pH and Pac1 control development and antifungal activity in Trichoderma harzianum.

In Trichoderma harzianum CECT 2413 external pH regulates essential functions such as growth rate, sporulation, cell and colony morphology, pattern of secreted proteins, gene expression or mycoparasitism-related enzymatic activities. pH regulation is mediated by the transcriptional factor Pac1 (homologous to PacC regulator in other fungi), encoded by pac1 whose expression increases with pH. Two pac1 mutants have been obtained from CECT 2413: P2.32, that possesses an allele of pac1 active at any pH, and R13, that does not express pac1 due to interferent RNA. Cell morphology, sporulation and growth rate are optimal for strain P2.32 at pH 7.5 and for strain R13 at pH 3.0, mimicking alkaline and acidic conditions, respectively. Pac1 regulates some genes involved in antagonism: chit42 chitinase, papA protease, gtt1 glucose permease, and qid74 cell wall protein. Furthermore, Pac1 modulates T. harzianum antifungal activity since wild type and mutants inhibit several phytopathogenic fungal strains at various degrees in different assays.

Microscopic and transcriptome analyses of early colonization of tomato roots by Trichoderma harzianum.

The capacity of the fungus Trichoderma harzianum CECT 2413 to colonize roots and stimulate plant growth was analyzed. Tobacco seedlings (Nicotiana benthamiana) transferred to Petri dishes inoculated with T. harzianum conidia showed increased plant fresh weight (140%) and foliar area (300%), as well as the proliferation of secondary roots (300%) and true leaves (140%). The interaction between strain CECT 2413 and the tomato-root system was also studied during the early stages of root colonization by the fungus. When T. harzianum conidia were inoculated into the liquid medium of hydroponically grown tomato plants (Lycopersicum esculentum), profuse adhesion of hyphae to the plant roots as well as colonization of the root epidermis and cortex were observed. Confocal microscopy of a T. harzianum transformant that expressed the green fluorescent protein (GFP) revealed intercellular hyphal growth and the formation of plant-induced papilla-like hyphal tips. Analysis of the T. harzianum-tomato interaction in soil indicated that the contact between T. harzianum and the roots persisted over a long period of time. This interaction was characterized by the presence of yeast-like cells, a novel and previously undescribed developmental change. To study the molecular mechanism underlying fungal ability to colonize the tomato-root system, the T. harzianum transcriptome was analyzed during the early stages of the plant-fungus interaction. The expression of fungal genes related to redox reactions, lipid metabolism, detoxification, and sugar or amino-acid transport increased when T. harzianum colonized tomato roots. These observations are similar to those regarding the interactions of mycorrhiza and pathogenic fungi with plants.

Microscopic and transcriptome analyses of early colonization of tomato roots by Trichoderma harzianum

The capacity of the fungus Trichoderma harzianum CECT 2413 to colonize roots and stimulate plant growth was analyzed. Tobacco seedlings (Nicotiana benthamiana) transferred to Petri dishes inoculated with T. harzianum conidia showed increased plant fresh weight (140%) and foliar area (300%), as well as the proliferation of secondary roots (300%) and true leaves (140%). The interaction between strain CECT 2413 and the tomato-root system was also studied during the early stages of root colonization by the fungus. When T. harzianum conidia were inoculated into the liquid medium of hydroponically grown tomato plants (Lycopersicum esculentum), profuse adhesion of hyphae to the plant roots as well as colonization of the root epidermis and cortex were observed. Confocal microscopy of a T. harzianum transformant that expressed the green fluorescent protein (GFP) revealed intercellular hyphal growth and the formation of plant-induced papilla-like hyphal tips. Analysis of the T. harzianum-tomato interaction in soil indicated that the contact between T. harzianum and the roots persisted over a long period of time. This interaction was characterized by the presence of yeast-like cells, a novel and previously undescribed developmental change. To study the molecular mechanism underlying fungal ability to colonize the tomato-root system, the T. harzianum transcriptome was analyzed during the early stages of the plant-fungus interaction. The expression of fungal genes related to redox reactions, lipid metabolism, detoxification, and sugar or amino-acid transport increased when T. harzianum colonized tomato roots. These observations are similar to those regarding the interactions of mycorrhiza and pathogenic fungi with plants (AU)

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Distinct signalling pathways coordinately contribute to virulence of Fusarium oxysporum on mammalian hosts.

