Plenary Session: Gene Regulation and Metabolism (Chair: Claudio Scazzocchio)
pH regulation in Aspergillus nidulans .
Miguel Angel Penalva, Centro de Investigaciones Biologicas CSIC, Madrid, Spain
The zinc-finger transcription factor PacC mediates pH regulation in Aspergillus nidulans and other
ascomycetes. The 678-residue PacC primary translation product is inactive in structural gene regulation. Under
ambient alkaline pH conditions, a signal provided by the six pal -gene pathway causes an unknown modification in
the protein which makes it accessible to a proteolytic processing step. This limited proteolysis eliminates the ~60%
residues of PacC at the carboxyl side. The processed protein (~residues 1 - 270) is fully active in structural gene
regulation, activating alkaline-expressed genes (through 5'-GCCARG-3' sites) and repressing acid-expressed
genes. Thus, the conformational change in PacC resulting from pH signal reception is the pH-sensitive step in the
regulatory circuit. The C-terminal moiety of PacC mediates its negative action at least by masking PacC domains
involved in transcriptional activation and presumably by preventing nuclear import of the full length protein, but
does not appear to impair DNA binding. Point mutations and C-terminal truncations disrupt the interactions
between the amino- and carboxyl-terminal moieties of the protein. pacC is itself an alkaline-expressed gene, but its
mode of regulation is different from that of alkaline structural genes.
Developmental regulation of catalases in Aspergillus nidulans.
Rosa E. Navarro, Laura Kawasaki and Jesus Aguirre, Instituto de Fisiologia Celular, Univ Nacional Autonoma de Mexico.
A general hypothesis to explain microbial cell differentiation as a response to hyperoxidant states was
derived from studies on sporulation in Neurospora crassa. Since catalases are ubiquitous enzymes that are central
to cellular antioxidant responses, we have now approached this hypothesis by studying the function and regulation
of catalases during A. nidulans asexual sporulation (conidiation). We have found two catalases in this fungus
encoded by the catA and catB genes, whose predicted polypeptides are as similar between them, as they are to E.
coli HPII catalase. The catA and catB expression is differentially regulated during growth and development. The
catA mRNA and protein appear during sporulation and are accumulated in both, sexual and asexual spores
independently of the brlA regulatory gene, in a process that involves transcriptional and translational controls. In
contrast, the catB mRNA and protein are very low in spores, accumulate in mature hyphae throughout conidiation
and in response to H202 both catalases can offer protection against H20, at different stages of the life cycle.
Although the increase in CatB activity and the appearance of CatA during sporulation is consistent with the
occurrence of oxidative stress during development, our data indicate the operation of efficient alternative pathways
for H202 detoxification.
Genetic Regulation of Aspergillus Mycotoxin Biosynthesis.
Nancy P. Kellerl and Thomas H. Adams2, lDept. of Plant Pathology and Microbiology, 2Dept. of Biology, Texas A&M
University, College Station, TX 77845.
Aflatoxin (AF) and sterigmatocystin (ST) are polyketide mycotoxins derived from the same biochemical
pathway found in several seed contaminating Aspergillus spp. The AF/ST biosynthesis genes are clustered in a
~60-75 kb DNA segment in all species examined to date. The magnitude of this cluster is well illustrated by the
characterization of the A. nidulans ST gene cluster which contains 25 coordinately regulated transcripts most of
which encode enzymes with functions required for AF and ST biosynthesis. In each cluster there is a positive-acting pathway specific regulatory gene, aflR, that encodes a sequence-specific DNA binding protein required for
cluster gene expression. Regulation of this cluster is complex and involves nutritional factors, pH and life cycle
controls. This complexity is illustrated by the fact that the ability of AflR to activate ST/AF gene expression is
linked to regulation of asexual sporulation through a requirement for inactivation of an heterotrimeric G protein
mediated signal transduction pathway.
White collar 1 and White collar 2 are partners in the blue light transduction pathway in Neurospora crassa.
