Evidence for sexuality in the opportunistic fungal pathogen Aspergillus fumigatus

Aspergillus fumigatus is a medically important opportunistic pathogen and a major cause of respiratory allergy. The species has long been considered an asexual organism. However, genome analysis has revealed the presence of genes associated with sexual reproduction, including a MAT-2 high-mobility group mating-type gene and genes for pheromone production and detection (Galagan et al., personal communication; Nierman et al., personal communication). We now demonstrate that A. fumigatus has other key characteristics of a sexual species. We reveal the existence of isolates containing a complementary MAT-1 alpha box mating-type gene and show that the MAT locus has an idiomorph structure characteristic of heterothallic (obligate sexual outbreeding) fungi. Analysis of 290 worldwide clinical and environmental isolates with a multiplex-PCR assay revealed the presence of MAT1-1 and MAT1-2 genotypes in similar proportions (43% and 57%, respectively). Further population genetic analyses provided evidence of recombination across a global sampling and within North American and European subpopulations. We also show that mating-type, pheromone-precursor, and pheromone-receptor genes are expressed during mycelial growth. These results indicate that A. fumigatus has a recent evolutionary history of sexual recombination and might have the potential for sexual reproduction. The possible presence of a sexual cycle is highly significant for the population biology and disease management of the species.     

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.     

The mating-type locus B alpha 1 of Schizophyllum commune contains a pheromone receptor gene and putative pheromone genes.

Analysis of the multispecific B alpha mating-type locus of Schizophyllum commune provided evidence that pheromones and pheromone receptors govern recognition of self versus non-self and sexual development in this homobasidiomycetous fungus. Four subclones of an 8.2 kb genomic fragment carrying B alpha 1 specificity induced B-regulated sexual morphogenesis when introduced into a strain with one of the eight compatible B alpha specificities that are known to exist in nature. One of these clones, which activated all other B alpha specificities, contains a gene termed bar1. The predicted protein product of bar1, as well as that of bar2, a homologous gene isolated from a B alpha 2 strain, has significant homology to known fungal pheromone receptor proteins in the rhodopsin-like superfamily of G protein-linked receptors. The other three active B alpha 1 clones were subcloned further to identify the minimal active element in each clone. Every active subclone contains a putative pheromone gene ending in a signal for possible isoprenylation. A message of approximately 600 bp was observed for one of these genes, bap1(1). This paper presents the first evidence for a system of multiple pheromones and pheromone receptors as a basis for multispecific mating types in a fungus.     

Cloning and analysis of the mating-type idiomorphs from the barley pathogen <Emphasis Type="Italic"> Septoria passerinii</Emphasis>

The genus Septoria contains more than 1000 species of plant pathogenic fungi, most of which have no known sexual stage. Species of Septoria without a known sexual stage could be recent derivatives of sexual species that have lost the ability to mate. To test this hypothesis, the mating-type region of S. passerinii, a species with no known sexual stage, was cloned, sequenced, and compared to that of its close relative S. tritici (sexual stage: Mycosphaerella graminicola). Both of the S. passerinii mating-type idiomorphs were approximately 3 kb in size and contained a single reading frame interrupted by one (MAT-2) or two (MAT-1) putative introns. The putative products of MAT-1 and MAT-2 are characterized by alpha-box and high-mobility-group sequences, respectively, similar to those in the mating-type genes of M. graminicolaand other fungi. The mating-type genes of S. passerinii and M. graminicolaare evolving rapidly, approximately ten times faster than the internal transcribed spacer region of the ribosomal DNA, and are not closely related to those from Cochliobolusor other loculoascomycetes in the order Pleosporales. Therefore, the class Loculoascomycetes may be polyphyletic. Furthermore, differences between the phylogenetic trees may indicate separate evolutionary histories for the MAT-1 and MAT-2 idiomorphs. A three-primer multiplex-PCR technique was developed that allowed rapid identification of the mating types of isolates of S. passerinii. Both mating types were present in approximately equal frequencies and often on the same leaf in fields in Minnesota and North Dakota. Analyses with isozyme and random amplified polymorphic DNA markers revealed that each isolate had a unique genotype. The common occurrence of both mating types on the same leaf and the high levels of genotypic diversity indicate that S. passerinii is almost certainly not an asexual derivative of a sexual fungus. Instead, sexual reproduction probably plays an integral role in the life cycle of S. passerinii and may be much more important than previously believed in this (and possibly other) "asexual" species of Septoria.     

Expression of the BAR1 gene in Saccharomyces cerevisiae: induction by the alpha mating pheromone of an activity associated with a secreted protein.

