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.     

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).     

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.     

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.     

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.     

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.     

Pheromones are essential for male fertility and sufficient to direct chemotropic polarized growth of trichogynes during mating in Neurospora crassa

Neurospora crassa is a self-sterile filamentous fungus with two mating types, mat A and mat a. Its mating involves chemotropic polarized growth of female-specific hyphae (trichogynes) toward male cells of the opposite mating type in a process involving pheromones and receptors. mat A cells express the ccg-4 pheromone and the pre-1 receptor, while mat a strains produce mRNA for the pheromone mfa-1 and the pre-2 receptor; MFA-1 and CCG-4 are the predicted ligands for PRE-1 and PRE-2, respectively. In this study, we generated Deltaccg-4 and Deltamfa-1 mutants and engineered a mat a strain to coexpress ccg-4 and its receptor, pre-2. As males, Deltaccg-4 mat A and Deltamfa-1 mat a mutants were unable to attract mat a and mat A trichogynes, respectively, and consequently failed to initiate fruiting body (perithecial) development or produce meiotic spores (ascospores). In contrast, Deltaccg-4 mat a and Deltamfa-1 mat A mutants exhibited normal chemotropic attraction and male fertility. Deltaccg-4 Deltamfa-1 double mutants displayed defective chemotropism and male sterility in both mating types. Heterologous expression of ccg-4 enabled mat a males to attract mat a trichogynes, although subsequent perithecial differentiation did not occur. Expression of ccg-4 and pre-2 in the same strain triggered self-stimulation, resulting in formation of barren perithecia with no ascospores. Our results indicate that CCG-4 and MFA-1 are required for mating-type-specific male fertility and that pheromones (and receptors) are initial determinants for sexual identity during mate recognition. Furthermore, a self-attraction signal can be transmitted within a strain that expresses a pheromone and its cognate receptor.     

桦纤孔菌有性生殖相关特征及交配型位点分析

对桦纤孔菌菌株MDJCBS88的显微形态、菌丝及担孢子核相进行了观察。采用棉籽壳培养基对担孢子萌发形成的菌株进行栽培试验,筛选出不形成子实体或子实体发育不完整的菌株,将这些菌株在平板上进行了亲和试验,分析桦纤孔菌的有性生殖方式;并基于基因组序列进行交配型基因克隆验证,分析桦纤孔菌的交配型位点结构。显微观察发现,桦纤孔菌菌丝没有锁状联合结构,菌丝细胞无核到多核;子实层担孢子可含0-4个不等的细胞核,不同时期弹射的担孢子含有的细胞核数量不同。桦纤孔菌担孢子萌发率极低,能萌发的担孢子多为早期弹射的担孢子;培养基也影响担孢子的萌发率,与PDA培养基和CYM培养基相比,桦木屑培养基最适合桦纤孔菌担孢子萌发,萌发率为4.55%。从担孢子萌发的96个菌株中获得了2个不结实菌株和9个结实不产孢菌株,占11.5%,这些菌株间亲和试验出现不同的表现特征,包括形成产孢子实体,产生菌丝纽结,相互融合和相互拮抗等现象,认为桦纤孔菌的有性生殖以次级同宗结合为主,并受交配型基因控制。交配型位点克隆测序后分析发现,桦纤孔菌交配型A位点共14 034 bp,含有一个MIP基因和两组HD1和HD2基因;交配型B位点包含3个疑似信息素受体基因和1个信息素前体编码基因。