Role of gliotoxin in the symbiotic and pathogenic interactions of Trichoderma virens.

Using a gene disruption strategy, we generated mutants in the gliP locus of the plant-beneficial fungus Trichoderma virens that were no longer capable of producing gliotoxin. Phenotypic assays demonstrated that the gliP-disrupted mutants grew faster, were more sensitive to oxidative stress and exhibited a sparse colony edge compared with the WT strain. In a plate confrontation assay, the mutants deficient in gliotoxin production were ineffective as mycoparasites against the oomycete, Pythium ultimum, and the necrotrophic fungal pathogen, Sclerotinia sclerotiorum, but retained mycoparasitic ability against Rhizoctonia solani. Biocontrol assays in soil showed that the mutants were incapable of protecting cotton seedlings from attack by P. ultimum, against which the WT strain was highly effective. The mutants, however, were as effective as the WT strain in protecting cotton seedlings against R. solani. Loss of gliotoxin production also resulted in a reduced ability of the mutants to attack the sclerotia of S. sclerotiorum compared with the WT. The addition of exogenous gliotoxin to the sclerotia colonized by the mutants partially restored their degradative abilities. Interestingly, as in Aspergillus fumigatus, an opportunistic human pathogen, gliotoxin was found to be involved in pathogenicity of T. virens against larvae of the wax moth, Galleria mellonella. The loss of gliotoxin production in T. virens was restored by complementation with the gliP gene from A. fumigatus. We have, thus, demonstrated that the putative gliP cluster of T. virens is responsible for the biosynthesis of gliotoxin, and gliotoxin is involved in mycoparasitism and biocontrol properties of this plant-beneficial fungus.

Best practices for fungal germplasm repositories and perspectives on their implementation.

In over 50 years, the Fungal Genetics Stock Center has grown to become a world-recognized biological resource center. Along with this growth comes the development and implementation of myriad practices for the management and curation of a diverse collection of filamentous fungi, yeast, and molecular genetic tools for working with the fungi. These practices include techniques for the testing, manipulation, and preservation of individual fungal isolates as well as for processing of thousands of isolates in parallel. In addition to providing accurate record keeping, an electronic managements system allows the observation of trends in strain distribution and in sample characteristics. Because many ex situ fungal germplasm repositories around the world share similar objectives, best-practice guidelines have been developed by a number of organizations such as the Organization for Economic Cooperation and Development or the International Society for Biological and Environmental Repositories. These best-practice guidelines provide a framework for the successful operation of collections and promote the development and interactions of biological resource centers around the world.

Rediscovery by Whole Genome Sequencing: Classical Mutations and Genome Polymorphisms in Neurospora crassa.

Classical forward genetics has been foundational to modern biology, and has been the paradigm for characterizing the role of genes in shaping phenotypes for decades. In recent years, reverse genetics has been used to identify the functions of genes, via the intentional introduction of variation and subsequent evaluation in physiological, molecular, and even population contexts. These approaches are complementary and whole genome analysis serves as a bridge between the two. We report in this article the whole genome sequencing of eighteen classical mutant strains of Neurospora crassa and the putative identification of the mutations associated with corresponding mutant phenotypes. Although some strains carry multiple unique nonsynonymous, nonsense, or frameshift mutations, the combined power of limiting the scope of the search based on genetic markers and of using a comparative analysis among the eighteen genomes provides strong support for the association between mutation and phenotype. For ten of the mutants, the mutant phenotype is recapitulated in classical or gene deletion mutants in Neurospora or other filamentous fungi. From thirteen to 137 nonsense mutations are present in each strain and indel sizes are shown to be highly skewed in gene coding sequence. Significant additional genetic variation was found in the eighteen mutant strains, and this variability defines multiple alleles of many genes. These alleles may be useful in further genetic and molecular analysis of known and yet-to-be-discovered functions and they invite new interpretations of molecular and genetic interactions in classical mutant strains.

Two classes of new peptaibols are synthesized by a single non-ribosomal peptide synthetase of Trichoderma virens.

Peptaibols are a group of small peptides having a high α-aminoisobutyric acid (Aib) content and produced by filamentous fungi, especially by the members of the genus Trichoderma (anamorph Hypocrea). These antibiotics are economically important for their anti-microbial and anti-cancer properties as well as ability to induce systemic resistance in plants against microbial invasion. In this study we present sequences of two classes (11-residue and 14-residue) of peptaibols produced by the biocontrol fungus Trichoderma virens. Of the 35 11-residue peptaibols sequenced, 18 are hitherto not described, and all the 53 14-residue sequences described by us here are new. We have also identified a peptaibol synthetase (non-ribosomal peptide synthetase, NRPS) with 14 complete modules in the genome of this fungus and disruption of this single gene (designated as tex2) resulted in the loss of both the classes of peptaibols. We, thus present here an unprecedented case where a single NRPS encodes for two classes of peptaibols. The new peptaibols identified here could have applications as therapeutic agents for the management of human and plant health.

Characterization of the temperature-sensitive mutations un-7 and png-1 in Neurospora crassa.

