GqPCR-stimulated dephosphorylation of AKT is induced by an IGBP1-mediated PP2A switch.

BACKGROUND: G protein-coupled receptors (GPCRs) usually regulate cellular processes via activation of intracellular signaling pathways. However, we have previously shown that in several cell lines, GqPCRs induce immediate inactivation of the AKT pathway, which leads to JNK-dependent apoptosis. This apoptosis-inducing AKT inactivation is essential for physiological functions of several GqPCRs, including those for PGF2α and GnRH. METHODS: Here we used kinase activity assays of PI3K and followed phosphorylation state of proteins using specific antibodies. In addition, we used coimmunoprecipitation and proximity ligation assays to follow protein-protein interactions. Apoptosis was detected by TUNEL assay and PARP1 cleavage. RESULTS: We identified the mechanism that allows the unique stimulated inactivation of AKT and show that the main regulator of this process is the phosphatase PP2A, operating with the non-canonical regulatory subunit IGBP1. In resting cells, an IGBP1-PP2Ac dimer binds to PI3K, dephosphorylates the inhibitory pSer608-p85 of PI3K and thus maintains its high basal activity. Upon GqPCR activation, the PP2Ac-IGBP1 dimer detaches from PI3K and thus allows the inhibitory dephosphorylation. At this stage, the free PP2Ac together with IGBP1 and PP2Aa binds to AKT, causing its dephosphorylation and inactivation. CONCLUSION: Our results show a stimulated shift of PP2Ac from PI3K to AKT termed "PP2A switch" that represses the PI3K/AKT pathway, providing a unique mechanism of GPCR-stimulated dephosphorylation. Video Abstract.

Secretome Analysis of Arabidopsis-Trichoderma atroviride Interaction Unveils New Roles for the Plant Glutamate:Glyoxylate Aminotransferase GGAT1 in Plant Growth Induced by the Fungus and Resistance against Botrytis cinerea.

The establishment of plant-fungus mutualistic interaction requires bidirectional molecular crosstalk. Therefore, the analysis of the interacting organisms secretomes would help to understand how such relationships are established. Here, a gel-free shotgun proteomics approach was used to identify the secreted proteins of the plant Arabidopsis thaliana and the mutualistic fungus Trichoderma atroviride during their interaction. A total of 126 proteins of Arabidopsis and 1027 of T. atroviride were identified. Among them, 118 and 780 were differentially modulated, respectively. Bioinformatic analysis unveiled that both organisms' secretomes were enriched with enzymes. In T. atroviride, glycosidases, aspartic endopeptidases, and dehydrogenases increased in response to Arabidopsis. Additionally, amidases, protein-serine/threonine kinases, and hydro-lyases showed decreased levels. Furthermore, peroxidases, cysteine endopeptidases, and enzymes related to the catabolism of secondary metabolites increased in the plant secretome. In contrast, pathogenesis-related proteins and protease inhibitors decreased in response to the fungus. Notably, the glutamate:glyoxylate aminotransferase GGAT1 was secreted by Arabidopsis during its interaction with T. atroviride. Our study showed that GGAT1 is partially required for plant growth stimulation and on the induction of the plant systemic resistance by T. atroviride. Additionally, GGAT1 seems to participate in the negative regulation of the plant systemic resistance against B. cinerea through a mechanism involving H2O2 production.

Deletion of the Trichoderma virens NRPS, Tex7, induces accumulation of the anti-cancer compound heptelidic acid.

The anticancer antibiotic heptelidic acid is a sesquiterpene lactone produced by the beneficial plant fungus Trichoderma virens. This species has been separated into two strains, referred to as P and Q, based on its biosynthesis of secondary metabolites; notably, only P-strains were reported to produce heptelidic acid. While characterizing a Q-strain of T. virens containing a directed mutation in the non-ribosomal peptide synthetase encoding gene Tex7, the appearance of an unknown compound in anomalously large quantities was visualized by TLC. Using a combination of HPLC, LC-MS/MS, and NMR spectroscopy, this compound was identified as heptelidic acid. This discovery alters the strain classification structure of T. virens. Additionally, the Tex7 mutants inhibited growth of maize seedlings, while retaining the ability to induce systemic resistance against the foliar fungal pathogen, Cochliobolus heterostrophus.

The AP-1-like transcription factor ChAP1 balances tolerance and cell death in the response of the maize pathogen Cochliobolus heterostrophus to a plant phenolic.

