Biochemistry and Secondary Metabolism

1. Salt stress mediated regulation of glucose 6 phosphate dehydrogenase and glyceraldehyde 3 phosphate dehydrogenase in halotolerant Aspergillus repens. Phullara Shelat, Surobhi Lahri, Rajiv Vaidya and H.S. Chhatpar Department of Microbiology and Biotechnology Centre, Faculty of Science, M.S. University of Baroda, Vadodara - 390 002, Gujarat, India.

Aspergillus repens which was isolated earlier from salt pans was found to show significant growth under salinity conditions (2M). Significant increase was observed in the activities of glyceraldehyde 3 phosphate dehydrogenase and glucose 6 phosphate dehydrogenase under salt stress conditions as compared to control. Osmolytes betaine and proline did not have any influence on G6PD activity from control whereas under salt stress conditions, both these osmolytes caused reduction in enzyme activities. Glycine and glycerol however were found to decrease the activity in both control and salt stress conditions. Asp. repens was earlier found to accumulate Na⁺ and K⁺ when grown under salt stress conditions. In vitro conditions sodium and potassium were found to increase the activity of glyceraldehyde 3 Phosphate dehydrogenase, high concentrations were however found to be inhibitory. Potassium, magnesium and calcium were found to be inhibitory to glucose 6 P dehydrogenase activity from both control and salt stress conditions. Fatty acids like oleic and myristic were found to be inhibitory to glucose 6 phosphate dehydrogenase activity. Two dimensional electrophoresis showed induction and repression of some proteins under salt stress condition.

2. Yeast as a tool to study the effects of saponins on fungi. Veronika Simons and Anne Osbourn. John Innes Centre, Norwich, UK.

Saponins are antifungal secondary metabolites (glycosylated steroids, steroidal alkaloids or triterpenoids) that are associated with plant defence. Fungal pathogens of saponin-containing plants are generally insensitive to the saponins of their hosts owing to production of saponin glycosyl hydrolases or by non-degradative mechanisms. We have established (Saccharomyces cerevisiae) as a model to investigate the effects of saponins and to identify genes required for saponin resistance/sensitivity. Representatives of two different classes of saponins (the tomato steroidal glycoalkaloid alpha-tomatine and the oat root triterpenoid saponin avenacin A-1) inhibit the growth of S. cerevisiae at micromolar concentrations. The two saponins do not have identical effects on yeast. The degree of membrane permeabilization associated with inhibition of growth is substantially greater for alpha-tomatine than for avenacin A-1. Also some sterol-deficient mutants of yeast show differential sensitivity to the two saponins (for example, the erg6 mutant has reduced sensitivity towards alpha-tomatine but is hypersensitive to avenacin A-1). Importantly, the aglycones of alpha-tomatine and other steroidal alkaloids are potent growth inhibitors but this inhibition is not associated with electrolyte leakage. Analysis is being carried out using Affymetrix gene chips to assess the effects of avenacin A-1, alpha-tomatine and tomatidine on gene expression. A complete set of yeast deletion mutants is also being screened for altered saponin sensitivity. These experiments are expected to give insight into the effects of saponins on membranes and cellular processes.

3. Aspergillus parasiticus AFLJ interacts with AFLR and regulates transcription of aflatoxin biosynthetic pathway genes. Perng-Kuang Chang, Southern Regional Research Center, Agricultural Research Service, U. S. Department of Agriculture.

 The Aspergillus parasiticus aflJ gene, located in the aflatoxin biosynthetic gene cluster and divergently transcribed from the aflatoxin pathway regulatory gene aflR, encodes a 438-amino acid protein. Disruption of aflJ resulted in non-pigmented mutants that lost the ability to synthesize aflatoxin intermediates. Transcript profiling by real time RT-PCR indicated that a lack of the aflJ transcript in the aflJ knockout mutants significantly decreased the transcript levels of the genes of the early (pksA andnor1), middle (ver1) and later (omtA) steps of aflatoxin biosynthetic pathway, respectively, and the reduction ranged from 5 to 20-fold. Deletion of aflJ, however, did not correlate with changes in the aflR transcript level and vice versa. Two-hybrid assays showed that AFLJ did not interact with aflatoxin biosynthetic enzymes, including NOR1, VER1, OMTA and ORDA. But AFLJ interacted with full-length AFLR, and the DNA-binding domain of AFLR was not essential for the interaction. Simultaneous substitutions of Arg427, Arg429, and Arg431 at the carboxyl terminus of AFLR with Leu abolished its interaction with AFLJ. Substitution of Asp436, previously shown to be crucial for AFLR's activation activity, with His had little effect on the interaction. Deletions in most regions of AFLJ appeared to destroy its function despite the fact that random amino acid substitution(s) at its carboxyl terminus did not drastically affect its capacity to interact with AFLR. The results show that aflJ is involved in the expression of aflatoxin structural genes and support the hypothesis that aflJ is a coactivator gene.

4. Homologs of aflatoxin biosynthetic genes in a cluster of genes involved in the biosynthesis of a perithecial red pigment in Nectria haematococca. Stéphane Graziani, Christelle Vasnier and Marie-josée Daboussi. Institut de Génétique et Microbiologie, Université Paris-Sud, 91405 Orsay Cedex, France

Previous work led to the identification of a polyketide synthase gene (pksN1)required for synthesis of a perithecial red pigment in Nectria haematococca. Phylogenetic analysis of this PKSN based on ketoacylacyl synthase and acyltransferase domains revealed that it belongs to the WA-type PKS which includes PKS involved in spore and mycelium pigmentation and aflatoxin pathway. The clustering of other genes with (pksN1)was expected in view of the gene clustering seen in secondary metabolism pathways. Subcloning and partial sequencing of the cosmid containing (pksN1)allowed the identification of three other ORFs with similarities to genes involved in aflatoxin (AF) and sterigmatocystin (ST) gene clusters. Their predicted activity are O-methltransferase, P450 monooxygenase and an homologue of AflJ. By similarity with the organisation of AF cluster which is bordered by PKSA and OMTA genes, we suspected that all or most of the genes involved in the biosynthesis of the red pigment are present in the cosmid and define a new cluster (PP for perithecial pigment). The nucleotide sequence of the genes were analyzed for matches to the AFLR regulatory protein binding motif found in A. parasiticus aflatoxin genes. At least, two ORFs have matching sequence to recognition sites of transcriptional factors. Further work will allow comparative studies between the two pathways.

5. Identification of beta-lactam production related ABC transporters in Penicillium chrysogenum. Melchior Evers¹, Hein Trip¹, Jeroen Nijland¹, Marco van den Berg², Roel Bovenberg², and Arnold Driessen¹.¹Biological Center, Haren, The Netherlands. ²DSM Anti-infectives, Delft, The Netherlands.

The pathway of the biosynthesis of penicillin and the cellular localization of the critical enzymatic steps has been elucidated. Little is known about the transport processes that play a role in beta-lactam excretion by P. chrysogenum. Since secondary metabolites are often secreted by dedicated ABC transporters, we have used a PCR-based cloning strategy to identify transporters involved in beta-lactam transport. By means of sets of degenerate primers, a set of ABC transporters were identified that are expressed under conditions of beta-lactam production. These systems were cloned and sequenced and found to belong to the family of multidrug transporters. Two of these systems are highly expressed when cells are challenged with extracellular added beta-lactams suggesting that they are involved in the secretion of beta-lactams.

6. The CRG1 gene required for resistance to the singlet oxygen-generating cercosporin toxin in Cercospora nicotianaeencodes a putative fungal transcription factor. Kuang-Ren Chung¹, Margaret E. Daub², and Christoph Schuller³. ¹University of Florida, IFAS, Citrus Research and Education Center, 700 Experiment Station Road, Lake Alfred, FL 33850, ²Department of Botany, North Carolina State University, Raleigh, NC 27695, ³Institute of Medical Biochemistry, Department of Molecular Genetics, University and BioCenter Vienna, Dr. Bohr-Gasse 9/2, A-1030 Vienna, Austria

The Cercospora nicotianae CRG1 gene is involved in cellular resistance to the perylenequinone toxin, cercosporin that generates highly toxic singlet oxygen upon exposure to light. CRG1 gene contains an ORF of 1950 bp including a 65-bp intron. CRG1 protein with 650 amino acids contains a Cys6Zn2 binuclear cluster DNA-binding motif with homology to various fungal regulatory proteins, indicating that CRG1 may act functionally as a transcription activator. Targeted gene disruption of CRG1 resulted in mutants that are partially sensitive to cercosporin and down-regulated in cercosporin production ( <30-55%). Genetic complementation revealed that CRG1 fully restored cercosporin resistance, but only slightly restored cercosporin production in a UV-derived mutant (CS10). Complementation of a crg1 -null mutant, however, yielded strains that are similar to wild type in both phenotypes. These results indicated that the transcription regulator CRG1 is involved in activation of genes associated with cercosporin resistance and production in the fungus C. nicotianae.

