Regulation of conidiation and antagonistic properties of the soil-borne plant beneficial fungus Trichoderma virens by a novel proline-, glycine-, tyrosine-rich protein and a GPI-anchored cell wall protein.

Trichoderma spp. are widely used as commercial biofungicides, and most commercial formulations are conidia based. Identification of genes that regulate conidiation would thus be of help in genetic reprogramming of these species to optimize sporulation. In this study, we constructed an SSH (suppression subtractive hybridization) library from RNA samples of the wild type strain and a non-conidiating mutant, M7, grown under constant illumination for 2 days. We identified several genes that are underexpressed in the mutant. Some of these genes are related to secondary metabolism, and a few could be associated with conidiation. Genes coding for the following proteins, among others, were identified: O-methyl transferase, ATPase, alpha/beta-hydrolase, WD repeat containing protein, dehydrogenase, thioesterase, translationally controlled tumour protein, and a proline-glycine-tyrosine-rich protein (PGYRP) that has been annotated in T.reesei as a signalling protein. Two of these genes, encoding Pgy1, a novel PGYRP, and Ecm33, a GPI-anchored cell wall protein, were further studied in detail by generation of deletion mutants. We demonstrate here that both these genes not only regulate radial growth and conidiation in Trichoderma virens, but are also involved in antagonism against soil-borne wide host range plant pathogens. Furthermore, deletion of ecm33 affected hydrophobicity and cell wall integrity.

Trichoderma virens exerts herbicidal effect on Arabidopsis thaliana via modulation of amino acid metabolism.

Trichoderma virens is a plant beneficial fungus well-known for its biocontrol, herbicidal and growth promotion activity. Earlier, we identified HAS (HA-synthase, a terpene cyclase) and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) to be involved in the production of multiple non-volatiles and non-volatile+volatile metabolites, respectively. The present study delineates the function of HAS and GAPDH in regulating herbicidal activity, using the model plant Arabidopsis thaliana. Under axenic conditions, rosette-biomass of seedlings co-cultivated with ΔHAS (HASR) and ΔGAPDH (GAPDHR) was higher than WT-Trichoderma (WTR) as well as non-colonized control (NoTR), even though the root colonization ability was reduced. However, HASR biomass was still higher than those of GAPDHR, indicating that blocking volatiles will not provide any additional contribution over non-volatile metabolites for Trichoderma-induced herbicidal activity. LC-MS analysis revealed that loss of herbicidal activity of ΔHAS/ΔGAPDH was associated with an increase in the levels of amino acids, which coincided with reduced expression levels of amino-acid catabolism and anabolism related genes in HASR/GAPDHR. RNAi-mediated suppression of an oxidoreductase gene, VDN5, specifically prevented viridin-to-viridiol conversion. Additionally, vdn5 mimics ΔHAS, in terms of amino-acid metabolism gene expression and partially abolishes the herbicidal property of WT-Trichoderma. Thus, the study provides mechanistic frame-work for better utilization of Trichoderma virens for biocontrol purposes, balancing between plant growth promotion and herbicidal activity.

Genetic Evidence in Favor of a Polyketide Origin of Acremeremophilanes, the Fungal "Sesquiterpene" Metabolites.

