Talks: Sexual and Asexual Reproduction

Sexual reproduction in ascomycetes: roles of the N. crassa mating type polypeptides

Melissa L. Philley, Adlane Ferreira*, N. Louise Glass* and Chuck Staben University of Kentucky and *University of British Columbia

The N. crassa mating type idiomorphs encode polypeptides that play crucial roles in vegetative incompatibility, mating, and formation of ascogenous hyphae. RIP experiments also suggest a role for the mating type genes within the ascogenous hyphae. The sole product of the mt a idiomorph, MT a-1, is a sequence-specific DNA binding protein of the HMG box family. The presumed regulatory targets of MT a-1 include genes necessary for mating pheromone biosynthesis and response. MT a-1 DNA binding activity is not necessary for vegetative incompatibility function, but it is necessary for mating functions. The mt A idiomorph encodes at least three products. One of these products, MT A-3, is also a member of the HMG box family. MT A-3 produced in E. coli binds DNA fragments that are also bound by MT a-1. The interactions of the mating type polypeptides with DNA and with each other as well as the biochemical and biological consequences of such interactions are under investigation.

Genes involved in vegetative incompatibility reaction in Podospora anserina

B. Turcq, P. Balhadere, A. Groppi, M. Paoletti, C. Clav and J. Bgueret. Laboratoire de Gntique CNRS-UPR 9026, Talence, France.

The three genes, het-c, het-e and het-d, involved in the two nonallelic. incompatibility het- c/het-e and het-c/het-d systems have been cloned. HET-C protein is similar to a glycolipid transfer protein. A strain containing a disrupted het-c locus is affected in ascospores production. A het-e gene encodes a 1356-amino acid protein which exhibits two domains. In the N-part of the protein, there is a P-loop motif characteristic of GTP binding protein. In the C-part of the protein, there are ten direct repeats of a motif of 42 amino aids which are characteristic of beta-transducin like protein. Mutation in the P-loop motif results in a loss of the incompatibility phenotype. It has been shown in P. anserina that mutation in some genes named mod genes results in the suppression of a barrage reaction. Moreover, resulting strains have defects in secondary ramification and reproductive female organ production. On the basis of the wild type phenotype restoration of a modD mutant strain, a gene has been cloned. It encodes a polypeplide which has homology with the N. crassa adenylate cyclase. But, by RFLP, the modD locus and the adenylate cyclase locus are distinct. Transcriptional and/or translational regulation of genetic expression and enhancement of the proteolytic activity had been shown in cells undergoing cell death associated to incompatibility. Two genes under transcriptional regulation have been cloned. Study of their expression is in progress. The cloned gene papA, encoding an aspartylprotease, has been disrupted.

Mating type regulation of sexual reproduction in Coprinus

Lorna Casselton, University of Oxford, Oxford, UK

Mating compatibility in the basidiomycete Coprinus cinereus is determined by multiallelic genes at two complex loci termed A and B. These genes regulate a developmental programme that converts the asexual monokaryon into a fertile dikaryon. It is estimated that there are some 160 versions of A and 79 versions of B which would generate more than 12000 cross compatible mating types in nature. We have a fascinating biological problem in trying to resolve how such large numbers of A and B specificities are derived and how it is that so many versions can interact to trigger the same developmental pathway. The A locus contains several genes that encode multiple versions of two dissimilar homeodomain proteins. Specific combinatorial interactions between these proteins promotes the A-regulated part of the pathway; we are interested in why certain pairs of proteins can heterodimerize but others cannot. The B locus also contains several genes of different classes; preliminary data indicate that these play a role in signal transduction. Rare mutations that result in self-compatibility arise within the A and B loci and molecular analysis of these provides novel insights into the function of the genes.

The multispecific B mating-type genes of Schizophyllum commune encode complexes of putative pheromones and pheromone receptors

Lisa J. Vaillancourt and Carlene A. Raper, Department of Microbiology and Molecular Genetics, University of Vermont, Burlington VT

Molecular genetic analyses of the B alpha and B beta mating-type loci have produced evidence that a multispecific system of pheromones and pheromone receptors govern self/nonself recognition and sexual development in the Homobasidiomycete Schizophyllum commune. Two of the nine known specificities at the B alpha locus (alpha 1 and alpha 3) and two of the nine known specificities at the linked B beta locus (beta 1 and beta 2) have been cloned and are being examined. A synthesis of data to date, from DNA sequencing, nucleic acid hybridizations, and functional tests via DNA-mediated transformation experiments, suggests that B alpha 1 and B beta 1 each contains several genes encoding pheromones and at least one gene encoding a pheromone receptor similar to those known in Ustilago maydis and Saccharomyces cerevisiae. Each of these loci spans approximately 8 kb of genomic DNA and includes multiple elements capable of differentially activating B-regulated development in strains representing the other eight specificities at the relevant locus. None of these elements can elicit a response in strains carrying the self B mating-type specificity. This is the first evidence for a multispecific system of pheromones and pheromone receptors governing mating in a fungus. Comparison of pheromone receptor genes in Schizophyllum commune Jurgen Wendland, Jorg Hegner and Erika Kothe, University of Marburg, Germany The mating type locus Ba1 of S. commune contains a pheromone receptor and putative pheromone genes. The pheromone receptor Bar1 shows homology to other pheromone receptors such as Ste2 and Ste3 of Saccharomyces cerevisiae and pral and pra2 of Ustilago maydis. It also contains sites conserved among other G protein-linked receptors of the seven transmembrane domain family. The fact,that the mating system in S. commune provides nine different allelic specificities at the mating type locus B-alpha makes it a model system to investigate ligand interaction between different allelic specificities of pheromones and receptors. For S. commune multiple isolates for each mating specificity exist. In order to obtain evidence for functional domains, different alleles for the BAR1 gene will be compared. Southern hybridization experiments could show that strains sharing the specificity B-alpha1 exhibit strong hybridization signals. This indicates higher homology of the alleles of BAR1, all sharing the mating specificity B-alpha1, as compared to strains with other mating specificities, which also hybridize, but to a lesser extent.

