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PAUL TUDZYNSKI Institut für Botanik, Westf. Wilhelms-Universität, Schlossgarten 3, D-48149 Münster

For about 70 years genetic analyses of Claviceps purpurea have been performed (e.g. Kirchhoff, 1929); nevertheless, genetic data available are still rather scarce, mainly due to intrinsic experimental problems, which place this fungus far apart from being a genetic model organism: the generation time is extremely long and—since it involves parasitic culture of the fungus—the conditions of obtaining sexual progeny are rather complex; biotechnologically relevant strains normally are imperfect and even do not produce conidia; and, last but not least, C. purpurea strains tend to be instable during prolonged vegetative cultivation. Nevertheless, due to the development of molecular genetic approaches, and due to growing interest in C. purpurea as a pathogen, in recent years genetic research in C. purpurea has been intensified. In some recent review articles on C. purpurea also genetic data have been included (e.g. Soèiè and Gaberc-Porekar, 1992; Tudzynski et al., 1995; Lohmeyer and Tudzynski, 1997).

4.1. CLASSICAL GENETICS Compared to other biotrophic parasites, C. purpurea can be rather easily handled: most laboratory strains and field isolates grow well and sporulate abundantly in axenic culture. Since conidia normally are mononuclear, mutants can be easily obtained by standard procedures; heterokaryons are readily formed and can be used for parasexual approaches; and sexual crosses are possible, though time consuming. 4.1.1. Mutants Numerous stable mutants of C. purpurea have been obtained, mainly by UVmutagenesis. They include various auxotrophic mutants (e.g. Strnadová, 1964; Tudzynski et al., 1982), for only a few of which the metabolic block was determined exactly, e.g. for 5 arg- mutants (Kus et al., 1987) and several pyrmutants (Smit and Tudzynski, 1992). The latter mutants were obtained in an “intelligent” screening approach, using resistance against the toxic analogue 5fluoro-orotate (FOA) as selective principle; FOA-resistant mutants were obtained after UV-mutagenesis at a rate of 3.2×10-6; from these, 12.5% were pyrimidine auxotrophs, about half of these were shown to be defective in the
Copyright © 1999 OPA (Overseas Publishers Association) N.V. Published by license under the Harwood Academic Publishers imprint, part of The Gordon and Breach Publishing Group.

g. Already Kirchhoff (1929) found segegration within an F1 progeny of albino sclerotia. Jungehülsing. Heterokaryosis Most field isolates and industrial strains of C. 1995) or cercobin (Tudzynki et al. Esser and Tudzynski (1978) could demonstrate that homokaryotic mycelia can produce alkaloids. in contrast to earlier observations (e. Also several fungicide resistant mutants have been described. unpubl. purpurea (at least the isolates tested sofar) obviously is homothallic. The main effort over the years. reasons for this heterogenicity could be mixed infections in the field strains and generally a high mutation rate. . 1986. inspite of the mononuclear status of their conidia. purpurea. Recently a novel and promising approach to generate mutants by random integration of transforming vector DNA has been successfully used to obtain auxotrophic and pathogenicity mutants in C.g. Müller.1.g. 1995).). haploidization experiments using low doses of benomyl applied on two different isolates yielded derivatives with only 50% of relative DNA content. purpurea have been found to be heterokaryotic. Recently also recombinant DNA technology has been applied to mark strains with antibiotic resistance genes and use them in crosses (Jungehülsing. DidekBrumec et al. purpurea (Voß. unpubl.. heterokaryosis is not required for completion of the life-cycle: Strnadová and Kybal (1974) and Esser and Tudzynski (1978) could prove that homokaryotic strains can perform the parasitic cycle and produce ascospores. we found a high degree of morphological variation. however. 1982). e. Obviously these different Copyright © 1999 OPA (Overseas Publishers Association) N. Stehling and Tudzynski.. 1987. Determination of relative DNA content per nucleus by DAPI staining showed a high level of variation among different isolates. 1995. in one case about 15 different phenotypes (Tudzynski. When we analysed selfing F1 progeny of field isolates. 1967) that only heterokaryotic strains were good producers. 1956). C. 4. has been laid on mutagenesis programs concerning alkaloid biosynthesis. 1996). Amici et al. 1978). often using successive steps of various mutagens. and that benomyl treatment represents a good tool for the generation of bona-fide haploid strains for mutagenesis programs and certain molecular studies (see below). Despite this high level of heterokaryosis in field isolates. Despite these numerous successfull reports on mutagenesis in C. Kobel and Sanglier. confirming a heterokaryotic status of these sclerotia (see also Békésy.2. part of The Gordon and Breach Publishing Group.80 PAUL TUDZYNSKI orotidine-monophosphate-carboxylase (OMPD) gene (the rest probably was defective in the OMP pyrophosphorylase gene).. indicating various degrees of aneuploidy in these field isolates.V. Published by license under the Harwood Academic Publishers imprint. we found that several field isolates of this fungus were unexpectedly highly recalcitrant against mutagenesis. various mutant strains showing increased or decreased amount or a different spectrum of alkaloids have been described (e.. see below). confirming the idea that the original strains were not haploid (Jungehülsing.. against benomyl (Kus et al.

