Isolation of Pheromone Precursor Genes of Magnaporthe grisea

In heterothallic ascomycetes one mating partner serves as the source of female tissue and is fertilized with spermatia from a partner of the opposite mating type. The role of pheromone signaling in mating is thought to involve recognition of cells of the opposite mating type. We have isolated two putative pheromone precursor genes of Magnaporthe grisea. The genes are present in both mating types of the fungus but they are expressed in a mating type-specific manner. The MF1-1 gene, expressed in Mat1-1 strains, is predicted to encode a 26-amino-acid polypeptide that is processed to produce a lipopeptide pheromone. The MF2-1 gene, expressed in Mat1-2 strains, is predicted to encode a precursor polypeptide that is processed by a Kex2-like protease to yield a pheromone with striking similarity to the predicted pheromone sequence of a close relative, Cryphonectria parasitica. Expression of the M. grisea putative pheromone precursor genes was observed under defined nutritional conditions and in field isolates. This suggests that the requirement for complex media for mating and the poor fertility of field isolates may not be due to limitation of pheromone precursor gene expression. Detection of putative pheromone precursor gene mRNA in conidia suggests that pheromones may be important for the fertility of conidia acting as spermatia.     

Isolation of the Mating-Type Genes of the Phytopathogenic Fungus Magnaporthe Grisea Using Genomic Subtraction

Using genomic subtraction, we isolated the mating-type genes (Mat1-1 and Mat1-2) of the rice blast fungus, Magnaporthe grisea. Transformation of M. grisea strains of one mating type with a linearized cosmid clone carrying the opposite mating-type gene resulted in many ``dual maters,' strains that contain both mating-type genes and successfully mate with both Mat1-1 and Mat1-2 testers. Dual maters differed in the frequency of production of perithecia in pure culture. Ascospores isolated from these homothallic crosses were either Mat1-1 or Mat1-2, but there were no dual maters. Most conidia from dual maters also had one or the other of the mating-type genes, but not both. Thus, dual maters appear to lose one of the mating-type genes during vegetative growth. The incidence of self-mating in dual maters appears to depend on the co-occurrence of strains with each mating type in vegetative cultures. In rare transformants, the incoming sequences had replaced the resident mating-type gene. Nearly isogenic pairs produced from three M. grisea laboratory strains were mated to investigate their fertility. One transformant with switched mating type appears to have a mutation that impairs the development of asci when its mating partner has a similar genetic background. The M. grisea Mat1-1 and Mat1-2 genes are idiomorphs approximately 2.5 and 3.5 kb in length, respectively.     

Role of the Mf1-1 pheromone precursor gene of the filamentous ascomycete Cryphonectria parasitica

Site-directed recombination was used to obtain a Cryphonectria parasitica strain carrying deletions at the Mf1-1 gene locus. Macroscopic features such as growth rate and conidia production were unaffected by Mf1-1 deletions, but, when a strain containing a complete deletion of Mf1-1 was used as spermatia it was male sterile. The same strain was fully competent as a female parent. Deletion of three of the seven putative pheromone peptide repeats within the gene had no effect on mating. Male fertility of the complete deletion strain was restored when an ectopic copy of the Mf1-1 gene was returned by transformation. Expression of the mating type specific pheromone precursor gene Mf1-1 was stimulated by growth in nutritionally poor liquid media. It was found that age and source of inoculum of liquid cultures influences pheromone precusor gene expression, i.e., conidia did not express Mf1-1 and cultures derived from conidia were significantly delayed in expression of this gene, as were cultures derived from young mycelium. Cultures inoculated with older hyphae, however, expressed Mf1-1 within 1 day after inoculation.     

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.     