We have demonstrated and partially characterized the genetic control and pheromonal regulation of a soluble activity, produced only by mating-type a cells, that inhibits the action of the alpha mating pheromone, alpha-factor, on mating-type a cells. This activity was found to be associated with a heat-stable protein and to be secreted by MATa BAR1, mat alpha 2 BAR1, and mat alpha 1 mat alpha 2 BAR1 strains, but not by MAT alpha BAR1, MATa/MAT alpha BAR1, mat alpha 1 BAR1, or MATa barl strains, demonstrating that it is under the control of both the MAT alpha 2 and the BAR1 genes. Secretion of this activity was also found to be stimulated to as much as five times the basal level by exposure of the cells to alpha-factor. This stimulation was maximal after 6 h at a pheromone concentration of approximately 2 U/ml. An assay for this activity was developed by using a refined, quantitative assay for alpha-factor. The pheromone activity of samples added to wells in an agar plate was related to the size of the halo of growth inhibition produced in a lawn of mutant cells that are abnormally sensitive. The alpha-factor-inhibiting activity was related to a reduction of the halo size when active samples were added to the lawn. Although the assay for alpha-factor was found to be relatively insensitive to pH over a range of several units, the alpha-factor-inhibiting activity displayed a sharp pH optimum at approximately 6.5. The properties of this activity have important implications concerning the role of the BAR1 gene product in recovery of mating-type a cells from cell division arrest by alpha-factor.     

Cloning and analysis of the MAT1-2-1 gene from the traditional Chinese medicinal fungus Ophiocordyceps sinensis

The entomopathogenic fungus Ophiocordyceps sinensis has been important in traditional Chinese medicine but has yet to be commercially cultivated. One bottleneck is the very low frequency of stromata formation from artificially infected moth larvae. The mating system of fungi is the determining factor for sexual reproduction, but mating-type genes of O. sinensis have not been previously investigated. In this study, the putative mating-type gene MAT1-2-1 within the MAT1-2 idiomorph was amplified by polymerase chain reaction (PCR) and was determined to consist of 859 nucleotides that encode 249 amino acids; genes within the MAT1-1 idiomorph, however, were not determined. The MAT1-2-1 gene contained the conserved high-mobility group (HMG) box, and MAT1-2-1 flanking sequences were subsequently obtained. Although no putative open reading frames of the MAT1-1 idiomorph were detected within the ca. 8-kb flanking sequences of MAT1-2-1, a putative DNA lyase gene (which is present next to both idiomorphs in some heterothallic fungi) was found ca. 3.0 kb downstream of MAT1-2-1. The intervening distance between MAT1-2-1 and the DNA lyase gene in O. sinensis is larger than that in Cordyceps militaris and Cordyceps takaomontana. In addition, O. sinensis showed low sequence similarities with C. militaris and C. takaomontana in both MAT1-2-1 and the DNA lyase gene. In the phylogenetic tree, different MAT1-2-1 haplotypes of O. sinensis clustered together with high bootstrap support. As a single-copy gene, MAT1-2-1 was detected in all examined O. sinensis isolates including tissue cultures and single-ascospore cultures. This report describes, for the first time, a mating-type gene of O. sinensis.     

Characterization and population analysis of the mating-type genes in Aspergillus flavus and Aspergillus parasiticus

We characterize the mating-type genes in Aspergillus flavus,Aspergillus parasiticus and Petromyces alliaceus. A single MAT1-1 or MAT1-2 gene was detected in the genomes of A. flavus and A. parasiticus, which is consistent with a potential heterothallic organization of MAT genes in these species. In contrast, the only known, functionally homothallic species in Aspergillus section Flavi, P. alliaceus, has tightly linked (<2kb) MAT1-1 and MAT1-2 genes, typical of other self-fertile homothallic euascomycetes. This is the first example of linked MAT genes within a homothallic species of Aspergillus. We tested the null hypothesis of no significant difference in the frequency of MAT1-1 and MAT1-2 in A. flavus and A. parasiticus sampled from a single peanut field in Georgia. For each species, mating-type frequencies were determined for the total population samples and for samples that were clone-corrected based on vegetative compatibility groups (VCGs) and aflatoxin gene cluster haplotypes. There was no significant difference in the frequency of the two mating types for A. flavus and A. parasiticus in either VCG or haplotype clone-corrected samples. The existence of both mating-type genes in equal proportions in A. flavus and A. parasiticus populations, coupled with their expression at the mRNA level and the high amino acid sequence identity of MAT1-1 (77%) and MAT1-2 (83%) with corresponding homologs in P. alliaceus, indicates the potential functionality of these genes and the possible existence of a sexual state in these agriculturally important species.     