Regulatory interactions that control multicellular development during Aspergillus sporulation

Bruce L. Miller, Karen Y. Miller and James R. Dutton. Dept. of Microbiol., Mol. Biology and Biochem., University of Idaho.

Multicellular development in the filamentous fungus Aspergillus nidulans requires that spatiotemporal regulation of cell-specific gene expression be coupled to alterations in cell cycle regulation. A genetic hierarchy consisting of five essential regulatory genes controls the transition from multinucleate filamentous growth to uninucleate budding growth, the assembly of a multicellular reproductive structure (conidiophore) and the production of dormant conidia (spores). Complex regulatory interactions provide a mechanism for establishing overlapping gradients in these transcription factors which, in turn, coordinately activate hundreds of conidiation-specific genes. One class of genes (brlA, abaA, wetA) establishes a linear regulatory pathway that directs formation of the terminally differentiated conidia. A second class modifies these events to allow multicellular development. The modifier gene medusa (medA) is required for commitment to the developmental program. MedAp physically interacts with BrlAp, functioning as a co-activator of a subset of BrlAp target genes that includes abaA. The stunted (stuA) gene encodes a member of a family of transcription factors that control cell cycle progression and fungal development. Overexpression of StuAp blocks the ability of BrlAp to drive terminal differentiation and promotes a pseudohyphal growth pattern. During conidiation, cell-specific morphology is established by a balance between BrlAp, AbaAp and MedAp driving terminal differentiaton and StuAp driving uninuclear cell divisions by regulating expression of brlA, a subset of BrlAp target genes and possibly cell cycle regulators.

The first non-mammalian homologue of the PAFl gene (Zellweger syndrome) discovered as a gene involved in caryogamy in Podospora anserina

V. Berteaux-Lecellier, A. Adoutte-Panvier, M. Picard, C. Thompson-Coffe and D. Zickler. I.G.M. CNRS, URA. D-1354. Bt. 400. U.P.S. F-91405 Orsay. Cedex. France.
The fusion of haploid nuclei (caryogamy) is a vital part of the sexual cycle. To date, in fungi, this process has been well studied only in S. cerevisiae. Caryogamy in filamentous ascomycetes is more complex than in unicellular yeasts. It does not occur immnediately after fertilization but it is a deferred stage occuring in specialized cells. The car1 mutants of P. anserina, completely defective for caryogamy, have been isolated during a systematic search for sporulation deficient mutants. The car1 gene was cloned by complementation. Surprisingly, this gene encodes a protein that shows similarity to the mammalian PAF1 protein (Zellweger syndrome). Altogether, the molecular, physiological (inability for the car1 mutants to grow on oleic acid), genetical (revertants analysis) and ultrastuctural approaches (immunofluorescence and electron microscopical analyses) gave evidence that the P. anserina car1 protein is actually a peroxisomal protein. Therefore, this study shows that peroxisomes are required at a specific stage of sexual development, at least in P. anserina, and that a functional homologue of the PAF1 gene is present in a lower eucaryote. The data will be discussed in terms of the possible functional role of peroxisomes in the caryogamy process or, more likely, in the cell determination or differentiation programs.

Control of DNA replication and mitotic checkpoint function by the Aspergillus nidulans nimO gene

Steven W. James, Gettysburg College.

We are investigating the role of the Aspergillus nimO gene in DNA replication and mitotic checkpoint function. Flow cytometric and cytological analyses showed that when conidia of a temperature sensitive lethal nimO18 mutant were germinated at the restrictive temperature (44 C), they were unable to replicate their DNA, but 35-40% of nuclei nevertheless progressed into mitosis and arrested with condensed chromatin. These results identify nimO as a candidate regulator of G1/S or S phase, and further suggest that this gene influences the checkpoint that prevents mitosis (M) from occurring until DNA synthesis has been completed. Sequencing of a nimO cDNA revealed a predicted protein of 72.8 kd that shares 29% identity with DBF4, a budding yeast G1/S regulator which associates with origins of replication and with the CDC7 kinase in order to initiate DNA synthesis. nimO and DBF4 are especially strongly conserved in a C-terminal domain containing a single, novel motif which bears strong resemblance to Cys2-His2 zinc fingers. A deletion of this 60 bp motif from a nimO gene fused with the inducible alcA gene promoter rescued the ts-lethality of nimO18 for growth, but not conidiation, when high-level expression was induced by ethanol. On glycerol, which permits low basal expression, the strains grew poorly and were likewise unable to conidiate. Thus, the putative zinc finger appears non-essential for mycelial growth when the variant gene is highly expressed, but may be necessary for the accelerated cell cycling that must occur during conidiation. We are continuing to examine the function of this motif and to test nimO function through gene disruption.

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