Signal Transduction

A. nidulans calmodulin-dependent protein kinase (ACMPK), exhibits tyrosine kinase activity.

B.M. Manolas, Rose Antilus and D.C. Bartelt. Department of Biological Sciences, St. John's University, Jamaica, NY.
Previous studies have shown that ACMPK has substrate specificity similar to calmodulin (CaM)-dependent protein kinase II (CaMKII). Like other CaM-dependent protein kinases, the optimal substrate consensus sequence for ACMPK is R/K-X-X-S/T, a sequence present in autocamtide 2 (K-K-A-L-R-R-Q-E-T-V-D-A-L). Other protein S/T kinases including cAMP- dependent protein kinase and phosphorylase kinase have recently been shown to phosphorylate tyrosine (Y) residues in peptide substrates such as angiotensin II (N-R-V-Y-V-H-P-F). Protein tyrosine kinase activity is Mn²⁺ dependent. We have examined the ability of ACMPK to phosphorylate tyrosine-containing peptides and studied the effects of divalent cations on kinase activity. ACMPK phosphorylates both angiotensin 11 and alpha-neo endorphin (Y-G-G-F-L-R-K-Y-P-K) but not a peptide containing the phosphorylation site of pp6O^src (R-R-L-I-E-D-A-E-Y-A-A-R-G). Mn²⁺ enhances both the threonine and tyrosine kinase activity of ACMPK. Phosphorylation of Threonine/serine and tyrsolne by ACMPK is Ca²⁺/CaM- dependent.

Supported by the American Cancer Society, and by NIGMS 5 S06 GM50780 (MBRS program).

1. Protein kinase C, a novel component of blue light transduction pathway in Neurospora crassa.

G. Arpaia, C. Catalanotto, F. Cerri and G. Macino.

In the ascomyces Neurospora crassa blue light influences developmental processes, production of carotenoids and the entrainment of the circadian rhythm. Genetic aproach for the identification of components of the blue light transduction pathway, never gave clear indications on the characteristics of new molecules other than wc-1 and wc-2. In the present study we used a pharmacological approach to screen a wide range of second messengers and chemical compounds capable to interfere with the activity of well known signal transducers in vivo. By this aproach we identify the Protein Kinase C as an additional component of the light transduction cellular machinery. We tested specific inhibitors (Calphostin C and Chelerythrine chloride) or activators of PKC (1,2-Dihexanoyl-sn-Glycerol) and monitored the effect on the blue light-stimulated transcription of the gene albino-3.

PKC role is carried out both during the vegetative growth and conidiation. In the first case PKC is responsible for the desensitization to light, an adaptative response: inhibitors of the enzyme inhibit the decay-phase in the kinetic of light induction of the al-3(m) transcript. During conidiation, instead, PKC activity is necessary for the light induction of the al-3(c) conidiation-specific transcript. In this phase, PKC inhibitors totally abolish light-induced al-3(c) expression, but not conidiation-driven transcription of the gene.

We cloned the gene coding for the Neurospora crassa PKC and its characterization is actually under way.

2. Blue light signal transduction: The white collar-1/white collar-2 dimerization system.

P.Ballario, H.Linden*, D.Gallie G. Macino* Universita' "La Sapienza" Roma Italy.

Both White collar-1 and White collar-2 genes products are necessary for light perception in Neurospora crassa since mutations in either one of the two genes cause the total blindness of the fungus. As the wc mutants are defective only in blue light induced processes, their products are considered to be specific elements of the blue light transduction pathway. Extensive screenings for other mutants with a "full blind" phenotype have been unsuccessful. The WC-1 and WC-2 genes have been recentely isolated (1, 2 ) and although no overall homology between them was identified they share some common domains. WC proteins are putative transcriptional factors characterized by amino terminal activation regions and a carboxy terminal Zn finger DNA binding domain. Both Zn finger binding domains expressed in E.coli, are able to bind in vitro to the promoter of the light inducible al-3 gene. In addition both WC proteins show homology to the dimerization domain termed PAS (Per,Arnt,Sim) (3). PAS domains are present in several proteins, mostly transcriptional factors, able to form homo and heterodimers. The human AHR (aryl hydrocarbon receptor) containing a PAS domain shows the highest degree of amino acid conservation with WC-1 PAS or WC-2 PAS. This receptor is able upon the binding with xenobiotic metabolites (i.e. dioxin) to dimerize with ARNT (its nuclear translocator protein).The heterodimeric protein once translocated in the nucleus, recognized the XRE (xenbiotic recognition element) of certain promoters through the bHLH binding domains present in both proteins. AHR and ARNT are both able to homo and heterodimerize. We have in vitro evidences that WC-1 and WC-2 are able to form homo and heterodimers, and also to be engaged in heterodimeization with AHR in vitro. The implications of this finding for the the construction of a model of blue light transduction in Neurospora will be discussed.

