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Rust fungi like Puccinia graminis f. sp. tritici are known to change their cell wall properties upon entering the plant tissue. Immunohistochemistry revealed the cellular localization of two class III chitin synthase isoforms in rust mycelia developing on and in the host plant. Isoform IIIa is restricted to fungal infection structures growing on the surface of the plant, such as germ tubes and, predominantly, appressoria. Isoform IIIb is found exclusively in haustoria developed inside the plant. Thus, the rust fungus uses at least two chitin synthase isoforms with specialized functions in the differentiation of infection structures during the biotrophic plant-pathogen-interaction.

This study presents novel information useful for addressing the question how species of the genus Alcanivorax discharge triacylglycerols (TAG) and/or wax esters (WE). The observed structures were referred as "blebs" according to Gauthier et al. [1] to avoid confusion with other discharging phenomena. The cells were aerobically cultivated on solid media and not in liquid media to maintain the cells in the native state, and were investigated by transmission electron microscopic (TEM) and scanning electron microscopic (SEM) methods to document the surface structures of the cells. The phenomenon of lipid export could be allocated to three phases: phase I: protrusion formation of the cell membrane occurred; phase II: discharging progressed further with blebs becoming larger; and phase III: the blebs at the cell surface were separated from the cells. Using freeze-fracture micrographs by TEM, vesicle experiments and TLC, we have shown that the blebs contained TAG and WE. The results shown in this study will support further research to unravel the unknown discharging mechanism. In addition, the formation of an extensive extracellular matrix was observed by SEM.

Streptomyces sp. strain K30 was isolated from soil next to a city high way in Münster (Germany) according to its ability to degrade natural and synthetic poly(cis-1,4-isoprene) rubber and to form clear zones on natural rubber latex agar plates. The clear zone forming phenotype was used to clone the responsible gene by phenotypic complementation of a clear zone negative mutant. An open reading frame (lcp) of 1,191 bp was identified, which was preceded by a putative signal sequence and restored the capability to form clear zones on natural rubber latex in the mutant. The putative translation product exhibited strong homologies (50% aa identity) to a putative secreted protein from Streptomyces coelicolor strain A3(2), another clear zone forming strain. Heterologous expression of lcp of Streptomyces sp. strain K30 in Streptomyces lividans strain TK23 enabled the latter to form clear zones on latex-overlay agar plates and to accumulate a degradation product of about 12 kDa containing aldehyde groups. Two ORFs putatively encoding a heterodimeric molybdenum hydroxylase (oxiAB) were identified downstream of lcp in Streptomyces sp. strain K30 strain which exerted a positive effect on clear zone formation and enabled the strain to oxidize the resulting aldehydes. Heterologous expression of a fragment harboring lcp plus oxiAB in S. lividans TK23 resulted in accumulation of aldehydes only in the presence of 10 mM tungstate. Determination of protein content during cultivation on poly(cis-1,4-isoprene) revealed an increase of the cellular protein, and gel permeation chromatography analysis indicated a shift of the molecular weight distribution of the rubber to lower values in the transgenic S. lividans strains and in the wild type, thus confirming utilization and degradation of rubber. Therefore, for the first time, genes responsible for clear zone formation on natural rubber latex and synthetic cis-1,4-polyisoprene degradation in Gram-positive bacteria were identified and characterized.

SUMMARY The oxidative burst, a transient and rapid accumulation of reactive oxygen species (ROS), is a widespread defence mechanism of higher plants against pathogen attack. There is increasing evidence that the necrotrophic fungal pathogen Botrytis cinerea itself generates ROS, and that this capability could contribute to the virulence of the fungus. Two potential H(2)O(2)-generating systems were studied with respect to their impact on the interaction of B. cinerea and its host plant Phaseolus vulgaris. A Cu-Zn-superoxide dismutase gene (bcsod1) and a putative glucose oxidase gene (bcgod1) were cloned and characterized, and deletion mutants were created using a gene-replacement methodology. Whereas the Deltabcgod1-mutants displayed normal virulence on bean leaves, the Deltabcsod1 mutants showed a significantly retarded development of lesions, indicating that the Cu-Zn SOD-activity is an important single virulence factor in this interaction system. Whether dismutation of (fungal or host) superoxide, or generation of H(2)O(2) (or both), are important for pathogenesis in this system remains to be elucidated.

