Characterization of mat A-2, mat A-3 and deltamatA mating-type mutants of Neurospora crassa.

The mating-type locus of Neurospora crassa regulates mating identity and entry into the sexual cycle. The mat A idiomorph encodes three genes, mat A-1, mat A-2, and mat A-3. Mutations in mat A-1 result in strains that have lost mating identity and vegetative incompatibility with mat a strains. A strain containing mutations in both mat A-2 and mat A-3 is able to mate, but forms few ascospores. In this study, we describe the isolation and characterization of a mutant deleted for mat (deltamatA), as well as mutants in either mat A-2 or mat A-3. The deltamatA strain is morphologically wild type during vegetative growth, but it is sterile and heterokaryon compatible with both mat A and mat a strains. The mat A-2 and mat A-3 mutants are also normal during vegetative growth, mate as a mat A strain, and produce abundant biparental asci in crosses with mat a, and are thus indistinguishable from a wild-type mat A strain. These data and the fact that the mat A-2 mat A-3 double mutant makes few asci with ascospores indicate that MAT A-2 and MAT A-3 are redundant and may function in the same pathway. Analysis of the expression of two genes (sdv-1 and sdv-4) in the various mat mutants suggests that the mat A polypeptides function in concert to regulate the expression of some sexual development genes.     

Sex-linked transcriptional divergence in the hermaphrodite fungus Neurospora tetrasperma

In the filamentous ascomycete Neurospora tetrasperma, a large (approx. 7 Mbp) region of suppressed recombination surrounds the mating-type (mat) locus. While the remainder of the genome is largely homoallelic, this region of recombinational suppression, extending over 1500 genes, is associated with sequence divergence. Here, we used microarrays to examine how the molecular phenotype of gene expression level is linked to this divergent region, and thus to the mating type. Culturing N. tetrasperma on agar media that induce sexual/female or vegetative/male tissue, we found 196 genes significantly differentially expressed between mat A and mat a mating types. Our data show that the genes exhibiting mat-linked expression are enriched in the region genetically linked to mating type, and sequence and expression divergence are positively correlated. Our results indicate that the phenotype of mat A strains is optimized for traits promoting sexual/female development and the phenotype of mat a strains for vegetative/male development. This discovery of differentially expressed genes associated with mating type provides a link between genotypic and phenotypic divergence in this taxon and illustrates a fungal analogue to sexual dimorphism found among animals and plants.     

The Neurospora crassa pheromone precursor genes are regulated by the mating type locus and the circadian clock

Pheromones play important roles in female and male behaviour in the filamentous ascomycete fungi. To begin to explore the role of pheromones in mating, we have identified the genes encoding the sex pheromones of the heterothallic species Neurospora crassa. One gene, expressed exclusively in mat A strains, encodes a polypeptide containing multiple repeats of a putative pheromone sequence bordered by Kex2 processing sites. Strains of the opposite mating type, mat a, express a pheromone precursor gene whose polypeptide contains a C-terminal CAAX motif predicted to produce a mature pheromone with a C-terminal carboxy-methyl isoprenylated cysteine. The predicted sequences of the pheromones are remarkably similar to those encoded by other filamentous ascomycetes. The expression of the pheromone precursor genes is mating type specific and is under the control of the mating type locus. Furthermore, the genes are highly expressed in conidia and under conditions that favour sexual development. Both pheromone precursor genes are also regulated by the endogenous circadian clock in a time-of-day-specific fashion, supporting a role for the clock in mating.     

Large-scale analysis of yeast filamentous growth by systematic gene disruption and overexpression

Under certain conditions of nutrient stress, the budding yeast Saccharomyces cerevisiae initiates a striking developmental transition to a filamentous form of growth, resembling developmental transitions required for virulence in closely related pathogenic fungi. In yeast, filamentous growth involves known mitogen-activated protein kinase and protein kinase A signaling modules, but the full scope of this extensive filamentous response has not been delineated. Accordingly, we have undertaken the first systematic gene disruption and overexpression analysis of yeast filamentous growth. Standard laboratory strains of yeast are nonfilamentous; thus, we constructed a unique set of reagents in the filamentous Σ1278b strain, encompassing 3627 integrated transposon insertion alleles and 2043 overexpression constructs. Collectively, we analyzed 4528 yeast genes with these reagents and identified 487 genes conferring mutant filamentous phenotypes upon transposon insertion and/or gene overexpression. Using a fluorescent protein reporter integrated at the MUC1 locus, we further assayed each filamentous growth mutant for aberrant protein levels of the key flocculence factor Muc1p. Our results indicate a variety of genes and pathways affecting filamentous growth. In total, this filamentous growth gene set represents a wealth of yeast biology, highlighting 84 genes of uncharacterized function and an underappreciated role for the mitochondrial retrograde signaling pathway as an inhibitor of filamentous growth.     

The evolutionary trajectory of the mating-type (<Emphasis Type="Italic">mat</Emphasis>) genes in <Emphasis Type="Italic">Neurospora</Emphasis>relates to reproductive behavior of taxa

Background  

Comparative sequencing studies among a wide range of taxonomic groups, including fungi, have led to the discovery that reproductive genes evolve more rapidly than other genes. However, for fungal reproductive genes the question has remained whether the rapid evolution is a result of stochastic or deterministic processes. The mating-type (mat) genes constitute the master regulators of sexual reproduction in filamentous ascomycetes and here we present a study of the molecular evolution of the four mat -genes (mat a-1, mat A-1, mat A-2 and mat A-3) of 20 Neurospora taxa.     

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

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.     

The fission yeast homologue of CENP-B, Abp1, regulates directionality of mating-type switching

In fission yeast, mating-type switching involves replacing genetic information contained at the expressed mat1 locus by that of either the mat2P or mat3M donor loci. Donor selection is nonrandom, as mat1P cells preferentially use mat3M for switching, whereas mat1M cells use mat2P. Switching directionality is determined by the cell-type-specific distribution of the Swi2-Swi5 complex that, in mat1P cells, localises to mat3M and, only in mat1M cells, spreads to mat2P in a heterochromatin-dependent manner. Mechanisms regulating spreading of Swi2-Swi5 across heterochromatin are not fully understood. Here, we show that the fission yeast homologue of CENP-B, Abp1, binds to the silent domain of the mating-type locus and regulates directionality of switching. Deletion of abp1 prevents utilisation of mat2P, as when heterochromatin is disrupted and spreading of Swi2-Swi5 is impaired. Our results show that, indeed, deletion of abp1 abolishes spreading of Swi2-Swi5 to mat2P. However, in abp1Delta cells, heterochromatin organisation at the mating-type locus is preserved, indicating that Abp1 is actually required for efficient spreading of Swi2-Swi5 through heterochromatin. Cbh1 and Cbh2, which are also homologous to CENP-B, have only a minor contribution to the regulation of directionality of switching, which is in contrast with the strong effects observed for Abp1.     

Sex in fungi: lessons of gene regulation

Fungi have been very useful for gene regulation studies. Mating implicates in a series of events influenced by many types of environmental input that are interpreted into regulatory pathways, including signal transduction. Although various aspects of mating and signal transduction in the yeast Saccharomyces cerevisiae have long been characterized, recent findings in filamentous fungi indicate that pheromones and pheromone receptors may be essential for mating partner recognition and also for nucleus recognition in sorting before meiosis. A brief overview on mating-type genes of ascomycete fungi and recent contributions to the understanding of their role in the regulation of multicellularity and sexual dimorphism is presented in this review.