Identification of genes involved in fungal responses to strigolactones using mutants from fungal pathogens.

Strigolactones (SLs) as components of root exudates induce hyphal branching of arbuscular mycorrhizal (AM) fungi which is thought to favor the establishment of the beneficial symbiosis. Little is known on how AM fungi respond to SLs. Since AM fungi are poor model systems due to their obligate biotrophism and the lack of genetic transformation protocols, we took advantage of the sensitivity of several phytopathogenic fungi to GR24, a synthetic SLs analog. With the aim to identify the molecular determinants involved in SLs response in AM fungi and assuming conserved mechanisms in the fungal kingdom, we exploited the fungal pathogens Botrytis cinerea and Cryphonectria parasitica, for which mutant collections are available. Exposure of B. cinerea and C. parasitica to GR24 embedded in solid medium led to reduction of fungal radial growth. We set up the screening of a set of well-characterized gene deletion mutants to isolate genotypes with altered responses to SLs. Two B. cinerea mutants (defective of BcTrr1, a thioredoxin reductase and BcLTF1, a GATA transcription factor) turned out to be less responsive to GR24. One feature shared by the two mutants is the overproduction of reactive oxygen species (ROS). Indeed, an oxidizing effect was observed in a B. cinerea strain expressing a redox-sensitive GFP2 in the mitochondrial intermembrane space upon exposure to GR24. ROS and mitochondria are, therefore, emerging as mediators of SLs actions.

Identification of pathogenesis-associated genes by T-DNA-mediated insertional mutagenesis in Botrytis cinerea: a type 2A phosphoprotein phosphatase and an SPT3 transcription factor have significant impact on virulence.

Agrobacterium tumefaciens-mediated transformation (ATMT) was used to generate an insertional mutant library of the gray mold fungus Botrytis cinerea. From a total of 2,367 transformants, 68 mutants showing significant reduction in virulence on tomato and bean plants were analyzed in detail. As reported for other fungal ATMT libraries, integrations were mostly single copy, occurred preferentially in noncoding (regulatory) regions, and were frequently accompanied by small deletions of the target sequences and loss of parts of the border sequence. Two T-DNA integration events that were found to be linked to virulence were characterized in more detail: a catalytic subunit of a PP2A serine/threonine protein phosphatase (BcPP2Ac) and the SPT3 subunit of a Spt-Ada-Gcn5-acetyltransferase (SAGA-like) transcriptional regulator complex. Gene replacement and silencing approaches revealed that both Bcpp2Ac and SPT3 are crucial for virulence, growth, and differentiation as well as for resistance to H(2)O(2) in B. cinerea.

The biotrophic, non-appressorium-forming grass pathogen Claviceps purpurea needs a Fus3/Pmk1 homologous mitogen-activated protein kinase for colonization of rye ovarian tissue.

Claviceps purpurea is a common pathogen of a wide range of grasses and cereals that is able to establish a stable, balanced interaction with its host plant and is considered a biotroph. It does not form special penetration structures such as appressoria. To study the signaling processes involved in this special host-pathogen interaction, we have cloned a gene, cpmk1, encoding a mitogen-activated protein (MAP) kinase that shows significant homology to Fus3 of Saccharomyces cerevisiae and to pmk1 of Magnaporthe grisea. Using a gene-replacement approach, we isolated a Acpmk1 mutant and characterized it in detail. Loss of CPMK1 has no obvious effect on vegetative properties (such as growth rate, morphology, and conidia formation); however, infection tests on rye show that the mutant is unable to colonize rye tissue, i.e., it appears to be completely nonpathogenic. Complementation of the mutant with a wild-type copy of cpmk1 fully restores its pathogenicity, confirming that this MAP kinase is essential for infection of rye by C. purpurea. Transformation of the delta pmk1 mutant of M. grisea with a complete copy of cpmk1 (including the C. purpurea promoter) fully restored its ability to form appressoria and its pathogenicity on barley. Although both fungi drastically differ in their pathogenic strategies, this result indicates that the signal pathway involving CPMK1 is highly conserved.

Biotechnology and genetics of ergot alkaloids.