Summary Cpmk2 , encoding a mitogen-activated protein (MAP) kinase from the ascomycete Claviceps purpurea , is an orthologue of SLT2 from Saccharomyces cerevisiae , the first isolated from a biotrophic, non-appressorium-forming pathogen. Deletion mutants obtained by a gene replacement approach show impaired vegetative properties (no conidiation) and a significantly reduced virulence, although they retain a limited ability to colonize the host tissue. Increased sensitivity to protoplasting enzymes indicates that the cell wall structure of the mutants may be altered. As the phenotypes of these mutants are similar to those observed in strains of the rice pathogen, Magnaporthe grisea , that have been deprived of their MAP kinase gene mps1 , the ability of cpmk2 to complement the defects of Δ mps1 was investigated. Interestingly, the C. purpurea gene, under the control of its own promoter, was able to complement the M. grisea mutant phenotype: transformants were able to sporulate and form infection hyphae on onion epidermis and were fully pathogenic on barley leaves. This indicates that, despite the differences in infection strategies, which include host and organ specificity, mode of penetration and colonization of host tissue, CPMK2 / MPS1 defines a second MAP kinase cascade (after the Fus3p/PMK1 cascade) essential for fungal pathogenicity.

Summary There is evidence that the necrotrophic fungal pathogen Botrytis cinerea is exposed to oxidative processes within plant tissues. The pathogen itself also generates active oxygen species and H(2)O(2) as pathogenicity factors. Our aim was to study how the pathogen may defend itself against cellular damage caused by the accumulation of H(2)O(2) and the role of an extracellular catalase in its detoxification during the infection of tomato and bean plants by B. cinerea. Chloronaphthol staining followed by light microscopy showed that H(2)O(2) accumulates in the infection zone in tomato and bean leaves. An extracellular catalase gene (denominated Bccat2) was cloned from B. cinerea. Exposure of mycelium to H(2)O(2) in liquid culture resulted in increased Bccat2 mRNA levels in a concentration-dependent manner. Bccat2 mRNA was detected at early stages of tomato leaf infection, suggesting that B. cinerea experiences oxidative stress. Bccat2-deficient mutants were generated by transformation-mediated gene disruption. Mutants were more sensitive then the wild-type strain to H(2)O(2)in vitro, but they partly compensated for the absence of BcCAT2 by activating other protective mechanisms in the presence of H(2)O(2). Bccat2-deficient mutants did not display a consistent reduction of virulence on bean and tomato leaves. Cerium chloride staining of infected leaf tissue for ultrastructural studies showed that Bccat2-deficient mutants were exposed to H(2)O(2) comparably to the wild-type. The results suggest that B. cinerea is a robust pathogen adapted to growing in hostile oxidizing environments in host tissues.

Immunogold labelling and electron microscopy were used to investigate β-1,3-glucanase secretion by Claviceps purpurea during ergot disease of rye in situ. Molecular cytology allowed us to explore the hypothesis that this enzyme might degrade host phloem callose to maintain flow of assimilates for fungal nutrition and therefore β-1,3-glucanase could play a role in pathogenicity. An extracellular endo-β-1,3-glucanase was purified from axenic culture and an antibody was raised. Enzyme activity staining and immunoblotting showed that the antibody was monospecific for β-1,3-glucanase present in fungal protein populations. Mycelia printings of plate-grown cultures displayed spot-like and streaky immuno-signals suggesting a secretion of β-1,3-glucanase at hyphal tips and young hyphae. The enzyme was immuno gold localized predominantly in cell walls of mycelia from axenic culture. In Western blots of honeydew fractions, one β-1,3-glucanase was immunoreactive. In inoculated plants, immunogold labelling was found in all infection stages and limited to the host-pathogen interface. Gold labelling was detected over fungal protoplasts in vacuoles and in tubularvesicular complexes and multivesicular bodies, which fused with the fungal plasma membrane, indicating that they are part of the secretion pathway. The labelling of the fungal secretory organelles and lack of labelling in any host area apart from the interface verified the fungal origin of β-1,3-glucanase immuno-detected in infected ovaries. Antigenic sites were located in external, subcuticular, penetrating and intercellular hyphae, indicating that the enzyme was secreted throughout the colonization process in planta. Intense labelling regularly associated with fungal cell walls extended into adjacent host walls, indicating a migration of the fungal β-1,3-glucanase into the host apoplast. The gold labelling over host periplasmic spaces showed that the enzyme reached the deposition sites of callose, pointing to an enzymatic suppression of putative plant defense reactions. The host phloem was colonized inter- and intracellularly. Hyphae penetrated into the pectic middle lamella of sieve plates and intense immuno-labelling for β-1,3-glucanase in this area supports a phloem unblocking hypothesis.

