Elsevier

Fungal Biology

Volume 125, Issue 6, June 2021, Pages 427-434
Fungal Biology

Unidirectional mating-type switching confers self-fertility to Thielaviopsis cerberus, the only homothallic species in the genus

https://doi.org/10.1016/j.funbio.2020.12.007Get rights and content

Abstract

Sexual reproduction is ubiquitous in nature, and nowhere is this more so than in the fungi. Heterothallic behaviour is observed when there is a strict requirement of contact between two individuals of opposite mating type for sexual reproduction to occur. In contrast, a homothallic species can complete the entire sexual cycle in isolation, although several genetic mechanisms underpin this self-fertility. These can be inferred by characterising the structure and gene-content of the mating-type locus, which contains genes that are involved in the regulation of sexual reproduction. In this study, the genetic basis of homothallism in Thielaviopsis cerberus was investigated, the only known self-fertile species within this genus. Using genome sequencing and conventional molecular techniques, two versions of the mating-type locus were identified in this species. This is typical of species that have a unidirectional mating-type switching reproductive strategy. The first version was a self-fertile locus that contained four known mating-type genes, while the second was a self-sterile version with a single mating-type gene. The conversion from a self-fertile to a self-sterile locus is likely mediated by a homologous recombination event at two direct repeats present in the self-fertile locus, resulting in the deletion of three mating-type genes and one of the repeats. Both locus versions were present in isolates that were self-fertile, while self-sterility was caused by the presence of only a switched locus. This study provides a clear example of the architectural fluidity in the mating-type loci that is common among even closely related fungal species.

Introduction

Fungi represent fascinating systems of sexual reproduction (Heitman et al., 2013; Ni et al., 2011) because they employ a magnitude of reproductive strategies (Coppin et al., 1997; Glass et al., 1990; Haber, 2012; Turgeon and Yoder, 2000). Information gained from studying sexual processes in model fungi has facilitated significant progress in unravelling the mating systems of various non-model species. However, many non-model fungi have novel mating strategies, thus highlighting that much remains unknown about the diversity and associated mechanisms underlying sexual reproduction in fungi (Harrington et al., 1998; Kanematsu et al., 2007; Wilson et al., 2015b; Xu et al., 2016).

Fungal mating strategies are regarded as either heterothallic or homothallic. In heterothallism, initiation of the sexual cycle is defined by the need for contact between two isolates of opposite mating type (Blakeslee, 1904). Mating-type identity is conferred by the genes present at the mating-type (MAT1) locus that controls many aspects of the sexual process (Yoder et al., 1986; Yun et al., 1999). In heterothallic species, each mating partner has either a MAT1-1 or MAT1-2 “allelic” or idiomorphic form of the locus (Metzenberg and Glass, 1990). Sexual reproduction takes place when the products of both these idiomorphs are present in a single cell, which is achieved in heterothallic species by fusion of the two interacting partners. At the genetic level, heterothallism is thus synonymous with obligate outcrossing (Glass and Nelson, 1994).

Homothallism is an umbrella term used to describe sexual reproduction in the absence of a mating partner (Blakeslee, 1904; Whitehouse, 1949; Wilson et al., 2015b). In other words, a homothallic individual is self-fertile and this condition may be achieved through various genetic mechanisms or homothallic behaviours (Wilson et al., 2015b). Most forms of homothallism are conferred by both MAT1-1 and MAT1-2 genes being present in a single cell (Aanen and Hoekstra, 2007). For example, in primary homothallic species, both these gene types are present at the MAT1 locus (Blakeslee, 1904; Nasmyth, 1982). In the case of pseudohomothallic fungi, a single spore can contain two haploid nuclei of opposite mating-type and their subsequent germination produces heterokaryons capable of self-fertility (Lin and Heitman, 2007; Raju, 1992). In species capable of unidirectional mating-type switching, homothallism is mechanistically more complex (Harrington and McNew, 1997; Perkins, 1987; Wilken et al., 2014; Xu et al., 2016; Yun et al., 2017). The MAT1 locus of these fungi is usually structured such that the MAT1-2 genes are flanked by MAT1-1 genes (Wilken et al., 2014; Xu et al., 2016; Yun et al., 2017). In certain cells, homologous recombination between direct repeats then eliminates the MAT1-2 genes, thereby effectively restructuring their MAT1 locus (Wilken et al., 2014; Xu et al., 2016; Yun et al., 2017). This confers MAT1-1 identity to such cells, causing homokaryotic isolates harbouring them to be self-sterile (Yun et al., 2017). At the gene and genomic levels, these different mechanisms for achieving homothallism may be distinguished by analysis of the architecture of the MAT1 locus, because they are each associated with a distinct architecture (Coppin et al., 1997; Nelson, 1996; Yun et al., 2000).

