135. Genetics and breeding of Agaricus.
Leo Calvo-Bado, Simon Cutler, Mike Challen and Tim Elliott, Horticulture Research International, Wellesboume, Warwick, CV35 9EF, UK.
The genus Agaricus comprises c. 100 species and includes the cultivated mushroom A. bisporus.
HRI has a genetics programme to develop technologies and approaches of value in strain
improvement. Current aims are to develop a reproducible transformation system and to characterise
and exploit the diversity present in the genus. Although transformation has been achieved in A.
bisporus using the pAN7.1 vector, the system is not reproducible. We are developing transforming
vectors, controlled by regulatory sequences from Agaricus genes, or based on Agaricus genes
themselves. Putative regulatory sequences have been defined and vectors constructed in which
promoter sequences have been fused to reporters. Protoplast-mediated transformation remains a
problem with Agaricus. We have therefore developed ballistic delivery of DNA for use in
mushrooms. Other Agaricus species provide unexploited genetic diversity; some may be directly
cultivable whilst others may provide genes of agronomic value that can be utilised in A. bisporus. We
are analysing Agaricus breeding systems using classical and molecular methodologies. These studies
have revealed an unexpected complexity in some breeding systems where single spore isolates can
fruit "normally" but heterokaryosis is still regulated by mating-type. Our poster illustrates the range
and variety of studies being carried out at HRI.
136. The ATPase 6 gene in Agaricus, a comparison of a structural gene with the plasmid, pEM.
Becky Chiang, Mary Robison, and Paul A. Horgen. University of Toronto.
The ATPase 6 gene has been sequenced and analyzed from a representative sample of the genus,
Agaricus, and from numerous isolates of A.bisporus (with different phenotypes, geographical
location, and reproductive strategies). We have integrated PCR with that of DNA cycle sequencing.
DNA sequence comparison across the genus suggested a considerable amount of variability with
transversions (pydmidine purine) being more common than transitions (purine purine or
pyrimidine pyrimidine). We have compared the differences in primary nucleotide and primary
amino acid sequence in the representative isolates across the genus. Within the 6 isolates of A.
bisporus examined, one variable site was detected. Within this small sample, the difference was
observed in the U3 cultivar, and a heterothallic isolate from the Sonoran Desert. From this small, but
representative sample of the genus, we have constructed a phylogenetic tree comparing the sequences
of the structural gene of ATPase 6 with the extra chromosomal plasmid, pEM. The results of this
analysis suggests that the evolution of these two mitochondrial. genetic elements occurred quite
separate from one another. The results from this analysis further supports the hypothesis that the
integration of pEM homologous sequences into the mitochonddal genome was an ancient event
probably occurring before the speciation of the genus. If pEM were of mitochondrial origin, one
would predict that the evolutionary pattern should be similar to that of ATPase 6.
137. Cytological localization of an epitope-tagged Frtl fusion protein in the Basidiomycete Schizophyllum commune.
Gail E. Palmer, J. Stephen Horton, Dept. of Biological Sciences, Union College, Schenectady, NY, USA
Fruiting bodies (mushrooms) can be induced to form in normally nonfruiting homokaryotic strains
of the Basidiomycete fungus Schizophyllum commune by the ectopic genomic integration of a cloned
gene called Frtl. The Frtl gene encodes a predicted polypeptide of 192 amino acids, which has
motifs which suggest that it is an ATP-binding protein. An important step in elucidating the
mechanism of how Frt1 influences mushroom development is to determine the cellular location of
its protein product. A plasmid construct was made in which a triple hemagglutinin tag was fused inframe to the N-terminus of Frt1. In vivo function of the tagged transgene was confirmed by its ability
to induce fruiting in homokaryotic transformation recipients. Hyphal cells from transformants were
analyzed by immuno-EM and immunofluorescent microscopy, using a monoclonal antibody against
the HA tag. The results of these experiments indicate that the Frtl protein is localized in the cellular
membrane. The authors speculate that Frtl may be involved in a signal transduction pathway
controlling the development of fruiting bodies in S. commune.
138. Parasitic interactions between thn and wild-type mycelia of Schizophyllum commune.
Frank H.J. Schuren, Swiss Federal Institute of Technology, Zurich, Switzerland.
