Rosa Hermosa

Comparative genome sequence analysis underscores mycoparasitism as the ancestral life style of Trichoderma

BackgroundMycoparasitism, a lifestyle where one fungus is parasitic on another fungus, has special relevance when the prey is a plant pathogen, providing a strategy for biological control of pests for plant protection. Probably, the most studied biocontrol agents are species of the genus Hypocrea/Trichoderma.ResultsHere we report an analysis of the genome sequences of the two biocontrol species Trichoderma atroviride (teleomorph Hypocrea atroviridis) and Trichoderma virens (formerly Gliocladium virens, teleomorph Hypocrea virens), and a comparison with Trichoderma reesei (teleomorph Hypocrea jecorina). These three Trichoderma species display a remarkable conservation of gene order (78 to 96%), and a lack of active mobile elements probably due to repeat-induced point mutation. Several gene families are expanded in the two mycoparasitic species relative to T. reesei or other ascomycetes, and are overrepresented in non-syntenic genome regions. A phylogenetic analysis shows that T. reesei and T. virens are derived relative to T. atroviride. The mycoparasitism-specific genes thus arose in a common Trichoderma ancestor but were subsequently lost in T. reesei.ConclusionsThe data offer a better understanding of mycoparasitism, and thus enforce the development of improved biocontrol strains for efficient and environmentally friendly protection of plants.

Plant-beneficial effects of Trichoderma and of its genes.

Trichoderma (teleomorph Hypocrea) is a fungal genus found in many ecosystems. Trichoderma spp. can reduce the severity of plant diseases by inhibiting plant pathogens in the soil through their highly potent antagonistic and mycoparasitic activity. Moreover, as revealed by research in recent decades, some Trichoderma strains can interact directly with roots, increasing plant growth potential, resistance to disease and tolerance to abiotic stresses. This mini-review summarizes the main findings concerning the Trichoderma-plant interaction, the molecular dialogue between the two organisms, and the dramatic changes induced by the beneficial fungus in the plant. Efforts to enhance plant resistance and tolerance to a broad range of stresses by expressing Trichoderma genes in the plant genome are also addressed.

Transgenic expression of the Trichoderma harzianum hsp70 gene increases Arabidopsis resistance to heat and other abiotic stresses.

The ability of some Trichoderma strains, a biological control agent, to overcome extreme environmental conditions has previously been reported and related to heat-shock proteins (HSPs). These proteins are induced environmentally and are involved in important processes, acting as molecular chaperones in all organisms. In a previous study, we demonstrated, by overexpression, that the Trichoderma harzianum hsp70 gene conferred tolerance to heat and other abiotic stresses to this fungus. In this work, we investigate the function of the T. harzianum T34 hsp70 gene in Arabidopsis thaliana. We analyze transgenic plant responses under adverse environmental conditions and the expression levels of a set of seven stress genes, using quantitative RT-PCR. As expected, transgenic plants expressing the T. harzianum hsp70 gene exhibited enhanced tolerance to heat stress. In addition, they did not show growth inhibition and, after heat pre-treatment, transgenic seedlings were more tolerant to osmotic, salt and oxidative stresses with respect to the wild-type behavior. Transgenic lines also had increased transcript levels of the Na(+)/H(+) exchanger 1 (SOS1) and ascorbate peroxidase 1 (APX1) genes, involved in salt and oxidative stress responses, respectively. However, the heat-shock factor (HSF) and four HSP genes tested were down-regulated in 35S:hsp70 plants. Overall, our results indicate that hsp70 confers tolerance to heat and other abiotic stresses and that the fungal HSP70 protein acts as a negative regulator of the HSF transcriptional activity in Arabidopsis.

