| Fusarium head blight, caused by the fungus Fusarium graminearum, is a major disease on wheat and barley worldwide, leading to a reduction in grain yield and quality. Colletotrichum lagenarium is the causal agent of anthracnose diseases on watermelon and cucumber. However, the molecular basis of pathogenicity for these two fungal pathogens remains unclear yet. Isolation of pathogenicity mutants and identification of the corresponding genes associated with pathogenicity through functional genomics approach will definitely be helpful in accelerating our understanding of the molecular mechanisms of the pathogenicity in these two important fungal pathogens. The objectives of this study were to (1) construct a binary vector, which is suitable for Agrobacterium-mediated transformation of pathogenic fungi and contains a reporter gene cassette, (2) establish and optimise Agrobacterium-mediated transformation of Fusarium graminearum and Colletotrichum lagenarium, (3) isolate and characterize T-DNA insertional mutants of Fusarium graminearum and Colletotrichum lagenarium. To construct a binary vector, which is suitable for Agrobacterium-mediated transformation of fungi and contains a reporter gene cassette, we first constructed a cassette that contains GFP-GUS fusion gene driven by Aspergillus nidulans trpC promoter and terminated by Aspergillus nidulans trpC terminator, and cloned this cassette into the T-DNA region of pBIG2RHPH2, a binary vector used for Agrobacterium-mediated transformation of fungi and carrying the bacterial hygromycin B phosphotransferase gene cassette under the control of Aspergillus nidulans trpC promoter as a selection marker. This led to construction of a new binary vector pBlG2RHPH2-GUS-GFP. The transformants from transformation with pBIG2RHPH2-GUS-GFP can grow on medium containing hygromycin B and show GFP and GUS activities.To establish the Agrobacterium-mediated transformation for F. graminearum and C. lagenarium, we first analysed the effect of co-cultivation factors on the transformation effciency, including period of agrobacteria and spores of the tested fungi, pH of the co-cultivation media, concentrations of agrobacteria and spores, antibiotics and their concentrations, to optimise these factors for improving transformation efficiency. Our optimized transformation system was as following: The agrobacteria culture (OD600=0.12-0.15) containing 25 μmol/L acetosyringone (AS) with equal volume of the fungal spores and incubated at 25 ℃ with shaking for 24 h, The mixture was spread onto a nylon membrane and placed on the surface of MM medium containing 100 μmol/L AS, and then incubated at 24 ℃ in dark for 24 h, The mixture of agrobacteria and spores from nylon membrane was collected and spread onto PDA selection plates containing 50 μg/ml ampicillin, 200 μg/ml cefotaxime and 200 μg/ml hygromycin B. Using our optimized transformation system, we obtained more than 200 transformants of F. graminearum and 150-200 transfromants of C. lagenarium from transformation of 1 × 106 spores.We further characterized the hygromycin-resistant transfromants of F. graminearum and C. lagenarium by PCR amplification of the GUS gene fragment and detection of GFP and GUS activity. Our results showed that the GUS fragment could be amplified from all transfromants tested with genomic DNA as template in PCR, indicating that the T-DNA in the binary vector pBIG2RHPH2-GUS-GFP had been integrated into genomes of F. graminearum and C. lagenarium. Microscope observation and GUS staining also confirmed that all transformants tested showed GFP in mycelium and spores and GUS activity. The T-DNA inserted transformants of F. graminearum and C. lagenarium appeared to be stable as they showed hrgromycin resistance, GFP fluorescence and GUS activity after 4 generations.The morphology of the F graminearum T-DNA insertional mutants was characterized by comparing the colony color, growth, and spore morphology and germination with those of the wild type strain used in this study. T-DNA insertional mutants FG-44, 47, 55 and 57 have different colony colour, showing primrose or gray as compared with damask in the wild type strain. Mycelium growth and spore... |
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