Ergot alkaloids, i.e. ergoline-derived toxic metabolites, are produced by a wide range of fungi, predominantly by members of the grass-parasitizing family of the Clavicipitaceae. Naturally occurring alkaloids like the D-lysergic acid amides, produced by the "ergot fungus" Claviceps purpurea, have been used as medicinal agents for a long time. The pharmacological effects of the various ergot alkaloids and their derivatives are due to the structural similarity of the tetracyclic ring system to neurotransmitters such as noradrenaline, dopamine or serotonin. In addition to "classical" indications, e.g. migraine or blood pressure regulation, there is a wide spectrum of potential new applications of this interesting group of compounds. The biotechnology of ergot alkaloids has a long tradition, and efficient parasitic and submerse production processes have been developed; the biochemistry of the pathway and the physiology of production have been worked out in detail. The recent identification of a cluster of genes involved in ergot alkaloid biosynthesis in C. purpurea and the availability of molecular genetic techniques allow the development of strategies for rational drug design of ergoline-related drugs by enzyme engineering and by biocombinatorial approaches.

The role of G protein alpha subunits in the infection process of the gray mold fungus Botrytis cinerea.

To identify signal transduction pathways of the gray mold fungus Botrytis cinerea involved in host infection, we used heterologous hybridization and a polymerase chain reaction (PCR)-based approach to isolate two genes (bcg1 and bcg2) encoding alpha subunits of heterotrimeric GTP-binding proteins. Both genes have homologues in other fungi: bcg1 is a member of the G alpha(i) class, whereas bcg2 has similarities to the magC gene of Magnaporthe grisea and the gna-2 gene of Neurospora crassa. Reverse-transcription (RT)-PCR experiments showed clearly that both genes are expressed at very early stages in infected bean leaves. Gene replacement experiments were performed for both genes. bcg1 null mutants differ in colony morphology from the wild-type strain, do not secrete extracellular proteases, and show clearly reduced pathogenicity on bean and tomato. Conidia germination and penetration of plant tissue is not disturbed in bcg1 mutants, but the infection process stops after formation of primary lesions. In contrast, bcg2 mutants show wild-type colony morphology in axenic culture and are only slightly reduced in pathogenicity. Complementation of bcg1 mutants with the wild-type gene copy led to the full recovery of colony morphology, protease secretion, and pathogenicity on both host plants. Application of exogenous cyclic AMP restored the wild-type growth pattern of bcg1 mutants, but not the protease secretion, implicating an essential role of BCG1 in different signaling pathways.

Identification and characterization of a tri-partite hydrophobin from Claviceps fusiformis. A novel type of class II hydrophobin.

A new type of hydrophobin is encoded by an abundant mRNA of Claviceps fusiformis. The predicted amino-acid sequence of the protein, dubbed CFTH1, shows a putative signal sequence for secretion, followed by three class II hydrophobin domains each preceded by glycine/asparagine rich regions. SDS/PAGE analysis of 60% ethanol extractions of C. fusiformis mycelia from shaken cultures showed CFTH1 at the 50-55-kDa position. N-terminal sequencing of both untreated mature CFTH1 and of a fragment obtained by trypsin digestion revealed that CFTH1 is not processed between the hydrophobin domains. Mass spectroscopy showed a mass of about 36 500 Da, which is about 1500 Da higher than the mass predicted from the constituent amino acids, indicating post-translational modification but not glycosylation. Purified CFTH1 self-assembled at hydrophilic/hydrophobic interfaces and, after assembly at a water/air interface, it was found to be highly surface active. Antibodies raised against CFTH1 localized the protein in a mucilageous coat surrounding submerged vegetative hyphae in liquid shaken culture and, as a discrete layer of about 10 nm thickness at the surface of aerial hyphae of standing cultures, suggesting a role in the formation of aerial hyphae.

Evidence for an ergot alkaloid gene cluster in Claviceps purpurea.

