Poster Abstracts, Fungal-host- interaction and signal transduction

Molecular Analysis of Cytokinesis Mutants in Ustilago maydis

Michael Bolker and Regine Kahmann. Institut fur Genetik und Mikrobiologie, Universitat Munchen Maria- Ward-Str. la, 80638 Munchen, Germany

The phytopathogenic fungus Ustilago maydis exhibits a dimorphic life style. Haploid sporidia grow yeast-like by budding and are nonpathogenic. The dikaryon grows filamentous and is able to induce tumors in maize plants. To identify genes that are involved in this morphogenetic switch we have isolated a number of mutants with aberrant morphology. Among these we could identify two mutants that are affected in cytokinesis. Both the don1 and don3 mutants show normal nuclear division but the mutant cells fail to separate after bud formation. Cells remain connected through a septum that can be stained by calcofluor. Molecular analysis of these genes revealed that the final step of cytokinesis seems to be regulated by a rho/rac GTPase. The don1 gene shows high similarity to the family of rho/rac guanine exchange factors, the don3 gene codes for a kinase that is homologous to the yeast STE20 and the mammalian pak/p65 kinase. Since these kinases are known to be activated by the active form of the rho/rac GTPase we propose that a similar signaling pathway is involved in the regulation of cytokinesis.

Differentiation-specific Expression of Chitin Deacetylase in Infection Structures of Uromyces viciae-fabae H. Deising1 and J. Siegrist2 1 Universitat Konstanz, Fakultat fur Biologie, Phytopathologic, D-78434 Konstanz, and 2Universitat Hohenheim, Institut fur Phytomedizin, D-70593 Stuttgart, Germany

Uredosporelings of rust fungi differentiate complex infection structures in order to penetrate the host leaf through the stomatal openings. Probing with FITC-conjugated wheat germ agglutinin revealed that chitin is a major cell wall component only of infection structures formed on the plant surface, but not of those differentiated in the intercellular space of the plant.

Radiometric enzyme assays and analyses of extracellular proteins of different developmental stages by substrate inclusion-SDS PAGE showed that at least five chitin deacetylases are secreted, beginning with the penetration process. These enzymes remove the acetyl groups from the chitin molecule to give rise to chitosan. Since chitosan is resistant to degradation by plant chitinases, secretion of chitin deacetylases and enzyme-mediated cell wall modifications may represent a mechanism to avoid hyphal lysis after penetration into the plant.

A fragment of the cda (chitin deacetylase) cDNA of Mucor rouxii was used to screen a gt 1 0 cDNA library of Uromyces viciae-fabae. Two gt 1 0 clones hybridizing to the heterologous probe were purified. The cDNA inserts were amplified by PCR and subcloned into plasmid vectors. Exonuclease deletions were made for sequence analyses, and sequencing of the rust cDNAs is currently in progress.

Characterization of P-glycoprotein Encoding Genes in Aspergillus nidulans

Giovanni del Sorbo1 and Maarten A. de Waard 2. 1Institute of Plant Pathology, Via Universita 100, 80055 Portici

(Naples), Italy . 2Department of Phytopathology, P.O.Box 8025, 6700 EE Wageningen, The Netherlands.

P-glycoproteins are members of the ATP-Binding Cassette (ABC) superfamily of transporters. They consist of two transmembrane domains (each with six membrane spanning -helices) and two ATP-binding domains that couple ATP hydrolysis to transport of substrates. These substrates can be either non-toxic (e.g. the a-factor pheromone of Saccharomyces cerevisae) or toxic (e.g. antibiotics). P-glycoprotein transporters became especially known for their role in multidrug resistance (MDR) of mammalian tumor cells. MDR is defined as the simultaneous resistance against a wide variety of both natural toxic compounds and synthetic drugs. MDR can be ascribed to increased efflux of the drugs caused by increased expression of P-glycoprotein encoding genes and resulting in decreased cellular accumulation. Phenotypic, genetic and biochemical evidence indicates that MDR also plays a role in resistance of Aspergillus nidulans to fungicides which inhibit sterol demethylation (DMIS) and non-related compounds such as cycloheximide.

The research presented in our poster gives a characterization of P-glycoprotein encoding genes of A. nidulans and a functional analysis of these genes in fungal physiology and MDR.

Heterologous hybridization of a genomic library of A. nidulans resulted in cloning of two P-glycoprotein encoding genes, coded Anpgpl and Anpgp2. Both genes were fully sequenced and their deduced gene products show a high degree of homology with other members of the ABC superfamily. Northern analysis experiments demonstrated that the basic level of expression of Anpgpl is higher in DMI-resistant mutants than in the wild-type isolate. Expression could strongly be induced by treatment. of mycelium with DMIs and cycloheximide, especially in some of the DMI-resistant mutants. The basic level of expression of Anpgp2 was the same for wild-type and DMI-resistant mutants, but again transcription could be induced by a several nonrelated toxicants among which DMIs. Time-course experiments demonstrated that transcription of both genes was already induced within 5-15 min of treatment of mycelium with the toxicants. These results strongly suggest that the cloned Anpgp genes indeed play a role in MDR of A. nidulans. The natural physiological role of the encoded gene products may be to protect this saprophytic fungus against naturally toxic compounds which do occur in the environment.

The Putative Role of P-glycoproteins in Pathogenesis of Botrytis cinerea

Giovanni del Sorbo' and Maarten A. de Waard 2. 'Institute of Plant Pathology, Via Universita 100, 80055 Portici (Naples), Italy. 2 Department of Phytopathology, P.O.Box 8025, 6700 EE Wageningen, The Netherlands.

P-glycoproteins are members of the ATP-Binding Cassette (ABC) superfamily of transporters. They consist of two transmembrane domains (each with six membrane spanning -helices) and two ATP-binding domains that couple ATP hydrolysis to transport of substrates. The enzymes have a low substrate specificity and can simultaneously secrete a variety of toxic, hydrophobic compounds. Therefore, it is believed that P-glycoproteins play a major role in protection of both pro- and eukaryotic organisms against naturally toxic products. The transporters can be regarded as "membrane or cytoplasmic vacuum cleaners" which expel toxicants as soon as they are detected in the plasma membrane or cytoplasm.

In view of these considerations we propose that P-glycoproteins of the filamentous plant pathogen Botrytis cinerea can play a role in pathogenesis. This fungus has a wide host range which may contain a wide variety of phytoncides and phytoalexins. The pathogen is obviously able to cope with these toxins and it is proposed that the mechanisms involved is based on secretion by P-glycoproteins as soon as they accumulate in the pathogen during plant penetration and further colonization. In addition, it may also be possible that P-glycoproteins are involved in secretion of fungal toxins involved in pathogenesis. In order to test these hypotheses we started to characterize P-glycoprotein encoding genes in B. cinerea. Heterologous hybridization of a genomic library resulted in the isolation of three independent clones. One of them has been fully sequenced and contained a P-glycoprotein encoding gene of which the deduced amino acid sequence shows a high degree of homology with other members of the ABC superfamily. Northern analysis experiments indicated that transcription of the gene can be induced by various toxic compounds. Time-course experiments showed that induction takes place within 15 min after mycelial treatment, thus suggesting that P-glycoproteins of B. cinerea may be involved in secretion of plant toxins preventing their accumulation in fungal cells. This would implicate that P-glycoproteins can be regarded as a new pathogenicity factor. The validity of this hypothesis will be further tested by studying the pathogenicity of isolates with disrupted P-glycoprotein encoding gene(s).

Variation in Conidial Size of Some Pyricularia spp.

Kumari Devulapalle and S. Suryanarayanan. CAS in Botany, University of Madras, Guindy Campus, Madras-600025. India

The size of the conidia has been used as an indicator of ploidy levels in fungi. Conidial size and volume have been used to detect diploids since diploid spores are larger than haploid spores. The size of conidia of six forms of Pyricularia (from Oryza sativa, Eleusine coracana, Setaria italica, Pennisetum typhoideum, Panicum repens and Leersia hexandra) produced in culture and on their respective host lesions were compared. The study revealed certain interesting features about conidial widths in Pyricularia that have not been so far recognized. Regardless of the isolate, populations of conidia could be grouped into three classes based on their width (6, 9 and 12 um) and their proportion varied with the isolate and source of conidia. In general, 9 um conidia predominated in populations derived from culture or host lesions. Among the six isolates studied here, the 12 um wide conidia were present to a greater extent with Leersia isolate than others both in culture and on lesions. The presumptive mean volumes of the three classes of conidia revealed that the volume of the widest conidia (12 um) was 1.5-2.0 times the volume of the less wide conidia (9 um) in five of the six isolates studied. In culture, the S. italica isolate showed the greatest variation in length (15-45 um) and the E. coracana isolate, the least (18-30 um). On host lesions, however, the S. italica isolate showed the least variability (21-30 um) and P. typhoideum isolate, the greatest variability (21-45 um). It seems possible to differentiate different forms and isolates of Pyricularia on the basis of frequency distribution of their conidial lengths as demonstrated here for the first time. A bimodal distribution of conidial lengths was evident with certain monoconidial isolates of Pyricularia which was considered to indicate the heterokaryotic nature of the isolates. Although conidia of Pyricularia spp. could not be distinguished on the basis of their widths, a study of the frequency distribution of lengths of 9 um wide conidia showed differences in the frequency patterns which appeared to be adequate to differentiate conidia of Pyricularia spp.