This paper describes the first purification method for crystalline inclusions (cores) from plant peroxisomes and an ultrastructural characterization of these isolated cores. 5-day-old sunflower (Helianthus annuus L.) cotyledon fractions which were highly enriched in cores showed negligible activity of the matrix enzyme glycolate oxidase but high catalase activity. As proven by electron microscopy, crystalline particles were surrounded neither by matrix material nor by membranes. Their geometrical outlines and ultrastructure were identical to those of cores in tissue sections, as was their reactivity with three different polyclonal catalase antibodies in the immunogold technique. Three-dimensional reconstruction, based on the geometrical outlines and ultrastructure of sectioned isolated cores from sunflower, suggested that they were quadrangular blocks. Ultrastructural analysis revealed an even periodic arrangement of repeating units which are probably cubes with 20 nm long edges. Isolated peroxisomal cores from potato (Solanum tuberosum L.) tubers had outlines which suggested that they were even rhomboidal prisms. They showed a granular ultrastructure without any repeating units and contained catalase, demonstrated by immunogold labelling and enzyme activity measurement. The results presented here suggested the hypothesis that the structural elements in plant peroxisomal cores are made of enzymatically active catalase, although the substructure may vary from species to species.

Two different antibodies against bovine serum albumin (BSA)-p-coumaric acid-conjugates were produced and used to localize phenolic compounds in exines of pollen from different species,p-Coumaric acid (pC) was coupled to BSA either via the carboxy group (BSA-pC) or directly to the aromatic ring system (BSA-azopC). The polyclonal antibodies raised in rabbits were characterized by ELISA with homologous and heterologous antigens using turkey ovalbumin as carrier protein. The results showed that the two immune sera directed against BSA-pC and BSA-azo-pC, respectively, were specific forp-coumaric acid and structurally similar compounds. Only a very poor binding by acetic acid-ovalbumin-conjugates and no binding by turkey ovalbumin was detectable. The antibodies reacted with partially purified pollen walls and with highly purified exines. The intensity of the immune reaction was proved to be dependent upon the pollen source and the preparation of the pollen walls. Using light and electron microscopy, it was shown for the first time that, in the exines ofCucurbita maxima, antibody binding was predominantly observed in the region of the germ pore apertures, the outer foot layers, and in the micro- and macrospines. We conclude from this and other earlier published data that phenols are important structural compounds of sporopollenin.

At the host-pathogen interface of hyphae penetrating host cell walls in the rye ovary, a lack of cellulase-gold labeling of β-1,4-glucan in host cell walls indicates that enzymatic 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 cellulase gene (cel1) was isolated from a genomic library of the C. purpurea strain T5. The coding region of 1,616 bp contains two introns and a putative signal peptidase cleavage site, leaving a coding capacity of 437 amino acids for the mature protein. The derived amino acid sequence shares significant homology with other fungal cellobiohydrolases and lacks the substrate binding domain. Expression analysis using reverse transcriptase-polymerase chain reaction (RT-PCR) shows that cel1 is induced during the first days of infection of rye by C. purpurea. It may be involved in the penetration and degradation of host cell walls by depolymerizing plant β-1,4-glucan and, therefore, play a role in the infection process.

