transformation rate, were optimized. These factors include selection medium, concentration of F. graminearum spores and acetosyingone (AS), time of co-cultivation, pH of co-cultivation medium, A. tumefaciens concentration, and the best time of spores of F. graminearum. Our optimized transformation system is to (1) combine agrobacteria (OD₆₀₀=0.12⁰.15) containing 75 μmol/L AS with equal volume of fungal spores at concentration of 10⁶ spores/mL and incubate at 25℃ with shaking for 24 h, (2) spread the mixture onto a nylon membrane placed on the surface of MM medium containing 75 μmol/L AS and incubate at 24℃ in dark for 24 h;(3) collect the mixture of agrobacteria and spores from nylon membrane and spread onto PDA selection plates containing 50 μg/mL streptomycin, 200 μg/mL cefotaxime,100 μg/mL spectinomycin and 75 μg/mL hygromycin B. Using the optimized transformation system, we obtained 100 to 200 transformants from transformation of 1x10⁶ spores of F. graminearum.2. The hygromycin-resistant transfromants of F. graminearum were characterized by PCR amplification of the GUS gene fragment with genomic DNA of 10 randomly-selected transformants as templates. The result showed that the GUS fragment could be amplified from all transfromants tested and thus the T-DNA in the binary vector used for transformation had been integrated into genomes of F. graminearum.3. To examine the genetic stability of the transformants, 10 randomly-selected hygromycin-resistant transformants were cultivated on. PDA or PDA containing 75μg/mL hygromcin B for 4 generations, and observed for GFP fluorescence .The results showed that the 5th generation could grow on the selected plates containing hygromycin B and could show GFP, suggesting that all tested transformants were genetically stable.4. Phenotypes of the hygromycin-resistant transfromants were analyzed for their changes in colony color, growth, spore production and pathogenicity. The results showed that most of the transformants were similar to the original strain of F. graminearum in their colony color, growth and pathogenicity. However, some transformants showed altered phenotypes as compared with those of the original strain. For example, the colony color of some transformants was changed to primrose yellow, pink red or dark red from damask (the original strain). Some transformantsshowed reduced growth or spore production.5. Pathogenicity tests for the T -DNA insertional mutants of F. graminearwn were performed using spike inoculation and caryopsis inoculation methods. Several putative pathogenicity mutants were identified. T-DNA insertional mutants 1-5, 1-11, 1-27,2-5 showed reduced pathogenicity.Further characterization of the F. graminearwn T-DNA insertional mutants with reduced growth phenotype or reduced pathogenicity and cloning of the flanking sequences flanked the T-DNA in the mutants by TAIL-PCR are in progress. The establishment of the Agrobacterium-mediated transformation of F.graminearum will facilitate the construction of the T-DNA inserted mutant population, screening of mutants with altered pathogenicity and identification of pathogenicity-related genes, which in turn will be helpful in dissecting the molecular basis of the pathogenicity in both of the fungal pathogens.