Eremophilanes are a large group of "sesquiterpenes" produced by plants and fungi, with more than 180 compounds being known in fungi alone. Many of these compounds are phytotoxic, antimicrobial, anticancer and immunomodulators, and hence are of great economic values. Acremeremophilanes A to O have earlier been reported in a marine isolate of Acremonium sp. We report here the presence of Acremeremophilane I, G, K, N, and O, in a plant beneficial fungus Trichoderma virens, in a strain-specific manner. We also describe a novel, P strain-specific polyketide synthase (PKS) gene cluster in T. virens. This gene cluster, designated amm cluster, is absent in the genome of a Q strain of T. virens, and in other Trichoderma spp.; instead, a near identical cluster is present in the genome of the toxic mold Stachybotrys chartarum. Using gene knockout, we provide evidence that acremeremophilanes are biosynthesized via a polyketide route, and not via the mevalonate/terpene synthesis route as believed. We propose here that the 10-carbon skeleton is a product of polyketide synthase, to which a five-carbon isoprene unit is added by a prenyl transferase (PT), a gene for which is present next to the PKS gene in the genome. Based on this evidence, we propose that at least some of the eremophilanes classified in literature as sesquiterpenes (catalyzed by terpene cyclase) are actually meroterpenes (catalyzed by PKSs and PTs), and that the core moiety is not a sesquiterpene, but a hybrid polyketide/isoprene unit. IMPORTANCE The article contradicts the established fact that acremeremophilane metabolites produced by fungi are sesquiterpenes; instead, our findings suggest that at least some of these well-studied metabolites are of polyketide origin. Acremeremophilane metabolites are of medicinal significance, and the present findings have implications for the metabolic engineering of these metabolites and also their overproduction in microbial cell factories.

Structure-function analysis reveals Trichoderma virens Tsp1 to be a novel fungal effector protein modulating plant defence.

Trichoderma virens colonizes roots and develops a symbiotic relationship with plants where the fungal partner derives nutrients from plants and offers defence, in return. Tsp1, a small secreted cysteine-rich protein, was earlier found to be upregulated in co-cultivation of T. virens with maize roots. Tsp1 is well conserved in Ascomycota division of fungi, but none of its homologs have been studied yet. We have expressed and purified recombinant Tsp1, and resolved its structure to 1.25 Å resolutions, from two crystal forms, using Se-SAD methods. The Tsp1 adopts a ß barrel fold and forms dimer in structure as well as in solution form. DALI based structure analysis revealed the structure similarity with two known fungal effector proteins: Alt a1 and PevD1. Structure and evolutionary analysis suggested that Tsp1 belongs to a novel effector protein family. Tsp1 acted as an inducer of salicylic acid mediated susceptibility in plants, rendering maize plants more susceptible to a necrotrophic pathogen Cochliobolus heterostrophus, as observed using plant defence assay and RT-qPCR analysis.

Dual role of a dedicated GAPDH in the biosynthesis of volatile and non-volatile metabolites- novel insights into the regulation of secondary metabolism in Trichoderma virens.

Trichoderma virens produces viridin/viridiol, heptelidic (koningic) acid, several volatile sesquiterpenes and gliotoxin (Q strains) or gliovirin (P strains). We earlier reported that deletion of the terpene cyclase vir4 and a glyceraldehyde-3-phosphate dehydrogenase (GAPDH, designated as vGPD) associated with the "vir" cluster abrogated the biosynthesis of several volatile sesquiterpene metabolites. Here we show that, the deletion of this GAPDH also impairs the biosynthesis of heptelidic acid (a non-volatile sesquiterpene), viridin (steroid) and gliovirin (non-ribosomal peptide), indicating regulation of non-volatile metabolite biosynthesis by this GAPDH that is associated with a secondary metabolism gene cluster. To gain further insights into the details of this novel form of regulation, we identified the terpene cyclase gene responsible for heptelidic acid biosynthesis (hereafter designated as has1) and prove that the expression of this gene is regulated by vGPD. Interestingly, deletion of has1 impaired biosynthesis of heptelidic acid (HA), viridin and gliovirin, but not of volatile sesquiterpenes. Deletion of the vir cluster associated terpene cyclase gene (vir4), located next to the vGPD gene, did not impair biosynthesis of HA, viridin or gliovirin. We thus unveil a novel circuitry of regulation of secondary metabolism where an HA-tolerant GAPDH isoform (vGPD) regulates HA biosynthesis through the transcriptional regulation of the HA-synthase gene (which is not part of the "vir" cluster). Interestingly, impairment of HA biosynthesis leads to the down-regulation of biosynthesis of other non-volatile secondary metabolites, but not of volatile secondary metabolites. We thus provide evidence that the "vir" cluster associated, HA-tolerant GAPDH in T. virens participates in the biosynthesis of volatile sesquiterpenes as a biosynthetic enzyme, and regulates the production of non-volatile metabolites via regulation of HA biosynthesis. The orthologue of the "vir" cluster in Aspergillus oryzae was earlier reported to synthesize HA by another group. Our study thus proves that the same gene cluster can code for unrelated metabolites in different species.