1)P.Ballario, G.Macino (1996) EMBO J , 15,1650-1657

2)Linden H. and Macino G.(1997)EMBO J, 16, 98-109.

3)Huang Z.J., Edery I., Rosbash M.(1993)Nature, 364, 259-262

3. Characterization of Neurospora crassa transport mutant nap.

Tatiana A. Belozerskaya, Tatiana V. Potapova, Natalla N. Levina, Natalia E. Petrova and Yuri V. Ershov. A.N. Bach Inst Biochem., RAS, *A.N. Beloversky Inst Phys.-Chem Biol., MSU Moscow, Russia.

To elucidate the role of the plasma membrane transport systems in the blue light signal transduction chain leading to biosynthesis of carotenoid pigments, electrophysiological characteristics, intracellular ATP content and ability to synthesize carotenoids have been studied in the N. crassa transport mutant nap. Resting membrane potential value of the mutant nap measured with the aid of intracellular microelectrodes turned out to be about 1.5 times lower than the wild-type. In spite of this fact the plasma membranes of the mutant nap responded to the blue light treatment by hyperpolarozation. Its value was the same as in the wild type and appeared to be energy dependent and thus connected with the functioning of H⁺ ATPase. Intracellular ATP content of the mutant strain was about 1.5 times higher than in the wild type. Proportional about two times increase of all the carotenoid fractions was observed in the mutant nap. Thus the intensity of transport processes throught the plasma membrane does not seem to be limiting for the light induced carotenoid acccumulation in N. crassa. The critical factor for the process of carotenogenesis appear to be the photoinduced changes in the electrical properties of the plasma membrane.

4. Cloning and sequencing of the gene encoding the catalytic subunit of a cAMP-dependent protein kinase in Candida albicans.

Monikca Cloutier*, Beatrice B. Magee and Luc *. Laval University*, Quebec, Canada; University of Minnesota, MN, USA.

The cloning and sequence analysis of a gene that encodes the catalytic subunit of a cAMP-dependent protein kinase (PKA-C) in the human pathogen Candida albicans is reported. Two highly conserved amino acid segments (IYRDLKP and GTHEYLAPE; respectively subdomains VI and VIII of the conserved regions from catalytic domains through the kinase family) served to design two oligonucleotides that were used to PCR amplify a 150 pb fragment. Sequence analysis of this fragment showed it to be 83% identical to that of the Saccharomyces cerevisiae TPK2 gene. This amplicon was used to screen a C. albicans genomic fosmid library. One of the three positive cosmids obtained was mapped using several restrictions endonucleases. The PKA-C gene was localized by Southern analysis. Two overlapping DNA fragments (a 1.7 kpb PstI and a 3.0 kpb EcoRI/KpnI) were subcloned and used to determined the sequence of the gene. An ORF of about 1,2 kb was detected by sequence analysis. Comparison of the predicted amino acid sequence showed it to be 80% identical to that of TPK2 from S. cerevisiae. Construction of homozygous null mutant for PKA-C is underway using the "URA-blaster" technique. This should allow us to verify the implication of PKA-C in cellular morphogenesis and virulence in C. albicans. (Supported by MRC grant #MT-12892).

5. Spore germination and trehalose metabolism in Aspergillus nidulans.

Christophe d' Enfert and Thierry Fontaine. Labratoire des Aspergillus, Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris, Cedex 15.