7. Analysis of altered G-protein subunit accumulation in Cryphonectria parasitica reveals a third G-alpha homologue. Gert C. Segers, Todd B. Parsley, Donald L. Nuss and Angus L. Dawe. UMBI-CBR, 5115 Plant Sciences Bldg, College Park MD

Heterotrimeric G-proteins mediate many responses of eukaryotic cells to external stimuli and have been shown to be important for fungal pathogenicity. In this study, we explored the accumulation of G-protein subunits of the chestnut blight fungus Cryphonectria parasitica in mutant strains deleted for one or more putative partner subunits. Using a series of extraction buffers and immunoblot end-point dilution analysis we have established a convenient method to assess the relative abundance of these membrane-associated proteins. Disruption of either cpg-1, which encodes the G-alpha subunit CPG-1, or cpgb-1, the G-beta subunit CPGB-1, consistently reduced the level of its presumptive partner protein. This was not observed in the case of a second G-alpha subunit, CPG-2, suggesting that CPG-1 and CPGB-1 regulate each other's stability. Further, analysis of transcript levels indicated that the G-alpha and G-beta protein turnover rates were increased in the mutant strains. Additionally, a previously unidentified protein that was cross-reactive with anti-CPG-1 antiserum was found to be enhanced in liquid culture. We describe the sequence of a new G-alpha subunit, CPG-3, that is most similar to three other filamentous fungal G-alpha proteins that form a phylogenetically distinct grouping.

8. Is the aconitase from Aspergillus nidulans involved in the conversion of 2-methylcitrate to 2-methylisocitrate? Claudia Maerker¹, Wolfgang Buckel², Matthias Brock¹. ¹Institut für Mikrobiologie Universität Hannover, Germany. ²Laboratorium für Mikrobiologie Phillips-Universität Marburg, Germany

Most if not all filamentous fungi are able to use propionate as sole carbon and energy source. We were able to identify the pathway responsible through purification of one of the key enzymes, the 2-methylcitrate synthase from Aspergillus nidulans (Brock et al., 2000) and from Aspergillus fumigatus. 2-Methylcitrate is converted to 2-methyl-cis-aconitate by a specific dehydratase. Furthermore, a 2-methylisocitrate lyase was identified, which cleaves 2-methylisocitrate into succinate and pyruvate, completing the methylcitrate cycle. However, the enzyme converting 2-methyl-cis-aconitate into 2-methylisocitrate is still unknown. Investigations on Escherichia coli, which uses the same pathway have shown that this hydration is performed by the well-known citric acid cycle aconitase AcnB (Brock et al., 2002). Therfore, the first attempts at purification were made from crude extracts of Aspergillus nidulans, which displayed 2-methylisocitrate dehydratase activity. Analysis by SDS-PAGE showed a band of 90 kDa in all active fractions. This size is in agreement with the deduced molecular mass of an aconitase of Aspergillus terreus. For further analysis a cosmid library of A. nidulans was screened, and identified an insert, containing the entire sequence of an aconitase. Subcloning of the gene under the control of the inducible alcA-promotor should facilitate the purification and biochemical characterization of the encoded protein.

Literature: Brock, M., Fischer, R. Linder, D., Buckel, W. (2000) Methylcitrate synthase from Aspergillus nidulans: implications for propionate as an antifungal agent. Mol. Microbiol. 35, 961-973.

Brock, M., Maerker, C., Schütz, A., Völker, U., Buckel, W. (2002) Oxidation of propionate to pyruvate in Escherichia coli. Eur. J. Biochem. 269, 6184-94.

9. Regulation of vitamin B₂ overproduction – a stress response in Ashbya gossypii. Thomas Schloesser¹, Cornelia Gaetgens¹ and K.-Peter Stahmann²¹Institut für Biotechnologie 1, Forschungszentrum Juelich, Germany²Technische Mikrobiologie, FH Lausitz, Senftenberg, Germany

The ascomycete Ashbya gossypii is a natural overproducer of riboflavin (vitamin B₂). We focused on the regulation of the genes involved in biosynthesis of riboflavin. To study expression of the RIB-genes RT-PCR experiments were performed. Interestingly, one branch of the divided riboflavin biosynthesis pathway was found to be strongly transcriptionally regulated. Reporter studies with a RIB3-promoter-lacZ fusion showed an 8-fold increase in enzyme specific activity in the production phase. Since time courses of batch fermentations suggested a riboflavin production at low growth rates chemostatic cultivations were performed. Surprisingly, riboflavin overproduction was not detectable at constant dilution rates. This fitted with a weak expression of RIB3-lacZ. But, a peak of reporter expression and riboflavin overproduction was detected after down-shifts in dilution rate. Shifts from D = 0.4 to 0.02 h^-1 resulted in an increase of reporter activity from 0.03 to 2.5 U per mg protein and an increase in riboflavin concentration from -1. We conclude that a decline in growth rate triggers riboflavin overproduction as a stress response. This result is in line with the observation that riboflavin overproduction is linked to spore formation as a mechanism to protect the hyaline spores against UV-light.

10. The siderophore system is essential for viability of Aspergillus nidulans: it is the major iron uptake system and its lack causes oxidative stress. Hubertus Haas, Martin Eisendle, Ivo Zadra and Harald Oberegger Department of Molecular Biology, University of Innsbruck, Fritz-Pregl-Str. 3, A-6020 Innsbruck, Austria

The filamentous ascomycete A. nidulans produces three major siderophores: it excretes fusigen and triacetylfusarinine C to capture iron, and uses ferricrocin as a cellular iron storage compound. Here we report the characterization of two siderophore biosynthetic genes, sidA and sidC,encoding L-ornithine N5-monooxygenase and a nonribosomal peptide synthetase, respectively. Deletion of sidA resulted in a complete lack of siderophore biosynthesis. Such strains were unable to germinate and grow unless the growth medium was supplemented with siderophores. These results suggests that the siderophore system is the major iron assimilatory system of A. nidulans. Growth-stimulation of the siderophore-deficient mutant by a high concentration of ferrous salts suggested the presence of an additional ferrous transport system. Disruption of sidCresulted in the loss of the cellular siderophore ferricrocin and decreased conidiation. The intracellular labile iron pool, monitored with calcein, was significantly increased in ferricrocin-lacking mutants. Consistently, these mutants showed increased expression of genes encoding antioxidative enzymes (sodA, catB,and cycA) and elevated sensitivity to the redox cycler paraquat demonstrating that the lack of ferricrocin causes oxidative stress. Remarkably, the SidA-deficient mutant synthesized ferricrocin when feeded with triacetylfusarinine C proving that A. nidulans possesses the enzymatic machinery to regenerate L-hydroxyornithine from this siderophore.

This work was supported by Austrian Science Foundation grant FWF-P13202-MOB (to H.H.) and Austrian National Bank (OENB) grant 8750 (to H.H.).

11. Characterization of Tri16 from Fusarium sporotrichioides and F. graminearum. Nancy J. Alexander¹, Susan P. McCormick¹, Troy M. Larson^1,2 and James E. Jurgenson³¹Mycotoxin Research Unit, USDA/ARS National Center for Agricultural Utilization Research, Peoria, IL; ²Dept. of Biol., Bradley University, Peoria, IL;³Dept. of Biol., University of Northern Iowa, Cedar Falls, IA

Many of the genes involved in the trichothecene biosynthetic pathway in Fusarium have now been identified within a 29 kb section of DNA. Within this cluster are 10 genes encoding either structural or regulatory genes of the pathway. In the search for the remaining trichothecene genes, the use of an EST library from a toxin over-producing strain carrying an altered Tri10 has identified Tri16, a gene believed to be involved with trichothecene biosynthesis. We isolated and cloned this gene from F. sporotrichioides and F. graminearum, then formed disruption vectors through insertional and truncated disruption. Transformants were tested by PCR and Southern hybridization for disruption events and analyzed for toxin production. None of the disruptants showed an altered toxin phenotype. Northern analyses suggest that Tri16 is regulated like a secondary metabolite as are several of the other toxin biosynthetic genes. Tri16 is physically located on linkage group 2 whereas the main trichothecene cluster is on linkage group 1. Even though Tri16 is found in the EST library, these studies show that Tri16 is not necessary for toxin production.

12. Mycotoxin PKSs as Models for Fungal Polyketide Biosynthesis. Fengan Yu, Xiacheng Zhu, Ravi Bojja, Han Yi, Kathia Zaleta-Rivera and Liangcheng Du. Chemistry Department, University of Nebraska, Lincoln, NE 68588.

