Truncation of MAT1-2-7 deregulates developmental pathways associated with sexual reproduction in Huntiella omanensis

The MAT1-1-1 and MAT1-2-1 genes are thought to be the master regulators of sexual development in most ascomycete fungi, and they are often essential for this process. In contrast, it has been suggested that the secondary mating-type genes act to calibrate the sexual cycle and can be dispensable. Recent functional characterization of genes such as Aspergillus fumigatus MAT1-2-4, Huntiella omanensis MAT1-2-7, and Botrytis cinerea MAT1-1-5 has, however, shown that these secondary genes may play more central roles in the sexual pathway and are essential for the production of mature fruiting structures. We used a comparative transcriptome sequencing (RNAseq) experiment to show that the truncation of MAT1-2-7 in the wood inhabiting H. omanensis residing in the Ceratocystidaceae is associated with the differential expression of approximately 25% of all the genes present in the genome, including the transcriptional regulators ste12, wc-2, sub1, VeA, HMG8, and pro1. This suggests that MAT1-2-7 may act as a transcription factor and that DMAT1-2-7 mutant sterility is the result of layered deregulation of a variety of signaling and developmental pathways. This study is one of only a few that details the functional characterization of a secondary MAT gene in a nonmodel species. Given that this gene is present in other Ceratocystidaceae species and that there are diverse secondary MAT genes present throughout the Pezizomycotina, further investigation into this gene and others like it will provide a clearer understanding of sexual development in these eukaryotes. IMPORTANCE Secondary mating-type genes are being described almost as quickly as new fungal genomes are being sequenced. Understanding the functions of these genes has lagged behind their description, in part due to limited taxonomic distribution, lack of conserved functional domains, and difficulties with regard to genetic manipulation protocols. This study aimed to address this by investigating a novel mating-type gene, MAT1- 2-7, for which two independent mutant strains were generated in a previous study. We characterized the molecular response to the truncation of this gene in a nonmodel, wood-infecting fungus and showed that it resulted in widespread differential expression throughout the transcriptome of this fungus. This suggests that secondary MAT genes may play a more important role than previously thought. This study also emphasizes the need for further research into the life cycles of nonmodel fungi, which often exhibit unique features that are very different from the systems understood from model species.The University of Pretoria, the Department of Science and Technology (DST)/National Research Foundation (NRF) Centre of Excellence in Tree Health Biotechnology (CTHB) as well as B.D. Wingfield’s DST/NRF SARChI chair in Fungal Genomics and A. M. Wilson’s DST/NRF Scarce Skills Postdoctoral Fellowship.https://journals.asm.org/journal/spectrumam2023BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant Patholog

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.https://journals.asm.org/journal/mmbrhj2022BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant Patholog

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.The South African Department of Science and Innovation’s South African Research Chair Initiative (SARChI); Botanical Resources Australia—Agricultural Services, Pty. Ltd; post-doctoral grant from the University of Pretoria, South Africa; Melbourne International Fee Remission and Melbourne International Research Scholarships from the University of Melbourne, Australia.http://www.g3journal.orgam2022BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant Patholog

Blast from the past : a study of decades-old fungal cultures resolves a long-standing tree disease mystery

DATA AVAILABILITY : The sequences that were generated for this study have been deposited at the NCBI. The accession numbers will be made available upon publication.A root disease in plantations of Pinus radiata and Pinus pinaster, where trees died in distinct patches, was present in the Western Cape province of South Africa during the 1970s and 1980s. Phytophthora cinnamomi was initially believed to be the cause, but the disease was later ascribed to the insect-associated fungus Leptographium serpens, a fungal species residing in the Ophiostomatales. Doubt regarding the cause of the disease was raised in a later study due to the fact that most Leptographium spp., particularly those that colonise ray parenchyma tissues, which is the case for L. serpens, are not typically primary disease agents. In this study, cultures of an unidentified sterile fungus collected from the dying trees were revived and identified using DNA sequencing methods, which were not available when the disease was first studied. These cultures were identified as the pyrophillic pathogen Rhizina undulata, well-known to cause patch death of conifers in South Africa and elsewhere in the world. While the patches of dying trees no longer exist and the disease cannot be newly studied, it is most likely that the tree death originally thought to be caused by L. serpens was due primarily to R. undulata. The study provides a vivid example of the value of preserving cultures of fungi for later study and the power of modern techniques to identify fungal pathogens.The members of the Tree Protection Co-operative Programme (TPCP) and the National Research Foundation (NRF) in South Africa. Open access funding provided by University of Pretoria.https://link.springer.com/journal/42161hj2023BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant PathologyPlant Production and Soil Scienc