Characterisation of the mating-type locus in the genus Xanthoria (lichen-forming ascomycetes, Lecanoromycetes)

Conserved regions of mating-type genes were amplified in four representatives of the genus Xanthoria (X. parietina, X. polycarpa, X. flammea, and X. elegans) using PCR-based methods. The complete MAT locus, containing one ORF (MAT1-2-1) coding for a truncated HMG-box protein, and two partial flanking genes, were cloned by screening a genomic lambda phage library of the homothallic X. parietina. The flanking genes, a homologue of SLA2 of Saccharomyces cerevisiae and a DNA lyase gene, served to amplify the two idiomorphs of the X. polycarpa MAT locus. Each idiomorph contains a single gene: MAT1-2-1 codes for a HMG-box protein, MAT1-1-1 encodes an alpha domain protein. The occurrence of mating-type genes in eight single spore isolates derived from one ascus was studied with a PCR assay. In the homothallic X. parietina a HMG fragment, but no alpha box fragment was found in all isolates, whereas in X. elegans, another homothallic species, all tested isolates contained a fragment of both idiomorphs. Conversely, isolates of the heterothallic X. polycarpa contained either a HMG or an alpha box fragment, but never both.     

Breeding systems in the lichen-forming fungal genus Cladonia

The breeding systems of three species of the lichen-forming fungal genus Cladonia were investigated. Cladonia floerkeana, Cladonia galindezii, and Cladonia portentosa were selected due to their contrasting ecologies and reproductive strategies, and because they belong to the Lecanorales, the major lichen-forming order. Sibling single-spore progeny were collected from apothecia and used to establish axenic cultures. Two experimental approaches were used to determine breeding systems. First, RAPD-PCR and AFLP fingerprinting revealed that spores from the same apothecium were not genetically uniform, indicating heterothallism in each of these species. Second, segregation of a MAT-2 mating-type gene was assessed using degenerate PCR primers designed to amplify the high-mobility group region. A MAT-2 gene occurred in 40-60% of progeny, consistent with a heterothallic breeding system. The PCR product from C. galindezii was cloned and sequenced, and confirmed to have the characteristic motifs of a MAT-2 HMG gene. This is thought to be the first report of the use of segregation of a mating-type gene among ascospore progeny to determine the breeding system of a fungal species. The ecological significance of the results is discussed.     

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.     

An STE12 homolog from the asexual, dimorphic fungus Penicillium marneffei complements the defect in sexual development of an Aspergillus nidulans steA mutant

Penicillium marneffei is an opportunistic fungal pathogen of humans and the only dimorphic species identified in its genus. At 25 degrees P. marneffei exhibits true filamentous growth, while at 37 degrees P. marneffei undergoes a dimorphic transition to produce uninucleate yeast cells that divide by fission. Members of the STE12 family of regulators are involved in controlling mating and yeast-hyphal transitions in a number of fungi. We have cloned a homolog of the S. cerevisiae STE12 gene from P. marneffei, stlA, which is highly conserved. The stlA gene, along with the A. nidulans steA and Cryptococcus neoformans STE12alpha genes, form a distinct subclass of STE12 homologs that have a C2H2 zinc-finger motif in addition to the homeobox domain that defines STE12 genes. To examine the function of stlA in P. marneffei, we isolated a number of mutants in the P. marneffei-type strain and, in combination with selectable markers, developed a highly efficient DNA-mediated transformation procedure and gene deletion strategy. Deletion of the stlA gene had no detectable effect on vegetative growth, asexual development, or dimorphic switching in P. marneffei. Despite the lack of a detectable function, the P. marneffei stlA gene complemented the sexual defect of an A. nidulans steA mutant. In addition, substitution rate estimates indicate that there is a significant bias against nonsynonymous substitutions. These data suggest that P. marneffei may have a previously unidentified cryptic sexual cycle.     

Isolation and characterization of the mating-type locus of the barley pathogen Pyrenophora teres and frequencies of mating-type idiomorphs within and among fungal populations collected from barley landraces.

Pyrenophora teres f. sp. teres mating-type genes (MAT-1: 1190 bp; MAT-2: 1055 bp) have been identified. Their predicted proteins, measuring 379 and 333 amino acids, respectively, are similar to those of other Pleosporales, such as Pleospora sp., Cochliobolus sp., Alternaria alternata, Leptosphaeria maculans, and Phaeosphaeria nodorum. The structure of the MAT locus is discussed in comparison with those of other fungi. A mating-type PCR assay has also been developed; with this assay we have analyzed 150 isolates that were collected from 6 Sardinian barley landrace populations. Of these, 68 were P. teres f. sp. teres (net form; NF) and 82 were P. teres f. sp. maculata (spot form; SF). Within each mating type, the NF and SF amplification products were of the same length and were highly similar in sequence. The 2 mating types were present in both the NF and the SF populations at the field level, indicating that they have all maintained the potential for sexual reproduction. Despite the 2 forms being sympatric in 5 fields, no intermediate isolates were detected with amplified fragment length polymorphism (AFLP) analysis. These results suggest that the 2 forms are genetically isolated under the field conditions. In all of the samples of P. teres, the ratio of the 2 mating types was consistently in accord with the 1:1 null hypothesis. This ratio is expected when segregation distortion and clonal selection among mating types are absent or asexual reproduction is rare. Overall, sexual reproduction appears to be the major process that equalizes the frequencies of the 2 mating types within populations.     

