Trichoderma-plant Interactions Research Articles

New Insights into the Mechanism of Trichoderma virens-Induced Developmental Effects on Agrostis stolonifera Disease Resistance against Dollar Spot Infection.

Trichoderma is internationally recognized as a biocontrol fungus for its broad-spectrum antimicrobial activity. Intriguingly, the crosstalk mechanism between the plant and Trichoderma is dynamic, depending on the Trichoderma strains and the plant species. In our previous study, the Trichoderma virens 192-45 strain showed better pathogen inhibition through the secretive non-volatile and volatile substrates. Therefore, we studied transcriptional and metabolic responses altered in creeping bentgrass (Agrostis stolonifera L.) with T. virens colonization prior to a challenge with Clarireedia homoeocarpa. This fungal pathogen causes dollar spot on various turfgrasses. When the pathogen is deficient, the importance of T. virens to the enhancement of plant growth can be seen in hormonal production and microbe signaling, such as indole-3-acrylic acid. Therefore, these substrates secreted by T. virens and induced genes related to plant growth can be the 'pre-defense' for ensuing pathogen attacks. During C. homoeocarpa infection, the Trichoderma-plant interaction activates defense responses through the SA- and/or JA-dependent pathway, induced by T. virens and its respective exudates, such as oleic, citric, and stearic acid. Thus, we will anticipate a combination of genetic engineering and exogenous application targeting these genes and metabolites, which could make creeping bentgrass more resistant to dollar spot and other pathogens.

Nitrogen availability determines plant growth promotion and the induction of root branching by the probiotic fungus Trichoderma atroviride in Arabidopsis seedlings.

Plant growth-promoting fungi are integral components of the root microbiome that help the host resist biotic and abiotic stress while improving nutrient acquisition. Trichoderma atroviride is a common inhabitant of the rhizosphere, which establishes a perdurable symbiosis with plants through the emission of volatiles, diffusible compounds, and robust colonization. Currently, little is known on how the environment influences the Trichoderma-plant interaction. In this report, we assessed plant growth and root architectural reconfiguration of Arabidopsis seedlings grown in physical contact with T. atroviride under contrasting nitrate and ammonium availability. The shoot and root biomass accumulation and lateral root formation triggered by the fungus required high nitrogen supplements and involved nitrate reduction via AtNIA1 and NIA2. Ammonium supplementation did not restore biomass production boosted by T. atroviride in nia1nia2 double mutant, but instead fungal inoculation increased nitric oxide accumulation in Arabidopsis primary root tips depending upon nitrate supplements. N deprived seedlings were largely resistant to the effects of nitric oxide donor SNP triggering lateral root formation. T. atroviride enhanced expression of CHL1:GUS in root tips, particularly under high N supplements and required an intact CHL1 nitrate transporter to promote lateral root formation in Arabidopsis seedlings. These data imply that the developmental programs strengthened by Trichoderma and the underlying growth promotion in plants are dependent upon adequate nitrate nutrition and may involve nitric oxide as a second messenger.

The fungal NADPH oxidase is an essential element for the molecular dialog between Trichoderma and Arabidopsis.

Members of the fungal genus Trichoderma stimulate growth and reinforce plant immunity. Nevertheless, how fungal signaling elements mediate the establishment of a successful Trichoderma-plant interaction is largely unknown. In this work, we analyzed growth, root architecture and defense in an Arabidopsis-Trichoderma co-cultivation system, including the wild-type (WT) strain of the fungus and mutants affected in NADPH oxidase. Global gene expression profiles were assessed in both the plant and the fungus during the establishment of the interaction. Trichoderma atroviride WT improved root branching and growth of seedling as previously reported. This effect diminished in co-cultivation with the ∆nox1, ∆nox2 and ∆noxR null mutants. The data gathered of the Arabidopsis interaction with the ∆noxR strain showed that the seedlings had a heightened immune response linked to jasmonic acid in roots and shoots. In the fungus, we observed repression of genes involved in complex carbohydrate degradation in the presence of the plant before contact. However, in the absence of NoxR, such repression was lost, apparently due to a poor ability to adequately utilize simple carbon sources such as sucrose, a typical plant exudate. Our results unveiled the critical role played by the Trichoderma NoxR in the establishment of a fine-tuned communication between the plant and the fungus even before physical contact. In this dialog, the fungus appears to respond to the plant by adjusting its metabolism, while in the plant, fungal perception determines a delicate growth-defense balance.

