Birgitt Oeser

Polygalacturonase is a pathogenicity factor in the Claviceps purpurea/rye interaction

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 linear mitochondrial plasmid pClK1 of the phytopathogenic fungus Claviceps purpurea may code for a DNA polymerase and an RNA polymerase.

SummaryPlasmid pClK1, a linear mitochondrial plasmid of Claviceps purpurea, was completely sequenced. The sequence contains two long open reading frames (ORF1, 3291 bp; ORF2, 2910 bp), and at least four smaller ORFs. The potential polypeptide derived from ORF1 shows homology to the family B type DNA polymerases. The product of ORF2 has significant homology to the mitochondrial RNA polymerase of yeast and RNA polymerases from bacteriophages. ORF1 and ORF2 show homology to URF3 and URF1 of the maize plasmids S1 and S2, respectively. No homology to any published protein sequence was found for the smaller ORFs. The origin of the terminal protein attached to the 5′ ends of pClK1 remains open; several alternatives for its origin are discussed. The sequence data as a whole confirm the virus-like character of pClK1 already postulated from structural properties. Thus pClK1 together with S plasmids of maize and several other linear plasmids make up a distinct class of DNA species of plants and fungi probably derived from a common virus-like ancestor.

CPTF1, a CREB-like transcription factor, is involved in the oxidative stress response in the phytopathogen Claviceps purpurea and modulates ROS level in its host Secale cereale.

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 delta cptf1 mutant. Biochemical analyses indicated that CPTF1 is a general regulator of catalase activity. Delta cptf1 mutants showed significantly reduced virulence on rye. Electron microscopical in situ localization revealed significant amounts of H2O2 in delta cptf1-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 plants defense reactions.

BcAtf1, a global regulator, controls various differentiation processes and phytotoxin production in Botrytis cinerea

Atf1-homologous basic region leucine zipper (bZIP) transcription factors are known to act downstream of the stress-activated mitogen-activated protein kinase (SAPK) cascade in mammals, as well as in several fungi; they regulate the transcription of genes involved in the general stress response. Functional analyses of BcAtf1 in Botrytis cinerea show that it is also connected to the SAPK BcSak1, as it shares several stress response target genes. However, Δbcatf1 mutants are not hypersensitive to osmotic or oxidative stress, as are Δbcsak1 mutants. Both BcSak1 and BcAtf1 are regulators of differentiation, but their roles in these processes are almost inverse as, in contrast with Δbcsak1, Δbcatf1 mutants are significantly impaired in conidia production and do not differentiate any sclerotia. They show extremely vigorous growth in axenic culture, with a thick layer of aerial hyphae and a marked increase in colonization efficiency on different host plants and tissues. In addition, the sensitivity to cell wall-interfering agents is increased strongly. Microarray analyses demonstrate that the loss of BcAtf1 leads to extensive transcriptional changes: apart from stress response genes, the expression of a broad set of genes, probably involved in primary metabolism, cell wall synthesis and development, is affected by BcAtf1. Unexpectedly, BcAtf1 also controls secondary metabolism: the mutant contains significantly elevated levels of phytotoxins. These data indicate that BcAtf1 controls a diversity of cellular processes and has broad regulatory functions.

Extrachromosomal genetics of Cephalosporium acremonium

SummaryA hybrid vector carrying a 1.9 kb ars from the mitochondrial DNA (mtDNA) of Cephalosporium acremonium was shown to be relatively stable in yeast even without selective pressure. Subcloning of parts of this 1.9 kb fragment indicated that ars activity (i.e., high transformation rate) is associated with a 675 bp HinfI-fragment. Sequence analysis of the ars-subfragment revealed several ars-typical features, a long open reading frame and, most interestingly, homology to a mitochondrial origin of replication.

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.

De novo biosynthesis of cytokinins in the biotrophic fungus Claviceps purpurea

Disease symptoms of some phytopathogenic fungi are associated with changes in cytokinin (CK) levels. Here, we show that the CK profile of ergot-infected rye plants is also altered, although no pronounced changes occur in the expression of the host plants CK biosynthesis genes. Instead, we demonstrate a clearly different mechanism: we report on the first fungal de novo CK biosynthesis genes, prove their functions and constitute a biosynthetic pathway. The ergot fungus Claviceps purpurea produces substantial quantities of CKs in culture and, like plants, expresses enzymes containing the isopentenyltransferase and lonely guy domains necessary for de novo isopentenyladenine production. Uniquely, two of these domains are combined in one bifunctional enzyme, CpIPT-LOG, depicting a novel and potent mechanism for CK production. The fungus also forms trans-zeatin, a reaction catalysed by a CK-specific cytochrome P450 monooxygenase, which is encoded by cpp450 forming a small cluster with cpipt-log. Deletion of cpipt-log and cpp450 did not affect virulence of the fungus, but Δcpp450 mutants exhibit a hyper-sporulating phenotype, implying that CKs are environmental factors influencing fungal development.

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

Homology of two linear, mitochondrial (mt) Claviceps purpurea plasmids, pC1K1 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.

Structural and functional analysis of an oligomeric hydrophobin gene from Claviceps purpurea

SUMMARY Fungal hydrophobins are small hydrophobic proteins containing eight cysteine residues at conserved positions which have the ability to form amphipathic polymers. We have characterized a gene from the phytopathogenic ascomycete Claviceps purpurea, cpph1, which encodes a modular-type hydrophobin. It consists of five units, each showing a significant homology to class II hydrophobins. The units are separated by GN-repeat regions, which could form amphipathic alpha-helices; the amino terminus contains a glycine-rich region which could be involved in attaching the protein to the cell wall. The presence of long direct repeats within cpph1, and the high homology of the three internal modules suggest a recent generation of this gene from a tripartite precursor. Although sequencing of cDNA clones indicated that recombination could be mediated via the direct repeats, the majority of the transcripts appear to be full-sized. This was confirmed by Northern blot analysis, which showed the presence of a full-sized transcript in axenic culture. The high molecular weight pentahydrophobin was detected by Western blot analysis, indicating that CPPH1 is not processed into monomeric subunits. Targeted deletion of cpph1 did not lead to differences in morphology, growth rate, sporulation, or hydrophobicity of spores. Furthermore, the cpph1 deletion mutants showed no reduction in virulence on rye.

Expressed sequence tags from the flower pathogen Claviceps purpurea

SUMMARY The ascomycete Claviceps purpurea (ergot) is a biotrophic flower pathogen of rye and other grasses. The deleterious toxic effects of infected rye seeds on humans and grazing animals have been known since the Middle Ages. To gain further insight into the molecular basis of this disease, we generated about 10 000 expressed sequence tags (ESTs)-about 25% originating from axenic fungal culture and about 75% from tissues collected 6-20 days after infection of rye spikes. The pattern of axenic vs. in planta gene expression was compared. About 200 putative plant genes were identified within the in planta library. A high percentage of these were predicted to function in plant defence against the ergot fungus and other pathogens, for example pathogenesis-related proteins. Potential fungal pathogenicity and virulence genes were found via comparison with the pathogen-host interaction database (PHI-base; http://www.phi-base.org) and with genes known to be highly expressed in the haustoria of the bean rust fungus. Comparative analysis of Claviceps and two other fungal flower pathogens (necrotrophic Fusarium graminearum and biotrophic Ustilago maydis) highlighted similarities and differences in their lifestyles, for example all three fungi have signalling components and cell wall-degrading enzymes in their arsenal. In summary, the analysis of axenic and in planta ESTs yielded a collection of candidate genes to be evaluated for functional roles in this plant-microbe interaction.