Purification and Cloning of Protein Phosphatases from Neurospora crassa

Balázs Szö r1, Zsigmond Feher2, Oded Yarden3, Gabor Szabo2, Pal Gergelyl1 and Viktor Dombradi1. Departments of Medical Chemistry1 and Biology2, University Medical School of Debrecen, H-4012 Hungary and Department of Plant Pathology and Microbiology3, The Hebrew University of Jerusalem, Rehovot, Israel

Protein phosphorylation is a universal regulatory mechanism in eukaryotic cells. The phosphorylated state of a protein is affected by the conflicting activities of protein kinases and phosphatases. Nearly all Ser/Thr specific dephosphorylation reactions can be attributed to four classes of protein phosphatases (PP's): PPI, PP2A, PP2B, PP2C, which are differentiated on the basis of inhibitor sensitivity and metal ion dependence. We found that all the four main classes of Ser/Thr protein phosphatases are present in N. crassa. The catalytic subunits of PP2A and PPI (PP2AC and PPIC) were successfully purified to near homogeneity by using ammonium sulfate-ethanol precipitation, DEAE Sephacel, Heparin-Sepharose and MonoQ FPLC chromatography. The molecular mass of PP2AC proved to be 33 kD by SDS PAGE. PP2AC,. was completely inhibited by 1 nM okadaic acid, was insensitive to rabbit muscle inhibitor 2, and was specific for the -subunit of rabbit muscle phosphorylase kinase as substrate. Antipeptide antibodies raised against the N-terminal and C-terminal ends of human PP2A. did not cross-react with N. crassa PP2AC .The catalytic subunit of PPI was found to be 33 kD in size, was inhibited by okadaic acid and inhibitor-2. It could be established that PPI and PP2A of N. crassa showed striking similarity to their mammalian counterparts with respect to biochemical characteristics. The pSV50 cosmid library of N. crassa was screened using a Drosophila PPI CDNA probe. Four recombinant cosmids of different restriction patterns were isolated. The heterologous CDNA probe hybridized to 4 - 5 BamHl and Hindlll fragments in a Southern hybridization experiment. Subclones from two recombinant cosmids were sequenced with primers homologous to the conserved regions of PPI. One of the sequences proved to be a truncated version of the other. The obtained sequence showed high degree of homology to Saccharomyces cerevisae PPZI and PPZ2 enzymes which are considerably larger (75 and 78 kD) than PPI and may play a role in osmotic stability and the PKC-mediated signal transduction pathway. The new N. crassa protein phosphatase gene was mapped by RFLP mapping to the left arm of chromosome 1.

This work was supported by OTKA grant #T6005 and FEFA grant # 1559.

Molecular Characterisation of Plant-induced Genes in the Take-all Fungus Gaeumannomyces graminis

P.Garosi, M.Daniels, A.Osbourn. Sainsbury Laboratory, John Innes Centre, Norwich Research Park, Norwich NR4

Our work focuses on the use of different approaches for the isolation of genes controlling pathogenicity in Gaeumannomyces graminis. One approach involves the identification of fungal genes that are induced in planta by differential screening of a library of fungal genomic DNA. This approach has allowed the isolation of nine in planta- induced genes from the potato blight fungus Phytophthora infestans (Pieterse et al.,1993). In the use of this approach it is very important to solve some problems that arise in the production of the infected material:

1) To imitate the natural conditions of infection

2) To use inoculation procedures which give uniform infection

3) To find a time point at which to harvest infected material for RNA extraction

4) To isolate a constitutively expressed fungal gene to use as a standard in Northern blot analysis.

The first two points have been solved with the use of different inoculation procedures in parallel, and the third point has been partially solved with microscopic observations of the infected roots harvested at four different time points.

To solve the last point it is necessary to characterize a constitutively expressed gene of the fungus to use as an internal standard in Northern blots, since the signal obtained with this probe should give an indication of the proportion of fungal RNA in the mixture of RNA of plant and fungal origin.

Genes encoding actin and the elongation factor EF- I a have been shown to be very useful markers. (Mahe et al.,1992; Pieterse et al,, 1993).

Since it has been demonstrated that many plant-induced genes can also be induced in vitro by starvation conditions (Oliver et al., 1994), we want also to screen a genomic library of Gaeumannomyces graminis with cDNA from the fungus grown under condition of nitrogen and/or carbon starvation.

Pieterse C.MJ., Riach M.B.R., Bleker T., Van den Berg-Velthius G.C.M. and Govers F. (1993). Isolation of putative pathogenicity genes of the potato late blight fungus Phytophthora infestans by differential screening of a genomic library. Physiol. Mol. Plant Pathology 43, 69-79.

Mahe A., Grisvard J., Dron M. (1992). Fungal and plant-specific gene markers to follow the bean anthracnose infection process and normalize a bean chitinase mRNA induction. Mol.Plant-Microbe Inter. 5, 242-248.

Oliver R.P., Coleman MJ., Faber B., Horskins A. and Arriau J. (1994). Isolation of plant-induced genes of Cladosporium fulvum. Abstract n. 428, 7th Int. Symp. Mol.Plant-Microbe Interactions. Edinburgh, June 26th-July 2nd.

Role of in Planta-expressed Genes in Biotrophic Nutrient Uptake of the Rust Fungus Uromyces viciae-fabae

M. Hahn, C. Struck, U. Neef, J. Sohn and K. Afendgen. Universitat Konstanz, Fakultit Biologie, D-78434 Konstanz, Germany

Haustoria are specialized hyphae of biotrophic fungi which play a crucial role in parasitic nutrition. To identify proteins involved in nutrient uptake, we have isolated genes which are specifically expressed in rust haustoria. Differential screening of a haustorial cDNA library revealed a high proportion (approx. 20%) of haustoria-specific cDNAs. Some of these cDNAs (PIGs = in planta induced genes) were strongly expressed in haustoria (each 1% of the total haustorial mRNA). By sequencing, one PIG was identified which encodes a membrane protein with high similarity to yeast amino acid permeases. With antipeptide antibodies, the protein was localized to haustorial membranes. Heterologous expression of the protein in yeast was achieved, but no complementation of amino acid transport mutants was observed. We are currently using Xenopus oocytes to determine the substrate specificity of the putative amino acid transporter.

In rust haustoria, the activity of the plasma membrane H+-ATPase is strongly increased compared to rust spores and germlings, indicating that this enzyme generates an electrochemical potential across the haustorial plasma membrane. We have cloned and sequenced a full-length cDNA of the H+-ATPase, and have studied its expression using Northern analysis and RT-PCR (see poster by Wernitz et al.). Taken together, our data suggest an active, carrier-mediated uptake of nutrients by rust haustoria.

The Pheromone Response Factor Coordinates Filamentous Growth and Pathogenicity in Ustilago maydis

A.Hartmann, M.Urban, M.Bolker, and R.Kahmann. Institut fur Genetik und Mikrobiologie der Universitat Munchen, Maria Ward Str.1a, D-80638 Munchen, Germany

Dimorphic growth and pathogenic development of the corn smut fungus Ustilago maydis is regulated by the two unlinked mating type loci a and b. Haploid cells grow by budding and are non pathogenic. Cells which differ in both mating type loci can fuse and form the filamentous growing dikaryon, which is able to infect corn plants. Cell fusion is controlled by the biallelic a locus, which encodes a pheromone based cell recognition system. After fusion, pathogenicity is triggered by heterodimers of homeodomain proteins encoded by the multiallelic b locus. Filamentous growth of the dikaryon requires an activated pheromone pathway in addition to the active heterodimer.

Transcription of all genes in the a and b mating type loci is induced in response to pheromone. We have isolated an HMG box protein (Prfl) which binds to short pheromone response elements present in both loci. prf1 mutants do not express the pheromone and receptor genes even after pheromone stimulation and thus are sterile. Disruption of prf1 in pathogenic haploid strains results in loss of pathogenicity, which can be restored by constitutive expression of the b genes. In addition these strains grow filamentous in the absence of pheromone stimulation. We present a model in which Prfl coordinates cell fusion, pathogenicity and filamentous growth by regulating the expression of the a and b genes in response to different stimuli.

Assimilatory Nitrate Reductase in the Arbuscular Fungus Glomus

M. Kaldorf, S. Sonnwald, E. Schmelzer*, H. Bothe. Botanisches Institut der Universitat K61n, D-50931 K61n and * Max Planck Institut fur Zuchtungsforschung, K61n-Vogelsang

There is ample but circumstantial evidence that nitrogen is among the nutrients supplied by arbuscular mycorrhizal (AM) fungi to the host. Nitrogen is mainly taken up as nitrate and then reduced by assimilatory nitrate reductase. By PCR, a part of the gene coding for the apoprotein of nitrate reductase could be amplified from the fungi Aspergillus nidulans, Pythium intermedium, Phytophtora infestans, Phytophtora megasperma, Glomus D13, Glomus fasciculatum, Glomus intraradices and Glomus mosseae. Sequencing of four of the amplificates as well as DNA hybridizations revealed strong homologies with the nitrate reductase gene in all cases. The digoxigenin labeled amplificate from Glomus D 13 hybridized with DNA isolated from Glomus spores (Kaldorf et al., Mycorrhiza 5, 23-28, 1994).

Expression of the nitrate reductase genes of maize and Glomus has been investigated by Northern hybridization experiments with gene probes from maize and Glomus. Expression of maize nitrate reductase is about tenfold lower in the roots of plants infected with Glomus intraradices compared to noninfected roots. The difference in the level of maize nitrate reductase mRNA is smaller but also detectable in the leaves of infected and noninfected maize plants.

Hybridization with the gene probe for the nitrate reductase from Glomus intraradices gave a clear signal with RNA from infected maize roots. RNA from roots and leaves of control plants also gave a hybridization signal which was, however, distinctly weaker than the signal in infected plants and might be the result of cross-hybridization.

In situ hybridization experiments with the gene probe from Glomus D13 have been performed with infected maize roots. A significantly enhanced labeling was obtained in arbuscules but not in vesicles of Glomus D13 indicating that the arbuscules are the active sites of nitrate reduction. Experiments with the gene probe for maize nitrate reductase are currently under way.