genetic analysis of C.g. purpurea strain and a clavine producing C. A major technical problem hampering such genetic analyses is the low germination rate of sclerotia. It could be shown by several groups that formation of heterokaryons between strains of different genetic background could indeed significantly increase or generally alter alkaloid biosynthesis. 4. 1984). 1984). such mixed infections lead to a high rate of mixed sclerotia yielding recombinant ascospores (e. Copyright © 1999 OPA (Overseas Publishers Association) N. since e. nevertheless the process cannot be considered as being easily reproducible in any lab. Taber. respectively. fusion of strains and generation of defined heterokaryons represent a powerful tool for improvement of the (normally imperfect) alkaloid production strains.g. Matings are obtained by inoculating young rye florets with mixed conidial suspensions of both strains. In short. conditions for induction of this germination process have been studied in detail (e. Published by license under the Harwood Academic Publishers imprint. Spalla et al. gene dosis. surface-sterilized by HgCl2 or formaline treatment. fusiformis strain (Robbers. purpurea the formation and germination of sclerotia (see chapter 3 “Biology of Claviceps”) is obligatory. Brauer and Robbers (1987) could demonstrate that in heterokaryotic strains parasexual processes take place.GENETICS OF CLAVICEPS 81 data reflect differences in the genetic background of the strains used in these studies.1.V.g. most auxotrophic mutants turned out to be non-pathogenic (Tudzynski et al. Even interspecies fusions have been successfully used to obtain different spectra of alkaloids. 1982. placed on wet sand or vermiculite. leading to recombination.g.. the heterokaryon produced all 3 alkaloids. the fusion product synthesizing both types of alkaloids. Therefore. here probably effects like heterosis. are important. sclerotia are harvested. the use of auxotrophic markers in crosses is limited. since the needle-like ascospores are tightly intertwined and cannot be isolated without breakage from an intact ascus.3. Békésy. 1929. After transfer to 14–17°C (white light) sclerotia (hopefully) germinate to form perithecial stromata. purpurea strains producing ergocristine and ergocornine/ ergokryptine. they germinate within a few hours. and kept for at least 3 months at about 0°C. complementation.. inspite of the homothallism mentioned above. (1976) produced a forced heterokaryon (which had to be kept on minimal medium) between C. Isolation of tetrads is not possible. Sexual Cycle and Recombination For completion of the sexual cycle in C. between an ergotamin-producing C. 1984). Since—despite intensive efforts of several groups—so far induction of sclerotia formation in axenic culture never has been achieved. purpurea requires facilities to grow Secale cereale (or another host plant) under defined ± sterile conditions. Ejected ascospores are trapped on agar-covered glass slides and isolated. 1982). etc. 1956. Taber. . part of The Gordon and Breach Publishing Group. e. Another problem is that virulence/pathogenicity is an essential prerequisite for successful matings. Kirchhoff. Tudzynski et al.

