125. MCH1, a gene encoding Sch9 homolog kinase of Magnaporthe grisea. Kiichi Adachi1, Martin Urban2, and John E. Hamer 1. 1Purdue University, Biological Sciences, West Lafayette, IN, USA. 2Monsanto Europe, Belgium.
In the rice blast fungus Magnaporthe grisea cAMP signaling pathways seem to control a variety of processes such as vegetative growth, asexual and sexual development and appressorium formation. Molecular genetic analysis of genes encoding adenylate cyclase (MAC1), PKA regulatory subunit (SUM1) and PKA catalytic subunit (CPKA) suggests that cAMP signaling pathways diverge at the level of PKA regulation. Two lines of evidence support this hypothesis. First, mac1 knockout mutants exhibit pleiotropic defects in growth, conidiation, mating and appressorium formation, whereas cpkA knockout mutants have a specific defect only in appressorium penetration. Second, the defects of mac1 knockout mutants are suppressed by the point mutation in SUM1. In order to identify cAMP effectors other than CpkA, we carried out PCR screening using degenerate primers designed to identify conserved subdomains in PKA catalytic subunits. However, we failed to obtain PCR products similar to PKA catalytic subunits other than CpkA. Under less stringent conditions, we identified a PCR product with high homology to the Sch9 kinase of Saccharomyces cerevisiae. Sch9 kinase has overlapping functions with PKA catalytic subunits in S. cerevisiae but is distinct from typical PKA catalytic subunits. Using the PCR product as a hybridization probe, genomic and cDNA clones of M. grisea were isolated and sequenced. MCH1 (Magnaporthe Sch9 homolog 1) encodes 714-amino acid protein with 47% identity to Sch9 kinase. Results of gene knockout experiments with MCH1 are in progress and will be presented.
126. Identification of a homeobox gene in the Euascomycete Podospora anserina.Sylvie Arnaise, and Robert Debuchy. Universte Paris XI, IGM, Orsay, Essonne, France.
The homeobox-containing genes are widely represented among eukaryotic organisms, nevertheless until now none has been identify in the filamentous ascomycetes. Here we report the discovery of a homeobox gene in the filamentous ascomycete Podospora anserina. Assuming conservation of the homeodomain motifs in eukaryotic proteins we performed PCR experiments using degenerate primers corresponding to homeodomain motifs and P. anserina cDNA. This gave an amplification product of an expected size of 120 bp. This fragment was used as a probe to screen a P. anserina library and allowed us to isolate a 3.9 kb PstI fragment which contains a putative gene with two introns and coding for a protein of 354 aa. This protein displays significant similarities with other homeodomain proteins suggesting that the 3.9 kb fragment contains a homeobox gene. Isolation of the corresponding cDNA indicated that the putative homeobox gene is transcribed and confirmed the position of the introns. To investigate the function of this gene two kinds of mutants were constructed. First the resident gene was deleted by substituting it with a recombinant gene in which the entire coding region was replaced by the bacterial hygromycin resistance gene. Second, to overexpress the gene, we replaced its 5'UTR by the higly expressed glyceraldehyde-3-phosphate dehydrogenase promoter of P. anserina. The phenotypes of these two kinds of mutant strains are being investigated and will be presented.
127. Interaction between and transactivation by mating type polypeptides of Neurospora crassa. Tom C. Badgett and Chuck Staben. University of Kentucky, Biological Sciences, Lexington, KY, USA.
The polypeptides encoded by the mating type idiomorphs of Neurospora crassa control diverse aspects of the fungal life cycle. Biochemical characterization of the MAT a-1, MAT A-1, and MAT A-3 reveal new activities that correlate with important biological activities of the polypeptides. All three polypeptides have domains capable of activating transcription in a yeast reporter system. The transcriptional activation domains of both MAT a-1 and MAT A-1 are not critical for either mating or vegetative incompatibility activities in Neurospora. Two hybrid assays establish the ability of MAT a-1 to interact with MAT A-1. Mutations that interfere with this interaction correlate with mutations that eliminate vegetative incompatibility, but not mating, in Neurospora. These results suggest the hypothesis the interaction of MAT a-1 with MAT A-1 stimulates vegetative incompatibility. Two-hybrid interaction screens of Neurospora cDNA libraries with MAT a-1 have identified other polypeptides likely to be important in the various activities associated with the mating type locus.
128. The fluffy gene of Neurospora crassa regulates macroconidiation and con gene expression.Lori Bailey Shrode, and Daniel J. Ebbole. Texas A&M University, Plant Pathology & Microbiology, College Station, Texas, USA.
Strains of an otherwise wild type background expressing a cpc-1-fl fusion gene
were induced to conidiate in minimal medium. Expression of the fl fusion gene in
the acon-2 and acon-3 mutant backgrounds demonstrated that these
genes are required for development. However, overexpression of fl combined with
nitrogen starvation was sufficient to induce conidiophore morphogenesis in the
acon-2 mutant strain. The morphogenesis induced by fl overexpression
closely resembles typical conidiophore development, however, some conidiation-induced genes
were not expressed. fl expression was always found to be sufficient for expression of
eas. However, high level expression of con-6 and con-10
was only observed in the wild type strain background and then only with growth conditions that
normally induce conidiation and con-6 and con-10 expression. Thus,
elevated fl expression is sufficient to induce conidiophore morphogenesis and
eas expression, but is not sufficient to induce con-6 and
130. Neurospora mating pheromone precursor genes.
130. Neurospora mating pheromone precursor genes.Piotr Bobrowicz1, Rebecca Lowe2, Amy Miller1, Wei-Chiang Shen1, Pam Kazmierczak1, Lori Bailey Shrode1, Daniel J. Ebbole1, Deborah Bell-Pedersen2, and Neal K. Van Alfen1. 1Texas A&M; University, Plant Pathol. & Microbiol, College Station, Texas, USA. 2Texas A&M; University, Biology, College Station, Texas, USA.
The mating type loci of N. crassa encode regulators that control expression of genes involved in sexual fertility and development. We have begun to analyze the genes encoding the sex pheromones of N. crassa. One gene, expressed in mat-A strains, encodes a polypeptide containing multiple repeats of a putative pheromone sequence bordered by KEX2 processing sites. The predicted sequence of the pheromone is remarkably similar to those encoded by the rice blast fungus, Magnaporthe grisea, and the chestnut blight fungus, Cryphonectria parasitica. mat-a strains express a pheromone precursor gene whose polypeptide contains a C-terminal CAAX motif predicted to produce a mature pheromone with a C-terminal carboxy-methyl isoprenylated cysteine. The pheromone precursor genes are regulated by nutrients, macroconidiation, and the circadian clock, but display strict mating type specificity. They are repressed by the RCO1 repressor, but repression by RCO1 is not required to maintain mating type specificity.
131. A Tc1-like Transposable Element in Coccidioides immitis.Michael A. Bolaris1, Tao Peng2, Kris I. Orsborn2, John N. Galgiani2, and Marc J. Orbach 1.1University of Arizona, Plant Pathology, Tucson, Arizona, USA. 2VA Medical Center, Infectious Diseases, Tucson, Arizona, USA.
We report the isolation, characterization and distribution of a Tc1-like transposable element in Coccidioides immitis, a dimorphic fungal pathogen of mammals known as the Valley Fever fungus. During the analysis of a Proline Rich Antigen (PRA) gene which is being used for vaccine development, a Tc1 transposable element was discovered upstream of the PRA open reading frame (ORF). A complete copy of this element has been cloned from the Silveira strain of C. immitis and designated Tcim1 for transposon C. immitis 1. The transposon is approximately 1.8 kb in size, and contains a single open reading frame with homology to the Tc1 class of transposases. The transposase ORF is interrupted by two introns. Inverted terminal repeats of 29 bp are present at the ends of this transposon, a characteristic of this class of element. DNA hybridization analyses have shown variation in the copy number among strains of C. immitis, ranging from no copies to eight copies. Comparisons of Tcim1 banding patterns between Arizona and California isolates will be presented. Preliminary results suggest that there may be different intensities of hybridization among these strains and this may represent sequence divergence as a result of geographic isolation. This is the first transoposon isolated from this human pathogen and may be a useful tool for typing strains and for the study of C. immitis populations. Very little research has been conducted with field isolates of C. immitis, with most strains used in research being clinical isolates. We are using a PCR-based method to identify C. immitis soil isolates, to compare their Tcim1 profiles to those of clinical isolates.
132. Cloning and characterisation of an Aspergillus nidulans abaA homologue in the dimorphic fungus Penicillium marneffei. Anthony R. Borneman, Michael J. Hynes, and Alex Andrianopoulos. University of Melbourne, Gentics, Parkville, Victoria, Australia.
Penicillium marneffei like many other pathogenic species of fungi is dimorphic.
At 25 C, P. marneffei exhibits filamentous growth morphology, reproducing through
the production of asexual spores borne on conidiophores. At 37C a yeast-like growth form
replaces the filamentous morphology with single cells reproducing via fission. This form is
pathogenic, and is associated with many cases of human and animal infection. Here we describe
the cloning and characterisation of a P. marneffei gene which encodes an ATTS
DNA binding domain protein - PmabaA. In Aspergillus nidulans,
AbaA (an ATTS protein) regulates development of sporogenous cells during
conidiation. In Saccharomyces cerevisiae, Tec1p, an AbaA homologue
regulates pseudohyphal development. This implies a conserved role for ATTS proteins in
regulating fungal development systems. A P. marneffei homologue may also have a
regulatory role during development in this species, perhaps during dimorphic growth.
PmabaA was therefore cloned via low stringency hybridisation to the A.
nidulans abaA gene. A high level of homology between the two is apparent at the protein
level, with complete conservation of the DNA binding motif. Amino acid conservation is limited
at the C-termini of the proteins and may suggest functional divergence in the P.
marneffei homologue however it is able to complement the abaA mutation in
A. nidulans indicating functional conservation. A transformation protocol for this
fungus was developed and enabled the creation of a PmabaA mutant by targeted
deletion. The aconidial mutant phenotype at 25C indicates that PmabaA, like its
A. nidulans homologue plays a crucial role in regulating cellular morphogenesis.
The cloned abaA genes from both P. marneffei and A.
nidulans complement the mutant phenotype, indicating functional interchangeability
between the two proteins in vivo.
133. Two fungal galectins: their expression, localization and secretion during fruitbody
133. Two fungal galectins: their expression, localization and secretion during fruitbody development. Robert P. Boulianne1, Ursula Kües1, Benjamin C. Lu2, and Markus Aebi2. 1ETH, Institut für Mikrobiologi, Zurich, Zurich, Switzerland. 2University of Guelph, Molecular Biology and Gen, Guelph, Ontario, Canada.
Fruitbody development in Coprinus cinereus requires a transition from a linear radiating pattern of hyphal growth to the development of multiple hyphal:hyphal interactions that comprise the fruitbody. These initial stages of fruitbody morphogenesis occur during knot, initial and primordial stages of development. We isolated two fungal galectins, a pair of low molecular weight lectins that specifically bind beta-galactose residues and their derivatives. We show that these proteins are essentially fruitbody specific proteins and their expression is differentially regulated with respect to light, nutrition and the presence of compatible A mating type genes. The dimeric galectins are believed to mediate hyphal:hyphal interactions, since they are present within the cell walls and the extracellular matrix. While it is clear that galectins are secreted, they lack typical secretion signal sequences and are not glycosylated, even though potential N-linked glycosylation sites are present. In order to test whether galectins are secreted independently of the classical secretory pathway, we examined the secretion of galectins when expressed (heterologously) in Saccharomyces cerevisiae. The Coprinus galectins were secreted to the cell wall in yeast. In mutants blocked in the transport of proteins from the ER to the Golgi (sec18) the galectins were still found extracellularly. These results show for the first time that a filamentous fungal protein can be secreted independently of the classical secretory pathway.
134. Elucidating the role of linoleic acid in Aspergillus development. Ana M. Calvo-Byrd1, Lori L. Hinze1, Harold W. Gardner2, and Nancy P. Keller1. 1Texas A&M; University, Plant Pathology and Micro, College Station, Texas, USA. 2 USDA-ARS, NCAUR, Peoria, Illinois, USA.
Development of conidia and overwintering bodies (cleistothecia and sclerotia) of Aspergillus nidulans, Aspergillus flavus and Aspergillus parasiticus is affected by linoleic acid and light. Specific morphological effects of linoleic acid include inducing precocious and increased asexual spore development in A. flavus and A. parasiticus strains and altered sclerotial production in some A. flavus strains where sclerotial production decreased in the light but increased in the dark. In A. nidulans, the asexual to sexual spore ratio increased with increasing amounts of linoleic acid. Spore development was also induced in all three spp. by hydroperoxylinoleic acids, linoleic acid seed-derivatives produced during fungal colonization. The sporogenic effects of these linoleic compounds on A. nidulans are similar to the sporogenic effects of A. nidulans psi factor, an endogenous mixture of hydroxylinoleic acid moieties. Light significantly increased asexual production in all three spp. The sporogenic effects of light, psi factor, linoleic acid and linoleic acid derivatives on A. nidulans required an intact veA gene. To better study the function of linoleic acid in Aspergillus development, the gene required for the formation of linoleic acid, odeA, that encodes a delta-12 desaturase, has been cloned and disrupted in A. nidulans veA+ and veA1 strains. A better understanding of the molecular mechanisms governing Aspergillus lipid metabolism could contribute to the design of control strategies to reduce the survival and spread of seed-colonizing aspergilli.