Althought conidial germination is a key developmental stage in the life cycle of Aspergillus species, it remains poorly understood at the molecular level. Trehalose is a non-reducing disaccharide found at high concentrations in Aspergillus conidia and rapidly degraded upon induction of conidial germination. Trehalose-degradation is concomitant with the accumulation of a glycerol pool in the conidia. Our aim is to understand the role of the trehalose and glycerol pools wth respect to conidial germination and to identily the signal transduction pathways that are responsible for activating trehalose breakdown and that might control other events required for the achievement of conidial germination. Two A. nidulans genes encoding trehalases have been cloned and sequenced and A. nidulans strains with a null mutation in either gene have been obtained. treA encodes an acid trehalsae that is localized in the conidial wall and that is similar to S. cereviasiae vacuole trehalase. Disruption of treA results in A. nidulans strains that are unable to grow on trehalose as a carbon source and that are not affected in the mobilasation of intracellular trehalose during conidial germination. treB encodes a cytoplasmic neutral trehalase highly homologous to the two S. cerevisiae neutral trehalases that are activated by the cAMP-dependent pootein kinase (PKA) and catalyze intracellular trehalose breakdown in budding yeasts. Disruption of treB results in A. nidulans strains that are unable to degrade intracellular trehalose at the onset of conidial germination and that do not show the transient accumulation of a glycerol pool, suggesting that this pool results from the entry into glycolysis of the glucose formed from trehalose. The involvement of a neutral trehalase is consistent with a role for the PKA pathway during the early events of conidial germination.

6. Four protein kinsase homologs in Ustilago maydis.

Ge Yang, Franz Durenberger, Ann B. Orth* and James W. Kronstad. Biotechnology Laboratory, University of British Columbia, Vancouver, BC V6T 1Z3 Canada and *DowElanco discovery Research, Indianapolis, IN 46268-1054 USA

Recently, the cyclic AMP pathway and a protein kinase (adr-1) in U. maydis have been implicated in dimorphism and fungicide resistance. We are interested in additional homologs of protein kinase A (PKA) and protein kinase C (PKC) for their potential roles in morphogenesis and as fungicide tergets. A 150 bp PCR fragment, which contains a PKC homolog, was used as a probe to isolate four cosmid clones carrying additional protein kinase genes. The four cosmids have been subcloned and partially sequenced. The sequence information indicates that one of the genes may encode another catalytic subunit of PKA. The other three genes appera to encode PKC homologs, with strongest homology to ypk1, ypk2 and sch9 genes in Saccharomyces cerevisiae. The disruption constructs have been made to knock out the four genes in diploids and/or haploids. The results of the gene disruption experiments will be reported.

7. G protein a subunit genes of Trichoderma harzianum and Cochlioholus heterostrophus.

Benjamin A. Horwitz, Department of Biology, Technion, Haifa, Israel.

G genes with features of the Gi class have been isolated from T. harzianum¹ and C. heterostrophus² . Both have high homology to gna1 of Neurospora crassa; intron positions are conserved between the two species, and there is no evidence for more than one gene in either. Trichoderma species are soil saprophytes or mycoparasites.A brief pulse of blue light (200 umol m^-2, delivered over ns to min) induces synchronous conidiation of T harzianum even under otherwise favorable conditions. Loops, coils and branches are formed in response to the proximity of host hyphae. Experiments with activators of animal cell G proteins suggest that G- is involved in mycoparasitism rather than in the blue light response. In C. heterostrophus, a foliar pathogen of corn, disruption of the G subunit gene by homologous recombination results in the loss of the ability to form appressoria on a glass surface. Furthermore, crosses in which even one member of the pair lacks G produce infertile pseudothecia. The pleiotropic nature of the G null mutants suggests that the unique consequence of each signal is determined not by G, but by proteins with which it interacts.

1. V, Rocha, J. Inbar, I. Chet, A. Herrera-Estrella and B.A. Horwitz (in preparation)

2. B.A. Horwitz., A. Sharon, S. Lu, O. C. Yoder and B.G. Turgeon (in preparation)

8. Biochemical Analysis of Heterotrimeric G Protein Regulated Events in the Filamentous Fungus Neurospora crassa.

F. Douglas Ivey and Katherine A. Borkovich. University of Texas Medical School- Houston.

Heterotrimeric guanine nucleotide binding proteins (G proteins), consisting of of , and polypeptides, regulate a vast range of processes from muscle contraction and vision in mammals to mating in yeast. Through coupling to seven helix transmembrane receptors, G proteins relay ligand/receptor binding events to downstream proteins or enzymes known collectively as effectors. Neurospora crassa possesses at least three known G subunits, Gna-1, Gna-2, and Gna3. Gna- 1 was the 1st reported microbial subunit to be a member of any mammalian G family. The G_i subfamily members are thought to participate in diverse functions including controlling ion channels and regulating phospholipase activity. It was previously shown that deletion of gna-1 in N. crassa results in vegetative growth defects that include sensitivity to hyperosmotic media and abnormal formation of aerial hyphae. More importantly, deletion of gna-1 in N. crassa results in female sterility. Recently, the levels of second messenger molecules have been investigated using several approaches. The deletion of gna-1 appears to lead to observable changes in hyphal-tip calcium levels as observed using chlor-tetracycline fluorescence. Because the levels of inositol 3-phoshate affect Ca²⁺ levels in many eukaryotic cell types the levels Of EP3 in both gna-1 mutants and in control strains were measured. In addition, pharmacologic agents that act on cellular Ca2+ levels appear to differentially affect the growth of gna-1 and control strains. Currently, biochemical methods are being applied to determine if any of the observed gna-1 mutant phenotypes are related to changes in intracellular or extracellular cAMP levels. The goal of this research is to correlate changes in second messenger molecules with G proteinmediated signalling events.