Fungi are rich sources for polyketide natural products. These products are synthesized by a group of unique polyketide synthases (PKSs), the iterative modular PKSs. Because the enzymes only have a single set of domains, it has been a mystery how the PKSs control product structural variations. We have been studying the biosynthesis of fungal polyketides by using a group of mycotoxin PKSs as a model system. These compounds have a linear carbon chain with various lengths. The single-modular PKSs, when combined, can be regarded as a ¡§heterogeneous multi-modular¡̈ system, thus, can be manipulated by the genetic approaches developed for bacterial non-iterative modular PKSs. We have made an active site mutation of the methyltransferase (MT) domain, which adds methyl groups to the polyketide carbon chain, of FUM5 from Fusarium verticillioides. FUM5 gene encodes a PKS catalyzing the biosynthesis of the carbon chain of fumonisins, a group of mycotoxins that impair animal health. We have obtained several MT domain mutants and are currently working on the product identification. We have also performed a domain swapping experiment by exchanging the FUM5 ketosynthase domain with the corresponding region on PKS1 from Cochliobolus heterotrophus, a pathogen virulent toward Texas male sterile maize. PKS1 gene encodes a PKS responsible for the biosynthesis of T-toxins, a family of long-chain (C35 to C41) polyketides. The completion of these experiments would shed lights on the biosynthetic mechanism of fungal PKSs.

13. Characterization of a Verticillium dahliae hydrophobin. Anna Klimes ¹ and Katherine Dobinson ^1,2. ¹University of Western Ontario, London, Ontario, Canada. ² Agriculture and AgriFood Canada, London, Ontario, Canada.

The soil-borne fungus Verticillium dahliae is the causal agent of an economically significant vascular wilt disease that affects a wide range of hosts in the temperate areas of the world. The pathogen persists in the soil in the form of highly resistant resting structures known as microsclerotia. Microsclerotia serve as the primary source of disease inoculum and their formation represents a critical process in the fungal life cycle. We have identified a hydrophobin gene homologue that appears to be involved in microsclerotial development. Northern analyses indicate that the V. dahliae hydrophobin gene, vdh1, is preferentially expressed during growth at an air/solid interface. This expression pattern is consistent with the pattern of microsclerotial development in planta. Targetted disruption of the vdh1 gene appears to result in a delay, or inhibition, of microsclerotial development. Studies are underway to characterize vdh1 expression in greater detail, and to quantify the effects of vdh1 gene disruption on microsclerotial development and on rates of disease progression in tomato plants.

14. White Collar-1, a DNA Binding Transcription Factor and a Light Sensor. Qiyang He*, Ping Cheng*, Yuhong Yang, Lixing Wang, Kevin H. Gardner, Yi Liu. UTSouthwestern Medical Center, Department of Physiology, Dallas, TX

 Blue light regulates many physiological processes in fungi, but their photoreceptors are not known. In Neurospora crassa, all light responses depend on the Per-Arnt-Sim (PAS) domain-containing transcription factor white collar-1 (wc-1). By removing the WC-1 light, oxygen, or voltage domain, a specialized PAS domain that binds flavin mononucleotide in plant phototropins, we show that light responses are abolished, including light entrainment of the circadian clock. However, the WC-1-mediated dark activation of frq remains normal in this mutant, and the circadian clock can be entrained by temperature. Furthermore, we demonstrate that the purified Neurospora WC-1-WC-2 protein complex is associated with stoichiometric amounts of the chromophore flavin-adenine dinucleotide. Together, these observations suggest that WC-1 is the blue-light photoreceptor for the circadian clock and other light responses in Neurospora. * These authors contributed equally to this work.

15. Molecular cloning and genetic analysis of a symbiosis expressed gene cluster for lolitrem biosynthesis. Carolyn Young¹, Mike Christensen², Brian Tapper², Greg Bryan² and Barry Scott¹. ¹Institute of Molecular BioSciences, Massey University, Palmerston North, New Zealand. ²AgResearch, Palmerston North, New Zealand.

 The indole-diterpene, lolitrem B, is well documented as the cause of ‘ryegrass staggers' in animals grazing on Neotyphodium lolii-infected ryegrass. Although much is known about how lolitrem B is produced, the genes and proteins responsible have not been isolated. We have recently cloned a cluster of genes from Penicillium paxilli required for the synthesis of paxilline, an analogue of lolitrem B (Young et al. 2001) This cluster comprises a set of core genes required for indole-diterpene biosynthesis including paxG, a geranylgeranyl diphosphate synthase (GGPPS), paxC, a prenyl transferase, paxM, an FAD-dependent monooxygenase, and two cytochrome P450 monooxygenases, paxP and paxQ. The orthologue (ltmG) of paxG has been isolated from N. lolii and other related endophytes, by degenerate PCR using primers designed to conserved regions of fungal GGPP synthases. Sequence analysis of a lambda clone isolated with ltmG identified orthologues of paxM (ltmM) and paxP (ltmP) linked to ltmG. RT-PCR analysis showed that the ltm genes are highly expressed in planta. A targeted deletion of ltmM has been constructed in Epichloë festucae, and artificial associations of wild-type and the ltmM mutant established with perennial ryegrass. Analysis of the lolitrem phenotype of these associations has established that this gene cluster is required for lolitrem B production.

16. dffA gene from Aspergillus oryzae encodes L-ornithineN⁵-oxygenase and is indispensable for deferriferrichrysin biosynthesis. Osamu Yamada¹, Suthamas Na Nan¹, Takeshi Akao¹, Mihoko Tominaga¹, Hisayuki Watanabe², Toshitsugu Satoh², Hitoshi Enei², and Osamu Akita¹¹National Research Institute of Brewing, Hiroshima, Japan. ²Iwate Biotechnology Research Center, Iwate, Japan.

We identified the dffA gene from Aspergillus oryzae which encodes L-ornithineN⁵-oxygenase involved in the biosynthesis of deferriferrichrysin, a low-molecular-weight iron chelating compound. From more than 20,000 A. oryzae EST library, we found only one clone encoding a protein that exhibited homology to the U. maydis sid1 and P. aeruginosa pvdA protein. The complete gene sequence shows that the dffA gene encodes 502 amino acids with putative FAD-binding, NADP-binding, and 'FATGY' motifs, which are conserved in N-hydroxylating enzymes. Northern analysis showed that this gene expression was induced under iron-limited conditions, and the promoter region has several GATA-type transcription regulator binding motifs. When the dffA gene was expressed under the control of the alpha-amylase promoter in A. oryzae, transformants revealed high L-ornithine N⁵-oxygenase activities. In addition, a dffA gene disruptant showed no deferriferrichrysin production even under iron-limited conditions. These results suggest that the dffA gene is indispensable for deferriferrichrysin biosynthesis in A. oryzae.

17. Isolation and analyses of polyketide synthase genes from Exserohilum monoceras. Hisanori Tatewaki, Chihiro Tanaka, Mitsuya Tsuda. Agriculture, Kyoto University, Kyoto, Japan.

 Polyketides are one of the major fungal metabolites. Some of them are well known as mycotoxins and phytotoxins, others are used for drug precursors. They are synthesized by polyketide synthase (PKS). The chemical diversity of polyketides is due to the structural diversity of PKSs. But little is known about the relation between the structure and the function of fungal PKS. Therefore, we conducted to elucidate the characteristics of PKS genes of graminicolous plant pathogenic fungi, Exserohilum monoceras. We have obtained at least three putative PKS genes (emp1–emp3) from E. monoceras by degenerate PCR. Emp1–emp3 are armatic type PKS, and especially, emp1 is closely related to PKS1 involved in melanin biosynthesis in Colletotrichum lagenarium. Southern blot analyses using emp1–emp3 as probe have indicated that Bipolaris maydis which is phylogenicaly closely related to E. monoceras has same set of PKS genes. We ascertained the functions of PKS genes by comparing the metabolites of wild type and PKS gene disrupted mutants. The disruptant of emp1 lacks melanin production. The chemical profiles of other disrupted mutants are now being analyzed.

18. The NADPH: cytochrome P450 reductase gene from Gibberella fujikuroi is essential for gibberellin biosynthesis. S. Malonek and B. Tudzynski. Institut für Botanik der Westfälischen Wilhelms-Universität Münster, Schloßgarten 3, D-48149 Münster, Germany

G. fujikuroi is famous for its production of large amounts of gibberellins (GAs). Four of the seven GA biosynthetic genes encode cytochrome P450 monooxygenases. This group of enzymes depend on NADPH: cytochrome P450 reductases (CPRs) which catalyze the transfer of electrons from NADPH via FAD and FMN to the prosthetic heme group of the P450 monoxygenase. A cpr gene (cpr-Gf) was cloned from G. fujikuroi that contains the conserved FAD, FMN, and NADPH-binding functional domains. Gene disruption resulted in total loss of GA production demonstrating that CPR-Gf is essential as electron donor to the four P450 monooxygenases involved in GA biosynthesis. In addition, cpr mutants show a reduced growth rate and are much more sensitive to benzoate due to the lost activation of another P450 monooxygenase, the detoxifying benzoate para-hydroxylase by CPR. The UV mutant G. fujikuroi SG138 which was shown to be blocked at most of the GA biosynthetic steps catalyzed by P450 monoxygenases, revealed the same phenotype. Sequence analysis of the mutant cpr allele revealed a nonsense mutation at amino acid 627. The mutant was complemented with the cpr-Gf and the A. nidulans cprA genes, and both genes restored the ability to produce GAs. Northern blot analysis revealed a co-regulated expression of the cpr-Gf gene and the GA biosynthetic genes under GA production conditions (nitrogen starvation). In addition, with benzoate in the medium, expression of cpr is induced by benzoate only, but not by nitrogen starvation anymore. These results indicate that CPR-Gf is the main but not the only electron donor for several P450 monooxygenases from primary and secondary metabolism.