Expression and function of sex pheromones and receptors in the homothallic ascomycete Gibberella zeae

In heterothallic ascomycete fungi, idiomorphic alleles at the MAT locus control two sex pheromone-receptor pairs that function in the recognition and chemoattraction of strains with opposite mating types. In the ascomycete Gibberella zeae, the MAT locus is rearranged such that both alleles are adjacent on the same chromosome. Strains of G. zeae are self-fertile but can outcross facultatively. Our objective was to determine if pheromones retain a role in sexual reproduction in this homothallic fungus. Putative pheromone precursor genes (ppg1 and ppg2) and their corresponding pheromone receptor genes (pre2 and pre1) were identified in the genomic sequence of G. zeae by sequence similarity and microsynteny with other ascomycetes. ppg1, a homolog of the Saccharomyces alpha-factor pheromone precursor gene, was expressed in germinating conidia and mature ascospores. Expression of ppg2, a homolog of the a-factor pheromone precursor gene, was not detected in any cells. pre2 was expressed in all cells, but pre1 was expressed weakly and only in mature ascospores. ppg1 or pre2 deletion mutations reduced fertility in self-fertilization tests by approximately 50%. Deltappg1 reduced male fertility and Deltapre2 reduced female fertility in outcrossing tests. In contrast, Deltappg2 and Deltapre1 had no discernible effects on sexual function. Deltappg1/Deltappg2 and Deltapre1/Deltapre2 double mutants had the same phenotype as the Deltappg1 and Deltapre2 single mutants. Thus, one of the putative pheromone-receptor pairs (ppg1/pre2) enhances, but is not essential for, selfing and outcrossing in G. zeae whereas no functional role was found for the other pair (ppg2/pre1).     

Molecular analysis of the pheromone and pheromone receptor genes of Ustilago hordei

Cell-cell signaling is an integral part of the sexual and disease cycles of the smut fungi, which must mate to be pathogenic. This study reports the cloning and characterization of the pheromone genes Uhmfa1 and Uhmfa2 from MAT-1 and MAT-2 mating types of U. hordei, respectively, and the pheromone receptor gene Uhpra2 from MAT-2 cells. Similar to other fungal pheromone genes, Uhmfa1 and Uhmfa2 encode precursor peptides. Uhpra2 encodes a protein with sequence similarity to the 7-transmembrane class of G-protein coupled receptors. Deletion of Uhmfa1 and Uhpra1, and their subsequent replacement, confirmed the role of these genes in initiation of the sexual cycle. Uhmfa1 and Uhmfa2 were differentially expressed in various cell types and when opposite mating-type cells were grown together. The predicted mature pheromones of each mating type were synthesized, and each specifically induced conjugation tube formation in cells of the opposite mating type.     

A putative pheromone signaling pathway is dispensable for self-fertility in the homothallic ascomycete Gibberella zeae

Gibberella zeae, a homothallic ascomycetous fungus, does not seek a partner for mating. Here, we focused on the role(s) of putative pheromone and receptor genes during sexual development in G. zeae. Orthologs of two pheromone precursor genes (GzPPG1 and GzPPG2), and their cognate receptor genes (GzPRE2 and GzPRE1) were transcribed during sexual development. The expression of these genes was controlled by the mating-type (MAT) locus and a MAP kinase gene, but not in a MAT-specific manner. Targeted gene deletion and subsequent outcrosses generated G. zeae strains lacking these putative pheromone/receptor genes in various combinations (from single to quadruple deletions). All G. zeae deletion strains were similar to the self-fertile progenitor in both male- and female fertility and other traits. Sometimes, the deletions including ΔGzPPG1;ΔGzPRE2 caused increased numbers of immature perithecia. Taken together, it is clear that these putative pheromones/receptors play a non-essential role in the sexual development of G. zeae.     