9. Withdrawn

10. Fil1, a G-protein subunit that mediates dimorphic switching of Ustilago hordei.

Amnon Lichter and Dallice Mills. Department of Botany & Plant Pathology, Oregon State University, 2082 Cordley Hall Corvallis, Oregon, 97331-2902,

A constitutive mutation, fil1, causing filamentous growth in the haplophase of the dimorphic smut fungus Ustilago hordei, was previously shown to be genetically associated with a 50 kb deletion in a 940 kb chromosome. The effect of this mutation could be transiently relieved by adding cAMP, or adenylyl cyclase stimulators, or a cAMP phosphodiesterase inhibitor to the growth media, suggesting that a gene that functioned upstream in the cAMP cascade was deleted. Representational difference analysis (RDA) was modified to construct a chromosome subtraction library enriched for deletion-specific sequences that were used to identify homologous genomic DNA in a cosmid library. The cosmid clone, pOSU2100 and a derivative plasmid with a 2.1 kb insert, converted the transformants from the filamentous to the sporidial cell type. The 2.1 kb insert contained a single open reading frame of 354 amino acids that encodes a putative subunit of the heterotrimeric G-proteins. FIL1 displayed high amino acid sequence identity to Gpa1 of the basidiomycete Cryptococcus neoformans, CPG-2 of the ascomycete Cryphonectria parasitica and intermediate homology to Gpa2 of Saccharomyces cerevisiae. FIL1 was verified to be deleted in the mutant and to be present as a single-copy gene in the wild-type. Wild-type strains transformed with FIL1 were suppressed for filament production which can normally be induced in minimal medium with limiting levels of nitrogen. These strains, including the complemented mutant were mating-competent with a wild-type partner. The effect of FIL1 on the pigmentation pattern of U. hordei is demonstrated.

11. Fruit body development of Sordaria macrospora: Isolation and molecular characterization of a regulatory gene.

S. Masloff, S. Pöggeler and U. Kück Lehrstuhl fur Allgemeine Botanik, Ruhr-Universitat, D-44780 Bochum, Germany

We have chosen the homothallic ascomycete Sordaria macrospora as a model organism for investigating fruit body development. Here we present the molecular characterization of the sterile mutant prol, which formes only protoperithecia. Complementation transformation of this mutant was performed with an indexed genomic cosmid-library [1, 2], leading to the isolation of two overlapping cosmid clones. The minimal complementing region was defined on a 2.3 kb DNA fragment, which subsequently was sequenced. This analysis was completed by the generation and molecular characterization of partial cDNA clones. Restriction and hybridization analysis of mutant pro1 indicates a deletion of more than 11 kb, including the complementing region. Database search of the deduced amino acid sequence of the complementing DNA fragment reveals a region of significant homology to a DNA-binding motif found in the GAL4 transcription factor from yeast [3]. Functional studies are underway to identify domains in the polypeptide that are involved in fruit body formation of this ascomycete.

[1] Walz M, Kuck U (1995) Curr Genet 29:88-95

[2] Poggeler S, Nowrousian M, Jacobsen S, Kuck U, submitted

[3] Pan T, Coleman JE (1990) Proc Natl Acad Sci USA 87:2077-2081

12. A ras homolog of Neurospora crassa regulates apical growth.

T. Murayama (Kanto-Gakuin Univ., Yokohama), S. Tanabe (Tohoku Univ., Sendai), and A. Kana-uchi (Tokyo Medical and Dental University School of Medicine).