The filamentous fungus Fusarium oxysporum causes vascular wilt on a wide range of plant species and is an emerging pathogen of humans. A mitogen-activated protein kinase, Fmk1, and a G protein beta subunit, Fgb1, control pathogenicity of F. oxysporum on plants through distinct signalling pathways. In the present report, we studied the genetic interaction between fmk1 and fgb1 and their role in virulence on a mammalian host. The delta fmk1 or delta fgb1 single mutants exhibited similar virulence patterns as the wild type strain in an immunodepressed mouse model. By contrast, double mutants lacking both genes had dramatically reduced virulence. All mutants showed similar in vitro growth or tolerance to temperature and osmotic stress as the wild type strain. However, the delta fgb1 and delta fmk1 strains were reduced in specific extracellular protease activity or adhesion to fibronectin, respectively, two factors previously associated with fungal virulence. Thus, Fmk1 and Fgb1 are components of distinct signalling pathways which collectively control virulence of F. oxysporum on mammalian hosts.

Generation, annotation and analysis of ESTs from Trichoderma harzianum CECT 2413.

BACKGROUND: The filamentous fungus Trichoderma harzianum is used as biological control agent of several plant-pathogenic fungi. In order to study the genome of this fungus, a functional genomics project called "TrichoEST" was developed to give insights into genes involved in biological control activities using an approach based on the generation of expressed sequence tags (ESTs). RESULTS: Eight different cDNA libraries from T. harzianum strain CECT 2413 were constructed. Different growth conditions involving mainly different nutrient conditions and/or stresses were used. We here present the analysis of the 8,710 ESTs generated. A total of 3,478 unique sequences were identified of which 81.4% had sequence similarity with GenBank entries, using the BLASTX algorithm. Using the Gene Ontology hierarchy, we performed the annotation of 51.1% of the unique sequences and compared its distribution among the gene libraries. Additionally, the InterProScan algorithm was used in order to further characterize the sequences. The identification of the putatively secreted proteins was also carried out. Later, based on the EST abundance, we examined the highly expressed genes and a hydrophobin was identified as the gene expressed at the highest level. We compared our collection of ESTs with the previous collections obtained from Trichoderma species and we also compared our sequence set with different complete eukaryotic genomes from several animals, plants and fungi. Accordingly, the presence of similar sequences in different kingdoms was also studied. CONCLUSION: This EST collection and its annotation provide a significant resource for basic and applied research on T. harzianum, a fungus with a high biotechnological interest.

ThHog1 controls the hyperosmotic stress response in Trichoderma harzianum.

Trichoderma harzianum is a widespread mycoparasitic fungus, able to successfully colonize a wide range of substrates under different environmental conditions. Transcript profiling revealed a subset of genes induced in T. harzianum under hyperosmotic shock. The hog1 gene, a homologue of the MAPK HOG1 gene that controls the hyperosmotic stress response in Saccharomyces cerevisiae, was characterized. T. harzianum hog1 complemented the hog1Delta mutation in S. cerevisiae, but showed different features to yeast alleles: improved osmoresistance by expression of the hog1 allele and a lack of lethality when the hog1(F315S) allele was overexpressed. ThHog1 protein was phosphorylated in T. harzianum under different stress conditions such as hyperosmotic or oxidative stress, among others. By using a ThHog1-GFP fusion, the protein was shown to be localized in nuclei under these stress conditions. Two mutant strains of T. harzianum were constructed: one carrying the hog1(F315S) allele, and a knockdown hog1-silenced strain. The silenced strain was highly sensitive to osmotic stress, and showed intermediate levels of resistance against oxidative stress, indicating that the main role of ThHog1 protein is in the hyperosmotic stress response. Stress cross-resistance experiments showed evidences of a secondary role of ThHog1 in oxidative stress. The strain carrying the hog1(F315S) allele was highly resistant to the calcineurin inhibitor cyclosporin A, which suggests the existence of links between the two pathways. The two mutant strains showed a strongly reduced antagonistic activity against the plant pathogens Phoma betae and Colletotrichum acutatum, which points to a role of ThHog1 protein in fungus-fungus interactions.

