Cross-talk of the biotrophic pathogen Claviceps purpurea and its host Secale cereale.

BACKGROUND: The economically important Ergot fungus Claviceps purpurea is an interesting biotrophic model system because of its strict organ specificity (grass ovaries) and the lack of any detectable plant defense reactions. Though several virulence factors were identified, the exact infection mechanisms are unknown, e.g. how the fungus masks its attack and if the host detects the infection at all. RESULTS: We present a first dual transcriptome analysis using an RNA-Seq approach. We studied both, fungal and plant gene expression in young ovaries infected by the wild-type and two virulence-attenuated mutants. We can show that the plant recognizes the fungus, since defense related genes are upregulated, especially several phytohormone genes. We present a survey of in planta expressed fungal genes, among them several confirmed virulence genes. Interestingly, the set of most highly expressed genes includes a high proportion of genes encoding putative effectors, small secreted proteins which might be involved in masking the fungal attack or interfering with host defense reactions. As known from several other phytopathogens, the C. purpurea genome contains more than 400 of such genes, many of them clustered and probably highly redundant. Since the lack of effective defense reactions in spite of recognition of the fungus could very well be achieved by effectors, we started a functional analysis of some of the most highly expressed candidates. However, the redundancy of the system made the identification of a drastic effect of a single gene most unlikely. We can show that at least one candidate accumulates in the plant apoplast. Deletion of some candidates led to a reduced virulence of C. purpurea on rye, indicating a role of the respective proteins during the infection process. CONCLUSIONS: We show for the first time that- despite the absence of effective plant defense reactions- the biotrophic pathogen C. purpurea is detected by its host. This points to a role of effectors in modulation of the effective plant response. Indeed, several putative effector genes are among the highest expressed genes in planta.

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 plant's 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.

Plant-symbiotic fungi as chemical engineers: multi-genome analysis of the clavicipitaceae reveals dynamics of alkaloid loci.

The fungal family Clavicipitaceae includes plant symbionts and parasites that produce several psychoactive and bioprotective alkaloids. The family includes grass symbionts in the epichloae clade (Epichloë and Neotyphodium species), which are extraordinarily diverse both in their host interactions and in their alkaloid profiles. Epichloae produce alkaloids of four distinct classes, all of which deter insects, and some-including the infamous ergot alkaloids-have potent effects on mammals. The exceptional chemotypic diversity of the epichloae may relate to their broad range of host interactions, whereby some are pathogenic and contagious, others are mutualistic and vertically transmitted (seed-borne), and still others vary in pathogenic or mutualistic behavior. We profiled the alkaloids and sequenced the genomes of 10 epichloae, three ergot fungi (Claviceps species), a morning-glory symbiont (Periglandula ipomoeae), and a bamboo pathogen (Aciculosporium take), and compared the gene clusters for four classes of alkaloids. Results indicated a strong tendency for alkaloid loci to have conserved cores that specify the skeleton structures and peripheral genes that determine chemical variations that are known to affect their pharmacological specificities. Generally, gene locations in cluster peripheries positioned them near to transposon-derived, AT-rich repeat blocks, which were probably involved in gene losses, duplications, and neofunctionalizations. The alkaloid loci in the epichloae had unusual structures riddled with large, complex, and dynamic repeat blocks. This feature was not reflective of overall differences in repeat contents in the genomes, nor was it characteristic of most other specialized metabolism loci. The organization and dynamics of alkaloid loci and abundant repeat blocks in the epichloae suggested that these fungi are under selection for alkaloid diversification. We suggest that such selection is related to the variable life histories of the epichloae, their protective roles as symbionts, and their associations with the highly speciose and ecologically diverse cool-season grasses.

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.

Identification and functional characterization of indole-3-acetamide-mediated IAA biosynthesis in plant-associated Fusarium species.

The plant hormone indole-3-acetic acid (IAA) can be synthesized from tryptophan via the intermediate indole-3-acetamide (IAM). The two genes, IaaM (encoding tryptophan monooxygenase) and IaaH (encoding indole-3-acetamide hydrolase) that constitute the IAM pathway have been described in plant-associated bacteria. We have identified putative homologs of the bacterial IaaM and IaaH genes in four Fusarium species -Fusarium proliferatum, Fusarium verticillioides, Fusarium fujikuroi, and Fusarium oxysporum. In all four species the two genes are organized next to each other in a head to head orientation and are separated by a short non-coding region. However, the pathway is fully functional only in the orchid endophytic strain F. proliferatum ET1, which produces significant amounts of IAM and IAA. Minor amounts of IAM are produced by the corn pathogen F. verticillioides strain 149, while in the two other species, the rice pathogen F. fujikuroi strain m567 and the tomato pathogen F. oxysporum f. sp. lycopersici strain 42-87 the IAM pathway is inactive. Deletion of the entire gene locus in F. proliferatum ET1 resulted in drastic reduction of IAA production. Conversely, transgenic strains of F. fujikuroi over-expressing the F. proliferatum IAM genes produced elevated levels of both IAM and IAA. Analysis of the intergenic promoter region in F. proliferatum showed that transcriptional activation in direction of the IaaH gene is about 3-fold stronger than in direction of the IaaM gene. The regulation of the IAM genes and the limiting factors of IAA production via the IAM pathway are discussed.

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.

Cross-species hybridization with Fusarium verticillioides microarrays reveals new insights into Fusarium fujikuroi nitrogen regulation and the role of AreA and NMR.

In filamentous fungi, the GATA-type transcription factor AreA plays a major role in the transcriptional activation of genes needed to utilize poor nitrogen sources. In Fusarium fujikuroi, AreA also controls genes involved in the biosynthesis of gibberellins, a family of diterpenoid plant hormones. To identify more genes responding to nitrogen limitation or sufficiency in an AreA-dependent or -independent manner, we examined changes in gene expression of F. fujikuroi wild-type and DeltaareA strains by use of a Fusarium verticillioides microarray representing approximately 9,300 genes. Analysis of the array data revealed sets of genes significantly down- and upregulated in the areA mutant under both N starvation and N-sufficient conditions. Among the downregulated genes are those involved in nitrogen metabolism, e.g., those encoding glutamine synthetase and nitrogen permeases, but also those involved in secondary metabolism. Besides AreA-dependent genes, we found an even larger set of genes responding to N starvation and N-sufficient conditions in an AreA-independent manner. To study the impact of NMR on AreA activity, we examined the expression of several AreA target genes in the wild type and in areA and nmr deletion and overexpression mutants. We show that NMR interacts with AreA as expected but affects gene expression only in early growth stages. This is the first report on genome-wide expression studies examining the influence of AreA on nitrogen-responsive gene expression in a genome-wide manner in filamentous fungi.

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 plant's defense reactions.

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.

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.