We cloned and characterized a ras homologue gene, termed NC-ras2. The predicted protein product of this gene is composed of 229 amino acid residues and contains all the consensus sequences shared by the ras protein family. An NC-ras2 disruptant showed morphological characteristics very similar to that of the smco7 mutant. Nucleotide sequence analysis revealed that the smco7 mutant harbored a single base deletion in the NC-ras2 gene which is predicted to result in the truncation of the protein product. Introduction into the smco7 mutant of an NC-ras2 clone yielded stable transformants with the wild type phenotype. The smco7 mutant exhibited very slow hyphal growth. The smco7 mutation also caused both the changes in the pattern of hyphal growth and the defects in cell wall synthesis. Both the diameter and the length of the apical compartment were shorten in the hyphae of the smco7 mutant.

These results suggest that NC-ras2 is identical to smco7, and that the signal transduction pathway mediated by the NC-ras2 protein regulates the apical growth of hypha by regulating the transport of apical vesicles containing cell wall material in N. crassa.

13. Cloning and sequencing of the gene encoding the regulatory subunit of a cAMP-dependent protein kinase in Candida albicans.

Marc Parrot*, Beatrice B. Magee  and Luc Giasson*. Laval University*, Quebec, Canada; University of Minnesota , MN

We have isolated and sequenced the gene encoding a cAMP-dependent protein kinase regulatory subunit (PKA-R) from the opportunistic human pathogen Candida albicans. This gene is suspected to be involved in the control of cellular dimorphism, a putative virulence trait. A genomic cosmid library was screened with a probe (generated by PCR) derived from two almost perfectly conserved amino acid segments of the cAMP binding sites from Saccharomyces cerevisiae, Blastocladiella emersonii and mouse PKA-R genes. Five positive clones were recovered. The PKA-R gene was localized by Southern hybridization. A 5.5 kb PstI fragment was isolated from one of the positive fosmids, subcloned in pUC19 and mapped using several restriction enzymes. Two EcoRI subfragments (0.75 and 1.85 kb), each one containing part of the PKA-R gene, were subcloned in pCRScript and sequenced. The PKA-R gene is 1380 nucleotides long with a single copy per haploid genome. Nucleotide sequence analysis has shown that the predicted protein comprises 459 amino acids, giving an average molecular mass of 55 kDa with an overall sequence identity of 62% with its homologue in S. cerevisiae. The promoter region contains the canonical CAAAT and TATA boxes. The presence of a serine residue in the phosphorylation site suggests that this Candida gene encodes a type II regulatory subunit. We will also report on the work in progress to inactivate the PKA-R gene in C. albicans. (Supported by MRC grant #MT-12892).

14. Characterization of the role of the cAMP pathway in vinclozolin resistance of Ustilago maydis.

Marilee A. Ramesh, Ann B. Orth*, and James W. Kronstad. University of British Columbia, Vancouver, B.C. V6T lZ3, Canada. *DowElanco Discovery Research, Indianapolis, IN 46268-1054 USA

Ustilago maydis, the basidiomycete smut pathogen of maize, grows as a yeast while haploid and filamentously when dikaryotic. Recent work from this laboratory has shown that the cAMP pathway plays a role in the dimorphic switch. Mutants defective in adenylate cyclase and protein kinase A (PKA) have altered cell morphology. In addition, it has been reported that a catalytic subunit of PKA, encoded by adr1, may also play a role in fungicide resistance. To determine whether other components of the cAMP pathway could play a role in vinclozolin resistance, mutants in adenylate cyclase and PKA, which were previously generated by our laboratory, were grown in media containing various concentrations of vinclozolin. A disruption mutant of the ubc1 gene, which encodes the regulatory subunit of PKA, showed increased resistance tovinclozolin compared to wild type cells. To explore the role of the cAMP pathway in vinclozolin resistance, the ubc1 disruption mutant strain has been mutagenized and several mutants have been isolated which show decreased resistance to vinclozolin. Further analysis of these mutants will provide a better understanding to the role of the cAMP pathway in fungicide resistance.

15. Signal transduction in Trichoderma harzianum: Role of a G protein.

Victor Rocha-Ramirez*, Benjamin A. Horowitz** and Alfredo Herrera-Estrella*. *CINVESTAV-IPN, Unidad Irapuato, Mexico. **Department of Biology, Technion, Haifa, Israel.

Trichoderma harzianum a biocontrol agent of phytopathogenic fungi which is attractive as a model for the study of diferentiation processes, such as sporulation and apresorium formation. Biochemical evidences suggests that a trimeric G protein regulates apressoria formation and induction of conidiation by light.

