1. Mating system of Glomerella cingulata.
Cindy R. Cisar and David O. TeBeest, Department of Plant Pathology, University of Arkansas, Fayetteville, AR 72701.
Heterothallic filamentous ascomycetes typically have a one locus, two allele (idiomorph)
mating system. In this study five heterothallic strains of Glomerella cingulata (anamorph:
Colletotrichum gloeosporioides) from pecan were crossed in all possible combinations. Four of
the isolates behaved as if they had a bipolar mating system while one isolate mated with all four of
the other isolates suggesting that the mating system of G. cingulata is more complex. To further
investigate the genetics of this mating system single ascospore progeny were isolated from each of
the successful crosses and backcrossed with the original parental strains. In addition, a subset of
Fl isolates was crossed with all five isolates from pecan and in all possible combinations with each
other. The results indicate that progeny stably inherited the mating pattern of one of the original
parents and that in each of the original crosses, a single locus which controls mating segregated
1:1. A model for the mating system of G. cingulata consistent with these data will be presented.
2. How nuclei with different mating types can recognize each other in a plurinucleate cell?
Sylvie Arnaise, Evelyne Coppin, Robert Debuchy, Denise Zickler and Marguerite Picard. Institut de Genetique et Microbiologie. URA CNRS 1354. Bdt. 400. F-91405 Orsay cedex, France.
In the heterothallic fungus Podospora anserina, fertilization leads to the formation of plurinucleate cells which contain a mixture of parental nuclei of opposite mating type termed mat+ and mat-. Then mat+ and mat- nuclei undergo a possible recognition which is followed by the development of specialized dikaryotic hyphae in which each cell contains a mat+ and a mat- nucleus. Caryogamy, meiosis and ascospore formation occur in these specialized hyphae.
Data indicates that there is indeed an intemuclear recognition process which is controled by the single mat+ gene and two of the three mat- genes. It also indicates that these mating-type genes, which encode transcription factors, have a nucleus restricted expression. We propose that this property allows the transcription factors to control the expression of specific markers on mat+ and mat- nuclei. These specific nuclear markers would be recognized by the cytoskeleton which would help to associate compatible nuclei during the recognition process, prior to their migration into the specialized hyphae.
We have demonstrated that one of the three mat- genes is not a bonafide mating-type
gene, because it may be present in either or both parental nuclei without altering the progeny.
Several lines of evidence suggest that this gene is required after the intemuclear recognition
process, when the specialized dikaryotic hyphae start their development.
3. Expression of the mating-type genes in Podospora anserina.
Evelyne Coppin and Robert Debuchy. Institut de Genetique et Microbiologie. URA CNRS 1354. Bat. 400. F-91405 Orsay cedex, France.
The heterothallic fungus Podospora anserina has two mating-type alleles termed mat+ and mat-. The mat+ sequence contains one gene, FPR1 while mat- contains three genes : FMR1, SMR1 and SMR2. FPR1 and FMR1 are required for fertilization which is followed by mitotic divisions of the two parental nuclei inside the female organ. This leads to the formation of plurinucleate cells. FPR1, FMR1 and SMR2 control the recognition between nuclei of opposite mating type inside the syncytium. Then, pairs of compatible nuclei migrate into specialized hyphae, of which initial development requires SMR1.
Mature transcripts of the mating-type genes are detectable only in the fertilized female organ. We supposed that ectopic expression of FMR1, SMR2 and FPR1 would be deleterious to P. anserina in inducing untimely nuclear recognition. However, fusion of the mat gene coding sequences to the gpd promoter of Aspergillus nidulans does not impair their function nor have deleterious effect on P. anserina. Further characterization of vegetative expression of the mat genes by fusing their 5'untranslated region to a reporter gene shows that FPR1 , FMR1 and SMR2 are transcribed constitutively. The absence of their mature transcripts in the mycelium indicates that either they are degraded or the primary transcripts are not spliced at this stage. SMR1 is the only mat gene specifically transcribed in the fruiting body.
The hypothesis of deleterious effect of ectopic expression of FPR1, FMR1 and SMR2
could not be tested with the help of a constitutive promoter because it does not overcome the
post-transcriptional control of these genes.
