Keisuke Hayashi

Bcmfs1, a Novel Major Facilitator Superfamily Transporter from Botrytis cinerea, Provides Tolerance towards the Natural Toxic Compounds Camptothecin and Cercosporin and towards Fungicides

ABSTRACT Bcmfs1, a novel major facilitator superfamily gene from Botrytis cinerea, was cloned, and replacement and overexpression mutants were constructed to study its function. Replacement mutants showed increased sensitivity to the natural toxic compounds camptothecin and cercosporin, produced by the plant Camptotheca acuminata and the plant pathogenic fungus Cercospora kikuchii, respectively. Overexpression mutants displayed decreased sensitivity to these compounds and to structurally unrelated fungicides, such as sterol demethylation inhibitors (DMIs). A double-replacement mutant of Bcmfs1 and the ATP-binding cassette (ABC) transporter gene BcatrD was more sensitive to DMI fungicides than a single-replacement mutant of BcatrD, known to encode an important ABC transporter of DMIs. The sensitivity of the wild-type strain and mutants to DMI fungicides correlated with Bcmfs1 expression levels and with the initial accumulation of oxpoconazole by germlings of these isolates. The results indicate that Bcmfs1 is a major facilitator superfamily multidrug transporter involved in protection against natural toxins and fungicides and has a substrate specificity that overlaps with the ABC transporter BcatrD. Bcmfs1 may be involved in protection of B. cinerea against plant defense compounds during the pathogenic phase of growth on host plants and against fungitoxic antimicrobial metabolites during its saprophytic phase of growth.

Expression of the ABC transporter BcatrD from Botrytis cinerea reduces sensitivity to sterol demethylation inhibitor fungicides

The ATP-binding cassette (ABC) transporter gene BcatrD from Botrytis cinerea was cloned and characterized. The open reading frame of BcatrD contains seven introns and encodes a putative protein of 1502 amino acids. The function of BcatrD was analyzed by phenotypic characterization of gene-replacement and overexpression mutants. Replacement mutants of BcatrD displayed a higher sensitivity to sterol demethylation inhibitor (DMI) fungicides as compared to the parental isolate. Gene replacement mutants also showed a relatively high accumulation of the DMI fungicide oxpoconazole. Overexpression mutants showed increased levels of basal and oxpoconazole-induced expression of BcatrD. Mutants with the highest expression level displayed the highest decrease in sensitivity to oxpoconazole and a relatively low accumulation of the compound. These results indicate a relation between oxpoconazole sensitivity, expression of BcatrD, and accumulation of oxpoconazole and demonstrate that the ABC transporter BcatrD is a determinant of the sensitivity of B. cinerea to DMI fungicides.