The ThPG1 Endopolygalacturonase Is Required for the Trichoderma harzianum-Plant Beneficial Interaction

Considering the complexity of the in vivo interactions established by a mycoparasitic biocontrol agent at the plant rhizosphere, proteomic, genomic, and transcriptomic approaches were used to study a novel Trichoderma gene coding for a plant cell wall (PCW)-degrading enzyme. A proteome analysis, using a three-component (Trichoderma spp.-tomato plantlets-pathogen) system, allowed us to identify a differentially expressed Trichoderma harzianum endopolygalacturonase (endoPG). Spot 0303 remarkably increased only in the presence of the soilborne pathogens Rhizoctonia solani and Pythium ultimum, and corresponded to an expressed sequence tag from a T. harzianum T34 cDNA library that was constructed in the presence of PCW polymers and used to isolate the Thpg1 gene. Compared with the wild-type strain, Thpg1-silenced transformants showed lower PG activity, less growth on pectin medium, and reduced capability to colonize tomato roots. These results were combined with microarray comparative data from the transcriptome of Arabidopsis plants inoculated with the wild type or a Thpg1-silenced transformant (ePG5). The endoPG-encoding gene was found to be required for active root colonization and plant defense induction by T. harzianum T34. In vivo assays showed that Botrytis cinerea leaf necrotic lesions were slightly smaller in plants colonized by ePG5, although no statistically significant differences were observed.

Transcriptomic response of Arabidopsis thaliana after 24 h incubation with the biocontrol fungus Trichoderma harzianum.

Trichoderma harzianum is a fungus used as biocontrol agent using its antagonistic abilities against phytopathogenic fungi, although it has also direct effects on plants, increasing or accelerating their growth and resistance to diseases and the tolerance to abiotic stresses. We analyzed Arabidopsis thaliana gene expression changes after 24 h of incubation in the presence of T. harzianum T34 using the Affymetrix GeneChip Arabidopsis ATH1. Because this microarray contains more than 22,500 probe sets representing approximately 24,000 genes, we were able to construct a global picture of the molecular physiology of the plant at 24 h of T. harzianum-Arabidopsis interaction. We identified several differentially expressed genes that are involved in plant responses to stress, regulation of transcription, signal transduction or plant metabolism. Our data support the hypothesis that salicylic acid- and jasmonic acid-related genes were down-regulated in A. thaliana after 24 h of incubation in the presence of T. harzianum T34, while several genes related to abiotic stress responses were up-regulated. These systemic changes elicited by T. harzianum in Arabidopsis are discussed.

Involvement of Trichoderma trichothecenes in the biocontrol activity and induction of plant defense-related genes.

ABSTRACT Trichoderma species produce trichothecenes, most notably trichodermin and harzianum A (HA), by a biosynthetic pathway in which several of the involved proteins have significant differences in functionality compared to their Fusarium orthologues. In addition, the genes encoding these proteins show a genomic organization differing from that of the Fusarium tri clusters. Here we describe the isolation of Trichoderma arundinaceum IBT 40837 transformants which have a disrupted or silenced tri4 gene, a gene encoding a cytochrome P450 monooxygenase that oxygenates trichodiene to give rise to isotrichodiol, and the effect of tri4 gene disruption and silencing on the expression of other tri genes. Our results indicate that the tri4 gene disruption resulted in a reduced antifungal activity against Botrytis cinerea and Rhizoctonia solani and also in a reduced ability to induce the expression of tomato plant defense-related genes belonging to the salicylic acid (SA) and jasmonate (JA) pathways against B. cinerea, in comparison to the wild-type strain, indicating that HA plays an important function in the sensitization of Trichoderma-pretreated plants against this fungal pathogen. Additionally, the effect of the interaction of T. arundinaceum with B. cinerea or R. solani and with tomato seedlings on the expressions of the tri genes was studied.

Thctf1 transcription factor of Trichoderma harzianum is involved in 6-pentyl-2H-pyran-2-one production and antifungal activity.

We describe the cloning and characterization of the Trichoderma harzianum Thctf1 gene, which shows high sequence identity with a transcription factor gene of Fusarium solani f. sp. pisi. In T. harzianum, disruption of the Thctf1 gene by homologous recombination gave rise to transformants that in plate experiments did not show the yellow pigmentation observed in the wild-type strain. In several Trichoderma spp. a yellow pigmentation and a coconut aroma have been related to the production of 6-pentyl-2H-pyran-2-one (6PP) compounds. Prompted by this, we explored whether the loss of pigmentation in the Thctf1 null mutants of T. harzianum might be related to the synthesis of 6PP. Chromatographic and spectroscopic analyses revealed that the disruptants did not produce two secondary metabolites, derived from 6PP and not previously described in the Trichoderma genus, that are present in wild-type culture filtrates. Since 6PP is a recognized antifungal compound, this ability was analyzed in both the disruptants and wild-type, observing that the Thctf1 null mutants of T. harzianum had reduced antimicrobial capacity. Our results point to the significant role of THCTF1 in the production of secondary metabolites and in the antifungal activity of T. harzianum.