The Trichoderma harzianum Kelch Protein ThKEL1 Plays a Key Role in Root Colonization and the Induction of Systemic Defense in Brassicaceae Plants.

The fungal genus Trichoderma includes strains with biocontrol and/or biostimulant potential and is recognized as a source of genes with biotechnological value. In a previous study the Kelch domain protein, encoded by the Thkel1 gene of Trichoderma harzianum T34, was found to confer tolerance to salt stress when expressed in plants of Arabidopsis thaliana. In the present work, we have overexpressed Thkel1 in rapeseed plants in order to generate an additional biotechnological tool for analyzing the role of this gene in Trichoderma-plant interactions. The overexpression of this gene in Brassicaceae plants improves responses to pathogens through the induction of systemic defenses mediated by jasmonic acid, facilitates root colonization by modulating the myrosinase activity, and, as a result, increases plant productivity. These effects were also observed in Thkel1 overexpressing plants subjected to abiotic stress conditions. Additionally, the differences detected in root colonization levels by T. harzianum wild type and Thkel1 silenced transformants between Arabidopsis or rapeseed and tomato plants indicate that ThKEL1 interacts in different ways in Brassicaceae and non-Brassicaceae plants.

Tal6 From Trichoderma atroviride Is a LysM Effector Involved in Mycoparasitism and Plant Association.

LysM effectors play a relevant role during the plant colonization by successful phytopathogenic fungi, since they enable them to avoid either the triggering of plant defense mechanisms or their attack effects. Tal6, a LysM protein from Trichoderma atroviride, is capable of binding to complex chitin. However, until now its biological function is not completely known, particularly its participation in plant–Trichoderma interactions. We obtained T. atroviride Tal6 null mutant and Tal6 overexpressing strains and determined the role played by this protein during Trichoderma-plant interaction and mycoparasitism. LysM effector Tal6 from T. atroviride protects the hyphae from chitinases by binding to chitin of the fungal cell wall, increases the fungus mycoparasitic capacity, and modulates the activation of the plant defense system. These results show that beneficial fungi also employ LysM effectors to improve their association with plants.

TgSWO from Trichoderma guizhouense NJAU4742 promotes growth in cucumber plants by modifying the root morphology and the cell wall architecture

BackgroundColonization of Trichoderma spp. is essential for exerting their beneficial functions on the plant. However, the interactions between Trichoderma spp. and plant roots are still not completely understood. The aim of this study was to investigate how TgSWO affect Trichoderma guizhouense to establish themselves in the plant rhizosphere and promote plant growth. In this study, we deeply analyzed the molecular mechanism by which the functional characterization of the TgSWO by expressing different functional region deletion proteins (FRDP) of TgSWO.ResultsRoot scanning analysis results showed that TgSWO could dramatically increase root density and promote growth. In addition, we also found that TgSWO could expand root cell walls, subsequently increase root colonization. Moreover, knockout of TgSWO mutants (KO) or overexpression of TgSWO mutants (OE) produced greatly reduced or increased the number of cucumber root, respectively. To clarify the molecular mechanism of TgSWO in plant-growth-promotion, we analyzed the ability of different FRDP to expand the root cell wall. The root cell wall architecture were considerably altered when treated by ΔCBD protein (the TgSWO gene of lacking in the CBD domain was cloned and heterologously expressed), in correlation with the present YoaJ domain of TgSWO. In contrast, neither the expansion of cell walls nor the increase of roots was detectable in ΔYoaJ protein.ConclusionsOur results emphasize the YoaJ domain is the most critical functional area of TgSWO during the alteration of cell wall architecture. Simultaneously, the results obtained in this study also indicate that TgSWO might play a plant-growth-promotion role in the Trichoderma-plant interactions by targeting the root cell wall.

Elicitor hydrophobin Hyd1 interacts with Ubiquilin1-like to induce maize systemic resistance.

