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 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.     

Identification of the cAD1 sex pheromone precursor in Enterococcus faecalis

The Enterococcus faecalis virulence plasmid pAD1 encodes a mating response induced by exposure to an octapeptide sex pheromone, cAD1, secreted by plasmid-free enterococci. The determinant for the pheromone in E. faecalis FA2-2, designated cad, was found to encode a 309-amino-acid lipoprotein precursor with the last 8 residues of its 22-amino acid signal sequence representing the cAD1 moiety. The lipoprotein moiety contained two 77-amino-acid repeats (70% identity) separated by 45 residues. The nonisogenic E. faecalis strain V583 determinant encodes a homologous precursor protein, but it differs at two amino acid positions, both of which are located within the pheromone peptide moiety (positions 2 and 8). Construction of a variant of strain FA2-2 containing the differences present in V583 resulted in cells that did not produce detectable cAD1. The mutant appeared normal under laboratory growth conditions, and while significantly reduced in recipient potential, when carrying pAD1 it exhibited a normal mating response. A mutant of FA2-2 with a truncated lipoprotein moiety appeared normal with respect to recipient potential and, when carrying plasmid DNA, donor potential. A gene encoding a protein designated Eep, believed to be a zinc metalloprotease, had been previously identified as required for pheromone biosynthesis and was believed to be involved in the processing of a pheromone precursor. Our new observation that the pAD1-encoded inhibitor peptide, iAD1, whose precursor is itself a signal sequence, is also dependent on Eep is consistent with the likelihood that such processing occurs at the amino terminus of the cAD1 moiety.     

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.     

A potential positive feedback loop controlling CLN1 and CLN2 gene expression at the start of the yeast cell cycle

The CLN1, CLN2, and CLN3 genes of S. cerevisiae form a redundant family essential for the G1-to-S phase transition. CLN1 and CLN2 mRNAs were previously shown to be negatively regulated by mating pheromone and by cell cycle progression out of G1, whereas CLN3 mRNA is not. The CLN3-2 (DAF1-1) allele prevents both cell cycle arrest and the turnoff of CLN1 and CLN2 mRNAs in response to mating pheromone, but only in the presence of an active CDC28 gene. An internally deleted nonfunctional cln2 gene was used as a reporter gene to demonstrate that in the absence of mating pheromone, efficient expression of cln2 mRNA requires both an active CDC28 gene and at least one functional CLN gene. mRNA from a nonfunctional cln1 gene was regulated similarly. Thus, CLN function and CDC28 activity jointly stimulate CLN1 and CLN2 mRNA levels, potentially forming a positive feedback loop for CLN1 and CLN2 expression.     

Competition is the mechanism of biocontrol of brown rot in stone fruit by <Emphasis Type="Italic">Penicillium frequentans</Emphasis>

The mechanism of biocontrol of brown rot in stone fruit by Penicillium frequentans Westling (Pf909) was investigated using in vitro and in vivo growth assays and a benomyl-resistant strain of Monilinia fructicola (G Winter) Honey (Mf3C). For the in vitro assays, Pf909 and Mf3C conidia were suspended in Czapek-Dox broth, which was amended or not amended with a skin extract of mature peaches. The growth and germination of Pf909 and Mf3C conidia were determined by counting the number of colony-forming units on potato dextrose agar plates, which were amended or not amended with 0.5 g ml^?1 benomyl. In some of the assays, germinated Pf909 conidia were used before their exposure to Mf3C conidia. For the in vivo assays, healthy cherries were inoculated with Mf3C conidia before and after applying Pf909 conidia on the cherry surface and the incidence of brown rot was recorded for seven days. Since we found that Pf909 conidia compete with Mf3C conidia for space and nutrients in the different assays, we concluded that competition is the probable primary mechanism of biocontrol of Pf909.     

Characterization of TUP1, a mediator of glucose repression in Saccharomyces cerevisiae.

The TUP1 and CYC8 (= SSN6) genes of Saccharomyces cerevisiae play a major role in glucose repression. Mutations in either TUP1 or CYC8 eliminate or reduce glucose repression of many repressible genes and induce other phenotypes, including flocculence, failure to sporulate, and sterility of MAT alpha cells. The TUP1 gene was isolated in a screen for genes that regulate mating type (V.L. MacKay, Methods Enzymol. 101:325-343, 1983). We found that a 3.5-kb restriction fragment was sufficient for complete complementation of tup1-100. The gene was further localized by insertional mutagenesis and RNA mapping. Sequence analysis of 2.9 kb of DNA including TUP1 revealed only one long open reading frame which predicts a protein of molecular weight 78,221. The predicted protein is rich in serine, threonine, and glutamine. In the carboxyl region there are six repeats of a pattern of about 43 amino acids. This same pattern of conserved residues is seen in the beta subunit of transducin and the yeast CDC4 gene product. Insertion and deletion mutants are viable, with the same range of phenotypes as for point mutants. Deletions of the 3' end of the coding region produced the same mutant phenotypes as did total deletions, suggesting that the C terminus is critical for TUP1 function. Strains with deletions in both the CYC8 and TUP1 genes are viable, with phenotypes similar to those of strains with a single deletion. A deletion mutation of TUP1 was able to suppress the snf1 mutation block on expression of the SUC2 gene encoding invertase.     