19. An osmosensing histidine kinase mediates dicarboximide fungicide resistance in Botrytis cinerea. Wei Cui¹, Ross E. Beever², Stephanie L. Parkes² and Matthew D. Templeton¹. ¹ HortResearch, Private Bag 92169, Auckland, New Zealand. ² Landcare Research, Private Bag 92170, Auckland, New Zealand.

A two-component histidine protein kinase (HK) gene, homologous to os-1 from Neurospora crassa, was cloned and sequenced from Botrytis cinerea (Botryotinia fuckeliana) strain A1. A series of nine spontaneous laboratory mutants resistant to dicarboximide fungicides was selected from this strain and crossed to a sensitive strain. All behaved as single gene mutants and were highly osmotically sensitive but could be divided into two groups based on phenotype. Six strains showed high-level dicarboximide resistance (DafHR) and three strains showed low-level dicarboximide resistance (DafLO). Full or partial DNA sequencing of the HK gene detected single point mutations in the coiled-coil region of all nine mutants although only two of the mutants showed the same change. Genetic crosses of dicarboximide resistant isolates from the field have shown they map at a single locus, Daf1. Crosses between selected laboratory mutants and an authentic Daf1 strain showed the laboratory mutants map to this same locus. We conclude that Daf1 encodes an osmosensing HK, which mediates dicarboximide resistance, and we propose that the fungicides target this enzyme. This putative HK was fully or partially sequenced from four field dicarboximide-sensitive (DafS) strains and found to be polymorphic. Sequence studies of 27 field resistant strains (DafR) showed predicted amino acid differences from the sensitive strains in the coiled-coil region. The 27 strains could be grouped into four classes, with three classes differing from the sensitive strains by one amino acid and one class differing by two amino acids.

20. Asparaginase gene from Aspergillus nidulans.  Tania de la Fuente, Darryl Yorkey and Patricia M. Shaffer, Department of Chemistry, University of San Diego, San Diego, CA, 92110, USA

L-asparaginase is an amidohydrolase that catalyzes the hydrolysis of asparagine to aspartic acid and ammonia.  Asparaginases are classified into two categories, type I and type II, the latter being regulated.  Aspergillus nidulans has two asparaginase genes, apnA (on chromosome II) and ahrA (on chromosome VIII).  The enzyme expressed by the ahrA gene is categorized as a type II asparaginase [Shaffer et al. (1988) Mol. Gen. Genet. 212, 337-341]. Since Cereon Genomics, LLC (subsidiary of Monsanto), had sequenced the entire A.nidulans genome, we supplied them with the sequences of five asparaginases (type II) and received a single matching sequence (contig ANIC1307).  From this sequence we prepared PCR primers and will use them with genomic DNA to produce a nucleotide sequence containing one of the L-asparaginase genes.  We will clone this sequence into a vector in order both to amplify the DNA for sequencing and express it for enzymatic testing of this possible asparaginase gene (apnA orahrA).  Since type II asparaginases are used as a cure for childhood acute lymphoblastic leukemia, this research may have some pharmaceutical significance.

21. A global regulator of secondary metabolism in Aspergillus. Jin Woo Bok and Nancy P. Keller Department of Plant Pathology, University of Wisconsin-Madison, 1630 Linden Dr., Madison, WI 53706

 Secondary metabolites are low molecular weight natural products that are not essential to the producing cells but likely have a survival function in nature. They are of intense interest to humankind due to their pharmaceutical and/or toxic properties. We have identified a novelAspergillus nidulans protein, LaeA, which regulates gene expression of several secondary metabolites. This protein is conserved in A. fumigatus and likely other ascomycetes. LaeA is a nuclear located protein that positively regulates sterigmatocystin biosynthesis, mycelial pigment formation, penicillin biosynthesis, lovastatin biosynthesis and asexual spore pigment biosynthesis in A. nidulans, and mycelial pigment formation, asexual spore pigment biosynthesis and gliotoxin biosynthesis in A. fumigatus. mRNA studies of both deltalaeA and over expression laeA strains suggest this regulation is transcriptional. Two signal transduction molecules (protein kinase A and rasA) which negatively regulate sterigmatocystin biosynthesis and asexual sporulation in A. nidulans also negatively regulate laeA expression. However delta laeA strains show little difference in asexual spore production from wild type thus suggesting the primary role of LaeA is to regulate secondary metabolism. Current studies are aimed at deciphering the mechanism of this regulation.

22. Reconciliation of the genetically defined Fum3 locus with the molecularly definedFUM9 gene in Gibberella moniliformis. Robert A. E. Butchko, Ronald D. Plattner, and Robert H. Proctor. National Center for Agricultural Resource Utilization, ARS, USDA, 1815 N. University St., Peoria, IL 61604.

Gibberella moniliformis causes ear and stalk rot of maize and can produce the polyketide-derived mycotoxins fumonisin B₁, B₂, B₃ and B₄. Fumonisins disrupt sphingolipid biosynthesis in animal cells, cause leukoencephalomalacia and pulmonary edema in horses and swine respectively, and are associated with liver and kidney cancer in laboratory rodents. A fumonisin biosynthetic gene (FUM) cluster, consisting of 15 co-regulated genes, was recently described in G. moniliformis. BLAST comparisons indicate that most genes in the cluster are likely to encode proteins involved in fumonisin biosynthetic reactions. During our ongoing deletion analysis of FUM genes, we have been able to determine the molecular basis of some naturally occurring and induced G. moniliformis mutants defective in fumonisin production. Here, we describe evidence for the molecular basis of one of these mutations at the genetically defined Fum3 locus. Deletion mutants of FUM9, a cluster gene predicted to encode a dioxygenase, produce only fumonisins B3 and B4, which lack a hydroxyl group at carbon 5. This is the same phenotype exhibited by strains with mutations in the Fum3 locus. We sequenced the region corresponding to FUM9 in aFum3 mutant and identified a transition mutation that introduces a stop codon early in theFUM9 protein-coding region. Together the results indicate that the FUM9 gene is equivalent to the Fum3 locus. Currently, we are attempting to complement theFum3 mutant by transformation with a wild-type FUM9 allele.

23. Identification of Antibiotic Binding Sites in the Vacuolar ATPase. Barry Bowman, Marija Draskovic and Emma Jean Bowman, Department of Molecular, Cell, and Developmental Biology, University of California, Santa Cruz, CA USA 95064

The macrolide antibiotics bafilomycin and concanamycin are potent inhibitors of V- ATPases. To identify the binding site of bafilomycin we selected mutant strains of Neurospora crassa (named bfr) that are resistant to this antibiotic. In one class of bfr strains the V-ATPase was resistant to inhibition in vitro. These strains had five different point mutations in the vma-3 gene, which encodes the hydrophobic c subunit of the vacuolar ATPase. Two mutations were in the region between the 1st and 2nd transmembrane helix. The other three sites were in the 4th membrane helix, near the putative proton-binding site (glu-138). Thus, the mutated sites appear to be on the outer face of the "rotor" sector of the enzyme, a region hypothesized to form an interface with the "a" subunit.

Surprisingly, the bfr strains had little resistance to concanamycin, which has a similar structure. By further mutagenizing one of the bfr strains we obtained four new strains that were resistant to both antibiotics. Each of these had two altered residues in the c subunit. Thus, concanamycin does appear to bind to the same region, but we have been unable to obtain a concanamycin-resistant strain that has only one altered residue.

The positions of two of the mutated residues in the bfr strains correspond precisely to the positions of mutated residues in the homologous c subunit of the mitochondrial ATPase that confer resistance to oligomycin. These results suggest that vacuolar and mitochondrial ATPases have an ancient, conserved antibiotic binding site. As the sequences of the polypeptides have diverged, new antibiotics that target the same vulnerable site in this family of enzymes have arisen. The data also provide support for the hypothesis that the tertiary structure of the c subunit of the V-ATPase is very similar to that of the c subunit of the F-ATPase.