4. Cloning of a putative mating-type gene from the Discomycete fungus Tapesia yallundae.
Paul S. Dyer1, Paul Bowyer2, John A. Lucas2, John F. Peberdy1. 1University of Nottingham, 2IACR Long Ashton, United Kingdom.
The Discomycete species Tapesia yallundae, causal agent of eyespot disease of cereals,
exhibits a two-allele heterothallic mating system. Work is in progress to clone the mating-type
genes (MAT1-1, MAT1-2) from T. yallundae for use in developing molecular markers to
determine the mating-type of field isolates of the pathogen. A PCR-based strategy has been
adopted using degenerate primers designed to amplify a partially conserved 270 - 280 bp region
of the MAT1-2 locus from other Ascomycete species (N. crassa, P. anserina, C. heterostrophus,
S. pombe). These primers were used successfully to amplify a 280bp fragment from one
mating-type (MAT1-1) of T. yallundae which was cloned and sequenced. It contains a
characteristic HMG-box motif and exhibits 35-40% amino acid homology to other Ascomycete
MAT1-2 sequences in the partially conserved regions of the gene. The PCR fragment was used to
probe Southern blots of genomic DNA from MAT1-1 isolates of T. yallundae and a single
strongly hybridising band was detected. However, a similar signal was also detected in blots of
MAT1-2 genomic DNA, indicating the presence of similar sequences in MAT1-2 isolates.
Genomic libraries of T. yallundae MAT1-1 and MAT1-2 isolates have been constructed in lambda
Zap and are now being screened with the PCR fragment to isolate the entire gene.
5. Neurospora crassa mating type: evidence for activation and repression by mating-type products.
Adlane V.B. Ferreira1, Robert L. Metzenberg2 and N. Louise Glass1. 1 Dept. of Botany, Biotechnology Lab, Univ. British Columbia, Vancouver, BC and 2Dept. Biol. Sciences, Stanford University, Stanford, CA.
The Neurospora crassa mt A idiomorph contains three genes mt A-1, mt A 2 and mt A-3,
that have amino acid sequence characteristics of transcriptional factors. mt A-1 controls mating
identity and mating-type associated heterokaryon incompatibility. Mutations in mt A-2 and mt A-3 genes were identified in a previously isolated A RIP mutant which is affected in post fertilisation
functions. We have isolated specific mt A-2 and mt A-3 mutants using Repeat Induced Point
mutation (RIP). These mutants (A-2 RIP and A-3 RIP) are able to mate and produce abundant
ascospores, in contrast to the A-2/A-3RIP mutant. Heterokaryon tests showed that mt A-2 and mt
A-3 do not affect heterokaryon incompatibility. Northern analyses of genes expressed during
sexual development (sdv) were performed using strains mutated in the mt A and mt a genes and a
strain in which the mating-type sequences were deleted. Our results confirm that mt A-1 acts as
an activator and indicate that mt A-2 acts as a repressor of most genes analysed. Some genes are
expressed in the deletion strain suggesting that repression is necessary for regulation. The
mating-type products may form transcriptional complexes to specifically regulate several genes.
Identification of target genes of the mating-type products is the first step towards understanding
gene interactions and how mating type controls sexual development in N. crassa.
6. Clues to the molecular basis of mutant B2-mating types of Schizophyllum commune. Thomas J. Fowler,* Michael F. Mitton,* Natasha Motchoulskaia, * Lisa J. Vaillancourt1 and Carlene A. Raper*. *University of Vermont and 1University of Kentucky.
