Unclassified abstracts

Abstract numbers > 380

Unclassified abstracts

380. Osmotin, an antifungal plant defense protein, induces apoptosis-like cell death in yeast.Barbara Damsz, Meena L. Narasimhan, Jose I. Ibeas, Paul M. Hasegawa and Ray A. Bressan. Center for Plant Environmental Stress Physiology . Purdue University. West Lafayette, IN.

Osmotin is a tobacco pathogenesis-related, protein (PR-5) that is antifungal. It is implicated in plant defense against invading fungi. Cell death induced in the unicellular Ascomycete, Saccharomyces cerevisiae, by osmotin exhibits several features of apoptosis. First, cells can be rescued after brief osmotin treatments but not after prolonged treatments. Cytosol shrinkage, an early marker of apoptosis, was observed in cells after brief treatments, with greater than 50% of the cells exhibiting cytosol shrinkage after prolonged treatment when most cells had lost viability. Plasma membrane blebbing, unusual mitochondrial morphology (enlarged, with swollen inner membrane) cytoplasmic blebbing and apoptotic bodies were observed in the osmotin-treated cells that were losing viability. Nuclear fragmentation in 14-25% of the cells was observed in this population by DAPI staining. Chromatin fragmentation was also detected using terminal nucleotidyl transferase. Osmotin-induced cell death was clearly different from necrosis that was induced by poly-L-lysine in low ionic strength medium.

381. G proteins mediate the fungal cytotoxicity of osmotin, a plant defense protein.Maria A. Coca, Meena L. Narasimhan, Dae-Jin Yun, Paul M. Hasegawa and Ray A. Bressan. Center for Plant Environmental Stress Physiology . Purdue University. West Lafayette, IN.

Osmotin is a pathogenesis-related protein1, that has in vitro and in vivo antifungal activity against a broad range of fungi, including several plant pathogens2. Tobacco osmotin induces rapid cell death in some strains of Saccharomyces cerevisiae3. We have shown that the cytotoxic effect of osmotin in yeast cells is mediated by a heterotrimeric G protein and a MAP kinase based signaling pathway4. We report here that G proteins are also required for the cytotoxic action of osmotin in Aspergillus nidulans. Osmotin induces spore lysis, inhibits spore germination and inhibits hyphal tip elongation of A. nidulans. Deletion of the flbA gene, which encodes an A. nidulans RGS (for regulator of G protein signaling) protein5, results in osmotin resistance. Also, a dominant interfering mutation in the fadA gene, that encodes the subunit of a heterotrimeric G protein, results in osmotin resistance. Moreover, the addition of the guanidine nucleotide GDPS, that locks G-proteins in a GDP-bound inactive form inhibits the cytotoxic effect of osmotin on A. nidulans spores. The G proteins regulate osmotin sensitivity by altering the porosity of the cell wall. These results suggest that the mode of osmotin action elucidated in S. cerevisiae can be applied to filamentous fungi.1. Yun DJ, Bressan RA, Hasegawa PM. (1997). Plant antifungal proteins. In Plant Breeding Reviews, J. Janick, ed. (New York:John Wiley & Sons, INc.), 14, 39-88.2. Abad LR, et al. . (1996). Plant Sci. 118, 11-23.3. Yun DJ, et al. (1997). Proc. Natl. Acad. Sci. USA 94, 7082-7087.4. Yun DJ, et al. (1998). Molecular Cell 1, 807-817.5. Lee BN and Adams TH. (1994). Mol. Microbiol., 14, 323-334.6. Yu JH, Wieser J and Adams TH. (1996). EMBO J. (15),19, 5184-5190.

382. A genetic analysis of H. capsulatum pathogenesis. Anita Sil. Department of Microbiology and Immunology, University of California San Francisco, San Francisco, CA.