Giuseppe Macino, Hartmut Linden, Paola Ballario. Universita "La Sapienza" Roma, Italy.
A saturating genetic dissection of "blind" mutants in Neurospora crassa has identified a total of two non-redundant loci (wc1 and wc2 ) each of which is required for blue light perception/signal transduction. Previously,
we demonstrated that wc1 is a putative zinc-finger transcription factor able to bind specifically to a light regulated
promoter. We have recently demonstrated using mutation analysis and in vitro DNA binding assays that wc2 is the
second partner of this two component light signal transduction system and encodes a functional zinc-finger DNA-binding protein with a putative PAS dimerization domain and transcription activation domain. This molecular-genetic dissection of the two components of this light signal transduction system has elucidated a model whereby
wc1 and wc2 interact via homologous PAS domains, bind to promoters of light-regulated genes and activate
transcription. As such, this study provides the first insight into two interacting partners in blue light signal
transduction in any organism and provides the molecular tools with which to dissect this engimatic process.
The circadian clock in Neurospora: light resetting of the oscillator and the control of gene expression.
Jennifer Loros, Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755.
An important property of circadian oscillators is their ability to entrain to the daily light/dark cycle. The clock is a negative feedback loop wherein the frq gene, known component of the clock in Neurospora crassa encodes the FRQ protein which feeds back to turn off the gene, yielding the oscillation in frq transcript and FRQ protein that is the Neurospora clock. Light delivered at any point within the circadian cycle acts rapidly to increase the level of frq transcript. The magnitude of the light-induced increase in frq mRNA and the extent of clock resetting are correlated and the threshold, kinetics and magnitude of this response indicate elevation of the level of frq transcript in the cell is the initial clock-specific even involved in resetting of the clock by light. We are examining the roles of two photo blind strains, band;white collar-1 (bd;wc-1) and white collar-2 ;band;(wc-2 ;bd) in light-resetting of the clock. By Northern analysis we find the transient, light induced accumulation of frq is blocked in bd;wc-1 but not in wc-2;bd .
In constant light, frq mRNA remains at high levels in the bd strain, thereby suppressing the frq cycle. This
sustained, light driven increase is blocked in both bd;wc strains. Both of the bd;wc strains failed to show a
rhythmic phenotype when entrained by either light or temperature steps. In a separate effort aimed at
understanding light regulation of rhythmic processes, circadianly expressed genes downstream of the clock, eas
(ccg-2 ) and ccg-1, have been examined in a clock null strain and foun not to require a functional clock. Deletion
analyses of the eas (ccg-2 ) promoter has localized cis-acting elements mediating clock, light, and developmental
control. Primary sequence determinants of a positive activating clock element were found reside in a 45-base pair
region close to the TATA box, and spatially distinct from sequence elements governing light regulation.
Plenary Session: Cell Biology and Pathogenesis (Chair: Hans VanEtten)
Animal pathogenesis.
David. W. Holden, Royal Postgraduate Medical School, London, UK.
Systemic fungal infections of humans caused by Candida spp., Aspergillus spp. and
Cryptococcus neoformans are increasing in incidence, mainly in patients with impaired immune
responses. Patient mortality is high because of difficulties in early diagnosis and the lack of
effective and safe antifungal drugs. These fungi therefore pose a serious threat to human health,
and although we have some understanding of the host defences which prevent infection in
immunocompetent individuals, little is known of the molecular mechanisms of fungal
pathogenesis. The antiphagocytic capsule of C. neoformans and melanin production by this
fungus are important virulence determinants. A range of physiological attributes and specific gene
products has also been proposed as virulence determinants of Candida albicans, but in most cases
compelling evidence has been lacking because the molecular genetics of this diploid fungus have
only been developed in recent years. The cell wall component chitin and a mannosyl transferase
are required for the fungus to cause disease in experimental models of infection. We have
identified a multigene family encoding six chitin synthases in A. fumigatus. Four of these genes
have been disrupted and although two of the mutations lead to hyphal abnormalities, the mutant
strains infect the lungs of immunosuppressed mice efficiently. We have developed a technique
called signature-tagged mutagenesis (STM) which allows large numbers of uncharacterised
insertional mutants to be tested simultaneously for loss of virulence. STM was originally applied
to the bacterial pathogen Salmonella typhimurium, and has now been adapted for virulence gene
identification in A. fumigatus and Candida glabrata as well as other bacterial pathogens.