Overexpression of the trichodiene synthase gene tri5 increases trichodermin production and antimicrobial activity in Trichoderma brevicompactum

Trichoderma brevicompactum produces trichodermin, a simple trichothecene-type toxin that shares the first steps of the sesquiterpene biosynthetic pathway with other phytotoxic trichothecenes from Fusarium spp. Trichodiene synthase catalyses the conversion of farnesyl pyrophosphate to trichodiene and it is encoded by the tri5 gene that was cloned and analysed functionally by homologous overexpression in T. brevicompactum. tri5 expression was up-regulated in media with glucose, H(2)O(2) or glycerol. tri5 repression was observed in cultures supplemented with the antioxidants ferulic acid and tyrosol. Acetone extracts of tri5-overexpressing transformants displayed higher antifungal activity than those from the wild-type. Chromatographic and spectroscopic analyses revealed that tri5 overexpression led to an increased production of trichodermin and tyrosol. Agar diffusion assays with these two purified metabolites from the tri5-overexpressing transformant T. brevicompactum Tb41tri5 showed that only trichodermin had antifungal activity against Saccharomyces cerevisiae, Kluyveromyces marxianus, Candida albicans, Candida glabrata, Candida tropicalis and Aspergillus fumigatus, in most cases such activity being higher than that observed for amphotericin B and hygromycin. Our results point to the significant role of tri5 in the production of trichodermin and in the antifungal activity of T. brevicompactum.

The contribution of Trichoderma to balancing the costs of plant growth and defense

Trichoderma is a fungal genus of cosmopolitan distribution and high biotechnological value, with several species currently used as biological control agents. Additionally, the enzyme systems of the fungus are widely applied in industry. Species of Trichoderma protect plants against the attack of soil-borne plant pathogens by competing for nutrients and inhibiting or killing plant pathogenic fungi and oomycetes, through the production of antibiotics and/or hydrolytic enzymes. In addition to the role of Trichoderma spp. as biocontrol agents, they have other beneficial effects on plants, including the stimulation of plant defenses and the promotion of plant growth. In this review, we focus on the complex plant defense signaling network that allows the recognition of fungi as non-hostile microbes, including microbial-associated molecular patterns (MAMPs), damage-associated molecular patterns (DAMPs) and secreted elicitors. We also examine how fungal interactions with plant receptors can activate induced resistance by priming and balancing plant defense and growth responses. Our observations are integrated into a model describing Trichoderma-plant hormone signaling network interactions.

Cell wall-degrading isoenzyme profiles of Trichoderma biocontrol strains show correlation with rDNA taxonomic species

Trichoderma is known for being the most frequently used biocontrol agent in agriculture. A fundamental part of the Trichoderma antifungal system relies on a series of genes coding for a variety of extracellular lytic enzymes. Characterization of the polymorphism between five putative isoenzymatic activities [β-1,3-glucanase (EC 3.2.1.39, EC 3.2.1.58), β-1,6-glucanase (EC 3.2.1.75), cellulase (EC 3.2.1.4; EC 3.2.1.21, EC 3.2.1.91), chitinase (EC 3.2.1.30, EC 3.2.1.52), protease (EC 3.4.11; EC 3.4.13–19; EC 3.4.21–24, EC 3.4.99)] was carried out using 18 strains from three sections of Trichoderma. Of these, seven strains were from T. sect. Pachybasium, nine from T. sect. Trichoderma and two from T. sect. Longibrachiatum. Thirty-seven different alleles in total were identified: 13 for β-1,3-glucanase, four for β-1,6-glucanase, three for cellulase, eight for chitinase and nine for protease activity. A dendrogram (constructed by the unweighted pair group method with arithmetic averages) based on isoenzymatic data separated the 18 strains into three main enzymatic groups: T. harzianum, T. atroviride/T. viride/T. koningii and T. asperellum/T. hamatum/T. longibrachiatum. Isoenzymatic groupings obtained from biocontrol strains are discussed in relation to their phylogenetic location, based on their sequence of internal transcribed spacer 1 in ribosomal DNA and their antifungal activities.