135. Analysis of white collar 2 mutants reveals a central component of the Neurospora circadian clock and distinct mechanisms regulating frequency in light and dark. Mike A. Collett, Jay C. Dunlap, and Jennifer J. Loros. Dartmouth Medical School, Dept of Biochemistry, Hanover, NH, USA.
White collar 2 (wc-2), encoding a PAS domain containing, Zn-finger
transcription factor, is essential for almost all blue light responses in Neurospora. It
was predicted that wc-2 would be a component of the Neurospora
circadian clock, acting by increasing levels of the central clock component,
frequency (Crosthwaite et al., Science 276: 763-769, 1997). To test this we
examined the effect on the clock of several wc-2 alleles thought to be partially
functional. It was expected that if wc-2 was a clock component the partially
functional wc-2 alleles might yield an altered period length as well as lowered levels
of frq transcript and protein in constant darkness. Indeed, compared to wild type,
strains containing one of these partially functional alleles, wc-2 (ER24), displayed
increased period length, reduced temperature compensation and reduced levels of frq
mRNA and FRQ protein in constant darkness. We also examined photoinduction of
frq transcript, which in a wild-type strain is robustly induced in response to light, in
these mutant strains. In either constant light, or after a light pulse, strain wc-2
(ER24) displayed frq mRNA and protein levels virtually identical to wild type.
However, presumptive loss of function alleles of wc-2 had reduced levels of
frq mRNA under all conditions tested. These data suggest that wc-2's
function in frq regulation is essential and limiting in constant darkness. However,
although wc-2 is required for full frq mRNA induction by light, it is not
limiting in the light signal transduction pathway leading to frq transcription.
136. Vegetative coexpression of the mating-type genes determining nuclear identity is lethal
in Podospora anserina.
136. Vegetative coexpression of the mating-type genes determining nuclear identity is lethal in Podospora anserina.Evelyne Coppin and Robert Debuchy. Université Paris Sud - CNRS URA 2225, Batiment400, Orsay, 91405, FRANCE.
The mat+ and mat- mating types control a recognition process between cells during fertilization and between nuclei after fertilization, when reproductive male and female nuclei which have divided in a common cytoplasm form a mat+/mat- pair within the ascogenous hyphae. Four genes encoding transcriptional factors have been characterized, FPR1 in the mat+ locus, FMR1, SMR1 and SMR2 in the alternative mat- locus. Fertilization is controlled by FPR1and FMR1 while internuclear recognition inside the fruiting body is controlled by FPR1 which confers the mat+ nuclear identity and FMR1 and SMR2which confer the mat- nuclear identity.SMR1 would be necessary to restore growth of ascogenous hyphae after the nuclei have paired. By using the RT-PCR method, mature transcripts of the four mat genes were detected in the fertilized female organs whereas only FPR1 and FMR1 transcripts were detected in vegetative mycelium. Increased or induced vegetative expression of the four mat genes by replacing their natural promoter by the constitutive Aspergillus nidulans gpd promoter has no vegetative effect when the recombined gene is alone in the wild-type strain. However, mat+ ascospores carrying both a gpd::FMR1 fusion and a constitutively transcribed SMR2 transgene are unable to germinate. Ascospore lethality is suppressed by introduction of the gpd::SMR1 fusion. This suggests that the vegetative coexpression of the three nuclear identity genes triggers the recognition mechanism at an inappropriate time and thus interferes with some essential growth process and this confirms the role of SMR1 in growth restoration following internuclear recognition.
137. Addressing clonality in Magnaporthe grisea using vegetative compatibility. James C. Correll1, Tyler L. Harp1, Fleet N. Lee1, and Robert S. Zeigler2. 1University of Arkansas, Plant Pathology, Fayetteville, AR, USA. 2Kansas State University, Plant Pathology, Manhattan, KS, USA.
Spontaneous nitrate nonutilizing (nit) and sulfate non-utilizing (sul) mutants were recovered from P. grisea and used to characterize a collection of archived and contemporary rice-infecting field isolates into vegetative compatibility groups (VCGs). Pairing of certain phenotypically distinct nit mutants yielded robust heterokaryons as did certain nit x sul pairings. However, some cross-feeding was evident in some nit x sulpairings indicating that caution should be exercised in the interpretation of such reactions. Four distinct VCGs were identified among archival and contemporary rice-infecting isolates of P. grisea collected from Arkansas, Texas, Louisiana, and California. Each VCG corresponded with a distinct MGR586 group (or lineage) among the contemporary isolates in all but one case; VCG US-004 contained contemporary isolates collected from Arkansas belonging to MGR586 group D and contemporary isolates recovered from California in 1997 which belonged to group H. VCG US-004 also contained two isolates recovered from Texas and Arkansas in the mid 1970's which belonged to MGR586 group H. The data indicate that distinct barriers to vegetative compatibility are present in P. grisea and that certain VCGs may represent genetically distinct populations in Arkansas. However, the impact vegetative compatibility barriers have on horizontal gene transfer in P. grisea is unknown. A preliminary genetic analysis of vegetative compatibility with the sexually compatible strains Guy11 and 2539 of P. grisea indicated that multiple vegetative incompatibility (vic) loci were present. The use of VCGs may be very helpful in characterizing the population structure of this important pathogen worldwide.
138. An efficient genetic system for analysis of Gibberella circinata. Sarah F. Covert, Angela Briley, and M. Margaret Wallace. University of Georgia, Forest Resources, Athens, GA, USA.
Genetic analysis of Gibberella circinata, the causative agent of pitch
canker disease of pines, has been thwarted by a low frequency of mating success in the
laboratory. We describe two findings which should facilitate genetic analysis of this fungus and
related species. First, we determined that previously described degenerate primers can be used to
amplify a portion of the MAT-2 mating type gene from G. circinata.
This led to cloning and sequencing of a fragment of the MAT-2 gene, which in turn made it
possible to distinguish between G. circinata isolates of opposite mating
types either by hybridization or PCR amplification. Second, we discovered that out of the 18
G. circinata isolates in our collection, the one female fertile isolate expressed its
fertility at 20°C, but not at 25°C, the temperature typically used for crossing the
closely related Gibberella fujikuroi mating populations. It is evident,
therefore, that when sexual reproduction in other closely related species is initially being
investigated, the crosses should be established at a variety of temperatures. Once we learned that
female fertility in this G. circinata isolate was expressed at 20°C, a high
frequency of mating success was achieved. Eleven out of seventeen isolates crossed successfully
with this isolate. PCR analysis indicated that five of the six non-mating isolates were of the same
mating type as the female fertile isolate, and thus expected to be incompatible with it.
139. cAMP-dependent neutral trehalases catalyze intracellular trehalose breakdown during
spore germination in Aspergillus nidulans and Neurospora crassa.
139. cAMP-dependent neutral trehalases catalyze intracellular trehalose breakdown during spore germination in Aspergillus nidulans and Neurospora crassa.Christophe d'Enfert 1, Beatriz M. Bonini2, Pio D.A. Zapella3, Thierry Fontaine4, Marie-Kim Chaveroche1, Aline M. da Silva3, and Hector F. Terenzi2. 1Institut Pasteur, Physiologie Cellulaire, Paris, , France. 2Universidade de Sao Paulo, Departamento de Biologia, Ribeirao Preto, SP, Brazil. 3Universidade de Sao Paulo, Departamento de Bioquimic, Sao Paulo, SP, Brazil. 4Institut Pasteur, Laboratoire Aspergillus, Paris, France.
A cAMP-activable Ca 2+ -dependent neutral trehalase was identified in germinating conidia of Aspergillus nidulans and Neurospora crassa. Using a PCR approach, Aspergillus nidulans and Neurospora crassa genes encoding homologues of the neutral trehalases found in several yeasts were cloned and sequenced. Disruption of the AntreB gene encoding A. nidulans neutral trehalase revealed that it is responsible for intracellular trehalose mobilization at the onset of conidial germination and that this phenomenon is partially involved in the transient accumulation of glycerol in the germinating conidia. While trehalose mobilization is not essential for the completion of spore germination and filamentous growth in A. nidulans, it is required to achieve wild-type germination rates under carbon limitation suggesting that intracellular trehalose can partially contribute the energy requirements of spore germination. Furthermore, it was shown that trehalose accumulation in A. nidulans can protect germinating conidia against an otherwise lethal heat shock. Since transcription of the treB genes is not increased following a heat-shock but induced upon heat-shock recovery, it is proposed that, in filamentous fungi, mobilization of trehalose during the return to appropriate growth is promoted by transcriptional and post-translational regulatory mechanisms, in particular cAMP-dependent protein kinase-mediated phosphorylation.
140. A molecular dissection of signaling cascades regulating mating and virulence in Cryptococcus neoformans. Robert C. Davidson, and Joseph Heitman. Duke University, Genetics, Durham, NC, USA.
Recent findings reveal that a pheromone induced mating pathway operates in the human fungal pathogen Cryptococcus neoformans; similar to the mating pathway characterized in Saccharomyces cerevisiae. Mating in S. cerevisiae is mediated by pheromones recognized by membrane bound receptors which trigger a MAP kinase cascade, culminating in transcription of mating response genes. We have cloned a gene, CPK1, which encodes a homolog of the FUS3/KSS1 MAP kinases in the pheromone response pathway of S. cerevisiae. In contrast to several other homologs of the mating response pathway found in C. neoformans, which are mating type specific, the CPK1 gene is present in both MATa and MATalpha cells. This indicates that the gene is not located within the MATalpha locus that contains the MFalpha1 and MFalpha2 pheromone genes, and the STE11 and STE12 homologs. Furthermore, this observation suggests that at least this element of the signaling cascade is shared by the two mating types. Disruption of the CPK1 gene by homologous recombination reveals that the CPK1 MAP kinase is required for mating in both mating types and is required for MATa cells to autorespond to the MFalpha1 pheromone. This finding suggests that MATa cells may contain divergent homologs of STE11 and STE12involved in mating. In addition a MATalpha cpk1 mutant strain is defective in haploid fruiting. The cpk1 defect in haploid fruiting is suppressed by overexpression of CnSTE12, indicating that CPK1 functions upstream of STE12 in this cascade. Furthermore, conserved MAP kinases play an essential role as virulence factors in pathogenesis of the rice blast fungus Magnaporthe grisea and the human pathogen Candida albicans, and CPK1 may play a similar role in C. neoformans.
141. Nuclear positioning model and internuclear recognition in Podospora anserina. Robert Debuchy. Université Paris-Sud, IGM Bâtiment 400, Orsay, France, France.
In heterothallic Euascomycetes, fertilization is followed by mitotic divisions of parental
nuclei, resulting in a plurinucleate stage. Nuclei of opposite mating type then recognize one
another and form dikaryons which undergo karyogamy and meiosis within ascogenous hypha.
Genetic analysis indicated that internuclear recognition (IR) in P. anserina is
controlled by mating-type genes. These genes encode transcription factors which have
nucleus-limited expression. This allows nuclei with different mating type to express a pattern of
specific proteins directly involved in IR. As the molecular nature of these proteins is unknown,
the exact mechanism of internuclear recognition remains elusive. Schuurs et al.
(1998) have proposed that internuclear distance affects gene expression through a
pheromone/receptor system in Schizophyllum commune. A similar system is
proposed to control IR in P. anserina. Each nucleus controls the expression and
localization of specific pheromones and receptors for the opposite mating-type pheromone in a
portion of the wall and membrane respectively. When two compatible nuclei are close enough to
superimpose their specific domains, this permits the binding of pheromones to the responsive
receptors and generates a spatial cue.This cue triggers the formation of the ascogenous hypha and
the migration of the two compatible nuclei inside the budding ascogenous hypha. Genetic data
suggest that the genes controling IR could also control degradation systems which help the nuclei
to preserve their domain from contamination by compatible pheromones. Schuurs TA, Dalstra
HJP, Scheer JMJ and Wessels JGH. 1998. Fung. Genet. Biol. 23: 150-161.
142. Interfertility among self-sterile strains of Glomerella
142. Interfertility among self-sterile strains of Glomerella graminicola. Meizhu Du1, Juan Wang2, Robert Hanau2, and Lisa Vaillancourt1. 1University of Kentucky, Plant Pathology, Lexington, KY, USA. 2Purdue University, Botany and Plant Patholog, West Lafayette, IN, USA.
Although the ascomycete Glomerella graminicola was originally described as homothallic, self-sterile but cross-fertile strains are common. Furthermore there appear to be multiple mating types among these cross-fertile isolates. The mechanism of this unusual mating system is unknown, but it is proposed that it involves complementary mutations in the developmental pathway for self-fertility. Analysis of random spore and tetrad progeny from a cross of two self-sterile isolates of G. graminicola indicated that sexual compatibility between these strains is controlled by two loci. One of the loci appeared to confer self-nonself recognition, a characteristic reminiscent of the mating type idiomorphs that regulate interfertility in heterothallic ascomycetes such as Neurospora crassa and Cochliobolus heterostrophus A degenerate PCR approach designed to amplify a DNA fragment which is highly conserved among ascomycete mating type idiomorphs was performed using the two G. graminicola isolates. The conserved DNA fragment was present in both of the compatible self-sterile isolates of G. graminicola , suggesting that mating type idiomorphs are not primarily responsible for cross-fertility between these strains. Experiments are currently underway to determine whether the conserved sequence is at the same locus in both strains, and also to evaluate the distribution of this sequence among self-sterile and self-fertile strains of G. graminicola
143. Molecular links between morphogenesis and pathogenicity in Ustilago maydis. Flora Banuett. Department of Biochemistry and Biophyscs. University of California. San Francisco, CA 94143.