Fungal pathogens need to contend with stresses including oxidants and antimicrobial chemicals resulting from host defenses. ChAP1 of Cochliobolus heterostrophus, agent of Southern corn leaf blight, encodes an ortholog of yeast YAP1. ChAP1 is retained in the nucleus in response to plant-derived phenolic acids, in addition to its well-studied activation by oxidants. Here, we used transcriptome profiling to ask which genes are regulated in response to ChAP1 activation by ferulic acid (FA), a phenolic abundant in the maize host. Nuclearization of ChAP1 in response to phenolics is not followed by strong expression of genes needed for oxidative stress tolerance. We, therefore, compared the transcriptomes of the wild-type pathogen and a ChAP1 deletion mutant, to study the function of ChAP1 in response to FA. We hypothesized that if ChAP1 is retained in the nucleus under plant-related stress conditions yet in the absence of obvious oxidant stress, it should have additional regulatory functions. The transcriptional signature in response to FA in the wild type compared to the mutant sheds light on the signaling mechanisms and response pathways by which ChAP1 can mediate tolerance to ferulic acid, distinct from its previously known role in the antioxidant response. The ChAP1-dependent FA regulon consists mainly of two large clusters. The enrichment of transport and metabolism-related genes in cluster 1 indicates that C. heterostrophus degrades FA and removes it from the cell. When this fails at increasing stress levels, FA provides a signal for cell death, indicated by the enrichment of cell death-related genes in cluster 2. By quantitation of survival and by TUNEL assays, we show that ChAP1 promotes survival and mitigates cell death. Growth rate data show a time window in which the mutant colony expands faster than the wild type. The results delineate a transcriptional regulatory pattern in which ChAP1 helps balance a survival response for tolerance to FA, against a pathway promoting cell death in the pathogen. A general model for the transition from a phase where the return to homeostasis dominates to a phase leading to the onset of cell death provides a context for understanding these findings.

Regulation of conidiation and antagonistic properties of the soil-borne plant beneficial fungus Trichoderma virens by a novel proline-, glycine-, tyrosine-rich protein and a GPI-anchored cell wall protein.

Trichoderma spp. are widely used as commercial biofungicides, and most commercial formulations are conidia based. Identification of genes that regulate conidiation would thus be of help in genetic reprogramming of these species to optimize sporulation. In this study, we constructed an SSH (suppression subtractive hybridization) library from RNA samples of the wild type strain and a non-conidiating mutant, M7, grown under constant illumination for 2 days. We identified several genes that are underexpressed in the mutant. Some of these genes are related to secondary metabolism, and a few could be associated with conidiation. Genes coding for the following proteins, among others, were identified: O-methyl transferase, ATPase, alpha/beta-hydrolase, WD repeat containing protein, dehydrogenase, thioesterase, translationally controlled tumour protein, and a proline-glycine-tyrosine-rich protein (PGYRP) that has been annotated in T.reesei as a signalling protein. Two of these genes, encoding Pgy1, a novel PGYRP, and Ecm33, a GPI-anchored cell wall protein, were further studied in detail by generation of deletion mutants. We demonstrate here that both these genes not only regulate radial growth and conidiation in Trichoderma virens, but are also involved in antagonism against soil-borne wide host range plant pathogens. Furthermore, deletion of ecm33 affected hydrophobicity and cell wall integrity.

Secretome of Trichoderma interacting with maize roots: role in induced systemic resistance.

Trichoderma virens is a biocontrol agent used in agriculture to antagonize pathogens of crop plants. In addition to direct mycoparasitism of soil-borne fungal pathogens, T. virens interacts with roots. This interaction induces systemic resistance (ISR), which reduces disease in above-ground parts of the plant. In the molecular dialog between fungus and plant leading to ISR, proteins secreted by T. virens provide signals. Only a few such proteins have been characterized previously. To study the secretome, proteins were characterized from hydroponic culture systems with T. virens alone or with maize seedlings, and combined with a bioassay for ISR in maize leaves infected by the pathogen Cochliobolus heterostrophus. The secreted protein fraction from coculture of maize roots and T. virens (Tv+M) was found to have a higher ISR activity than from T. virens grown alone (Tv). A total of 280 fungal proteins were identified, 66 showing significant differences in abundance between the two conditions: 32 were higher in Tv+M and 34 were higher in Tv. Among the 34 found in higher abundance in Tv and negatively regulated by roots were 13 SSCPs (small, secreted, cysteine rich proteins), known to be important in the molecular dialog between plants and fungi. The role of four SSCPs in ISR was studied by gene knockout. All four knockout lines showed better ISR activity than WT without affecting colonization of maize roots. Furthermore, the secreted protein fraction from each of the mutant lines showed improved ISR activity compared with WT. These SSCPs, apparently, act as negative effectors reducing the defense levels in the plant and may be important for the fine tuning of ISR by Trichoderma. The down-regulation of SSCPs in interaction with plant roots implies a revision of the current model for the Trichoderma-plant symbiosis and its induction of resistance to pathogens.