Trichoderma harzianum is a plant-beneficial fungus that secretes small cysteine-rich proteins that induce plant defense responses; however, the molecular mechanism involved in this induction is largely unknown. Here, we report that the class II hydrophobin ThHyd1 acts as an elicitor of induced systemic resistance (ISR) in plants. Immunogold labeling and immunofluorescence revealed ThHyd1 localized on maize (Zea mays) root cell plasma membranes. To identify host plant protein interactors of Hyd1, we screened a maize B73 root cDNA library. ThHyd1 interacted directly with ubiquilin 1-like (UBL). Furthermore, the N-terminal fragment of UBL was primarily responsible for binding with Hyd1 and the eight-cysteine amino acid of Hyd1 participated in the protein-protein interactions. Hyd1 from T. harzianum (Thhyd1) and ubl from maize were co-expressed in Arabidopsis thaliana, they synergistically promoted plant resistance against Botrytis cinerea. RNA-sequencing analysis of global gene expression in maize leaves 24 h after spraying with Curvularia lunata spore suspension showed that Thhyd1-induced systemic resistance was primarily associated with brassinosteroid signaling, likely mediated through BAK1. Jasmonate/ethylene (JA/ET) signaling was also involved to some extent in this response. Our results suggest that the Hyd1-UBL axis might play a key role in inducing systemic resistance as a result of Trichoderma-plant interactions.

Influence of three different soil types on the interaction of two strains of Trichoderma harzianum with Brassica rapa subsp. sylvestris cv. esculenta, under soil mineral fertilization

The influence of Trichoderma fungi on growth, yield and nutrient status of food crops has been widely studied. However, a lack of knowledge still exists on the effect of agricultural soils with different geological nature, land use and management on Trichoderma-plant interaction. This study aims to evaluate the influence of three soil types, with different physicochemical properties and under fertilization by NPK-based nutrient solutions, on the efficacy of two T. harzianum (T22 and M10) strains on the growth, nutrient uptake and glucosinolate concentration in Brassica rapa subsp. sylvestris cv. esculenta. Overall data showed that the biofertilization by Trichoderma strains is very effective to enhance the fertility and productivity of the nutrient-poor soils even in the absence of fertilization by NPK-nutrient solutions, as well as to stimulate the production of glucosinolates by foliar biomass of B. rapa, although this effect was strongly dependent on the soil type.

Ferricrocin, the intracellular siderophore of Trichoderma virens, is involved in growth, conidiation, gliotoxin biosynthesis and induction of systemic resistance in maize

Fungal siderophores are known to be involved in iron acquisition and storage, as well as pathogenicity of mammals and plants. As avirulent plant symbionts, Trichoderma spp. colonize roots and induce resistance responses both locally and systemically. To study the role of intracellular siderophore(s) in Trichoderma-plant interactions, we have obtained mutants in a non-ribosomal peptide synthetase, TvTex10, that was predicted to be involved in intracellular siderophore(s) biosynthesis. This gene has a detectable basal level of expression and is also upregulated under iron-deplete conditions. This is unlike two other siderophore-encoding genes, which are tightly regulated by iron. Disruption of tex10 gene using homologous recombination resulted in mutants with enhanced growth rate, reduced conidiation and hyper-sensitivity to oxidative stress as compared to wildtype strain. The mutants also produced reduced levels of gliotoxin and dimethyl gliotoxin but have enhanced ability to colonize maize seedling roots. The mutants were also impaired in induction of induced systemic resistance (ISR) in maize against the foliar pathogen Cochliobolus heterostrophus.

Functional Characterization of TvCyt2, a Member of the p450 Monooxygenases From Trichoderma virens Relevant During the Association With Plants and Mycoparasitism.

Secondary metabolites are crucial for the establishment of interactions between plants and microbes, as in the case of Trichoderma-plant interactions. In the biosynthetic pathway of secondary metabolites, specific enzymes participate in the formation of hydroxyl and epoxy groups, belonging to the p450 monooxygenases family. Here, we show that the product of the gene TvCyt2 from Trichoderma virens encodes a new protein homologous to the cytochrome p450, which is down-regulated at the beginning of Trichoderma-Arabidopsis interaction. To investigate its role in the interactions established by Trichoderma spp., we analyzed the metabolic profile obtained from the overexpressing (OETvCyt2) and null mutant (Δtvcyt2) strains, observing that the OETvCyt2 strains produce a higher concentration of some metabolites than the wild-type (WT) strain. Δtvcyt2 strains showed a decreased antagonistic activity against Rhizoctonia solani in antibiosis assays. Arabidopsis plants cocultivated with the OETvCyt2 strains showed stronger induction of systemic acquired resistance than plants cocultivated with the WT strain, as well as increases in biomass and fitness. Our data suggest that the product of the TvCyt2 gene is involved in secondary metabolite biosynthesis, which can increase antagonistic activity with phytopathogenic fungi and the capacity to promote plant growth.