Ustilago maydis, the causal agent of corn smut disease, exhibits three basic cell morphologies during its life cycle: 1) a unicellular haploid form, 2) a filamentous dikaryotic form, and 3) a round cell type, the teliospore. The teliospore, which exhibits no vegetative growth, undergoes meiosis and gives rise to the unicellular haploid form. The unicellular form is cigar-shaped, grows by budding, and is not pathogenic. Haploids of different mating type fuse to give rise to the filamentous dikaryon.This form is pathogenic and is characterized by cylindrical cells separated by septa. It undergoes a discrete developmental pathway in the plant resulting in formation of round teliospores, which are surrounded by a specialized cell wall resistant to adverse environmental conditions. Genetic programming by the mating type loci (a and b), cell-cell signalling, plant signalling, and environmental conditions govern these morphological transitions. I am interested in the molecular mechanisms that govern morphology of the different forms in U. maydis and how the machinery responsible for maintenance of shape affects pathogenicity. In order to identify the machinery responsible for morphology, I have taken a multiprongued approach: isolation of morphological mutants and a candidate gene approach. A mutation that alters cell shape and the developmental program in the plant has led to the identification of a gene with similarity to samB of A. nidulans. This and other results will be described.
144. Biogeographical relationships and microevolutionary patterns in the Tricholoma matsutake species complex. Matteo Garbelotto and Ignacio H. Chapela. Department of Environmental Science, Policy and Management, University of California, Berkeley, CA, USA.
Matsutakes, a taxonomically challenging complex of related Tricholoma species, are intensively harvested in the Northern Hemisphere as valuable non-timber forest products. Ribosomal DNA sequencing and Amplified Fragment Length Polymorphism (AFLP) analyses were employed to search for a clarification of (a) species delimitation, (b) biogeographical relationships, and (c) evolutionary patterns within this group. Twenty-five taxa, traditionally regarded as very close relatives, fell into one of two sister clades, one including the commercial matsutakes, and one including species from the zelleri-focale-robustum complex. We find that species complexes in both clades appear to have their origin in an angiosperm-related ectomycorrhizal symbiosis, and that tracking of hosts in evolving forests in North America and Eurasia led secondarily to a conifer association. Commercial matsutakes are all part of a recently derived, conifer-associated lineage. Sequencing and AFLP data indicate that this lineage contains natural groupings which are not consistent with either accepted taxonomic arrangements nor with the market's price-structure: Eurasian matsutakes are vicariantly related to Eastern North American and Mexican fungi but surprisingly not to populations from the North American Pacific North West. The North American continent, by including all three commercial taxa, is the likely region of speciation for the complex. This study elucidates evolutionary relationships among different matsutakes species, and suggests a model for their present-day distribution and population structure. We interpret the new understanding of the systematics in this narrow group as an expression of a dynamic geographic mosaic of ectomycorrhizal forests in North America, where Eocene climatic changes led to a confrontation, and then separation, of interacting fungal populations in angiosperm and coniferous hosts.
145. Sexual development of Aspergillus nidulans tryptophan auxotrophic strains. Sabine E. Eckert and Gerhard Braus. Univ. of Goettingen, Molecular Microbiology, Goettingen, Niedersachsen, Germany.
A study on the interplay between sexual development and tryptophan biosynthesis in the ascomycete Aspergillus nidulans was initiated. To elucidate the connection, strains were examined which are unable to regulate their tryptophan biosynthesis and are dependent on external supply. Therefore, the auxotrophic mutants trpA, trpB, trpC and trpD were tested for growth and differentiation. These strains which carry mutations in the structural genes for the biosynthetic enzymes of each step in the formation of tryptophan from chorismate, are unable to form fruitbodies on medium containing low tryptophan concentrations. We show that cleistothecia formation can be restored by high tryptophan supplementation. Cleistothecia formation of the trpA and trpD strains can also be restored by high concentrations of either the tryptophan precursors anthranilate and indole or auxin. Fertilty of ascospores of the strains can only be restored to about one hundredth of the wild-type fertility. Oversupplementation with tryptophan inhibits cleistothecia formation, but not conidiation. Tryptophan supplementation was found to result in auxin production of A. nidulans. trpC transcript levels and enzyme activities remain stable during development whereas transcript levels and enzyme activities of another biosynthetic gene as control are reduced. We conclude an extensive connection between a balanced tryptophan biosynthesis and sexual development in Aspergillus nidulans .
146. Identification of mating regulated cDNAs in Phytophthora infestans. Anna-Liisa Fabritius, and Howard S. Judelson. University of California, Plant Pathology, Riverside, California, USA.
Sexual development in the heterothallic oomycete, Phytophthora infestans, is a complex process that involves the differentiation of male and female gametangia, meiosis, fertilization, and formation of a diploid sexual spore called the oospore. To study molecular events underlying this development, we have compared gene expression between mating and vegetative cultures. The suppression subtractive hybridization technique was used to obtain cDNAs that showed differential gene expression. Subtracted cDNA libraries of 7500 to 8000 clones were constructed from mating and non-mating cDNAs, and screening for differential gene expression is underway. In preliminary screening, three genes were identified that are up-regulated and two which are down-regulated during mating as suggested by Northern blot analysis; additional clones are currently being characterized. Also, two cDNAs were cloned that were expressed in either A1 or A2 mating type strains used in this study but were not present in the genomic DNA. The origin of these cDNAs may be extrachromosomal and is currently being investigated. Sequencing of differentially expressed cDNAs suggested that one of the down-regulated cDNAs is very similar to sorbitol dehydrogenases (P<= 1.4 x 10-15). All other cDNA clones represent novel genes since no significant matches were found in databases. To study the structure and function of the corresponding genes, genomic clones have been isolated from the bacterial artificial chromosome (BAC) library and subcloned
147. The cyclin homologue PclA: A new regulatory protein for asexual development of Aspergillus nidulans. Niklas Schier, Ralf Liese and Reinhard Fischer. Max-Planck-Institut für terrestrische Mikrobiologie, Karl-von-Frisch-Str., D-35043 Marburg, Germany.
Cyclins comprise a family of proteins, which interact with cyclin-dependent kinases (CDK), the key regulators of the eukaryotic cell cycle. Here, we show that a yeast Pcl1 homologue, named PclA, is involved in the regulation of asexual development of the filamentous fungus Aspergillus nidulans. A new developmental mutant derived from insertional mutagenesis was genetically and phenotypically characterized. The mutant produces multiple layers of phialides and thus resembles the abaA mutant. It fails to produce chains of conidiospores. Vegetative growth and sexual development is not affected in the mutant. The mutation was mapped to linkage group VIII and the mutant phenotype was complemented by transformation with a chromosome VIII specific cosmid library of A. nidulans. A 2.1 kb EcoRI-fragment complemented the mutation in trans and hybridized to a 2.3 kb transcript in RNA extracted from vegetative and developmental cells. The ORF of the cDNA encodes a putative 420 aa protein, which is 34 % identical to the yeast cyclins Pcl1 and Pcl2 (279 aa and 308 aa in length). An N-terminally truncated PclA derivative (271 aa) was sufficient for rescuing the mutant phenotype. Determination of the pclA1 mutation revealed a Protein kinase C phosphorylation site to be essential for the biological function of PclA. These results suggest a cyclin-dependent phosphorylation cascade triggering the late stages of asexual development of A. nidulans.
148. Determining fungal phylogenies by the use of microsatellite markers. Matthew C. Fisher, and John W. Taylor. UC Berkeley, Plant Biology, Berkeley, CA, USA.
Uncovering the correct phylogeny of closely related fungal species requires data from multiple gene genealogies. However, this requires considerable investment in sequencing or SSCP strategies because polymorphisms are rare and taxon sampling needs to be as inclusive as possible. An alternative to using sequencing strategies is the use of short tandem repeats (STRs, or microsatellites) as phylogenetic markers. These markers are highly polymorphic, theoretically enabling the determination of recently evolved lineages, and easily scored. However, a concern in their use in phylogenetic reconstruction is the potential for constraints on allele sizes to occur, resulting in homoplastic distributions of alleles. Here, we report the development of a panel of microsatellites from the pathogenic fungus Coccidioides immitis and analyze the mutational processes that lead to the patterns of genetic diversity seen in this species complex.
149. Population level genetic differences observed between geographically distant populations of Ustilago maydis. James R. Garton. University of Minnesota, Plant Biology, St. Paul , MN, USA.
Genetic analysis of Ustilago maydis populations shows differences in the structure of these populations. Analysis was performed with RFLP probes generated by a partial restriction digest of U. maydis. Fungal isolates from Minnesota, Illinois, Nebraska, North Carolina, Ohio, and Uruguay were analyzed with these probes. Data gathered in this analysis shows that populations separated by greater geographical distances were also less similar to each other genetically. This preliminary data suggests that gene flow does not occur at a high level in this pathogenic species.
150. Small GTPases and cytoskeletal organization at the mating of Schizophyllum.Markus Gorfer, Marjukka Uuskallio, and Marjatta Raudaskoski. Department of Biosciences, University of Helsinki, Helsinki, Finland.
Homobasidiomycetes, such as Schizophyllum commune, respond to potential mating partners by recognizing specific pheromones secreted by cells of opposite mating-type. Recently it has been shown that the pheromones are recognized by serpentine 7TM receptors probably linked to a heterotrimeric G-protein. The perception of the pheromone signal leads to reciprocal exchange and migration of nuclei between the mates, which implies hyphal fusions, septal break-down and reorganization of cytoskeletal components. In animal and yeast cells small GTPases, such as the products of the rho-, ras- and cdc42-gene families are known to affect the cytoskeletal organization. We have used the polymerase chain reaction to isolate members of these gene families from S. commune. The cloned PCR- products were further used for screening the cDNA library prepared from 8 h mating between compatible strains, at which time the expression of the pheromone and receptor encoding genes is high. This procedure has led us to isolate cDNAs of a rho and ras gene from S. commune highly homologous with Saccharomyces cerevisiae RH03 and Neurospora crassa ras2 genes, respectively. The Northern hybridization revealed an increase in the rho3 but not in the ras2 expression at the mating in comparison with that in the nonmated homokaryons. A genomic clone of rho3 was isolated and mutagenized at amino acids corresponding to the mutations G12V and T17N in p21 to create an constitutively activated GTP-bound and a dominant negative GPD-bound form of rho3, respectively. Effects of expression of these mutant rho3-alleles in a wild-type background should allow the elucidation of RHO3-function in signal transduction and cytoskeletal organization at the intercellular nuclear migration during mating of compatible homokaryons of S. commune.
151.Characterization of two swo mutants involved in polarity establishment and maintenance in Aspergillus nidulans. Debra L. Haas and Michelle Momany, University of Georgia, Athens, GA, 30602 .
The germination of asexual propagules of Aspergillus nidulans employs two distinct growth modes: isotropic growth and polarized growth. When spores of A. nidulans initiate germination, they first grow isotropically adding cell wall material uniformly in every direction. Later they switch to polarized growth, with new wall components added to the tips of emerging germ tubes. This polarized growth continues via apical extension and results in hyphae and ultimately a vegetative colony. We previously screened for mutants defective in the germination process and identified 8 non-allelic mutants, swoA-H. (swo for swollen phenotype) Here we describe the initial characterization of 2 swo mutants, swoD and swoF. The kinetics of germination and septation of swoD and swoF mutants, as well as the assignment of linkage groups and initial cloning of the swoD and swoF genes are presented.
152. The nsdD gene encoding a putative GATA type transcription factor regulates positively the sexual development of Aspergillus nidulans. Kap-Hoon Han, and Dong-Min Han. Wonkwang University, Division of Life Science, Iksan, Chonbuk, South Korea.
The nsdD (never in sexual development) gene encodes a putative GATA type transcription factor that has a type IVb zinc finger DNA binding domain at the C-terminus. Null mutants that the total nsdD ORF was deleted showed the typical NsdD phenotype. However, being cultured for a week or more after inoculation onto the center of the plate, a few of cleistothecia bearing mature ascospores could be found in null mutant strains. On the other hand, the nsdD19 mutant strain which contains a nonsense mutation within the nsdD coding region never developed cleistothecia at all under the same conditions, suggesting a dominant negative effect of the truncated NSDD proteins. When NSDD was over-expressed under the niiA promoter with nitrate, the number of cleistothecium was dramatically increased, implying a positive regulatory role of NSDD on sexual development. Northern blot analysis using total RNAs from a wild-type strain grown in minimal media revealed that the nsdD gene was expressed throughout the life cycle. However, its expression was repressed under the certain conditions, such as in the presence of high concentrations of salts, under which sexual development was inhibited. It was also found that the phenotypes of reduced or delayed sexual development in veA1 or nsdB5 could be suppressed when multiple copies of the nsdD gene are present, or the gene is over-expressed.
153. Reactive oxygen species and development in N. crassa. Wilhelm Hansberg, Fernando Lledías, Adelaida Díaz, and Pablo Rangel. Universidad Nacional Autónoma de México, Biochemistry, México, D.F., Mexico.