The Fungal Cytoskeleton.
Berl R. Oakley, The Ohio State University, Columbus.
Microtubules and microfilaments comprise the two major cytoskeletal systems of
filamentous fungi. Together they are essential for mitosis, meiosis, organellar movement,
septation and polarized growth. For these two systems to function correctly, their assembly must
be regulated spatially and temporally. The microtubule cytoskeleton is regulated in part by
microtubule organizing centers such as the spindle pole body (SPB). SPBs nucleate microtubule
assembly and are presumed to establish microtubule polarity. Their ability to nucleate
microtubule assembly changes through the cell cycle. One of the major questions in microtubule
research has been identity of the components of the SPB that nucleate microtubule assembly. We
have discovered a novel protein, -tubulin that is ubiquitous in eukaryotes and is located most
obviously at microtubule organizing centers such as the SPB. Disruption of the -tubulin gene of
Aspergillus nidulans leads to blockage of nuclear division, failure of mitotic spindle formation and
a transient blockage with condensed chromosomes. Accumulating data indicates that -tubulin
rings at microtubule organizing centers nucleate microtubule assembly. In A. nidulans, the
majority of the -tubulin is associated with the nucleus (and thus, presumably, with the spindle
pole body). Approximately 30% of the -tubulin, however, is present in the cytoplasm in
complexes of a variety of sizes. We hypothesize that these complexes may be intermediates in the
formation of -tubulin ring complexes. Supported by grant GM31837 from the NIH.
Motors and filamentous fungi.
Mike Plamann, In Hyung Lee, Peter Minke, and John Tinsley. Department of Biology, Texas A&M University, College Station, TX.
Mechanochemical enzymes ("motors") hydrolyze ATP and generate force relative to either
actin filaments or microtubules. Actin-based movement is dependent on the myosin superfamily of
motor proteins, while microtubule-based movement relies on the kinesin and dynein superfamilies
of motors. Hyphae of filamentous fungi exhibit highly polarized growth which requires the
directed movement of secretory vesicles to hyphal tips and the movement and positioning of
organelles such as nuclei and mitochondria. Members of all three classes of motor proteins are
currently being studied in filamentous fungi and have been shown to be required for a diverse
array of functions including polarized growth and secretion, nuclear movement, and establishment
of the mitotic spindle. We have been conducting a genetic analysis of cytoplasmic dynein, the
most complex of the cytoplasmic motor proteins which is required for nuclear positioning in
fungi. We have shown previously that the Neurospora crassa ro-1, ro-3, and ro-4 genes encode
subunits of either cytoplasmic dynein or the dynein-associated complex dynactin. We report here
that five additional ropy genes encode novel proteins, one of which is required for the proper
intracellular localization of cytoplasmic dynein and dynactin.
Cell wall biosynthesis in Filamentous fungi.
Oded Yarden, Adi Beth Din, Vered Ziv, Einat Yatzkan, Klaas Sjollema* and Hans Sietsma*, Dept. of Plant Pathology and Microbiology, The Hebrew University of Jerusalem, Rehovot, 76100, Israel and *Dept. of Plant Biology, University of Groningen, NI-9715 NN Haren, The Netherlands.
The biosynthesis of the fungal cell wall, the major determinant of the fungal cell shape, is
spatially and temporally regulated. Components of the cell wall are synthesized in the cytoplasm,
the plasma membrane and the wall itself. One of the components of the primary cell wall is chitin.