A Gene Encoding a Protein Elicitor of Phytophthora infestans is Down-regulated During Infection of Potato

Sophien Kamoun, Pieter van West, Anke J. de Jong, Grardy van den Berg-Velthuis and Francine Govers. Department of Phytopathology, Wageningen Agricultural University, POB 8025, 6700 EE, Wageningen, The Netherlands

Most species of the genus Phytophthora produce 10 kDa extracellular protein elicitors, collectively termed eliciting. Elicitins induce hypersensitive response specifically in the Solananceae family. The role of eliciting in the interaction between Phytophthora infestans and potato was investigated. A cDNA encoding INFI, the major secreted elicitin of P. infestans, was isolated and characterized. The expression of the inf1 gene in relation to the disease cycle of P. infestans was analyzed. inf1 was shown to be expressed in mycelium grown in various conditions, whereas it was not expressed in sporangiospores, zoospores, cysts, and germinating cysts. During the early stages of infection of potato, inf1 expression was downregulated compared to in-vitro and late (sporulating) stages of infection. The low level of expression of inf1 during infection could be an adaptation of P. infestans to evade effective defense responses, and could be typical for elicitor or avirulence genes encoded by multiple copies. In order to test this hypothesis, transformation experiments using constitutive promoters fused to inf1 are being conducted.

Nip1, a Bifunctional Signal Molecule from the Barley Pathogen, Rhynchosporium secalis

Volkhart Li, Marion Fiegen, Angela Gierlich and Wolfgang Knogge. Dept. of Biochemistry, Max-Planck-Institut fur Zuchtungsforschung, D-50829 Koln, Germany

The imperfect fungus Rhynchosporium secalis is the causal agent of barley leaf scald. The interaction of this pathogen with its host complies with the gene-for-gene hypothesis. R. secalis secretes a family of small toxic proteins (necrosis inducing proteins, NIPS) which exert unspecific activity towards mono- and dicot plants. Toxicity is at least in part mediated through a stimulation of the plant plasmalemma H+-ATPase. In addition, one of these proteins, NIP1, was found to be a race-specific elicitor of defense reactions in Rrs1-barley at concentrations 2-3 orders of magnitude lower than those needed for toxic activity.

Genetic complementation and gene disruption demonstrated that NIP1 is encoded by the fungal avirulence gene, AvrRrs1, that is complementary to the barley resistance gene, Rrs1. Fungal races virulent on Rrs1-barley either lack the nip1 gene or secrete a gene product that lacks elicitor activity due to a single amino acid alteration. However, while NIP1 producing races are highly virulent on rrs1-barley, nip1-deficient races show a lower degree of virulence on both rrsl- and Rrsi-barley. A similar phenotype was found with transformants in which the functional nip1 gene was replaced by a non-functional gene through homologous recombination. This indicates that in addition to its avirulence function the nip1 gene also plays a significant role in the development of virulence.

We have synthesized 3 oligopeptides spanning the amino acid primary sequence of NIP1. These peptides, alone or in all possible combinations, were elicitor-inactive. However, combinations including the central and the C-terminal peptide were toxic on Rrs1- as well as on rrsl-barley indicating that the structural constraints for toxicity are higher than those for elicitor activity. In addition, one of the amino acids that is essential for elicitor activity of the mature protein is located in the non-toxic N-terminal peptide indicating that different parts of the molecule appear to be involved in its two functions. Furthermore, the receptor mediating the toxic activity may be different from the elicitor receptor. Models including 1 or 2 NIPI receptors will be discussed.

Cutinase: a Virulence Factor in the Interaction Chickpea / Ascochyta rabiei?

G. Kohler and W. Barz. Institut fur Biochemie und Biotechnologie der Pflanzen, 48143 Munster, Germany

Ascochyta rabiei, the main pathogen of chickpea (Cicer arietinum L.), penetrates its host directly through the cuticle (Hohl, 1992). Therefore, the cuticle is the first barrier that must be overcome by A. rabiei. Our aim is to construct a cutinase-deficient A. rabiei strain via transformation mediated gene replacement. The cutinase gene from A. rabiei had been cloned (Tenhaken, 1992) and a gene replacement vector had been constructed. Several transformants were analyzed with respect to the integration pattern of the vector and the cutinase enzyme activity. Infection studies of such transformants in comparison to the wildtype with resistant and susceptible chickpea cultivars will provide information on the importance of the cutinase in this interaction.

About 70% of the transformants carried the replacement vector in the "downstream" homologous integration site; but the gene was not replaced, which was supported by Southern- and PCR-analyses. Nevertheless, this integration of the vector into the left border fragment of the gene led to a substantial loss of exogenous esterase activity in comparison with activities of wildtype and ectopic transformants. These transformants are not impaired in virulence (infection of the susceptible cultivar ILC 1929 with transformants carrying homologous integrated vector or ectopic integration). Disease symptoms developed as quickly and as intensively as in the control infection with the wildtype. This was also observed, when the spore inoculum was decreased from 1.0*105/ml to 1.0*104/ml. Interaction with the resistant cultivar ILC 3279 was also not affected.

Mycoparasitic Interaction Relieves Binding of the Cre1 Carbon Catabolite Repressor Protein to Promoter Sequences of the Ech42 (Endochitinase-encoding) Gene in Trichoderma harzianum

Matteo Lorito * , Clemens K. Peterbauer#, Robert L. Mach#, Joseph Strauss# and Christian P. Kubicek# *Istituto di Patologia Vegetale, Universita degli Studi di Napoli, Via Universita 100, 80055 Portici (NA), Italy; and # Abteilung fur Mikrobielle Biochemie, Institut fur Biochemische Technologie und Mikrobiologie, TU Wien, A-1060 Wien, Austria

The fungus Trichoderma harzianum is a potent mycoparasite against various plant pathogenic fungi. In order to study the molecular regulation of mycoparasitism. we have used Botrytis cinerea as a model plant pathogen, confronted it on agar plates with T harzianum PI, isolated cell-free extracts from various stages of mycoparasitism, and used them in electrophoretic mobility shift assays (EMSAs) with two promoter fragments of the ech-42 (42 kDa endochitinase encoding)-gene of T harzianum. This gene was chosen since ist expression has been demonstrated to be triggered during mycoparasitic interaction (Carsolio et al.. 1995-, Proc. Natl. Acad. Sci. USA 91, 10903 ff.). Cell-free extracts from T harzianum mycelia, harvested before contact with B. cinerea. and those harvested upon mycoparasitism on B. cinerea both produced high-molecular weight protein-DNA complexes. The complex obtained from mycoparasitic mycelia exhibited a smaller size. Competition experiments, using oligonucleotides containing functional and non-functional consensus sites for binding of the carbon catabolite repressor Crel provided evidence that the complex from nonmycoparasitic mycelia involves binding of Crel to both ech-42 promoter fragments. These findings are consistent with the presence of two consensus sites for binding of Crel in the ech-42 promoter. In contrast, the protein-DNA complex from mycoparasitic mycelia does not involve Crel as its formation is unaffected by the addition of the competing oligonucleotides. The mycoparasitic complex can. however. be changed into the non-mycoparasitic complex by the addition of equal amounts of protein of cell-free extracts from non-mycoparasitic mycelia, or by the addition of the purified Crel-.:glutathione-S-transferase protein. These findings suggest that a model for regulation of ech-42 expression in T. harzianum which involves at least (a) binding of Crel to two single sites in the ech-42 promoter-, (b) binding of a “mycoparasitic" protein/protein complex to the ech-42 promoter in close vicinity of the Crel binding sites- and (c) functional inactivation of Crel upon mycoparasitic interaction to enable the formation of the “mycoparasitic" protein-DNA complex.

Phytophthora infestans Population Divergence on Potato and Tomato in Moscow Region

J. Maleva, A Dolgova*, A. Smirnov*, S. Bagirova*, A. Kolesnikov, D. Shaw**, Y Dyakov*

Molecular Biology Dept. and *Mycology and Algology Dept, Biological Fad, Moscow State University, Moscow 119899 Russia. **School of Biological Sciences, Univ of Wales, Bangor, Gwynedd, LL57 2UW, UK

Earlier parasitic specialization of T0 and T1 races to potato and tomato and a difference for phenotypes rates among P. infestans isolates collected from the host plants were detected. In 1993 130 isolates were collected from adjacent fields and shown to be of A1 mating type. In contrast to potato, 7% of isolates from tomato were metalaxyl-resistant , 90% were of T1 race and 50% carried “old” Ib type of mtDNA. Potato isolates carried “new” Ia and IIa mtDNA and had higher mean number of virulent genes than the tomato isolates. According to frequencies of different pathotypes the isolates from potato and tomato could be grouped separately. Difference in frequencies of mtDNA types and pathotypes were found in the isolates from tomato fruits and leaves. Population divergence induced by specialization of P. infestans was genetically fixed by a larger fertility of T1 race hybrids in comparison with T0 and T0x T1 races

This work was supported by grants of RFBR RAS, the Royal Society and European Science Foundation.

Mitotic Stability of Transforming DNA in Antagonistic Fusarium oxysporum in Vitro and After Release in Soil Microcosms

O. Migheli, D. Del Tedesco, 0. Friard, MR. Musso. Dipartimento di Valorizzazione e Protezione delle Risorse Agroforestali, Universita di Torino, Via Giuria 15, I-10126, Torino, Italy

Before planning the large scale release of both genetically manipulated and wild type organisms, their behavior in natural and agricultural environments should be carefully evaluated for risk assessment. One of the main factors for consideration to predict the environmental risk of transformed biocontrol fungi is their genetic stability: any evidence for genetic instability in contained preliminary experiments should prompt a re-evaluation of the antagonist and its deliberate release, as unpredictable changes in the transforming sequences may lead to erroneous risk evaluation. A benomyl resistant, dark red-pigmented Fusarium oxysporum mutant antagonistic against phytopathogenic formae speciales of F oxysporum was transformed with the Escherichia coli hygromycin B phosphotransferase gene (hph), conferring hygromycin B resistance. Hybridization with the complete plasmid suggested that the integration had generally occurred in a multiple-tandem array at multiple sites. Both hygromycin B resistance and mitotic stability of nine transformants were evaluated after in vitro growth and after release of the transformants in soil microcosms. Three of the transformants were mitotically stable after four rounds of vegetative growth with no selective pressure, while six showed various changes in the integration pattern, mainly consisting in excision and rearrangement of plasmid copies. One transformant had lost the ability to grow in the presence of hygromycin B. Four weeks after release in soil microcosms all the transformants maintained the hygromycin B resistant phenotype, but six of them showed rearrangement of transforming DNA. Only one strain underwent no obvious rearrangement of the transforming DNA both after in vitro growth and after recovery from the soil microcosm. The construction of genetically engineered strains of antagonistic F. oxysporum marked with altered pigmentation and with double resistance to benomyl and hygromycin B will be very helpful for further studies on biological activity, population dynamics and behavior of this biocontrol agent under various conditions. This will provide additional knowledge to the build-up of a risk assessment protocol to predict the fate and effects of genetically altered microbial antagonists in agricultural environments.

