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.     

Characterization of a mitogen-activated protein kinase gene from cucumber required for trichoderma-conferred plant resistance

The fungal biocontrol agent Trichoderma asperellum has been recently shown to induce systemic resistance in plants through a mechanism that employs jasmonic acid and ethylene signal transduction pathways. Mitogen-activated protein kinase (MAPK) proteins have been implicated in the signal transduction of a wide variety of plant stress responses. Here we report the identification and characterization of a Trichoderma-induced MAPK (TIPK) gene function in cucumber (Cucumis sativus). Similar to its homologs, wound-induced protein kinase, MPK3, and MPK3a, TIPK is also induced by wounding. Normally, preinoculation of roots with Trichoderma activates plant defense mechanisms, which result in resistance to the leaf pathogen Pseudomonas syringae pv lachrymans. We used a unique attenuated virus vector, Zucchini yellow mosaic virus (ZYMV-AGII), to overexpress TIPK protein and antisense (AS) RNA. Plants overexpressing TIPK were more resistant to pathogenic bacterial attack than control plants, even in the absence of Trichoderma preinoculation. On the other hand, plants expressing TIPK-AS revealed increased sensitivity to pathogen attack. Moreover, Trichoderma preinoculation could not protect these AS plants against subsequent pathogen attack. We therefore demonstrate that Trichoderma exerts its protective effect on plants through activation of the TIPK gene, a MAPK that is involved in signal transduction pathways of defense responses.     

Role of the 4-phosphopantetheinyl transferase of Trichoderma virens in secondary metabolism and induction of plant defense responses

Trichoderma virens is a ubiquitous soil fungus successfully used in biological control due to its efficient colonization of plant roots. In fungi, 4-phosphopantetheinyl transferases (PPTases) activate enzymes involved in primary and secondary metabolism. Therefore, we cloned the PPTase gene ppt1 from T. virens and generated PPTase-deficient (?ppt1) and overexpressing strains to investigate the role of this enzyme in biocontrol and induction of plant defense responses. The ?ppt1 mutants were auxotrophic for lysine, produced nonpigmented conidia, and were unable to synthesize nonribosomal peptides. Although spore germination was severely compromised under both low and high iron availability, mycelial growth occurred faster than the wild type, and the mutants were able to efficiently colonize plant roots. The ?ppt1 mutants were unable of inhibiting growth of phytopathogenic fungi in vitro. Arabidopsis thaliana seedlings co-cultivated with wild-type T. virens showed increased expression of pPr1a:uidA and pLox2:uidA markers, which correlated with enhanced accumulation of salicylic acid (SA), jasmonic acid, camalexin, and resistance to Botrytis cinerea. Co-cultivation of A. thaliana seedlings with ?ppt1 mutants compromised the SA and camalexin responses, resulting in decreased protection against the pathogen. Our data reveal an important role of T. virens PPT1 in antibiosis and induction of SA and camalexin-dependent plant defense responses.     

Sm1, a proteinaceous elicitor secreted by the biocontrol fungus Trichoderma virens induces plant defense responses and systemic resistance

The soilborne filamentous fungus Trichoderma virens is a biocontrol agent with a well-known ability to produce antibiotics, parasitize pathogenic fungi, and induce systemic resistance in plants. Even though a plant-mediated response has been confirmed as a component of bioprotection by Trichoderma spp., the molecular mechanisms involved remain largely unknown. Here, we report the identification, purification, and characterization of an elicitor secreted by T. virens, a small protein designated Sm1 (small protein 1). Sm1 lacks toxic activity against plants and microbes. Instead, native, purified Sm1 triggers production of reactive oxygen species in monocot and dicot seedlings, rice, and cotton, and induces the expression of defense-related genes both locally and systemically in cotton. Gene expression analysis revealed that SM1 is expressed throughout fungal development under different nutrient conditions and in the presence of a host plant. Using an axenic hydroponic system, we show that SM1 expression and secretion of the protein is significantly higher in the presence of the plant. Pretreatment of cotton cotyledons with Sm1 provided high levels of protection to the foliar pathogen Colletotrichum sp. These results indicate that Sm1 is involved in the induction of resistance by Trichoderma spp. through the activation of plant defense mechanisms.     