The absence of chitin from plant and mammalian species makes the biosynthesis of this polymer
an attractive target for developing strategies to interfere with fungal growth. In filamentous fungi
the chitin synthase gene family is comprised of 5-7 genes, suggesting that chitin biosynthesis is
complex. The regulation of chitin synthase activity involves both transcriptional and post-translational regulation. Compartmentalization may be one form of chitin synthase activity
regulation, as at least some chitin syntheses are conveyed within vesicles in the fungal cell. Gene
inactivation experiments indicate that not all chitin syntheses contribute equally to fungal growth
or are essential for fungal development or pathogenesis. As filamentous proliferation is in most
cases polarized, coordination of cell wall biosynthesis must be tightly regulated and in concert
with other cellular activities. Therefore, the study of genes and their products involved in the
biosynthesis of cell wall components (e.g., chitin and glucan syntheses, hydrophobins) should be
coupled with the analysis of other factors governing hyphal growth. Various elements potentially
involved in transduction of signals governing cell wall biosynthesis and cell shape determination
are being studied. Evidence concerning the involvement of ser/thr as well as other kinases and
phosphatases in governing cell wall biosynthesis is accumulating, advancing our understanding of
the linkage between cell wall biosynthesis and other cellular processes.
Plenary Session: Evolution and Population Genetics (Chair: John Taylor)
Fungal transposable elements and genome evolution.
M.J. Daboussi, Universite Paris-Sud, Orsay, France.
Many different classes of transposable elements which reflect the whole spectrum of
eukaryotic transposons are now known in fungi. Most of our knowledge comes from species
representing different ecological situations: plant pathogens, industrial and field strains, most of
them lacking the sexual stage. A number of changes in gene structure and function have been
shown to be TE-mediated: inactivation of gene expression upon insertion, DNA sequence
variation through excision and probably by inducing extensive chromosomal rearrangements.
Moreover, TEs may have other roles in evolution related to their ability to be transferred
horizontally and to capture and transpose chromosomal host sequences thus providing a
mechanism for dispersing sequences to new sites. However, the activity of transposable elements
and consequently their proliferation within a host genome can be affected in some fungal species
which undergo meiosis by silencing processes. It is now clear that transposable elements are
ancient and ubiquitous components of the fungal genome with the potential to influence many
aspects of fungal genome evolution. Our understanding of the biological effects of TEs on the
fungal genome has increased dramatically in the past few years but much remains to be learned to
understand the long term effects of TEs on genomes, populations and species.
Phylogenetics, RDNA polymorphisms and phylogeography of the phytopathogen Fusarium.
Kerry O'Donnell and Elizabeth Cigelnik, MPR, National Center for Agricultural Utilization Research, USDA/ARS, Peoria IL.
As part of a phylogenetically-based revision of Fusarium, we investigated the evolutionary
history of the phytopathogenic Gibberella fujikuroi species complex by parsimony analysis of
DNA sequences from multiple loci. Gene phylogenies inferred from 5 loci were generally
concordant, providing strong support for a fully resolved phylogeny of most species. The
biogeographic hypothesis proposed from the phylogenetic evidence is based primarily on the
formation of natural barriers associated with the fragmentation of the ancient super-continent
Gondwanaland over the last 100 million years. Discordance of the nuclear RDNA internal
transcribed spacer 2 (ITS2) gene tree with trees from the other loci is due to nonorthologous
ITS2 sequences. The molecular evidence suggests that two highly divergent ITS2 types were
combined by an ancient interspecies hybridization or gene duplication that occurred early in the
evolutionary radiation of the Gibberella lineage of Fusarium. Only one of the two ITS2 types is
discernible within each species when conserved ITS primers were used; however, the second
ITS2 type was recovered from every strain tested with ITS2 type-specific PCR primers.