Summary There is evidence that the necrotrophic fungal pathogen Botrytis cinerea is exposed to oxidative processes within plant tissues. The pathogen itself also generates active oxygen species and H(2)O(2) as pathogenicity factors. Our aim was to study how the pathogen may defend itself against cellular damage caused by the accumulation of H(2)O(2) and the role of an extracellular catalase in its detoxification during the infection of tomato and bean plants by B. cinerea. Chloronaphthol staining followed by light microscopy showed that H(2)O(2) accumulates in the infection zone in tomato and bean leaves. An extracellular catalase gene (denominated Bccat2) was cloned from B. cinerea. Exposure of mycelium to H(2)O(2) in liquid culture resulted in increased Bccat2 mRNA levels in a concentration-dependent manner. Bccat2 mRNA was detected at early stages of tomato leaf infection, suggesting that B. cinerea experiences oxidative stress. Bccat2-deficient mutants were generated by transformation-mediated gene disruption. Mutants were more sensitive then the wild-type strain to H(2)O(2)in vitro, but they partly compensated for the absence of BcCAT2 by activating other protective mechanisms in the presence of H(2)O(2). Bccat2-deficient mutants did not display a consistent reduction of virulence on bean and tomato leaves. Cerium chloride staining of infected leaf tissue for ultrastructural studies showed that Bccat2-deficient mutants were exposed to H(2)O(2) comparably to the wild-type. The results suggest that B. cinerea is a robust pathogen adapted to growing in hostile oxidizing environments in host tissues.

The outer epidermal wall of Picea abies needles was investigated throughout its life-span. Several cytochemical procedures at the light and electron microscope level including enzyme–gold affinity labeling and elemental mapping by electron energy loss spectroscopy were applied to gain detailed information about its fine structure. The adult outer epidermal wall averages 9 μm in thickness and occupies half the cell height. It is composed of five main layers and additional epicuticular waxes. In the cuticular membrane a thin homogeneous cuticle proper is clearly distinguishable from the cuticular layer, which is composed of an exterior striated, a middle reticulate, and an interior arborescent sublayer. Cellulose and mannans were localized and identified among other polysaccharides in the cuticular layer in situ. Arborescent structures extend to an adjacent, usually lignified layer, containing numerous calcium crystals. Beneath this is an electron-dense layer, formed mainly of hemicelluloses. The innermost wall layer comprises several alternately arranged lamellae formed of several types of polysaccharides and differing mainly in their polyphenol content. During ontogeny of the outer epidermal wall, lasting a whole year, five phases were distinguished: a protodermal phase, one of even growth, one of uneven growth, and one of lignification and further growth. With the fifth phase the outer epidermal wall of current needles achieves maturity in late autumn. No differences in ultrastructure and development of the outer epidermal wall between apparently healthy and damaged needles were observed. Key words: Picea abies, outer epidermal wall, light and electron microscopical cytochemistry, enzyme–gold technique, polysaccharides, calcium crystals.

Hydrogen peroxide of the host origin accumulates in plant apoplasts in response to pathogen attack and probably functions directly in defense reactions or in signaling, according to a previous study. Since Claviceps purpurea produces compatible interactions with hundreds of host species, we hypothesized that the fungus might interfere with H2O2-mediated defense by means of secreted catalases. In axenic culture of C. purpurea, catalase activity accumulated in the medium and was inhibited by the catalase inhibitor aminotriazole. Polyacrylamide gel electrophoresis followed by diaminobenzidine (DAB)-mediated activity staining showed that one specific catalase found in culture filtrate was also present in rye ovaries infected with C. purpurea and in honeydew. This catalase form is probably induced during infection. In situ activity staining, using DAB-mediated enzyme-cytochemistry in electron microscopy, located catalase activity in hyphal walls during both axenic culture and infection of rye. Activity staining accumulated in periplasmic spaces and was especially strong at hyphal surfaces; control staining after aminotriazole inhibition was negative. Intracellular activity staining in organelles of the fungal secretory pathway substantiated that catalase was secreted by C. purpurea. With molecular cytology, anticatalase epitopes were localized with different heterologous catalase antibodies at sites corresponding to the activity staining pattern. In all infection phases, immunogold labeling indicated that the putative catalase was secreted via multivesicular bodies into the fungal wall and diffused into the host apoplast exclusively at the hostpathogen interface. The secretion of fungal catalase is a novel finding in phytopathology, and we discuss its role in the ubiquitous ergot disease.