digested with four different restriction endonucleases.V. purpurea in recent years and have considerably widened the options for genetic research: they allow detailed molecular characterization of strains—this is important for “typing” of strains and systematic analyses—. Published by license under the Harwood Academic Publishers imprint. for ribosomal DNA).e. disease diagnosis. part of The Gordon and Breach Publishing Group. All these techniques have been successfully applied also for the genus Claviceps (see Pazoutova.). (see recent compilation by Tudzynski and Tudzynski. 1986). could be valuable Copyright © 1999 OPA (Overseas Publishers Association) N. Tudzynski and Düvell. . 1983. 1992). purpurea (Luerweg and Tudzynski. RAPD) or specific primers (e. Therefore the sexual cycle and meiotic recombination could form a valuable basis for molecular genetic approaches.. inspite of the pitfalls and technical problems of the system.2. etc. and. are not strictly strain-specific: identical plasmids can occur in strains from different geographic origin (Tudzynski and Esser. by enhancing expression of genes of interest or by creating defined deletion mutants. MOLECULAR GENETICS Molecular genetic techniques now readily available also for fungal systems have been successfully applied to C. definition of compatibility groups/subspecies. Mt plasmids present in most field isolates. i.g. however. Molecular Characterization of Strains Molecular methods like restriction-fragment-length-polymorphism (RFLP) analysis. Genomic DNA from two non-related secale-derived field isolates. which lists corresponding data for more than 40 species of phytopathogenic fungi). 4. (a) RFLP: The first investigations on RFLPs in C. polymerase-chain-reaction (PCR) based techniques using random primers (random-amplified-polymorphic DNA. and pulsed field gel electrophoresis (PFGE) for the separation of fungal chromosomes have been widely used to characterize fungal isolates with respect to aspects of evolution. especially of the alkaloid pathway. 4. 1985). purpurea used mitochondrial DNA (mtDNA): it could be shown that the restriction pattern of mtDNA is strain specific. they open the possibility to create recombinant strains.1. Recently in our lab we started a genomic RFLP program with the aim to establish a genetic map of C. they facilitate and speed up the evaluation of crosses and the establishment of a genetic map considerably. they provide the tools for detailed regulatory studies. population biology. last not least. purpurea strain T5 (Smit and Tudzynski. 50% of the clones checked obviously contained repetitive DNA and could not be used for genetic analyses (nevertheless.2. unpubl. 1996. this volume). was blotted and hybridized to randomly chosen lambda clones from a genomic library of C. allows the identification of specific isolates (Tudzynski et al.82 PAUL TUDZYNSKI In our lab recently the construction of a (preliminary) genetic map was achieved by analysing the progeny of a cross between two fungicide/antibiotic resistant strains by molecular methods (see below).