24. Functional variation of Tri8 in Gibberella zeae. Seung Hoon Lee¹, Theresa Lee¹, Sung-Hwan Yun², and Yin-Won Lee¹. ¹School of Agricultural Biotechnology, Seoul National University, Suwon, 441-744, Korea. ²Division of Life Sciences, Soonchunhyang University, Asan, 336-745, Korea

Gibberella zeae, a cereal head blight fungus, produces sesquiterpene epoxides, 8-ketotrichothecenes such as deoxynivalenol (DON) and nivalenol (NIV). In most cases, this fungus produces acetylated derivatives along with the 8-ketotrichothecenes. The DON chemotype isolates co-produces 3-acetyl-DON (3-ADON) or 15-acetyldeoxynivalenol (15-ADON) with DON and the NIV chemotype co-produces 4-acetyl-NIV (4-ANIV) with NIV. Molecular studies have so far revealed that Tri8 and Tri7, located at a gene cluster of G. zeae, were responsible for production of these acetylated compounds:Tri8 for deacetylation at C-3 in a 15-ADON-producing strain and Tri7 for acetylation at C-4 in 4-ANIV-producing isolates. In this study, we have focused on theTri8 genes as a possible genetic element for structural variation at the C-3 position of 8-ketotrichothecenes produced by G. zeae. First, we have compared amino acid sequences of Tri8 from three Korean strains. Sequence analyses, however, showed that SCD2, 3-ADON-producing strain, carried an intact copy of Tri8 open reading frame (ORF) as 15-ADON-producing (H-11) and NIV-producing (88-1) strains, both lacking the acetyl group at the C-3 position. To prove the role of Tri8, an internal region of Tri8ORF was disrupted in each strain. Transgenic H-11 strains created by disruption of Tri8 accumulated 3, 15-ADON rather than 15-ADON in solid culture as previously reported. However, disruption of Tri8 in both SCD2 and 88-1 caused no alteration in trichothecene biosynthesis. These results suggest that, unlike the H-11 Tri8, the SCD2 Tri8 protein lacks the ability to remove the acetyl group at the C-3 in trichothecene biosynthesis. In addition, the result that the 88-1 Tri8 is dispensable may suggest other pathway for deacetylation of the C-3 in NIV chemotype of G. zeae isolates. Further functional studies to support these hypotheses are in progress.

25. Pathways for synthesis of polyunsaturated fatty acids in the oleaginous Zygomycete Mortierella alpina. Chris Lounds, Adrian Watson, Marcos Alcocer, Andrew Carter*, Donald MacKenzie* and David Archer School of Life and Environmental Science, University of Nottingham, Nottingham NG7 2RD, UK. *Institute of Food Research, Norwich NR4 7UA, UK.

Linoleic acid (C18:2,n-6) and alpha-linolenic acid (C18:3,n-3) are essential components of the mammalian diet. Humans lack the ability to produce these PUFAs de novo due to a lack of key fatty acid desaturases (specifically the delta-12 and delta-15). These essential fatty acids are elongated and undergo further desaturations to produce a range of n-6 and n-3 polyunsaturated fatty acids (PUFAs) including arachidonic acid (C20:4,n-6). The supply of arachidonic acid can be limiting, for example in babies and infants, so some baby milk formulations are supplemented with arachidonic acid. The Zygomycete Mortierella alpina produces up to 50% of its dry weight as oil, of which 40% can be arachidonic acid, providing a source for supplementation of formula milk.

We have developed genetic transformation systems in two strains of M. alpina in order to be able to study the regulation of the pathways of oil accumulation and to manipulate the metabolic pathway of PUFA biosynthesis. We have also studied the impact of culture conditions on the yield and type of PUFAs synthesised. High C, N-limited media favour the accumulation of oils and a range of transcripts have been monitored to determine which genes are likely to be regulated transcriptionally under these conditions. Two of the three fatty acid delta-9 desaturase genes identified in this fungus, for example, are shown to be transcriptionally regulated by high carbon to nitrogen levels. In addition to the regulation of individual genes by nutrients we are examining the role of epigenetic mechanisms in transcriptional regulation in M. alpina and have shown that M. alpina has methylated DNA.

26. Biosynthesis of sulfur amino-acids in Magnaporthe grisea: molecular and biochemical characterization of cystathionine gamma-lyase. Audrey Beaurepaire¹, Pascale Balhadhère², Nick Talbot², Marc-Henri Lebrun¹, Michel Droux¹. ¹1932 CNRS-Bayer CropScience, Lyon, France. ²University of Exeter, Exeter, United Kingdom

Mutant characterization in N. crassa and E. nidulans indicates that sulfur assimilation involves different pathways than those found either in plants or in the yeast S. cerevisae. Using the recently released draft genome sequence of the rice blast fungus (M. grisea), we identified most of the genes involved in sulfate assimilation and in cysteine/methionine biosynthesis. Biosynthesis of sulfur amino-acids is essential for the pathogenic development of M. grisea. Our goal is to understand at a molecular and biochemical level, the role of sulfur assimilation in the development of the fungus during plant infection, and to compare this to sulfur metabolism in the host plant. We have characterised cystathionine gamma-lyase, which is involved in the reversed transsulfuration sequence from homocysteine to cysteine (the S. cerevisae homologue). The corresponding cDNA was over-expressed in recombinant bacteria and the resulting protein purified to homogeneity. Biochemical characterization of the proteins will be presented and the data were compared to enzymes of this family catalyzing a similar reaction.

27. Screening of Fusarium graminearum mutants for loss of zearalenone production using yeast bioassays. Naser Safaie^1,2, Michaela Peruci¹, Herwig Bachmann¹, Rudolf Mitterbauer¹, Frances Trail³ and Gerhard Adam¹. ¹Center of Applied Genetics, University of Agricultural Sciences, Vienna, Austria. ²Tarbiat Modarres University, Tehran, Iran (present address),³Michigan State University, MI, USA.

Zearalenone (ZON) is a mycotoxin produced by several species of Fusarium , which is problematic due to its xeno-hormone activity in animals and humans. As plants do not possess an estrogen receptor, the role of ZON and its derivatives in the plant-pathogen interaction is unknown. We have developed yeast bioassays that allow cost effective detection and quantification of the estrogenic activity present in biological samples. Growth of strain YZRM7 is only possible, when ZON is present in the yeast medium in concentrations higher than 1 ppb (microgram/kg). We have also constructed an ABC transporter deficient strain (YZHB817) expressing a hybrid transcription activator consisting of the DNA binding domain of the yeast Gal4p and the hormone dependent activation domain of the human estrogen receptor, allowing quantitative determination of 5-100 ppb ZON with a GAL7-lacZ reporter gene. Using these two assays we have screened 1000 insertional mutants of F. graminearum. Although the wild type strain PH-1 produces low amounts of ZON on plates, we could easily detect it in agar plugs. Several promising candidates producing very little or undetectable amounts of ZON when grown on plates or on liquid starch glutamate medium have been identified. The mutants should be valuable tools for identification of ZON biosynthetic genes and for virulence testing.

28. Peroxisomal Origins of Aflatoxin/Sterigmatocystin Biosynthesis. Lori A. Maggio-Hall, Richard A. Wilson and Nancy P. Keller Department of Plant Pathology, University of Wisconsin-Madison

The Aspergillus mycotoxins, aflatoxin (AF) and its precursor sterigmatocystin (ST), are carcinogenic polyketides assembled from acetyl-coenzyme A units. We are interested in how the acetyl-CoA pool is controlled and made available for secondary metabolic pathways, particularly the AF/ST pathway. One important cellular mechanism for controlling this pool is its compartmentalization into different organelles. Here we present evidence that one of these organelles, the peroxisome, plays a role in the synthesis of ST in A. nidulans. We have found that a mutant lacking the delta-12 desaturase gene (odeA) shows signs of peroxisomal proliferation, accumulates oleic acid, overexpresses beta-oxidation and ST biosynthetic genes and makes substantially more ST than a wild type strain. Growth medium containing oleic acid supports a similar phenotype in the wild type strain. Fatty acid beta-oxidation is the major endogenous source of acetyl-CoA units in this organelle. Disruption of fox2, the gene encoding the multifunctional beta-oxidation protein of A. nidulans, results in a strain unable to grow on oleic acid as the sole carbon and energy source and crippled in the ability to synthesize ST. However, expression levels of AF/ST biosynthetic (stc) genes were not affected by the mutation. Finally, microscopic studies showed that the AF/ST precursor norsolorinic acid (NOR) appears to accumulate in the peroxisomes of NOR-accumulating mutant strains. Since NOR is the first stable intermediate after the polyketide synthase step in the pathway, it is possible that acetyl-CoA-requiring steps occur in the peroxisome. We are currently exploring the subcellular localization of these steps which require the polyketide synthase (StcA) and fatty acid synthase (StcJK), and analyzing the effect of mutations that alter carbon flow into and/or out of the peroxisome on the synthesis of ST.