A gene (cpd1) coding for the dimethylallyltryptophan synthase (DMATS) that catalyzes the first specific step in the biosynthesis of ergot alkaloids, was cloned from a strain of Claviceps purpurea that produces alkaloids in axenic culture. The derived gene product (CPD1) shows only 70% similarity to the corresponding gene previously isolated from Claviceps strain ATCC 26245, which is likely to be an isolate of C. fusiformis. Therefore, the related cpd1 most probably represents the first C. purpurea gene coding for an enzymatic step of the alkaloid biosynthetic pathway to be cloned. Analysis of the 3'-flanking region of cpd1 revealed a second, closely linked ergot alkaloid biosynthetic gene named cpps1, which codes for a 356-kDa polypeptide showing significant similarity to fungal modular peptide synthetases. The protein contains three amino acid-activating modules, and in the second module a sequence is found which matches that of an internal peptide (17 amino acids in length) obtained from a tryptic digest of lysergyl peptide synthetase 1 (LPS1) of C. purpurea, thus confirming that cpps1 encodes LPS1. LPS1 activates the three amino acids of the peptide portion of ergot peptide alkaloids during D-lysergyl peptide assembly. Chromosome walking revealed the presence of additional genes upstream of cpd1 which are probably also involved in ergot alkaloid biosynthesis: cpox1 probably codes for an FAD-dependent oxidoreductase (which could represent the chanoclavine cyclase), and a second putative oxidoreductase gene, cpox2, is closely linked to it in inverse orientation. RT-PCR experiments confirm that all four genes are expressed under conditions of peptide alkaloid biosynthesis. These results strongly suggest that at least some genes of ergot alkaloid biosynthesis in C. purpurea are clustered, opening the way for a detailed molecular genetic analysis of the pathway.

Cloning, characterization, and targeted disruption of cpcat1, coding for an in planta secreted catalase of Claviceps purpurea.

Claviceps purpurea has been shown to secrete catalases in axenic and parasitic culture. In order to determine the importance of these enzymes in the host-parasite interaction, especially their role in overcoming oxidative stress imposed on the pathogen by the plant's defense system, the catR gene from A. niger was used to isolate a putative catalase gene from a genomic library of C. purpurea, cpcat1 consists of an open reading frame of 2,148 bp that is interrupted by five introns. Its derived gene product shows significant homology to fungal catalases and contains a putative signal peptide of 19 amino acids and three putative N-glycosylation sites, which indicates that CPCAT1 is a secreted catalase. Disruption of the gene by a gene replacement approach resulted in the loss of two catalase isoforms, CATC and CATD, strongly suggesting that they are both encoded by cpcat1. CATD is the major secreted catalase of C. purpurea and is furthermore the only catalase present in the honeydew of infected rye ears. Deletion mutants of cpcat1 were inoculated on rye plants and showed no significant reduction in virulence. Ovarian tissue and honeydew of plants inoculated with the mutants lacked CATD, confirming that this catalase is not essential for colonization of the host tissue by C. purpurea.

The Xylanolytic System of Claviceps purpurea: Cytological Evidence for Secretion of Xylanases in Infected Rye Tissue and Molecular Characterization of Two Xylanase Genes.

ABSTRACT 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-beta-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 (Deltacpxyl1) had significantly reduced xylanase activity; Western analyses confirmed that it lacked a polypeptide of approximately 23 kDa.

Identification of genes induced in alkaloid-producing cultures of Claviceps sp.

In order to identify genes which are expressed during alkaloid synthesis in an axenic culture of Claviceps sp. (strain ATCC 26245), a cDNA library from a producing culture was differentially screened with cDNA from producing (cDNA+) and non-producing (cDNA-) cultures, respectively. Altogether, ten cDNA clones were obtained, the alkaloid-synthesis-correlated expression of which was confirmed by Northern analyses. Evaluation of their nucleotide and derived amino-acid sequences identified one gene unequivocally, coding for dimethylallyltryptophan-synthase (DMAT-S), the initial enzyme of the specific alkaloid pathway. For two other genes significant homologies to known fungal genes were detected: one clone showed homology to the Neurospora crassa ccg1 gene, coding for a clock-regulated putative general stress protein; seven cDNA clones, derived from the same gene, which is highly expressed under these conditions, contained typical hydrophobin domains and long stretches of asparagine/glycine repeats (like QID3 from Trichoderma harzianum), thus probably representing a cell-wall constituent. These data show that this is not only a successful approach to clone genes specific for the alkaloid-pathway of C. purpurea, but also of genes which might be involved in the differentiation of sclerotial hyphae, the prerequisite for alkaloid synthesis.