The model filamentous fungus Neurospora crassa has been studied for over fifty years and many temperature-sensitive mutants have been generated. While most of these have been mapped genetically, many remain anonymous. The mutation in the N. crassa temperature-sensitive lethal mutant un-7 was identified by a complementation based approach as being in the open reading frame designated NCU00651 on linkage group I. Other mutations in this gene have been identified that lead to a temperature-sensitive morphological phenotype called png-1. The mutations underlying un-7 result in a serine to phenylalanine change at position 273 and an isoleucine to valine change at position 390, while the mutation in png-1 was found to result in a serine to leucine change at position 279 although there were other conservative changes in this allele. The overall morphology of the strain carrying the un-7 mutation is compared to strains carrying the png-1 mutation and these mutations are evaluated in the context of other temperature-sensitive mutants in Neurospora.

A proteinaceous elicitor Sm1 from the beneficial fungus Trichoderma virens is required for induced systemic resistance in maize.

We have previously shown that the beneficial filamentous fungus Trichoderma virens secretes the highly effective hydrophobin-like elicitor Sm1 that induces systemic disease resistance in the dicot cotton (Gossypium hirsutum). In this study we tested whether colonization of roots by T. virens can induce systemic protection against a foliar pathogen in the monocot maize (Zea mays), and we further demonstrated the importance of Sm1 during maize-fungal interactions using a functional genomics approach. Maize seedlings were inoculated with T. virens Gv29-8 wild type and transformants in which SM1 was disrupted or constitutively overexpressed in a hydroponic system or in soil-grown maize seedlings challenged with the pathogen Colletotrichum graminicola. We show that similar to dicot plants, colonization of maize roots by T. virens induces systemic protection of the leaves inoculated with C. graminicola. This protection was associated with notable induction of jasmonic acid- and green leaf volatile-biosynthetic genes. Neither deletion nor overexpression of SM1 affected normal growth or development of T. virens, conidial germination, production of gliotoxin, hyphal coiling, hydrophobicity, or the ability to colonize maize roots. Plant bioassays showed that maize grown with SM1-deletion strains exhibited the same levels of systemic protection as non-Trichoderma-treated plants. Moreover, deletion and overexpression of SM1 resulted in significantly reduced and enhanced levels of disease protection, respectively, compared to the wild type. These data together indicate that T. virens is able to effectively activate systemic disease protection in maize and that the functional Sm1 elicitor is required for this activity.

The 18mer peptaibols from Trichoderma virens elicit plant defence responses.

SUMMARY Peptaibols, the products of non-ribosomal peptide synthetases (NRPS), are linear peptide antibiotics produced by Trichoderma and other fungal genera. Trichoderma virens strain Gv29-8, a well-known biocontrol agent and inducer of plant defence responses, produces three lengths of peptaibols, 11, 14 and 18 residues long, with several isoforms of each. Disruption of the NRPS gene, tex1, encoded by a 62.8-kb uninterrupted open reading frame, results in the loss of production of all forms of 18-residue peptaibols. Tex1 is expressed during all Trichoderma developmental stages (germinating conidia, sporulating and non-sporulating mycelia) examined on solid media. Expression analysis by reverse transcriptase PCR shows that in Gv29-8 wild-type the abundance of tex1 transcript is greater during co-cultivation with cucumber seedling roots than when grown alone. Cucumber plants co-cultivated with T. virens strains disrupted in tex1 show a significantly reduced systemic resistance response against the leaf pathogen Pseudomonas syringae pv. lachrymans, and reduced ability to produce phenolic compounds with inhibitory activity to the bacteria as compared with plants grown in the presence of wild-type. Two synthetic 18-amino-acid peptaibol isoforms (TvBI and TvBII) from Gv29-8 when applied to cucumber seedlings through the transpiration stream can alone induce systemic protection to the leaf pathogenic bacteria, induce antimicrobial compounds in cucumber cotyledons and up-regulate hydroxyperoxide lyase (hpl), phenylalanine ammonia lyase (pal1) and peroxidase (prx) gene expression. These data strongly suggest that the 18mer peptaibols are critical in the chemical communication between Trichoderma and plants as triggers of non-cultivar-specific defence responses.

Identification of peptaibols from Trichoderma virens and cloning of a peptaibol synthetase.

The fungus Trichoderma virens is a ubiquitous soil saprophyte that has been applied as a biological control agent to protect plants from fungal pathogens. One mechanism of biocontrol is mycoparasitism, and T. virens produces antifungal compounds to assist in killing its fungal targets. Peptide synthetases produce a wide variety of peptide secondary metabolites in bacteria and fungi. Many of these are known to possess antibiotic activities. Peptaibols form a class of antibiotics known for their high alpha-aminoisobutyric acid content and their synthesis as a mixture of isoforms ranging from 7 to 20 amino acids in length. Here we report preliminary characterization of a 62.8-kb continuous open reading frame encoding a peptaibol synthetase from T. virens. The predicted protein structure consists of 18 peptide synthetase modules with additional modifying domains at the N- and C-termini. T. virens was shown to produce a mixture of peptaibols, with the largest peptides being 18 residues. Mutation of the gene eliminated production of all peptaibol isoforms. Identification of the gene responsible for peptaibol production will facilitate studies of the structure and function of peptaibol antibiotics and their contribution to biocontrol activity.