Our goal is to identify fungal genes that are required for the pathogenesis of Histoplasma capsulatum. This organism grows in hyphal or conidial forms in the soil; hyphal fragments or microconidia are inhaled by the host. At 37 C, the cells undergo a morphogenetic switch and grow as a budding yeast form which parasitizes macrophages, multiplying within their phagolysosomes. How H. capsulatum is able to escape killing by macrophages and colonize the phagolysosome, an intracellular niche that is normally hostile to microbes, is a mystery. The recent development of molecular genetic tools now makes it possible to use genetics and molecular biology to dissect the mechanism of interaction between H. capsulatum and the host cell.We are using molecular genetic methodology to identify H. capsulatum genes that are necessary for the organism to parasitize macrophages. We are employing a previously developed cell culture assay in which H. capsulatum cells are co-cultured with a monolayer of mouse macrophages (Eissenberg et al., Infect Immun, 1991. 59(5)). H. capsulatum is quickly internalized by the macrophages and lyses the macrophage monolayer after 3-5 days. EMS mutagenesis will be used to generate a bank of mutant yeasts; we will then use the monolayer lysis assay to identify mutant strains that fail to lyse macrophages. Microscopic assays will be used to sort the mutants into the following classes: those that affect adherence of the fungus to macrophages, entry into macrophages, evasion of killing by macrophages, replication within macrophages, and lysis of macrophages. Genes that are defective in the mutant strains will be cloned by complementation of the lysis phenotype in the monolayer assay. The gene products so identified will be analyzed using sequence comparison, expression pattern, and sub-cellular localization. We believe that molecular genetics, which has been key to guiding our understanding of biology in other organisms, will shed light on the pathogenesis of this fascinating fungus

383. The hydrophobin cryparin strengthens cell walls of the ascomycete Cryphonectria parasitica. P. Kazmierczak, D.H. Kim, P.M. McCabe, and N.K. Van Alfen. Department of Plant Pathology and Microbiology, Texas A&M University, College Station, TX 77843-2132.

During stationary growth of the ascomycete Cryphonectria parasitica in liquid culture up to 1% of the dry weight of the fungus is the Class II hydrophobin cryparin. The amino acid sequence of cryparin is remarkably similar to that of the hydrophobin produced by Ophiostoma ulmi (53% of the amino acids are conserved) . To better understand why this type of hydrophobin would be so highly conserved in these fungi, the gene encoding cryparin was deleted and the consequent phenotype characterized. As observed for most hydrophobins, the lack of cryparin resulted in a wettable aerial hyphae phenotype. Since aerial hyphae play no known role in the biology of this fungus, other phenotypic changes were also sought. Antibody detection methods have demonstrated that when the fungus grows on its natural substrate, chestnut trees, the hydrophobin is found only in fruiting body walls. We found no evidence that cryparin plays a role in virulence of the fungus, sexual sporulation, or asexual sporulation in culture. We have found that the hyphal cell walls are reduced in strength without cryparin and that the strength can be restored by exogenous addition of cryparin. We postulate that cryparin plays an important role in the eruption of pycnidia in stroma of infected trees.

384. Perturbation of cryparin secretion by the Cryphonectria hypovirus. P. M. McCabe and N. K. Van Alfen. Department of Plant Pathology and Microbiology, Texas A&M University, College Station, Texas. 77843-2132

Strains of the chestnut blight fungus, Cryphonectria parasitica, infected with a dsRNA virus, CHV1, show a reduction in pigmentation, conidial formation, sexual reproduction and virulence. Virus infection also results in the accumulation of small membranous vesicles which are many fold more abundant in the infected strains than the uninfected ones. Viral replication in vitro is correlated with this vesicle fraction and the putative CHV1 polymerase was present in this fraction. A number of host genes have been shown to be differentially regulated as a result of viral infection. We have characterised three secreted proteins that are down-regulated by the virus: a laccase, a hydrophobin and a mating type specific pheromone. All contain signal peptides followed by a pro region with recognition sequences for cleavage by a protease similar to Kex2p.One of these proteins, the hydrophobin cryparin, is present as a cargo protein in the vesicle fraction on which CHV1 replicates. Using pulse-chase methods to study the secretion of cryparin we have found that the virus is utilising a host secretory system for replication, causing an accumulation of vesicles and the cargo protein cryparin, with a resultant decrease in the rate transport of the cargo protein.