棘孢木霉丝裂原活化蛋白激酶基因task1的克隆及序列分析

为深入研究丝裂原活化蛋白激酶(Mitogen-activated protein kinase,MAPK)的功能,从棘孢木霉(Tricho-derma asperellum)中克隆了丝裂原活化蛋白激酶(MAPK)基因task1,并对其序列进行分析。该基因编码355个氨基酸,全长1 757 bp,理论分子质量41.1 kD,理论等电点为6.64,与深绿木霉(T.atroviride)MAPK基因tmk1、里氏木霉(T.reesei)MAPK基因tmkA和绿色木霉(T.virens)MAPK基因tmkA在氨基酸和核苷酸水平上同源性都很高,蛋白结构预测为丝氨酸/苏氨酸蛋白激酶。     

Homologous expression of a mutated beta-tubulin gene does not confer benomyl resistance on Trichoderma virens

AIMS: To clone the beta-tubulins and to induce resistance to benzimidazoles in the biocontrol fungus Trichoderma virens through site-directed mutagenesis. METHODS AND RESULTS: Two beta-tubulin genes have been cloned using PCR amplification followed by the screening of a T. virens cDNA library. The full-length cDNA clones, coding for 445 and 446 amino acids, have been designated as T. virens tub1 and T. virens tub2. A sequence alignment of these two tubulins with tubulins from other filamentous fungi has shown the presence of some unique amino acid sequences not found in those positions in other beta-tubulins. Constitutive expression of the tub2 gene with a histidine to tyrosine substitution at position 6 (known to impart benomyl/methyl benzimadazol-2-yl carbamate resistance in other fungi), under the Pgpd promoter of Aspergillus nidulans, did not impart resistance to benomyl. CONCLUSIONS: The homologous expression of tub2 gene with a histidine to tyrosine mutation at position +6, which is known to impart benomyl tolerance in other fungi, does not impart resistance in T. virens. SIGNIFICANCE AND IMPACT OF THE STUDY: Unlike other Trichoderma spp., T. virens, has been difficult to mutate for benomyl tolerance. The present study, through site-directed mutagenesis, shows that a mutation known to impart benomyl tolerance in T. viride and other fungi does not impart resistance in this fungus. Understanding the mechanisms of this phenomenon will have a profound impact in plant-disease management, as many plant pathogenic fungi develop resistance to this group of fungicides forcing its withdrawal after a short period of use.     

Induction of defense responses in cucumber plants (Cucumis sativus L. ) By the biocontrol agent trichoderma harzianum

The potential of the biocontrol agent Trichoderma harzianum T-203 to trigger plant defense responses was investigated by inoculating roots of cucumber seedlings with Trichoderma in an aseptic, hydroponic system. Trichoderma-treated plants were more developed than nontreated plants throughout the experiment. Electron microscopy of ultrathin sections from Trichoderma-treated roots revealed penetration of Trichoderma into the roots, restricted mainly to the epidermis and outer cortex. Strengthening of the epidermal and cortical cell walls was observed, as was the deposition of newly formed barriers. These typical host reactions were found beyond the sites of potential fungal penetration. Wall appositions contained large amounts of callose and infiltrations of cellulose. The wall-bound chitin in Trichoderma hyphae was preserved, even when the hyphae had undergone substantial disorganization. Biochemical analyses revealed that inoculation with Trichoderma initiated increased peroxidase and chitinase activities within 48 and 72 h, respectively. These results were observed for both the roots and the leaves of treated seedlings, providing evidence that T. harzianum may induce systemic resistance mechanisms in cucumber plants.     

Proteome, salicylic acid, and jasmonic acid changes in cucumber plants inoculated with Trichoderma asperellum strain T34

Trichoderma spp. is one of the most commonly used biological control agents against plant pathogens. This fungus produces changes in plant metabolism, thus increasing growth and enhancing resistance to biotic and abiotic stresses. However, its modes of action remain to be defined. In the first hours of interaction between cucumber plant roots and Trichoderma asperellum strain T34, salicylic and jasmonic acid levels and typical antipathogenic peroxidase activity increase in the cotyledons to different degrees depending on the applied concentration of the fungi. The use of 2-DE protein profiling and MS analysis allowed us to identify 28 proteins whose expression was affected in cotyledons after cucumber root colonization by Trichoderma applied at high concentrations: 17 were found to be up-regulated while 11 were down-regulated. Proteins involved in ROS scavenging, stress response, isoprenoid and ethylene biosynthesis, and in photosynthesis, photorespiration, and carbohydrate metabolism were differentially regulated by Trichoderma. The proteome changes found in this study help to give an understanding of how Trichoderma-treated plants become more resistant to pathogen attacks through the changes in expression of a set of defence-oriented proteins which can directly protect the plant or switch the metabolism to a defensive, nonassimilatory state.     