Isolation of the Ashbya gossypii Genes AgLEU2 and AgSTE7 by Heterologous Complementation and Characterization of Non-reverting Mutants

Ch. Mohr, R. Pohlmann, P. Philippsen. Department of Applied Microbiology, Biozentrum, University of Basel, Klingelbergstr. 70, CH-4056 Basel, Switzerland

The filamentous ascomycete Ashbya gossypii was isolated as a phytopathogenic fungus of cotton. A. gossypii can naturally excrete riboflavin (vitamin B2) and improved strains are used for the commercial production of this vitamin. Systematic molecular genetic investigations with this fungus started only a few years ago. A transformation system based on the resistance to Geneticin by expression of the bacterial Kanamycin-resistance gene was established. Surprisingly free replication of Saccharomyces cerevisiae ARS plasmids and efficient homologous recombination takes place in this fungus.

Two A. gossypii genes were isolated by heterologous complementation of S. cerevisiae mutations with a genomic A. gossypii library. The cloning of the AgLEU2 gene was an essential prerequisite for the construction of A. gossypii strains with a non-reverting leu2 deletion. This was achieved by one step gene replacement and direct repeat induced homologous excision. The AgLEU2 gene was successfully used as a selection marker on freely replicating plasmids and as a selection marker for one-step gene disruption. We also have started to study the biological function of an A. gossypii gene which is a structural homolog of the S. cerevisiae STE7 gene. The STE7 gene of S. cerevisiae codes for a protein kinase and is involved in mating of haploids and in control of pseudohyphal growth in certain diploids under nitrogen limitation. The A. gossypii homolog (AgSTE7) could be cloned by heterologous complementation and a knock-out mutation (Agste7) was constructed using the AgLEU2 marker gene. The AgSTE7 protein shows significant homology to serine/threonine protein kinases. The AgSTE7 gene is non-essential and does not influence sporulation, germination of the spores and growth on full medium. However, on minimal medium with asparagine as N-source the Agste7 mutant strain grows significantly slower than the wild type strain.

Mating Populations of Gibberella fujikuroi (Fusarium Section Liseola) from European and Their Maize and Their Toxigenic Profile

A. Moretti1, A. Logrieco1, A. Ritietii2 , A. Bottalico3. 1 Istitiito Tossine e micotossine da Parassiti Vegetali, C.N.R., Bari, 70125. 2Dipartimetito di Scienza degli Alimenti dell'Universita degli studi di Napoli “Federico II”, Portici, 80055, Napoli. 3 Istituto di Patologia Vegetale, dell'Unversita degli studi di Sassari, Sassari, 07100.

Gibberella fujikuroi (Saw.) Ito (Fusarium section Liseola), a fungus distributed worldwide occurring on a variety of host, is one of the most important pathogens of maize. On the basis of the fertility, G. fujikuroi has been subdivided into at least six different, genetically distinct mating populations, termed "A"-"F". We tested members of the Liseola section isolated from maize from some European countries (Austria, Hungary, Italy, Poland) for their capability both to form sexual stage and to produce toxins fumonisin BI, beauvericin and fusaproliferin. Strains were identified as belonging to the "A","D", and "E" mating populations. Fumonisin B, was produced by isolates belonging to "A" and "D" mating populations. Beauvericin was produced at high level by isolates belonging to "D", "E", , whereas isolates of the "A" mating populations produced by isolates little, if any, of this toxin. Fusaproliferin was produced by isolates from the "D" and "E" mating populations, but not by isolates from the"A". These results revealed the occurrence of three different mating populations of G. fujikuroi from European, maize and support the fact that each mating population has a characteristic toxicological profile.

Isolation and Replacement of a Cellulase Gene from the Fungal Pathogen Claviceps purpurea

Ulrike Muller and Paul Tudzynski. Westfalische Wilhelms-Universitat, lnstitut fur Botanik, Schlossgarten 3, 48149 Munster, Germany

Cytological analyses indicate that the degradation of cellulose might be an important factor during the infection of rye by Claviceps purpurea.

Using cbhI from Trichoderma reesei as a probe, a putative cellulose gene (CEL 1) was isolated from a genomic library of C. purpurea strain T5. The derived amino acid sequence shows significant homology to other fungal cellobiohydrolases, but it obviously lacks the substrate binding domain, which is also regarded for the cellulose encoding genes cbh1-1 from Phanerochaete chrysosporium (Covert et al. 1992) and CEL 1 from Cochliobolus carbonum (Sposato 1995).

Transcription analyses by northern indicate that CEL 1 is induced by crystalline cellulose on solid medium.

CEL 1 was inactivated by targeted gene replacement. Transformants with homologous integration of a deleted CEL 1 gene still show cellulolytic activity in axenic culture, but no transcription of CEL1 could be detected after induction with crystalline cellulose.

To reveal the role of the CEL 1 gene in pathogenicity of C purpurea infection studies with the isolated transformants on rye were performed. Evaluation of the data will be presented.

A CEL 1 complementation vector was constructed by cloning the CEL1 gene with adjacent promotor and terminator regions in the hygromycin resistance vector pAN7- 1. Complementation of the CEL 1 mutants with this vector will show, if the reduced pathogenicity of the transformants is due to the absence of the CEL 1 gene product. The complementation vector could also be useful to obtain multi copy CEL1 transformants for further analysis of the regulation of the CEL 1 gene and the corresponding protein.

References: Covert et al. (1992), Appl.Env.Microbiol. 58:2168-2175, Sposato et al. (1995), MPMI 8(4):602-609.

Clustering of Trichothecene Producing Fusarium Strain Determined from Partial rDNA Sequences

G.Mule1, A. Logrieco1, G. Stea1, MT Sinisi1 & A. Bottalico. 1 Istituto Tossine e micotossine da Parassiti Vegetali, CN.R., Viale Eitiaudi 51, 70125, Bari. 2 Istituto di Patologia Vegetale, dell'Universiti degli studi di Sassari. Via E. De Nicola, 07100, Sassari, Italy.

Fusarium genus includes several species which produce trichothecenes, toxigenic secondary metabolites involved in plant and animal disease. The genetic relationship among trichothecene producing Fusarium species was investigated by PCR amplification and by analysis of partial sequences of a variable region at the 5' end of the larger nuclear rDNA. All trichothecene producing strains clustered together and two principal monophyletic groups were resolved. The first clade includes strains of F acumitialtim, F. sambucinum, F. tumidum, F conipactitm, F. camptoceras (red pigmented), F sporotrichioides and F venenottini which produced type A trichothecenes (T-2 toxin, HT-2 toxin, neosolaniol and diacetotoxyscirpenol). A second clade consist of F. crookwellense, F. culmorum and F graminearum producing type B trichothecenes (fusarenone-X nivalenol and deoxynivalenol). Such study shows a good relationship between genetic affinity and mycotoxin profile in Fusarium genus. Moreover, this region appears quite reliable for a rapid determination and phylogenetic arrangement of uncertain and atypical toxigenic Fusarium strains.

Ectomycorrhizal Fungus Talks to Plant - Up-regulation of the par Gene Expression of Eucalyptus globtilus by Pisolithus hypaphorine During the Symbiosis Development

Nehls, U *., Beguiristain T., Lapeyrie F., Martin F. Equipe de Microbiologie Forestiere. I.N.R.A, Centre de Nancy, 54280 Champenoux, France *Present address: Universitat Tubingen, Physiolog. Ekol. der Pflanzen, Auf der Morgenstelle 1, 72076 Tubingen, Germany

Several soil fungi are able to colonize roots of woody plants to develop a symibiotic association, the ectomycorrhiza. In this symbiosis both, root and fungus function no longer independently, but form a novel organ with adapted metabolic pathways controling the exclhange of metabolites between both partners. The most pronounced morphological changes of the plant occurring during ectomycorrhisa development are an enhanced formation of secondary roote, the loss of root hairs and swelling of the apex of infected roots. Comparable morphological changes of the root architecture are obtained by, the application of exogenous auxins to plant.

To investigate the molecular mechanisms which take place during the ectomycorrhiza development, we have isolated a full length cDNA clone from Eucalyptus globulus which shares homology with an auxin-induced gene, the parC from tobacco and Arabidopsis. In these plants. the expression of' the parC gene coincides with cell divisions accompanying the initiation of root primordia. The eucalypt parC gene was expressed at a constitutive level in roots and other tissues of the eucalypt seedlings. However, the steady state of the parC gene was several fold up-regulated in eucalypt roots during the early steps of the interaction with the ectomycorrhizal Gasteromycete Pisolithus tinctorius.

An enhanced level of the parC transcripts was also obtained in roots of seedlings incubated in growth medium containing micromolar concentrations of IAA or its analogs. A dramatic up-regulation of the level of parC transcripts was also obscrved in roots incubated in the presence of either Pisolillitis extracts or the indolic compound hypaphorine, a tryptophane betaine, abundantly excreted by this fungus. The latter data indicated that the fungal hypaphorine is able to triger gene expression of the host plant and may act as an auxin derivative in eucalypt roots. This is the first report of an alteration of the host plant gene expression by a difusible signal from an ectomycorrhizal fungus.