Cellular and molecular insight into the inhibition of primary root growth of Arabidopsis induced by peptaibols, a class of linear peptide antibiotics mainly produced by Trichoderma spp.

Trichoderma spp. are well known biocontrol agents that produce a variety of antibiotics. Peptaibols are a class of linear peptide antibiotics mainly produced by Trichoderma Alamethicin, the most studied peptaibol, is reported as toxic to plants at certain concentrations, while the mechanisms involved are unclear. We illustrated the toxic mechanisms of peptaibols by studying the growth-inhibitory effect of Trichokonin VI (TK VI), a peptaibol from Trichoderma longibrachiatum SMF2, on Arabidopsis primary roots. TK VI inhibited root growth by suppressing cell division and cell elongation, and disrupting root stem cell niche maintenance. TK VI increased auxin content and disrupted auxin response gradients in root tips. Further, we screened the Arabidopsis TK VI-resistant mutant tkr1. tkr1 harbors a point mutation in GORK, which encodes gated outwardly rectifying K(+)channel proteins. This mutation alleviated TK VI-induced suppression of K(+)efflux in roots, thereby stabilizing the auxin gradient. The tkr1 mutant also resisted the phytotoxicity of alamethicin. Our results indicate that GORK channels play a key role in peptaibol-plant interaction and that there is an inter-relationship between GORK channels and maintenance of auxin homeostasis. The cellular and molecular insight into the peptaibol-induced inhibition of plant root growth advances our understanding of Trichoderma-plant interactions.

The toolbox of Trichoderma spp. in the biocontrol of Botrytis cinerea disease.

Botrytis cinerea is a necrotrophic fungal pathogen causing disease in many plant species, leading to economically important crop losses. So far, fungicides have been widely used to control this pathogen. However, in addition to their detrimental effects on the environment and potential risks for human health, increasing fungicide resistance has been observed in the B. cinerea population. Biological control, that is the application of microbial organisms to reduce disease, has gained importance as an alternative or complementary approach to fungicides. In this respect, the genus Trichoderma constitutes a promising pool of organisms with potential for B. cinerea control. In the first part of this article, we review the specific mechanisms involved in the direct interaction between the two fungi, including mycoparasitism, the production of antimicrobial compounds and enzymes (collectively called antagonism), and competition for nutrients and space. In addition, biocontrol has also been observed when Trichoderma is physically separated from the pathogen, thus implying an indirect systemic plant defence response. Therefore, in the second part, we describe the consecutive steps leading to induced systemic resistance (ISR), starting with the initial Trichoderma-plant interaction and followed by the activation of downstream signal transduction pathways and, ultimately, the defence response resulting in ISR (ISR-prime phase). Finally, we discuss the ISR-boost phase, representing the effect of ISR priming by Trichoderma spp. on plant responses after additional challenge with B. cinerea.

Overexpression of erg1 gene in Trichoderma harzianum CECT 2413: effect on the induction of tomato defence-related genes.

To investigate the effect of the overexpression of erg1 gene of Trichoderma harzianum CECT 2413 (T34) on the Trichoderma-plant interactions and in the biocontrol ability of this fungus. Transformants of T34 strain overexpressing erg1 gene did not show effect on the ergosterol level, although a drastic decrease in the squalene level was observed in the transformants at 96 h of growth. During interaction with plants, the erg1 overexpression resulted in a reduction of the priming ability of several tomato defence-related genes belonging to the salicylate pathway, and also of the TomLoxA gene, which is related to the jasmonate pathway. Interestingly, other jasmonate-related genes, such as PINI and PINII, were slightly induced. The erg1 overexpressed transformants also showed a reduced ability to colonize tomato roots. The ergosterol biosynthetic pathway might play an important role in regulating Trichoderma-plant interactions, although this role does not seem to be restricted to the final product; instead, other intermediates such as squalene, whose role in the Trichoderma-plant interaction has not been characterized, would also play an important role. The functional analysis of genes involved in the synthesis of ergosterol could provide additional strategies to improve the ability of biocontrol of the Trichoderma strains and their interaction with 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.