Doing it alone : unisexual reproduction in filamentous ascomycete fungi

Unisexuality in fungi is the result of sexual reproduction in a single isolate that harbors genes associated with only a single mating type. To date, unisexual reproduction has been described in only three genera of filamentous fungi. Consequently, our understanding of this unusual pathway is limited. In this critical review, we compare genetic, genomic and transcriptomic data from a variety of unisexual species to similar data from their primary homothallic and heterothallic relatives. These analyses show that unisexual reproduction is likely derived from heterothallism via the mutation of genes involved in the initiation of sexual reproduction. We show that significant changes in mating-type genes, pheromone precursor genes and pheromone receptor genes are common in unisexual species, but that similar changes are not evident in their primary homothallic or heterothallic relatives. These findings are particularly notable because the unisexual species are accommodated in unrelated genera, illustrating that a similar transition to unisexuality has likely occurred independently in their lineages.The University of Pretoria, the Department of Science and Innovation (DSI)/National Research Foundation (NRF) Centre of Excellence in Tree Health Biotechnology and the Genomics Research Institute (University of Pretoria Institutional Research Theme).http://www.elsevier.com/locate/fbr2022-01-13hj2021BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant Patholog

IMA Genome - F16 : Draft genome assemblies of Fusarium marasasianum, Huntiella abstrusa, two Immersiporthe knoxdaviesiana isolates, Macrophomina pseudophaseolina, Macrophomina phaseolina, Naganishia randhawae, and Pseudocercospora cruenta

Draft genome assemblies of Fusarium marasasianum, Huntiella abstrusa, two Immersiporthe knoxdaviesiana isolates, Macrophomina pseudophaseolina, Macrophomina phaseolina, Naganishia randhawae, and Pseudocercospora cruenta.Department of Science and Technology (DSI) , South Africa National Research Foundation (NRF) , South Africa Centre of Excellence in Tree Health Biotechnology, South Africa.https://imafungus.biomedcentral.comBiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant Patholog

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 University of Pretoria, the Department of Science and Technology (DST)/National Research Foundation (NRF) Centre of Excellence in Tree Health Biotechnology (CTHB). The project was additionally supported by Prof BD Wingfield’s DST/NRF SARChI chair in Fungal Genomics (Grant number: 98353) as well as Dr AM Wilson’s NRF PhD bursary (108548).https://www.jove.comam2020BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant Patholog

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

ADDITIONAL FILE 1 : FIG S1. RNA mapping to confirm the single nucleotide deletion in the H. moniliformis MAT1-2-7. FIG. S2. The structure of the Huntiella a-factor pheromone proteins and the sequence of the putative mature repeats. FIG. S3. An alignment of the a1 a-factor pheromone factor genes from all eight Huntiella species considered in this study. FIG. S4. RNA mapping to confirm in frame stop codons in the a1 and a6 a-factor pheromone factor genes from the unisexual H. moniliformis. FIG. S5. An alignment of the a3 a-factor pheromone from all eight Huntiella species considered in this study. FIG. S6. RNA mapping to determine expression of the multiple a-factor pheromone genes from H. abstrusa. FIG. S7. RNA mapping to determine expression of the multiple a-factor pheromone genes from H. omanensis. FIG. S8. RNA mapping to determine expression of the multiple a-factor pheromone genes from H. moniliformis. FIG. S9. The structure of the Huntiella α-factor pheromones from the various Huntiella species along with the sequences of the putative mature repeats.ADDITIONAL FILE 2 : SUPPLEMENTARY FILE 1. All of the scripts and parameters used for the genome assemblies produced in this study.ADDITIONAL FILE 3 : SUPPLEMENTARY FILE 2. The gene alignments and all parameters for the phylogenetic analysis conducted in this study.ADDITIONAL FILE 4 : TABLE S1. Genome sequencing and assembly statistics. TABLE S2. Comparisons of Huntiella genome statistics. TABLE S3. Gene present at the second a-factor pheromone locus.DATA AVAILABILITY : All data used and generated in this study are available either in the Genome or Sequence Read Archive (SRA) databases of the NCBI or in the Supplementary Data associated with this manuscript. The accession numbers of the genomes are as follows: H. abstrusa (JAJNMT000000000.1), H. omanensis (JSUI00000000.1), H. bhutanensis (MJMS00000000.1), H. decipiens (NETU00000000.1), H. savannae (LCZG00000000.1), H. moniliformis (JMSH00000000.1), H. fecunda (JAPHQJ000000000.1) and H. tyalla (JAPHQI000000000.1). The BioProject numbers of the RNAseq raw reads are as follows: H. abstrusa (PRJNA894346), H. omanensis (PRJNA385659) and H. moniliformis (PRJNA385659).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.DSI/NRF Scarce Skills Postdoctoral Fellowship; DSI/NRF SARChI chair in Fungal Genomics and University of Pretoria.https://bmcgenomics.biomedcentral.comBiochemistryForestry and Agricultural Biotechnology Institute (FABI)Genetic

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.The University of Pretoria, the Department of Science and Technology (DST)/National Research Foundation (NRF) Centre of Excellence in Tree Health Biotechnology (CTHB). The project was additionally supported by Prof BD Wingfield’s DST/NRF SARChI chair in Fungal Genomics (Grant number: 98353).http://www.elsevier.com/locate/yfgbi2021-04-01hj2020BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant Patholog