Isolation and Characterization of Phase Specific Clones of the Grass Pathogen Claviceps purpurea

B. Oeser, A. Julich and P. Tudzynski, lnstitut f. Botanik, Westf. Wilhelms Univ., SchloBgarten 3, D-48149 Munster

The ergot fungus Claviceps purpurea infects the florets of rye and other grasses . The infected ovary is fully replaced by fungal tissue. During this process a balanced biotrophic interaction and a distinct border between fungus and plant is established; the fungus switches from a conidium producing form (sphacelium) to an alkaloid producing resting structure (sclerotium). We are characterizing the interaction between C.purpurea and its host Secale cereale. We have isolated two clones out of a genomic gene bank of C. purpurea which seem to carry regions which are expressed in the honeydew(plant colonization, conidia producing) phase, but not in axenic culture. Both clones were sequenced, and the transcribed regions were localized. The identification of phase specific ORFs turned out to be difficult. As an additional approach we are now trying to construct at least a partial cDNA bank from infected plant florets.

Expression of Septoria lycopersici Tomatinase in Heterologous Fungi

R. Melton#, L. Flegg*, R. Oliver*, M. Daniels# and A. Osbourn# #Sainsbury Laboratory, John Innes Centre and *University of East Anglia,

Norwich Research Park, Norwich NR4 7UH, UK.

The steroidal glycoalkaloid saponin -tomatine has been implicated in the resistance of tomato to attack by saponin-sensitive fungi (Schonbeck and Schlosser, 1976). In general, fungal pathogens of tomato are more resistant to this compound than fungi which do not infect tomato (Arneson and Durbin, 1968). A number of tomato-infecting fungi produce extracellular enzymes known as tomatinases, which detoxify -tomatine by the removal of sugars (for review see Osbourn, 1995). The tomatinase enzyme produced by the tomato leaf spot fungus Septoria lycopersici has recently been purified and the cognate gene cloned (Osbourn et al., 1995; Sandrock et al., 1995). Experiments are currently in progress to test the role of this enzyme in pathogenicity of S. lycopersici to tomato, using the techniques of targeted gene disruption. This poster will describe the expression of S. lycopersici tomatinase in the heterologous fungi Neurospora crassa and Cladosporium fulvum, neither of which produce -tomatine-degrading enzymes. The effects of tomatinase expression on the ability of N. crassa to colonize green and ripe tomato fruits, and on the compatible and incompatible interactions of races of C. fulvum with tomato cultivars, will be presented.

Arneson, P.A. and Durbin, R.D. (1967). Phytopathology 57:1358-1360. Osbourn, A.E. (1995). Trends in Plant Science, in press.

Osbourn, A.E., Bowyer, P., Lunness, P., Clarke, B.R. and Daniels, M.J. (1995). Molecular-Plant Microbe Interactions, in press.

Sandrock, R.W., DellaPenna, D. and VanEtten, H.D. (1995). Molecular-Plant Microbe Interactions, in press.

Schonbeck, F. and Schlosser, E. (1976). In: Heitefuss, R., Williams, P.H. (Eds), Physiological Plant Pathology. Springer-Verlag, Berlin, pp. 653-678.

Characterization of a Repetitive DNA Sequence from Erysiphe graminis fsp. tritici

A.C. Payne1, M-C. Grosjean-Cournoyer2, D.W. Hollomon1 1 Institute of Arable Crop Research-Long Ashton, Dept. of Agricultural Sciences, Univ. of Bristol, Bristol BS18 9AF, UK. 2 Rhone-Poulenc AGRO, 14, rue P. Baizet, 69009 Lyon, France

Erysiphe graminis is an obligate pathogen causing powdery mildew of cereals. It adapts rapidly to new environmental factors and is able to overcome both host-plant resistance and fungicide control measures. Increased understanding of genome organization of E. graminis should aid both the development of techniques leading to the cloning and analysis of pathogenicity and fungicide resistance genes and improved disease management. Repeated DNA sequences have already been described in filamentous fungi including E. graminis fsp. hordei (Rasmussen et al. Mol. Gen. Genet., 1993, 239: 298-303). The ability of these repetitive elements to move throughout the genome may be involved in the adaptive potential of powdery mildew.

We have previously described a genomic clone from E. graminis fsp. tritici (pAT5B, Grosjean-Cournoyer et al. ECFG2, 1994, D4) which contained a repetitive element which showed some homology to pBTEG20, the clone containing the B-tubulin gene of E. graminis fsp. hordei (Sherwood and Somerville, Nucleic Acid Res., 1990, 18: 1052). We have further characterized this repetitive sequence and determined by Southern analysis, that it is at least 700 bp in size. Slot blot analysis estimated the repetitive element to be present in several hundred copies per genome. The nucleotide sequence of the cloned repetitive sequence has been determined and this showed no structural similarity with repetitive elements described in other organisms. Restriction analysis and Southern hybridization of several different genomic clones from a E. graminis fsp. tritici genomic library indicated that this repetitive element is distributed throughout the genome and has identified sequence differences within a population of repetitive elements.

We are currently working to define the exact size of the repetitive element. Sequence variation within this repetitive element family in E. graminis fsp. tritici will be investigated further by Southern analysis of restricted genomic clones. To complement these approaches a second repetitive element from the E. graminis fsp. tritici genomic library will be isolated and sequenced.

Cloning the Tomatinase Gene from Fusarium oxysporum f.sp. lycopercisi.

A. Perez-Espinosa, N.Anaya, K Lairini and M. Ruiz-Rubio. Departamento de Genetica, Universidad de Cordoba, 14071 Cordoba, Spain.

A possible determinant of resistance of tomato to fungi has been attributed to the presence in the plant of a preformed inhibitor of fungal growth: the -tomatine. Previous studies have shown that tomato pathogens are less sensitive to -tomatine that are most non-pathogenic fungi. In this way some fungi are resistant to tomatine because of their membrane composition, while others produce specific tomatine-detoxifying enzymes known as tomatinases. F oxysporum f. sp. lycopersici produce an inducible tomatinase (1). The significance of -tomatine detoxification by this fungus in the pathogenic process has not been determined. In order to clone the tomatinase coding gone different approaches were used. One of them was to utilize as a probe a fragment of cDNA of the avenacinase gene from the oat pathogen Gaeumannomyces graminis var. avenae (2). Avenacinase degrade the oat saponine avenacine, a compound related to -tomatine. A genomic library from F oxysporum. f.sp. lycopersici constructed in the vector -EMBL3 was screened against the heterologous probe. Several clones were isolated that hybridised specifically with the cDNA fragment from the avenacinase gene. The pattern of hybridization indicated that the positive clones corresponded at least to four different DNA genomic fragments. Hybridising segments have been subcloned characterized and sequenced. We found that three of them were represented as unique copies in the genome. The second approach was to construct a cDNA library of the fungus using mRNA obtained from mycelia induced with -tomatine. A degenerate oligonucleotide deduced from the aminoend of the purified tomatinase was used as a probe to screen the library. The nucleotide sequence of the fragments containing the putative genes are being determined and their expression under different induction conditions are being studied.

(1) Ford J E, D J McCance, R B Drysdale (1977) Phytochemistry 16:545-546

(2)Bowyer P, B R Clarke, P Lunnes, M J Daniels, A E Osbourn (1995) Science 267:371-374

Differential Screening of a Genomic Library to Isolate Botrytis cinerea Genes Induced During Infection of Tomato.

Theo W. Prins, Ernesto P. Benito and Jan A.L. van Kan -Dept of Phytopathology, Wageningen Agricultural University, P.O. Box 8025, 6700 EE Wageningen, The Netherlands.

The Ascomycete Botrytis cinerea, a.k.a. Grey Mould, causes severe pre and post-harvest crop loss to a wide range of ornamentals and other plants, but there is a lack of fundamental insight into molecular aspects of the infection process.

By definition, a prerequisite for pathogenicity factors is, that they are expressed during penetration and invasion of the host plant. Examples of such factors could be genes coding for extracellular hydrolases (cutinase, pectolytic enzymes), toxins or other, as yet unidentified, genes.

In the B. cinerea-tomato interaction, the isolation of genes expressed during infection is carried out by means of a non-biassed approach. This is performed by differential screening of a genomic library of B. cinerea. For the probes, poly(A)' RNA was isolated from B. cinerea grown in vitro and the tomato-Botrytis interaction, and reverse transcribed into cDNA in the presence of 32P- dATP. Since the fungal poly(A)+ RNA in the interaction sample only represents 3%, the in vitro probe was supplemented with a tenfold excess of tomato poly(A)+ RNA to establish a better comparison and to exclude fungal clones hybridizing to tomato cDNAs.

Phages which hybridized differentially after the first and second screening were used to isolate DNA. Hybridizing bands, selected on Southern blots, were cloned and checked on genomic blots containing B. cinerea DNA and tomato DNA to confirm the origin. Finally, the expression inplanta and in vitro is checked on northern blots containing RNA from B. cinerea grown in vitro, Botrytis-tomato interaction RNA and appropriate controls. First results of this screening procedure will be presented.

Detoxification of -tomatine by the Phytopathogenic Fungus Botrytis cinerea - Molecular and Biochemical Studies

Thomas Quidde and Paul Tudzynsk. Inst. f Botanik, Westfalische Wilheims-Universitat Muenster, Germany

Botrytis cinerea is the causal agent of "grey mould", a severe disease of many economically important fruits, vegetables and flowers. Our main interest is in the mechanisms of virulence and pathogenesis during the interaction process between B. cinerea and its hosts. Since the detoxification of saponins - preformed fungitoxic compounds involved in plant defense against pathogens - has been shown to determine host-specificity in the Gaeumannomyces-Avena pathosystem (Osbourn et al., 1994a, 1994b), we started to investigate the importance of saponin-detoxification in the interaction of B. cinerea with its hosts, especially tomato.

B. cinerea has been described to detoxify -tomatine - a saponin from tomato by deglycosylation (Verhoeff and Liem, 1975). In contrast to literature data, we identified the detoxification product as 1-tomatine, suggesting a xylose removing activity of the Botrytis tomatinase. Analyses of field isolates from different hosts indicate that not all of them are able to detoxify -tomatine. A tomatinase deficient isolate showed enhanced sensitivity towards -tomatine in in-vitro assays compared to tomatinase producing isolates. Infection tests on detached tomato leaves with both tomatinase-producing and non-producing strains showed significantly reduced virulence in the nonproducing strain.