Claviceps purpurea is a common phytopathogenic fungus that colonizes ovarian tissue of grasses. A concerted approach involving cytological and molecular techniques was initiated to investigate the role of the fungus' xylanolytic system in the interaction. Using enzyme-gold and immuno-gold electron-microscopic techniques, the presence of arabinoxylans in cell walls of rye ovarian tissues (i.e., along the usual path of infection of C. purpurea) was confirmed; tissue-print and immunostaining analyses indicated the presence of xylanase(s) exclusively in ovaries infected with C. purpurea. This strongly suggests that C. purpurea secretes xylanase while colonizing its host. Two xylanase genes (cpxyl1 and cpxyl2) were isolated from a genomic library of C. purpurea using genes from Cochliobolus carbonum (xyl1) and Magnaporthe grisea (xyn33) as heterologous probes. cpxyl1 of C. purpurea had an open reading frame (ORF) of 832 bp interrupted by a 181-bp intron. The derived gene product (CPXYL1) had a molecular mass of 21.5 kDa and an pI of 8.88; it showed significant homology to family G endo-β-1,4-xylanases. The cpxyl2 ORF (1,144 bp) contained two introns (76 and 90 bp) and coded for a polypeptide of 33.8 kDa with an pI of 7.01; CPXYL2 belonged to family F xylanases. Southern analyses with genomic DNA demonstrated that both genes were single-copy genes. Using reverse transcription polymerase chain reaction, it could be shown that both genes were expressed in vitro and in planta (during all infection stages). Inactivation of cpxyl1 was achieved by a gene-replacement approach. The mutant strain (Δcpxyl1) had significantly reduced xylanase activity; Western analyses confirmed that it lacked a polypeptide of approximately 23 kDa.

Gutta percha, the trans-isomer of polyisoprene, is being used for several technical applications due to its resistance to biological degradation. In the past, several attempts to isolate micro-organisms capable of degrading chemically pure poly(trans-1,4-isoprene) have failed. This is the first report on axenic cultures of bacteria capable of degrading gutta percha. From about 100 different habitats and enrichment cultures, six bacterial strains were isolated which utilize synthetic poly(trans-1,4-isoprene) as sole carbon and energy source for growth. All isolates were assigned to the genus Nocardia based on 16S rRNA gene sequences. Four isolates were identified as strains of Nocardia nova (L1b, SH22a, SEI2b and SEII5a), one isolate was identified as a strain of Nocardia jiangxiensis (SM1) and the other as a strain of Nocardia takedensis (WE30). In addition, the type strain of N. takedensis obtained from a culture collection (DSM 44801(T)) was shown to degrade poly(trans-1,4-isoprene). Degradation of poly(trans-1,4-isoprene) by these seven strains was verified in mineralization experiments by determining the release of CO(2). All seven strains were also capable of mineralizing poly(cis-1,4-isoprene) and to use this polyisoprenoid as a carbon and energy source for growth. Mineralization of poly(trans-1,4-isoprene) after 80 days varied from 3 % (strain SM1) to 54 % (strain SEI2b) and from 34 % (strain L1b) to 43 % (strain SH22a) for the cis-isomer after 78 days. In contrast, Gordonia polyisoprenivorans strain VH2, which was previously isolated as a potent poly(cis-1,4-isoprene)-degrading bacterium, was unable to degrade poly(trans-1,4-isoprene). Scanning electron microscopy revealed cavities in solid materials prepared from poly(trans-1,4-isoprene) and also from poly(cis-1,4-isoprene) after incubation with N. takedensis strain WE30 or with N. nova strain L1b, whereas solid poly(trans-1,4-isoprene) material remained unaffected if incubated with G. polyisoprenivorans strain VH2 or under sterile conditions.