Previous molecular work on the Schizophyllum commune B mating-type locus shows
that the locus is complex, encoding several pheromones and at least one pheromone-receptor. In
the mating process, pheromones facilitate the donation of migrating nuclei from an individual to a
compatible mate while the pheromone-receptor regulates the acceptance of migrating nuclei from
a mate. Normally, each B specificity is self incompatible. Clones containing wild type B2
mating-type genes were isolated and subclones were derived that confer either B2 pheromone or
pheromone-receptor activity. Mutations within B2 yield a variety of phenotypes from
constitutively self-activating to loss of some or all B2 activities. We are using functional assays
and DNA sequencing to define the lesions. We hypothesize that B2(l), a mutant with
constitutively self-activating B function, is the result of an altered gene that produces a
pheromone able to stimulate the resident B2 pheromone-receptor. Mutation of the B2(l) strain
resulted in a self-incompatible strain able to donate migrating nuclei to other specificities including
its grand progenitor, B2, but unable to accept migrating nuclei from test mates. We suggest that
this secondary mutant retains the primary gain of function mutation but has lost B2 pheromonereceptor function. We tested this hypothesis using the secondary mutant as a transformation
recipient for a subclone from the B2 wild type locus that has pheromone-receptor activity. This
resulted in constitutively self-activated transformants, confirming that pheromone-receptor
activity was missing from the secondary mutant. Other mutants are being examined similarly.
7. Cloning and analysis of the mating-type genes from the homothallic ascomycete Sordaria macrospora.
Stefanie Pöggeler, Siegfried Risch, Heinz Dieter Osiewacz*, Ulrich Kuck. Lehrstuhl fur Allgemeine Botanik, RuhrUniversitat, 44780 Bochum, Germany; *AK Entwicklungsbiologie, J.-W.-GoetheUniversitat, Marie-Curie-Str. 9, 60439 Frankfurt, Germany
In the homothallic ascomycete Sordaria macrospora (Sordariaceae) a single ascospore gives rise to hyphae that are able to enter the sexual pathway and to produce fruiting bodies (perithecia) that enclose the meiotic ascospore progeny. To better understand the molecular basis of homothallism and to elucidate the role of mating-products during fruting body development, we cloned and sequenced the mating-type locus of Sordaria macrospora. In the S. macrospora mating-type region we were able to identify four open reading frames called Smta-1, SmtA-1, SmtA-2 and SmtA-3. Comparison of the Sordaria mating type locus to the functional regions of the mating-type idiomorphs from the heterothallic ascomycetes Neurospora crassa and Podospora anserina revealed that sequences from both idiomorphs (A/a and mat-/mat+, respectively) are closely linked in S. macrospora. For further investigation of the functional conservation of the Sordaria mating-type genes we transformed cosmid clones containing the mating-type locus of S. macrospora into mat- and mat+ strains of the closeley related heterothallic fungus P. anserina. The Sordaria mating-type genes were partially able to complement fruiting body formation in both mat- and mat+ strains of P. anserina.
8. Multiple B mating types generated by three sets of multiallelic genes.
John R. Halsall, Pushpalata T. Chaure, Ian F. Connerton and Loma A. Casselton, University of Oxford, Oxford, UK.
Pheromone signalling plays an essential role during and after mating in mushroom
Coprinus cinereus. The genes that encode the pheromones and their cognate receptors are
sequestered in the B mating type locus. Two asexual monokaryons must have different versions
of the B locus to be compatible mating partners and thus generate the fertile dikaryon.
Remarkably there are a predicted 79 versions of the B locus. By cloning and sequencing two loci,
B6 and B42, w show that multiple B mating specificities are derived from three sets of paralogous
genes, all of which are multiallelic. Each set of genes encodes a receptor and two pheromones
and any one of these genes is effective in altering B mating specificity if introduced by
transformation into a host monokaryon that has a different allele of the gene. Judged by
transformation, B6 and the uncloned B3 locus have different alleles of all three sets of genes
whereas B42 shares one set with B6 and one set with B3. We show that there is insufficient DNA
homology between different allelic versions of each gene and immediate flanking sequences to
permit cross-hybridization. This enabled us to use Southern analyses to demonstrate that five
other uncloned B loci share at least one set of genes with B42 or B6. We will compare sequences
of allelic and non-allelic genes and describe experiments designed to determine how this large
family of receptor proteins can be so specific in ligand recognition.
9. Targets for the bE and bW homeodomain proteins in Ustilago maydis.
Tina Romeis, Regine Kahmann and Jorg Kamper, Institut fur Genetik, Munchen, Germany.
