We have isolated the 3-phosphoglycerate kinase (PGK) gene of the yeastYarrowia lipolytica by probing a genomic library with a PCR fragment amplified with primers deduced from two highly conserved regions of various PGKs. It is a unique sequence encoding a polypeptide of 417 residues with extensive homology to other PGKs, especially to that ofAspergillus nidulans (76% identity). The expression of theY. lipolytica PGK1 gene proved to be higher on gluconeogenic substrates than on glycolytic ones. Haploid strains harboring a disrupted allele were able to grow on mixtures of a gluconeogenic carbon source and of a glycolytic one, but required proline supplementation in the presence of glucose, and were inhibited by glycerol.

Destruxins are among the most exhaustively researched secondary metabolites of entomopathogenic fungi, yet definitive evidence for their roles in pathogenicity and virulence has yet to be shown. To establish the genetic bases for the biosynthesis of this family of depsipeptides, we identified a 23,792-bp gene in Metarhizium robertsii ARSEF 2575 containing six complete nonribosomal peptide synthetase modules, with an N-methyltransferase domain in each of the last two modules. This domain arrangement is consistent with the positioning of the adjacent amino acids N-methyl-l-valine and N-methyl-l-alanine within the depsipeptide structure of destruxin. DXS expression levels in vitro and in vivo exhibited comparable patterns, beginning at low levels during the early growth phases and increasing with time. Targeted gene knockout using Agrobacterium-mediated transformation produced mutants that failed to synthesize destruxins, in comparison with wild type and ectopic control strains, indicating the involvement of this gene in destruxin biosynthesis. The destruxin synthetase (DXS) disruption mutant was as virulent as the control strain when conidial inoculum was topically applied to larvae of Spodoptera exigua, Galleria mellonella, and Tenebrio molitor indicating that destruxins are dispensable for virulence in these insect hosts. The DXS mutants exhibited no other detectable changes in morphology and development.

Sexual reproduction of fungi is governed by genes located on the mating-type (MAT) locus. To analyze the MAT locus of the genus Diaporthe (anamorph: Phomopsis), a large genera within the ascomycetous class Sordariomycetes, we cloned and sequenced loci MAT1-1 and MAT1-2 from two heterothallic Diaporthe species, designated as Diaporthe W- and G-types (four isolates in total). The mating-type loci structures of Diaporthe W- and G-types were similar; MAT1-1 isolates had a MAT locus containing three genes, MAT1-1-1, MAT1-1-2 and MAT1-1-3, as was the case with other Sordariomycetes, and in contrast to other Sordariomycetes, MAT1-2 isolates had genes homologous to MAT1-1-2 and MAT1-1-3, in addition to MAT1-2-1. Expression analysis by RT-PCR revealed that all the mating-type genes of Diaporthe W-type were transcriptionally active during vegetative growth. The structure of MAT loci of Diaporthe W- and G-types is distinct from that in other heterothallic filamentous ascomycetes, which have dissimilar gene structure in opposite mating-type loci. This unique structure is informative to discussing the evolutionary history and function of mating-type genes of Sordariomycete fungi.

Hydrophobins are small, cysteine-rich, secreted proteins, ubiquitously produced by filamentous fungi that are speculated to function in fungal growth, cell surface properties, and development, although this has been rigorously tested for only a few species. Herein, we report identification of three hydrophobin genes from the entomopathogenic fungus, Metarhizium brunneum, and functional characterization of strains lacking these genes. One gene (HYD1/ssgA) encodes a class I hydrophobin identified previously. Two new genes, HYD3 and HYD2, encode a class I and class II hydrophobin, respectively. To examine function, we deleted all three separately, from the M. brunneum strain KTU-60 genome, using Agrobacterium tumefaciens-mediated transformation. Deletion strains were screened for alterations in developmental phenotypes including growth, sporulation, pigmentation, colony surface properties, and virulence to insects. All deletion strains were reduced in their ability to sporulate and showed alterations in wild-type pigmentation, but all retained wild-type hydrophobicity, except for one individual hyd3 mutant. Complementation with the wild-type HYD3 gene restored hydrophobicity. Each gene, present as a single copy in the genome, showed differential expression patterns dependent on the developmental stage of the fungus. When Spodoptera exigua (beet armyworm) larvae were treated with either conidia or blastospores of each hyd mutant, reductions in virulence and delayed mortality were observed as compared to WT. Together, these results suggest that hydrophobins are differentially expressed and may have distinct, but compensating roles, in conidiation, pigmentation, hydrophobicity, and virulence.

This work reports the cloning and sequencing of pkpA, a gene of the filamentous fungus Phycomyces blakesleeanus, whose expression seems to be coupled to vegetative growth. This gene encodes a putative serine/threonine-specific protein kinase, whose sequence is related to that of the yeast protein STE20, involved in pheromone-response pathways, and to a number of MAPK kinase proteins. However, detailed analysis of the kinase sequence suggests that PkpA is a novel serine/threonine protein kinase that probably participates as an intermediate in an intracellular system controlling nuclear proliferation in P. blakesleeanus.