Expression, purification, crystallization and X-ray diffraction studies of a novel root-induced secreted protein from Trichoderma virens.

Small secreted cysteine-rich proteins (SSCPs) from fungi play an important role in fungi-host interactions. The plant-beneficial fungi Trichoderma spp. are in use worldwide as biocontrol agents and protect the host plant from soil-borne as well as foliar pathogens. Recently, a novel SSCP, Tsp1, has been identified in the secreted protein pool of T. virens and is overinduced upon its interaction with the roots of the maize plant. The protein was observed to be well conserved in the Ascomycota division of fungi, and its homologs are present in many plant-pathogenic fungi such as Fusarium oxysporum and Magnaporthe oryzae. However, none of these homologs have yet been characterized. Recombinant Tsp1 protein has been expressed and purified using an Escherichia coli expression system. The protein, with four conserved cysteines, forms a dimer in solution as observed by size-exclusion chromatography. The dimerization, however, does not involve disulfide bonds. Circular-dichroism data suggested that the protein has a ß-strand-rich secondary structure that matched well with the secondary structure predicted using bioinformatics methods. The protein was crystallized using sodium malonate as a precipitant. The crystals diffracted X-rays to 1.7 Šresolution and belonged to the orthorhombic space group P212121 (Rmeas = 5.4%), with unit-cell parameters a = 46.3, b = 67.0, c = 173.2 Å. The Matthews coefficient (VM) of the crystal is 2.32 Å3 Da-1, which corresponds to nearly 47% solvent content with four subunits of Tsp1 protein in the asymmetric unit. This is the first report of the structural study of any homolog of the novel Tsp1 protein. These structural studies will help in understanding the classification and function of the protein.

Genome-wide analysis of cytochrome P450s of Trichoderma spp.: annotation and evolutionary relationships.

BACKGROUND: Cytochrome P450s form an important group of enzymes involved in xenobiotics degradation and metabolism, both primary and secondary. These enzymes are also useful in industry as biotechnological tools for bioconversion and a few are reported to be involved in pathogenicity. Trichoderma spp. are widely used in industry and agriculture and are known for their biosynthetic potential of a large number of secondary metabolites. For realising the full biosynthetic potential of an organism, it is important to do a genome-wide annotation and cataloguing of these enzymes. RESULTS: Here, we have studied the genomes of seven species (T. asperellum, T. atroviride, T. citrinoviride, T. longibrachiatum, T. reesei , T. harzianum and T. virens) and identified a total of 477 cytochrome P450s. We present here the classification, evolution and structure as well as predicted function of these proteins. This study would pave the way for functional characterization of these groups of enzymes and will also help in realization of their full economic potential. CONCLUSION: Our CYPome annotation and evolutionary studies of the seven Trichoderma species now provides opportunities for exploration of research-driven strategies to select Trichoderma species for various applications especially in relation to secondary metabolism and degradation of environmental pollutants.

The Terpenoid Biosynthesis Toolkit of Trichoderma.

The widely used biotechnologically important fungi belonging to the genus Trichoderma are rich sources of secondary metabolites. Even though the genomes of several Trichoderma spp. have been published, and data are available on the genes involved in biosynthesis of non-ribosomal peptide synthetases and polyketide synthases, no genome-wide data are available for the terpenoid biosynthesis machinery in these organisms. In the present study, we have identified the genes involved in terpene biosynthesis in the genomes of three Trichoderma spp., viz., T. virens, T. atroviride and T. reesei. While the genes involved in the condensation steps are highly conserved across the three species, these fungi differed in the number and organization of terpene cyclases. T. virens genome harbours eleven terpene cyclases, while T. atroviride harbours seven, and T. reeseisix in their genomes; seven, three and two being part of putative secondary metabolism related gene clusters.