Cel1, probably encoding a cellobiohydrolase lacking the substrate binding domain, is expressed in the initial infection phase of Claviceps purpurea on Secale cereale.

At the host-pathogen interface of hyphae penetrating host cell walls in the rye ovary, a lack of cellulase-gold labeling of beta-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 cbh1 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 beta-1, 4-glucan and, therefore, play a role in the infection process.

Variations in ploidy among isolates of Botrytis cinerea: implications for genetic and molecular analyses.

Field isolates and laboratory strains of Botrytis cinerea, an ascomycetous fungus causing considerable economic losses, e.g., as "grey mould" of vine, were compared for differences in ploidy level by determining their DNA content per nucleus. Strain SAS56, an ascospore line used routinely for genetic analyses, is probably polyploid, since treatment with benomyl causes a significant reduction in DNA content per nucleus. This conclusion is substantiated by the increased sensitivity of the putative haploid derivatives to mutagens (UV and EMS). Molecular analyses (RAPD) of the haploidized strains indicate a very limited degree of heterozygosis of the parent strain SAS56. Analysis of field isolates of B. cinerea showed that their DNA content per nucleus varied considerably, indicating that aneuploidy/polyploidy is a widespread phenomenon in this species. This can explain both the variability and phenotypic instability of many field isolates of this fungus and the unusual difficulties faced by researchers in recovering stable recessive laboratory mutants. Since the haploid derivatives of SAS56 resemble the parent strain in their parasitic and physiological properties they should provide a good basis for classical and molecular genetic studies.

The Claviceps purpurea glyceraldehyde-3-phosphate dehydrogenase gene: cloning, characterization, and use for the improvement of a dominant selection system.

The GPD 1 gene of Claviceps purpurea coding for glyceraldehyde-3-phosphate dehydrogenase was cloned and sequenced, including 1,800 bp of its 5' upstream region. This gene shows an identical structure to the gpd gene of Podospora anserina and Cryphonectria parasitica (one intron at an identical position) with high homology at both the DNA and amino-acid levels. Two fragments of the promoter spanning from the ATG to -500 bp and to -1,400 bp were fused to the phleomycin-resistance gene. Both constructs transformed C. purpurea at a high rate. The enhanced expression of the long vector construct indicates the presence of additional elements between -500 bp and -1,400 bp upstream of the initiation codon.

A DNA-polymerase-related reading frame (pol-r) in the mtDNA of Secale cereale.

Mitochondrial (mt)DNA of Secale cereale contains an open reading frame (pol-r), the potential translation product of which shows significant homology to the type-B DNA polymerase encoded by the S1 plasmid of Zea mays; it contains the highly-conserved domains IIa to V of family B polymerases. The pol-r ORF is transcribed, as proven by RT-PCR, but the transcript is not edited. Upstream of the putative start codon a potential promoter motif was detected, fitting well into the postulated consensus sequence of the transcription initiation regions of Z. mays and Triticum aestivum. The pol-r ORF occurs in mtDNA of the fertile rye variety "Halo" and the cytoplasmic male-sterile (CMS) line "Pampa". Both ORFs are almost identical, apart from the 3' terminus; pol-r from Halo can code for 289 amino acids, pol-r from Pampa for 312 amino acids. Based on codon usage and the lack of editing, pol-r is considered to be a "young" gene, probably introduced in the mtDNA of rye by recombination with an mt plasmid.

CMS in rye: comparative RFLP and transcript analyses of mitochondria from fertile and male-sterile plants.