Plant phenolic acids induce programmed cell death of a fungal pathogen: MAPK signaling and survival of Cochliobolus heterostrophus.

Plant aromatic compounds provide signals and a nutrient source to pathogens, and also act as stressors. Structure-activity relationships suggest two pathways sensing these compounds in the maize pathogen Cochliobolus heterostrophus, one triggering a stress response, and one inducing enzymes for their degradation. Focusing on the stress pathway, we found that ferulic acid causes rapid appearance of TUNEL-positive nuclei, dispersion of histone H1:GFP, hyphal shrinkage, and eventually membrane damage. These hallmarks of programmed cell death (PCD) were not seen upon exposure to caffeic acid, a very similar compound. Exposure to ferulic acid dephosphorylated two MAP kinases: Hog1 (stress activated) and Chk1 (pathogenicity related), while increasing phosphorylation of Mps1 (cell integrity related). Mutants lacking Hog1 or Chk1 are hypersensitive to ferulic acid while Mps1 mutants are not. These results implicate three MAPK pathways in the stress response. Ferulic acid and the antifungal fludioxonil have opposite additive effects on survival and on dephosphorylation of Hog1, which is thus implicated in survival. The results may explain why some fungal pathogens of plants undergo cell death early in host invasion, when phenolics are released from plant tissue.

Host-specific transcriptomic pattern of Trichoderma virens during interaction with maize or tomato roots.

BACKGROUND: Members of the fungal genus Trichoderma directly antagonize soil-borne fungal pathogens, and an increasing number of species are studied for their potential in biocontrol of plant pathogens in agriculture. Some species also colonize plant roots, promoting systemic resistance. The Trichoderma-root interaction is hosted by a wide range of plant species, including monocots and dicots. RESULTS: To test the hypothesis that gene expression by the fungal partner in this beneficial interaction is modulated by the plant, Trichoderma virens was co-cultured with maize or tomato in a hydroponic system allowing interaction with the roots. The transcriptomes for T. virens alone were compared with fungus-inoculated tomato or maize roots by hybridization on microarrays of 11645 unique oligonucleotides designed from the predicted protein-coding gene models. Transcript levels of 210 genes were modulated by interaction with roots. Almost all were up-regulated. Glycoside hydrolases and transporters were highly represented among transcripts induced by co-culture with roots. Of the genes up-regulated on either or both host plants, 35 differed significantly in their expression levels between maize and tomato. Ten of these were expressed higher in the fungus in co-culture with tomato roots than with maize. Average transcript levels for these genes ranged from 1.9 fold higher on tomato than on maize to 60.9 fold for the most tomato-specific gene. The other 25 host-specific transcripts were expressed more strongly in co-culture with maize than with tomato. Average transcript levels for these genes were 2.5 to 196 fold higher on maize than on tomato. CONCLUSIONS: Based on the relevant role of Trichoderma virens as a biological control agent this study provides a better knowledge of its crosstalk with plants in a host-specific manner. The differentially expressed genes encode proteins belonging to several functional classes including enzymes, transporters and small secreted proteins. Among them, glycoside hydrolases and transporters are highlighted by their abundance and suggest an important factor in the metabolism of host cell walls during colonization of the outer root layers. Host-specific gene expression may contribute to the ability of T. virens to colonize the roots of a wide range of plant species.

Plant phenolic compounds and oxidative stress: integrated signals in fungal-plant interactions.

Upon invasion of a host, fungal pathogens are exposed to a variety of stresses. Plants release reactive oxygen species, and mount a variety of preformed and induced chemical defenses. Phenolic compounds are one example: they are ubiquitous in plants, and an invading pathogen encounters them already at the leaf surface, or for soil-borne pathogens, in the rhizosphere. Phenolic and related aromatic compounds show varying degrees of toxicity to cells. Some compounds are quite readily metabolized, and others less so. It was known already from classical studies that phenolic substrates induce the expression of the enzymes for their degradation. Recently, the ability to degrade phenolics was shown to be a virulence factor. Conversely, phenolic compounds can increase the effectiveness of antifungals. Phenolics are known antioxidants, yet they have been shown to elicit cellular responses that would usually be triggered to counter oxidant stress. Here, we review the evidence for a connection between the fungal response to phenolics as small-molecule signals, and the response to oxidants. The connections proposed here should enable genetic screens to identify specific fungal receptors for plant phenolics. Furthermore, understanding how the pathogen detects plant phenolic compounds as a stress signal may facilitate new antifungal strategies.