Using the tomatinase cDNA of the tomato pathogen Septoria lycopersici (Osbourn, pers. comm.) as a heterologous probe we isolated a corresponding gene (toml) from a genomic library of B. cinerea SAS 56. The putative tomatinase gene showed significant homology with the probe and with the avenacinase gene of Gaeumannomyces graminis on both nucleotide and amino acid level.

Analyses of B. cinerea field isolates derived from different hosts showed that the cloned gene of the reference strain is present in all isolates.

The importance of -tomatine detoxification for phytopathogenicity is under investigation using complementation of a tomatinase deficient wild type strain with the toml-locus of strain SAS 56, together with gene replacement in the tomatinase-positive strain B. 05. 1 0.

The G Protein Alpha Subunit Gpa3 Is Involved in the Pheromone Response of Ustilago maydis

Erika Regenfelder, Michael Bolker and Regine Kahmann. Institut fur Genetik und Mikrobiologie der Ludwig-Maximilians-Universitat Munchen, Maria-Ward-Str. I a, 80638 Munchen, Germany

In the maize pathogenic fungus Ustilago maydis mating and sexual development are regulated by the a and b mating type loci. The multiallelic b locus governs pathogenicity while the a locus, which occurs in the alleles a1 and a2, is responsible for cell-recognition and fusion. The cloning and molecular analysis of the a locus revealed the existence of genes for pheromone precursors (mfal and mfa2) and their respective receptors (pral and pra2). These pheromone receptors show significant homologies to serpentine receptors, structurally characterized by seven transmembrane spanning domains and functionally coupled to heterotrimeric G proteins. Thus, the signal transduction of the pheromone stimulus in Ustilago maydis is expected to occur via a G protein mediated mechanism. Using cross hybridization studies and PCR of genomic DNA with degenerated primers three genes coding for alpha subunits of G proteins could be identified. These genes have been designated gpal, gpa2 and gpa3. Null mutants were generated for all three genes and tested for mating competence. Only the gpa3 null mutants showed an alteration in morphology and were sterile. In haploid gpa3 strains the basal expression level of the pheromone inducible mfal gene was unchanged. However, upon pheromone induction no increase of the mfal expression could be detected in gpa3 strains. This result indicates that the loss of gpa3 abolishes pheromone stimulation of mfal gene transcription. By side directed mutagenesis we have created a constitutively active gpa3 allele. This mutation causes a significant increase in mfal gene expression in haploid strains. We take this to indicate that Gpa3 plays an active role during the pheromone-signaling in Ustilago maydis.

Stress-inducible Cell Wall Proteins from the Mycoparasitic Fungus Trichoderma harzianum.

Manuel Rey, Jose A. Pintor-Toro*, Antonio LLobell** and Tahia Benitez.

Depariamento de Genetica, Universidad de Sevilla. *IRNA and **IBVF, CSIC, Sevilla.

cDNA clones encoding cell wall proteins (QID3 and QID74) were isolated from a library prepared from chitin-induced mRNA in cultures of Trichoderma harzianum. Whereas QID3 gene was induced by , at least, chitin, fungal cell walls and nutrient-stress. QID74 was also induced by abiotic stress conditions such as heavy metals (Cu and Zn), saline solutions and high temperatures, even in the presence of glucose.

When QID3 protein was introduced in yeast, the growth rate was similar to that of the control but the protein gave rise to cell division arrested in cytokinesis and cell separation. Qid74 protein does not alter the phenotype of the yeast cell. They divided normally but those cells which expressed the protein seemed to be more resistant to mechanical forces such as vacuum: under the scanning microscope, the cells were more turgent and well preserved in comparison with the control. In addition, both conjugation and sporulation were slowed down and the yield was also lower. Western analysis has shown QID74 protein to be located in the cell wall.

The sequence of the QID3 protein shows similarities both with cell wall plant proteins and fungal hydrophobins: small cell wall proteins highly hydrophobic which contribute to the fungal morphogenesis as well as cell-cell attachment and/or pathogen recognition and appresories formation. The sequence of QID74 has homologies with cell wall proteins involved in resistance mechanisms such as extensines or the Balbiani Ring Proteins.

When the expression pattern of QID74 gene was compared in two T. harzinaum mycoparasit-ic strains, one of them (CECT 2413) which produces high amounts of hydrolytic enzymes and the other one (IMI 206040) whose levels of enzyme production are very low QID74 was strongly induced only in the former strain.

Results points to QID74 and QID3 proteins being cell wall components of T. harzinaum, expressible under stress conditions and probably with different function: QID74 as a defense mechanism against biotic (lytic enzymes) and abiotic stress (i.e. heavy metals) and QID3 in cell-cell recognition by increasing cell hydrophobicity.

The Reproductive Biology of Cryphonectria parasitica at the Edge of the Chestnut Blight Epidemic in Europe.

Daniel Rigling, Martin Bissegger and Ursula Heiniger. Department of Forest Ecology, Swiss Federal Institute for Forest, Snow and Landscape Research. 8903 Birmensdorf, Switzerland.

Chestnut blight caused by the introduced ascomycete Cryphonectria parasitica is still spreading in Europe. We have used vegetative compatibility (VC) and mating type to characterize three populations that have recently infested chestnut stands in northern Switzerland. The dsRNA virus that causes hypovirulence was not found in these populations. VC and mating type markers indicated that each population was founded by only one or two genotypes. Many isolates of two populations produced perithecia in the presence of both mating type testers and were also able to self-fertilize. Single ascospore and conidiospore isolates derived from two selfing strains showed segregation for mating type. Only few cultures of both spore types again self-fertilized. We conclude that selfing strains are heterokaryons for mating type probably as a result of mating type switching. The biological significance of these phenomenon may lay in 1) the production of ascospores important for long distance dissemination, 2)the possibility to outcross in the second generation of otherwise sexually incompatible strains and thus increasing genetic diversity, and 3) the defense against viruses which are not transmitted into ascospores.

ITS-sequence Analysis Shows That Flower Bulb Attacking Rhizoctonia solani Strains Are a Distinct Subgroup in Anastomosis Group 2- 1.

Oscar Salazar1, Hans Schneider2, Matise Borjal, Jaap Keijer3 and Victor Rubio1 . Centro Nacional de Biotecnologia (CSIC-UAM).

Campus Cantoblanco, Universidad Autonoma de Madrid. Madrid, Spain. 2 DLO-Research Institute for Plant Protection

(IPO-DLO), Wageningen, The Netherlands. 3DLO-State Institute for Quality Control of Agricultural Products (RIKILT-DLO),

Wageningen, The Netherlands.

Rhizoctonia solani Kuhn (Thanatephorus cucumeris (Frank) Donk) occurs word-wide and is a soilborne plant pathogen with almost unlimited host range. Many isolates exist that are saprotrophs or mycorrhizal on orchids or other plants. The Rhizoctonia solani species complex is currently divided in 12 Anastomosis Groups (AG, designated AG 1 to 11 and AG BI), and several of these AGs can be divided in subgroups. For insight in pathogenic behavior and for the design of management control strategies it Is necessary to understand the population structure and the relationship between different isolates. Today molecular identification methods can be used to resolve questions on relationship.

AG2 can be divided into three subgroups. AG 2-1, AG 2-2 and AG 2-3, based on hyphal fusion frequency. AG2-2 can be subdivided in AG2-2-HGIII and AG-2-2-HGrV based on DNA base sequence homology. In addition, isolates belonging to AG-2 can anastomose in low frequency with isolates belonging to AG-8 and AG BI (bridging isolates). AG-2-1 is a typical pathogen of crucifers, but some strains defined as AG21 are also able to attack flower bulb.

34 strains belonging to AG 2 have been analyzed at sequence level, by asymmetrically PCR amplified ITS regions. The analyzed strains have been isolated from different diseased plants in The Netherlands, Japan and Italy. Phylogenetic trees were constructed according to ITS sequence variations (with the 5.8s rDNA being identical in all the isolates). All strains attacking flower bulb cluster together as a separate group within AG-2- 1. These results confirm that AG-2-t (the strains attacking flower bulb) is a specific subgroup within AG-2.

Inducible Tomatinase Activities in Different Formae Specialis of Fusarium oxysporum Not Pathogenic to Tomato Plant

K. Lairinil, A. Perez-Espinosal, N. Anayal, FJG.Muriana2 and M. Ruiz-Rubiol. ldepartamento de Genetica, Facultad de Ciencias, Universidad de 10071 Cordoba, Spain y 2CSIC Instituto de la Grasa, 41012 Sevilla, Spain.

Fusarium oxysporum is a very common and worldwide existent soilborne plant pathogen that causes severe losses in a broad range of agricultural crops. Within the species there is a considerable host specificity with over 120 described formae speciales and races capable of causing vascular wilt diseases on particular hosts. Plants have evolved different defense mechanisms to protect themselves against a great variety of invasive pathogens. The antifungal compound -tomatine, present in tomato plants, has been reported providing a preformed chemical barrier against phytopathogenic fungi. F oxysporum f. sp. lycopersici, a tomato pathogen, produces an inducible extracellular enzyme able to detoxify -tomatine. We only detected a unique inducible protein with tomatinase activity in extracellular filtrates. This protein was purified and characterized and was found to be a monomer of 50 kDa. The native tomatinase showed at least five isoforms with pi’s ranging from 4.8 to 5.8. Treatment with Nglycosidase F gave a single protein band of 45 kDa, indicating that the 50 kDa protein was N-glycosylated. This result suggests that there is only one gene coding tomatinase. Tomatinase degrading activity was also inducible in other formae speciales not pathogenic on tomato such as melonis, niveum and tuberose. The host plant of these formae speciales (muskmelon, watermelon, potato) do not produce -tomatine. On the other hand tomatinase was absent in other formae speciales (e.g. lini and conglutinans). These enzymes have molecular weights similar to the enzyme from F oxysporum f. sp. lycopersici. The mechanism of action of these enzymes was identical; all of them detoxify -tomatine by cleaving the glycoalkaloid into the tetrasaccharide lycotetraoside and tomatidine. This finding suggests a possible evolutive parentage between different formae speciales of F oxysporum.