385. Mixing Signals: Schizophyllum pheromones and receptors initiate pheromone response in Saccharomyces, providing a means to study receptor/ligand interaction. Thomas J. Fowler, Susan M. Desimone, Michael F. Mitton, Janet Kurjan and Carlene A. Raper, Dept of Microbiology and Molecular Genetics, University of Vermont, Burlington, VT USA

The homobasidiomycete Schizophyllum commune encodes about eighteen receptors and possibly more than fifty pheromones among the eighteen variants of its Ba and Bb mating-type loci. These seven-transmembrane-domain receptors and compatible lipopeptide pheromone ligands trigger multiple downstream events during mating. We asked whether Schizophyllum receptors and pheromones could substitute for their yeast counterparts to initiate the Saccharomyces cerevisiae pheromone response pathway, and if so, was the receptor/ligand specificity of Schizophyllum maintained in the heterologous yeast system. Genes encoding these Schizophyllum molecules were separately introduced via plasmids into different strains of yeast. Their expression and the ability of their products to interact was assayed through several pheromone-inducible events in yeast: transcriptional induction of a pheromone-responsive gene, cell-cycle arrest, mating projection display, and mating. Four molecules - Schizophyllum receptors Bbr1 and Bbr2, and pheromones Bbp1(1) and Bbp2(4) - were successfully expressed in yeast. Of the four possible combinations of these pheromones and receptors, only the two naturally compatible Schizophyllum pairs initiated downstream events of yeast pheromone response. Further investigations showed that the compatible pair Bbp2(4)/Bbr1 signals through a pathway similar to that activated by yeast pheromones and receptors. Interestingly, Schizophyllum pheromone Bbp2(4) processing and secretion does not require many of the proteins used in biogenesis of the yeast lipopeptide a-factor, although the Schizophyllum pheromone does appear to require farnesylation. We are now in a position to exploit the natural variation of pheromones and receptors that exists in Schizophyllum in order to understand the recognition process of these molecules. We can also take advantage of the excellent genetic and biochemical tools available for yeast to introduce changes in both the receptors and the pheromone ligands in order to better understand how they work.

386. Molecular analysis of the cellular reaction of Fusarium sambucinum to defence compounds of potato.K. Loser, J. Wessels, P. Becker and K.-M. Weltring. Westf. Wilhelms-Universität, Inst. für Botanik, 48149 Münster, Germany.

Fusarium sambucinum (teleomorph Gibberella pulicaris) is a causal agent of potato dry rot. The fungus enters the tubers via wounds where it has to cope with phytoanticipins like the saponins -chaconine and -solanine, and phytoalexins such as the sesquiterpenes rishitin and lubimin. F. sambucinum is able to detoxify all of these plant defence compounds. To learn more about the cellular reaction of the fungus to rishitin, apart from detoxification, we set out to identify genes specifically expressed in response to rishitin exposure. By differential cDNA screening we isolated several rishitin induced (rin) genes which showed increased expression in response to the phytoalexin. Sequence comparison of the isolated genes with known genes revealed homology, among others, to a polyubiquitin and a HMG14/17 gene as well as to multi-facilitator-superfamiliy (MFS) proteins of the multi-drug-resistance type found in yeast. Based on the deduced function of the identified genes a new model of the cellular reaction of F. sambucinum to rishitin is proposed. The model is supplemeted by our investigations of the metabolism of the phytoanticipins -chaconine and -solanine. We have purified and characterized the enzyme -chaconinase, which on one hand is inducible by other saponins like -solanine and -tomatine, but on the other hand is highly specific for the removal of the 1,2-bound rhamnose of -chaconine. We are currently in the process of isolating the corresponding gene to study its regulation and to perform knock-out experiments to evaulate the importance of saponin metabolism for pathogenicity of F. sambucinum on potatoes.

387. Translation Elongation Factor Three: a unique target for anti-fungal drugs.Andrew Hopkins, Dina Shayevich, Jennifer Luebke-Wheeler and Elizabeth Rute. Alverno College, Milwaukee, Wisconsin.