29. Non-ribosomally synthesized peptides in Fusarium culmorum. Carsten T. Tobiasen and Henriette Giese Institute of Ecology, Section of Genetics, Royal Veterinary and Agricultural University (KVL). Thorvaldsensvej 40, 1870 Frb. C, DK.

Fusarium culmorum is the most frequent Fusarium species in Danish soils and is believed to be the major cause of Fusarium head blight of barley in Denmark. Peptides of non-ribosomally origin have been reported to be important in plant pathogenesis. An example of such a peptide is enniatin (Burmeister and Plattner, 1987), which consist of different analogs and is one of the most well-described non-ribosomally synthesized peptides (NRSP). Enniatin production has been reported by many different Fusarium spp., but F. culmorum do not synthesize enniatin. To obtain an idea of the peptides present in F. culmorum a chemical purification is performed. The initial screening was done by HPLC and one peak of interest was selected for further characterization. The compound were purified on a Sephadex LH20 column and by preparative HPLC, while LC-MS and NMR will be used for structure elucidation. To identify NRPS genes in F. culmorum we apply two different approaches. A modified gene specific differential display technique is used as well as screens of cDNA libraries that will compliment the DD-technique. Fragments of an adenylation- and condensationdomain have already been cloned and are used for optimal primer design and as probes for the library screens. These approaches will help us identifying the final module of the NRPS gene and facilitate the identification of the encoded peptide.

Ref.: Burmeister H.R. and Plattner R.D. (1987). Enniatin production by Fusarium tricinctum and its effect on germinating wheat seeds. Physiology and Biochemistry, Vol. 77 (10) p. 1483-1487.

30. Investigation of the pentose phosphate pathway in Trichoderma reesei: disruption of the phosphoglucose isomerase gene. M. Carmen Limón, Jaana Uusitalo, Tiina Pakula, Markku Saloheimo and Merja Penttilä. VTT Biotechnology, P.O. Box 1500, FIN-02044 VTT, Finland.

T. reesei is widely used for industrial protein production, very little is however known about its physiology and primary metabolism. In order to study the role and strength of the T. reesei pentose phosphate pathway (PPP), we have generated a phosphoglucose isomerase (PGII) disruptant in the strain RutC30. The disruption of this gene blocks glycolysis at the second reaction step from glucose-6-P to fructose-6-P and directs the major carbon flux to PPP. The gene disruptants show a clearly different phenotype from the parental strain. They do not grow with fructose, glycerol or xylose as the sole carbon source but growth is restored if glucose is added to the media. This indicates that glucose is needed in the cells for e.g. lipid and cell wall component synthesis. When glucose is the only carbon source, the disruptants display small colonies on plates and pellets in liquid media but they are able to grow at different concentrations of glucose. Moreover, the disruptants have an altered morphology. Glycolytic enzymes such as pyruvate kinase had lower activity in the pgi1 disruptants than in the parental strain. On the other hand, glucose-6P-dehydrogenase that directs glucose to PPP had somewhat higher activity in the disruptants. The results of this study indicate that T. reesei has a relatively active pentose phosphate pathway and in this respect it resembles more Kluyveromyces lactis than S. cerevisiae.

31. veA is necessary for normal secondary metabolism in Aspergillus nidulans. Kato N, Brooks W and Calvo AM. Biological Sciences, Northern Illinois University, DeKalb, IL

Aspergillus spp. mycotoxins affect corn, peanuts, cotton, sorghum and tree nuts. Controlling mycotoxin biosynthesis or fungal dissemination could eliminate impact on health and the economy. Because signaling pathways tend to be conserved in Aspergillus spp., the model system Aspergillus nidulans is used to study regulation of mycotoxin biosynthesis and development. Most studies on regulation of morphological differentiation inAspergillus nidulans focus on conidiation. Some pathways regulating asexual development also regulate mycotoxin biosynthesis. Aspergillus spp. also produce resistant structures: fruiting bodies called cleistothecia in Aspergillus nidulans or sclerotia in Aspergillus flavus and Aspergillus parasiticus. Said structures allow survival in adverse conditions. Because it is posited that sclerotia derive from cleistothecia, it is likely that conserved signaling pathways controlling cleistothecial development also control sclerotial formation in Aspergillus flavus and Aspergillus parasiticus, the major aflatoxin producers. Molecular studies on control of cleistothecial or sclerotial development are limited. Few regulatory genes have been identified in Aspergillus nidulans. Deletion of one of these genes, veA, blocks cleistothecial production. We found that veAdeletion alters secondary metabolism, preventing synthesis of the mycotoxin sterigmatocystin and of penicillin. We identified a veA homolog in Aspergillus parasiticus and we are characterizing its function.

32. Investigating the acquisition of HC-toxin-resistant histone deacetylase activity in the maize pathogen Cochliobolus carbonum. Pierre-Henri Clergeot¹, Jennifer A. Bieszke¹, Dipnath Baidyaroy² and Jonathan D. Walton¹.¹MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, USA. ²Current address: Dow Chemical Company, San Diego, CA 92121, USA.

HC-toxin, a cyclic peptide produced by the filamentous ascomycete C. carbonum, is a major determinant of pathogenicity towards maize lines homozygous recessive at the HM1 locus. In vitro, it is a broad spectrum inhibitor of histone deacetylases (HDACs) in plants, animals, and fungi. With age, still cultures of C. carbonum acquire HC-toxin-resistant HDAC activity (Brosch et al., 2001, Biochemistry 40:12855). Genetic evidence shows that acquisition of resistance is independent of toxin biosynthesis, but linked to the TOX2 locus (Baidyaroy et al., 2002, Eukaryotic Cell 1:538). Furthermore, resistance was found to be independent of three of the four HDAC genes from C. carbonum, HDC1, HDC3 and HDC4. Fractionation of both HC-toxin-sensitive and resistant crude HDAC extracts by gel filtration showed that acquisition of resistance correlates with a shift of HDAC activity from high to low molecular weight proteins. Western blotting shows that resistance correlates with a structural modification of Hdc2 to a form that is no longer recognized by an antibody raised against its C-terminus. Both HC-toxin-resistant HDAC activity and structural modification of Hdc2 can be gained by a sensitive crude HDAC extract upon mixing with a resistant one. These results are consistent with resistance being due to a post-translational modification of Hdc2. Making the hypothesis that the processing of Hdc2 might be critical for the self-protection of C. carbonum against its own toxin, we are currently working to isolate the putative processing factor or factors.

33. Gene Clusters Associated with Production of 1-aminopyrrolizidine (Loline) Alkaloids in the Grass Endophyte Neotyphodium uncinatum. Spiering, Martin J., Moon, Christina D., and Schardl, Christopher L. Department of Plant Pathology, University of Kentucky, Lexington, Kentucky

Fungi of the genus Epichloë (anamorphs, Neotyphodium) are endophytic symbionts of grasses, frequently promoting grass persistence. In certainEpichloë/Neotyphodium--grass associations, insecticidal 1-aminopyrrolizidine (loline) alkaloids are produced, which hold promise as natural plant protectants. The objective of our research is to identify genes involved in loline production. Several genes differentially expressed during loline accumulation in N. uncinatum cultures were isolated by suppression subtractive hybridization. Two genes isolated, lolA and lolC, had similarity to genes encoding aspartate kinases and homocysteine synthase, respectively, enzymes in methionine biosynthesis. lolA and lolC were highly expressed during loline production in culture, and were present only in endophytes with a loline-producing phenotype. Long PCR indicated linkage of lolA and lolC in the N. uncinatum genome, and genome walking showed that lolA and lolC are part of a cluster of 10 putative genes in a genomic region of 24 kb. Moreover, N. uncinatum has two allelic lol clusters, and expression of six of these genes in loline-producing N. uncinatum cultures was confirmed. The putative lol genes are related to known genes for monooxygenases, oxidoreductases, epoxidases, and pyridoxal phosphate-containing enzymes. Many of these genes related to the putative lol genes are also present in secondary-metabolite gene clusters of fungi. Gene-disruption experiments are now underway to test involvement of the gene cluster in loline alkaloid production.

34. Evolution of a secondary metabolite gene cluster implicated in loline alkaloid biosynthesis of grass-endophytes (Epichloë and Neotyphodium spp.). Kutil, Brandi L., Martin J. Spiering, Christopher L. Schardl and Heather H. Wilkinson. Plant Pathology, Texas A&M University, College Station, TX. Plant Pathology, University of Kentucky, Lexington, KY.