The mitochondrial (mt) genomes of rye (Secale cereale L.) lines with "normal" and cytoplasmic male sterility (CMS) inducing "Pampa" cytoplasm were compared by detailed restriction fragment length polymorphism (RFLP) and Northern analyses. RFLP analyses using several heterologous mt genes as probes revealed considerable differences in the overall structure of the two mt genomes. With cob and atpA, the data indicate intragenic recombination and/or different copy numbers of these genes in the two cytoplasms. In spite of this heterogeneity at DNA level, the transcriptional patterns of nine out of ten mitochondrial genes analysed are unaffected. The exception is in the "Pampa" cytoplasm which contains an additional cob-homologous transcript. Since this transcript is strongly reduced in the presence of restorer genes, it might causally be correlated to the CMS phenotype.

pClK1 and pClT5--two linear mitochondrial plasmids from unrelated Claviceps purpurea strains: a comparison.

pClT5, a linear mitochondrial (mt) plasmid from Claviceps purpurea, strain T5, was sequenced and compared to pClK1, a linear mt plasmid from an unrelated C. purpurea strain. Both plasmids have terminal proteins (TPs) at their inverted terminal repeats (TlR). The TlRs of both plasmids show short conserved sequences, which are probably involved in plasmid transcription and replication. The coding capacity of pClT5 and pClK1 is similar: there are two large ORFs (ORF1 and ORF2) homologous to the DNA and RNA polymerase ORFs of pClK1 and several small hydrophobic ORFs. ORF3 shows homology to a small ORF of the Neurospora crassa mt plasmid maranhar and is transcribed. ORF6 of pClT5 is homologous to ORF4 of pClK1; both are transcribed and are possible candidates for the TP encoding ORF.

Interaction between mitochondrial DNA and mitochondrial plasmids in Claviceps purpurea: analysis of plasmid-homologous sequences upstream of the lrRNA-gene.

Homology of two linear, mitochondrial (mt) Claviceps purpurea plasmids, pClK1 and pClT5, to the upstream region of the large ribosomal RNA gene in the mtDNA of three strains (W3, T5 and K) has been investigated in detail to explore the widespread phenomenon of homology between mt plasmids and mtDNA in C. purpurea. Sequence comparison indicates that recombination between free plasmids and mtDNA is the cause of the observed homology. The process is similar to the integration of the structurally related adenoviruses into the mammalian genome. As in other fungi, palindromic sequences seem to be involved in this mitochondrial recombination process.

Efficient transformation of Claviceps purpurea using pyrimidine auxotrophic mutants: cloning of the OMP decarboxylase gene.

A homologous transformation system was developed for the phytopathogenic fungus Claviceps purpurea. Orotidine-5'-monophosphate decarboxylase (OMPD)-deficient mutants were obtained by UV mutagenesis and selection for resistance against 5-fluoroorotate. These mutants could be complemented well by the corresponding genes of Aspergillus niger (pyrA) and Neurospora crassa (pyr4), yielding significantly higher transformation rates (and lower copy numbers per transformant) than the phleomycin resistance system. The homologous OMPD gene was isolated from a lambda genomic library by heterologous hybridization with the pyr4 gene of N. crassa, identified by complementation of Aspergillus and Claviceps mutants, and used to confirm homologous integration in Claviceps. The pyr transformation system also proved to be very efficient in cotransformation experiments using the bacterial beta-glucuronidase gene (uidA) as a reporter gene, which was also efficiently expressed during the parasitic cycle: honeydew produced by plants infected with pyr/uidA cotransformants was shown to contain significant levels of beta-glucuronidase activity.

Transcripts and translation products of a mitochondrial plasmid of Claviceps purpurea.

Expression of the linear mitochondrial (mt) plasmid pClK1 of strain K of the ascomycete Claviceps purpurea was studied at the RNA and protein level. Using strand-specific probes two major transcripts were detected, corresponding to ORF1 and ORF2 of the plasmid, which most likely code for a DNA-polymerase and an RNA-polymerase, respectively. Primer extension experiments showed that both transcripts start at identical positions from opposite sides within the terminal inverted repeat (TIR). The initiation sequence corresponds to the proposed general mt initiation consensus-sequence. Conserved parts of the putative polymerase ORFs were expressed in an E. coli system and used to prepare antisera. In Western experiments the presence of corresponding proteins was demonstrated in a strain carrying plasmid pClK1, whereas a plasmid-free strain lacked these polypeptides. The sizes of these proteins are in good accordance with the sizes derived from the coding capacity of ORF1 and 2.