see Table 1). The two strains. RAPD can be useful for the construction of a genetic map. We tested the use of RAPD bands as molecular markers in genetic crosses. purpurea (see also chapter 3. however.or dekanukleotides as primers (Williams et al. Figure 1 shows the segregation pattern of an RFLP detected by the cpxyl gene of C. which carried resistance markers (benomyl resistance. purpurea strains. about a quarter of the RAPD markers tested segregated 1:1 and could be used as genetic markers (Jungehülsing. unpubl. Tudzynski and Tudzynski. since they showed significant differences. With some of these primers each of the 29 isolates showed a unique RAPD pattern. Most of them segregated in a Mendelian way (1:1). Published by license under the Harwood Academic Publishers imprint. coding for a xylanase (Giesbert and Tudzynski. each RAPD marker. respectively). purpurea chromosomes (see also Tudzynski et al. due to their comparatively small sizes (for review see Skinner et al. A preliminary tree analysis based on these 9 RAPD primers gave indications for some degree of host specificity or beginning sub-species formation (though this data must be interpreted cautiously).. Therefore. 16 of these primers yielded a reproducible RAPD pattern. RAPD without any doubt is a very useful tool for the typing of strains and for systematic analyses in C. has to be checked carefully for its Mendelian behaviour. 1995). and a transformation introduced phleomycin resistance.V. (b) RAPD: This widely applied technique is based on the polymerasechainreaction (PCR) using single random nona. 9 were used for comparative analysis. depending on the primers used. Inclusion of additional markers and combination with PFGE (see below) will allow to fill in the gaps and to assign these linkage groups to specific chromosomes. (c) PFGE: Pulsed-field-gel-electrophoresis has turned out to be an effective method to separate fungal chromosomes.. confirming that this technique can be used for the identification of C. Only 6% of the single copy clones yielded no RFLPs.. showing numerous chromosome length polymorphisms (the size varying between 7 and Copyright © 1999 OPA (Overseas Publishers Association) N. purpurea from various host-plants. A preliminary genetic map was obtained (see Figure 2). RAPD allows a fingerprint-like characterization of strains. Jungehülsing and Tudzynski (1996) tested 58 different random decamer primers using as template genomic DNA from 29 field isolates of C. Unexpectedly the karyotypes of the field isolates differed considerably. Altogether 72 RFLP markers (and the two resistance markers) were used for a linkage analysis.. 1996). purpurea.GENETICS OF CLAVICEPS 83 for fingerprint studies!). 1995). using the randomly selected lambda clones and several cloned genes from C. purpurea. and that others yielded unusual segregation patterns. 1991. part of The Gordon and Breach Publishing Group. . Jungehülsing (1995) successfully optimized electrophoresis conditions for the separation of C. this volume). 1991). were crossed and the progeny (56 randomly isolated ascospore lines) were evaluated by RFLP. the rest showed one or more RFLPs. nevertheless. comprising 13 linkage groups (and two unlinked markers). it turned out that more than 50% of the RAPD fragment were derived from repetitive DNA.

purpurea. Genomic DNA of the parent strains (T5 B17R and BiP5-1) and F1 progeny strains (KTB) was digested with BamHI. mating between strains with different karyotypes yielded viable progeny. Published by license under the Harwood Academic Publishers imprint. indicating a high degree of interchromosomal recombinations (translocations. purpurea strain tested sofar showed a unique karyotype. part of The Gordon and Breach Publishing Group.V. showing Copyright © 1999 OPA (Overseas Publishers Association) N.). Actually each C. etc.84 PAUL TUDZYNSKI Figure 1 Example for an RFLP analysis in C.8 Mb) and differences in numbers (3–7). blotted and hybridized with a labelled fragment of the cpxyl gene of C. purpurea (see Table 1) 1. Interestingly. . separated in an agarose gel.

. Copyright © 1999 OPA (Overseas Publishers Association) N. Published by license under the Harwood Academic Publishers imprint. cDNA-clones obtained (from a cDNA library from a glucose-derepressed culture) during the screening for other genes.V. 85 1: 2: differential screening of a cDNA library from an alkaloid-producing culture with cDNA from a non-producing (cDNA-) and a producing culture (cDNA+).GENETICS OF CLAVICEPS Table 1 Genes (or fragment of genes) available from Claviceps sp. part of The Gordon and Breach Publishing Group.

V. the distance of the various markers (random lambda clones from a EMBL library from C. data) Copyright © 1999 OPA (Overseas Publishers Association) N. Linkage groups are designed with roman numbers.86 PAUL TUDZYNSKI Figure 2 Preliminary genomic map of C. purpurea as listed in Table 1) is indicated in map units (Frank Luerweg. part of The Gordon and Breach Publishing Group. purpurea T5 and various genes of C. purpurea. Published by license under the Harwood Academic Publishers imprint. . unpubl.