As a first step towards the elucidation of the possible signal transduction pathway in Trichoderma we have cloned the gene encoding the Ga subunit, PCR technology was applied to generate a small fragment of the gene using oligonucleotides designed based on two of the highly conserved regions of Ga proteins. This product was used as a probe to obtain a T. harzianum genomic clone and its corresponding cDNA. The expression pattern of the gene under several conditions including those used for the induction of sporulation and appresorium formation was analyzed. Additionally, transgenic Trichoderma strains were obtained using a series of plasmids which will allow us to determine the role of the G protein in light induced conidiation and apresorium formation.

16. Dissecting the signaling pathways between oxalic acid production and sclerotia development in Sclerotinia sclerotiorum.

J.A. Rollins and M.B. Dickman. University of Nebraska-Lincoln.

The production of oxalic acid has been demonstrated previously to be an essential pathogenicity determinant in disease interactions between Sclerotinia sclerotiorum and its hosts Phaseolus vulgaris and Arabidopsis thaliana. UV induced mutants of S. sclerotiorum selected for their inability to produce oxalic acid, are pleiotropically deficient in sclerotia development. We have initiated studies to determine the molecular basis for these observations and to dissect the molecular relationships between these two phenotypes. To determine if oxalic acid plays a direct role in sclerotia development, wild type S. sclerotiorum has been transformed to express a bacterial oxalyl decarboxylase gene to lower or eliminate oxalic acid accumulation. Several sclerotia minus transformants have been obtained by this approach and are currently being evaluated to determine if they express the transgene and whether they have altered levels of oxalic acid accumulation. Additional work has focused on endogenous signal transduction as related to development. In these studies, several classes of signal transduction effectors, including drugs which affect cAMP levels, protein kinase activities, and calcium homeostasis, were examined for their effects on oxalic acid production and sclerotia morphogenesis. Addition of exogenous 8-Br-cAMP, or treatment with caffeine, 3-isobutyl-l-methylxanthine, or sodium fluoride, which raise endogenous levels of cAMP, resulted in the reduction or complete inhibition of sclerotia development without affecting the production of oxalic acid. These results suggest that cAMP mediated signaling may also play a role in the switch from mycelial growth to sclerotial development.

17. Lovastatin may interfere with the functions of MRasl and 3 and triggers a cell death process resembling apoptotis in Mucor racemosus.

Ludmila V. Roze and John E. Linz, Michigan State University, East Lansing.

The filamentous fungus Mucor racemosus possesses three Ras genes, MRasl, 2 and 3, which have striking similarity in nucteotide sequence to each other and to mammalian Ras genes. The Ras gene family is implicated in the pathophysiology of cell transformation and the pathogenesis of human cancer. Differences between MRasl and 3 have been observed in the pattern and level of transcript and protein accumulation, post-translational modification as well as the protein complexes formed in vivo, suggesting that the each MRas gene may have a different function. Lovastatin, an indirect inhibitor of protein farnesylation, affected the processing of as MRas1, blocked the expression of MRas3 and caused the complex MRasl/ p2O to disappear. Concurrently it blocked sporangiospore germination and decreased the growth rate of Mucor racemosus. Lovastatin at high concentration (250 uM) caused the loss of cell viability accompanied with cell shrinkage, increased cell density and cytoplasm condensation and triggered intensive DNA fragmentation to nucleosomes and nucleosome multimers with profound laddering at late stages. The specific morphological and biochemical events seen in Mucor cell death, particularly DNA fragmentation, resemble the best known characteristics of classical apoptosis in mammalian cells and to our knowledge represents the first observation of programmed cell death in filamentous fungi. These findings provide insights into the origin of programmed cell death and support the idea that controlled cell suicide machinery originated in unicellular organisms and was selected through evolution for the optimal adaptation to the environment.

18. Isolation, functional analysis and overexpression in mammalian cells of a ras gene from Colletotrichum trifolii.

Gina Truesdell, Clinton Jones, Todd Holt and Marty Dickman, University of Nebraska-Lincoln.