Distribution of the major ITS2 type within the species lineages obscures true phylogenetic
relationships because the major ITS2 type switched between a type I and type II sequence at least
twice during the evolution of the G.fujikuroi complex. Remarkably, the two intragenomic ITS2
types have escaped concerted evolution within this lineage of Fusarium.
Clonality and Sex in Basidiomycete Populations.
Jim Anderson, University of Toronto.
The population structure of hymenomycete fungi is largely determined by two processes:
vegetative growth and sexual reproduction. These fungi are territorial; each genetic individual
arises in a unique mating event and then grows vegetatively to colonize a territory that can vary in
size and shape. Diploid genetic individuals of Armillaria gallica thought to be at least one million
mitotic cell generations old (ca. 1000 years) offer a rare opportunity to detect and estimate the
rates of mutation, gene conversion, and mitotic crossing over in selected genomic regions under
natural conditions. In effect, the history of genetic change within the individual should be evident
in a spatial pattern reflecting growth from a point of origin. In contrast, most previous estimates
of mutation rate in natural populations have been indirect; they are based on the frequencies of
alleles whose precise origins in the population are unknown. In the sexual population of A.
gallica, from which genetic individuals arise, genotype frequencies at nuclear loci do not deviate
significantly from Hardy-Weinberg expectations and allele frequencies are not significantly
different between localities in eastern North America. These observations are consistent with a
high rate of gene flow among localities. Even mtDNA genotypes reflect the prevalence of genetic
exchange and recombination in natural populations. Most pairs of non-contiguous mtDNA loci
marked by RFLPs or nucleotide substitutions show no significant linkage disequilibrium.
Population genetics of Cryphonectria parasitica: Evolutionary inferences from studies of population structure.
Michael G. Milgroom, Cornell University, Ithaca, NY.
Analysis of multilocus genetic structure within populations can be used for making
inferences about reproductive biology and the extent to which recombination shapes populations.
The chestnut blight fungus, Cryphonectria parasitica, has much variation in its multilocus genetic
structure. A few populations are panmictic, with little evidence of nonrandom associations
between loci (gametic disequilibrium). Other populations are nearly exclusively clonal, but the
majority of populations are intermediate in structure. The structure of 11 populations of C.
parasitica in Italy were analyzed based on frequencies of vegetative compatibility (vc) types.
Genetic analyses were done to determine vegetative incompatibility (vic) genotypes for each vc
type found in Italy. We found six independent, polymorphic vic loci, each with two alleles,
however, only 20 of the 64 possible vc types were found in Italy. Most populations had
significantly fewer vc types and less genotypic diversity than would be expected under random
mating. Furthermore, 10 of the 11 populations had significant, but varying degrees of gametic
disequilibrium. Very few recombinant genotypes were found in some populations, even with
large samples, although sexual structures were commonly found in almost all populations.
Approximately 25% of the perithecia of C. parasitica sampled in field populations appear to be
self-fertilized, and even among outcrossed perithecia, there is evidence for inbreeding. The future
challenge is to understand the genetic and environmental regulation of the breeding system in this
fungus.
Pseudohomothallism and evolution of Neurospora tetrasperma.
Donald O. Natvig, David J. Jacobson*, Marian P. Skupski and Alena Gallegos. University of New Mexico and *Stanford University.
N. tetrasperma is self fertile as a consequence of each ascospore receiving one nucleus of
each mating type. Spindle overlap at the second meiotic division and programmed nuclear
movements lead to four heterokaryotic ascospores per ascus, instead of the eight homokaryotic
ascospores typical of N. crassa. This reproductive system has been termed pseudohomothahism.
Occasional single mating-type spores are self sterile and heterothalhc, suggesting the potential for
outcrossing in nature. N. tetrasperma provides examples of both the power of evolutionary
innovation and the constraints imposed by life-cycle biology. Recombination between N.
tetrasperma mating-type chromosomes is suppressed relative to that observed for N. crassa. This
suppressed recombination appears to reflect selection against homokaryotic (self-sterile)
ascospores that would result from crossovers between mating-type genes and centromeres.