Translation Elongation Factor Three (EF-3) was originally identified in Saccharomyces cerevisiae. It has been shown to be absolutely required for translation in S. cerevisiae and Candida albicans in addition to the two translation elongation factors required for protein synthesis in other eukaryotes: EF-1 & EF-2. The requirement for this factor appears to be uniquely limited to the fungal kingdom. We have identified homologues of EF-3 in two pathogenic fungi which infect immuno-compromised patients: Aspergillus fumigatus and Histoplasma capsulatum. We have sequenced approximately 80% of the A. fumigatus gene. It is most similar to the previously sequenced Candida albicans gene. The unique requirement for EF-3 presents a very attractive target for anti-fungal drugs. Determination of the structure and function of EF-3 will facilitate the design of drugs targeted at this essential factor for protein synthesis in fungi. We recently identified a homologue of EF-3 in the thermophilic fungus Thermomyces lanuginosus. We are in the process of sequencing the gene for the EF-3 protein in this fungus, with a view to obtaining adequate quantities for crystallization and 3-dimensional structure determination.

388. Maintenance of Iron Homeostasis in Ustilago maydis. Sally A. Leong,1,2 W. M. Yuan,2 and L. Kearney2. 1USDA, ARS Plant Disease Resistance Research Unit, University of Wisconsin, Madison, WI USA. 2 Department of Plant Pathology, University of Wisconsin, Madison, WI 53706

Iron present in loosely bound or unchelated forms within cells can act catalytically to produce DNA damaging and lipid peroxidizing oxygen radicals. Living cells regulate the transport and storage of iron to minimize free radical damage. When this regulation is disrupted, the consequences on cellular growth and survival are dramatic. Mutations in fur, a gene regulating high affinity iron uptake in Escherichia coli, lead to 8-fold elevated levels of intracellular iron (Keyer and Imlay, 1996) and increased rates of cell death and mutagenesis during aerobic growth (Keyer and Imlay, 1996; Touati et al., 1995). Likewise, an AFT1up mutation in yeast leads to deregulated iron uptake and reduced cell growth in iron-replete medium (Yamaguchi-Iwai et al., 1995). In both systems, the effects of iron overload are aggravated by mutations in DNA repair systems demonstrating that DNA damage is prevalent under these conditions (Touati et al., 1995; Philpott et al., 1996). In humans, hemachromatosis leds to iron loading in the liver, heart and pancreas, resulting in cirrhosis of the liver, pancreatic fibrosis and cardiac dysfunction, as well as increased risk of hepatocellular carcinoma (Crawford et al., 1996). Moreover, an iron-unresponsive mutant of the IRP protein, which normally controls iron uptake and storage at the cellular level, results in diminished cell survival (DeRusso et al., 1995). Despite the wealth of information on the negative impact of iron overload in cells, surprising little is known about the molecular basis of iron-mediated, transcriptional control of iron uptake in eucaryotes where transcription occurs on a chromatin template. In response to iron starvation, the basidiomycete fungus Ustilago maydis produces two cyclic hydroxamate siderophores ferrichrome and ferrichrome A. Three genes required for siderophore biosynthesis and regulation have been characterized: sid1 encodes ornithine-N5-oxygenase, the first enzyme in the ferrichrome biosynthetic pathway; sid2 encodes a putative peptide synthetase required for ferrichrome biosynthesis; and urbs1 encodes a transcription repressor that interacts via its C-terminal finger domain with GATA sequences in the sid1 (Leong and Winkelmann, 1998). Our working hypothesis is that iron modulates siderophore gene expression at one or more levels. Iron may act as a corepressor of genes regulated by Urbs1 and as a physiological effector that indirectly affects expression of siderophore genes by altering the structure and/or cellular location of Urbs1. As with Fur, the procaryotic analog of Urbs1, iron may directly activate Urbs1 in DNA binding. Urbs1 may repress expression of sid1 through positioning of nucleosomes and/or by formation of a DNA loop in the sid1 upstream region. Consistent with this hypothesis, discrete and iron-dependent, nuclease hypersensitivity sites have been identified in the sid1/sid2 intervening promoter region. To investigate the role of additional genes in iron-mediated regulation of iron homeostasis, we are developing an iron-dependent screen for auxotrophy. Progress on the development of this screening method will be presented.

389. Overexpression of the penicillin biosynthetic genes in Penicillium chrysogenum. M.A. van den Berg, D.J. Hillenga and R.A.L. Bovenberg. Gist-brocades, IPPD/PDD. Delft, The Netherlands.