CPTF1, a transcription factor with significant homology to ATF/CREB bZIP factors, was identified during an expressed sequence tag (EST) analysis of in planta-expressed genes of the phytopathogen Claviceps purpurea. Using a gene-replacement approach, deletion mutants of cptf1 were created. Expression studies in axenic culture showed that the H2O2-inducible gene cpcat1 (encoding a secreted catalase) had a reduced basal expression level and no longer responded to oxidative stress in the Δcptf1 mutant. Biochemical analyses indicated that CPTF1 is a general regulator of catalase activity. Δcptf1 mutants showed significantly reduced virulence on rye. Electron microscopical in situ localization revealed significant amounts of H2O2 in Δcptf1-infected rye epidermal tissues, indicating that the plant tissue displayed an oxidative burst-like reaction, an event not detected in wild-type infections. These data indicate that CPTF1 is involved not only in oxidative stress response in the fungus but also in modulation of the plant's defense reactions.

Cpmk2, encoding a mitogen-activated protein (MAP) kinase from the ascomycete Claviceps purpurea, is an orthologue of SLT2 from Saccharomyces cerevisiae, the first isolated from a biotrophic, non-appressorium-forming pathogen. Deletion mutants obtained by a gene replacement approach show impaired vegetative properties (no conidiation) and a significantly reduced virulence, although they retain a limited ability to colonize the host tissue. Increased sensitivity to protoplasting enzymes indicates that the cell wall structure of the mutants may be altered. As the phenotypes of these mutants are similar to those observed in strains of the rice pathogen, Magnaporthe grisea, that have been deprived of their MAP kinase gene mps1, the ability of cpmk2 to complement the defects of delta mps1 was investigated. Interestingly, the C. purpurea gene, under the control of its own promoter, was able to complement the M. grisea mutant phenotype: transformants were able to sporulate and form infection hyphae on onion epidermis and were fully pathogenic on barley leaves. This indicates that, despite the differences in infection strategies, which include host and organ specificity, mode of penetration and colonization of host tissue, CPMK2/MPS1 defines a second MAP kinase cascade (after the Fus3p/PMK1 cascade) essential for fungal pathogenicity.

Claviceps purpurea is a biotrophic, organ-specific pathogen of grasses and cereals, attacking exclusively young ovaries. We have previously shown that its mainly intercellular growth is accompanied by degradation of pectin, and that two endopolygalacturonase genes (cppg1/cppg2) are expressed throughout all stages of infection. We report here on a functional analysis of these genes using a gene-replacement approach. Mutants lacking both polygalacturonase genes are not affected in their vegetative properties, but they are nearly nonpathogenic on rye. Complementation of the mutants with wild-type copies of cppg1 and cppg2 fully restored pathogenicity, proving that the endopolygalacturonases encoded by cppg1 and cppg2 represent pathogenicity factors in the interaction system C. purpurea/Secale cereale, the first unequivocally identified so far in this system.

The plant cell wall functions as a barrier to biotic and abiotic agents. Plant pathogenic bacteria and fungi produce cell wall degrading enzymes (CWDEs) which are believed to degrade this barrier, thereby facilitating both inter- and intracellular growth and providing nutrients to the invader. A pectate lyase from the bacterium Erwinia chrysanthemi was the first CWDE that was shown to be required for full virulence (Roeder and Colmer 1985). Subsequent molecular genetic studies have shown that many other bacterial CWDEs are virulence factors (reviewed by Hugouvieux-Cotte-Pattat et al. 1996). It took many years before similar evidence was obtained for the involvement of fungal CWDEs in pathogenesis, in spite of several efforts (reviewed by Walton 1994; Annis and Goodwin 1997). Eventually, an endopolygalacturonase from Aspergillus fiavus was shown to play a role in the invasion of cotton bolls (Shieh et al. 1997).

Botrytis cinerea Persoon: Fries (known as “grey mould fungus”) causes serious preand post-harvest diseases in at least 235 plant species (Jarvis, 1977), including a range of agronomically important crops, such as grapevine, tomato, strawberry, cucumber, bulb flowers and ornamental plants. Graminaceous monocots are generally considered as poor hosts for grey mould. Disease control frequently relies on chemicals, although efforts to develop biological control strategies are increasingly successful (e.g. Kohl et al., 1995; Elad, 1996) and biocontrol agents are marketed. The name of the asexual stage or anamorph, Botrytis cinerea, is preferred to the name of the teleomorph, Botryotinia fuckeliana (de Bary) Whetzel (XIth International Botrytis Symposium, 1996, Wageningen, The Netherlands). The teleomorph has rarely been detected in the field during the last century, but molecular population studies recently provided clear evidence that sexual reproduction occurs more frequently than previously anticipated (Giraud et al., 1997). The pathogen is a typical necrotroph, inducing host cell death resulting in serious damage to plant tissues, culminating in rot of the plant or the harvested product. There are extensive descriptions of microscopic and biochemical studies on infection mechanisms (reviewed by Staples and Mayer, 1995). Comprehensive insight in the infection process, however, is hampered by the fact that various groups used different fungal strains and different host species for their studies.