Here we studied the MAT1 locus of Thielaviopsis cerberus (Ascomycota, Ceratocystidaceae), the only homothallic species within a genus of heterothallic fungi (De Beer et al., 2014; Mbenoun et al., 2014). Thielaviopsis species are known as pathogens on mainly monocotyledonous plants including sweet pea, pineapple and cacao tree (De Beer et al., 2014). Detailed analyses showed that all these fungi, except for T. cerberus, were obligately outcrossing in culture and their MAT1 locus structure were typical of heterothallic species, with two idiomorphs (MAT1-1 and MAT1-2) present in the population (Wilken et al., 2018). This previous study also showed the unusual position of a MAT1-1 specific gene (i.e., MAT1-1-2) in the MAT1-2 idiomorph (Wilken et al., 2018). In the case of T. cerberus, isolates typically showed self-fertility in culture by producing ascomata and ascospores in isolation (Mbenoun et al., 2014). Although nothing was known about the MAT1 locus of this species, homothallism is widespread in the Ceratocystidaceae (Liu et al., 2018; Simpson et al., 2018; Wilken et al., 2018; Wilson et al., 2015a). For example, all species of Ceratocystis and Endoconidiophora, as well as Davidsoniella virescens achieve homothallism via unidirectional mating-type switching (Baker et al., 2003; Harrington and McNew, 1997; Wilken et al., 2014; Witthuhn et al., 2000).

The aim of the current study was to elucidate the genetic basis of homothallism in T. cerberus. To do this, a draft genome sequence was generated for this species to identify and annotate its MAT1 locus. Single ascospore progeny were used to experimentally validate the structure and gene content of the locus, while RNA sequence data were generated and used to confirm the predicted gene models. This provides the basis for a model that explains the changes in locus configuration that accompany the transition between heterothallism and homothallism, as well as contributes to a larger effort to study sexual reproduction in the Ceratocystidaceae.

Section snippets

Thielaviopsis cerberus isolates and genome sequencing

For this study, two isolates (CMW36641 and CMW36653) of T. cerberus were obtained from the culture collection of the Forestry and Agricultural Biotechnology Institute (FABI) at the University of Pretoria. These were specifically selected for this study as they represent self-sterile and self-fertile individuals respectively, both of which were previously isolated from oil palm (Elaeis guineensis) in Cameroon (Mbenoun et al., 2014). The fungi were grown and maintained at 25 °C on medium (MEA-TS)

Genome sequence of Thielaviopsis cerberus isolate CMW36653

A total of 3 024 097 single-reads with an average length of 274.362 bases were produced. These were assembled into a draft genome sequence of 29,1 Mbp contained in 9307 contigs. Of these, 4089 contigs were larger than 1000 bp, with the largest being 58 730 bp in length. The genome had a GC-content of 49.7 %, N50 and L50 of 10 338 bp and 821 contigs, respectively, and 255.89 ambiguous nucleotides per 100 000 bp. AUGUSTUS predicted that the genome contained 8308 protein coding genes. The results

Discussion

The findings presented here showed that T. cerberus and its MAT1 locus bear all of the hallmarks associated with species that achieve homothallism via unidirectional mating-type switching (Harrington and McNew, 1997; Wilken et al., 2014; Xu et al., 2016; Yun et al., 2017). Similar to what was observed previously (Mbenoun et al., 2014), some isolates of T. cerberus were self-fertile and capable of producing ascomata and ascospores in the absence of a mating partner. Also, about half of the

Declaration of competing interest

The authors declare no conflicts of interest.

Acknowledgements

This project was funded by the University of Pretoria, as well as the Department of Science and Innovation (DSI)/National Research Foundation (NRF) Centre of Excellence in Plant Health Biotechnology and the South African Research Chairs Initiative (SARChI) in Fungal Genomics. The Grant holders acknowledge that opinions, findings and conclusions or recommendations expressed in any publication generated by the NRF supported research are that of the author(s), and that the NRF accepts no liability

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