Molecular dissection of Alternaria brassicicola pathogenesis mechanisms

Alternaria brassicicola is a necrotrophic fungal pathogen that causes black spot disease on cruciferous plants including economically important Brassica species and the model plant Arabidopsis thaliana. The purpose of this dissertation was to gain a better understanding of the pathogenesis mechanisms employed by A. brassicicola by identifying and cloning candidate pathogenicity factors. A functional genomics based approach was used to identify candidate pathogenicity genes. Suppression subtractive hybridization (SSH) and expressed sequence tag (EST) analysis were used to identify a total of 2,002 A. brassicicola genes expressed during infection of Brassica oleraceae var. capitata and the model plant Arabidopsis thaliana. In addition, 1554 B. oleraceae sequences were identified that were expressed during A. brassicicola infection. Reverse-transcriptase (RT) PCR and cDNA Northern blots were used to further analyze the expression of selected genes involved in the fungal-plant interaction. Many fungal genes that were upregulated during plant infection had significant homology with known fungal pathogenicity genes based on BLAST analyses. These included genes such as: cyanide hydratase, ABC transporters, MAP kinases, polyketide synthases, serine proteases, and various plant cell wall degrading enzymes. Plant genes identified known to be involved in plant defense responses included: catalase, peroxidase, chitinase, CC-NBS-LRR disease resistance proteins, WRKY transcription factors, pathogenesis related proteins (PR genes), and polygalacturonase inhibiting proteins. Sequence and phylogenetic analysis were conducted for fungal genes including a major Alternaria allergen Alt a 1 homolog, and two MAP kinases with homology to FUS3/KSS1 and SLT2. Functional analysis of the A. brassicicola cyanide hydratase was conducted by creating a gene disruption mutant via homologous recombination. Mutants with a disrupted cyanide hydratase locus displayed increased sensitivity to cyanide in vitro, but were still capable of causing disease symptoms on Brassica species indicating that this cyanide hydratase is not likely required for A. brassicicola pathogenesis. Taken together, these results represent the first large scale analysis of genetic factors involved in A. brassicicola pathogenesis. Thus, the results of this study have established a foundation for future studies on the Alternaria brassicicola-Brassicaceae interaction.