The fungal pathogen Cochliobolus heterostrophus responds to maize phenolics: novel small molecule signals in a plant-fungal interaction.

The transcription factor ChAP1 of the fungal pathogen of maize, Cochliobolus heterostrophus, responds to oxidative stress by migration to the nucleus and activation of antioxidant genes. Phenolic and related compounds found naturally in the host also trigger nuclear localization of ChAP1, but only slight upregulation of some antioxidant genes. ChAP1 thus senses phenolic compounds without triggering a strong antioxidant response. We therefore searched for genes whose expression is regulated by phenolic compounds and/or ChAP1. The C. heterostrophus genome contains a cluster of genes for metabolism of phenolics. One such gene, catechol dioxygenase CCHD1, was induced at least 10-fold by caffeic and coumaric acids. At high phenolic concentrations (≥ 1.6 mM), ChAP1 is needed for maximum CCHD1 expression. At micromolar levels of phenolics CCHD1 is as strongly induced in chap1 mutants as in the wild type. The pathogen thus detects phenolics by at least two signalling pathways: one causing nuclear retention of ChAP1, and another triggering induction of CCHD1 expression. The low concentrations required for induction of CCHD1 indicate fungal receptors for plant phenolics. Symbiotic and pathogenic bacteria are known to detect phenolics, and our findings generalize this to a eukaryotic pathogen. Phenolics and related compounds thus provide a ubiquitous plant-derived signal.

Distinct and combined roles of the MAP kinases of Cochliobolus heterostrophus in virulence and stress responses.

Pathogenicity mitogen-activated protein kinases (MAPKs), related to yeast FUS3/KSS1, are essential for virulence in fungi, including Cochliobolus heterostrophus, a necrotrophic pathogen causing Southern corn leaf blight. We compared the phenotypes of mutants in three MAPK genes: HOG1, MPS1, and CHK1. The chk1 and mps1 mutants show autolytic appearance, light pigmentation, and dramatic reduction in virulence and conidiation. Similarity of mps1 and chk1 mutants is reflected by coregulation by these two MAPKs of several genes. Unlike chk1, mps1 mutants are female-fertile and form normal-looking appressoria. HOG1 mediates resistance to hyperosmotic and, to a lesser extent, oxidative stress, and is required for stress upregulation of glycerol-3-phosphate phosphatase, transaldolase, and a monosaccharide transporter. Hog1, but not Mps1 or Chk1, was rapidly phosphorylated in response to increased osmolarity. The hog1 mutants have smaller appressoria and cause decreased disease symptoms on maize leaves. Surprisingly, loss of MPS1 in a wild-type or hog1 background improved resistance to some stresses. All three MAPKs contribute to the regulation of central developmental functions under normal and stress conditions, and full virulence cannot be achieved without appropriate input from all three pathways.

MRSP1, encoding a novel Trichoderma secreted protein, is negatively regulated by MAPK.

A novel gene, MRSP1 (MAP kinase repressed secreted protein 1) is strongly overexpressed in the tmkA MAPK mutant of the biocontrol fungus Trichoderma virens. Transcriptional regulation of MRSP1 is determined by presence or absence of TmkA, rather than by light or sporulation, making it a molecular marker for the unusual, negative, regulation by TmkA. The predicted protein is 15.9 kDa, has a secretory signal, and the four-cysteine pattern, C-X29-CP(G)C-X31-C, may define a new cysteine-rich motif. This is a novel protein with functions not known from any other organism. Conservation in ascomycete, basidiomycete, and Dictyostelium homologs, as well as tight MAPK regulation, might indicate important cellular functions.

A secondary metabolite biosynthesis cluster in Trichoderma virens: evidence from analysis of genes underexpressed in a mutant defective in morphogenesis and antibiotic production.

A transcriptional comparison of wild type and a secondary metabolite deficient Trichoderma virens mutant resulted in the identification of six genes similar to those involved in secondary metabolism in other fungi, including four cytochrome P450 genes, one O-methyl transferase and one terpene cylase. Four of the genes (three cytochrome P450s and the cyclase) are located as a cluster. Transcript levels of three of the P450 genes, the O-methyl transferase and the terpene cyclase were measured. These genes are underexpressed in the mutant, which lacks the major secondary metabolites produced by this strain, viridin and viridiol. Expression levels of clones from the differential library with similarity to fungal trehalose synthase and a hydrophobin were also underexpressed in the mutant, while a heat shock protein hsp98 homolog was not. Based on the gene expression pattern and associated secondary metabolite profile, along with similarity to other secondary metabolism pathways in related fungi, we predict that the cluster is associated with the production of a terpene. The terpene could be viridin. This is the first report on cloning of secondary metabolism related genes from T. virens, and of their organization in a cluster, in this biocontrol fungus.

