Structure, function, and phylogeny of the mating locus in the Rhizopus oryzae complex

Abstract

The Rhizopus oryzae species complex is a group of zygomycete fungi that are common, cosmopolitan saprotrophs. Some strains are used beneficially for production of Asian fermented foods but they can also act as opportunistic human pathogens. Although R. oryzae reportedly has a heterothallic (+/-) mating system, most strains have not been observed to undergo sexual reproduction and the genetic structure of its mating locus has not been characterized. Here we report on the mating behavior and genetic structure of the mating locus for 54 isolates of the R. oryzae complex. All 54 strains have a mating locus similar in overall organization to Phycomyces blakesleeanus and Mucor circinelloides (Mucoromycotina, Zygomycota). In all of these fungi, the minus (-) allele features the SexM high mobility group (HMG) gene flanked by an RNA helicase gene and a TP transporter gene (TPT). Within the R. oryzae complex, the plus (+) mating allele includes an inserted region that codes for a BTB/POZ domain gene and the SexP HMG gene. Phylogenetic analyses of multiple genes, including the mating loci (HMG, TPT, RNA helicase), ITS1-5.8S-ITS2 rDNA, RPB2, and LDH genes, identified two distinct groups of strains. These correspond to previously described sibling species R. oryzae sensu stricto and R. delemar. Within each species, discordant gene phylogenies among multiple loci suggest an outcrossing population structure. The hypothesis of random-mating is also supported by a 50:50 ratio of plus and minus mating types in both cryptic species. When crossed with tester strains of the opposite mating type, most isolates of R. delemar failed to produce zygospores, while isolates of R. oryzae produced sterile zygospores. In spite of the reluctance of most strains to mate in vitro, the conserved sex locus structure and evidence for outcrossing suggest that a normal sexual cycle occurs in both species.

Figure 1. Zygospores of Rhizopus oryzae.

A cross between CBS346.36 (+) and CBS110.17 (−) was analyzed with light microscopy (A) and scanning electron microscopy (B). Zygospores (white arrow heads) are attached to an asymmetric suspensor (black arrow heads). Scale = 20 µm. Asterisks indicate conjugated hyphae during early stage of zygospore formation.

Figure 2. Comparison of sex locus structure in the Mucorales (Mucoromycotina, Zygomycota).

(A) Dot plot showing the divergent DNA sequence of sexP and sexM genes in Rhizopus oryzae, whereas the TPT and RNA helicase genes are highly conserved. The orientations of sexP and sexM genes are the same in R. oryzae. (B) sex locus structures of P. blakesleeanus and M. circinelloides are compared with R. oryzae (this study). Relative to R. oryzae, the TPT gene in M. circinelloides and both the TPT gene and sexP in P. blakesleeanus have an inverted orientation. The (+) allele of R. oryzae contains an additional gene between the TPT gene and sexP that is not found in the other two species or in the (−) allele of R. oryzae. Black boxes show repetitive sequence tracts, arrows show gene orientation, and lines represent intergenic sequence.

Figure 3. Maximum Likelihood phylogenies of the Rhizopus oryzae complex clearly delimit the two cryptic species, Rhizopus oryzae s. s. and Rhizopus delemar.

(A) sexP alleles, (B) sexM alleles, (C) concatenated phylogram of rDNA ITS1-5.8S-ITS2, RPB2, TPT gene, and RNA helicase gene, and (D) four-gene strict consensus tree. Analysis included a total of 458 (+), 635 (−) and 3064 (MLS) nucleotide characters. ML bootstrap proportions higher than 70 are shown above the nodes. Asterisks (*) indicate strains that produced zygospores. Filled circles indicate a single R. delemar strain, CBS329.47, closest to the ancestral state.

Figure 4. Range of sporangiospore sizes for isolates from the Rhizopus oryzae complex.

Naming convention includes the species prefix (Ro – Rhizopus oryzae, Rd – Rhizopus delemar) followed by the strain number. Bars indicate the 95% confidence interval.

Figure 5. Main pattern of asexual sporangiospore micromorphology.

(A) Rhizopus delemar NRRL3562. (B) R. oryzae NRRL2908. Arrows show the variation in spore size on the same sporangium (×5000). Scale bar = 1 µm.

Figure 6. Different copy numbers of lactate dehydrogenase (LDH) in Rhizopus oryzae s. s. and Rhizopus delemar.

(A) LHDA has a longer the 3′ end compared to LDHB. Primers P1 and P2 recognize both LDHA and LDHB producing 238 bp DNA fragments in PCR, whereas P3 only recognizes the 3′ end of the LDHA producing 525 bp DNA fragments in PCR with P1. Gene sizes are not to scale. (B) Rhizopus oryzae s. s. strains produces 238 bp and 525 bp bands in a PCR reaction with the three primers P1, P2, and P3, however, R. delemar strains produce only 238 bp bands with the same PCR conditions. Non-specific amplification of 900 bp bands occurred with several R. oryzae s. s. strains. Size marker is 100 bp ladder (NEB, Ipswich, MA, USA). Rhizopus oryzae s. s. strains are (from left to right) NRRL2908, CBS346.36, NRRLA-336, CBS110.17, NRRL1897, NRRL1501 and Rhizopus delemar strains are (from left to right) ATCC34612, NRRL2871, NRRL2005, NRRL1528, NRRL21447, NRRL13908.