Functional analysis of non-ribosomal peptide synthetases (NRPSs) in Trichoderma virens reveals a polyketide synthase (PKS)/NRPS hybrid enzyme involved in the induced systemic resistance response in maize

Trichoderma virens genome harbours genes encoding 22 non-ribosomal peptide synthetases (NRPSs) with at least one complete module (containing adenylation, thiolation and condensation domains) and four PKS/NRPS (polyketide synthase/NRPS) hybrid enzymes. After a primary screen for expression of these 26 genes when mycelia of T. virens are in contact with maize roots, seven genes that are upregulated were selected for further study. Using homologous recombination, loss-of-function mutants in six of these were obtained (the seventh, tex2, was acquired from our previous studies). Plant assays in a hydroponics system revealed that all seven mutants retained the ability to internally colonize maize roots. However, a mutation in one of the PKS/NRPS hybrid genes impaired the ability of T. virens to induce the defence response gene pal (phenylalanine ammonia lyase), suggesting a putative role for the associated metabolite product in induced systemic resistance. Interestingly, the mutant retained its ability to induce another defence response gene aos (allene oxide synthase). We thus provide evidence that a PKS/NRPS hybrid enzyme is involved in Trichoderma-plant interactions resulting in induction of defence responses.

Gene expression analysis of the biocontrol fungus Trichoderma harzianum in the presence of tomato plants, chitin, or glucose using a high-density oligonucleotide microarray.

BackgroundIt has recently been shown that the Trichoderma fungal species used for biocontrol of plant diseases are capable of interacting with plant roots directly, behaving as symbiotic microorganisms. With a view to providing further information at transcriptomic level about the early response of Trichoderma to a host plant, we developed a high-density oligonucleotide (HDO) microarray encompassing 14,081 Expressed Sequence Tag (EST)-based transcripts from eight Trichoderma spp. and 9,121 genome-derived transcripts of T. reesei, and we have used this microarray to examine the gene expression of T. harzianum either alone or in the presence of tomato plants, chitin, or glucose.ResultsGlobal microarray analysis revealed 1,617 probe sets showing differential expression in T. harzianum mycelia under at least one of the culture conditions tested as compared with one another. Hierarchical clustering and heat map representation showed that the expression patterns obtained in glucose medium clustered separately from the expression patterns observed in the presence of tomato plants and chitin. Annotations using the Blast2GO suite identified 85 of the 257 transcripts whose probe sets afforded up-regulated expression in response to tomato plants. Some of these transcripts were predicted to encode proteins related to Trichoderma-host (fungus or plant) associations, such as Sm1/Elp1 protein, proteases P6281 and PRA1, enchochitinase CHIT42, or QID74 protein, although previously uncharacterized genes were also identified, including those responsible for the possible biosynthesis of nitric oxide, xenobiotic detoxification, mycelium development, or those related to the formation of infection structures in plant tissues.ConclusionThe effectiveness of the Trichoderma HDO microarray to detect different gene responses under different growth conditions in the fungus T. harzianum strongly indicates that this tool should be useful for further assays that include different stages of plant colonization, as well as for expression studies in other Trichoderma spp. represented on it. Using this microarray, we have been able to define a number of genes probably involved in the transcriptional response of T. harzianum within the first hours of contact with tomato plant roots, which may provide new insights into the mechanisms and roles of this fungus in the Trichoderma-plant interaction.

Role of Swollenin, an Expansin-Like Protein fromTrichoderma, in Plant Root Colonization

Swollenin, a protein first characterized in the saprophytic fungus Trichoderma reesei, contains an N-terminal carbohydrate-binding module family 1 domain (CBD) with cellulose-binding function and a C-terminal expansin-like domain. This protein was identified by liquid chromatography-mass spectrometry among many other cellulolytic proteins secreted in the coculture hydroponics medium of cucumber (Cucumis sativus) seedlings and Trichoderma asperellum, a well-known biocontrol agent and inducer of plant defense responses. The swollenin gene was isolated and its coding region was overexpressed in the same strain under the control of the constitutive pki1 promoter. Trichoderma transformants showed a remarkably increased ability to colonize cucumber roots within 6 h after inoculation. On the other hand, overexpressors of a truncated swollenin sequence bearing a 36-amino acid deletion of the CBD did not differ from the wild type, showing in vivo that this domain is necessary for full protein activity. Root colonization rates were reduced in transformants silenced in swollenin gene expression. A synthetic 36-mer swollenin CBD peptide was shown to be capable of stimulating local defense responses in cucumber roots and leaves and to afford local protection toward Botrytis cinerea and Pseudomonas syringae pv lachrymans infection. This indicates that the CBD domain might be recognized by the plant as a microbe-associated molecular pattern in the Trichoderma-plant interaction.