Mutations in cell division cycle genes CDC36 and CDC39 activate the Saccharomyces cerevisiae mating pheromone response pathway.

Conditional mutations in the genes CDC36 and CDC39 cause arrest in the G1 phase of the Saccharomyces cerevisiae cell cycle at the restrictive temperature. We present evidence that this arrest is a consequence of a mutational activation of the mating pheromone response. cdc36 and cdc39 mutants expressed pheromone-inducible genes in the absence of pheromone and conjugated in the absence of a mating pheromone receptor. On the other hand, cells lacking the G beta subunit or overproducing the G alpha subunit of the transducing G protein that couples the receptor to the pheromone response pathway prevented constitutive activation of the pathway in cdc36 and cdc39 mutants. These epistasis relationships imply that the CDC36 and CDC39 gene products act at the level of the transducing G protein. The CDC36 and CDC39 gene products have a role in cellular processes other than the mating pheromone response. A mating-type heterozygous diploid cell, homozygous for either the cdc36 or cdc39 mutation, does not exhibit the G1 arrest phenotype but arrests asynchronously with respect to the cell cycle. A similar asynchronous arrest was observed in cdc36 and cdc39 cells where the pheromone response pathway had been inactivated by mutations in the transducing G protein. Furthermore, cdc36 and cdc39 mutants, when grown on carbon catabolite-derepressing medium, did not arrest in G1 and did not induce pheromone-specific genes at the restrictive temperature.     

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.     

The Pheromone Cell Signaling Components of the Ustilago a Mating-Type Loci Determine Intercompatibility between Species

The MAT region of Ustilago hordei, a bipolar barley pathogen, harbors distinct mating functions (a and b loci). Here, we show that the b locus is essential for mating and pathogenicity, and can induce pathogenicity when introduced into a strain carrying a b locus of opposite specificity. Transformation experiments using components of the a1 locus and analysis of resulting dual mating phenotypes revealed that this locus harbors a pheromone receptor gene (Uhpra1) and a pheromone gene (Uhmfa1). These U. hordei a1 genes, when introduced by transformation, are necessary and sufficient to make U. maydis, a tetrapolar corn pathogen, intercompatible with U. hordei MAT-2, but not MAT-1, strains. U. hordei strains transformed with the U. maydis a1 locus also become intercompatible with U. maydis a2, but not a1, strains. The interspecies hybrids produced dikaryotic hyphae but were not fully virulent on either corn or barley. Partial, natural intercompatibility was shown to exist between the sugarcane smut U. scitaminea and both U. hordei and U. maydis. These results show that the signal transduction pathway for mating responses is conserved between different smut species. We conclude that, apart from intraspecies compatibility, the Ustilago a locus also dictates intercompatibility in this group of fungi.     

Isolation and characterization of the mating-type idiomorphs from the wheat septoria leaf blotch fungus Mycosphaerella graminicola

Both mating-type loci from the wheat septoria leaf blotch pathogen Mycosphaerella graminicola have been cloned and sequenced. The MAT1-2 gene was identified by screening a genomic library from the MAT1-2 isolate IPO94269 with a heterologous probe from Tapesia yallundae. The MAT1-2 idiomorph is 2772 bp and contains a single gene encoding a putative high-mobility-group protein of 394 amino acids. The opposite idiomorph was obtained from isolate IPO323, which has the complementary mating type, by long-range PCR using primers derived from sequences flanking the MAT1-2 idiomorph. The MAT1-1 locus is 2839 bp in size and contains a single open reading frame encoding a putative alpha1-domain protein of 297 amino acids. Within the nonidiomorphic sequences, homology was found with palI, encoding a membrane receptor from Aspergillus nidulans, and a gene encoding a putative component of the anaphase-promoting complex from Schizosaccharomyces pombe and a DNA-(apurinic or apyrimidinic) lyase from S. pombe. For each of the MAT genes specific primers were designed and tested on an F1 mapping population that was generated from a cross between IPO323 and IPO94269. An absolute correlation was found between the amplified allele-specific fragments and the mating type as determined by backcrosses of each F1 progeny isolate to the parental isolates. The primers were also used to screen a collection of field isolates in a multiplex PCR. An equal distribution of MAT1-1 and MAT1-2 alleles was found for most geographic origins examined.