Asexual cephalosporin C producer Acremonium chrysogenum carries a functional mating type locus

Acremonium chrysogenum, the fungal producer of the pharmaceutically relevant beta-lactam antibiotic cephalosporin C, is classified as asexual because no direct observation of mating or meiosis has yet been reported. To assess the potential of A. chrysogenum for sexual reproduction, we screened an expressed sequence tag library from A. chrysogenum for the expression of mating type (MAT) genes, which are the key regulators of sexual reproduction. We identified two putative mating type genes that are homologues of the alpha-box domain gene, MAT1-1-1 and MAT1-1-2, encoding an HPG domain protein defined by the presence of the three invariant amino acids histidine, proline, and glycine. In addition, cDNAs encoding a putative pheromone receptor and pheromone-processing enzymes, as well as components of a pheromone response pathway, were found. Moreover, the entire A. chrysogenum MAT1-1 (AcMAT1-1) gene and regions flanking the MAT region were obtained from a genomic cosmid library, and sequence analysis revealed that in addition to AcMAT1-1-1 and AcMAT1-1-2, the AcMAT1-1 locus comprises a third mating type gene, AcMAT1-1-3, encoding a high-mobility-group domain protein. The alpha-box domain sequence of AcMAT1-1-1 was used to determine the phylogenetic relationships of A. chrysogenum to other ascomycetes. To determine the functionality of the AcMAT1-1 locus, the entire MAT locus was transferred into a MAT deletion strain of the heterothallic ascomycete Podospora anserina (the PaDeltaMAT strain). After fertilization with a P. anserina MAT1-2 (MAT(+)) strain, the corresponding transformants developed fruiting bodies with mature ascospores. Thus, the results of our functional analysis of the AcMAT1-1 locus provide strong evidence to hypothesize a sexual cycle in A. chrysogenum.     

Effect of liquid culture media on morphology,growth, propagule production,and pathogenic activity of the Hyphomycete,Metarhizium flavoviride

Two isolates of Metarhizium spp. were studied for propagule production, because of their pathogenic activity towards locusts and grasshoppers (Mf189 = M. flavoviride (or M. anisopliae var. acridum) strain IMI 330189, and Mf324 = M. flavoviride strain ARSEF324). Both isolates were grown in seven different liquid media, which have been developed for mass production of various Hyphomycetes, considered as candidates for microbial control of noxious insects. Shake-flask experiments were carried out at 28 °C in the dark. Production was quantified for 72 h and the effects of the tested media were evaluated on propagule concentration, morphology and pathogenicity. Based on preliminary experiments, all tested media were supplemented with 0.4% Tween 80 to avoid the formation of pellets and to produce unicellular propagules. Submerged propagule yields were higher withMf189 than with Mf324 in all seven media. While high concentrations of propagules (1.4 to 2.4 × 10⁸ propagules ml^-1 for MF189 and1.4 to 8.3 × 10⁷ propagules ml^-1 for Mf324) were produced in four media (Adamek, Catroux, Jackson, and Jenkins–Prior media), production of propagules was lower in the three other media (Goral, Kondryatiev, and Paris media). Both isolates produced oblong blastospore-like propagules, except in Kondryatiev medium in which they provided ovoid propagules. In this case, Mf189 submerged propagules looked like aerial conidia, but scanning observations did not demonstrate a typical conidiogenesis via phialides. In Kondryatiev medium, Mf324 submerged propagules were significantly smaller than aerial conidia. Infection potential of submerged propagules was assayed on Schistocerca gregaria. Second-instar larvae fed for 48 h on fresh wheat previously contaminated by a spraying suspension of each inoculum titrated at 10⁷ propagules ml^-1. All seven media produced submerged propagules that were highly infectious for S. gregaria larvae. Shake flask culture assays permitted us to select three low-costmedia, Adamek, Jenkins–Prior, and Catroux for improving scale-up of liquid fermentation focused on mass-production of Metarhizium propagules for mycoinsecticides devoted to locust control. This revised version was published online in June 2006 with corrections to the Cover Date.