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

Highlights

• A genome editing protocol has been developed for use in Huntiella species.

• MAT1-2-7, a novel mating gene, is a true mating gene in Huntiella omanensis.

• ΔMAT1-2-7 strains show delays in protoascomatal development and hyphal growth.

• MAT1-2-7 is essential for ascomatal maturation.

Abstract

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.

Introduction

Sexual reproduction in ascomycetes is controlled almost entirely by genes present at the mating type (MAT) locus. At minimum, this includes the primary MAT genes, MAT1-1-1 and MAT1-2-1 (Debuchy et al., 2010, Dyer et al., 2016). However, other secondary genes can also be present, often in a lineage-specific manner. As the nomenclature suggests, the MAT1-1-1 gene is the defining feature of the MAT1-1 idiomorph, while the same is true of the MAT1-2-1 gene at the MAT1-2 idiomorph (Turgeon and Yoder, 2000). In heterothallic, or self-sterile, fungi, a single individual can be assigned a mating type based on the genic content of its MAT locus, with the MAT1-1 idiomorph conferring the MAT1-1 mating type and the MAT1-2 idiomorph conferring the MAT1-2 mating type (Dyer et al., 2016).

At present, there is no clear definition of a MAT gene. This is in part due to the limited number of functional studies on these genes, particularly in non-model species, but also the fairly extensive variation in the genic content of the MAT locus (Debuchy et al., 2010). Some authors have classified a bona fide MAT protein as one that allows for internuclear recognition and that is functional only when expressed from the MAT locus (Arnaise et al., 1997). This definition is highly restrictive as it relies on the functional characterization of these genes as well as the ability to precisely track the expression and location of the protein during sexual reproduction. It also does not consider the many other processes involved in sexual reproduction that are not linked to recognition but remain essential for the production of recombinant offspring. Other authors define the MAT locus simply as a location in the genome responsible for mating, and thus a MAT gene is a gene that resides within this locus (Turgeon and Yoder, 2000, Wilken et al., 2017). This definition does not rely on functional characterization and is thus more relevant in non-model fungi where gene characterization has not yet been possible.

The broader, locus-dependent definition for a mating gene has led to the description of many MAT genes (as recently reviewed (Wilken et al., 2017)). Of these, the MAT1-1-1 and MAT1-2-1 genes fulfil an essential role in the mating process and are found in almost all studied species (Debuchy and Coppin, 1992, Paoletti et al., 2007, Staben and Yanofsky, 1990). They are thus considered the primary mating genes. In comparison, the secondary MAT genes are not as well-conserved and do not always have recognizable conserved domains (Wilken et al., 2017). These genes have been named numerically in the order of their discovery in various species (Turgeon and Yoder, 2000). Thus, for example, Cryphonectria parasitica harbours the MAT1-1-2 and MAT1-1-3 genes at the MAT1-1 idiomorph (McGuire et al., 2001); while the MAT1-2-2 and MAT1-2-9 genes are harboured in the MAT1-2 idiomorphs of Neurospora crassa (Pöggeler and Kück, 2000) and Fusarium fujikuroi (Martin et al., 2011, Wilken et al., 2017), respectively.

Functional characterization of the MAT genes in diverse fungal species has predominantly focused on MAT1-1-1 and MAT1-2-1, and in some cases, has even concentrated specifically on their functional domains. These genes have been shown to be essential for sexual reproduction in model and non-model species alike (Debuchy and Coppin, 1992, Ferreira et al., 1998, Paoletti et al., 2007, Staben and Yanofsky, 1990). The precise functions of MAT1-1-1 and MAT1-2-1 are also fairly well-conserved, with both primary genes often playing important roles during the initiation of sexual reproduction. However, while these genes are well-characterized, similar research is lacking with respect to the secondary MAT genes. The few examples that do exist include the Fusarium graminearum MAT1-1-2 and the Botrytis cinerea MAT1-1-5, both of which are important for the maturation of the ascomata (Kim et al., 2012, Rodenburg et al., 2018). In addition to their role in mating, MAT genes can also influence other important non-mating factors, such as pathogenicity, growth and vegetative incompatibility (Lee et al., 2015, Newmeyer et al., 1973).

Huntiella omanensis, a member of the Ceratocystidaceae (de Beer et al., 2014), has recently been the topic of genomic and transcriptomic studies with respect to its sexual development (Wilson et al., 2018, Wilson et al., 2015). MAT1-1 isolates of this fungus possess the MAT1-1-1 and MAT1-1-2 genes, while the MAT1-2 isolates harbour the MAT1-2-1 and MAT1-2-7 genes (Wilson et al., 2015). MAT1-1-1, MAT1-1-2 and MAT1-2-1 all encode proteins that are comparable to those encoded by this locus in other species and they each possess the expected functional domains associated with these proteins. In contrast, MAT1-2-7 was first detected in silico and showed no similarity to any other known genes. It also encoded a protein with no recognizable functional domains (Wilson et al., 2015). Later the gene was found to be expressed during sexual reproduction despite undetectable expression levels during the vegetative growth phase (Wilson et al., 2018). Its position in the MAT locus combined with its expression pattern thus suggested that this gene might have a role in the sexual process.

Huntiella moniliformis, a close relative of H. omanensis, possesses a significantly truncated and likely non-functional version of the MAT1-2-7 gene (Wilson et al., 2015). Interestingly, this species undergoes unisexual reproduction, unlike the many heterothallic species found in this genus (Wilson et al., 2015). It has thus been hypothesized that MAT1-2-7 plays a role in the regulation of sexual reproduction in these species and that its truncation in H. moniliformis leads, at least in part, to the homothallic behaviour observed in this species (Wilson et al., 2018). However, H. moniliformis also exhibits an interesting pheromone expression pattern, with a single isolate capable of producing both mating pheromones. This is unlike H. omanensis, which, similar to many heterothallic species, expresses these pheromones in a mating-type dependent manner (Wilson et al., 2018). It is not known whether the truncation of the MAT1-2-7 gene, the indiscriminate pheromone expression or a combination of both has led to unisexual reproduction.

The aim of this study was to characterize the H. omanensis MAT1-2-7 gene with respect to its involvement in sexual reproduction. Our objective was to mimic the truncation seen in the H. moniliformis MAT1-2-7 gene to determine whether MAT1-2-7 disruption can bring about unisexual behaviour in H. omanensis. This was achieved by developing a protoplast-based transformation and CRISPR-Cas9 genome editing protocol for use in the first successful genetic modification of any species within the genus Huntiella. The H. omanensis MAT1-2-7 gene was confirmed as an essential mating gene that plays an important role in ascomatal maturation.