A highly efficient gene-targeting system for Aspergillus parasiticus.
OBJECTIVE
To establish a system that greatly increases the gene-targeting frequency in Aspergillus parasiticus.


RESULTS
The ku70 gene, a gene of the nonhomologous end-joining (NHEJ) pathway, was replaced by the nitrate reductase gene (niaD) in A. parasiticus RHN1 that accumulates O-methylsterigmatocystin (OMST). The NHEJ-deficient strain, RHDeltaku70, produced conidia, sclerotia and OMST normally. It had identical sensitivity as RHN1 to the DNA-topoisomerase I complex inhibitor, camptothecin, and the DNA-damaging agent, melphalan. For targeting an aflatoxin biosynthetic pathway gene, adhA, partial restriction enzyme recognition sequences in its flanking regions were manipulated to create homologous ends for integration. Using a linearized DNA fragment that contained Aspergillus oryzae pyrithiamine resistance gene (ptr) marker the adhA-targeting frequency in RHDeltaku70 reached 96%.


CONCLUSIONS
The homologous recombination pathway is primarily responsible for repair of DNA damages in A. parasiticus. The NHEJ-deficient RHDeltaku70, easy creation of homologous ends for integration, and the ptr-based selection form a highly efficient gene-targeting system. It substantially reduces the time and workload necessary to obtain knockout strains for functional studies.


CONCLUSIONS
The developed system not only streamlines targeted gene replacement and disruption but also can be used to target specific chromosomal locations like promoters or intergenic regions. It will expedite the progresses in the functional genomic studies of A. parasiticus and Aspergilllus flavus.
Chang
P-K
PK
Southern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, New Orleans, LA 70124, USA. pkchang@srrc.ars.usda.gov
D000348:Aflatoxins / Q000235:genetics / Q000378:metabolism; D034961:Antigens, Nuclear / Q000235:genetics; D001230:Aspergillus / Q000187:drug effects / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics; D017353:Gene Deletion; D018390:Gene Targeting; D005822:Genetic Vectors; D000072200:Ku Autoantigen; D008826:Microbial Sensitivity Tests; D050901:Nitrate Reductase / Q000235:genetics; D011995:Recombination, Genetic; D014170:Transformation, Genetic
D000348:Aflatoxins; D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D050901:Nitrate Reductase; D000072200:Ku Autoantigen