Posters I: Signal Transduction

We investigated the function of calcineurin in N. crassa through the inductible antisense expression of the catalytic subunit. Three independent transformants were found to grow at wild-type rates on normal medium. However on induction medium at 37 C they displayed an "oscillating- growth phenotype". This was characterized by cycles of growth followed by several hours of arrest: after 20 hours of growth extensive hyphal branching and conidiation marked the end of each growth cycle. The mechanism involved is under investigation. The severity of the phenotype was temperature dependent, at 29 C the transformants exhibited hardly any growth once induction became effective. From this we conclude that calcineurin is essential for hyphal elongation in N. crassa. Antisense expression of the catalytic subunit of calcineurin correlates with sensitivity to the immunsuppressive drugs Cyclosporin A and FK506, respectively, indicating that calcineurin playes a role in the mechanism of drug action in Neurospora crassa.

42. Gna-2: a G-protein alpha subunit from Neurospora crassa.

Heterotrimeric G proteins which consist of three subunits, alpha, beta and gamma, regulate a variety of receptor-mediated signalling pathways. In fungi, G proteins have been shown to regulate mating, sexual sporulation and are also implicated in plant pathogenicity. Genes for two N. crassa G protein a subunits (gna-1 and gna-2) have been cloned and characterized by our laboratory. Based on amino acid identity and polyphyletic clustering, gna-2 is most related to gpa1 from Schizosaccharomyces pombe. S. pombe gpa1 is essential for sexual sporulation and mating but not for asexual growth. In contrast, our studies indicate that gna-2 might be essential for asexual growth of N. crassa. Attempts at recovering gna-2 homokaryotic strains were unsuccessful. To understand the function of Gna-2, we are currently using four approaches: 1) creating mutants using Repeat Induced Point Mutation, 2) placing gna-2 cDNA under the control of the inducible quinic acid qa-1 promoter, 3) utilizing the yeast dual hybrid system to identify proteins that interact with Gna-2, and 4) using site-directed mutagenesis to create specific point mutations that have been shown to constitutively activate G proteins in other systems. This last approach will allow us to investigate the outcome of Gna-2 overactivity. N. crassa gna-2 provides a unique chance for understanding the evolution of signalling pathways in fungi; gna-2 might be a related homologue of S. pombe gpa1 that during evolution has acquired an essential function for asexual growth.

43. Biochemical, genetic, and morphological study of a Neurospora crassa G protein-ai subunit deletion mutant

G-protein alpha-beta-gamma heterotrimers form the molecular switches that initiate signal transduction pathways. In response to extracellular cues such as light, nutrients, or sexual pheromones, G-proteins serve to transduce signals to downstream second-messenger-producing effectors. Our laboratory has cloned two G protein alpha-subunits from Neurospora crassa, gna-1+ and gna-2+. Gna-1 is 55% identical to members of the mammalian inhibitory class of G-alpha subunits (G-alpha-i) and is the first reported microbial G-alpha that has a mammalian homologue. Members of the mammalian G-alpha-i class are defined by possession of consensus sequences for myristoylation and ADP-ribosylation by pertussis toxin. The amino-acid transporter gene (mtr+) was used to mark a gene deletion/disruption of gna-1+ in Neurospora crassa. Southern analysis of gna-1::mtr strains showed the predicted pattern for a mtr+ insertion and loss of the wild-type gna-1+ pattern. Loss of the Gna-1 protein band on Western blots of mutant strain protein extracts and the loss of a pertussis toxin substrate in the same protein extracts verified that the strains were gna-1::mtr homokaryons. In addition, the pertussis toxin labeling experiments revealed that Gna-1 is the only G-alpha-i present in Neurospora crassa. The gna-1 mutation is pleiotropic, causing several morphological changes and slowing of apical extension. An effect of salt and temperature is also observed. Possible second messengers in the G protein signal transduction cascade are presently being investigated.