Fertility status and distribution of mating type alleles of the rice blast fungus, Magnaporthe grisea in northern Iran

This study was carried out using 155 monoconidial isolates collected from different areas of two major rice growing provinces in northern Iran, including 94 isolates from Guilan and 59 isolates from Mazandaran. Among 94 isolates from Guilan, 92 and two isolates recovered from rice and crabgrass (Digitaria sp.), respectively. All 61 rested isolates from Mazandaran were recovered from rice. All isolates were evaluated for in vitro sexual fertility and mating type status by pairing with Mat 1-1 and Mat 1-2 fertile standard hermaphrodite isolates including Br48 and Th12 (Mat 1-1) and KA9 and TH16 (Mat 1-2). Of 155 isolates, 98 (63.2%) were fertile and 57 (36.8%) were infertile and produced no perithecium when mated with standard isolates. Among 98 fertile isolates, 96 isolates were identified as Mat 1-1 and two isolates as Mat 1-2. All Mat 1-1 isolates were obtained from rice and two Mat 1-2 isolates obtained from crab grass. No Mat 1-2 isolate was identified from rice in this study. Both mating types were found in Guilan but all isolates recovered from Mazandaran were identified as Mat 1-1. Male fertility predominated in fertile Mat 1-1 and Mat 1-2 isolates from all sampling sites in northern Iran, and no female fertility was detected. This is the first report of existence of Mat 1-2 allele in Magnaporthe grisea population in Iran.     

Functional characterization of an alpha-factor-like Sordaria macrospora peptide pheromone and analysis of its interaction with its cognate receptor in Saccharomyces cerevisiae

The homothallic filamentous ascomycete Sordaria macrospora possesses genes which are thought to encode two pheromone precursors and two seven-transmembrane pheromone receptors. The pheromone precursor genes are termed ppg1 and ppg2. The putative products derived from the gene sequence show structural similarity to the alpha-factor precursors and a-factor precursors of the yeast Saccharomyces cerevisiae. Likewise, sequence similarity has been found between the putative products of the pheromone receptor genes pre2 and pre1 and the S. cerevisiae Ste2p alpha-factor receptor and Ste3p a-factor receptor, respectively. To investigate whether the alpha-factor-like pheromone-receptor pair of S. macrospora is functional, a heterologous yeast assay was used. Our results show that the S. macrospora alpha-factor-like pheromone precursor PPG1 is processed into an active pheromone by yeast MATalpha cells. The S. macrospora PRE2 protein was demonstrated to be a peptide pheromone receptor. In yeast MATa cells lacking the endogenous Ste2p receptor, the S. macrospora PRE2 receptor facilitated all aspects of the pheromone response. Using a synthetic peptide, we can now predict the sequence of one active form of the S. macrospora peptide pheromone. We proved that S. macrospora wild-type strains secrete an active pheromone into the culture medium and that disruption of the ppg1 gene in S. macrospora prevents pheromone production. However, loss of the ppg1 gene does not affect vegetative growth or fertility. Finally, we established the yeast assay as an easy and useful system for analyzing pheromone production in developmental mutants of S. macrospora.     

The STE4 and STE18 genes of yeast encode potential beta and gamma subunits of the mating factor receptor-coupled G protein

The STE4 and STE18 genes are required for haploid yeast cell mating. Sequencing of the cloned genes revealed that the STE4 polypeptide shows extensive homology to the beta subunits of mammalian G proteins, while the STE18 polypeptide shows weak similarity to the gamma subunit of transducin. Null mutations in either gene can suppress the haploid-specific cell-cycle arrest caused by mutations in the SCG1 gene (previously shown to encode a protein with similarity to the alpha subunit of G proteins). We propose that the products of the STE4 and STE18 genes comprise the beta and gamma subunits of a G protein complex coupled to the mating pheromone receptors. The genetic data suggest pheromone-receptor binding leads to the dissociation of the alpha subunit from beta gamma (as shown for mammalian G proteins), and the free beta gamma element initiates the pheromone response.     

A putative pheromone receptor gene is expressed in two distinct olfactory organs in goats

Mammals possess two anatomically and functionally distinct olfactory systems. The olfactory epithelium (OE) detects volatile odorants, while the vomeronasal organ (VNO) detects pheromones that elicit innate reproductive and social behavior within a species. In rodent VNO, three multigene families that encode the putative pheromone receptors, V1Rs, V2Rs and V3Rs, have been expressed. We have identified the V1R homologue genes from goat genomic DNA (gV1R genes). Deduced amino acid sequences of gV1R genes show 40-50% and 20-25% identity to those of rat and mouse V1R and V3R genes, respectively, suggesting that the newly isolated goat receptor genes are members of the V1R gene family. One gene (gV1R1 gene) has an open reading frame that encodes a polypeptide of 309 amino acids. It is expressed not only in VNO but also in OE. In situ hybridization analysis revealed that gV1R1 -expressing cells were localized in neuropithelial layers of VNO and OE. These results suggest that the goat may detect pheromone molecules through two distinct olfactory organs.     

