Trichoderma-plant root colonization: escaping early plant defense responses and activation of the antioxidant machinery for saline stress tolerance.

Trichoderma spp. are versatile opportunistic plant symbionts which can colonize the apoplast of plant roots. Microarrays analysis of Arabidopsis thaliana roots inoculated with Trichoderma asperelloides T203, coupled with qPCR analysis of 137 stress responsive genes and transcription factors, revealed wide gene transcript reprogramming, proceeded by a transient repression of the plant immune responses supposedly to allow root colonization. Enhancement in the expression of WRKY18 and WRKY40, which stimulate JA-signaling via suppression of JAZ repressors and negatively regulate the expression of the defense genes FMO1, PAD3 and CYP71A13, was detected in Arabidopsis roots upon Trichoderma colonization. Reduced root colonization was observed in the wrky18/wrky40 double mutant line, while partial phenotypic complementation was achieved by over-expressing WRKY40 in the wrky18 wrky40 background. On the other hand increased colonization rate was found in roots of the FMO1 knockout mutant. Trichoderma spp. stimulate plant growth and resistance to a wide range of adverse environmental conditions. Arabidopsis and cucumber (Cucumis sativus L.) plants treated with Trichoderma prior to salt stress imposition show significantly improved seed germination. In addition, Trichoderma treatment affects the expression of several genes related to osmo-protection and general oxidative stress in roots of both plants. The MDAR gene coding for monodehydroascorbate reductase is significantly up-regulated and, accordingly, the pool of reduced ascorbic acid was found to be increased in Trichoderma treated plants. 1-Aminocyclopropane-1-carboxylate (ACC)-deaminase silenced Trichoderma mutants were less effective in providing tolerance to salt stress, suggesting that Trichoderma, similarly to ACC deaminase producing bacteria, can ameliorate plant growth under conditions of abiotic stress, by lowering ameliorating increases in ethylene levels as well as promoting an elevated antioxidative capacity.

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.

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.

Transcript and metabolite analysis of the Trichoderma-induced systemic resistance response to Pseudomonas syringae in Arabidopsis thaliana.

In the present study we have assessed, by transcriptional and metabolic profiling, the systemic defence response of Arabidopsis thaliana plants to the leaf pathogen Pseudomonas syringae pv. tomato DC3000 (Pst) induced by the beneficial fungus Trichoderma asperelloides T203. Expression analysis (qPCR) of a set of 137 Arabidopsis genes related to Pst defence responses showed that T203 root colonization is not associated with major detectable transcriptomic changes in leaves. However, plants challenged with the bacterial pathogen showed quantitative differences in gene expression when pre-inoculated with T203, supporting priming of the plant by this beneficial fungus. Among the defence-related genes affected by T203, lipid transfer protein (LTP)4, which encodes a member of the lipid transfer pathogenesis-related family, is upregulated, whereas the WRKY40 transcription factor, known to contribute to Arabidopsis susceptibility to bacterial infection, shows reduced expression. On the other hand, root colonization by this beneficial fungus substantially alters the plant metabolic profile, including significant changes in amino acids, polyamines, sugars and citric acid cycle intermediates. This may in part reflect an increased energy supply required for the activation of plant defences and growth promotion effects mediated by Trichoderma species.

The qid74 gene from Trichoderma harzianum has a role in root architecture and plant biofertilization.

The Trichoderma harzianum qid74 gene encodes a cysteine-rich cell wall protein that has an important role in adherence to hydrophobic surfaces and cellular protection; this gene was upregulated in Trichoderma high-density oligonucleotide (HDO) microarrays in interaction with tomato roots. Using a collection of qid74-overexpressing and disrupted mutants the role of this gene in cucumber and tomato root architecture was analysed in hydroponic and soil systems under greenhouse conditions. No significant differences were found in the pattern of root colonization and the length of primary roots of cucumber or tomato plants inoculated by T. harzianum CECT 2413 wild-type (wt) strain or any of the qid74 transformants. However, compared to the wt treatment, lateral roots were significantly longer in plants inoculated with the overexpressing transformants, and shorter in those treated with the disruptant strains. Microscopic observations revealed more and longer secondary root hairs in cucumber plants treated with the qid74-overexpressing mutants and fewer and shorter hairs in roots treated with qid74-disrupted transformants, compared to those observed in plants inoculated with the wt strain. qid74-induced modifications in root architecture increased the total absorptive surface, facilitating nutrient uptake and translocation of nutrients in the shoots, resulting in increased plant biomass through an efficient use of NPK and micronutrients.

Marine isolates of Trichoderma spp. as potential halotolerant agents of biological control for arid-zone agriculture.