Since we have demonstrated specific enzyme oxidation under cell differentiation and stress conditions, we asked if antioxidant enzymes like catalases were also vulnerable to in vivo alteration by reactive oxygen species. Cat-1 is present in the whole vegetative life-cycle of N. crassa. Its specific activity increases step wise with each morphogenetic transition of the conidiation process. Cat-1 was modified in these transitions and under stress conditions. Purified Cat-1 was oxidized through a sequential reaction of the four monomers with singlet oxygen, giving rise to five active catalase conformers with increasingly acidic isoelectric points, similar to the ones observed in vivo. Cat-1 was modified with a pure source of singlet oxygen and modification was hindered by reducing agents or singlet oxygen scavengers. Cat-1 modification was traced to the heme. Heme molecular mass and asymmetry increased with modification. Bacterial, fungal, plant, and animal catalases were susceptible to modification by singlet oxygen. Reaction with singlet oxygen could explain in part the variety of catalases in several organisms and modifications detected in some catalases. Modification of catalases during development and under stress could indicate in vivo generation of singlet oxygen. The oxidation of Cat-1 was used to monitor generation of singlet oxygen during germination of conidia. Oxidation of total protein increased within 10 min of germination and was correlated with light intensity. Oxidized Cat-1 appeared during germination in relation to light intensity and correlated inversely with the amount of carotenes with eleven or more conjugated double bonds. Our results indicate that, as soon as conidia are hydrated, Cat-1 was oxidized, oxidized enzyme disappeared and Cat-1 was synthesized de novo; changes were more apparent in the carotene mutant strains than in the wild type. Acknowledge: supported in part by grants IN206097 and IN208994 from DGAPA, Universidad Nacional Autónoma de México and grant 2246P-N9508 from Consejo Nacional de Ciencia y Tecnología, México.
154. Mating type genes of Heterobasidion annosum, S-type. Nils O S. Högberg, and Tom Bruns. UC Berkeley, Plant & Microbial Biology, Berkeley, CA, USA.
Heterobasidion annosum (Fr.) Bref. is a heterothallic bipolar basidiomycete fungus with a large number of alleles at the mating type locus. It forms a species complex with several intersterility groups. The S-group of H. annosum is a pathogen of spruce and fir. The mating type locus in this species include homeodomain regions that could be amplified with primers based on homology with sequences from other basidiomycete mating types. The primer sequences were biased towards species in the genera of Coprinus and Schizophyllum. Nevertheless, the sequences obtained from H. annosum are very different from other basidiomycetes and more related to homeodomain sequences from various animal phyla. They co-segregate with the mating type. The results will be used for studies of the evolutionary history of the H. annosum species complex. The work is funded by a grant from the STINT-foundation.
155. Filamentous growth in a dimorphic fungus: Characterization of genes associated with the switch. Wei Hong1, David G. Smith1, Long Wang2, Kevin Smith1, and Michael H. Perlin1. 1University of Louisville, Department of Biology, Louisville, KY, USA. 2University of California, Neurosciences, San Francisco, CA, USA.
Many fungal pathogens utilize a switch between a yeast-like form and a filamentous form as an integral part of their overall strategy of disease production. Microbotryum violaceum is a fungal plant pathogen that infects over 200 host species in the carnation family (Caryophyllaceae), producing its spores in the anthers of infected plants. However, individual isolates are host-limited and can only infect one or a few host species. One important aspect of the infection process is that haploid yeast-like cells of opposite mating-type must conjugate and then, receiving a signal (normally host-derived) must differentiate to grow as dikaryotic hyphae. In addition to the host-provided signal, the transition from yeast-like to filamentous forms is dependent on other factors, including lower temperature (14o C) and reduced nutrient availability. The overall goal of the current work was to identify and characterize genes associated with the switch to filamentous growth in this fungal pathogen. It is likely that genes expressed upon mating and during the early stages of hyphal development are involved in controlling the switch and also in pathogenicity. The use of RNA differential display (RDD) in our laboratory successfully identified 3 genes whose expression is limited to the mating and/or hyphal stages; in addition, homologs of a high-affinity methylammonium permease were isolated. Besides the "normal" formation of dikaryotic hyphae, which requires a host-provided signal, we have also isolated filamentous diploid mutants, filamentous haploid mutants, and filamentous aneuploid strains. Several of these mutants produce what appear to be true hyphae, but which lack DAPI-stainable nuclear material throughout the hyphal extension. Genes expressed in mated and/or these additional filamentous forms but not in haploid yeast-like cells (and vice versa) are being identifed by RDD and expression of the genes already recovered will be examined in these "pseudohyphal" strains, as well. These experiments will enhance understanding of the process of dimorphism as it intersects that of disease production.
156. Regulation of dikaryon-expressed genes by FRT1 in Schizophyllum commune. Stephen J. Horton, Gail E. Palmer, and William J. Smith. Union College, Biological Sciences, Schenectady, NY, USA.
The gene FRT1 has previously been shown to induce homokaryotic fruiting in transformation recipients of the basidiomycete Schizophyllum commune. In this study, we demonstrate by gene disruption experiments that FRT1 is dispensable for dikaryotic fruiting. Non-fruiting homokaryotic FRT1 disruptant strains exhibited enhanced aerial growth of mycelia compared to wild-type. Introduction of a functional FRT1 allele into the disruptant restored the wild-type colony morphology. Transcript abundance of the dikaryon-expressed SC1 and SC4 hydrophobin genes, and the SC7 gene were greatly elevated in homokaryotic FRT1 disruptant strains. Growth of the disruptant strains under continuous light was found to inhibit the elevation of SC1 and SC4 transcript levels, but not of SC7 mRNA. These data suggest that the role of FRT1 in vegetatively-growing homokaryons is to act as a negative regulator of dikaryon-expressed genes.
157. Construction of an equalized cDNA library from Colletotrichum lagenarium and its application to isolation of differentially expressed genes. Atsuko Inagaki1, Yoshitaka Takano1, Yasuyuki Kubo2, Kazuyuki Mise1, and Iwao Furusawa1. 1Kyoto University, Agriculture, Kyoto, Kyoto, Japan. 2Kyoto Prefectural Univ., Agriculture, Kyoto, Kyoto, Japan.
For efficient isolation of differentially expressed genes of a phytopathogenic fungus Colletotrichum lagenarium, differential screening system by using an equalized (normalized) cDNA library is established. To isolate differentially expressed genes involved in infection process of conidia, mRNA from conidia undergoing appressorium differentiation was used for cDNA synthesis. The equalization of cDNA was performed twice by using kinetic method and the products were cloned into a plasmid vector. We evaluated the equalized cDNA library by colony hybridization with nine probes prepared from PKS1, SCD1, THR1, TUB1, CMR1 cDNA clones, and 4 randomly selected clones. The result showed a reduction in 'abundance variation' from 900-fold in the original library to 14-fold in the equalized cDNA library, indicating that the cDNAs were successfully equalized. Next, we performed differential screening of 1900cDNA clones in the equalized cDNA library. We identified 11 independent cDNA clones designated CAD1 through CAD11genes that were expressed in conidia differentiating appressoria but not in vegetative mycelia. The expression of CAD1, CAD2, CAD3, and CAD4 was investigated in more detail. The transcripts of CAD1 and CAD2 hardly accumulated in preincubated conidia whereas those of CAD3 and CAD4 accumulated highly and slightly, respectively. The accumulation of the four CAD transcripts increased after the start of appressorium differentiation process. In our wild type strain of C. lagenarium, developments of conidia could be controlled by incubation conditions of conidia. Conidia incubated in water at 32C germinated and elongated germ tubes without appressorium differentiation. Conidia in 0.1% yeast extract solution at 32C elongated vegetative hyphae. The four CAD transcripts accumulated in conidia elongating germ tubes as same as in conidia differentiating appressoria. On the other hand, the four CAD transcripts remarkably reduced in conidia elongating vegetative hyphae.
158. Phylogenetic study of Histoplasma capsulatum. Takao Kasuga1, Gina Koenig1, Thomas J. White1, and John W. Taylor2. 1Roche Molecular Systems, Clinical Micology, Alameda, CA, USA. 2UC Berkeley, Plant & Microbial Biology, Berkeley, California, USA.
An ascomycetous fungus Histoplasma capsulatum is the etiologic agent of histoplasmosis. The fungus has been isolated from all over the world, and several distinct varieties are known which show difference in virulence and host specificity. It is not clear whether H. capsulatum varieties form genetically isolated subspecies or the fungus has a more homogeneous panmictic population structure and difference in pathogenicity among varieties is due to difference in hosts. We have applied phylogenetic analysis on 64 geographically diverse Histoplasma capsulatum isolates representing the three varieties capsulatum, duboisii and farciminosum using partial DNA sequences of four protein coding genes. Parsimony and distance analysis of the separate genes were generally congruent and analysis of the combined data identified six clades: (1) class 1 North American H. c. var. capsulatum, (2) class 2 North American H. c. var. capsulatum, (3) Panamanian H. c. var. capsulatum, (4) South American H. c. var. capsulatum group A, (5) South American H. c. var. capsulatum group B, and (6) H. c. var. duboisii. H. c. var. farciminosum was found within the South American H. c. var. capsulatum group A clade. With the exception of the South American H. c. var. capsulatum group A clade, genetic distances within clades were an order of magnitude lower than those between clades and each clade was supported by a number of shared, derived nucleotide substitutions, leading to the conclusion that each clade was genetically isolated from the others. Under a phylogenetic species concept based on possession of multiple shared derived characters as well as concordance of four gene genealogies, H. capsulatum could be considered to harbor six species instead of three varieties.
159. A G protein alpha subunit, GNA-3, mediates conidiation in Neurospora crassa by regulating adenylyl cyclase. Ann Marlena Kays, Patricia S. Rowley, Rudeina Baasiri, and Katherine A. Borkovich. University of Texas Medical School - Houston, Microbiology, Houston, TX, USA.
Extracellular signals mediated by G protein coupled receptors are transmitted to heterotrimeric guanine nucleotide binding proteins to generate a cellular response. Our lab previously identified and characterized two G protein alpha subunits, gna-1 and gna-2. A third G alpha subunit, gna-3, cloned in Neurospora crassa is most identical at the peptide sequence level to CPG2 (90.7%) and MAGA (86.5%) from Cryphonectria parasitica and Magnaporthe grisea, respectively. A deletion of gna-3 has been marked by resistance to the bacterial hygromycin B phosphotransferase (gna-3::hph). Western analysis indicates that GNA-1 and GNA-2 are present at wild-type levels in the gna-3 mutant. The sexual cycle of the gna-3 mutant is defective in homozygous crosses and produces a large proportion of inviable ascospores. During vegetative growth on solid medium, the gna-3 mutant exhibits denser conidiation and stunted aerial hyphae. The addition of exogenous cAMP to solid cultures and standing liquid cultures restores the strain to wild-type morphology. Grown in submerged cultures, the gna-3 mutant conidiates abundantly; this phenotype is suppressed by exogenous peptone, but not cAMP. Intracellular cAMP levels of the gna-3 mutant are reduced 65% in submerged culture and 90% during vegetative and sexual growth on solid surfaces, relative to wild type. Adenylyl cyclase activity in gna-3 mutants is reduced 70%; however, the decreased activity results from a loss of active enzyme and not from a loss of GNA-3 stimulation of adenylyl cyclase. Phenotypic and biochemical results suggest that GNA-3 mediates conidiation in Neurospora crassa by regulating the amount of active adenylyl cyclase enzyme.
160. The role of a Myb-like regulator of fungal development. Ellen M. Kellner, Tom H. Adams. Cereon Genomics, LLC, Microbial Discovery, Cambridge, MA, USA.
Conidiation of the filamentous ascomycete Aspergillus nidulans follows a developmental program of cell differentiation which culminates in the production of spore-bearing structures called conidiophores. This developmental program serves as model system for the study of cellular differentiation and inhibition of proliferative growth pathways. The flbD gene product has been proposed to function in an initiation pathway which leads to development. Strains which harbor a deletion of the flbD locus exhibit a fluffy phenotype associated with delayed conidiation and uncontrolled proliferative growth. In order to further understand the pathways in which flbD functions, a screen for mutations which suppress the phenotype of a flbD null mutant were sought. Twenty such mutations were isolated which restore wild-type timing of conidiation to a strain harboring a flbD deletion. These mutations map to two linkage groups designated sfdA and sfdB. Strains harboring mutations in sfdA or sfdB have been shown to exhibit a reduced growth phenotype on agar plates and unlike wild-type strains, are capable of forming conidiophores in submerged culture. The N-terminal region of FlbD is very similar to the DNA binding regions of the Myb family of transcription factors including the protooncogene c-myb. We show here that FlbD activates transcription from a yeast reporter gene containing consensus c-myb binding sites as upstream activating sequences. Partial funding for this work is provided by NIH National Research Service Award Number 1 F32 GM20072-01 to E.M.K.
161. Structural and functional characterization of the veA gene required for sexual development in Aspergillus nidulans. Hee-Seo Kim1, Kyung-Jin Kim1, Kwang-Yeop Jahng1, Keon-Sang Chae1, Dong Min Han2, and Suhn-Kee Chae3. 1Chonbuk National University, Faculty of Biol. Sci., Chonju, Chonbuk, South Korea. 2Wonkwang University, Div. Life Science, Iksan, Chonbuk, Korea. 3Paichai University, Div. Life Science, Taejon, Taejon, Korea.
