Fusarium species exhibit significant intrinsic resistance to most antifungal agents and fungicides, resulting in high mortality rates among immunocompromised patients. Consequently, a thorough characterization of the antifungal resistance mechanism is required for effective treatments and for preventing fungal infections and reducing antifungal resistance. In this study, an isolate of Fusarium oxysporum (wild-type) with broadly resistant to commonly antifungal agents was used to generate 1,450 T-DNA random insertion mutants via Agrobacterium tumefaciens-mediated transformation. Antifungal susceptibility test results revealed one mutant with increased sensitivity to azoles. Compared with the resistant wild-type, the mutant exhibited low MICs to KTZ, ITC, VRC, POS, and PCZ (0.125, 1, 0.06, 0.5, and 0.125μg/ml, respectively). The T-DNA insertion site of this mutant was characterized as involving two adjacent genes, one encoding a hypothetical protein with unknown function and the other encoding the NADPH-cytochrome P450 reductase, referred as CPR1. To confirm the involvement of these genes in the altered azole susceptibility, the independent deletion mutants were generated and the Cpr1 deletion mutant displayed the same phenotypes as the T-DNA random mutant. The deletion of Cpr1 significantly decreased ergosterol levels. Additionally, the expression of the downstream Cyp51 gene was affected, which likely contributed to the observed increased susceptibility to azoles. These findings verified the association between Cpr1 and azole susceptibility in F. oxysporum. Furthermore, this gene may be targeted to improve antifungal treatments.

BackgroundFusarium species are important pathogenic organisms, which can cause many diseases in plants and humans. Characterizing the mechanism underlying their pathogenicity and drug resistance is critical. Agrobacterium tumefaciens-mediated genetic transformation has been widely used for the molecular analysis of many species. ResultsIn this study, we constructed the pXEN recombinant plasmid carrying the neomycin phosphatase II gene (neo) and established a simple and efficient procedure for the transformation of resistant Fusarium oxysporum mediated by A. tumefaciens. The transformation efficiency was as high as 250 mutants per 104 conidia. A total of 1,450 stably transformed mutants were generated, resulting in a small-scale library of F. oxysporum mutants containing T-DNA tags. Some of the mutants exhibited phenotypic changes in growth, metabolism, and development. Additionally, the sequences flanking the inserted T-DNA were obtained by touchdown-TAIL PCR, the insertion sites and genes associated with the phenotypic changes could be determined.ConclusionsThe developed method may enable to analyze gene functions and study biological characteristics, which will lay the foundation for future analyses of the mechanism underlying F. oxysporum pathogenicity and resistance. Furthermore, it may be applicable to investigations of other important pathogenic fungi.