Submitted to: Molecular Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 1, 2010
Publication Date: March 20, 2011
Citation: Maruthachalam, K., Klosterman, S.J., Kang, S., Hayes, R.J., Subbarao, K.V. 2011. Identification of pathogenicity-related genes in the vascular wilt fungus verticillium dahliae by agrobacterium tumefaciens-mediated t-DNA insertional mutagenesis. Molecular Biotechnology. PMID:21424547. Interpretive Summary: The fungus Verticillium dahliae causes the disease Verticillium wilt on lettuce and other crops. The fungus can survive in the soil for years by producing hardened melanized survival structures. The identification of genes in the fungus that may be important to development of survival structures and the ability to cause disease may be useful to develop alternative control methods for Verticillium wilt on lettuce and other crops. The bacterium Agrobacterium tumefaciens is equipped to transfer DNA of interest from the bacterium into the fungal genome. Because the DNA transferred is of known sequence, the points of insertion into the fungal genome can be identified. In this study, DNA was randomly transferred into the fungal genome using the bacterium Agrobacterium tumefaciens, thereby potentially disrupting DNA sequences in the fungus that may encode genes important for survival or pathogenicity on lettuce. Following insertion of the DNA into the fungal genome, individual strains of the fungus were characterized for the ability to produce survival structures or to cause disease on lettuce. Disrupted sequences in the fungus were further characterized, enabling the identification of several genes that affected survival structure development and two genes that are important for the fungus to cause disease on lettuce.
Technical Abstract: Verticillium dahliae is the causal agent of vascular wilt in many economically important crops worldwide. Identification of genes that underpin pathogenicity or virulence may suggest targets for alternative control methods for this fungus. In this study, Agrobacterium tumefaciens-mediated transformation (ATMT) was applied for insertional mutagenesis of V. dahliae conidia. Southern blot analysis indicated that T-DNAs were inserted randomly into the V. dahliae genome and that 69% of the transformants were the result of single copy T-DNA insertion. DNA sequences flanking T-DNA insertion were isolated through inverse PCR (iPCR), and these sequences were aligned to the genome sequence to identify the genomic position of insertion. V. dahliae mutants of particular interest included those that had lost the ability to form microsclerotia. Also, based on the virulence assay of 181 transformants, we identified 20 mutant strains of V. dahliae that did not cause symptoms on lettuce plants. Among these mutants, T-DNA was inserted in genes encoding an endoglucase I (VdEg-I), a hydroxyl-methyl glutary-CoA synthase (VdHMGS1), a major facilitator superfamily (VdMFS1), and a glycosylphosphatidylinositol (GPI) mannosyltransferase (VdGPIM1). These results suggest that ATMT can effectively be used to identify genes associated with pathogenicity and other functions in V. dahliae.