The veA gene has been isolated by complementation of the veA1 mutation. The nucleotide sequences of the cDNA as well as its genomic DNA were determined, showing that there is one open reading frame (ORF) possibly coding for a 573 amino acid polypeptide in the gene interrupted by two introns. No similarities in the amino acid sequence as well as in the nucleotide sequence were found to anyone currently available in GenBank and SWISS-Prot, respectively. It had four putative PEST regions that contain large amounts of proline (P), glutamic acid (E), serine (S), and threonine (T), and a nuclear localization signal, PKRARAC. The nucleotide sequence of the veA1 mutant gene was also determined and was differed by one nucleotide from a wild type one. The mutant ORF seemed to have the 37th methionine codon of the wild type one as a new initiation codon. The expression of the gene started from just after 14 hours competence, peaked at 8 hr, and thereafter decreased, suggesting that it act at the early developmental stage. A null mutant of the gene constructed by replacing a part of its ORF with an argB gene never entered sexual development even under conditions where sexual development preferentially occurs. The induced expression of the gene in a veA1 mutant resulted in the formation of sexual structures even in a submerged culture where wild type strains never form the sexual structures. These results indicated that the gene is an activator for sexual development, although it also can be a negative regulator for asexual development.
162. Two novel genes highly expressed in sexual tissues of Neurospora crassa. Hyojeong Kim and Mary Anne. Nelson. Univ. of New Mexico, Biology, Albuquerque, New Mexico, USA.
Two novel and highly expressed genes have been isolated from starved and sexual tissues of
the filamentous fungus, Neurospora crassa. These genes were those most frequently
expressed in the starved mycelial and perithecial cDNA libraries of Neurospora Genome Project
(Nelson et al., Fungal Genetics and Biology 21, 348-363, 1997). The most abundantly expressed
of the two genes, NP2A8, has no easily identifiable ORF. The mRNA is mostly non-coding with
multiple stop codons and no region of good codon bias. NP2A8 mRNA also has an unusual
predicted secondary structure. The gene encodes a 10 kDa putative protein as determined by in
vitro transcription and translation, and it appears to be essential. An ORF with good codon bias
has been identified in the second most abundantly expressed gene, NM2H2. It encodes a 27 kDa
putative protein that contains a possible transmembrane helix and a possible signaling peptide
cleavage site. The putative protein also contains a novel 16 tandem repeat of 13-14 amino acid
residues. We are currently using repeat-induced point mutation (RIP) to analyze the functions of
these two genes in the development of N. crassa.
163. Assessment of clone size in Rhizopogon vinicolor based on PCR
amplification of microsatellite loci from mycorrhizal roots
163. Assessment of clone size in Rhizopogon vinicolor based on PCR amplification of microsatellite loci from mycorrhizal roots. Annette M. Kretzer1, Randolph Molina2, and Joseph W. Spatafora1. 1Oregon State University, Botany & Plant Pathology, Corvallis, Oregon, USA. 2USDA Forest Service, Corvallis, Oregon, USA.
We have identified ten microsatellite loci in the basidiomycete false-truffle Rhizopogon vinicolor T20787 (Boletales, Hymenomycetes). Four loci with the sequence motifs (GTG)7, (GTG)11, (TGG)6(CGG)5 and (TCG)4N24(TCG)6 were chosen and screened for length polymorphisms across individuals. Heterozygosity was estimated to range between 0.65 and 0.81. Based on the observed allele frequencies, chances to encounter the same allele combinations at all four loci at random are below 0.0009.We used these four polymorphic loci to assess the size of R. vinicolor clones within two plots located on Mary's Peak (Oregon Coast Range) and in the H.J. Andrews Experimental Forest (Oregon Cascades). Both sites are within mixed forests that are roughly 75-100 years old. We have chosen to sample mycorrhizae of R. vinicolor instead of sporocarps for the following reasons: (i) R. vinicolor is unique in forming tuberculate mycorrhizae that have a distinctive morphology and are fairly easy to sample due to their large size. (ii) The hypogeous sporocarps formed by R. vinicolor are no less cryptic than the mycorrhizae, but are less frequently encountered and are more seasonal in their production. Results are currently available from the Mary's Peak site and indicate the presence of one large clone (at least 15 m across) in addition to several potentially smaller ones.An analogous project is under way examining clone sizes in Suillus lakei, which is closely related to R. vinicolor but forms epigeous mushroom-like sporocarps. Results from the S. lakei clone study will be available at the time of the meeting. The long-term goal is to address differences in population dynamics between R. vinicolor and S. lakei as a function of their different reproductive life histories.
164. Microscopic analysis of spo11 and msh5 mutants of Coprinus cinereus. Sandra T. Merino, Martina Celerin, and Miriam E. Zolan. Indiana University, Biology, Bloomington, Indiana, USA.
We are using the fungus Coprinus cinereus to study genes necessary for meiosis and DNA repair. Because of its naturally synchronous meiosis, C. cinereus is an excellent organism to use in studying the relationships between meiotic chromosome behavior and other aspects of DNA metabolism. Mutants defective in meiosis have been identified using restriction enzyme-mediated integration (REMI) transformation. In our analysis, REMI mutants of two genes were characterized; one is a mutS homolog, msh5, and the other is a homolog of the Saccharomyces cerevisiae SPO11, which encodes a meiotic recombination-specific type II topoisomerase. Fluorescence in situ hybridization (FISH) has been used to examine pairing between homologs at 3 and 6 h post-karyogamy. The msh5 mutant (R1660) shows pairing at nearly wild-type levels, while the spo11mutant reveals less pairing than is seen for wild-type strains. Electron microscopic examination of silver-stained chromosomes revealed that R1660 forms full-length synaptonemal complexes (SCs) by K+3, and the chromatin appears to be as condensed as in wild-type. At K+6, the SCs remain, but the chromosomes are less condensed than those seen at K+3 and chromatin loops are apparently highly extended. Analysis of the spo11 mutant shows variable axial core formation and no visible synapsis at 6 hours after karyogamy. Immunolocalization of tubulin and DAPI stained DNA analysis indicate neither the msh5 mutant nor the spo11 mutant complete meiosis.
165. HMG domain proteins involved in mating and sexual development of Coprinus cinereus. Michael J. Milner. University of Oxford, Plant Sciences, Oxford, Oxon, UK.
Transcription factors possessing HMG domains play an important role in mating recognition and sexual development in both ascomycete and basidiomycete fungi. In the ascomycetes Schizosaccharomyces pombe, Neurospora crassa and Podospora anserina, genes encoding HMG domain proteins are found within the mating type loci themselves. In S. pombe and the basidiomycete Ustilago maydis, an HMG domain protein has also been implicated in regulating gene transcription in response to pheromone stimulation. Genes that are known to have pheromone responsive promoters in C. cinereus are the B mating type genes that encode the pheromones and pheromone receptors. Analysis of the promoter sequences identifies several copies of the motif ACAATGG which resembles the so- called pheromone response element in the promoters of pheromone responsive genes of both S. pombe and U. maydis. We have used degenerate primer PCR to isolate two genes encoding HMG domain proteins from Coprinus cinereus. hmg1 and hmg2 encode HMG domains with strong homology to those of the pheromone response factors of S. pombe (Ste11) and U. maydis (Prf1). A third gene encoding an HMG domain protein, pcc1, has been described by Murata, Fujii, Zolan and Kamada (Genetics 149,1753, 1998) and has a domain that more closely resembles that of the ascomycete mating type genes Mat-Mc, mta1, and FPR1. We will describe experiments designed to show the roles of these different proteins in mating and sexual development of C. cinereus.
166. The homothallic ascomycete Glomerella cingulata has at least one putative mating-type idiomorph. Agnieszka Mudge and Matt Templeton, Plant Defence Group, The Horticulture & Food Research Institute of New Zealand, Mt. Albert Research Centre, Private Bag 92-169, Auckland, New Zealand.
In most ascomycetes there are two mating types determined by two alleles at a single regulatory locus called MAT. For sexual reproduction to take place successfully the two mating types must be heterozygous at this locus. The alleles of the MAT locus lack sequence homology and hence have been termed "idiomorphs". MAT loci have been studied in only a handful of ascomycetes, but some common features include the presence of conserved DNA binding motifs, such as the HMG (High Mobility Group) box protein, most of which are also found in transcriptional regulators of other eukaryotes. These genes are found in single copy per genome, are constitutively expressed and control fertility. Other functions attributed to these genes include pheromone production, control of fruiting bodies and ascospore production, and self/non-self recognition at both cellular and nuclear levels. A single copy 300 bp HMG box from the homothallic ascomycete Glomerella cingulata has been amplified using published degenerate primers. An approximately 15 Kb genomic sequence containing the HMG box is currently being characterised.
167. Differential expression of catalase genes during stress and development in Aspergillus nidulans.Rosa E. G. Navarro, Laura W. Kawasaki, and Jesus L. Aguirre. Instituto de Fisiologia Celular-UNAM, Genetica Molecular, Mexico, D.F., Mexico.
Catalases are H2O2-detoxifying enzymes present in most living organisms that use H2O2 as a unique substrate. We study catalase gene expression as part of a general project aimed at demonstrating a causal relationship between oxidative stress and development (Hansberg and Aguirre, 1990). Two catalase genes, designated catA (Navarro et al., 1996) and catB (Kawasaki et al.,1997) have been characterized in A. nidulans. More recently, a third catalase (CatC) has been detected using catA/catB double mutants. Why are different enzymes required to carry out the same reaction?. Part of the answer is related to the fact that these catalases are differentially expressed both, in time and space, throughout the life cycle. The catA gene encodes catalase A, which is specifically localized to asexual and sexual spores, whereas catalase B is induced during stationary phase of growth, conidiation and oxidative stress. Part of catalases A and B have been immunolocalized to the cell wall of conidia and hyphae, respectively. Mutants affected in catA or catB are sensitive to H2O2 at different stages of A. nidulans life cycle. How is this differential regulation accomplished?. We have found that the catA mRNA accumulates in response to different types of stress. However, translation of the mRNA is coupled to the spore formation, in a process mediated by the catA mRNA 5' UTR region. On the other hand, the catB gene is regulated at the transcriptional level. A 580-bp region of the catB promoter necessary for regulation during stationary phase and oxidative stress has been defined. The study of catA, catB and eventually catC, will allow us to understand an important part of the antioxidant response and the mechanisms of gene regulation that operate during development. Partially supported by grant No. IN206097 from PAPIIT-UNAM, Mexico.
168. Cytological and molecular characterization of developmental mutants from Sordaria macrospora. Minou Nowrousian, Sandra Masloff, Stefanie Poeggeler, and Ulrich Kueck. Ruhr-Universitaet Bochum, Allgemeine Botanik, 44780 Bochum, NRW, Germany.
During sexual development, mycelial cells from most filamentous fungi differentiate into typical fruiting bodies. In our effort to elucidate this process of multicellular development, we chose the homothallic ascomycete Sordaria macrospora as a model organism and isolated several sterile mutants with defects in fruiting body formation. Using an indexed cosmid library of S. macrospora , we have been able to identify DNA sequences capable of restoring fertility in the mutant strains. Here we present the isolation and characterization of two sterile mutants, mutant pro1 which only forms protoperithecia, and mutant per5 with defects in fruiting body maturation. Mutant pro1 is complemented by the sdr1 gene (sexual development regulator 1) which encodes a protein with significant homology to the DNA binding domain from C6 zinc finger transcription factors. Therefore, SDR1 might be a transcriptional regulator of sexual development in S. macrospora. Using Southern hybridization and PCR, we identified homologous sequences in other homothallic as well as heterothallic ascomycetes. Mutant per5 is complemented by the acl1 gene which codes for a subunit of ATP citrate lyase (ACL). The protein was shown to be localized in the cytosol by immunological in situ detection of the epitope tagged ACL1 polypeptide . ACL is involved in the formation of acetyl-CoA for fatty acid and sterol biosynthesis, and the highest enzymatic activity is observed during the transition from vegetative growth to the formation of reproductive structures. The ascomycetes Aspergillus nidulans and Podospora anserina display a similar pattern of ACL activity, and we present a model explaining the involvement of ACL within differentiation processes in filamentous fungi.  J. Microbiol. Meth. (1997) 29: 49-61  Mol. Cell. Biol. (1999) 19: 450-460
169. Acquisition and analysis of an EST database of barley mildew: Signal transduction pathways. Richard P. Oliver1, Julia Kinane1, Lene Bindslev 1, Sussie Dalvin 1, Soeren w. Rasmussen 1, Jacques Rouster 1, Henrietta Giese2, Nick J. Talbot3, and Michael Kershaw3. 1Carlsberg Laboratory, Physiology, Valby 2500, Copenhagen, Denmark. 2Risoe National Laboratory, Plant Biology and Biogeo, Roskilde, DK-4000, Denmark. Exeter, Biological Sciences, Exeter, Devon, Britain.
