On the cover of this week’s Nature is a picture of Phycomyces blakesleeanus highlighting the discovery of the MAT locus in this Zygomycete fungus from Alex Idnurm and Joe Heitman and colleagues. While it was previously known that Zygomycetes (the Orange lineage represented by R. oryzae in the tree below) mate, the specific locus has until now, never been discovered. The authors in this study identified the MAT locus through a sequence search looking for HMG-box genes knowing that these are found the Mating Type locus in Basidiomycetes and Ascomycetes. They confirmed the identity through a through set of experiments that included PCR, sequencing and crosses of (+) and (-) strains of P. blakesleeanus, and Southern blots.
Other cloned MAT genes in fungi
The Saccharomyces MAT locus and the logic of the molecules, signaling network, and homothalism and molecular basis of the ability to switch mating types has been extensively worked on for several decades. The active and silent loci were cloned and shown to be responsible for the sexual identity in the early 1980’s. The MAT locus in the filamentous Neurospora crassa fungus was identified in 1990 (Staben and Yanofsky, Glass, Grotelueschen, and Metzenberg). However, it wasn’t until 1999 (Hull and Johnson) that the MAT locus in the human commensal fungus Candida albicans was cloned.
The Basidiomycete fungi had their own mystery to be discovered and is excellently reviewed by Casselton and Olesnicky. The tetrapolar mating systems seen in most Agarics (mushroom forming fungi) require two loci: A and B which each have two or more alleles in the population and mating occurs between individuals different at both loci effectively creating a huge number of “sexes”. This has been highlighted in work on Schizophyllum commune, Pleurotus sp, and Coprinus species. The loci controlling this were isolated in Ustilago maydis in the late 1980s (Kronstad and Leong, Schulz et al). Some basidiomycetes only have a bipolar mating system, one locus with two ideomorphs: A/alpha instead of A and B. The specific genes responsible for mating type identity in the human pathogenic and Cryptococcus neoformans were cloned in 2002 (Hull, Davidison, and Heitman) and the entire mating 100+ kb locus was sequenced for Cryptococcus in 2004 (Fraser et al) and additional projects have sequenced the genomes for several serotypes and species of the fungus. The evolution of a biopolar mating system. in Cryptococcus at least, was derived from a tetrapolar system where a fusion of A and B loci formed a single locus (now A or alpha).
The Zygomycete discovery
In the Idnurm et al study, the authors sought to find the MAT locus using what we’ve learned from Dikarya fungi (Ascos and Baisidios). In Zygomycetes the sexes are named (-) and (+) instead of A and alpha (just to keep it interesting!). To look for the potential MAT locus, they first used BLAST to search for sequences similar to A or alpha MAT-locus encoded transcription factors (i.e. MATalpha2) in the Phycomyces genome sequence, but this doesn’t find any significantly similar sequences that were reciprocal best hits (although RBS is a common approach to finding orthologs, it may not be sufficient for orthology detection anyways!). However, they widened the search to look for all genes containg HMG-box domains and found 10 candidates that were similar to Ascomycete HMG-box genes. The authors then used PCR (probably without the singing) to amplify the locus in strains of opposite mating type to see if any of the genes were present in the sequenced strain (mating type (-)) but not in the (+) strains. They found one out of the 10 loci that met this criteria and sequenced this region to determine the genes in the location from the opposite mating type.
The newly christened sexM (sex minus) and sexP (sex plus) genes were identified as distant homologs and were found to be the best homologs of each other in the genome, another prediction if there were indeed ideomorphs that controlled sexual identity. See SexM/SexP alignment for more details. The authors go on to prove these loci control sexual identity through a series of crosses because genetic manipulation via transformation is still not easily attainable in Phycomyces. Analysis of the 2,336 bp difference in size found insertion of repetitive sequence (which is reminiscent of what was found in the Cryptococcus loci).
In concluding statements about the overall significance of the locus finding, the authors argue that the repeats and single region controlling sexual identity is similar to formation of Y chromosome (an interpretation used in the Fraser et al paper) suggesting that commonality of formation of sex determining region in animals and fungi through inversions of simple repeats leading to a non-recombining region. They propose that the HMG-determining region was ancestral to at least the Mucorales (Zygomycete) lineages and the Dikaryka (Ascomycete and Basidiomycetes) and that deviations from this (like tetrapolar mating systems) in fungal lineages were subsequently derived.
The careful and thorough work in this paper has lead to a greater knowledge of the components and understanding of how sex and sexual identity evolved in fungi. Future experiments and analyses to see how well conserved the downstream elements from mating are (hopefully will be addressed in the Rhizopus and Phycomyces genome papers) and more experiments to understand if the circuitry that depends on the input from the MAPK and other pathways induced by mating to arrest the cell cycle, control invasive hyphal growth, melanization and other processes.