Needles in fungal haystacks: Discovery of a putative a-factor pheromone and a unique mating strategy in the Leotiomycetes.

The Leotiomycetes is a hugely diverse group of fungi, accommodating a wide variety of important plant and animal pathogens, ericoid mycorrhizal fungi, as well as producers of antibiotics. Despite their importance, the genetics of these fungi remain relatively understudied, particularly as they don't include model taxa. For example, sexual reproduction and the genetic mechanisms that underly this process are poorly understood in the Leotiomycetes. We exploited publicly available genomic and transcriptomic resources to identify genes of the mating-type locus and pheromone response pathway in an effort to characterize the mating strategies and behaviors of 124 Leotiomycete species. Our analyses identified a putative a-factor mating pheromone in these species. This significant finding represents the first identification of this gene in Pezizomycotina species outside of the Sordariomycetes. A unique mating strategy was also discovered in Lachnellula species that appear to have lost the need for the primary MAT1-1-1 protein. Ancestral state reconstruction enabled the identification of numerous transitions between homothallism and heterothallism in the Leotiomycetes and suggests a heterothallic ancestor for this group. This comprehensive catalog of mating-related genes from such a large group of fungi provides a rich resource from which in-depth, functional studies can be conducted in these economically and ecologically important species.

Structure and number of mating pheromone genes is closely linked to sexual reproductive strategy in Huntiella.

BACKGROUND: Huntiella resides in the Ceratocystidaceae, a family of fungi that accommodates important plant pathogens and insect-associated saprotrophs. Species in the genus have either heterothallic or unisexual (a form of homothallism) mating systems, providing an opportunity to investigate the genetic mechanisms that enable transitions between reproductive strategies in related species. Two newly sequenced Huntiella genomes are introduced in this study and comparative genomics and transcriptomics tools are used to investigate the differences between heterothallism and unisexuality across the genus. RESULTS: Heterothallic species harbored up to seven copies of the a-factor pheromone, each of which possessed numerous mature peptide repeats. In comparison, unisexual Huntiella species had only two or three copies of this gene, each with fewer repeats. Similarly, while the heterothallic species expressed up to 12 copies of the mature α-factor pheromone, unisexual species had up to six copies. These significant differences imply that unisexual Huntiella species do not rely on a mating partner recognition system in the same way that heterothallic fungi do. CONCLUSION: While it is suspected that mating type-independent pheromone expression is the mechanism allowing for unisexual reproduction in Huntiella species, our results suggest that the transition to unisexuality may also have been associated with changes in the genes governing the pheromone pathway. While these results are specifically related to Huntiella, they provide clues leading to a better understanding of sexual reproduction and the fluidity of mating strategies in fungi more broadly.

Genetic Networks That Govern Sexual Reproduction in the Pezizomycotina.

Sexual development in filamentous fungi is a complex process that relies on the precise control of and interaction between a variety of genetic networks and pathways. The mating-type (MAT) genes are the master regulators of this process and typically act as transcription factors, which control the expression of genes involved at all stages of the sexual cycle. In many fungi, the sexual cycle typically begins when the mating pheromones of one mating type are recognized by a compatible partner, followed by physical interaction and fertilization. Subsequently, highly specialized sexual structures are formed, within which the sexual spores develop after rounds of meiosis and mitosis. These spores are then released and germinate, forming new individuals that initiate new cycles of growth. This review provides an overview of the known genetic networks and pathways that are involved in each major stage of the sexual cycle in filamentous ascomycete fungi.

Unique patterns of mating pheromone presence and absence could result in the ambiguous sexual behaviors of Colletotrichum species.

Colletotrichum species are known to engage in unique sexual behaviors that differ significantly from the mating strategies of other filamentous ascomycete species. For example, most ascomycete fungi require the expression of both the MAT1-1-1 and MAT1-2-1 genes to induce sexual reproduction. In contrast, all isolates of Colletotrichum harbor only the MAT1-2-1 gene and yet, are capable of recognizing suitable mating partners and producing sexual progeny. The molecular mechanisms contributing to mating types and behaviors in Colletotrichum are, however, unknown. A comparative genomics approach analyzing 35 genomes, representing 31 Colletotrichum species and two Verticillium species, was used to elucidate a putative molecular mechanism underlying the unique sexual behaviors observed in Colletotrichum species. The existence of only the MAT1-2 idiomorph was confirmed across all species included in this study. Comparisons of the loci harboring the two mating pheromones and their cognate receptors revealed interesting patterns of gene presence and absence. The results showed that these genes have been lost multiple, independent times over the evolutionary history of this genus. These losses indicate that the pheromone pathway no longer plays an active role in mating type determination, suggesting an undiscovered mechanism by which mating partner recognition is controlled in these species. This further suggests that there has been a redirection of the underlying genetic mechanisms that regulate sexual development in Colletotrichum species. This research thus provides a foundation from which further interrogation of this topic can take place.