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 beneficial effect of Trichoderma spp. on tomato is modulated by the plant genotype

Rhizosphere-competent fungi of the genus Trichoderma are widely used as biofertilizers and biopesticides in commercial formulates because of the multiple beneficial effects on plant growth and disease resistance. In this work, we demonstrate that genetic variability among wild and cultivated tomato lines affects the outcome of the interaction with two 'elite' biocontrol strains of T. atroviride and T. harzianum. The beneficial response, which included enhanced growth and systemic resistance against Botrytis cinerea, was clearly evident for some, but not all, the tested lines. At least in one case (line M82), treatment with the biocontrol agents had no effect or was even detrimental. Expression studies on defence-related genes suggested that the fungus is able to trigger, in the responsive lines, a long-lasting up-regulation of the salicylic acid pathway in the absence of a pathogen, possibly activating a priming mechanism in the plant. Consequently, infection with B. cinerea on plants pretreated with Trichoderma is followed by enhanced activation of jasmonate-responsive genes, eventually boosting systemic resistance to the pathogen in a plant genotype-dependent manner. Our data indicate that, at least in tomato, the Trichoderma induced systemic resistance mechanism is much more complex than considered so far, and the ability of the plant to benefit from this symbiotic-like interaction can be genetically improved.     

Enhanced fungal resistance in transgenic cotton expressing an endochitinase gene from Trichoderma virens

Mycoparasitic fungi are proving to be rich sources of antifungal genes that can be utilized to genetically engineer important crops for resistance against fungal pathogens. We have transformed cotton and tobacco plants with a cDNA clone encoding a 42 kDa endochitinase from the mycoparasitic fungus, Trichoderma virens. Plants from 82 independently transformed callus lines of cotton were regenerated and analysed for transgene expression. Several primary transformants were identified with endochitinase activities that were significantly higher than the control values. Transgene integration and expression was confirmed by Southern and Northern blot analyses, respectively. The transgenic endochitinase activities were examined in the leaves of transgenic tobacco as well as in the leaves, roots, hypocotyls and seeds of transgenic cotton. Transgenic plants with elevated endochitinase activities also showed the expected 42 kDa endochitinase band in fluorescence, gel-based assays performed with the leaf extracts in both species. Homozygous T2 plants of the high endochitinase-expressing cotton lines were tested for disease resistance against a soil-borne pathogen, Rhizoctonia solani and a foliar pathogen, Alternaria alternata. Transgenic cotton plants showed significant resistance to both pathogens.     

Functional analysis of tvsp1, a serine protease-encoding gene in the biocontrol agent Trichoderma virens

Serine proteases are highly conserved among fungi and considered to play a key role in different aspects of fungal biology. These proteases can be involved in development and have been related to pathogenesis or biocontrol processes. A gene (tvsp1) encoding an extracellular serine protease was cloned from Trichoderma virens, a biocontrol agent effective against soilborne fungal pathogens. The gene was expressed in Escherichia coli and a polyclonal antibody was raised against the recombinant protein. The expression pattern of tvsp1 was determined and its physiological role was addressed by mutational analysis. Strains of T. virens in which tvsp1 was deleted (PKO) or constitutively overexpressed (POE) were not affected in growth rate, conidiation, extracellular protein accumulation, antibiotic profiles nor in their ability to induce phytoalexins in cotton seedlings. Tvsp1 overexpression, however, significantly increased the ability of some strains to protect cotton seedlings against Rhizoctonia solani. Our data show that Tvsp1 is not necessary for the normal growth or development of T. virens, but plays a role in the biocontrol process.     