Claviceps species are the causal agents of the ubiquitous ergot disease. About thirty-six different filamentous fungi constitute this genus of phytopathogenic ascomycetes. They parasitize more than 600 monocotyledonous plants of the families Poaceae, Juncaceae and Cyperaceae (Bove, 1970), including forage grasses and the leading cereals worldwide: wheat, rice, corn, barley, sorghum, oats, rye, millets (Baum et al., 1992). Being epidemic to a greater extent in semi-arid regions than in temperate zones, ergot is of increasing importance in India and Africa, where pearl millet and sorghum are essential crops (Frederickson et al., 1993). Although the fungi cause harvest losses due to replacement of host ovaries with the parasite’s resting structures, the ergotcalled sclerotia, the main problem is not a severe loss in seed quantity but arises from complete ruin of grain quality due to the alkaloid content of the sclerotia. Admittedly, ergot alkaloids are secondary metabolites of high pharmacological value and are, therefore, produced worldwide on a large scale, nevertheless, these toxins cause highly dangerous or even deadly ergotism when contaminated grains are fed to animals or are consumed by man. These are the reasons for a continuous interest for ages in ergot fungi and their persistent importance (see chapter 1 in this volume), which will remain valid as long as the main ubiquitous nutritional basis to man and herbivorous livestock is concerned. Worldwide reduction in grain yield and quality causes the permanent necessity for an expensive cleaning of attacked cereals to maintain a minimum of purity standard. A contamination of crops with ergots higher than 0.3% by weight spoils the grain even for feeding (Agrios, 1988). Specific measures for reliable control as well as utilization of positive capacities of ergot fungi closely depend on an overall understanding of host-and pathogen biology.

Two putative polygalacturonase (PG) genes were isolated from strain T5 of Claviceps purpurea, using the pgall gene of Aspergillus niger as a probe. The two genes (pg1 and pg2) are closely linked and arranged head - to-tail. They are highly homologous even in the upstream noncoding sequences, possess one intron each in the same position, and have cleavage sites for processing enzymes. They probably code for mature proteins of 343 and 344 amino acids, respectively, and share significant homology with endo-PGs of other filamentous fungi. Expression of pgl and pg2 in axenic culture and during various stages of infection of rye was demonstrated using reverse transcription-polymerase chain reaction. The potential substrate of the putative products of pg1 and pg2 (polygalacturonic acid), for the first time, was shown to be a component of the host cell walls in rye ovaries. This was achieved by immunogold transmission electron microscopy with the monoclonal antibody (MAb) JIM 5, specific for nonmethyl-esterified epitopes of pectin. This homogalacturonan was localized along the usual infection path in healthy carpels together with its methyl-esterified galacturonan type in the same cell walls with another MAb, JIM 7. At the interface of the penetrating hyphae and the host ovary epidermis, JIM 5 label density was locally enhanced and very high above hyphal sheaths. In the vicinity of intercellularly growing hyphae, label density was highly increased, and gold label occurred not only above the middle lamella area but also throughout the entire host cell wall. Chemical demethylation and immunogold labeling indicated a high total content of galacturonan and a conversion of pectic compounds at the host-parasite interface. During late infection phases, the lack of any JIM label, which previously occurred at the interface of intracellular hyphae, emphasized the complete utilization of homogalacturonan together with other plant polysaccharides. The observed host wall alterations provide evidence for secretion and activity of extracellular pectinolytic enzymes in planta. Both the expression of the two genes during infection of rye and the modification and degradation of homogalacturonan detected only at fungal sites indicate the fungal origin of pectinolytic enzymes, the activities of which have been documented previously in infected ovaries by B. I. Shaw and P. G. Mantle.