The scarcity of fresh water in the Mediterranean region necessitates the search for halotolerant agents of biological control of plant diseases that can be applied in arid-zone agriculture irrigated with saline water. Among 29 Trichoderma strains previously isolated from Mediterranean Psammocinia sp. sponges, the greatest number of isolates belong to the Trichoderma longibrachiatum-Hypocrea orientalis species pair (9), H. atroviridis/T. atroviride (9), and T. harzianum species complex (7), all of which are known for high mycoparasitic potential. In addition, one isolate of T. asperelloides and two putative new species, Trichoderma sp. O.Y. 14707 and O.Y. 2407, from Longibrachiatum and Strictipilosa clades, respectively, have been identified. In vitro salinity assays showed that the ability to tolerate increasing osmotic pressure (halotolerance) is a strain- or clade-specific property rather than a feature of a species. Only a few isolates were found to be sensitive to increased salinity, while others either were halotolerant or even demonstrated improved growth in increasingly saline conditions. In vitro antibiosis assays revealed strong antagonistic activity toward phytopathogens due to the production of both soluble and volatile metabolites. Two marine-derived Trichoderma isolates, identified as T. atroviride and T. asperelloides, respectively, effectively reduced Rhizoctonia solani damping-off disease on beans and also induced defense responses in cucumber seedlings against Pseudomonas syringae pv. lachrimans. This is the first inclusive evaluation of marine fungi as potential biocontrol agents.

Synthetic ultrashort cationic lipopeptides induce systemic plant defense responses against bacterial and fungal pathogens.

A new family of synthetic, membrane-active, ultrashort lipopeptides composed of only four amino acids linked to fatty acids was tested for the ability to induce systemic resistance and defense responses in plants. We found that two peptides wherein the third residue is a d-enantiomer (italic), C16-KKKK and C16-KLLK, can induce medium alkalinization of tobacco suspension-cultured cells and expression of defense-related genes in cucumber and Arabidopsis seedlings. Moreover, these compounds can prime systemic induction of antimicrobial compounds in cucumber leaves similarly to the plant-beneficial fungus Trichoderma asperellum T203 and provide systemic protection against the phytopathogens Botrytis cinerea B05, Pseudomonas syringae pv. lachrimans, and P. syringae pv. tomato DC3000. Thus, short cationic lipopeptides are a new category of compounds with potentially high utility in the induction of systemic resistance in plants.

Trichoderma species--opportunistic, avirulent plant symbionts.

Trichoderma spp. are free-living fungi that are common in soil and root ecosystems. Recent discoveries show that they are opportunistic, avirulent plant symbionts, as well as being parasites of other fungi. At least some strains establish robust and long-lasting colonizations of root surfaces and penetrate into the epidermis and a few cells below this level. They produce or release a variety of compounds that induce localized or systemic resistance responses, and this explains their lack of pathogenicity to plants. These root-microorganism associations cause substantial changes to the plant proteome and metabolism. Plants are protected from numerous classes of plant pathogen by responses that are similar to systemic acquired resistance and rhizobacteria-induced systemic resistance. Root colonization by Trichoderma spp. also frequently enhances root growth and development, crop productivity, resistance to abiotic stresses and the uptake and use of nutrients.

Isolation of two aspartyl proteases from Trichoderma asperellum expressed during colonization of cucumber roots.

Trichoderma asperellum and cucumber seedlings were used as a model to study the modulation of Trichoderma gene expression during plant root colonization. Seedlings were grown in an aseptic hydroponics medium and inoculated with Trichoderma spore suspension. Proteins differentially secreted into the medium were isolated. Three major proteins of fungal origin were identified: two arabinofuranosidases (Abf1 and Abf2) and an aspartyl protease. Differential mRNA display was conducted on Trichoderma mycelia interacting and non-interacting, with the plant roots. Among the differentially regulated clones another aspartyl protease was identified. Sequencing of the genes revealed that the first aspartyl protease is a close homologue of PapA from T. harzianum and the other, of AP1 from Botryotinia fuckeliana. RT-PCR analysis confirms that the proteases are induced in response to plant roots attachment and are expressed in planta. papA, but not papB, is also induced in plate confrontation assays with the plant pathogen Rhizoctonia solani. These data suggest that the identified proteases play a role in Trichoderma both as a mycoparasite and as a plant opportunistic symbiont.

The LysM receptor-like kinase LysM RLK1 is required to activate defense and abiotic-stress responses induced by overexpression of fungal chitinases in Arabidopsis plants.