44. Identification of proteins that interact with gna-1 during signal transduction in Neurospora crassa

Heterotrimeric GTP binding proteins, which consist of alpha, beta, and gamma subunits, are crucial intermediaries in signal transduction pathways. Neurospora crassa Gna-1 is a G protein a subunit which is a member of the Gi alpha family. The gna-1+ gene has been cloned and sequenced and gna-1 deletion mutants have been made by our laboratory. Recent results have shown that Gna-1 is involved in cell proliferation and differentiation in N. crassa. Proteins which interact with Gna-1 during signal transduction, such as receptors, beta subunits, gamma subunits and effectors, are currently being investigated. Gna-1 has been successfully immunoprecipitated from solubilized plasma membranes and coimmunoprecipitation of Gna-1-associated proteins is in progress. In addition, the Polymerase Chain Reaction is being applied as an alternative approach to identify receptor and beta subunit genes. In this case, degenerate primers have been designed according to the conserved portions of receptors and beta subunits from various organisms. Finally, the yeast two-hybrid system is being utilized in studies of protein-protein interactions. Elucidation of proteins which interact with Gna-1 will clarify the G protein signal transduction pathways in Neurospora crassa.

45. Exploring the role of protein kinase C and protein phosphorylation in hyphal development for a fungal plant pathogen

Ustilago violacea is a basidiomycete phytopathogen that infects members of the Carnation family (Pinks). When diploid teliospores of the fungus germinate on suitable surfaces, such as floral parts, they undergo meiosis to produce a linear tetrad of basidiospores which segregate two mating types, a1 and a2. Further development of the fungus and infectivity requires that sporidia of opposite mating type conjugate and then receive a signal from a suitable host plant to allow formation of dikaryotic hyphae. Compounds such as tocopherols and phytols stimulate hyphal growth of some conjugating strains at concentrations equivalent to hormonal action (as low as 10 uM). Moreover, such response is dominant: strains which are normally unaffected by alpha-tocopherol (VitE) when mated amongst themselves will support hyphal growth when conjugated with isolates that normally do respond. Hyphal growth is stimulated, to a lesser degree, by from 10-100 uM 1-o-hexadecyl-2-o-methyl-rac- glycerol (HEX), a specific inhibitor of protein kinase C (PKC). Phorbol ester, in the short term a specific activator of PKC, much later causes inhibition of the enzyme. After 6-12 h post exposure to concentrations of phorbol ester from 1.6-16 mM, conjugating sporidia produced abberant 3 and 4-cell mating patterns; when VitE was also present, conjugating sporidia showed a 50-75% reduction in hyphal production compared to those with VitE alone. After several days, massive hyphal production was eventually found in treatments with phorbol ester alone. Preliminary examinations of total cellular proteins suggest reduced phosphorylation in the presence of either VitE or HEX. Thus, level of phosphorylation of targets for PKC may control hyphal production in U. violacea.

46. G Protein alpha subunit of Pneumocystis carinii

Pneumocystis carinii pneumonia results in significant morbidity and mortality among immunocompromised individuals. A lack of understanding of the cellular or sexual biology of the organism has hampered rational development of new antimicrobial therapies. G-proteins are highly conserved member of the signal transduction pathway important in many intracellular signaling pathways including the mating cascade. To characterize signal transduction pathways in P. carinii, we have cloned the G protein alpha subunit of P. carinii. Genomic DNA from a candidate species of P. carinii from rat, P. carinii carinii, was used as target DNA. Primers were designed to a highly conserved region of the G alpha subunit, and a 280 bp product encoding the GTP binding domain of the alpha subunit was amplified. The amplified product hybridized to a 450 kb chromosome of P. c. carinii. Sequence analysis revealed an open reading frame interrupted by a single 46 bp intron in a position identical to one of the introns detected in GPA1 of Cryptococcus neoformans. The predicted peptide showed 57-61% identity with known fungal G alpha proteins with greatest homology to Schizosaccharomyces pombe GPA1 and 42-57% identity with known rat G-alpha proteins. The ORF had similar codon bias to other cloned P. carinii genes with 63% AT usage in the third nucleotide position of transcribed codons. Characterization of the genes involved in signal transduction will permit a better understanding of the reproductive capacity and other cellular processes in this family of organisms that cannot be continuously cultured.

