Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Biology

Supervisor

Katherine Dobinson

Abstract

The broad host range, soil borne fungus Verticillium dahliae Kleb. is the causal agent of an economically significant vascular wilt disease. This species produces persistent resting structures, known as microsclerotia, which are the primary source of disease inoculum in the field. Five hydrophobin-like proteins (VDH1 to 5) have been identified in the genome of V. dahliae. The results of bioinformatics analyses suggested secretion of these proteins, and that they are all class II hydrophobins. Gene expression analyses of VDH1 to 5 indicate that the transcript levels of the individual genes vary under different growth conditions. Additionally, the transcript levels of these genes differ from one another. These results suggest that the members of this protein family have different roles in V. dahliae. One of the hydrophobin genes, VDH5, contains an extra domain known as an extended N terminus (ENT) domain. For further functional analyses of this particular hydrophobin, Agrobacterium tumefaciens-mediated transformation was used to generate a targeted gene disruption (vdh5TN), and gene deletion mutants (Δvdh5). Both Δvdh5 and vdh5TN strains showed reduced spores and microsclerotial production, as well as a less aggressive pathogenic phenotype. Similarly, a strain carrying a gene lacking the ENT domain was produced (vdh5ΔENT). No differences in development or pathogenicity were found between the vdh5ΔENT and the WT strains. The response of pathogenesis related genes (PR1, PR3, and PR5) in tomato plants inoculated with the Δvdh5 mutant, and with the mutant of the previously characterize vdh1, was also studied. The lower transcript levels of these genes in response to infection with either vdh1 or vdh5 mutant strain, compared to that of WT-inoculated plants, suggest that the plant is responding specifically to these two hydrophobin genes. The study and characterization of hydrophobin genes has proven their importance in developmental processes and in pathogenicity, and the knowledge produced here may lead to novel disease control methods.


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