Rearrangements at a DNA Fingerprint Locus in the Rice Blast Fungus

Verel Shull a and John E. Hamer b . aDepartment of Plant Sciences, University of Oxford, Oxford OX I 3RB, United Kingdom. bDepartment of Biological Sciences, Purdue University, West Lafayette, IN 47907-1392, USA

A family of repetitive elements, called MGR586, are dispersed throughout the genomes of rice pathogens of Magnaporthe grisea, and are associated with a high incidence of restriction fragment length polymorphisms (RFLPs). The analysis of MGR586 RFLPs resolves collections of field isolates into a small number of clonal lineages. The asexual mechanisms that produce variation among members of a lineage, as well as different lineages, are not known. In the process of analyzing the segregation of certain MGR586 RFLPs we identified a novel polymorphism, called MGR586-P2, in one member (designated 4395-12-3) of a sister spore pair from a complete, tetrad. Molecular cloning suggests that MGR586-P2 was generated by a novel, long terminal repeat (LTR)-containing retrotransposon called fosbury. Genetic analysis shows that MGR586-P2 and its progenitor polymorphism (MGR586-PI) are alleles of a single genetic locus termed the MGR586 polymorphic locus or MGR586-PL. Surprisingly, we also found that strain 439512-3 and its clonal descendants produced strains containing as many as three allelic forms of MGR586-PL (i. e. these strains are heteronuclear). These results suggest a pathway that produces clonal variation in M. grisea in which recurrent nonrandom rearrangements at a single genetic locus lead to the formation of a heteronuclear mycelium. Sporulation of this heteronuclear strain may then produce an array of clonal variants.

Functional Analysis of the Cladosporium fulvum Avr9 Promoter

Sandor Snoeijers1, Theo Goosen1, Henk van den Broek1 and Pierre de Wit2

1 Department of Genetics, Wageningen Agricultural University, Dreijenlaan 2,6703 HA Wageningen, the Netherlands and 2Department of Phytopathology, Wageningen Agricultural University, Binnehaven 9,6709 PD, Wageningen, The Netherlands

Physiological and biochemical aspects of the interaction between the fungal pathogen Cladosporium fulvum and tomato have been studied quite intensively. From C fulvum several in planta induced genes have been cloned: the avirulence genes Avr9 and Avr4 and the putative pathogenicity genes ecp1 and ecp2. The avirulence gene products e.g. the race-specific elicitor proteins AVR9 and AVR4, are thought to interact with the gene products of the complementary resistance gene Cf9 and Cf4, resp.) in tomato, thus giving rise to a hypersensitive-response (HR) which is a characteristic resistance reaction of the plant against pathogens. The Avr9 gene is proven to be responsible for race-specific resistance by transformation and replacement studies (Van den Ackerveken et al. 1992) Expression of the Avr9 gene is induced upon growth in plaina. When grown on liquid medium, expression of the Avr9 gene could be induced by limitation of amount of nitrogen (present as nitrate, ammonium, glutamate or glutamine Van den Ackerveken et al. 1994)

Detailed sequence analysis of the Avr9 promoter showed the presence of several putative regulatory elements with the consensus sequence (GATA) of tile binding site for major wide domain nitrogen regulator proteins like NIT2 of Neurospora crassa and AREA of Aspergillus nidulans. Using the Avr9 promoter-GUS fusion in combination with the argB site directed integration system for A. nidulans, data have been obtained which suggest that functional sites for the binding of a regulatory protein homologous to Nin and AREA are present, and that the initial analysis of the in vitro induced mutations in the potential regulatory elements in the Avr9 promoter is feasible in A. nidulans.

Van den Ackerveken GFJM, Van Kan JAL, De Wit PJGM (1992) Molecular analysis of the avirulence gene avr9 of the fungal tomato pathogen Cladosporium fulvum fully supports the gene-for-gene hypothesis. Plant J 2:359-366

Van den Ackerveken GFJM, Dunn RM, Cozijnsen TJ, Vossen P, Van den Broek HWJ, De Wit PJGM (1994) Nitrogen limitation induces expression of the avirulence gene avr9 in the tomato pathogen Cladosporium fulvum . Mol Gen Genet 243:277-285

Molecular Genetics of Infection-Related Development by the Rice Blast Fungus Magnaporthe grisea

Nicholas J. Talbot, Michael J. Kershaw, Barbara J. McCormack and Heather R.K. McCafferty

Department of Biological Sciences, University of Exeter, Washington Singer Laboratories, Perry Road, Exeter, EX4 4QG, UK.

The blast fungus Magnaporthe grisea infects rice by elaborating infection structures known as appressoria. We are investigating appressorial development and the mechanism of appressorial infection by using a combination of biochemical and genetic approaches.

First we are studying the pathogenicity gene MPG1 which is highly expressed during appressorial development and is required for efficient appressorial formation. MPG1 encodes a class I hydrophobin and we have evidence that it encodes a novel rodlet protein composed of 5nm interwoven rodlets. This protein appears to interact with the rice surface during the prepenetration phase. Attachment to the rice surface is a multi-component process but chemical extractions clearly indicate an MPGlp hydrophobin mediated interaction occurs. This suggests the presence of MPGlp at the fungal-rice interface is a pre-requisite for efficient appressorial development and may transfer the correct inductive signal(s) to facilitate morphogenesis. Functional relatedness of MPGlp to other hydrophobins is also being studied by cross-species complementation to determine the conserved features required for surface interaction, appressorial development and pathogenicity restoration.

Secondly, we are studying the mechanism of appressorial infection. Appressoria generate up to 8MPa of pressure during the infection process which is transferred into mechanical force to breach the host surface. We are studying the physiology and molecular genetics of turgor generation. We have determined that in order to generate such high pressure a solute accumulates within appressoria to concentrations in excess of 3M. Appressorial extractions have identified the solute and we are carrying out genetic studies to determine the pathway by which it is synthesized and the regulation of its accumulation.

Lastly we are carrying out a number of differential cDNA screens to identify genes expressed under nutrient limiting conditions which are also highly expressed during pathogenesis of M. grisea. We have identified and sequenced a number of cDNAs and mapped the corresponding loci and progress in this are will be reported.

Infection by the Ergot Fungus Claviceps purpurea: Structure and Function of Two Polygalacturonase Genes in Relation to Host Pectin Alteration

Klaus B. Tenbercie, Veronika Homann, Birgitt Oeser and Paul Tudzynski. Institut fur Botanik, Universitat Munster, Schloggarten 3, D-48149 Munster, Germany

C. purpurea is a biotrophic ascomycete infecting only florets of grasses. The fungus mainly colonizes the ovary by an intercellular mode of growthl. Therefore, pectinolytic enzymes have been thought to be of special importance2. Such activities had been detected in axenic and parasitic culture. We combined molecular genetics with ultrastructural studies in order to prove the fungal origin of such enzymes in infected ovaries and their operation on host pectin components.

Two putative polygalacturonase genes (pgl and pg2) were isolated from C. purpurea, using the pgall gene of A. niger as a probe. The two genes are closely linked head-to-tail and are highly homologous. They probably code for mature proteins of 343 and 344 amino acids, showing significant homology to endopolygalactuonases of filamentous fungi. We study the expression of pgl and pg2 in axenic and parasitic culture using RT PCR.

Their corresponding substrate, polygalacturonic acid, was demonstrated to be a component of the host cell walls in rye ovaries, using immunogold TEM with a monoclonal antibody (JIM5) specific for homo-galacturonic acid regions of pectin. JIM5 epitopes were localized along the usual infection path in healthy carpels. At the interface of penetrating and of intercellular hyphae, JIM5 label was heavily increased. The observed host wall alterations provide evidence for the secretion and activity of fungal extracellular enzymes in planta.

1 Tenberge, K.B., Tudzynski, P. (1994) BioEngineering 10 (S3/94), 22.

2 Tudzynski, P., Tenberge, K.B., Oeser, B. (1995) In: Pathogenesis and host specificity in plant diseases: histopathological, biochemical, genetic and molecular basis, Bd. 11, Eukaryotes, pp. 161-187, Kohmoto, K., Singh, U.S., Singh, R.P., eds., Oxford, Elsevier Science, Pergamon Press.

Functional Analysis of the in planta Induced Gene ipiO of Phytophthora infestans

Pieter van West, Anke J. de Jong and Francine Govers. Department of Phytopathology, Graduate School Experimental Plant Sciences, Wageningen Agricultural University, Binnenhaven 9, 6709 PD, Wageningen, The Netherlands.

An in planta induced gene, ipiO, of the potato late blight pathogen Phytophthora infestans, was isolated from a genomic library by differential hybridization. P. infestans has two ipiO genes, ipiO1 and ipiO2, which are very similar and closely linked. The encoded proteins, IPI-01 and IPI-02, have no homology with known protein sequences (Pieterse et al., Gene 138: 67-77). The ipiO genes are expressed at high levels in the early stages of the pathogenic interaction of P. infestans with its host plants potato and tomato suggesting that the IPI-0 proteins have a function in pathogenicity (Pieterse et al., MGG 244: 269-277). ipiO mRNA is also detectable prior to infection i.e. in zoospores, in cysts and in germinating cysts. In sporangiospores no ipiO mRNA is found, whereas germinating sporangiospores contain only little ipiO mRNA. In order to assay the role of IPI-0 in pathogenicity, we transformed P. infestans with constructs carrying a strong oomycete promoter fused to the ipiO coding sequence in anti-sense and sense orientations. In the anti-sense and sense transformants the endogenous ipiO mRNA level was determined and in addition, the transformants were tested for their ability to cause disease on potato leaves. Characterization of the transformants will be presented and the possible role of IPI-0 during pathogenesis of P. infestans on its hosts will be discussed.

