Comparative Phenotypic, Genomic, and Transcriptomic Analyses of Two Contrasting Strains of the Plant Beneficial Fungus Trichoderma virens.

Trichoderma virens is a beneficial fungus that helps plants fight pathogens and abiotic stresses and thereby enhances crop yields. Unlike other Trichoderma spp., there are two well-defined strains (P and Q) of T. virens, classified by secondary metabolites profiling, primarily the biosynthesis of the nonribosomal, strong antimicrobial agents gliotoxin (Q) and gliovirin (P). We have studied the phenotypic and biocontrol properties of two well-studied representative isolates (T. virens Gv29-8 and T. virens GvW/IMI304061) that represent a Q strain and a P strain of T. virens, respectively. We refined the genome assembly of the P strain using nanopore technology, and we compared it with the Q strain. The differences between the genomes include gene expansion in the Q strain. T. virens Gv29-8 is weaker than GvW as a mycoparasite on the broad host-range plant pathogen Sclerotium rolfsii, and it is ineffective as a biocontrol agent when applied to pathogen-infested soil. T. virens Gv29-8 proved to be phytotoxic to Arabidopsis seedlings, whereas the effect of T. virens GvW was not major. Both strains colonized the surface and outer cortex layer of tomato roots, with about 40% higher colonization by T. virens Gv29-8. T. virens Gv29-8 induced the expression of a larger set of tomato genes than did T. virens GvW, although some tomato genes were uniquely induced in response to T. virens GvW. We studied the comparative transcriptome response of T. virens Gv29-8 and T. virens GvW to S. rolfsii. A larger set of genes was regulated in T. virens GvW than in T. virens Gv29-8 in the presence of the plant pathogen. IMPORTANCE Trichoderma virens populations that were earlier classified into two strains (P and Q) based on secondary metabolites profiling are also phenotypically and genetically distinct, with the latter being ineffective in controlling the devastating, broad host range plant pathogen Sclerotium rolfsii. The two strains also provoke distinct as well as overlapping transcriptional responses to the presence of the plant and the pathogen. This study enriches our knowledge of Trichoderma-plant-pathogen interactions and identifies novel candidate genes for further research and deployment in agriculture.

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.

Whole Genome Sequencing Reveals Major Deletions in the Genome of M7, a Gamma Ray-Induced Mutant of Trichoderma virens That Is Repressed in Conidiation, Secondary Metabolism, and Mycoparasitism.

Trichoderma virens is a commercial biofungicide used in agriculture. We have earlier isolated a mutant of T. virens using gamma ray-induced mutagenesis. This mutant, designated as M7, is defective in morphogenesis, secondary metabolism, and mycoparasitism. The mutant does not produce conidia, and the colony is hydrophilic. M7 cannot utilize cellulose and chitin as a sole carbon source and is unable to parasitize the plant pathogens Rhizoctonia solani and Pythium aphanidermatum in confrontation assay. Several volatile (germacrenes, beta-caryophyllene, alloaromadendrene, gamma-muurolene) and non-volatile (viridin, viridiol, gliovirin, heptelidic acid) metabolites are not detected in M7. In transcriptome analysis, many genes related to secondary metabolism, carbohydrate metabolism, hydrophobicity, and transportation, among others, were found to be downregulated in the mutant. Using whole genome sequencing, we identified five deletions in the mutant genome, totaling about 250 kb (encompassing 71 predicted ORFs), which was confirmed by PCR. This study provides novel insight into genetics of morphogenesis, secondary metabolism, and mycoparasitism and eventually could lead to the identification of novel regulators of beneficial traits in plant beneficial fungi Trichoderma spp. We also suggest that this mutant can be developed as a microbial cell factory for the production of secondary metabolites and proteins.

Expression of a human cytochrome P4502E1 in Nicotiana tabacum enhances tolerance and remediation of γ-hexachlorocyclohexane.

Lindane (γ-hexachlorocyclohexane), a persistent organo-chlorine insecticide widely used in developing countries, has a negative effect as a polluting agent of soil and surface waters. Plants can be used for remediation of organic pollutants and their efficiency can be enhanced by introduction of heterologous genes. Mammalian cytochrome P4502E1 (CYP2E1), an important monooxygenase is involved in the degradation of a wide range of xenobiotics including environmental pollutants/herbicides and pesticides. Here, we report the development of transgenic tobacco plants expressing human CYP2E1 and the efficacy of plants for remediation of lindane. Transgenic tobacco plants with CYP2E1 showed enhanced tolerance to lindane when grown in hydroponic medium and soil compared to control plants. Remediation of (14)C-labeled lindane from hydroponic medium was higher in transgenic plants compared to that of control plants, with the best performing line showing 25% higher removal of lindane from solution than control plants. Similar results were seen in plants grown in soil spiked with lindane. The present study has shown that transgenic plants expressing CYP2E1 gene have potential use for remediation of lindane from contaminated solutions and soil.

Enhanced tolerance and remediation of anthracene by transgenic tobacco plants expressing a fungal glutathione transferase gene.

Plants can be used for remediation of polyaromatic hydrocarbons, which are known to be a major concern for human health. Metabolism of xenobiotic compounds in plants occurs in three phases and glutathione transferases (GST) mediate phase II of xenobiotic transformation. Plants, although have GSTs, they are not very efficient for degradation of exogenous recalcitrant xenobiotics including polyaromatic hydrocarbons. Hence, heterologous expression of efficient GSTs in plants may improve their remediation and degradation potential of xenobiotics. In the present study, we investigated the potential of transgenic tobacco plants expressing a Trichoderma virens GST for tolerance, remediation and degradation of anthracene-a recalcitrant polyaromatic hydrocarbon. Transgenic plants with fungal GST showed enhanced tolerance to anthracene compared to control plants. Remediation of (14)C uniformly labeled anthracene from solutions and soil by transgenic tobacco plants was higher compared to wild-type plants. Transgenic plants (T(0) and T(1)) degraded anthracene to naphthalene derivatives, while no such degradation was observed in wild-type plants. The present work has shown that in planta expression of a fungal GST in tobacco imparted enhanced tolerance as well as higher remediation potential of anthracene compared to wild-type plants.

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.