Identification of the mating-type (MAT) locus that controls sexual reproduction of Blastomyces dermatitidis

Abstract

Blastomyces dermatitidis is a dimorphic fungal pathogen that primarily causes blastomycosis in the midwestern and northern United States and Canada. While the genes controlling sexual development have been known for a long time, the genes controlling sexual reproduction of B. dermatitidis (teleomorph, Ajellomyces dermatitidis) are unknown. We identified the mating-type (MAT) locus in the B. dermatitidis genome by comparative genomic approaches. The B. dermatitidis MAT locus resembles those of other dimorphic fungi, containing either an alpha-box (MAT1-1) or an HMG domain (MAT1-2) gene linked to the APN2, SLA2, and COX13 genes. However, in some strains of B. dermatitidis, the MAT locus harbors transposable elements (TEs) that make it unusually large compared to the MAT locus of other dimorphic fungi. Based on the MAT locus sequences of B. dermatitidis, we designed specific primers for PCR determination of the mating type. Two B. dermatitidis isolates of opposite mating types were cocultured on mating medium. Immature sexual structures were observed starting at 3 weeks of coculture, with coiled-hyphae-containing cleistothecia developing over the next 3 to 6 weeks. Genetic recombination was detected in potential progeny by mating-type determination, PCR-restriction fragment length polymorphism (PCR-RFLP), and random amplification of polymorphic DNA (RAPD) analyses, suggesting that a meiotic sexual cycle might have been completed. The F1 progeny were sexually fertile when tested with strains of the opposite mating type. Our studies provide a model for the evolution of the MAT locus in the dimorphic and closely related fungi and open the door to classic genetic analysis and studies on the possible roles of mating and mating type in infection and virulence.

Fig 1

Syntenic comparison of the MAT locus of B. dermatitidis. Red bars/lines represent highly syntenic and similar DNA regions, and blue bars/lines represent highly syntenic and similar DNA regions with reverse orientation. The B. dermatitidis MAT locus is flanked by the SLA2, COX13, and APN2 genes, which share high sequence identity among these isolates. Isolate ATCC 18188 is designated plus (+) mating type due to the presence of the MAT1-1 locus, which is determined by an alpha-box gene. Isolates ER3, SLH-14081, and ATCC 26199 are designated minus (−) mating type, based on the identification of an HMG gene in the MAT1-2 locus. The genomic regions depicted with boxes in dashed outlines are the MAT locus. These TEs result in expansion of the MAT locus of B. dermatitidis. TEs are present in the MAT locus of three minus isolates ER3, SLH-14081, and ATCC 26199.

Fig 2

Sexual development of B. dermatitidis. After coculture of B. dermatitidis isolates ATCC 18188 (+) and ATCC 18187 (−) on SEA medium for 10 weeks, cleistothecia were scraped off the agar, disrupted in water, and fixed in formaldehyde prior to microscopic examination. (A) 20× light microscopy shows multiple disrupted cleistothecia, with highly spiraled hyphae. (B) 40× image showing thickened, coiled hyphae and specialized vegetative cells attached to the spiral hyphae. (C) 60× image shows multiple ascospores released from asci. The ascospores are characterized by a light halo around the cell.

Fig 3

(A and B) Mating-type determination of B. dermatitidis parental isolates (ATCC 18187 and ATCC 18188) and 22 potential progeny by PCR. Primers were designed to specifically amplify the alpha-box (plus mating type) and HMG (minus mating type) genes, which are characteristic of the B. dermatitidis MAT locus (see Table 1). (C and D) RAPD analysis of the progeny of isolates ATCC 18188 and ATCC 18187. Two RAPD primers, PI-10 (C) and PI-24 (D), reveal genetic variations (indicated by an asterisk) between these two parental isolates and the potential progeny. Lane 1, M1-1; lane 2, M1-2; lane 3, M1-3; lane 4, M1-4; lane 5, M1-5, lane 6, M1-6; lane 7, M1-7; lane 8, M1-8; lane 9, M1-9; lane 10, M1-10; lane 11, M2-1; lane 12, M2-2; lane 13, M2-3; lane 14, M2-4; lane 15, M2-6; lane 16, M2-7; lane 17, M2-11; lane 18, M2-16; lane 19, M2-17; lane 20, M2-18; lane 21, M2-19; lane 22, M2-20; lane 23, ATCC 18187; and lane 24, ATCC 18188.

Fig 4

PCR-RFLP analysis of the progeny of isolates ATCC 18188 (+) and ATCC 18187 (−). SNP and insertion/deletion between isolates ATCC 18188 and ATCC 18187 were used to design a PCR-RFLP analysis to detect genetic variation between these two isolates and the potential progeny. An SNP was identified between isolates ATCC 18188 and ATCC 18187 that abolished a single BamHI restriction site in ATCC 18188 (A). Note that for ATCC 18188, digestion results in two distinct bands, while digestion of ATCC 18187 fails to produce any novel fragments. Progeny were scored as either genotype 1 or 2, denoting their relationship to ATCC 18188 or ATCC 18187, respectively (see Table 3). A second SNP identified within a different region abolished an NdeI restriction site in ATCC 18187. However, sequences of the amplified region predicted an additional NdeI restriction site shared by both parents, which accounts for the faint low-molecular-weight band appearing after digestion of ATCC 18187 DNA (B). For panels A and B, parental DNAs representing the amplified region around the SNP prior to and after digestion are indicated by one asterisk and two asterisks, respectively. L, DNA ladder.