To analyse the effect of overproduction of the penicillin-biosynthetic genes in the fungus Penicillium chrysogenum, two strategies were applied. The strain Wisconsin 54-1255 strain was transformed with DNA-fragments containing the three penicillin biosynthetic genes. Transformations were done using four combinations: pcbAB, pcbAB+pcbC-penDE, pcbAB-pcbC-penDE en pcbC-penDE. The amdS selection was applied. Several strains with a substantially improved penicillin production were obtained. With the second approach we want to modulate the amount of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS), the first enzyme in the penicillin pathway. To obtain differential expression levels of the gene, the autonomous promoter of pcbAB, the gene encoding ACVS, should be replaced by several other promoters. As a host we used a P. chrysogenum strain in which the complete pcbAB promoter and a small part of the ORF (5' end) were removed. As selection marker the Tn5ble gene, encoding phleomycin resistance, and the cytC terminator were cloned in the same orientation as the pcbAB gene; behind the pcbAB promoter and just in front of the ORF. Two unique restriction sites just in front of the ATG were introduced for easy cloning of heterologous promoters. So far we obtained Penicillium transformants with P. chrysogenum pgkA and niaD promoters replacing the pcbAB promoter, which are currently investigated.

390. Characterization of the Penicillium paf gene promoter for gene expression in Penicillum chrysogenum. R. Kerkman, A.W.H. Vollebregt, R.A.L. Bovenberg. Gist-brocades, IPPD/PDD, Delft, The Netherlands.

In contrast to Aspergillus, only few Penicillium promoters have been characterised so far. In order to isolate and analyse strong promoters for gene expression in Penicillium we examined the relative strength of the promoters of the Penicillium pcbC and paf genes in P. chrysogenum. The promotor of pcbC, is one of most extensively investigated promoters and is considered to be a strong promotor. The paf gene, which encodes a 12-kDa protein with potential antifungal activity, is one of the most abundant proteins present in the culture medium (Vollebregt, ECFG2 abstract B48). To analyse the paf gene promotor in more detail integration vectors were constructed containing various parts/fragments of the promotor region fused to the Streptomyces clavuligerus cefE gene encoding expandase which can be used as a reporter gene. In the presence of suitable side chains e.g. adipate Penicillium transformants expressing expandase are able to convert penicillins to cephalosporins, which can be measured by using bio-assays and shake flask fermentations.Transformants were obtained by co-transformation using the Aspergillus amdS gene as a selectable marker. Selected transformants were tested for promoter activity by performing shake flask experiments and measuring formation of adipoyl- 7-ADCA. The results which will be presented indicate that the paf gene promotor is a strong/suitable promotor for heterologous gene expression in P. chrysogenum.

391. The Aspergillus nidulans spermidine synthase gene, spdA, is required for fungal differentiation.Doralinda Guzman-de-Peña1, Yuan Jin2 and Nancy Keller2. 1Laboratory of Mycotoxins. Unidad Irapuato CINVESTAV-IPN México, 2Dept. of Plant Pathology and Microbiology, Texas A&M University, College Station, TX 77843-2132.

The Aspergillus nidulans spdA gene, encoding for a putative spermidine synthase, was cloned, sequenced, and localized on chromosome VIII. The deduced amino acid sequence has a high similarity with Saccharomyces cerevisiae spermidine synthase encoded by the spE3 gene. In both yeast and the filamentous fungus Neurospora crassa, spermidine and other polyamines are required for fungal differentiation processes. To investigate the function of SpdA on Aspergillus development, we constructed a spdA null mutant by replacing a portion of the coding region with the argB gene. The DspdA strain, which accumulates the biosynthetic precursor of spermidine,putrescine, has an absolute requirement of spermidine for growth, sporulation and sterigmatocystin production. Wildtype growth and development could not be recovered in the DspdA strain by a high concentration (3mM) of either putrescine or spermine but was partiallyrecovered by a high concentration (3mM) of spermidine.

392. Apoptosis of meiotic mutants of Coprinus cinereus.Benjamin C. Lu and Ursula Kuees, Department of Molecular Biology and Genetics, University of Guelph, Guelph, Canada, and Institute of Mikrobiologie, ETH Zurich, Switzerland.

