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A High Throughput Targeted Gene Disruption Method for Alternaria brassicicola Functional Genomics Using Linear Minimal Element (LME) Constructs

¹Virginia Bioinformatics Institute, Blacksburg, VA 24061, U.S.A.; ²Department of Bioagricultural Sciences and Pest Management, Colorado State University, Fort Collins, CO 80523, U.S.A.; ³Department of Molecular Genetics and Microbiology, Duke University Medical Center, Duke University, Durham, NC 27708-9902, U.S.A.

Alternaria brassicicola causes black spot disease of cultivated Brassicas and has been used consistently as a necro-trophic fungal pathogen for studies with Arabidopsis. In A. brassicicola, mutant generation has been the most rate-limiting step for the functional analysis of individual genes due to low efficiency of both transformation and targeted integration. To improve the targeted gene disruption efficiency as well as to expedite gene disruption construct production, we used a short linear construct with minimal elements, an antibiotic resistance selectable marker gene, and a 250- to 600-bp-long partial target gene. The linear minimal element (LME) constructs consistently produced stable transformants for diverse categories of genes. Typically, 100% of the transformants were targeted gene disruption mutants when using the LME constructs, compared with inconsistent transformation and usually less than 10% targeted gene disruption with circular plasmid disruption constructs. Each mutant displayed a unique molecular signature thought to originate from endogenous exonuclease activities in fungal cells. Our data suggests that a DNA double-stranded break repair mechanism (DSBR) functions to increase targeting efficiency. This method is advantageous for high throughput gene disruption, overexpression, and reporter gene introduction within target genes, especially for asexual filamentous fungi where genetic approaches are unfavorable.