Submitted to: Current Genetics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/1/2005
Publication Date: 4/1/2006
Citation: White, D., and Chen, W. 2006. Genetic transformation of Ascochyta rabiei using Agrobacterium-mediated transformation. Current Genetics 49:272-280. Interpretive Summary: Ascochyta blight of chickpea caused by the fungal pathogen Ascochyta rabiei is a serious disease in many parts of the world. Control of this disease is mainly through breeding resistant cultivars and spraying fungicides. To achieve more effective and economical control, a better understanding of the pathogenic mechanisms of the pathogen would be necessary. Studies of the genetic mechanisms of the pathogen are hampered by a lack of suitable genetic tools to transform the pathogen. This research identified optimal conditions for efficient transform the chickpea blight pathogen Ascochyta rabiei. In addition, inverse PCR was found to be more efficient in recovering flanking sequences in transformants. A number of altered phenotypes including reduced capacity of conidial production and reduced virulence were observed among transformants. The findings will facilitate future investigations into the genetics mechanisms of Ascochyta blight.
Technical Abstract: The conditions for efficient transformation of Ascochyta rabiei using Agrobacterium-mediated transformation have been determined. A. tumefaciens cells were co-cultivated with conidia of A. rabiei strain AR628, highly virulent on chickpea. Hygromycin B resistance (hph) was found to be superior to geneticin resistance (nptII) for selecting transformants. In A. rabiei, the hph gene was more efficiently expressed by the Aspergillus nidulans trpC promoter than by the Cauliflower mosaic virus 35S promoter CaMV35S. Co-cultivation on solid media for 72 hours was optimal for generating transformants, whereas increasing the ratio of bacterial cells to conidia did not affect transformation efficiency. All hygromycin B-resistant transformants carried T-DNA as determined by PCR and the insertions appeared to be random and in single copy as detected by Southern hybridization. Inverse PCR was more efficient than TAIL-PCR in identifying flanking sequences from T-DNA borders. Upon integration, the T-DNA left border region contained deletions of up to 72 bps. Transformants remained resistant to hygromycin B in the absence selection, however variations in colony morphology were observed in the presence of hygromycin B under different culture conditions. This transformation technique will provide a useful tool for genetic studies of A. rabiei.