Activation of an AP1-like transcription factor of the maize pathogen Cochliobolus heterostrophus in response to oxidative stress and plant signals.

Redox sensing is a ubiquitous mechanism regulating cellular activity. Fungal pathogens face reactive oxygen species produced by the host plant's oxidative burst in addition to endogenous reactive oxygen species produced during aerobic metabolism. An array of preformed and induced detoxifying enzymes, including superoxide dismutase, catalases, and peroxidases, could allow fungi to infect plants despite the oxidative burst. We isolated a gene (CHAP1) encoding a redox-regulated transcription factor in Cochliobolus heterostrophus, a fungal pathogen of maize. CHAP1 is a bZIP protein that possesses two cysteine-rich domains structurally and functionally related to Saccharomyces cerevisiae YAP1. Deletion of CHAP1 in C. heterostrophus resulted in decreased resistance to oxidative stress caused by hydrogen peroxide and menadione, but the virulence of chap1 mutants was unaffected. Upon activation by oxidizing agents or plant signals, a green fluorescent protein (GFP)-CHAP1 fusion protein became localized in the nucleus. Expression of genes encoding antioxidant proteins was induced in the wild type but not in chap1 mutants. Activation of CHAP1 occurred from the earliest stage of plant infection, in conidial germ tubes on the leaf surface, and persisted during infection. Late in the course of infection, after extensive necrotic lesions were formed, GFP-CHAP1 redistributed to the cytosol in hyphae growing on the leaf surface. Localization of CHAP1 to the nucleus may, through changes in the redox state of the cell, provide a mechanism linking extracellular cues to transcriptional regulation during the plant-pathogen interaction.

G-protein beta subunit of Cochliobolus heterostrophus involved in virulence, asexual and sexual reproductive ability, and morphogenesis.

Previous work established that mutations in mitogen-activated protein (MAP) kinase (CHK1) and heterotrimeric G-protein alpha (Galpha) subunit (CGA1) genes affect the development of several stages of the life cycle of the maize pathogen Cochliobolus heterostrophus. The effects of mutating a third signal transduction pathway gene, CGB1, encoding the Gbeta subunit, are reported here. CGB1 is the sole Gbeta subunit-encoding gene in the genome of this organism. cgb1 mutants are nearly wild type in vegetative growth rate; however, Cgb1 is required for appressorium formation, female fertility, conidiation, regulation of hyphal pigmentation, and wild-type virulence on maize. Young hyphae of cgb1 mutants grow in a straight path, in contrast to those of the wild type, which grow in a wavy pattern. Some of the phenotypes conferred by mutations in CGA1 are found in cgb1 mutants, suggesting that Cgb1 functions in a heterotrimeric G protein; however, there are also differences. In contrast to the deletion of CGA1, the loss of CGB1 is not lethal for ascospores, evidence that there is a Gbeta subunit-independent signaling role for Cga1 in mating. Furthermore, not all of the phenotypes conferred by mutations in the MAP kinase CHK1 gene are found in cgb1 mutants, implying that the Gbeta heterodimer is not the only conduit for signals to the MAP kinase CHK1 module. The additional phenotypes of cgb1 mutants, including severe loss of virulence on maize and of the ability to produce conidia, are consistent with CGB1 being unique in the genome. Fluorescent DNA staining showed that there is often nuclear degradation in mature hyphae of cgb1 mutants, while comparable wild-type cells have intact nuclei. These data may be genetic evidence for a novel cell death-related function of the Gbeta subunit in filamentous fungi.

Host physiology and pathogenic variation of Cochliobolus heterostrophus strains with mutations in the G protein alpha subunit, CGA1.

Conserved eukaryotic signaling proteins participate in development and disease in plant-pathogenic fungi. Strains with mutations in CGA1, a heterotrimeric G protein G alpha subunit gene of the maize pathogen Cochliobolus heterostrophus, are defective in several developmental pathways. Conidia from CGA1 mutants germinate as abnormal, straight-growing germ tubes that form few appressoria, and the mutants are female sterile. Nevertheless, these mutants can cause normal lesions on plants, unlike other filamentous fungal plant pathogens in which functional homologues of CGA1 are required for full virulence. Deltacga1 mutants of C. heterostrophus were less infective of several maize varieties under most conditions, but not all, as virulence was nearly normal on detached leaves. This difference could be related to the rapid senescence of detached leaves, since delaying senescence with cytokinin also had differential effects on the virulence of the wild type and the Deltacga1 mutant. In particular, detached leaves may provide a more readily available nutrient source than attached leaves. Decreased fitness of Deltacga1 as a pathogen may reflect conditions under which full virulence requires signal transduction through CGA1-mediated pathways. The virulence of these signal transduction mutants is thus affected differentially by the physiological state of the host.

