Agrobacterium tumefaciens-mediated transformation of the dermatophyte,Trichophyton mentagrophytes: an efficient tool for gene transfer

Abstract

Agrobacterium tumefaciens-mediated transformation (ATMT) was used to facilitate gene transfer into the clinically important dermatophyte, Trichophyton mentagrophytes (teleomorph: Arthroderma vanbreuseghemii). A binary vector containing a hygromycin B resistance cassette was introduced into A. tumefaciens, and the resultant strain was co-cultivated with fungal small conidia. Transformation yielded a large number of hygromycin B-resistant transformants. Hybridization analysis showed that most of the transformants harboured a single copy of T-DNA randomly integrated into the genome. Transformation frequency was increased to more than 200 per 10(7) conidia by optimizing the co-cultivation time and temperature. ATMT was then used for targeted gene disruption mediated by homologous recombination. Using a PCR-based strategy, we isolated the areA/nit-2-like nitrogen regulatory gene (tnr:Trichophytonnitrogen regulator) from T. mentagrophytes. A binary vector containing two regions of the tnr locus flanking the hygromycin B resistance cassette was constructed and introduced into T. mentagrophytesvia ATMT. Transformants with disruption of the areA/nit-2-like gene (tnr) were obtained in three of four independent disruption experiments, most of which showed homologous recombination via double crossover without additional ectopic integration of the disruption construct.

Terbinafine Resistance of Trichophyton Clinical Isolates Caused by Specific Point Mutations in the Squalene Epoxidase Gene

“…A. vanbreuseghemii 1062Av1401 was transformed by the A. tumefaciens-mediated transformation (ATMT) method as described previously (29). After cocultiva- tion, nylon membranes were transferred onto SDA containing 250 g/ml G418 (Sigma-Aldrich) and 10 M CuSO 4 ·7H 2 O, and overlaid with 10 ml of SDA supplemented with the same concentration of G418 and CuSO 4 ·7H 2 O.…”

“…The desired transformants were finally screened by PCR, Southern blotting analyses, and nucleotide sequencing. Total DNA was extracted according to a method described previously (29). Aliquots of 50 to 100 ng of the total DNA were used as templates in the PCRs.…”

“…Microconidium formation was induced at 28°C using modified 1/10 Sabouraud dextrose agar (SDA) (27) supplemented with 500 g/ml cycloheximide (Wako Chemical) and 50 g/ml chloramphenicol (SigmaAldrich). Agrobacterium tumefaciens EHA105 (28) was maintained as previously described (29). Escherichia coli DH5␣ (Nippon Gene) was used for molecular cloning.…”

Targeted Gene Deletion and In Vivo Analysis of Putative Virulence Gene Function in the Pathogenic Dermatophyte Arthroderma benhamiae

“…In light of these benefits, it appeared timely to establish a genetic system for A. benhamiae, which allows isogenic strain construction and gene targeting, fundamental prerequisites for functional gene analysis. Basic improvements in dermatophyte genetics have recently been implemented by Yamada et al, who demonstrated the reproducible transformation of T. mentagrophytes and M. canis (32)(33)(34). Most filamentous fungi are known to only poorly support site-specific integration of linear DNA constructs in the genome via homologous recombination.…”

Site-Directed and Random Insertional Mutagenesis in Medically Important Fungi

“…In addition, T-DNA mutagenesis protocols have been developed for Coccidioides (Abuodeh et al, 2000), Trichoderma spp. (Cardoza et al, 2006;Dobrowolska and Staczek, 2009;Yamada et al, 2009), and Penicillium marneffei (Kummasook et al, 2010;Zhang et al, 2008). In C. neoformans, the use of T-DNA improved both the efficiency and the stability of transformation events.…”