In order to provide fundamental information about its pathogenicity, we have set out to sequence as many as possible of the barley mildew genes expressed throughout the interaction. Thus far, we have utilized two cDNA libraries, both of which are purely fungal. The first analysed is a germinated spore library (Justesen, Somerville & Giese, 1996) referred to as the CR3 library. The second is from "resting conidia", i.e. spores taken directly from sporulating leaves. Redundancy has been surprisingly low, especially for the CR3 library. There are significant differences in the identity of the genes found in the two libraries. Overall 50-60% of the clones have a clear similarity to known genes. Major catabolic activities appear to involve proteins, (a selection of) amino acids, lipid and glycogen breakdown. Major anabolism involves DNA, RNA, protein, lipid, sugar, a non-overlapping selection of amino acids and vitamins. A particular focus of interest is signal transduction. A protein kinase A catalytic subunit gene was found. This suggested a direct involvement of cAMP in the early stages of development. cAMP was found to inhibit germination and appressorium development on epidermal strips and artificial media. Subsequently, fragments of the regulatory subunit and adenylyl cyclase have been cloned by PCR. Amongst a wide range of potential activators tried, the most clear effect was with cholera toxin which stimulated germination on all surfaces tried. Cholera toxin may interact with one or more of the adenosyl-ribosylation factors which have been cloned in the library. Thus the components of paradigmatic signal transduction pathways have been identified. Their likely roles in pathogenicity will be discussed.
170. Mapping of intersterility genes in Heterobasidion annosum. Åke Olson, and Jan Stenlid. Swedish University of Agricultural Sciences, Forest Mycology, Uppsala, Uppland, Sweden.
Today there are three known genetic systems that control mating and somatic integrity among fungi: (i) somatic incompatibility (vegetative compatibility), which restrict free exchange of nuclei and cytoplasm between mycelia, (ii) mating type that allows individuals with different alleles at one or more mating type locus to mate (iii) intersterility, that define limits of biological species and allow mating between individuals with identical intersterility (IS) alleles at one or more of the intersterility loci. Very little is known about the system that restrict mating within IS groups. The best characterised IS system is in Heterobasidion annosum where Chase and Ullrich in 1990 showed that mating between and within IS groups is controlled by at least five genes, S, P, V1, V2 and V3 each with a + or a - allele. Mating requires at least one common + allele for both individuals at one or more of the five genes. Individuals with combinations of only +/- and -/- cannot mate. Our aim is to map and clone intresterility genes. Here we present results towards a linkage map of the H. annosum genome and the mapping of the IS genes S, P and V2. We have isolated 105 single spore progeny from a cross between the homokaryotic strains TC-32-1 and TC-122-12, with the genotypes S-, P+, V1*, V2+, and V3+ reps. S+,P-, V1-, V2-, and V3+ (*= not defined). About 100 polymorphic AFLP markers has been identified in the parental strains using five primer combinations. Analysis of the single spore isolates are in progress.
171. Alleic diversity at the het-c locus in Neurospora tetrasperma poses an evolutionary dilemma.Amy J. Powell1, Gregory S. Saenz1, John G. Stam1, David J. Jacobson2, and Donald O. Natvig1. 1University of New Mexico, Department of Biology, Albuquerque, NM, USA. 2Stanford University, Biological Sciences, Stanford, CA, USA.
Heterokaryon (het) incompatibility governs self/non-self recognition in the vegetative phase of filamentous fungi. To form stable heterokaryons, strains must be identical at all het loci. Eleven such loci, including mating-type, have been identified in Neurospora crassa. The pseudohomothallic species N. tetrasperma occurs naturally as a mating-type heterokaryon. Although adapted for selfing, N. tetrasperma possesses mechanisms for outcrossing that could combine incompatible het alleles into a single heterokaryon. Therefore, N. tetrasperma must in some way avoid het incompatibility. Mating-type het incompatibility is genetically suppressed in N. tetrasperma. In principle, incompatibility conferred by other loci could result either from suppression or from maintenance of het locus homoallelism through persistent selfing. Our survey of het-c sequences from diverse N. tetrasperma strains demonstrated (1) that individual wild-type strains are invariably homoallelic for het-c, but (2) closely-related strains within a population can possess functionally different het-c alleles. Three allele groups (het-cOR, het-cPA, and a variant of het-cPA) are present in N. tetrasperma and other species. Our results provide convincing evidence for both predominant selfing, which maintains homoallelism, and occasional outcrossing in nature. This suggests an evolutionary dilemma: Selection has maintained het-c variation in N. tetrasperma populations to provide vegetative individuals with self/non-self recognition. Conversely, crossing between strains with different het-c alleles should lead to decreased fitness through formation of heterokaryons with incompatible alleles, a prediction consistent with heterokaryon instability observed in laboratory outcrosses. Assuming het-c function is conserved in N. tetrasperma, it appears that outcrossing will create transient life cycle disruptions that must be resolved by additional matings to restore homoallelism.
172. Heterokaryons of Phytophthora infestans revealed by AFLP and RFLP markers. Andrew I. Purvis, David S. Shaw, and Susan J. Assinder.University of Wales, Biological Sciences, Bangor, Gwynedd, UK.
Populations of P. infestans in the UK and other countries are highly diverse; A1 and A2 mating types can be found at a single site. From an evolutionary viewpoint, the role of sexual mating and oospore formation is not clearly understood. Heterokaryosis and parasexuality have long been suspected as contributing to variation but this needs to be confirmed using reliable molecular markers. To identify these, a comparison was made of the usefulness of Restriction Fragment Length Polymorphisms (RFLPs) and Amplified Fragment Length Polymorphisms (AFLPs) for assessing variability. Isolates from around the UK were screened using the multi-locus fingerprinting probe, RG57. A subset of isolates, some with the same and others with different RFLP genotypes, were fingerprinted with two pairs of AFLP primers. UPGMA trees were constructed using the RFLP and AFLP data independently. On both the RFLP and AFLP trees, isolates of mitochondrial haplotype IIa (as opposed to type Ia) clustered on a unique branch. On the AFLP tree (but not the RFLP tree) isolates of A2 mating type clustered, indicating an extra level of resolution. To investigate the parasexual cycle, heterokaryons were selected by pairing isolates with complementary sensitive and resistant mutations to streptomycin, chloramphenicol or metalaxyl (A1 x A1 or A2 x A2) on rye agar plates as vegetative hyphae or mixtures of zoospores. The resultant colony was transferred to appropriate double-drugged rye agar. Double-drug resistant growths and their single-zoospore derivatives are being analysed for their RFLP/AFLP fingerprint patterns to determine whether they are heterokaryons of parental nuclei or somatic recombinants.
173. Incomplete chromosome pairing is correlated with a recombination block on the mating-type chromosome of Neurospora tetrasperma. Namboori B. Raju, and David J. Jacobson. Stanford University, Biological Sciences, Stanford, California, USA.
In N. tetrasperma, each ascospore encloses nuclei of opposite mating type. The
dual-mating-type ascospores are the direct result of segregation of mating types at the first
division of meiosis and overlapping spindles at the second and third divisions (Dev. Genet. 15:
104). Earlier genetic studies clearly show that there is little or no recombination between various
markers on the mating-type chromosome (linkage group I), whereas markers on the remaining
six chromosomes recombine as expected (Genetics 54: 293; Genetics 143: 789). We have
examined meiotic chromosome pairing in various laboratory and wild-collected N.
tetrasperma strains to see whether the recombination block is correlated with any
cytologically detectable chromosome pairing anomalies. Three- to four-day-old perithecia were
stained with the DNA-specific fluorochrome acriflavin and the young, developing asci were
observed under a fluorescence microscope (Mycologia 78: 901). All but the longest chromosome
showed intimate homologous pairing along the entire length. In contrast, the longest
chromosome, which bears the mating type locus, usually showed a long unpaired segment in the
middle, with normal pairing at both ends. In the sibling species N. crassa, all seven
chromosomes, including the mating-type chromosome, show complete homologous pairing. We
have not yet established a cause and effect relationship between the recombination block and
incomplete pairing on the longest chromosome in N. tetrasperma. However, our
observations are consistent with the notion that the recombination block between mating type
and centromere (possibly due to impaired pairing) is a significant correlate of normal production
of self-fertile ascospores in this pseudohomothallic species.
174. Programmed ascospore death in the homothallic ascomycete Coniochaeta
174. Programmed ascospore death in the homothallic ascomycete Coniochaeta tetraspora. Namboori B. Raju, and David D. Perkins. Stanford University, Biological Sciences, Stanford, California, USA.
Immature asci of Coniochaeta tetraspora originally contain eight uninucleate ascospores. Two ascospore pairs of each ascus mature and two degenerate. Arrangement of the two ascospore types in individual linear asci is what would be expected if death is controlled by a chromosomal factor segregating at the second meiotic division in about 50% of asci. Cultures originating from single ascospores or from single uninucleate conidia are self-fertile, producing eight-spored asci with four degenerating ascospores, generation after generation. These observations in C. tetraspora suggest that differentiation of nuclei into two types occurs de novo by epigenetic modification of a specific chromosomal locus prior to proliferation of ascogenous hyphae from the ascogonium, and that modified and unmodified nuclei behave as though they were of opposite mating type. In C. tetraspora, ascospores of one epigenotype die. A similar model might explain ascospore differentiation in Sclerotinia trifoliorum and Chromocrea spinulosa, in which all eight ascospores survive but four differ in size and in self-fertility. Sexual-phase-specific modification might also occur in ascogenous hyphae of homothallic species such as Neurospora africana and Sordaria macrospora, in which all eight ascospores are viable and morphologically identical. Nuclei might then be temporarily differentiated into two types that interact during crozier formation and karyogamy as though they were of opposite mating type.
175. Progress in identifying the mating type determinant(s) of Phytophthora infestans by positional cloning. Thomas A. Randall. University of California, Riverside, Plant Pathology, Riverside, CA, USA.
Phytophthora infestans, a heterothallic oomycete, has two mating types, A1 and A2. A current model suggests that the A1 type is heterozygous for alternate mating type alleles and the A2 type is a homozygote. The mating type locus segregates in a non-Mendelian fashion in the vast majority of crosses, appearing to be linked to a system of balanced lethals. A RAPD marker (B1) that co-segregates with the mating type locus (<0.5 cM) was previously identified. A collection of RFLP, RAPD, and cDNA clones that are physically linked to B1 are being mapped relative to mating type in several crosses to localize the mating type determinant. To help in positional cloning, a bacterial artificial chromosome (BAC) library of an A1 strain of P. infestans was constructed in a derivative of pBELOBACII containing a selectable marker for P. infestans transformation. The library contains 16,128 ordered clones with an average insert of 75 kb, representing a 4.6-fold coverage of the genome. This library was probed with the B1 marker and eight positive BACs were identified with inserts ranging from 43-135 kb. Several of these BACs were determined to be from the A1 homolog. DNA markers linked to mating type are being mapped on these BACs to further localize the mating type locus. BACs have been successfully transformed into P. infestans and we are using selected BACs to transform an A2 strain of P. infestans to help identify the mating type determinant by complementaion.
176. High levels of gene flow and heterozygote excess characterize Rhizoctonia solani AG-1 IA (Thanatephorus cucumeris) from Texas. U. Liane Rosewich1, Rodney Earl Pettway1, Bruce A. McDonald2, and H. Corby Kistler1. 1University of Florida, Plant Pathology, Gainesville, Florida, USA. 2ETH, Plant Sciences, Zuerich, Switzerland.
Seven single-copy nuclear RFLP probes differentiated 36 multilocus RFLP genotypes (MRGs) among 182 isolates of Rhizoctonia solani AG-1 IA, collected from six commercial rice fields in Texas. Population subdivision was assessed using FST, unbiased genetic distance (D) and analysis of molecular variance (AMOVA). Values near zero for clone-corrected data indicated a lack of population structure. Tests for Hardy-Weinberg equilibrium (HWE) among the 36 MRGs revealed that three out of seven loci significantly differed from HWE. Subsequent analysis demonstrated that departures from HWE were due to an excess of heterozygotes (and hence heterozygosity). Moreover, all loci displayed negative FIS, with values between -0.186 and -0.551. These data suggest that the population has experienced population bottlenecks (leading to excess heterozygosity) and/or that sexual reproduction is rare (leading to heterozygote excess). We propose that a combination of these two population genetic processes could explain these data. Historic and epidemiological observations support our hypothesis.
177. Peroxisomes and mitochondria : sexual development and retrograde regulation in Podospora anserina. Gwenaël Ruprich-Robert, Denise Zickler, Véronique Berteaux-Lecellier, Arlette Panvier-Adoutte, and Marguerite Picard.Université Paris-Sud, IGM, Orsay, Essonne, FRANCE.
The roles that organelles may play in developmental processes have been yet poorly investigated. We have previously shown that mutants of the Podospora anserina car1 gene, which encodes the peroxisomal membrane protein PEX2, are devoid of peroxisomes and exhibit pleiotropic defects. Unable to grow on oleic acid as sole carbon source, they are also unable to switch from the mitotic to the meiotic stages after fertilization : in homozygous crosses, crozier cells proliferate into new croziers instead of differentiating into meiotic ascal cells. Interestingly, in wild-type crosses, a massive proliferation of peroxisomes is observed in growing ascus cells (Cell, 1995, 81, 1043-1051). We have recently observed that mutants of the cit1 gene, which encodes the mitochondrial citrate synthase, also show a sexual defect : they are mainly blocked in a specific step of the first meiotic prophase, the diffuse stage preceeding diplotene. Most of the cit1mutants bear a nonsense or frameshift mutation. Noteworthingly, these mutations have been identified as suppressors of the metabolic defect of the car1 (pex 2) mutants. Our results may be explained through a retrograde regulation mechanism which involves a cross-talk between mitochondria and peroxisomes (via nuclear genes), as it has been observed in yeast (Can. J. Bot, 1995, suppl. 1, S205-S207) but not yet described in a filamentous fungus. Experiments are in progress to test this hypothesis and the two following ideas. First, a cytosolic (instead of peroxisomal) H2O2 production would have a mitogenic effect in the car1(pex 2) croziers. Second, a heterochronic peroxisomal activation (due to retrograde regulation) would be responsible of the meiotic cit1 phenotype.