Green fluorescent protein (GFP) as a vital marker for pathogenic development of the dermatophyte Trichophyton mentagrophytes.

Skin infections by dermatophytes of the genus Trichophyton are widespread, but methods to investigate the molecular basis of pathogenicity are only starting to be developed. The initial stages of growth on the host can only be studied by electron microscopy, which requires fixing the tissue. This paper shows that restriction-enzyme-mediated integration (REMI) provides stable expression of the green fluorescent protein (GFP) in a clinical isolate of Trichophyton mentagrophytes. Under control of a constitutively active fungal promoter, GFP renders the hyphae fluorescent both in culture and in a recently developed model using human skin explants. Stages of infection and penetration into the skin layers were visualized by confocal microscopy. The stages of infection can thus be followed using GFP as a vital marker, and this method will also provide, for the first time, a means to follow gene expression during infection of skin by dermatophyte fungi.

Role of two G-protein alpha subunits, TgaA and TgaB, in the antagonism of plant pathogens by Trichoderma virens.

G-protein alpha subunits are involved in transmission of signals for development, pathogenicity, and secondary metabolism in plant pathogenic and saprophytic fungi. We cloned two G-protein alpha subunit genes, tgaA and tgaB, from the biocontrol fungus Trichoderma virens. tgaA belongs to the fungal Galphai class, while tgaB belongs to the class defined by gna-2 of Neurospora crassa. We compared loss-of-function mutants of tgaA and tgaB with the wild type for radial growth, conidiation, germination of conidia, the ability to overgrow colonies of Rhizoctonia solani and Sclerotium rolfsii in confrontation assays, and the ability to colonize the sclerotia of these pathogens in soil. Both mutants grew as well as the wild type, sporulated normally, did not sporulate in the dark, and responded to blue light by forming a conidial ring. The tgaA mutants germinated by straight unbranched germ tubes, while tgaB mutants, like the wild type, germinated by wavy and highly branched germ tubes. In confrontation assays, both tgaA and tgaB mutants and the wild type overgrew, coiled, and lysed the mycelia of R. solani, but tgaA mutants had reduced ability to colonize S. rolfsii colonies. In the soil plate assay, both mutants parasitized the sclerotia of R. solani, but tgaA mutants were unable to parasitize the sclerotia of S. rolfsii. Thus, tgaA is involved in antagonism against S. rolfsii, but neither G protein subunit is involved in antagonism against R. solani. T. virens, which has a wide host range, thus employs a G-protein pathway in a host-specific manner.

TmkA, a mitogen-activated protein kinase of Trichoderma virens, is involved in biocontrol properties and repression of conidiation in the dark.

Trichoderma virens is a mycoparasitic fungus used in biocontrol of soilborne plant pathogens. It inhibits or kills plant-pathogenic fungi through production of antifungal antibiotics and parasitism of hyphae and sclerotia. Conidiation, or the production of asexual spores, an inducible process triggered by light or nutrient stress, is an important trait in survival and also development of formulation products. In many fungi, signaling pathways, including mitogen-activated protein kinase (MAPK) cascades, have been implicated in parasitism of host plants as well as in the production of asexual spores. Here, we have studied the role of a MAPK gene, that for TmkA, in conidiation and antagonistic properties of a biocontrol strain of T. virens. Through single- and double-crossover recombination, we obtained three tmkA loss-of-function mutants. The TmkA transcript was not detectable in these mutants. The mutants conidiated in the dark, although photoinduction was normal and the light sensitivities of the wild type and the mutant were the same. The mutants had, overall, normal colony morphology, but their radial growth rate was reduced by about 16%, with no decrease in biomass production. Against Rhizoctonia solani hyphae, the knockout mutants exhibited mycoparasitic coiling and lysis of host hyphae similar to that of the wild type. The mutants, however, were less effective in colonizing the sclerotia of R. solani. On Sclerotium rolfsii, the MAPK loss-of-function mutants had reduced antagonistic properties in confrontation assays and failed to parasitize the sclerotia. TmkA-dependent and -independent pathways are thus involved in antagonism against different hosts. Finally, in contrast to the case for other filamentous fungi studied so far, signaling through a MAPK represses, rather than induces, asexual sporulation.