This indicates that meiotic recombination considerably contributes to the heterogenicity in the karyotypes of field isolates. the dimethylallyltryptophansynthase (DMATS) was cloned... niger and C. crassa (a circadian clock regulated general stress protein. Recently. mostly by using heterologous probes. gpd. purpurea strain was pyr4. most of the others stem from molecular analyses of the pathogen C. Isolation of Genes The first gene that has been cloned from a C. but also by cDNA screening and PCR techniques (see Table 1). Qid3 from Trichoderma viride (Lora et al. a hydrophobin-like putative cell-wall protein showing high homology to other fungal hydrophobins. crassa as heterologous probe (Smit and Tudzynski. (see also Chapter 2 by Tenberge. purpurea have been cloned and characterized.. etc. purpurea. A major advantage of PFGE from the genetic point of view is the option to perform Southern hybridization experiments with PFGE gels. the identity of the gene was verified by expression in yeast.. 1996). however. Its function was confirmed by complementation analyses using defined OMPD-deficientmutants of A. Among the clones identified so far by this screening as being selectively expressed during alkaloid biosynthesis were clones coding for a DMATS (confirming that this appproach is useful for the isolation of genes of the alkaloid pathway). It can be used as an efficient selection marker in transformation experiments (see below). this volume). coding for a orotidine-monophosphate-decarboxylase (OMPD). e. and a protein showing homology to the ccg1 gene product of N.2. 1994). based on oligonucleotides derived from a partial aminoacid sequence of the purified enzyme. purpurea as recipients. Published by license under the Harwood Academic Publishers imprint. 4.g. In our group a differential cDNA screening approach was initiated (Arntz and Tudzynski. allowing to establish a link between the genetic and the physical genomic map. and the first genes have been characterized. Loros et al. part of The Gordon and Breach Publishing Group. . it was cloned from a genomic EMBL3 library of the rye isolate T5.). also studies on the molecular genetics of alkaloid biosynthesis were initiated. using the pyr4 gene of N. Some of these genes have been cloned because of their potential usefulness in transformation systems (pyr4. Since then a large number of genes from C. 1995) and cDNA preparations from an alkaloid producing and non-producing culture as probes. e.g. genes coding for plant cell wall degrading enzymes. The latter two clones indicate that this approach also yields genes that might be correlated with the physiological stress and different hyphal Copyright © 1999 OPA (Overseas Publishers Association) N. using a cDNA library (lambda Zap) from an alkaloid producing culture of strain ATCC 26245 (the same strain as used by Tsai et al. etc. 1995) the gene for the first specific enzyme of the alkaloid pathway. 1992).V.GENETICS OF CLAVICEPS 87 recombinant karyotypes including a high degree of “new” chromosome sizes. this opens the possibility to assign cloned genes or specific RFLP markers to single chromosomes.2. In the group of Schardl (Tsai et al. 1989).

1994). purpurea pyr4 gene (see above) was used to transform OMPD-deficient mutants of strain T5 to prototrophy. Van Engelenburg et al. Even better transformation rates were achieved using the homologous promoter of the C.. the transformation rate was poor (~ 1 transformant/µg DNA). . at a rate of more than 400 transformants/ µg DNA (Smit and Tudzynski. (1989) used the bacterial phleomycin resistance gene (phleoR) driven by the A.88 PAUL TUDZYNSKI morphology (sclerotial growth) typical for alkaloid producing cultures. which was efficiently expressed during the parasitic cycle of C. purpurea gpd gene: when an DNA fragment spanning 1. up to 300 transformants/µg DNA were obtained (Jungehülsing et al. tryptophan induction.4 kb of the non-coding upstream region of this gene was fused to the phleoR gene. In addition. purpurea have been characterized which might be involved in the last step of the pathway (Panaccione.. purpurea. (3) The identification of genes which might be correlated with the different hyphal morphology of producing cultures could lead to an understanding of the relation between this switch in morphology and induction of alkaloid synthesis. purpurea on rye: honeydew produced by plants infected with pyr4/uidA co-transformants was shown to contain significant levels of ß-glucuronidase activity (Smit and Tudzynski. and the prokaryotic hygromycin B-resistance gene unter the control of the A. The phleomycin selection system was further improved in our group by modifications of the selection process and by the use of stronger promoters. 1988). These preliminary data open up interesting new perspectives for the molecular analyses of ergot alkaloid biosynthesis: (1) the identification of the DMATS gene in this differential cDNA screening proves that at least this gene is regulated at the transcriptional level. 4.V. part of The Gordon and Breach Publishing Group.3. in which the pbleoR gene is controlled by the A. (1989). nidulans gpd promoter.2. Copyright © 1999 OPA (Overseas Publishers Association) N. (2) probably this approach will allow the identification of further genes of this pathway. nidulans. purpurea. a highly efficient homologous transformation system was developed based on the complementation of auxotrophic recipient strains. These data show that co-transformation is effective in C. The C. and the integration modus was complex (multicopy/multisite integration). The pyr4 transformation system also proved to be very efficient in co-transformation experiments using the bacterial ß-glucuronidase gene (uidA) as a reporter gene. Comparable results were obtained by Comino et al. 1992). This allows a detailed analysis of the different regulatory circuits influencing alkaloid biosynthesis (phosphate and nitrate regulation. Published by license under the Harwood Academic Publishers imprint.). and that transformation technology can be efficiently used to monitor the fungal development in planta. Recently also parts of a cyclic peptidase from C. Good results were obtained with vector pAN 8–1 (Mattern et al. 1992). etc. 1996). who used two other selection systems: the acetamidase (amds) gene of A. Transformation Several transformation systems have been developed for C. nidulans gpd promoter. nidulans trpC promoter for the first successfull transformation experiments.