Application of crab shell chitin or pentamer chitin oligosaccharide to Arabidopsis seedlings increased tolerance to salinity in wild-type but not in knockout mutants of the LysM Receptor-Like Kinase1 (CERK1/LysM RLK1) gene, known to play a critical role in signaling defense responses induced by exogenous chitin. Arabidopsis plants overexpressing the endochitinase chit36 and hexoaminidase excy1 genes from the fungus Trichoderma asperelleoides T203 showed increased tolerance to salinity, heavy-metal stresses, and Botrytis cinerea infection. Resistant lines, overexpressing fungal chitinases at different levels, were outcrossed to lysm rlk1 mutants. Independent homozygous hybrids lost resistance to biotic and abiotic stresses, despite enhanced chitinase activity. Expression analysis of 270 stress-related genes, including those induced by reactive oxygen species (ROS) and chitin, revealed constant up-regulation (at least twofold) of 10 genes in the chitinase-overexpressing line and an additional 76 salt-induced genes whose expression was not elevated in the lysm rlk1 knockout mutant or the hybrids harboring the mutation. These findings elucidate that chitin-induced signaling mediated by LysM RLK1 receptor is not limited to biotic stress response but also encompasses abiotic-stress signaling and can be conveyed by ectopic expression of chitinases in plants.

Characterization of ACC deaminase from the biocontrol and plant growth-promoting agent Trichoderma asperellum T203.

1-aminocyclopropane-1-carboxylate (ACC) deaminase activity was evaluated in the biocontrol and plant growth-promoting fungus Trichoderma asperellum T203. Fungal cultures grown with ACC as the sole nitrogen source showed high enzymatic activity. The enzyme encoding gene (Tas-acdS) was isolated, and an average 3.5-fold induction of the gene by 3 mM ACC was detected by real-time PCR. Escherichia coli bacteria carrying the intron-free cDNA of Tas-acdS cloned into the vector pAlter-EX1 under the control of the tac promoter revealed specific ACC deaminase (ACCD) activity and the ability to promote canola (Brassica napus) root elongation in pouch assays. RNAi silencing of the ACCD gene in T. asperellum showed decreased ability of the mutants to promote root elongation of canola seedlings. These data suggest a role for ACCD in the plant root growth-promotion effect by T. asperellum.

Role of swollenin, an expansin-like protein from Trichoderma, 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.

Inhibition of fungal and bacterial plant pathogens in vitro and in planta with ultrashort cationic lipopeptides.

Plant diseases constitute an emerging threat to global food security. Many of the currently available antimicrobial agents for agriculture are highly toxic and nonbiodegradable and cause extended environmental pollution. Moreover, an increasing number of phytopathogens develop resistance to them. Recently, we have reported on a new family of ultrashort antimicrobial lipopeptides which are composed of only four amino acids linked to fatty acids (A. Makovitzki, D. Avrahami, and Y. Shai, Proc. Natl. Acad. Sci. USA 103:15997-16002, 2006). Here, we investigated the activities in vitro and in planta and the modes of action of these short lipopeptides against plant-pathogenic bacteria and fungi. They act rapidly, at low micromolar concentrations, on the membranes of the microorganisms via a lytic mechanism. In vitro microscopic analysis revealed wide-scale damage to the microorganism's membrane, in addition to inhibition of pathogen growth. In planta potent antifungal activity was demonstrated on cucumber fruits and leaves infected with the pathogen Botrytis cinerea as well as on corn leaves infected with Cochliobolus heterostrophus. Similarly, treatment with the lipopeptides of Arabidopsis leaves infected with the bacterial leaf pathogen Pseudomonas syringae efficiently and rapidly reduced the number of bacteria. Importantly, in contrast to what occurred with many native lipopeptides, no toxicity was observed on the plant tissues. These data suggest that the ultrashort lipopeptides could serve as native-like antimicrobial agents economically feasible for use in plant protection.

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.

TasHyd1, a new hydrophobin gene from the biocontrol agent Trichoderma asperellum, is involved in plant root colonization.

SUMMARY A hydrophobin-like clone (TasHyd1) was isolated during a PCR differential mRNA display analysis conducted on Trichoderma asperellum mycelia interacting with plant roots. The open reading frame encodes a 145-amino-acid protein showing similarity to Pbhyd1, a Class I hydrophobin from the dimorphic fungus Paracoccidioides brasiliensis. TasHyd1 expression was detected in planta up to 5 days after Trichoderma root inoculation. TasHyd1 is constitutively expressed at low levels in mycelia in young cultures but gene expression is not detected in sporulating hyphae or in non-germinating spores. Carbon limitation stimulates expression of TasHyd1 whereas nitrogen or phosphate starvation down-regulate expression. TasHyd1 fused to an HA tag was over-expressed in Trichoderma and the protein was detected with an anti-HA antibody in the trifluoroacetic-acid-soluble fraction of mycelial cell walls. Over-expressor mutants were not affected in their mycoparasitic activity when tested in vitro against the plant pathogen Rhizoctonia solani and retained root colonization capacity comparable with that of the wild-type. TasHyd1 deletion mutants had no significant reduction in in vitro mycoparasitic activity but were altered in their wettability and were severely impaired in root attachment and colonization. These phenotypes were recovered by complementation of TasHyd1, indicating that the protein is a new hydrophobin that contributes to Trichoderma interaction with the plant.