Meiotic mutants of Coprinus cinereus were induced by the Restriction Enzyme Mediated transformation of the AmutBmut strain. These Remi mutants produced white caps with few basidiospore tetrads. This poster presents studies of meiotic proccesses leading to the formation of white caps as a consequence of apoptosis. Cytologically, the white-capped strains can be divided into two major groups: one that assembles perfect synaptonemal complelxes (SC) during meiotic prophase-I, and one that fails to do so. Both groups grogressed through meiotic prophase-I and entered meiotic metaphase-I. Those that assembled the SCs showed chromosome condensation and congregation, and those that did not assemble the SCs showed scatteredcondensed chromosomes. After a brief arrest at this stage, the basidia entered the path of apoptosis. Apoptosis is restricted to basidia which underwent DNA degradation and cellular disintegration, while the rest of fruiting cap continued to develop, showing cap expansion, stipe elongation, and the final stage of autolysis normally found in fruiting bodies of this species.

393. A new database supporting the collection at the Fungal Genetics Stock Center.Kevin McCluskey and Chris D. Kunce. FGSC, Department of Microbiology, University of Kansas Medical Center.

The FGSC has had a variety of databases throughout its life. The original database was the collection of deposit sheets that accompanied each strain as it was added to the collection. Key information was added to each sheet and was extracted by hand as the catalog was prepared. As the number of strains grew, this became overwhelming and when the FGSC moved to the University of Kansas, an electronic database was developed. The catalog was prepared electronically as well. With the advent of widespread use of the internet, the tables in this database were made available through a text-search capacity. While this is very useful, there is a great deal of information that is not readily accessible either to the FGSC staff or to clients. With the assistance of the Univ of Kansas Info-Tech staff, we have developed a relational database that allows more information to be presented to the staff and ultimately to users via the FGSC web-site. The on-line plasmid and bibliography searches are examples the features that are possible. The data model and future developments will be presented.

394. Virulence of Puccinia recondita f. sp. tritici on Lr26-resistance wheat: distribution and genetic originPardes E., Manistersky J., Ben Yehuda P., Kosman I., Anikster Y., and Sharon A. Institute of Cereal Crop Improvement, Tel Aviv University, Tel Aviv, Israel.

Leaf rust of wheat caused by Puccinia recondita f. sp. tritici is among the most widespread diseases of wheat. Fast distribution of the pathogen over long distances and development of epidemics, cause sever crop losses in wheat around the world. The sexual stage of the fungus takes place on Thalictrum apeciosissimum which had been eradicated in most wheat growing regions and thus in nature, P. recondita f. sp. tritici does not reproduce sexually. Despite the lack of a sexual stage, leaf rust epidemics are common around the world and build up of rust populations with the ability to infect resistant wheat varieties occur whenever a new resistance gene is introduced into cultivated wheat. We studied the genetic mechanisms that control the development of isolates virulent on wheat with the Lr26 resistance gene. Sixty isolates representing rust populations from Israel, Europe and the USA were characterized using molecular and phytopathological markers. Phylogenetic analyses showed, that isolates virulent on Lr26 had been evolved independently in several places around the world. The data suggest that Lr26-virulent isolates existed in low proportions in rust populations and their level increased following the introduction and extended use of wheat varieties with the Lr26 resistance gene. Our data also shed new light on the migration of rust. Two separate rust population were identified in Israel suggesting two different sources from which rust arrives in this region. The Israeli populations were found unrelated to the European population ruling out the current model that suggests transfer of wheat leaf rust between the middle east and Europe. Large differences were found between the results obtained using RAPD and phytopathological markers. The differences between the two methods suggest that the phenotypic markers may not be suitable for predicting the source of the rust isolates since they are markedly affected by the cultivars grown in the destination area.