178. Molecular analysis of laccase III of Aspergillus nidulans. Mario Scherer and Reinhard Fischer. Max-Planck-Institut für terrestrische Mikrobiologie, Karl-von-Frisch-Str., D-35043 Marburg, Germany.
Laccase is a copper containing polyphenol oxidase widely distributed among plants and fungi. Fungal laccases are involved in such divers processes as lignin degradation, morphogenesis and pathogenic interactions. Laccase I (yA) of Aspergillus nidulans is involved in pigment synthesis of asexual spores. In contrast, laccase II was described as differentially expressed during sexual spore development. During an approach to identify the laccase II gene of A. nidulans a partial laccase sequence appeared in the EST sequencing project (University of Oklahoma). We subsequently have cloned and sequenced the whole gene. The deduced amino acid sequence was not laccase II and thus the novel gene was named lac3. lac3 codes for a protein of 595 aa with highest homology to laccase I of A. nidulans (36% identical aa). Laccase III was immuno-cytochemically localized in the hyphal cell wall. Expression of the enzyme starts at the onset of conidiospore germination and highest concentrations of the protein were detected at the hyphal tip suggesting a crucial function for cell extension. Western blot analysis showed that laccase III is present throughout the vegetative and asexual stage of the fungus, whereas it could not be detected during late sexual development. Knock out and overexpression experiments are in progress to prove the role of laccase III in hyphal tip extension.
179. Magnaporthe grisea mating pheromone precursor genes. Wei-Chiang Shen, Piotr Bobrowicz, and Daniel J. Ebbole.Texas A&M; University, Plant Pathology. & Microbiology, College Station, Texas, USA
Magnaporthe grisea, the rice blast fungus, is a heterothallic ascomycete and as such individuals of opposite mating type must meet in order to undergo sexual reproduction. The first step in the mating process is for mating partners to recognize each other via mating pheromones. In Saccharomyces cerevisiae and Ustilago maydis, mating factors induce a pheromone response pathway and chemotropically attract cells of the opposite mating type to grow toward the pheromone source. In an effort to understand the pheromone response in a filamentous ascomycete, we have identified two putative pheromone precursor genes expressed in Mat1-1 and Mat1-2 strains of M. grisea. Mat1-1 strains express a pheromone precursor gene predicted to contain a farnesylated and carboxy-methylated C-terminal cysteine residue. Mat1-2 strains express a pheromone precursor gene that is related in gene organization to the alpha-factor pheromone of yeast. The sequence of the predicted pheromone bears strong sequence similarity those encoded by the putative pheromone genes of Neurospora and Cryphonectria. Expression analysis and characterization of the mating behavior of the Mat1-1 pheromone precursor mutant will be presented. This work was supported by a grant from the USDA.
180. Self-fertility in Phytophthora infestans. Christine D. Smart. Cornell University, Plant Pathology, Ithaca, NY, USA
Oospores were produced by some isolates of Phytophthora infestans growing on a medium containing V-8 juice, but not in all isolates. The number of oospores produced by these isolates in this fashion was much smaller than the number produced in an outcross (with the opposite mating type). Single zoospore isolates (n=128) derived from any of the field strains did not segregate for ability to produce or not produce oospores on a medium containing V-8 juice. Thus, this phenotype was not caused by heterokaryosis or mixtures of strains. The ability to produce oospores was maintained through repeated subculturing, and after passage through host tissue. Some of the oospores were germinated and progeny were observed to segregate for alleles at the glucose-6-phosphate isomerase - 1 locus. Thus, we conclude that this type of oospore production involved meiosis, and was not ameiotic apomixis. Analysis of a larger group of strains indicated that 96% (n=47) of A2 mating type strains and 6% (n=69) of A1 strains were self-fertile on V8 agar. The ecological implications of the different types of oospores are currently being investigated.
181. In Microbotryum violaceum there are two genes that encode methylammonium permeases associated with filamentous growth in yeast. David G. Smith, Wei Hong, and Michael H. Perlin. University of Louisville, Department of Biology, Louisville, KY, USA.
M. violaceum is a dimorphic fungus that is found as yeast-like haploid sporidia and as filamentous dikaryotic hyphae. For this plant pathogenic basidiomycete, the switch to filamentous growth normally only occurs after mating, and this only under conditions of nutrient starvation. In this study, homologues of signal transduction proteins involved in filamentous growth of yeast were sought. We had particular interest in the genes encoding methylammonium permeases in Saccharomyces cerevisiae. In S. cerevisiae there exists a family of proteins designated MEP1, MEP2, and MEP3. Only MEP2 (a high affinity ammonium transporter) was shown to regulate pseudohyphal growth under nitrogen starvation. Degenerate PCR primers were designed based on highly conserved hydrophobic amino acid sequences in the transmembrane regions. One set of primers amplified both an 800 bp and a 500 bp product. Sequence analysis of these cloned PCR products revealed that both products were highly homologous to MEP2. This is the first report to our knowledge of a methylammonium permease from a filamentous fungus. When used as probes these fragments hybridized at different positions in Southern hybridization analysis. This analysis suggested that these were the only ammonium permease genes present in this organism. Based on the fact that MEP2 has such low expression levels in cells grown on nutrient rich media we speculated that these genes would not produce a detectable signal using Northern hybridization. Thus, a more sensitive approach was to use RT-PCR to confirm the expression of this putative gene family. Sequence analysis of cDNA from both fragments confirmed the presence of three introns in the 800 bp fragment and no introns in the 500 bp fragment. 5' RACE was used to obtain the cDNA sequence upstream of the initial PCR fragments. The downstream region was amplified using a gene specific primer and an oligo dT primer on the 3' end. We are currently conducting expression studies of these genes under different conditions of growth.
182. Evidence that methylation is involved in the U. maydis mating response. Karen Snetselaar, Kevin Dougherty, Joseph Curry, and Michael McCann. St. Joseph's University, Biology, Philadelphia, PA, USA.
Ustilago maydis cells form mating hyphae when they are exposed to appropriate pheromone and nutrient conditions. These hyphae grow toward compatible pheromone-producing cells, and eventually they fuse to form a dikaryon that can infect maize plants. We have found that Ustilago methionine auxotrophs are impaired in their ability to form mating hyphae and locate compatible mating partners at a distance, although these auxotrophs can mate efficiently when they are close together. Other amino acid auxotrophs mate normally. Ethionine, a methionine analog known to inhibit activity of methyltransferases, mimics the mating deficient phenotype when added to wild-type cells. When ethionine is added to mating hyphae that have begun elongating in response to pheromone, they cease growth immediately. The same concentration of ethionine has little effect on the growth rate of vegetatively growing cells. This suggests that some type of methylation is involved in the regulation of mating filament formation in Ustilago.
183. Characterization of a new "fluffy" mutant isolated by REMI in Aspergillus nidulans. Gabriela Soid-Raggi, Olivia Sánchez, and Jesús Aguirre. UNAM, Instituto de Fisiología Celular, Molecular Genetics, México, D.F., México, México.
Aspergillus nidulans mutants showing delayed asexual sporulation (fluffy mutants) have been important to define signal transduction pathways needed for conidiation (Yu et al., 1996). A fluffy mutant named BL001 was isolated by REMI (Sánchez et al., 1998). The "fluffy" phenotype was shown to segregate as a monogenic mutation and to be linked to the argB marker used for REMI. Diploid complementation analysis and sexual crosses indicated that the mutation contained in BL001 is not allelic to other known "fluffy genes". The affected gene in BL001 has been provisionally named fluX. Plasmid pREMI1, containing the vector used for REMI as well as flanking sequences, was recovered from mutant BL001. pREMI1 insert was used to probe genomic DNA from wild type and fluffy strains derived from BL001. The hybridization patterns demonstrated that pREMI1 contains genomic sequences from A. nidulans and that these are altered in the BL001 mutant and its offspring. However, cosmids identified using pREMI1 insert failed to complement fluX1 phenotype. Sequence analysis of the insert showed that pREMI1 is a chimerical plasmid likely produced during in vitro ligation. Nevertheless, sequences adjacent to the REMI insertion point should represent the actual genomic arrangement present in BL001. Sequence comparision of the identified cosmids and pREMI1 indicate that fluffy mutant BL001 contains a deletion. The fluX gene as well as other genes whose mutation causes fluffy phenotypes are required for conidiation induced by nitrogen starvation, but not for coniciation induced by glucose starvation.
184. Clonality and genetic variation in Amylostereum aerolatum and A. chailletii. Jan Stenlid1, Rimvydas Vasiliauskas2, and Iben M. Thomsen3. 1Swedish Univ Agricultural Sciences, Forest Mycology and Path., Uppsala, S-750 07, Sweden. 2Lithuanian Univ Agricultu, Plant Protection, Kaunas, LT-4324, Lithuania. 3Danish Forest and Landsc., Research Institute, Horsholm, DK-2970, Denmark.
Genetic variation was investigated within and between vegetative compatibility groups (VCGs) of the basidiomycetes Amylostereum aerolatum (Fr.) Boid. and A. chailletii (Pers.:Fr.) Boid. DNA fingerprints were made using PCR and M13 core sequence as a primer. A total of 53 of A. aerolatum and 57 of A.chailletii from Sweden, Denmark, Lithuania, and Great Britain. In all cases, isolates belonging to the same VCG also showed identical fingerprints, suggesting that VCGs correspond to clones. In A. aerolatum, wide geographical spread of the VCGs was detected, corresponding well with oidial spread vectored by the wood wasp Sirex juvencus. In A chailletii, clones with restricted geographical distribution were detected, corresponding to a higher incidence of basidiospore spread, although oidial spread vectored by Urocerus gigas is also present.
185. Isolation and characterization of CMK1, a gene encoding MAP kinase of Colletotrichum lagenarium. Yoshitaka Takano1, Taisei Kikuchi1, Yasuyuki Kubo2, John E. Hamer3, Kazuyuki Mise1, and Iwao Furusawa1. 1Kyoto University, Agriculture, Kyoto, Kyoto, Japan. 2Kubo, Yasuyuki, Kyoto Prefectural Univ., Agriculture, Kyoto, Kyoto, Japan. 3Purdue University, Biological Sciences, West Lafayette, Indiana, USA.
Colletotrichum lagenarium is a phytopathogenic fungus that causes anthracnose of cucumber. C. lagenarium conidia differentiate well developed appressoria pigmented with melanin for penetration into its host plant. In Magnaporthe grisea, cAMP and MAP kinase signaling pathways play pivotal roles for pathogenicity including appressorium differentiation. M. grisea PMK1, homologous to Saccharomyces cerevisiae MAP kinases FUS3/KSS, is involved in appressorium differentiation and pathogenic growth. To investigate roles of MAP kinase signaling pathways in pathogenicity of C. lagenarium, we tried to isolate C. lagenarium MAP kinase genes by PCR-based screening. We identified a gene showing significant homology to M.grisea PMK1 and designated CMK1. Deduced amino acid sequences of CMK1 shared 96% homology with M. grisea PMK1, and 59% homology with S. cerevisiae FUS3. To examine functional complementation of PMK1 with CMK1, M. grisea PMK1 gene disruption mutant nn78 was transformed with C. lagenarium CMK1. CMK1introduced transformants of nn78 formed appressoria similar to those of M. grisea wild type whereas nn78 failed to form appressoria. This result suggests that C. lagenariumCMK1 MAP kinase could substitute for M. grisea PMK1. Currently, gene disruption analysis is in progress to characterize roles of signal transduction pathways involving the CMK1 MAP kinase in C. lagenarium.
186. Cloning and characterization of the colonial, sbr mutant of Neurospora crassa. John Vierula, Katrina Campsall, Paul Sallmen and Yanhua Yan, Department of Biology, Carleton University, Ottawa, Ontario, Canada..
The colonial, sbr strain of Neurospora crassa was recovered in a screen of morphological mutants generated by insertional inactivation with a hphr gene construct. This strain forms compact, slow-growing colonies which darken preceptibly with age but it does not produce conidiophores or conidia. Instead of hyphae, this strain initially forms sausage-shaped cell compartments which give rise to large, randomly positioned, spherical buds. In addition, the sbr mutation blocks ascospore germination. The hphrtag was used to clone the sbr gene from a genomic DNA library and subsequently, an apparent full length cDNA clone. Sequence analysis revealed that the sbr gene encodes a putative protein with a poly-glutamine domain, a cysteine-rich region and a helix-turn-helix motif suggestive of a function in transcriptional regulation.
187. Ascomycetal mating type genes. Cees Waalwijk1, Koen Venema2, Paul Dyer3, and Gert Kema1. 1DLO Research Institute for Plant ProtectionIPO-DLO, Mycology & Bacteriology, Wageningen, The Netherlands. 2Wageningen Agricultural U, Phytopathology, Wageningen, The Netherlands. 3University of Nottingham, School Biological Science, Nottingham, England, U.K.