47. Calmodulin-dependent multifunctional protein kinase (ACMPK) activity is essential for growth of Aspergillus nidulans

ACMPK is a monomeric calmodulin-dependent protein kinase in A. nidulans which exhibits a substrate specificity similar to mammalian CaMPKII. It is encoded by a single copy gene, cmkA, which is transcribed to yield a single 1.7 kb mRNA. Three strains of A. nidulans have been constructed in which ACMPK expression can be manipulated experimentally. The first, MG1, is a strain with an additional copy of cmkA under the control the inducible alcA promoter. In Ca+- depleted medium, MG1 cells overexpressing ACMPK exhibit distinct advantages in growth and maintenance of normal morphology over cells expressing normal amounts of the enzyme. In MG2 cells, induction of the expression of a truncated form of ACMPK lacking the CaM-binding domain leads to growth arrest after several rounds of nuclear division, despite the fact that these cells are constitutively expressing full length ACMPK. KS1 cells, in which a single copy of cmkA is under the control of alcA, grow normally in medium containing ethanol as the carbon source. KSl conidia are growth arrested following several mitotic divisions when germinated on medium containing glucose, which represses ACMPK expression, indicating that ACMPK is essential for viability. (Supported by the American Cancer Society).

48. Apoptotic cell death and G1 arrest in plant and animal cells induced by fumonisin

Fusarium monilforme is a major pathogen of corn. Most strains of this fungus produce several mycotoxins the most prominent of which is called fumonisin. Fumonisin causes a neurodegenerative disease in horses, induces hepatic cancer in rats, and pulmonary edema in swine. Studies have also demonstrated that fumonisin is correlated with increases of esophageal cancer. High levels of fumonisin can be detected in both healthy and diseased corn. Thus consumption of corn based products by both man and animals poses a serious health threat, Structurally, fumonisin resembles sphingolipids, although little is known concerning the mechanism by whlch fumonisin elicits its toxic effects. Recent findings in our laboratories indicated fumonisin alters transcription and signal transduction pathways in monkey cells. Fumonisin represses specific protein kinase C isoforms and AP-l dependent transcription. In contrast, fumonisin stimulated a simple promoter containing a single cycle AMP response element (CRE). Cells treated with fumonisin, showed inducible binding of nuclear factors with oligos containing a CRE or AP-l binding site. Furthermore, fumonisin induced programmed cell death (apoptosis) in both monkey and tomato and arrested those cells in the Gl phase of the cell cycle. Our working hypothesis is fumonisin activates or represses transcription of genes which regulate growth of animal cells. Consequently, abnormal growth occurs which contributes to the induction pathological changes. The ability of fumonisin to alter signal transduction pathways, cause Gl arrest and induce apoptosis is likely to be necessary for its carcinogenic and toxic effects.

49. The effect of neomycin on growth, inositol metabolism and protein synthesis in Neurospora crassa

Neomycin has been used to perturb the turnover of the inositol phospholipids in animal cells and inhibit protein synthesis in bacteria. In this study, the effects of neomycin on the growth, inositol metabolism and protein synthesis in Neurospora crassa were studied. Increasing levels of neomycin (2.2-8.8 mM) prevented growth. To determine how neomycin affected growth, high concentrations of the drug (5.5 mM) were added to cultures and at various times after the addition, mycelial samples were analyzed for changes in inositol metabolism and protein synthesis. Neomycin stimulated inositol uptake and metabolism. After the drug was added, inositol increased 125% within 1 h. Within 4 h, the phosphoinositides and inositol phosphates increased, on average, 43% and 103%, respectively. In contrast, the rate of protein synthesis declined dramatically after the addition of neomycin. Within 1 h, the rate of protein synthesis declined 34%. Within 3 h, the rate declined to 54%. A continued decline in protein synthesis caused by neomycin would prevent growth in Neurospora crassa. It is, however, unclear what the effect of increased inositol metabolism would have on growth and cell signalling.

50. Isolation and functional analysis of krev-1 gene, a ras-superfamily of N. crassa

The krev-1 gene is a member of the ras-superfamily, and this gene product suppresses oncogenic ras transformation in mammals. Recently, krev-1 homologs have been isolated from several species. We used polymerase chain reaction to isolate a new member of the ras superfamily of Neurospora, and obtained a krev-1 homolog. The Neurospora krev-1 gene encoded the polypeptide that was 57% identical to those of mammalian Krev-1 at the predicted amino acid level. RFLP mapping confirmed the chromosomal position of krev-1 on LGIL near nit-2. krev-1 is expressed constitutively at low level in vegetative growth phase. To disrupt krev-1 by the RIP phenomenon, cells carrying the duplicated krev-1 were crossed. Segregants carrying the mutations in krev-1, confirmed with Southern blot analysis, showed no characteristic phenotype. To examine krev-1 function, we used site-specific mutagenesis to alter conserved residues known to be important for the function of the ras- superfamily. We are now examining the functional relationship of Krev-1 homologs between N. crassa and S. cerevisiae.