Paradoxically, suppressed recombination, coupled with the unique ascospore development of N.
tetrasperma, has produced wild-type strains with high levels of heteroallelism on the mating-type
chromosome, but with virtually complete homoallelism for sequences on autosomes. Although N.
tetrasperma appears to be well suited for facultative outcrossing in nature, laboratory outcrosses
employing wild-type strains frequently result in high levels of sexual dysfunction, even when
strains are closely related. We hypothesize that the observed high levels of homoallelism on
autosomes reflects avoidance of heterokaryon incompatibility by N. tetrasperma, which is
typically found in nature as a heterokaryon. Sexual dysfunction may reflect activation of
heterokaryon incompatibility or other types of protoplasmic incompatibility. Efforts are underway
to better define the genetic basis of sexual dysfunction and explore its role in the evolution of N.
tetrasperma.
Plenary Session: Sexual/Asexual Reproduction (Chair:Mimi Zolan)
Meiotic initiation in Filamentous Ascomycetes.
Denise Zickler, Universite Paris-Sud, Orsay, France.
A cell lineage is part of the meiotic program of Podospora anserina, Neurospora crassa, N. tetrasperma, and Ascobolus immersus. After fertilization, only nuclei of opposite mating type form dikaryons which undergo karyogamy and meiosis, producing biparental progeny. Analyses of dikaryon formation showed that this process is a new example of asymmetric cell division linked to cell differentiation, including fixed patterns of nuclear division and migration with nuclear and cell death.
Functional mating type products are mainly required to distinguish self from non-self. When nuclei of P. anserina habor mutations in any one of the four genes that reside in the mating-type locus (one in mat+ and three in mat-, in collaboration with M. Picard's lab), the mutant nuclei have lost the controls that prevent haploid and uniparental diploid meiosis in wild-type strains. These nuclei can enter meiosis (haploid meiosis) and spores, demonstrating that heterozygosity of the mat genes is not a prerequisite for meiosis and sporulation.
The three components of the cytoskeleton are interdependently involved in ascus morphogenesis. They are also implicated in the segregation of non-self nuclei: (1) Recognition of opposite mating type nuclei could be mediated by chromosomal DNA located at the nuclear face of interphase spindle pole bodies (2) Three developmentally regulated MTOCs are required for proper migration of nuclei (3) Nuclei of opposite mating type that will be contained in a single cell form interconnected astral arrays of microtubules prior to the formation of that cell.
Peroxisomes are required at two steps: at the stage of meiotic cell differentiation, when
the haploid nuclei of opposite mating type undergo karyogamy, and for spore maturation. In
homozygous car1-rosses of P. anserina, the cells that would normally be committed to enter
meiosis do not differentiate and instead resume dividing.
Recognizing homology before and during meiosis.
Patricia J. Pukkila, Lesley S. Benyon, Sylvia A. Frazier, and Janet L. Knight. University of North Carolina at Chapel Hill.
The source of specificity for meiotic chromosome pairing is not known. We have shown
that prior to meiosis, a genome-wide homology search occurs in haploid nuclei of Coprinus
cinereus which results in the methylation of duplicated sequences, a process termed MIP
(methylation induced premeiotically). Genomic sequencing has confirmed that this methylation
occurs predominantly at CpG dinucleotides. MIP can be monitored in a variety of cell types that
never undergo meiosis including regenerated protoplasts recovered from dikaryotic mycelia,
asexual spores of the dikaryon (chlamydospores), and veil cells of fruit bodies. Thus, our system
is ideal to explore the nature and extent of genetic overlap between premeiotic and meiotic
homology recognition. We have analyzed two mutants that confer synaptic defects. The first
(rad3) is MIP-competent, while the second (bad27) is MIP-deficient. If DNAs methylated by
previous cycles of MIP are introduced into the bad27 mutant background, the methylation is
maintained. Our results suggest that either prior MIP is essential for synapsis, or that MIF and
meiotic pairing are genetically related.