In the phytopathogenic fungus Ustilago maydis the multiallelic b mating-type locus
controls sexual and pathogenic development. The b locus encodes the two unrelated
homeodomain proteins bE and bW. The bE and bW proteins form heterodimers via their highly
polymorphic N-terminal regions (variable domains) but only when they originate from different
alleles. The heterodimer is thought to act as a transcriptional regulator. We have constructed
different single chain fusions in which the bE protein is linked via a flexible kink region to the bW
protein (bE-k-bW). These single chain b-fusions are able to substitute for the active bE/bW
heterodimer. Our data demonstrate that allele-specific dimerization via the variable domains is non
longer prerequisite for activity if bE and bW proteins are fused; however, both homeodomains are
needed for function. We were able to show specific binding of a truncated bE/bW fusion protein
to the promoter regions of two genes in the a locus that had previously been shown genetically to
be regulated by the bE/bW heterodimer. The binding site for the b fusion protein was determined
using DNAse footprint and methylation interference techniques. Thus, the first targets for the
complex of two homeodomain proteins in a Basidiomycete have been identified.
10. Molecular analysis of sexual morphogenesis in Pyrenopeziza brassicae.
Gurjeet Singh & Alison M. Ashby. Department of Plant Sciences, University of Cambridge, UK.
Pyrenopeziza brassicae is a hemibiotrophic fungal pathogen of oilseed rape and other brassicas. It is heterothallic, having two mating types designated MAT1-1 and MAT1-2. Sexual reproduction involves a complex yet co-ordinated pathway of development involving early interactions of the two mating types.
Physiological studies have established the involvement of a potential morphogen, termed sex factor (SF) in controlling development in P. brassicae. SF is a lipoidal component produced during sexual development which has the capacity to promote fruiting body formation and inhibit asexual development.
Protein profiles of single mating type isolates induced with SF and individually picked fertile apothecia revealed the presence of a major protein termed sex factor induced (SFI 1) protein which was absent in asexually reproducing cultures. SFI 1 has been purified and internal amino acid sequence used to generate oligonucletide promers which generated a 700 bp PCR product against P. brassicae genomic DNA. When the 700 bp PCR product was used as a probe against genomic DNA of the two mating types of P. brassicae a different banding pattern was observed, indicating a physical linkage between the MAT loci and SFI 1 genes.
Cloning of the MAT loci from P. brassicae has involved heterologous probing using DNA probes from Neurospora crassa and Tapesia yallundae.
Current progress on cloning the MAT loci as well as cloning and further characterisation
of the SFI 1 gene will be described.
11. Y and Z A mating-type proteins of Schizophyllum commune bind in all combinations in vitro.
Robert C. Ullrich, Yasuhiko Asada, Changli Yue, Jian Wu, Guang-Ping Shen and Charles P. Novotny, University of Vermont, Burlington.
The A locus regulates sexual development via two proteins, Y and Z. Each A mating type encodes a unique Y and a unique Z protein. We have expressed two isoforms of Y (Y4 and Y5) and two isoforms of Z (Z4 and Z5) as GST and 6HIS fusion proteins. These proteins have been used for affinity chromatography assays of protein binding. Each full-length Y or Z protein binds in vitro to itself and other Y or Z proteins regardless of the A mating type from which they are encoded (i.e., mating-type independent binding). Use of partial length polypeptides identified mating-type dependent binding. Mating-type dependent binding is observed only between Y and Z proteins encoded from different A mating types (e.g., Y4+Z5 or Y5+Z4). N-terminal regions of Y and Z proteins are responsible for mating-type dependent binding; the determinants of mating-type independent binding are encoded elsewhere within the proteins. Deletion analysis shows that the Y4 specificity domain (the region conferring recognition uniqueness to the Y4 isoform) is essential for mating-type dependent binding.
12. The specificity determinant of the Y mating-type proteins is also essential for Y-Z protein binding.
Changli Yue, Charles P. Novotny, and Robert C. Ullrich, University of Vermont.