Ras proteins are ubiquitous molecular regulators of signal transduction pathways in eukaryotic cells. Ras proteins transduce external signals across the plasma membrane by cycling between inactive GDP-bound and active GTPbound states. The importance of Ras in regulating growth in mammalian cells is demonstrated by the finding that point mutations which cause constitutive activation of Ras lead to a transformed phenotype. To study the function of Ras in the life cycle of the alfalfa pathogen Colletotrichum trifolii, a cDNA encoding a Ras homolog, designated CT-ras, was cloned. CT-ras is a constitutively expressed single-copy gene and its deduced protein sequence is similar to Ras proteins from other organisms. To determine if CT-ras is functional, its activity was tested in a mammalian system. Activating mutations equivalent to those identified in animal tumors were introduced into the coding region by site-directed mutagenesis and the modified sequences were expressed in NIH 3T3 cells. Transfected cell lines expressing the modified fungal genes were identified. In vitro assays show these cells display characteristics of transformed cells, indicating activated CT-ras likely functions as an oncogene. The ability of these cells to form tumors in vivo is being tested. In addition, the consequence of constitutive Ras activation in C. trifolii is being investigated by expressing the modified genes in the fungus.

19. MAP Kinase Pathways in Magnaporthe grisea.

Jin-Rong -Xu and John E. Hamer, Purdue University, West Lafayeffe, IN 47907.

Many plant pathogenic fungi, including Magnaporthe grisea- the causative agent of the rice blast disease, develop specialized structures to invade their hosts and undergo dramatical morphological changes to grow invasively in plants. Our research objective is to study genetic mechanisms regulating this plant infection-related morphogenesis. We have isolated a MAP kinase PMKL (Pathogenicity MAP Kinase 1) from M. grisea which is essential for appressorium formation and invasive growth in plants. Further characterization of the PMKL MAP kinase pathway is in progress and will be presented. In addition to PMK1, we isolated two other M. grisea MAP kinases. PMK2 is 83% similar to S. cerevisiae HOGl gene, and 93% similar to S.pombe styl gene. PMK3 is 85% similar to S. cerevisiae SL T2 MAP kinase gene. Five PMK3 gene disruption mutants were isolated. pmk3 mutants are nonpathogenic on rice plants, but make melanized appressoria on Teflon membranes or slideglass with the addition of 10 mM cAMP. On onion epidermis, pmk3 appressoria fail to penetrate, but elicit autofluorescence and papilla formation in onion epidermal cells. pmk3 mutants are also dramatically reduced in conidiation, however, there is no obvious growth defect as measured by colony diameter on a variety of media. pmk3 mutants are not temperature sensitive or infertile. It appears that PMK3 regulates infection processes downstream of PMK1, and may play important roles in penetration, invasive hyphae differentiation and conidiation.

20. Analysis of the function of Gna-1, a G protein a subunit from Neurospora crassa.

Qi Yang and Katherine A. Borkovich, University of Texas Medical School, Houston, Texas.

Heterotrimeric GTP binding proteins, which consist of , and subunits, are crucial intermediates in signal transduction pathways. Upon ligand stimulation, exchange of GDP for GTP on the a subunit causes activation of the subunit and heterodimer. Hydrolysis of GTP by the subunit returns the subunit to its inactive conformation, causing it to reassociate with complex. An subunit with a mutation which causes defective GTPase activity is constitutively active. Neurospora crassa Gna-1 is a member of mammalian G 1 superfamily, which consists of G_i, G_o, G_t, and G_z subclasses. gna-1 mutants have a slower apical extension rate on solid medium and produce aberrant, infertile perithecia during the sexual cycle. We have introduced two kinds of GTPasedeficient mutations into a gna-1 mutant by targeted integration at the his-3 locus. In comparison to isogenic wild type controls, both of the activated alleles cause a dramatic increase in the amount and length of aerial hyphae. However, these strains are otherwise indistinguishable from wild type. The above results imply that Gna-1 has functions independent of subunits, since the gna-1 activated allele strains have different phenotypes from the gna-1 mutant strain. To investigate the evolutionary relationship between Gna-1 and mammalian G_i proteins, we transformed mammalian G_i, G_o, G_t, and G_z genes into the gna-1 strain. Preliminary data shows that some of the mammalian G subunits are able to partially complement the gna-1 defects in either the vegetative or sexual cycle.