51. A deletion mutant of NC-ras-2 in Neurospora crassa

One of the Ha-ras homologues of N. crassa, NC-ras-2, has been cloned and reported (Murayama and Kana-uchi; Fungal Genetics Conference, 1993). To study the roles of this gene, we tried to disrupt the gene through RIP (Rearrangement Induced Premeiotically) using transformants containing an additional copy of the genomic DNA carrying NC-ras-2 and qa-2. Among progenies of the crosses between the transformants and the wild type, one morphological mutant ras-17 which grew poorly was obtained. Morphology of ras-17 on the solid medium was compact and colonial. Southern blot analysis revealed that the ras-17 strain has large deletion in the coding region of NC-ras-2. To know the relationships between the adenylyl cyclase and NC-ras-2 protein in N.crassa, cAMP level in the ras-17 strain was measured. The roles of NC-ras-2 gene in N.crassa were discussed.

52. The isolation of mutants in the light signal transduction pathway of Neurospora crassa

In the filamentous fungus Neurospora crassa several photomorphogenic and biochemical events are regulated by blue light. During the asexual cycle, blue light regulates carotenogenesis in mycelium including the expression of the carotene biosynthesis genes al-1, al-2 and al-3. A new screening system has been developed in order to obtain mutants showing deficiencies in the blue light signal transduction. The gene of the neutral amino acid permease (mtr) has been cloned under the control of the al-3 promoter. This construct has been used to transform a Neurospora strain which carried mutations in the mtr and trp loci. In this transformant, thc expression of the amino acid permease was now found to be light regulated. In the presence of PFPA, a poisonous amino acid analogue, growth in the light was strongly inhibited due to the uptake of PFPA by the mtr permease. However, growth on low tryptophan concentrations was observed in the light only because of the light regulated uptake of the amino acid. Applying these two selection systems, several different mutants could be isolated after UV mutagenesis. Some of them were shown to have a "white collar" phenotype and to be "blind" for several blue light responses in Northern analysis. Using the positive selection instead, mutants were isolated which showed, in contrast to the wild type, a constitutive carotenoid biosynthesis even in the dark. The presented screening system can also be applied to recover the corresponding genes.

53. Regulation of conidial formation by cyclic AMP in Neurospora crassa

Cyclic AMP (cAMP) has been described as one of the most important mediators in the signal transduction pathway in various organisms. The reduction of nitrogen source and carbon source induced conidial formation, but the addition of cAMP to the medium repressed it. The level of cAMP decreascd quickly after being transferred to the carbon source- and nitrogen source-free medium. A mutant (cr-1) having defective adenylyl cyclase, which catalyzes the synthesis of cAMP from ATP, formed conidia before the reduction of the nutrients. A suppressor mutant hah which suppressed the colonial growth of cr-1 and had considerably elevated level of adenylyl cyclase gene transcript did not form conidia. These results suggest that reduction of cAMP induces conidial formation and that reduction of nitrogen source and carbon source induces formation of conidia through the reduction of cAMP.