Recently we have described the mating system of Mycosphaerella graminicola (anamorph Septoria tritici) as being bipolar and heterothallic. In search of the mating type genes universal primers for the HMG-box, as described by Arie et al., 1997, Fungal Gen. & Biol. 21:118-130, have been employed without success. However, using a heterologous probe from the mating type gene of Tapesia yallundae, the causal agent of eye-spot disease of wheat, the corresponding M. graminicola allele could be isolated. The most recent data on the genomic organization of this mating type locus will be presented. In Fusarium spp. both universal HMG primers as well as primers for the alpha domain produce amplicons in both heterothallic and homothallic species. Moreover, even in Fusarium oxysporum, generally recognized as an asexual species these regions could be amplified. Individual strains of this species contain either alpha domain- of HMG- sequences. Sequence analyses of amplicons indicate that only conservative mutations are allowed suggesting that sex may still occur, although rarely in F. oxysporum.
188. Characterization of four populations of Monilinia vaccinii-corymbosi using AFLPs. Lusike A. Wasilwa, and Peter V. Oudemans. Rutgers University, Blueberry/Cranberry Research Center. Chatsworth, NJ, USA.
The mummy-berry fungus, Monilinia vaccinii-corymbosi , is an ascomycete pathogen of blueberry. The sexual phenology (apothecium production) of the fungus is synchronous with the host ( Vaccinium corymbosum ) phenology and fungal populations from early and late blueberry cultivars have distinct phenologies. The purpose of this study was to determine the genetic structure and relationships among mummy-berry populations with similar phenologies. One hundred and twenty isolates representing four populations were evaluated using AFLPs. Thirty isolates from each population were restricted with Pst 1 and ligated to a Pst 1 adapter. The isolates were amplified with five Gibco BRL custom primers with 2-bp extension (GT, GA, GC, AC and CG) at the 3' end. PCR products were separated by electrophoresis using 6.5 % polyacrylamide gels. The primers generated unique fingerprint patterns. Between 10 to 30 bands were produced by the primers facilitating rapid identification. Fingerprints from three primers (GT, GA, GC) suggest genetic similarity between these four populations congruent with the phenology data. The low level of polymorphisms indicate that these populations are closely related. Several polymorphisms were detected with two primers (AC and CG) that would be used estimate mating systems and gene flow.
189. Relative action of RIP on complementary strands of a duplex and on tandem duplications of various lengths. Michael K. Watters1, Thomas A. Randall2, Brian S. Margolin3, Eric U. Selker3, and David R. Stadler4. 1Univ of British Columbia, Botany, Vancouver, BC, Canada. 2Univ. of Ca, Riverside, Plant Pathology, Riverside, Ca, USA. 3University of Oregon, Biology, Eugene, Or, USA. 4University of Washington, Genetics, Seattle, Wa, USA.
In Neurospora crassa, DNA sequence duplications are detected and altered efficiently during the sexual cycle by a process known as RIP (Repeat Induced Point mutation). To explore the pattern in which base changes are laid down by RIP, we examined two sets of strains. First, we examined the products of a presumptive spontaneous RIP event, that appeared to have been subjected to just a single round of RIP. Results of sequencing suggested that a single RIP event produces two distinct patterns of change, descended from the two strands of an affected DNA duplex. Equivalent results were obtained using an exceptional tetrad from a cross with a known duplication. The mtr sequence data was also used to further examine the basis for the differential severity of Cto T mutations on the coding and noncoding strands in genes. We find that the combination of a bias of RIP toward CA/TG sites in conjunction with the sequence bias of Neurospora can be used reliably to predict the distribution of RIP sites among codon positions, thus fully accounting for the differential effect. Finally, we used a collection of tandem repeats (from 16 to 935 bp in length) withing the mtr gene to examine the length requirement for RIP. No evidence of RIP was found with duplications shorter than 400 bp while all longer tandem duplications were frequently affected. A comparison of these results with vegetative reversion data for the same duplications supports the idea that reversion of long tandem duplications and RIP share a common step.
190. Isolation and characterization of Aspergillus nidulans delayed conidiation mutants. Jenny Wieser, and Thomas H. Adams. Texas A&M; University, Biology, College Station, TX, USA.
The formation of conidiophores in A. nidulans is a precisely regulated event that occurs within the context of a radially expanding colony. Conidiophores typically differentiate 1-2 mm behind the leading edge of vegetatively growing hyphae. We have previously characterized four A. nidulans genes flbB, flbC, flbD, and flbE that result in fluffy colonies that are delayed at least 24 hours in their ability to form conidiophores. This delay in conidiophore initiation gives rise to colonies in which conidiophore development occurs 12-15 mm behind the leading edge of the radially growing hyphae. flbB, flbC, flbD, and flbE were four of the six fluffy low brlA (FLB) expression mutants isolated in our original screen that identified 122 fluffy mutants with altered levels of brlA expression. In this screen, the delayed conidiation mutants were the most abundantly isolated phenotype indicating that proper timing of conidiophore development with in the context of a growing colony is a complex process that requires the combined activities of many genes. Twenty-nine delayed conidiation mutants that express higher levels of brlA fluffy medium brlA (FMB) and fluffy high brlA (FHB) but otherwise are phenotypically identical to flbB, flbC, flbD, and flbE were isolated. These FMB and FHB delayed conidiation mutants represent at least 4 new alleles and multiple alleles of three of the genes were isolated. Sequence analysis of three of these new delayed conidiation mutants is in progress and their genetic interactions with the previously characterized fluffy mutants flbA, fluG, flbB, flbC, flbD, and flbE will be presented.
191. Sugar sensing and conidiation in Neurospora. Xin Xie, Jed Parker, and Daniel J. Ebbole. Texas A&M; University, Plant Pathol. & Microbiol, College Station, Texas, USA.
The rco-3 gene was found to negatively regulate conidiation. rco-3 mutants conidiate in submerged aerated liquid culture without the usual requirement for carbon and nitrogen starvation. rco-3 was also found to be required for proper regulation of glucose transport activity and carbon catabolite repression. In addition, rco-3 is resistant to growth inhibition by 2-deoxyglucose and L-sorbose. rco-3 encodes a member of the sugar transporter superfamily and our characterization of rco-3 suggests that it functions as a sugar sensor rather than a transporter. As one approach to identifying genetic interactions with rco-3 we have isolated mutants that suppress sorbose resistance of rco-3 mutants.
192. Evolutionary divergence and recombination in the human fungal pathogen Cryptococcus neoformans. Jianping Xu1, Rytas Vilgalys2, and Thomas G. Mitchell1. 1Duke University, Microbiology, Durham, North Carolina, USA. 2 Duke University, Botany, Durham, NC, USA.
We applied a gene genealogical approach to investigate the evolutionary relationships among strains of the opportunistic human pathogen, Cryptococcus neoformans. Four genes, one from the mitochondrial genome (mtLrDNA) and three from the nucleus (ITS, Laccase and Ura 5), were sequenced for 34 strains obtained from diverse geographic locations. The strains included representatives of both varieties (vars. neoformans and gattii) and all 4 major serotypes (A,B,C,D). Phylogenetic analyses of individual gene sequences demonstrate considerable molecular divergence which largely concordant with traditional classifications of varieties and serotypes. However, significant incongruencies were observed among all gene genealogies. Both permutation compatibility test and partition homogeneity test provided evidence for recombination within and between varieties and among serotypes. Gene phylogenies also show little evidence for geographic patterns of molecular variation, which suggests that this species has the ability for long distance dispersal.
193. Molecular analyses of mating type loci of Gibberella/Fusarium spp. and Hypocrea spinulosa (Chromocrea spinulosa) .S-H. Yun1, T. Arie2, M. Klein 3, S-H Lee1, O. Yoder1, and G. Turgeon1. 1Cornell, Plant Pathology, Ithaca, NY, USA. 2 RIKEN, Japan. 3UC Davis, Davis, CA, USA.
We are examining structural and functional organization of MAT in heterothallic, homothallic and asexual members of the Gibberella/Fusarium group. The idiomorphs of heterothallic G. fujikuroi (F. moniliforme) have been cloned and sequenced. MAT-1 encodes three genes (MAT-1-1, MAT-1-2, MAT-1-3), while MAT-2 encodes one (MAT-2); all four are counterparts of the MATgenes encoded by Neurospora crassa and Podospora anserina. Asexual F. oxysporum encodes sequences corresponding to the four F. moniliforme MATgenes; the MAT-2 (Nc mat a-1, Pa FPR1) and MAT-1-1 (Nc mat A-1, Pa FMR1) genes have no obvious mutations, MAT-1-2 (Nc mat A-2, Pa SMR1) and MAT-1-3 (Nc mat A-3, Pa SMR2) sequences have several frame shift mutations. Seven diverse MAT-1 field isolates were sequenced and found to carry the same mutations. Heterologous expression studies of F. oxysporum genes in F. moniliforme are underway to test if they are functional. The MAT locus of homothallic G. zeae (F. graminearum) has been partially sequenced and a MAT-2 gene identified. Studies of MAT organization in the Gibberella/Fusarium group form the foundation for unraveling molecular mechanisms of mating in fungi that exhibit non-standard mating behavior. One example is Hypocrea spinulosa (Chromocrea spinulosa). Strains of this fungus are either strictly heterothallic or are homothallic. Crosses of heterothallic isolates by homothallic isolates or selfs of homothallic isolates produce 50% heterothallic and 50% homothallic progeny. Using sequence information from closely related Gibberella/Fusarium we have partially cloned and sequenced MAT genes of a homothallic H. spinulosa isolate and identified both MAT-2 and MAT-1-1 genes. Comparison of MAT from homothallic versus heterothallic strains will be used to analyze the mechanism by which this fungus produces both heterothallic and homothallic progeny from a single homothallic progenitor.
194. Mapping vegetative incompatibility (vic) loci in Gibberella fujikuroi (Fusarium moniliforme, MP-A) with AFLP markers. Kurt A. Zeller1, James E. Jurgenson2, and John F. Leslie1. 1Kansas State University, Dept. of Plant Pathology, Manhattan, KS, USA. 2University of N. Iowa, Dept. of Biology, Cedar Falls, IA, USA.There are 8-10 vegetative incompatibility (vic) believed to be segregating in the established mapping population of Gibberella fujikuroi (Fusarium moniliforme, MP-A). Selection for vegetative compatibility between two strains should simultaneously select for identity in all segregating vic loci and should skew segregation ratios for linked neutral markers that flank each vic locus. We crossed two nit1- strains (A0924 and A4644) derived from the mapping population's parental genetic backgrounds, and selected progeny from that cross that could form heterokaryons with individuals of known AFLP / RFLP genotype. We collected these heterokaryons, resolved each to recover the nit1-component, and retested each isolate to confirm its ability to form a heterokaryon. Once confirmed, we retained these isolates for AFLP analyses. We have used AFLP markers to localize genomic regions that display skewed segregation ratios in the vegetatively compatible progeny of this cross. In our preliminary analysis, we have identified 8-9 genomic regions that display segregation ratios significantly (alpha < 0.05) skewed from 1:1. Such regions are found on at least 7 of the 12 linkage groups of G. fujikuroi.
195. AFLP markers reveal genetic variation in Egyptian populations of Cephalosporium maydis. Kurt A. Zeller1, James E. Jurgenson2, and John F. Leslie1. 1Kansas State University, Dept. of Plant Pathology, Manhattan, KS, USA. 2University of N. Iowa, Dept. of Biology, Cedar Falls, IA, USA.Cephalosporium maydis causes late wilt; perhaps the most serious fungal disease of maize in Egypt. Genetic and pathogenic variation among isolates, and population structure in this fungal pathogen are poorly understood. Our objective was to identify molecular markers for use in studies of this pathogen. We characterized genetic diversity in C. maydis with AFLP (amplified fragment length polymorphism). We made AFLP fingerprints of 48 strains (10 reference + 38 field isolates) with 68 selective primer pair combinations. Average UPGMA similarity among all strains was about 89%. Statistical analyses indicate that these 48 strains cluster into four distinct subgroups (lineage groups), each containing 7-19 of the isolates. Pairwise UPGMA similarities within each lineage are generally greater than 95%, while similarities between these lineages are less than 91%. We have identified a subset of these selective AFLP primer-pairs that produce estimates of genetic distances that are highly correlated (r> 0.85) to that of the pooled data. We have begun to use data from these AFLP primer-pairs to characterize variation among additional C. maydis samples.
196. A long terminal repeat retrotransposon Cgret from the phytopathogenic fungus Colletotrichum gloeosporioides on cranberry. Peiliang Zhu, Peter V. Oudemans. Rutgers University, Plant Pathology, Chatsworth, New Jersey, USA.
A repetitive DNA element cloned from the cranberry fruit rot pathogen Colletotrichum gloeosporioides has been characterized. Sequence data indicate that it is a long terminal repeat (LTR) retrotransposon of 7916 base pairs in length. Long terminal repeat sequences of 544 base pairs occur at either end of an internal region 6828 base pairs in length. This element, designated Cgret (Colletotrichum gloeosporioides retrotransposon), encodes two putative polypeptides that have high homology to the gag and pol genes of other fungal retrotransposons. The sequence and structure suggest that Cgret is a member of the gypsy group of LTR retrotransposons. The Cgret retrotransposon was present in all of the cranberry isolates of the fungus C. gloeosporioides from New Jersey and Massachusetts, but not in the cranberry isolates from Wisconsin or Chile. None of the genomic DNA samples from isolates of the related fungus Colletotrichum acutatum hybridized with probes from this transposable element. Polymorphisms were detected among field isolates of C. gloeosporioides from various hosts using hybridization probes derived from the long terminal repeat and the reverse transcriptase domain of Cgret. The structural integrity of Cgret suggests that it is still a functional retrotransposon and may be used as a molecular marker to study the distribution of fungal genetic diversity.
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