Genetics of Chromosome Structure and Segregation.
Gregory S. May and Paul Anaya, Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030.
The condensation of chromatin into chromosomes prior to mitosis achieves several
important goals. 1) It ensures that the DNA is not broken during segregation at mitosis. 2) It
leads to the assembly of the kinetochore which interacts with the spindle microtubules and moves
the sister chromatids to the poles. How higher order chromosome structure is achieved is poorly
understood. Mitotic mutants and mutations that affect the fidelity of chromosome transmission
are likely to alter chromosomal constituents that help assemble the chromosome. The bimD6
mutation affects the ability of the chromosomes to interact with spindle microtubules. We have
identified four extragenic suppressor genes to the bimD6 mutation. One of these genes, sudA, is a
member of the SMC family of proteins. SMC proteins are widely distributed and are known to
function in chromosome condensation. A second gene, sudD, codes for another evolutionarily
conserved protein. We have cloned the budding yeast and human homologues of sudD and begun
working with these to determine this genes function. Curiously, the SUDD protein is cytoplasmic
and not nuclear as would be predicted for a suppressor of a mitotic mutant. Data will be
presented that suggests sudD and possibly sudB function not as chromosomal proteins but
upstream in the chromosome condensation pathway.
Regulation of sexual development in basidiomycete fungi.
Regine Kahmann, Erika Regenfelder, Andreas Hartmann, Julia Kruger, Tilman Spelling and Michael Bolker. Institut fur Genetik und Mikrobiologie der Universitat Munchen, Maria-Ward-Str. 1a, D-80638 Munchen, Germany.
In a number of basidiomycete fungi the mating type loci encoding pheromones and receptors have been identified. While the complex structural organization of these loci is now established the question how the pheromone signals are used to control diverse intracellular processes are just beginning to be understood.
Our work concentrates on the phytopathogenic fungus Ustilago maydis. This fungus
exhibits a dimorphic life style. Haploid sporidia grow yeast-like by budding and are non-pathogenic while the dikaryon grows filamentous and is able to induce tumors in maize plants. We
will review the known contributions of the mating type loci to this morphological transition and
then focus on an analysis of the pheromone signalling cascade. In particular we will describe the
characterisation of a G alpha subunit gene whose product plays an essential role in transmission of
the pheromone signal. The G alpha subunit required for pheromone signaling appears to be
essential for pathogenic development as well, althought this process does not require pheromone
stimulation. Interestingly, when constitutively active, this G alpha protein affects tumor
morphology. This may provide a handle to isolate molecules involved in the communication of U.
maydis with its host plant.
Vegetative incompatibility: A question of identity.
N. Louise Glass, Biotechnology Laboratory and Botany Department, University of British Columbia, Vancouver, B.C. V6T IW3, Canada.
Vegetative incompatibility is responsible for intraspecific self/nonself recognition in filamentous fungi during the assimilative phase of growth. In Neurospora crassa, vegetative incompatibility can be caused by heteroallelism at any of at least 11 het loci, including the mating type locus. The mating-type locus encodes putative transcriptional regulators that affect expression of various target genes. A suppressor of mating-type associated incompatibility, tol has been cloned and encodes a putative 922 aa polypeptide; RIP mutants are fully compatible with both A and a strains, but have no other observable phenotype. We have also cloned and characterized two additional het loci, het-c and het-6. The het-cor (for Oak Ridge) allele encodes a 966 aa coiled-coil, glycine-rich polypeptide that contains a signal sequence, suggesting that it is targeted to the fungal cell wall. RIP mutants in het-c are fully compatible, but have no other observable phenotype. Analysis of N. crassa strains indicates that het-c specificity is mediated by three alleles. Chimeric constructs localized the region determining het-c specificity to a highly variable domain of approximately 30 aa. Analysis of N. crassa strains, other Neurospora species and related genera show that the three allelic motifs that determine het-c specificity region are highly conserved.
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