Three different alleles of the β-isopropylmalate dehydrogenase gene were cloned and sequenced from a leucine auxotrophic mutant, G587, of Candida maltosa. The cloning of functionally-intact wild-type genes from this mutant strain suggests the presence of silent gene copies. An interallelic-divergence comparison has provided evidence for new regulatory mechanisms. Sequence data and karyotype analysis argue for a highly-aneuploid genome of C. maltosa. An interpretation for the spontaneous auxotrophy-prototrophy-auxotrophy sequence of mutations in C. maltosa is suggested.

The organisation of the URA1 gene of Schizosaccharomyces pombe was determined from the entire cDNA cloned by the transformation of an ATCase-deficient strain of Saccharomyces cerevisiae. The URA1 gene encodes the bifunctional protein GLNase/CPSase-ATCase which catalyses the first two steps of the pyrimidine biosynthesis pathway. The complete nucleotide sequence of the URA1 cDNA was elucidated and the deduced amino-acid sequence was used to define four domains in the protein; three functional domains, corresponding to GLNase (glutamine amidotransferase), CPSase (carbamoylphosphate synthetase) and ATCase (aspartate transcarbamoylase) activities, and one cryptic DHOase (dihydroorotase) domain. Genetic investigations confirmed that both GLNase/CPSase and ATCase activities are carried out by the same polypeptide. They are also both feedback-inhibited by UTP (uridine triphosphate). Its organization and regulation indicate that the S. pombe URA1 gene product appears very similar to the S. cerevisiae URA2 gene product.

The mes1⁺ gene of the fission yeast Schizosaccharomyces pombe is essential for the second meiotic division. We have cloned a 1.1-kb HindIII fragment containing mes1⁺ by complementation from an S. pombe genomic library. Sequencing of the genomic and cDNA fragments indicates the existence of one small intron of 75 nucleotides, although both the 5′ (G/GTTAGT) and 3′ (CAG/T) intron-exon junctions deviate from the consensus sequences proposed for S. pombe. The putative translation product of the mature mes1⁺ mRNA is a 11-kDa protein of 101 amino acids which has no significant homology to any previously-reported proteins. Disruption of mes1 has no effect on cell growth but causes an arrest of meiosis before the second meiotic division. Northern-blot analysis revealed that mes1⁺ was preferentially transcribed under conditions of nitrogen starvation. When a h⁹⁰ homothallic strain was shifted to a nitrogen-deficient medium, a pre-mRNA accumulated and then was gradually processed to generate a mature mRNA. This splicing did not occur in either a heterothallic haploid strain or in a homothallic mei2 mutant strain which was defective in the initiation of meiosis. Expression of the first exon alone was not able to suppress the mes1 null allele. These results indicate that mes1⁺ is required for the completion of meiosis, that splicing is required for the function of the mes1⁺ gene, and that this splicing requires the function of the mei2⁺ product.

Pyrenochaeta lycopersici, as other soil-transmitted fungal pathogens, generally received little attention compared to the pathogens affecting the aerial parts of the plants, although causing stunt and important fruit yield reduction of agronomic relevant crops. The scope of this study was to develop a system allowing to investigate the functional role of P. lycopersici genes putatively involved in the corky root rot of tomato. A genetic transformation system based on a split-marker approach was developed and tested to knock out a P. lycopersici gene encoding for a lytic polysaccharide monooxygenase (Plegl1) induced during the disease development. The regions flanking Plegl1 gene were fused with the overlapping parts of hygromycin marker gene, to favour homologous recombination. We were able to obtain four mutants not expressing the Plegl1 gene though, when tested on a susceptible tomato cultivar, Plegl1 mutants showed unaltered virulence, compared with the wild-type strain. The strategy illustrated in the present work demonstrated for the first time that homologous recombination occurs in P. lycopersici. Moreover, a transformation system mediated by Agrobacterium tumefaciens was established and stable genetic transformants have been obtained. The transformation systems developed represent important tools for investigating both the role of genes putatively involved in P. lycopersici interaction with host plant and the function of other physiological traits which emerged to be genetically expanded from the recent genome sequencing of this fungus.

A genomic library from the yeastPichia anomala has been constructed and employed to clone the gene encoding the sucrose-hydrolysing enzyme invertase by complementation of a sucrose non-fermenting mutant ofSaccharomyces cerevisiae. The cloned gene,INV1, was sequenced and found to encode a polypeptide of 550 amino acids which contained a 22 amino-acid signal sequence and ten potential glycosylation sites. The amino-acid sequence shows significant identity with other yeast invertases and also withKluyveromyces marxianus inulinase, a yeast β-fructofuranosidase which has a different substrate specificity. The nucleotide sequences of the 5′ and 3′ non-coding regions were found to contain several consensus motifs probably involved in the initiation and termination of gene transcription.