54. Purification and cloning of Ser/Thr protein phosphatases from Neurospora crassa

Protein phosphorylation is a universal regulatory mechanism in eukaryotic cells. The phosphorylated state of a protein is affected by the conflicting activities of protein kinases and phosphatases. Nearly all Ser/Thr specific dephosphorylation reactions can be attributed to four classes of protein phosphatases (PP's): PP1, PP2A, PP2B, PP2C, which are differentiated on the basis of inhibitor sensitivity and metal ion dependence. We found that all the four main classes of Ser/Thr protein phosphatases are present in N. crassa. The catalytic subunit of PP2A (PP2AC) was purified 100-fold by using ammonium sulfate-ethanol precipitation, DEAE Sephacel, Heparin-Sepharose and MonoQ FPLC chromatography. Its apparent molecular mass proved to be 35 kD in gel filtration experiment. PP2AC was completely inhibited by 1 nM okadaic acid, 4 nM microcystin and 20 mM NaF, was insensitive to rabbit muscle inhibitor-2, and was specific for the (alpha subunit of rabbit muscle phosphorylase kinase. The catalytic subunit of PP1 was found to be less stable because its activity was gradually decreasing during purification. The pSV50 cosmid library was screened using a Drosophila PP1 cDNA probe. Four recombinant cosmids of different restriction patterns were isolated. The heterologous cDNA probe hybridized to 4-5 BamHI and HindlII fragments in a Southern hybridization experiment. On the basis of these results we assume that a gene family of at least four PPI genes is present in N. crassa.

55. Inhibition of cAMP-dependent protein kinase prevents spore germination and appressorium development in Colletotrichum trifolii.

Colletotrichum trifolii, the causal agent of alfalfa anthracnose, is one of the most destructive fungal pathogens of alfalfa. Spore germination and the development of appressorium are requisite for this fungus to infect susceptible alfalfa cultivars, however, the signal transduction pathways responsible for triggering this developmental switch are poorly understood. We have found a relatively large amount of cAMP dependent protein kinase (PKA) activity in spores and germinated spores compared to mycelia and fully developed appressoria. Therefore, we investigated the involvement of PKA during this process. By using a PKA specific inhibitor, KT5720 at a physiologically relevant concentration, spore germination and appressorium development were virtually shut down. Further investigations using IBMX, a phosphodiesterase specific inhibitor and the cAMP analogue 8-bromo cAMP, which increase the endogenous level of cAMP, resulted in enhancement of spore germination and appressorium development. Our data strongly indicate that cAMP may act as a central mediator for differentiation by activating PKA and triggering a biochemical pathway resulting in both spore germination and appressorium development. Molecular cloning of the genes of PKA catalytic domain and regulatory domain are in progress.

56. Molecular characterization of a filamentous mutant of Ustilago horde

Successful infection of barley by Ustilago hordei, the causal agent of covered smut, depends on the formation of a filamentous dikaryon, which is produced after mating of compatible haploid cells, which are sporidial in appearance and nonpathogenic. We report the isolation of a filamentous, haploid mutant, fil1-1, isolated following heat shock treatment. Analysis by pulsed-field gel electrophoresis (PFGE) revealed a 50 kb deletion in a 940 kb chromosome. RFLP studies using a telomere-specific repeat (TTAGGG)18 from Fusarium oxysporum as a marker, place the deletion near the end of one arm of the 940 kb chromosome. In a cross wild type (sporidial) X mutant (filamentous), the fil1 mutation and the deletion were shown to invariably cosegregate 2:2 with the wild type morphology in an ordered tetrad. In the presence of cyclic AMP, the filamentous phenotype reverts to the sporidial wild type. The same effect was observed for analogs and stimulators of cyclic AMP and inhibitors of phosphodiesterase. These data suggest that the mutation is not in the structural gene encoding the adenylate cyclase, but is possibly in a gene encoding either a G protein or the receptor protein. The putative gene(s) controlling the dimorphic switch are currently being cloned.

57. Regulation of inositol synthesis in Cryptococcus neoformans

Cryptococcus neoformans is a basidiomycete that infects immunocompromised humans. C. neoformans grows in defined media with or without inositol supplementation. Inositol is a precursor of phosphatidylinositol (PI), an essential membrane lipid. The presence of PI in the membranes of C. neoformans grown in media without inositol supplementation indicates that this organism can synthesize inositol. The synthesis of PI in C. neoformans is different than in other yeast, however, in that it is unaffected by the presence of exogenous inositol. This study examines the regulation of inositol synthesis in C. neoformans. In eukaryotes glucose-6-phosphate is converted to inositol-1- phosphate by the enzyme inositol-1-phosphate synthase. Inositol-1-phosphate synthase was detected in crude extracts of C. neoformans. This activity was repressed by the provision of 75 uM inositol in the growth media. Regions of homology between sequences of the genes encoding inositol-1- phosphate synthase from two other yeasts and two plants were used to design synthetic oligonucleotides. PCR amplification of C. neoformans genomic DNA generated fragments that were used to probe Northern blots. Preliminary results reveal a transcript that is regulated in response to inositol in the growth media. Hence, the regulation of inositol synthesis in C. neoformans appears to be similar to other yeast, yet the regulation of PI synthesis is different. Perhaps the difference is due to this organism's unusual capacity to catabolize inositol. Clearly the regulation of inositol metabolism and the coordination of synthesis and catabolism are essential for balanced cell growth.