CRISPR-Cas9-Mediated Genome Editing in the Filamentous Ascomycete Huntiella omanensis.

The CRISPR-Cas9 genome editing system is a molecular tool that can be used to introduce precise changes into the genomes of model and non-model species alike. This technology can be used for a variety of genome editing approaches, from gene knockouts and knockins to more specific changes like the introduction of a few nucleotides at a targeted location. Genome editing can be used for a multitude of applications, including the partial functional characterization of genes, the production of transgenic organisms and the development of diagnostic tools. Compared to previously available gene editing strategies, the CRISPR-Cas9 system has been shown to be easy to establish in new species and boasts high efficiency and specificity. The primary reason for this is that the editing tool uses an RNA molecule to target the gene or sequence of interest, making target molecule design straightforward, given that standard base pairing rules can be exploited. Similar to other genome editing systems, CRISPR-Cas9-based methods also require efficient and effective transformation protocols as well as access to good quality sequence data for the design of the targeting RNA and DNA molecules. Since the introduction of this system in 2013, it has been used to genetically engineer a variety of model species, including Saccharomyces cerevisiae, Arabidopsis thaliana, Drosophila melanogaster and Mus musculus. Subsequently, researchers working on non-model species have taken advantage of the system and used it for the study of genes involved in processes as diverse as secondary metabolism in fungi, nematode growth and disease resistance in plants, among many others. This protocol detailed below describes the use of the CRISPR-Cas9 genome editing protocol for the truncation of a gene involved in the sexual cycle of Huntiella omanensis, a filamentous ascomycete fungus belonging to the Ceratocystidaceae family.

The novel Huntiella omanensis mating gene, MAT1-2-7, is essential for ascomatal maturation.

Sexual reproduction is a highly conserved feature of the eukaryotes, yet sexual compatibility is determined by a wide variety of mechanisms. In ascomycete fungi, sexual development is controlled by genes at the mating type (MAT) locus that confer either MAT1-1 or MAT1-2 mating identity. Although the locus harbours, at minimum, a single gene, the individual MAT loci of certain species, including Huntiella omanensis, encode for two or more genes. The MAT1-2 idiomorph of H. omanensis is made up of MAT1-2-1, a primary MAT gene that is highly conserved in the Pezizomycotina and possesses a well-characterized DNA binding motif, the HMG-box domain. The idiomorph also harbours a novel secondary MAT gene, named MAT1-2-7, with no recognizable functional domains. In this study, we developed a transformation and CRISPR-Cas9-based genome editing protocol to characterize the MAT1-2-7 gene with respect to its function in mating. We have shown that MAT1-2-7 is essential for sexual reproduction and that isolates carrying the truncated MAT1-2-7 gene are incapable of ascomatal maturation and further sexual development. MAT1-2-7 was also shown to influence the vegetative radial growth rate of H. omanensis, illustrating the pleiotropic effects often associated with MAT genes.

It's All in the Genes: The Regulatory Pathways of Sexual Reproduction in Filamentous Ascomycetes.

Sexual reproduction in filamentous ascomycete fungi results in the production of highly specialized sexual tissues, which arise from relatively simple, vegetative mycelia. This conversion takes place after the recognition of and response to a variety of exogenous and endogenous cues, and relies on very strictly regulated gene, protein, and metabolite pathways. This makes studying sexual development in fungi an interesting tool in which to study gene-gene, gene-protein, and protein-metabolite interactions. This review provides an overview of some of the most important genes involved in this process; from those involved in the conversion of mycelia into sexually-competent tissue, to those involved in the development of the ascomata, the asci, and ultimately, the ascospores.

Pheromone expression reveals putative mechanism of unisexuality in a saprobic ascomycete fungus.

Homothallism (self-fertility) describes a wide variety of sexual strategies that enable a fungus to reproduce in the absence of a mating partner. Unisexual reproduction, a form of homothallism, is a process whereby a fungus can progress through sexual reproduction in the absence of mating genes previously considered essential for self-fertility. In this study, we consider the molecular mechanisms that allow for this unique sexual behaviour in the saprotrophic ascomycete; Huntiella moniliformis. These molecular mechanisms are also compared to the underlying mechanisms that control sex in Huntiella omanensis, a closely related, but self-sterile, species. The main finding was that H. omanensis displayed mating-type dependent expression of the a- and α-pheromones. This was in contrast to H. moniliformis where both pheromones were co-expressed during vegetative growth and sexual development. Furthermore, H. moniliformis also expressed the receptors of both pheromones. Consequently, this fungus is likely able to recognize and respond to the endogenously produced pheromones, allowing for self-fertility in the absence of other key mating genes. Overall, these results are concomitant with those reported for other unisexual species, but represent the first detailed study considering the unisexual behaviour of a filamentous fungus.