The Differentiation of Leptosphaeria maculans Strains from Poland

M. Jedryczka1, K. Voigt 2, P. Kachlicki1, J. Wostemeyer 2. 1Institute of Plant Genetics, Polish Academy of Sciences, Poznan 60479, Poland 2Institute of Microbiology, Friedrich Schiller University, Jena 07743, Germany

The ascomycete Leptosphaeria maculans (Demn.) Ces. et de Not., together with its conidial stage Phoma lingam (Tode ex Fr.) Desm., is one of the most common and devastating fungi to oilseed winter rape (Brassica napus L.) in Poland. In the last decade the pathogen has spread from the northwestern part of Poland to all intensive rapeseed cultivation areas of the country.

Multidisciplinary studies to assess the distribution of aggressive pathotypes have been undertaken. Fifty strains collected from distant geographical areas were described in terms of colony morphology, sirodesmin production, RFLP and RAPD-PCR patterns. The results indicate the prevalence of non-aggressive isolates, with few aggressives - coming mainly from the northwestern part of Poland. However, the studies of potential isolates pathogenicity to rapeseed (1) suggest the high heterogeneity within “non-aggressive" group, varying from weakly to fairly harmful for rapeseed plants.

(1)Joqczka M., Lewartowska E., Frencel I. (1994). Phytopath.Polonica 7 XDC): 71-79

Molecular Analysis of Mixed Fungal Infections on Oilseed Rape (Brassica napus)

K. Voigt, S. Schleier, J. Wostemeyer. Institute of Microbiology, Friedrich Schiller University,

Jena 07743, Germany

Cultivation of oilseed rape has increased in importance during the past two decades especially in the European Community. This rise in popularity was accompanied by a succession of serious fungal epidemics caused by a variety of pathogens, which often occur in mixed infections. In Germany, probably the most important pathogen is Phoma lingam (sex. form: Leptosphaeria maculans), the causative agent of blackleg disease, leaf spot and stem canker. Other fungi commonly found in the blackleg complex are Cylindrosporium concentricium (light leaf spot), Sclerotinia sclerotiorum (stem rot), Fusarium moniliforme, A1ternaria brassicae and A. brassicicola (dark leaf and pod spot), Verticillium dahliae, Pythium sp. and Rhizoctonia solani. There is considerable interest in a fast and reliable procedure for analyzing these fungi within the blackleg complex.

We have identified RAPD-PCR amplificates which represent DNA sequences for most of these fungi. The species specificity was tested by eluting PCR bands from agarose gels and using them as probes in Southern blot experiments. For Phoma lingam, bands which turned out to be specific for the species and even for the major pathotype groups,”'aggressive” and "nonaggressive", have been characterized by sequencing. On the basis of these sequences, pathotype specific PCR primers have been constructed (1). This method allows the fast and reliable Phoma diagnosis without the known drawbacks of the RAPD-technique for routine analysis.

(1) Voigt, K., Wostmeyer, J. (1995). Microbiol. Res. 150, in press

Genetics and Toxicity of the Phytopathogenic Ascomycete Calonectria morganii (Imperfect State Cylindrocladium scoparium)

von Wallbrunn C., Overmeyer C., Lunnemann S. Meinhardt F. Institut for Mikrobiologie, Westfalische Wilhelms Universitat Munster, Germany

To study genetic diversity of the phytopathogenic Calonectria morganii (imperfect state Cylindrocladium scoparium), thirty two strains isolated from ericaceous hosts and two specimens from ATCC were examined by Random Amplification of Polymorphic DNA (RAPD) and Restriction Fragment Length Polymorphism (RFLP). Five oligonucleotides were chosen as primers to differentiate the isolates. RAPD patterns of the ATCC strains differ significantly from those of the field isolates. Diversity among field isolates is low. Results obtained in RFLP analysis using telomere repeats of Neurospora crassa as a probe were highly consistent with the RAPD data. To distinguish different mating populations isolates were paired in all possible combinations. Fertile perithecia occurred only in one combination, from which ascospores were analyzed by formal genetics and RAPD. A bipolar mechanism of homogenic incompatibility was found. Ascospore derived strains were much more variable than field isolates. Phylogenetic trees suggested a correlation to host plants from which strains were isolated.

Toxic compounds were detected in culture fluids as well as in mycelia. Among the strains tested toxin producers and non producers could be detected. The toxin was active in bacteria and also in eukaryotic organisms including yeasts, filamentous fungi, animals and plants. Preliminary results suggested the toxin to be a cyclic tetrapeptide carrying an epoxy decanoyl acid.

Molecular Genetic Mapping of Virulence in the Barley Net Blotch Pathogen, Pyrenophora teres

John J. Weiland1, Rick D. Cartwright2, Brian J. Steffenson1 and Robert K. Webster2 . 1 Department of Plant Pathology, North Dakota State University, Fargo, N.D. 58105 and 2 University of California-Davis, Davis, CA 95616 USA

Two strains of the fungal ascomycete Pyrenophora teres, one avirulent (strain 15A) and the other virulent (strain 0-1) on the barley cultivar 'Harbin', were mated and the progeny from the cross were isolated. Avirulence in the 76 progeny tested was observed to segregate at a near 1:1 ratio. Genomic DNA was prepared from fungal cultures of 15A, 0-1 and the cross progeny. Progeny DNA was combined into virulent and avirulent pools for bulked segregation analysis (BSA) using random amplified polymorphic DNA markers. Five markers were obtained that were amplified from DNA of avirulent progeny pools and the avirulent parent (1 5A), but not from the virulent progeny pools and parent (0-1). Estimates of marker order and marker genetic distance from the avirulence locus will be presented. The use of these markers in the potential cloning of the P. teres avirulence gene that is active on the barley cultivar 'Harbin' will be discussed.

Characterization of Two Genes of the Phytopathogenic Fungus Gibberella pulicaris, which are Induced by the Phytoalexin Rishitin

Klaus-Michael Weltring. Institut fur Botanik, Westfalische Wilhelms-Universitat, SchloBgarten 3, 48149 Munster, Germany

Gibberella pulicaris is one of the causal agents of potato dry rot. The fungus enters the tubers via wounds where it is confronted with different pre- and postinfectional inhibitors. These compounds like the phytoalexins rishitin and lubimin are produced by the tubers as part of a general defense reaction induced by wounding and invasion of microorganisms. In order to identify attributes of phytopathogenic fungi like G. pulicaris affecting their virulence and pathogenicity towards their host plants we have started to isolate genes induced by the above mentioned defense compounds. The goal is to study the regulation of these genes and their importance for the virulence of the fungus. By differential cDNA screening two clones of a genomic library of G. pulicaris have been isolated each carrying a gene induced by the phytoalexin rishitin. The first gene called rinl codes for a 400 bp transcript, which is also induced by the phytoalexin lubimin within 15 min. The second gene called rin-2 coding for a 500 bp transcript is induced 3-4 hours later and only by rishitin. Comparison of the sequences of both gene so far did not reveal any homology to know sequences. The construction of knock out transformants is under way to investigate the importance of these genes for tolerance of G. pulicaris of phytoalexins and their possible impact on the virulence of the fungus on different host plants.

Metabolism of the Saponins -tomatine, -solanine and -chaconine by Gibberella pulicaris

Judith Wessels and Klaus-Michael Weltring . Institut fur Botanik, Westfalische Wilhelms-Universitat, SchloBgarten 3, 48149 Munster

Gibberella pulicaris is a phytopathogenic ascomycete with a very broad host range. Among others the fungus infects potatoes and tomatoes. These plants are reported to accumulate saponins like -solanine, -chaconine, and -tomatine as preinfectional inhibitors in the vacuoles of their outer cell layers, from which they are liberated upon microbial attack or wounding. Therefore an invading fungus like G. pulicaris has to overcome this chemical barrier either by tolerating or by detoxifying these compounds.

G. pulicaris is able to metabolize -solanine and -chaconine by cleaving off part of the sugar moiety. In contrast, -tomatine is metabolized by removing the complete sugar residue resulting in the transient accumulation of the aglycon tomatidine. This compound is further hydroxylated to a product, which is not degraded any further.

A 2 kb BamHI fragment was isolated from a genomic cosmid clone of G. pulicaris by hybridization with the tomatinase gene of Septoria lycopersici. Partial sequence analysis revealed 62 % and 66 % homology to the tomatinase gene of S. lycopersici and the avenacinase gene of Gaeumannomyces graminis, respectively. Further sequences analysis and the production of knock out transformants are in progress to find out the function of the encoded enzyme.

The Plasma Membrane H+-ATPase of the Rust Fungus Uromyces viceae-fabae: Genetic and Functional Analysis

M. Wernitz, C. Siebels, C. Struck, K. Mendgen and M. Hahn. Universitat Konstanz, Fakultat Biologie, D-78434 Konstanz, Germany

Rust fungi are biotrophic parasites which differentiate haustoria within plant cells. We are interested in the mechanism of nutrient uptake by haustoria and the role played by the plasma membrane ATPase.

The activity of the vanadate-sensitive plasma membrane H+-ATPase was found to be severalfold higher in haustoria than in rust spores and germlings. This indicates that the H+-ATPase generates an electrochemical potential across the haustorial plasma membrane which drives the secondary active transport of nutrients. Using plasma membrane-enriched fractions, the biochemical properties of the enzyme were determined and found to be similar to those of other fungal and plant H+-ATPases. For purification and a full enzymatic characterization, we are planning to express the rust H+-ATPase gene in yeast. A full-length cDNA of the rust H+-ATPase was cloned and sequenced. Surprisingly, the deduced amino acid sequence showed significantly higher homology to the H+-ATPases of plants than to those of ascomycetous fungi. Northern analysis revealed in haustoria a transcript of different size as compared to the transcripts of rust hyphae growing in vitro. Nevertheless, no differences in the cDNAs sequences from in vitro grown hyphae and from haustoria were detected so far. We are now testing which kind of changes in H+-ATPase transcript processing are taking place during rust development.

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