Screening genes encoding transcription factors associated with pathogenesis in Alternaria brassicicola

The necrotrophic fungus Alternaria brassicicola causes black spot disease of brassicaceous plants, including green cabbage (Brassica oleracea) and the oil-producing B. napus. Pathogenesis is a multistep process that includes germination, penetration and colonization of host tissues, and survival structure formation. The de novo synthesis of various catabolic enzymes and secondary metabolites needed during each step in pathogenesis are under the regulation of transcription factors. Zinc finger DNA-binding domains containing transcription factor forms the largest family of transcription factors in eukaryotes. Based on the zinc binding motifs transcription factors are of three classes: Cys 2-His2, Cys2-Cys2, and the fungal specific, binuclear Zn(II)2Cys6His6. In the present study we made knock out mutants of all 184 C2H2 Zinc finger transcription factor encoding genes by targeted gene disruption and 35 Zn(II)2Cys 6His6 motif containing transcription factors through targeted gene deletion/replacement in Alternaria brassicicola. Our bioassays on detached leaves of green cabbage have identified twelve genes associated with early and late stages of plant infection. Among these twelve genes, two genes are pathogenicity factors as their mutants were nonpathogenic, while one showed increase in pathogenicity by almost 100%. Remaining nine genes were strong virulence factors whose mutants showed 50-90% reduction in disease symptoms compared to the wild type. We also discovered a unique gene whose mutants showed complete loss of conidia yet no effect on pathogenesis. We further report a gene encoding transcription factor associated with detoxification of phytoalexins, which is important for early plant colonization. All the discovered pathogenesis associated genes were novel pathogenicity or virulence factors and only one gene (PacC) was previously identified as a pathogenicity factor in other fungal species. The molecular mechanisms of pathogenesis and their regulation in necrotrophic fungi are in an early stage of research. This study sets the platform for discovery of downstream genes associated with pathogenesis and the characterization of their functions. Currently available data from this study indicate the importance of transcription factors as regulators of pathogenesis and as future targets for disease management.