Fusarium oxysporum G-protein beta subunit Fgb1 regulates hyphal growth, development, and virulence through multiple signalling pathways.

The vascular wilt fungus Fusarium oxysporum causes disease in a wide variety of crops. A signalling cascade controlled by the extracellular-regulated mitogen-activated protein kinase (MAPK) Fmk1 was previously found to be required for plant infection. To investigate the role of the heterotrimeric G-protein beta subunit Fgb1 as a putative upstream component of the Fmk1 signalling cascade, we generated F. oxysporum strains carrying either a Deltafgb1 loss-of-function allele or an fgb1(W115G) allele that mimicks the yeast STE4(W136G) mutation resulting in insensitivity to the cognate G-protein alpha subunit. Both types of mutants showed reduced virulence on tomato plants, similar to Deltafmk1 strains. However, in contrast to the latter, Deltafgb1 mutants displayed an abnormal hyphal growth phenotype with highly elongated cells, increased tip growth, a completely straight hyphal growth axis, and reduced subapical branching. Exogenous cAMP reversed part but not all of the Deltafgb1 growth phenotypes. Likewise, expression of the fgb1(W115G) allele only partly reversed growth phenotypes and failed to restore virulence on plants, whereas reintroduction of a functional fgb1 allele fully restored the wild type phenotype. Immunoblot analysis showed that levels of Fmk1 phosphorylation in fgb1 mutants were comparable to those in the wild type strain. Our results support a model in which Fgb1 controls hyphal growth, development and virulence in F. oxysporum both through cAMP-dependent and -independent pathways.

Glucose uptake in Trichoderma harzianum: role of gtt1.

Using a differential display technique, the gene gtt1, which codes for a high-affinity glucose transporter, has been cloned from the mycoparasite fungus Trichoderma harzianum CECT 2413. The deduced protein sequence of the gtt1 gene shows the 12 transmembrane domains typical of sugar transporters, together with certain residues involved in glucose uptake, such as a conserved arginine between domains IV and V and an aromatic residue (Phe) in the sequence of domain X. The gtt1 gene is transcriptionally regulated, being repressed at high levels of glucose. When carbon sources other than glucose are utilized, gtt1 repression is partially alleviated. Full derepression of gtt1 is obtained when the fungus is grown in the presence of low carbon source concentrations. This regulation pattern correlates with the role of this gene in glucose uptake during carbon starvation. Gene expression is also controlled by pH, so that the gtt1 gene is repressed at pH 6 but not at pH 3, a fact which represents a novel aspect of the influence of pH on the gene expression of transporters. pH also affects glucose transport, since a strongly acidic pH provokes a 40% decrease in glucose transport velocity. Biochemical characterization of the transport shows a very low K(m) value for glucose (12 micro M). A transformant strain that overexpresses the gtt1 gene shows a threefold increase in glucose but not galactose or xylose uptake, a finding which confirms the role of the gtt1 gene in glucose transport. The cloning of the first filamentous ascomycete glucose transporter is the first step in elucidating the mechanisms of glucose uptake and carbon repression in aerobic fungi.

New Saccharomyces cerevisiae baker's yeast displaying enhanced resistance to freezing.