Multiple Genes Encoding Pheromones and a Pheromone Receptor Define the Bβ1 Mating-Type Specificity in Schizophyllum Commune

The genes defining multiple B mating types in the wood-rotting mushroom Schizophyllum commune are predicted to encode multiple pheromones and pheromone receptors. These genes are clustered in each of two recombinable and independently functioning loci, Bα and Bβ. A difference in specificity at either locus between a mated pair of individuals initiates an identical series of events in sexual morphogenesis. The Bα1 locus was recently found to contain genes predicted to encode three lipopeptide pheromones and a pheromone receptor with a seven-transmembrane domain. These gene products interact in hetero-specific pairs, the pheromone of one Bα specificity with the receptor of any one of the other eight Bα specificities, and are likely to activate a signaling cascade similar to that known for mating in Saccharomyces cerevisiae. We report here that the Bβ1 locus also contains at least three pheromone genes and one pheromone receptor gene, which function similarly to the genes in the Bα1 locus, but only within the series of Bβ specificities. A comparison of the DNA sequences of the Bα1 and Bβ1 loci suggest that each arose from a common ancestral sequence, allowing us to speculate about the evolution of this unique series of regulatory genes.     

Mating type‐dependent partner sensing as mediated by VEL1 in Trichoderma reesei

Sexual development in the filamentous model ascomycete Trichoderma reesei (syn. Hypocrea jecorina) was described only a few years ago. In this study, we show a novel role for VELVET in fungi, which links light response, development and secondary metabolism. Vel1 is required for mating in darkness, normal growth and conidiation. In light, vel1 was dispensable for male fertility but essential for female fertility in both mating types. VEL1 impacted regulation of the pheromone system (hpr1, hpr2, hpp1, ppg1) in a mating type‐dependent manner and depending on the mating partner of a given strain. These partner effects only occurred for hpp1 and hpr2, the pheromone precursor and receptor genes associated with the MAT1‐2 mating type and for the mating type gene mat1‐2‐1. Analysis of secondary metabolite patterns secreted by wild type and mutants under asexual and sexual conditions revealed that even in the wild type, the patterns change upon encounter of a mating partner, with again distinct differences for wild type and vel1 mutants. Hence, T. reesei applies a language of pheromones and secondary metabolites to communicate with mating partners and that this communication is at least in part mediated by VEL1.     

Identification and characterization of MFA1, the gene encoding Candida albicans a-factor pheromone

In the opaque state, MTLa and MTLalpha strains of Candida albicans are able to mate, and this mating is directed by a pheromone-mediated signaling process. We have used comparisons of genome sequences to identify a C. albicans gene encoding a candidate a-specific mating factor. This gene is conserved in Candida dubliniensis and is similar to a three-gene family in the related fungus Candida parapsilosis but has extremely limited similarity to the Saccharomyces cerevisiae MFA1 (ScMFA1) and ScMFA2 genes. All these genes encode C-terminal CAAX box motifs characteristic of prenylated proteins. The C. albicans gene, designated CaMFA1, is found on chromosome 2 between ORF19.2165 and ORF19.2219. MFA1 encodes an open reading frame of 42 amino acids that is predicted to be processed to a 14-amino-acid prenylated mature pheromone. Microarray analysis shows that MFA1 is poorly expressed in opaque MTLa cells but is induced when the cells are treated with alpha-factor. Disruption of this C. albicans gene blocks the mating of MTLa cells but not MTLalpha cells, while the reintegration of the gene suppresses this cell-type-specific mating defect.