In Schizophyllum, tightly linked Y and Z mating-type genes do not promote development
in the combinations present in haploid strains (i.e., self combinations). When the Y and Z genes
from two different mating types are brought together by the fusion of two haploid cells, the Y and
Z proteins from different mating types recognize one another as nonself, form a complex and
activate development. We have made chimeric genes between Y1, Y3, Y4 and Y5 and examined
their mating-type specificities by transformation and mating tests. These studies show that the
specificity of Y protein recognized by Z protein is encoded within a short region of N-terminal
amino acids. The critical region is not precisely the same in each Y protein and in each Y-Z
protein interaction. For Y3 protein compared to Y4 protein the critical residues are in an N-terminal region of 56 amino acids (residues 17-72), with 40% identity and 65% similarity. Two-hybrid studies show that: (a) the first 144 amino acids of Y4 protein are sufficient to bind Z3 and
Z5 proteins, but not Z4 protein, and (b) proteins deleted of the Y4 specificity region do not bind
Z3, Z4 or Z5 protein. Thus the specificity determinant of the Y protein is essential for protein-protein recognition, Y-Z protein binding and mating activity.
13. Characterization of mating type genes in homothallic ascomycetes.
Sung Hwan Yuri, O.C. Yoder and B. Gillian Turgeon, Cornell Univ. Ithaca, NY.
Members of the genus Cocliliobolus have different reproductive life styles, i.e.,
homothallic (self fertile) vs. heterothallic (self sterile) or sexual vs. asexual. We are exploring
evolution of these strategies and have identified mating type genes (MAT) in heterothallic,
homothallic, and asexual species. Here we describe MAT organization in a homothallic (C
homomorphus), in a related heterothaflic (C heterostrophus) and in a distantly related homothallic
(Mycosphaerella zeae-maydis) species. MAT genes are arranged in tandem in both homothallics,
but differ strikingly in organization. In C. homomorphus, the counterparts of the C.
heterostrophus MAT genes are fused into a single reading frame [5'-(MAT-2/MAT-1)- 3']. This
finding is unprecedented and potentially useful since this novel gene organization may help
unravel the genetic mechanism by which this homothallic species arose. Furthermore, to date,
homothallic filamentous ascomycetes have been found to have either a single MAT gene, as in
certain Neurospora spp, or opposite MAT genes as in M. zeae-maydis and most members of the
Sordariaceae. The C. homomorphus MAT ORF encodes all of the C. heterostrophus MAT-2
homolog except for 9 amino acids at the 3' end and 8 amino acids at the 5' end of the MAT-1
homolog. M. zeae-maydis has complete versions of the C. heterostrophus counterparts, however
the MAT genes are separated by about 1 kb of unique, noncoding DNA; gene order is reversed
compared to C. homomorphus [5'-(MAT-1)-(MAT-2)- 3']. A second ORF in the flanking DNA is
conserved in both homothallic and heterothallic species. The remainder of the DNA flanking the
two homothallic MAT genes is unique. These variable MAT flanking regions may be determinants
of homothallism. Heterologous expression of homothallic MAT genes in a heterothallic genetic
background and vice versa is underway.
14. Perturbation of expression of sexual pheromone precursor genes of the ascomycete Cryphonectria parasitica by a hypovirus.
Lei Zhang, Patricia McCabe, Pam Kazmierczak, and Neal K. Van Alfen. Department of Plant Pathology and Microbiology, Texas A&M University, College Station, Texas.
There are a total of three pheromone precursor genes in both mating types of C. parasitica. In Mat I strains, only the Mfl-1 gene is expressed while in Mat2 strains the Mf2-1 and Mf2-2 genes are expressed. The putative coding regions of Mf2-1 and Mf2-2 are typical of the prenylated pheromones that have been described from yeasts and other filamentous fungi. The gene Mfl-1 has an ORF that encodes a protein of 530 amino acids. Within this ORF are seven repeats of the same decapeptide. Surrounding this decapeptide are the signals for post translational processing that have previously been described in the yeasts S. cerevisiae and S. pombe. Among these is the KR dipeptide sequence that directs cleavage by the Kex2p endoprotease. These three pheromone precursor genes are all down-regulated in strains infected with the hypovirus CHV I. The conserved sequences for Kex2p endoprotease maturation found in Mfl-I have also been identified in the leader peptide sequences of two other genes down-regulated by CHV1. One of these Kex2p-processed proteins that is down-regulated in virus infected strains has been found in host vesicles used by the virus for replication. We postulate that the virus perturbs expression of these genes by its replication on transport vesicles important for the processing of these proteins.