21. Molecular cloning and characterization of cAMP-dependent protein kinase (PKA) genes from Colletotrichum trifolii.

Zhonghui Yang and Martin Dickman,University of Nebraska-Lincoln, Nebraska 68583-0722

Colletotrichum trifolii is the causal agent of alfalfa anthracnose. Pharmacological data have strongly suggested that cAMP-dependent protein kinase (PKA) is involved in C.trifolii morphogenesis, including conidia germination and appressorial development. In order to dissect the function of PKA at molecular level, we have isolated and sequenced the genes for both PKA catalytic subunit and regulatory subunit, which are encoded by separate genes. Sequence analysis indicated high homology with PKA genes from other fungi. RNA blots using total RNA from different developmental fungal stages showed that the expression level is highest in conidia, relatively lower in germinated conidia and appressoria, and lowest level of expression was found in vegetatively grown mycelia. Antibodies were generated against the catalytic subunit, and similar expression patterns were also observed in a Western blot. These results indicated that PKA is likely to be important in C.trifolii morphogenesis and the expression of PKA genes are regulated at transcriptional level. A heterologous complementation experiment showed that C.trifolii PKA catalytic subunit could complement S.pombe PKA mutation. Further functional characterization is underway for antisense analysis of catalytic subunit gene and gene-replacement analysis of regulatory subunit gene.

22. Neurospora crassa type 2A protein phosphatase is involved in hyphal growth .

Einat Yatzkan and Oded Yarden, Department of Plant Pathology and Microbiology, Faculty of Agriculture, The Hebrew University of Jerusalem, Rehovot 76100, Israel.

The type 2A serine/threonine protein phosphatase (PP2A) holoenzyme consists of a core complex comprised of a 36 kDa catalytic subunit (C) tightly associated with a 65 kDa regulatory subunit (A). This dimeric core can be complexed with a third, variable, subunit (B), which in higher eukaryotes has been shown to control enzyme activity and specificity. In the presence of the PPI (type 1 phosphatase) and PP2A inhibitors cantharidin (100 mM) or calyculin A (250 nM), abundant cell leakage and abnormal swelling events were observed, respectively. Both inhibitors induced multiple branching of hyphae. We have isolated and analyzed two of the PP2A holoenzyme components. A PCR approach, employing the use of degenerate oligonucleotide mixtures, was used to isolate the genes encoding for the PP2A catalytic and variable regulatory subunits from N. crassa. In contrast to other organisms, N. crassa apparently has only one gene (designated pph-1) encoding for the C subunit. pph-1 was mapped to LG-IVR. Nonetheless, a cross-hybridizing, structurally related, gene encoding a novel PPT-like serine/threonine protein phosphatase was also identified (ppt-1, mapped to LG-VR). Transformants in which pph-1 had been disrupted could be maintained only as hetrokaryons which contained additional, intact, copies of pph-1. RNase protection and phosphatase activity assays showed differential pph-1 transcript and PP2A activity levels during conidial germination. Ectopic integration of pph-1 in a pph-1-disrupted strain brought about altered growth and sensitivity to cantharidin. rgb-1 (mapped to LG-IL), a gene encoding for the B regulatory subunit, is highlly similar to those found in other organisms. Attempts to obtain strains in which rgb-1 had been disrupted were unsuccessful, suggesting that this gene is essential.

23. Isolation and characterization of flbA suppressor mutants.

Jae-Hyuk Yu and Thomas H. Adams, Department of Biology, Texas A&M University, College Station, TX. 77843

Initiation of A. nidulans asexual reproductive development requires the control of proliferative growth and production of an extracellular signal that activates development. We have shown that two genes, flbA and fadA, have a major role in determining the balance between growth and sporulation. fadA encodes the - subunit for a heterotrimeric G protein and continuous activation of FadA blocks sporulation while stimulating proliferation. flbA encodes an A. nidulans RGS (regulator of G protein signaling) domain protein that antagonizes FadA-mediated signaling to reduce proliferation and allow development. Interestingly, this role for FlbA in negatively affecting FadA signaling is necessary for biosynthesis of the mycotoxin sterigmatocystin (ST) as well as sporulation. To better understand FlbA function and other aspects of FadA- mediated growth control, we have isolated and characterized mutations in five distinct loci (designated sfaA - sfaE) that suppress a flbA loss-of-function mutation (flbA98). These suppressors overcome the requirement for flbA in both sporulation and ST biosynthesis. sfaB8, sfaC67, sfaD82, and sfaE83 mutations are dominant to wild-type whereas sfaAl is semidominant. sfaA1 also differs from other suppressors in that it cannot suppress the flbA deletion (and is therefore allele specific) whereas all the dominant suppressors can. Only sfaE83 can suppress the dominant activating mutation in fadA indicating that sfaE may play a unique role in fadA-flbA interactions that control growth and sporulation. Finally, asexual sporulation directed by all suppressor mutations requiresflug, indicating that no suppressor can bypass the requirement for development-specific activation.