395. CAR 100 A Neurospora crassa mutant strain with a novel branching pattern Aleksandra Virag, UBC, Department of Botany, Vancouver, B. C. Canada

Tip growth and branching of hyphae have received considerable attention because of their importance in fungal development. Nevertheless, the mechanism of branching initiation is still not known. Previous research suggests that migration and incorporation of vesicles, changes in calcium concentration and redistribution of a cortical actin meshwork occur at branching sites. To test the involvement of actin in the initiation of hyphal branches in Neurospora crassa, conidia of the Oak-Ridge wild type strain 74-OR23-1A were irradiated with UV light and screened for new strains resistant to cytochalasin A, an F-actin antagonist. A strain named CAR 100 (cytochalasin A resistant) with a single gene mutation was isolated from this screen. Resistance of this strain to cytochalasin A is possibly a result of a change in the structure and function of an actin, or an actin associated protein. The branching pattern of CAR 100 is predominantly dichotomous and different from the lateral branching pattern of the wild type strain from which it derived. The presence of a differing branching pattern in CAR 100 together with the resistance to cytochalasin A supports the possible involvement of actin in branching. A reversible cold sensitivity detected in CAR 100 stands in favour of this involvement. Wild type strains have also showed a potential to change their branching pattern from lateral to dichotomous under low temperature conditions. Dichotomous branching was reported earlier in some strains, e.g. peak, but the mechanism was not investigated. The colony morphology of CAR 100 is abnormal. Growth from a conidium is similar to the wild type strain in the initial period, except for a mainly dichotomous branching pattern. After 18-28 hours the distance between branches rapidly decreases together with copious growth of aerial hyphae bearing numerous conidia. This stage is followed by a reduction in the abundance of aerial hyphae and a constant increase of the dense highly branched mycelium. The growth rate and biomass increase are slower than in the wild type. On the cytological level CAR 100 hyphae show small protrusions of the cell wall. Similar protrusions are present in the granular mutants, but the cause has not been investigated. cAMP and Ca2+ do not suppress the CAR 100 phenotype. The CAR 100 mutant strain gives a new possibility for investigating genes with a function in branching and tip growth.

396. Participation of NO and NO-Synthase in light-enhanced conidiation of Neurospora crassa and sporangiophorogenesis of Phycomyces blakesleeanus.
Helga Ninnemann and Josef Maier, Institut für Pflanzenbiochemie, Universität Tübingen, Germany.

We investigated the involvement of flavins, pterins and NO/NO-Synthase (NOS) in enhanced formation of conidia in Neurospora crassa and of sporangiophores in Phycomyces blakesleannus. Neurospora crassa albino mutant al-2, bd, grown on nitrate medium, shows light-promoted conidiation. A photoreceptor for the reaction is the FAD of nitrate reductase, which also contains molybdopterin (NR) (Klemm, E. and H. Ninnemann 1979 Photochem Photobiol 29:629; Ninnemann, H. 1991 J Photochem Photobiol B Biol 9:189; Ninnemann, H. 1991 J Plant Physiol 137:677). Additional pterins were shown by us in extracts of Neurospora, Phycomyces and other fungi and algae, including tetrahydrobiopterin (BH4) (Maier, J. and H. Ninnemann 1995 Photochem Photobiol 61:43). Inhibitors of pterin biosynthesis (e.g., 2,4-diamino-6-hydroxypyrimidine (DAHP) = against GTP-cyclohydrolase I) prevented light-promoted conidiation in Neurospora as well as light-enhanced formation of macrosporangiophores in Phycomyces. We assume that - beside the flavin of NR - a pterin acts as a photoreceptor or is part of the signal transduction chain. The NO-donor SNP (sodium nitroprusside) inhibited photoconidiation in Neurospora and formation of microsporangiophores in Phycomyces; NO enhanced formation of macrosporangiophores. Inhibitors of NOS like L-NA or L-NAME (NG-nitro-L-arginine and its methyl ester), but not D-NAME, had the opposite effect. In animals NO activates soluble guanylate cyclase producing a burst of cGMP. In Neurospora blue light depletes cGMP whereas in Phycomyces blue light activates production of cGMP in accordance with the observed light-regulated activities of NOS in both fungi. 3H-Citrulline production from 3H-arginine could be demonstrated in Phycomyces (1-10 pmol min-1 mg-1) and in Neurospora (10-100 pmol min-1 mg-1) (Ninnemann, H. and J. Maier 1996 Photochem Photobiol 61: 393). Light lowers the NOS activity in Neurospora and activates NOS in Phycomyces. NO was also detected after its reaction with ozone by chemiluminescence. However, the amount of NO was 10 to 100 times lower than the production of citrulline. In Neurospora the high activity of nitrite reductase will consume the nitrite produced from NO.

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