Assembly of a novel fungal secondary metabolite gene cluster likely involves recruitment of genes from other dispensable pathways and/or duplication and recruitment of genes from non-dispensable primary metabolism pathways. Fungal loline alkaloid (saturated 1-aminopyrrolizidine alkaloids with an oxygen bridge) production is associated exclusively with the closely related grass-endophyte species in the genera Epichloë and Neotyphodium. Recent identification and partial sequencing of gene clusters associated with lolines production (LOL) in Epichloë festucae and Neotyphodium uncinatum has revealed at least 7 ORFs implicated in the trait. N. uncinatum has two separate clusters with duplicates of some of the ORFs present at both loci. There is a high degree of microsynteny (gene order and orientation) among the E. festucae and N. uncinatum clusters. The ORFs show close homology to either genes normally associated with fungal secondary metabolism (e.g. cytochrome P450, oxidoreductase) or genes involved in polyamine biosynthesis (e.g. ornithine decarboxylase, homocysteine synthase). To address the evolutionary origins of the cluster we are investigating the phylogenetic relationships of particular lol ORFs in E. festucae to both orthologous genes in lolines expressing endophyte species and to paralogous genes (or gene families) in E. festucae, other endophytes, and model ascomycetes. Additionally, to determine whether there is evidence for seeding of the cluster with recruitment and/or duplication of linked genes, we are investigating the linkage of these same lol paralogs. While it is clear that these insecticidal compounds should convey a selective advantage, thus explaining maintenance of the trait, this analysis should provide a glimpse into the events that led to their origin.

35. Agrobacterium tumefaciens-mediated transformation of Mycosphaerella fijiensis, the black Sigatoka pathogen of bananas. Bruno Giuliano Garisto Donzelli and Alice C.L. Churchill, Boyce Thompson Institute for Plant Research at Cornell University, Ithaca, NY, 14853-1801, USA

Bananas and plantains (Musa sp.) are among the world's most important agricultural products, ranking fourth after rice, wheat, and maize for gross value of production. Black Sigatoka disease, caused by Mycosphaerella fijiensis, is currently the most destructive and economically important disease of Musa sp. worldwide. The disease is controlled in commercial plantations through the application of fungicides up to 45 times per growing season. Little is known of the molecular interactions between the pathogen and its Musa hosts. We have developed a method for Agrobacterium tumefaciens-mediated transformation (ATMT) of M. fijiensis to facilitate efficient genetic manipulations of this fungus. Transformations were carried out utilizing a mixture of spores and hyphal fragments as recipients for binary vectors, which carried hygromycin phosphotransferase as the selectable marker, as well as a gene for green fluorescent protein (GFP) expression in some cases. We obtained yields of up to 90 hygromycin-resistant colonies/plate after co-cultivation of the fungus with A. tumefaciens; transformants expressing GFP constitutively were also obtained. Most of the transformants resulted from single copy T-DNA insertions. Melanin shunt metabolites and other fungal toxins have been proposed to play a role in the disease by causing extensive leaf tissue necrosis. We have cloned fragments of several genes potentially involved in toxin production, including three genes encoding polyketide synthases (PKS), one THN-reductase, one alcohol oxidase, two alcohol dehydrogenases, and a transport facilitator protein. Molecular characterization of a putative melanin-type PKS gene by targeted gene disruption is in progress. Additionally, we have developed analytical HPLC methods to characterize changes in metabolic profiles of mutants of M. fijiensis.

36. The molecular profiles of low molecular metalloprotease from A. fumigatus. Youhei Yamagata, Takesi Kawamura, Ichiro Imao, Megumi Ose, Ohnishi Fumito, Keietsu Abe, Tasuku Nakajima. Agriculture, Tohoku Univ. Sendai, Japan.

Aspergillus fumigatus is one of the aspergillosis-causing fungi. The fungus produces some extracellular proteolytic enzyme, such as a serine protease and a metalloprotease. It has been thought that the proteolytic enzymes play important roles during early infection. Elastin occupies about 28% of lung proteins and it was reported that the elastinolytic activity was essential for infectious capacity of A. fumigatus. The serine protease has elastin degradation activity. Thermolysin like high molecular weight metalloprotease can digest collagen and gelatin but cannot hydrolyze elastin. The defective mutant of two proteases, however, maintaines infectious capacity and the expression level of the low molecular weight metalloprotease (MEP20) becomes higher than that of the wild type. MEP20 has been thought to be concerned with elastin digestion. We cloned the gene encoding a low molecular weight metalloprotease of A. fumigatusfrom Japanese aspergillosis patient and the enzyme was expressed by using A. oryzae as a host. We compared the amino acid sequences of MEP20s of A. fumigatus from Japan, America and Europe. The identities of amino acid sequences were shown only 87%, 59% and 56% between the enzymes from Japanese and American strains, Japanese and European strains, and American and European strains, respectively. The identity between the amino acid sequences of MEP20 from Japanese A. fumigatus and deutelolysin from Japanese fermentative strain A. oryzae was also only 60%. Amino acid sequences of MEP20s showed the diversification of depending on the location in A. fumigatus. We will discuss the enzymatic profiles of low molecular weight metalloproteases.

37. A 14-3-3 Homolog in Aspergillus flavus affects Aflatoxin Production. Ahmad M. Fakhoury and Gary A. Payne Department of Plant Pathology North Carolina State University

The filamentous fungus, Aspergillus flavus, is a pathogen of several crops including corn, cotton, peanuts and tree nuts. Upon infection, the fungus produces aflatoxins, a group of secondary metabolites known to cause cancer in animals. The biosynthesis of aflatoxin is influenced by environmental factors such as temperature, pH, carbon and nitrogen sources. A thorough understanding of the regulation of aflatoxin biosynthesis has proven to be a difficult task given the diversity of the cues involved. We identified a 14-3-3 homolog in an EST library made during aflatoxin biosynthesis. This class of proteins is thought to act as a switch coordinating the allocation of metabolites among different metabolic pathways. 14-3-3 proteins are ubiquitous in their distribution with functions ranging from regulating primary metabolism in plants, to controlling trafficking in cells. To determine if 14-3-3 plays a role in aflatoxin biosynthesis we disrupted the gene in A. flavus strain 86-10 by site-directed mutagenesis. Disruption of the gene in A. flavus resulted in subtle morphological changes in the generated mutant in comparison with the parent strain. These included a decrease in the rate of growth and in conidial germination and an increase in the sensitivity to temperature. Most interestingly, the strain with the disrupted 14-3-3 gene did not produce aflatoxin under several tested conditions. Complementing the mutation with a functional 14-3-3 gene rescued the ability of the fungus to produce aflatoxin.

38. Polyamines in Phytophthora sojae-Soybean Interactions. Marcus Chibucos & Paul Morris. Bowling Green State University, Bowling Green, OH USA.

Because zoospores are the predominant dispersal mechanism of the plant pathogenic oomycete Phytophthora sojae, an understanding of zoospore biology may contribute to new control mechanisms. In order to facilitate study of gene expression in swimming zoospores, cDNA was generated and PCR-amplified using different primers. A primer designed to amplify an ABC transporter yielded, due to mis-priming, a reproducible 1.5 kb product. A TBLASTX search at NCBI revealed 56% sequence identity and 67% sequence homology at the amino acid level to a putrescine transporter in Pseudomonas aeruginosa. High-stringency Southern analysis of an EcoRI-digested BAC clone with ³²P-labeled 1.5 kb product revealed 5 kb and 10 kb fragments. Fragments were cloned into pUC18, transposed with an EZ::TN^TM Kit (Epicentre) and sequenced. Because the putative transporter was identified in cDNA, suggesting functional expression, active putrescine assimilation was assessed in swimming zoospores. 1,4-¹⁴C-putrescine uptake experiments demonstrated constitutive transporter expression with K_M of 2 micromolar. Additional experiments have indicated that vegetative hyphae can utilize di- and polyamines as a sole nitrogen source. Demonstration of polyamine uptake by zoospores and hyphae, coupled with previous detection of soil polyamines, suggests importance of polyamines in rhizosphere-pathogen dynamics. Polyamine exudation by roots was confirmed by benzoylation of root secretions, followed by chromatographic separation by HPLC. Future research will explore the role of polyamines in P. sojae-soybean interactions.

39. Metabolic modeling of acetyl-CoA mutants of Aspergillus nidulans. Jessica H. Marshall, Harvey W. Blanch and Jay D. Keasling Department of Chemical Engineering, University of California, Berkeley, CA

Aspergillus species produce many secondary metabolites including the toxic, carcinogenic polyketide compounds aflatoxin (AF) and its precursor sterigmatocystin (ST). AF and ST are synthesized from acetyl-CoA. Mutations in genes associated with acetyl-CoA metabolism can dramatically affect the amount of ST produced in A. nidulans (N. Keller, personal communication). We are using metabolic modeling techniques, specifically metabolic flux and isotopomer analyses, to characterize the flow of carbon through primary and secondary metabolism in these mutant A. nidulans strains. Metabolic flux analysis calculates the metabolic fluxes through all reactions included in an organism's metabolic network, and thus allows quantification of the effect of genetic manipulations or changes to growth conditions on the entire metabolic network. In isotopomer analysis, ¹³C-labeled glucose is fed to the cells, and the label pattern of metabolites such as amino acids is measured and used as model input to improve flux calculations.