4. unpubl. leading to gene inactivation. confirming the usefulness of this method (Voß. Nevertheless. can be re-ligated.V. Though transposons occur in fungi (Oliver. The available molecular techniques should allow in the near future the cloning of important genes of the Copyright © 1999 OPA (Overseas Publishers Association) N. Stehling and Tudzynski. Three of 178 transformants obtained showed gene replacement at the cel1 locus. using the phleoR expression cassette (see above) flanked by the 5´ part of cell and the downstream non-coding region. The transformation assay includes significant amounts of a restriction enzyme. purpurea as in other fungi. The method mimics the transposon tagging approach in bacteria and plants. purpurea. leading to a random integration of vector DNA at these sites. 4 of about 500 tested mutants were auxotrophic. targeted gene inactivation by transformation is possible: Müller (1996) inactivated the cel1 gene of strain T5 by a gene replacement approach. The genomic DNA of transformants having a desired mutant phenotype is digested with an restriction enzyme having no site within the vector DNA. and sequenced. they could also be efficient tools for biotechnological purposes (see below). so far no transposon tagging method could be developed for hyphal fungi. 1992). Published by license under the Harwood Academic Publishers imprint. part of The Gordon and Breach Publishing Group. purpurea. REMI involves the use of a transforming vector without major homology to the host’s genome. the so-called REMI (restriction enzyme mediated integration) technique (Kuspa and Loomis. . recovered after transformation in E.). REMI is a transformation-based approach which leads to mutants tagged by an integrated plasmid. PERSPECTIVES The combination of classical and molecular genetic techniques opens interesting new perspectives of the analysis of ergot alkaloid biosynthesis in Claviceps and for the development of strain improvement programs. Gene deletions of this type allow a functional analysis of genes which are suspected to be pathogenicity factors (see also Chapter 2 by Tenberge. 1992). this volume). ectopic (non-homologous) integration is frequent in C. purpurea recently a REMI library of more than 1000 transformants has been established and so far 10 pathogenicity mutants have been identified.GENETICS OF CLAVICEPS 89 Homologous integration of transforming DNA in the genome obviously is a rare event in C. which randomly cuts the genomic DNA. Comparable results were obtained by Giesbert (in press) using the cpxyl1 gene of C. in addition. then the integrated vector plus parts of the tagged gene is excised.3. coli. Recently another promising application for transformation technology has been developed for filamentous fungi. As outlined above. Smit and Tudzynski (1992) found a rate of ~10% at the pyr4 locus. In C. The link between this “tag” and the observed mutant phenotype can be confirmed by using the rescued vector in knock out experiments with the recipient strain or by genetic linkage analysis. also this gene was successfully inactivated by a gene replacement approach.

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