Trichoderma mitogen-activated protein kinase signaling is involved in induction of plant systemic resistance.

The role of a mitogen-activated protein kinase (MAPK) TmkA in inducing systemic resistance in cucumber against the bacterial pathogen Pseudomonas syringae pv. lacrymans was investigated by using tmkA loss-of-function mutants of Trichoderma virens. In an assay where Trichoderma spores were germinated in proximity to cucumber roots, the mutants were able to colonize the plant roots as effectively as the wild-type strain but failed to induce full systemic resistance against the leaf pathogen. Interactions with the plant roots enhanced the level of tmkA transcript in T. virens and its homologue in Trichoderma asperellum. At the protein level, we could detect the activation of two forms reacting to the phospho-p44/42 MAPK antibody. Biocontrol experiments demonstrated that the tmkA mutants retain their biocontrol potential against Rhizoctonia solani in soil but are not effective against Sclerotium rolfsii in reducing disease incidence. Our results show that, unlike in many plant-pathogen interactions, Trichoderma TmkA MAPK is not involved in limited root colonization. Trichoderma, however, needs MAPK signaling in order to induce full systemic resistance in the plant.

Significance of lytic enzymes from Trichoderma spp. in the biocontrol of fungal plant pathogens.

The use of specific mycolytic soil microorganisms to control plant pathogens is an ecological approach to overcome the problems caused by standard chemical methods of plant protection. The ability to produce lytic enzymes is a widely distributed property of rhizosphere-competent fungi and bacteria. Due to the higher activity of Trichoderma spp. lytic enzymes as compared to the same class of enzymes from other microorganisms and plants, effort is being aimed at improving biocontrol agents and plants by introducing Trichoderma genes via genetic manipulations. An overview is presented of the data currently available on lytic enzymes from the mycoparasitic fungus Trichoderma.

Regulation of two homodimer hexosaminidases in the mycoparasitic fungus Trichoderma asperellum by glucosamine.

Trichoderma asperellum is a mycoparasitic fungus which is used as a biocontrol agent against plant pathogens. Its hydrolytic enzymes take part in its parasitic interaction, degrading the pathogen cell wall and thereby helping to control disease. One of those enzymes, beta- N-acetyl- d-glucosaminidase (GlcNAcase), degrades chitin, which is a major component of the cell wall of many plant-pathogenic fungi. Two GlcNAcases of T. asperellum T203, designated EXC1Y and EXC2Y, were purified, their genes and their promoters were sequenced, and their regulation was studied. The enzymes share homology (59% identity) but are easily distinguished by PAGE assay. Biochemical characterization, Edman degradation, and mass spectrometry demonstrated that EXC1Y and EXC2Y are both active as homodimers. Both genes are up-regulated by glucosamine (GlcN), in contrast to two endochitinases of this fungus. GlcN induces the secretion of several proteins (including a beta-glucosidase), among which EXC1Y is the most abundant. An exc2y knockout was constructed, to study the regulation of EXC1Y expression and secretion. The fungus has the ability to store a high amount of this enzyme in an active form and secrete it into the medium later.

Expression regulation of the endochitinase chit36 from Trichoderma asperellum (T. harzianum T-203).

The presence of the endochitinase CHIT36 from Trichoderma harzianum TM was assessed in several antagonistic Trichoderma strains belonging to different molecular taxonomic groups. CHIT37 from T. harzianum CECT 2413 was sequenced and found to display 89% homology with CHIT36 at the amino acid level. Northern analysis showed that chit36Y from T. asperellum is regulated both by glucose and nitrogen levels. Stress conditions, colloidal chitin and N-acetyl-glucosamine are effective inducers of this gene. The promoter of chit36Y was cloned and was used to direct expression of a gfp reporter gene in Trichoderma transformants. Confrontation experiments with the plant pathogen Rhizoctonia solani revealed that direct contact between the fungi is not necessary for gfp expression. The R. solani-inducing factor appears to be a soluble molecule capable of diffusing through a dialysis membrane (<12 kDa). CHIT36 recombinant protein from the yeast Pichia pastoris was active against different phytopathogens, confirming the importance of this endochitinase in the mycoparasitic activity of Trichoderma antagonistic strains.