Here we present flux calculations obtained from our model using measurements of steady-state biomass composition and amino acid ¹³C label distribution from continuous cultures of A. nidulans as inputs.

40. Trichothecene biosynthesis by Fusarium sporotrichioides requires a second biosynthetic gene cluster. Daren W. Brown, Robert H. Proctor and Ronald D. Plattner. Mycotoxin Research Unit, NCAUR, USDA/ARS, 1815 N. University St., Peoria, IL 61604

Fusarium species produce a variety of toxic trichothecenes including T-2 toxin, deoxynivalenol (DON) and nivalenol (NIV). These toxins are potent inhibitors of protein synthesis and are an agricultural problem due to their detrimental affect on human and animal health. For some Fusarium species, trichothecenes are a critical component of virulence on certain crop plants. Almost all of the genes so far characterized involved in trichothecene biosynthesis in F. sporotrichioides and F. graminearum are located in a cluster comprised of 10 to 12 open reading frames (ORFs). TRI101 is unlinked to the core cluster and is flanked by house-keeping genes. At present, the biochemical pathway leading from farnesyl pyrophosphate to T-2 toxin in F. sporotrichioides includes 15 steps of which 8 have been assigned to specific genes. This report describes the characterization of two new genes that are required for trichothecene biosynthesis. These two adjacent genes are not located near the previously characterized trichothecene genes and are flanked by ORFs that do not appear to be involved in trichothecene biosynthesis. These two genes represent a second, mini-cluster that is required for toxin synthesis.

41. Alterations in B versus C fumonisin production by transformation of the Fusarium oxysporum FUM8 coding region into Gibberella moniliformis. R.H.Proctor¹, R.D. Plattner¹, J.-A. Seo^1,2 and Y.-W. Lee². ¹USDA ARS National Center for Agricultural Utilization Research, Peoria, Illinois and ²Seoul National University, Suwon, Korea.

Fumonisins are carcinogenic mycotoxins produced by the maize pathogen Gibberella moniliformis (anamorph Fusarium verticillioides), several closely related Fusarium species, and at least one isolate of F. oxysporum. G. moniliformis produces predominantly B fumonisins, which are likely formed via the condensation of alanine and a 20-carbon polyketide. In contrast, the isolate of F. oxysporum produces predominantly C fumonisins, which are likely formed via the condensation of glycine and the same 20-carbon polyketide. The FUM8 gene is required for fumonisin production and its predicted protein is highly similar to the yeast sphingolipid biosynthetic enzyme that catalyzes the condensation of serine and palmitic acid. This similarity suggests the FUM8 protein catalyzes the condensation of alanine, or glycine, and the polyketide during fumonisin biosynthesis. To determine whether FUM8 is responsible for the different fumonisin production profiles of G. moniliformis and F. oxysporum, we constructed a hybrid gene consisting of the G. moniliformis FUM8 promoter region fused to the F. oxysporum FUM8 coding region. Transformation of this hybrid gene into G. moniliformis changed the fumonisin profile from predominantly B fumonisins to predominantly C fumonisins when the hybrid gene integrated at FUM8. In contrast, only slight changes in the fumonisin profile occurred when the hybrid gene integrated elsewhere. These results indicate that FUM8 is responsible for B versus C fumonisin production inFusarium/Gibberella.

42. A Fungal Specific Protein Domain Senses Arginine For Coordinate Feedback Inhibition of Two Enzymes. Catherine A. McKinstry and Richard L. Weiss. Department of Biochemistry UCLA, Los Angeles, CA

Arginine biosynthesis in Neurospora crassa is regulated primarily by feedback inhibition of the first two enzymes of the pathway, N-acetylglutamate synthase (AGS) and N-acetylglutmate kinase (AGK). AGS and AGK are encoded by unlinked genes, arg-14and arg-6. Previous genetic studies suggested a coordinate mechanism of inhibition mediated by interaction between AGS and AGK: mutations in the gene for AGK (arg-6) affect not only the activity and feedback sensitivity of AGK, but also of AGS. The yeast-two-hybrid system has been used to demonstrate direct interaction between these two enzymes and to define the interaction domain of AGK as a unique C-terminal region. This domain has been termed the fungal domain because it is not present in bacterial counterparts of this enzyme. Deletion of the fungal domain results in a catalytically active AGK, yet renders AGS insensitive to feedback inhibition by arginine. This truncated AGK can also activate AGS when transformed into strains with an otherwise inactive AGS. This supports an important role for the fungal domain in sensing arginine for coordinated feedback inhibition.

43. Identification of cosmids that functionally complement deficiencies in polyketide pigment biosynthesis in the chestnut blight fungus. Tara M. Sirvent and Alice C.L. Churchill, Boyce Thompson Institute for Plant Research, Cornell University, Ithaca, NY, USA.

Cryphonectria parasitica synthesizes a family of orange and yellow pigments, which are aromatic polyketides that exhibit numerous and diverse biological activities in vitro. These include antimicrobial and antiviral activities, cytotoxicity, apoptosis and cytochrome P450 induction, radical scavenging and active oxygen production, and tyrosine kinase inhibition. Our goal is to determine the role(s) of the polyketide pigments in the biology of C. parasitica by targeted disruption of the polyketide synthase (PKS) gene predicted to encode the first enzymatic step in the pigment biosynthetic pathway. We cloned ten unique PKS-like fragments from C. parasitica using PCR amplification with degenerate primers designed to conserved domains of known PKSs. These fragments were used as probes to a genomic cosmid library of a wild type, pigmented strain of the fungus. Forty-two unique cosmids hybridized to one or more PKS fragments; one fragment failed to hybridize to any members of the library. Cosmids were grouped according to their hybridization patterns to each PKS fragment, and four groups of five or six cosmids were transformed into a pigment- and sporulation-deficient ("white") C. parasitica mutant, which is also methionine-auxotrophic. Among many white hygromycin-resistant transformants, six independently isolated transformants produced orange pigment. Complementation occurred in the presence of transforming DNA from each of 3 independent cosmid groups. Orange-pigmented, monoconidial isolates were mitotically stable in the absence of hygromycin selection and exhibited increased sporulation in comparison with the white mutant. Efforts are underway to identify and sequence a single cosmid that confers orange pigmentation and sporulation to several independent pigment mutants of C. parasitica.

44. Characterization of the Arg-13 Mitochondrial Carrier Transport Protein. Rey Renato G. David and R. L. Weiss. UCLA, Los Angeles, California.

Metabolic processes take place in different compartments in eukaryotic cells. Intracellular compartments, such as the mitochondria, harbor enzymes and substrates that participate in specific metabolic pathways. Arginine biosynthesis in Neurospora crassa is an accessible model system to understand compartmentation. In N. crassa, glutamate is converted into citrulline inside the mitochondria, and citrulline is exported into the cytosol to be converted into arginine. We are investigating the role of the Arg-13 mitochondrial transport protein in the transport of arginine pathway metabolites across the mitochondrial membrane. The arg-13 gene has been cloned and inserted into a pET3a vector. Arg-13 has been overexpressed in BL21-SI cells and purified. Characterization of Arg-13 involves determining the submitochondrial localization of the protein using polyclonal antibodies and using proteoliposome transport assays to determine substrate(s) specificity, transport activity and mode of transport. Preliminary results suggest that Arg-13 is an ornithine transporter. Characterization of Arg-13 will help elucidate the role it plays in arginine metabolism and add another piece of information towards understanding the role of compartmentation in metabolic regulation.

45. Analysis of genes related with fumonisin production in Gibberella fujikuroi. González-Jaén, M.T, S. Mirete, E. Errasquin, B. Patiño, G. Mulè, C. Vázquez. Genetics, University Complutense of Madrid, Madrid, Spain

Toxin production is a complex process in which a number of genes are involved, such as biosynthetic genes or efflux pumps,and would be regulated by a net of interactions with other relevant processes in fungal development and stress responses. The reports on the organization of this genes in several cases reveals an impairment with the rest of nuclear genes in terms of similarity, for instance, and the possible involvement of horizontal transfer or transposon events. We have analyzed the occurrence and the expression of several genes included in the putative fumonisin biosynthetic cluster in species included in the G. fujikuroi species complex and particularly within F. verticillioides where two subpopulations could be differentiated. Those strains isolated from banana seemed to lack those fumonisin biosynthetic genes in the same way as other G. fujikuroi species, which generally are reported as fumonisin non producers (F. sacchari, F. subglutinans, F. thapsinum and F. circinatum). An analysis of efflux pump coding genes related with fumonisin production is also presented.