58. Inactivation of a single type 2A phosphoprotein phosphatase is lethal in Neurospora crassa

A PCR approach, employing the use of degenerate oligonucleotide mixtures, was used to isolate pph- 1, a type 2A protein phosphatase gene, from Neurospora crassa. The isolated single-copy gene is 1327 nucleotides in length, contains four putative introns and encodes a 310 amino acid polypeptide. pph-1 is located between pdx-1 and col-4 on the right arm of N. crassa linkage group IV. pph-1 transcript levels are highest during the first hours of conidial germination. Failure to obtain viable progeny in which pph-1 had been inactivated via the Repeat-Induced Point mutations process and evidence that nuclei harboring a disrupted pph-1 gene could only be maintained in a heterokaryon, indicate that a functional pph-1 gene is essential for fungal growth. This is the first report providing evidence that inactivation of a single type 2A protein phosphatase results in a lethal phenotype in fungi.

59. The NIMA protein kinase is hyper-phosphorylated and activated downstream of p34(cdc2)/cyclinB: coordination of two mitotic promoting kinases

Initiation of mitosis in Aspergillus nidulans requires activation of two protein kinases, p34(cdc2)/cyclinB and NIMA. Forced expression of NIMA, even when p34(cdc2) was inactivated, promoted chromatin condensation. NIMA may therefore directly cause mitotic chromosome condensation. However, the mitotic promoting function of NIMA is normally under control of p34(cdc2)/cyclinB as the active G2 form of NIMA is hyperphosphorylated and further activated by p34(cdc2)/cyclinB when cells initiate mitosis. To see the p34(cdc2)/cyclinB dependent activation of NIMA, okadaic acid had to be added to isolation buffers to prevent dephosphorylation of NIMA during isolation. Hyperphosphorylated NIMA contained the MPM-2 epitope and, in vitro, phosphorylation of NIMA by p34(cdc2)/cyclinB generated the MPM-2 epitope suggesting that NIMA is phosphorylated directly by p34(cdc2)/cyclinB during mitotic initiation. These two kinases, which are both essential for mitotic initiation, are therefore independently activated as protein kinases during G2. Then, to initiate mitosis, we suggest they each activate the other's mitotic promoting functions. This ensures that cells coordinately activate p34(cdc2)/cyclinB and NIMA to initiate mitosis only upon completion of all interphase events. Finally, we show that NIMA, like cyclinB, is degraded specifically during mitosis.

60. cAMP dependent protein kinase catalytic subunit (cpkA) gene is required for appressorium formation in Magnaporthe grisea

Successful infection of rice by the fungal pathogen Magnaporthe grisea depends upon the formation of a dome shaped, highly melanized infection structure, an appressorium. The differentiation of this unique, specialized cell from the tip of an emerging germ tube is a response to environmental stimuli. The exogenous and endogenous signalling mechanisms involved in surface recognition and the transfer of this information into the cell leading to infection related morphogenic events remain to be elucidated. We have shown that cyclic AMP, a second messenger involved in signal transduction systems, regulates appressorium formation. In other systems, the primary target for cAMP is cAMP dependent protein kinase. Activation of this kinase directly or indirectly results in specific developmental changes. To elucidate the mechanism of cAMP action in M. grisea, we have isolated, sequenced and disrupted the single gene encoding the catalytic subunit of cAMP dependent kinase (cpkA). Strains lacking the cpkA gene appear unaffected in their ability to grow and reproduce sexually and asexually. However, they are unable to form appressoria on rice or in the presence of cAMP and are non pathogenic. This is the first direct evidence that fungal pathogenesis is mediated via cAMP dependent protein kinase.

Return to the Asilomar 1995 page
Return to the Asilomar page
Return to the FGSC main page