Development of aerial structures in the basidiomycete Schizophyllum commune is regulated by the
THN1 gene. A frequently occurring spontaneous mutation in this gene prevents formation of aerial
hyphae in a monokaryon and, if homozygous, the formation of aerial mycelium and fruit bodies in a
dikaryon. Genes specifically expressed during formation of aerial structures (e.g. hydrophobin genes)
are not expressed in mutant colonies. Other characteristics are the typical wavy (sometimes
corkscrew-like) hyphae, faster radial growth rate than wild-type (although the biomass is only about
half of that of wt colonies) and a typical pungent smell. Thn colonies were shown to produce
excessive aerial mycelium when surrounded by an excess of wt colonies. When wt and thn colonies
were grown together the growth rate of thn colonies was clearly increased, resulting in complete
overgrowing of wt mycelium by thn hyphae when mixtures of both mycelia where grown from a
single inoculation point. Since the growth stimulation (but not the excessive formation of aerial
mycelium) could also be observed when thn and wt colonies were physically separated by a dialysis
membrane (cut-off 6-8 kDa) it was concluded that low-molecular-weigth molecules were responsible
for the observed effects.
139. Expression of heterologous genes in Schizophyllum commune is hampered by truncated transcripts.
Frank H.J. Schuren, Jeroen Bron and Joseph G.H. Wessels, University of Groningen, Groningen, The Netherlands.
GPD regulatory sequences were used to express a phleomycin resistance gene (Sh ble) in
Schizophyllum commune resulting in high numbers of phleomycin-resistant transformants. Attempts
to express heterologous genes coding for hygromycin B phosphotransferase (hph), geneticin, -glucuronidase (uidA) and B-galactosidase (lacZ) using the same regulatory sequences were not
successful and no mRNA could be detected. GC-methylation could not be detected in any of the
transformants. Cloning the hph and uidA genes in an internally deleted GPD gene resulted in
truncated transcripts which ended within the 5'-parts of the heterologous genes. Cloning of the same
genes as transcriptional fusions downstream of the Sh ble gene also resulted in truncated transcripts
ending in the 5'-parts of these heterologous genes. Based on analysis of terminator regions of S.
commune and the presented results, it is suggested that AT-rich sequences in heterologous genes are
involved in generating these truncated transcripts thereby preventing expression of these genes in S.
commune.
140. The molecular cloning and expression of laccases from the basidiomycete Coprinus cinereus.
Debbie S. Yaver, Sakari Kauppinen, Feng Xu, Kim Brown, Beth Nelson, Torben Halkier, Stephen Brown, Elizabeht Golightly, Sheryl Sandhal, and Palle Schneider, Novo Nordisk Biotech, Davis, CA and Novo Nordisk A/S, Denmark.
A laccase has been purified from the extracellular medium of a culture of the basidiomycete Coprinus cinereus. The protein is dimeric, comprised of two subunits of 63 kDa as determined by SDS-PAGE and ultra-filtration. On isoelectric focusing gels, two major bands with pIs of 3.7 and 4.0 are observed. The purified laccase has pH optima of 6 to 7 and 4 with syringaldazine and ABTS as substrates, respectively. Three laccase genes, lcc1, lcc2 and lcc3, have been cloned from C. cinereus. The nucleotide sequence of all 3 genes have been determined. Based on a comparison of the predicted proteins and the protein sequences determined from internal peptides of the purified enzyme, lcc1 encodes for the purified enzyme. After the predicted signal sequence cleavage the mature proteins are 521, 499 and 501 for lcc1, lcc2 and lcc3, respectively. Both partial cDNAs and full-length genomic clones have been isolated for all 3 genes, and based on the nucleotide sequences of the cDNA and genomic clones all 3 genes contain several introns. The lcc1 gene contains 7 introns while both lcc2 and lcc3 contain 13 introns. The predicted mature proteins share identity to one another ranging from a low of 58% to a high of 80%. The predicted mature laccases also share identities with other fungal laccases. Interestingly, the predicted mature lcc1 protein when compared to other fungal laccases contains a 23 amino acid C-terminal extension which is rich in arginine and lysine suggesting there maybe some C-terminal processing that occurs during its biosynthesis. The Lcc1 protein has been expressed in Aspergillus oryzae.