Trichoderma spp. Antagonism to the Dermatophyte Trichophyton rubrum: Implications in Treatment of Onychomycosis

Onychomycosis--the dermatophytic invasion of the nail--is difficult to eradicate with drug treatment. The hyphae of the main invading pathogen, Trichophyton rubrum, are often interwoven with the nail plate, preventing effective anti-mycotic agents from reaching its growing tips. An alternative approach to treat onychomycosis may possibly be the application of a biological control agent against the pathogen. In analogy with the success of biocontrol of phytopathogenic fungi, we screened a series of commercially available Trichoderma strains for potential antagonism between Trichoderma and Trichophyton spp. A wide spectrum of antagonism capacity, ranging from effective overgrowth to no interaction was found, with Trichoderma virens NRRL 26672 being the most effective against the Trichophyton strains tested e.g. T. rubrum NCPF118. Furthermore, T. virens NRRL 26672 grown with T. rubrum NCPF118 hyphae as a carbon source, exhibited enhanced induced secretion of active extracellular chitinases and beta-glucosidases, affecting lysis and sporulation on T. rubrum NCPF118 hyphae. Growth of Trichod. virens NRRL 26672 in poor medium also resulted in secretion of antibiotics active in arresting the growth of T. rubrum NCPF118 inoculum. Our findings may open new directions for the treatment of onychomycosis, either in combination with known medications or as a new "natural" route.     

Dimerization controls the activity of fungal elicitors that trigger systemic resistance in plants

The soilborne fungus Trichoderma virens secretes a small protein (Sm1) that induces local and systemic defenses in plants. This protein belongs to the ceratoplatanin protein family and is mainly present as a monomer in culture filtrates. However, Hypocrea atroviride (the telomorph form of Trichoderma atroviride) secretes an Sm1-homologous protein, Epl1, with high levels of dimerization. Nonetheless, the molecular mechanisms involved in recognition and the signaling pathways involved in the induction of systemic resistance in plants are still unclear. In this report, we demonstrate that Sm1 and Epl1 are mainly produced as monomer and a dimer, respectively, in the presence of maize seedlings. The results presented show that the ability to induce plant defenses reside only in the monomeric form of both Sm1 and Epl1, and we demonstrate for the first time that the monomeric form of Epl1, likewise Sm1, induces defenses in maize plants. Biochemical analyses indicate that monomeric Sm1 is produced as a glycoprotein, but the glycosyl moiety is missing from its dimeric form, and Epl1 is produced as a nonglycosylated protein. Moreover, for Sm1 homologues in various fungal strains, there is a negative correlation between the presence of the glycosylation site and their ability to aggregate. We propose a subdivision in the ceratoplatanin protein family according to the presence of the glycosylation site and the ability of the proteins to aggregate. The data presented suggest that the elicitor's aggregation may control the Trichoderma-plant molecular dialogue and block the activation of induced systemic resistance in plants.     

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.     

Histopathological studies of sclerotia of Rhizoctonia solani parasitized by the EGFP transformant of Trichoderma virens

Aims:  The aim of the study was to investigate the antagonistic interactions of Trichoderma species against Rhizoctonia solani sclerotia by enhanced green fluorescence protein (EGFP)-tagged transformant of Trichoderma virens TY009.
Methods and Results:  An EGFP was used as a report gene for transforming T. virens strain, and a stable EGFP transformant GF5 was obtained with the mycoparasitic activity against sclerotia of R. solani . Observation of parasitized sclerotia by fluorescence microscopy showed hyphae of transformant GF5 was able to invade into sclerotia and its colonization was mainly intercellar with uniformly distributed mycelium in sclerotia. The host cells were colonized, penetrated, and then the whole cells were replaced by transformant GF5 hyphae. Chlamydospores were seen after 10 days but mature ones after 20 days. Sclerotia became soft and decayed after 40 days but a few cells seemed not to be colonized completely.
Conclusions:  Trichoderma virens was able to parasitize sclerotia to make sclerotia soft and decayed, and its colonization was mainly intercellar in sclerotial tissues.
Significance and Impact of the Study:  This is first report of parasitism of sclerotia of R. solani by EGFP-tagged transformant, providing useful information for using T. virens as effective biocontrol agent.