Submitted to: Journal of Microbiological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 4, 2010
Publication Date: March 16, 2010
Citation: Chang, P., Scharfenstein, L.L., Wei, Q., Bhatnagar, D. 2010. Development and Refinement of a High-Efficiency Gene-Targeting System for Aspergillus flavus. Journal of Microbiological Methods. 81:240-246. Interpretive Summary: Aspergillus flavus produces aflatoxins and is a pathogen of economically important crops. It is also the second leading causative agent of invasive and non-invasive aspergillosis. Significant economic losses can result from aflatoxin contamination of food and feed. Genomic resources for A. flavus play an increasingly important role in understanding aflatoxin biosynthesis and fungus-plant interactions. Equally important is their impact on the understanding of A. flavus pathogenicity in humans and animals. An efficient gene-targeting system for A. flavus functional genomics was developed. The efficacy of this system was demonstrated by the disruption of four genes residing in different genome locations including two genes involved in spore color formation. This system will facilitate the elucidation of gene functions, which would lead to better strategies for reducing or eliminating aflatoxin contamination of agricultural commodities.
Technical Abstract: The development of an efficient gene-targeting system is a prerequisite for success in the functional genomics study of Aspergillus flavus, an aflatoxin-producing fungus of great economic importance. To this end, the ku70 gene, a gene of the nonhomologous end-joining pathway, was deleted to increase the homologous recombination frequency. The pyrG gene in the resulting strain was disrupted by a gene knock-in strategy for using the A. parasiticus pyrG gene as a selectable marker for transformation. Gene-targeting frequencies of A. flavus br1, br2, wA and ygA genes, homologous to genes reported to be involved in conidial pigment biosynthesis in A. fumigatus or A. nidulans, were examined in the 'ku70'pyrG strain. The br1 gene is located about 100 kb from the clustered br2 and wA genes on chromosome IV, and the ygA gene is located on chromosome I. The gene-targeting frequencies were achieved at 80 to 100% as confirmed by PCR screenings for altered genomic patterns in the transformants. Disruption of br2 (renamed as olgA) and wA resulted in mutants of olive-green and white conidial color, respectively. The br1 and ygA mutants retained the parental strain’s yellowish green conidial color. The gene targeting system described here substantially reduces the time and workload necessary to obtain knockout mutants, and should expedite the progresses in the functional genomic studies of A. flavus.