Three procedures were used to obtain new Saccharomyces cerevisiae baker's yeasts with increased storage stability at -20, 4, 22, and 30 degrees C. The first used mitochondria from highly ethanol-tolerant wine yeast, which were transferred to baker's strains. Viability of the heteroplasmons was improved shortly after freezing. However, after prolonged storage, viability dramatically decreased and was accompanied by an increase in the frequency of respiratory-deficient (petite) mutant formation. This indicated that mitochondria were not stable and were incompatible with the nucleus. The strains tested regained their original resistance to freezing after recovering their own mitochondria. The second procedure used hybrid formation after protoplast fusion and isolation on selective media of fusants from baker's yeast meiotic products resistant to parafluorphenylalanine and cycloheximide, respectively. No hybrids were obtained when using the parentals, probably due to the high ploidy of the baker's strains. Hybrids obtained from nonisogenic strains manifested in all cases a resistance to freezing intermediate between those of their parental strains. Hybrids from crosses between meiotic products of the same strain were always more sensitive than their parentals. The third method was used to develop baker's yeast mutants resistant to 2-deoxy-d-glucose (DOG) and deregulated for maltose and sucrose metabolism. Mutant DOG21 displayed a slight increase in trehalose content and viability both in frozen doughs and during storage at 4 and 22 degrees C. This mutant also displayed a capacity to ferment, under laboratory conditions, both lean and sweet fresh and frozen doughs. For industrial uses, fermented lean and sweet bakery products, both from fresh and frozen doughs obtained with mutant DOG21, were of better quality with regard to volume, texture, and organoleptic properties than those produced by the wild type.

Fusarium oxysporum: exploring the molecular arsenal of a vascular wilt fungus.

UNLABELLED: SUMMARY Taxonomy: Vascular wilt fungus; Ascomycete although sexual stage is yet to be found. The most closely related teleomorphic group, Gibberella, is classified within the Pyrenomycetes. HOST RANGE: Very broad at the species level. More than 120 different formae speciales have been identified based on specificity to host species belonging to a wide range of plant families. Disease symptoms: Initial symptoms of vascular wilt include vein clearing and leaf epinasty, followed by stunting, yellowing of the lower leafs, progressive wilting of leaves and stem, defoliation and finally death of the plant. In cross-sections of the stem, a brown ring is evident in the area of the vascular bundles. Some formae speciales are not primarily vascular pathogens but cause foot- and rootrot or bulbrot. Economic importance: Causes severe losses on most vegetables and flowers, several field crops such as cotton and tobacco, plantation crops such as banana, plantain, coffee and sugarcane, and a few shade trees. CONTROL: Use of resistant varieties is the only practical measure for controlling the disease in the field. Under greenhouse conditions, soil sterilization can be performed. Alternative control methods with potential for the future include soil solarization and biological control with antagonistic bacteria or fungi. USEFUL WEBSITES: http://www.fgsc.net/fus.htm, http://www-genome.wi.mit.edu/annotation/fungi/fusarium/, http://www.cbs.knaw.nl/fusarium/database.html.

Aspartyl protease from Trichoderma harzianum CECT 2413: cloning and characterization.

A gene that encodes an extracellular aspartyl protease from Trichoderma harzianum CECT 2413, papA, has been isolated and characterized. Based on several conserved regions of other fungal acid proteases, primers were designed to amplify a probe that was used to isolate the papA gene from a genomic library of T. harzianum. papA was an intronless ORF which encoded a polypeptide of 404 aa, including a prepropeptide at the N-terminal region formed by one putative signal peptide, a second peptide which could be cleaved to activate the enzyme and the active protease of calculated 36.7 kDa and pI 4.35. Northern experiments indicated that papA gene was pH regulated, repressed by ammonium, glucose and glycerol, and induced by organic nitrogen sources. The promoter possessed potential AreA, PacC and MYC sites for nitrogen, pH and mycoparasitism regulation respectively, but lacked potential CreA sites for carbon regulation. IEF and zymograms indicated that PAPA was a pepstatin-sensitive aspartyl protease of pI 4.5. Transformants from T. harzianum CECT 2413 cultivated in yeast extract-supplemented medium overexpressed papA and had a fourfold increase in protease activity compared to the wild-type, while transformants that overexpressed the beta-1,6-glucanase gene bgn16.2 and papA had an additional 30% increase in beta-1,6-glucanase activity compared to bgn16.2 single transformants. Overexpression of both genes in ammonium-supplemented medium did not result in higher levels of PAPA and/or BGN16.2 proteins. These results indicated that both PAPA and beta-1,6-glucanase undergo proteolysis in ammonium-supplemented medium but PAPA is not responsible for beta-1,6-glucanase degradation.