Sm2, a paralog of the Trichoderma cerato-platanin elicitor Sm1, is also highly important for plant protection conferred by the fungal-root interaction of Trichoderma with maize.

BACKGROUND: The proteins Sm1 and Sm2 from the biocontrol fungus Trichoderma virens belong to the cerato-platanin protein family. Members of this family are small, secreted proteins that are abundantly produced by filamentous fungi with all types of life-styles. Some species of the fungal genus Trichoderma are considered as biocontrol fungi because they are mycoparasites and are also able to directly interact with plants, thereby stimulating plant defense responses. It was previously shown that the cerato-platanin protein Sm1 from T. virens - and to a lesser extent its homologue Epl1 from Trichoderma atroviride - induce plant defense responses. The plant protection potential of other members of the cerato-platanin protein family in Trichoderma, however, has not yet been investigated. RESULTS: In order to analyze the function of the cerato-platanin protein Sm2, sm1 and sm2 knockout strains were generated and characterized. The effect of the lack of Sm1 and Sm2 in T. virens on inducing systemic resistance in maize seedlings, challenged with the plant pathogen Cochliobolus heterostrophus, was tested. These plant experiments were also performed with T. atroviride epl1 and epl2 knockout strains. In our plant-pathogen system T. virens was a more effective plant protectant than T. atroviride and the results with both Trichoderma species showed concordantly that the level of plant protection was more strongly reduced in plants treated with the sm2/epl2 knockout strains than with sm1/epl1 knockout strains. CONCLUSIONS: Although the cerato-platanin genes sm1/epl1 are more abundantly expressed than sm2/epl2 during fungal growth, Sm2/Epl2 are, interestingly, more important than Sm1/Epl1 for the promotion of plant protection conferred by Trichoderma in the maize-C. heterostrophus pathosystem.

Diverse lifestyles and strategies of plant pathogenesis encoded in the genomes of eighteen Dothideomycetes fungi.

The class Dothideomycetes is one of the largest groups of fungi with a high level of ecological diversity including many plant pathogens infecting a broad range of hosts. Here, we compare genome features of 18 members of this class, including 6 necrotrophs, 9 (hemi)biotrophs and 3 saprotrophs, to analyze genome structure, evolution, and the diverse strategies of pathogenesis. The Dothideomycetes most likely evolved from a common ancestor more than 280 million years ago. The 18 genome sequences differ dramatically in size due to variation in repetitive content, but show much less variation in number of (core) genes. Gene order appears to have been rearranged mostly within chromosomal boundaries by multiple inversions, in extant genomes frequently demarcated by adjacent simple repeats. Several Dothideomycetes contain one or more gene-poor, transposable element (TE)-rich putatively dispensable chromosomes of unknown function. The 18 Dothideomycetes offer an extensive catalogue of genes involved in cellulose degradation, proteolysis, secondary metabolism, and cysteine-rich small secreted proteins. Ancestors of the two major orders of plant pathogens in the Dothideomycetes, the Capnodiales and Pleosporales, may have had different modes of pathogenesis, with the former having fewer of these genes than the latter. Many of these genes are enriched in proximity to transposable elements, suggesting faster evolution because of the effects of repeat induced point (RIP) mutations. A syntenic block of genes, including oxidoreductases, is conserved in most Dothideomycetes and upregulated during infection in L. maculans, suggesting a possible function in response to oxidative stress.

Iron, oxidative stress, and virulence: roles of iron-sensitive transcription factor Sre1 and the redox sensor ChAp1 in the maize pathogen Cochliobolus heterostrophus.

The gene SRE1, encoding the GATA transcription factor siderophore biosynthesis repressor (Sre1), was identified in the genome of the maize pathogen Cochliobolus heterostrophus and deleted. Mutants were altered in sensitivity to iron, oxidative stress, and virulence to the host. To gain insight into mechanisms of this combined regulation, genetic interactions among SRE1 (the nonribosomal peptide synthetase encoding gene NPS6, which is responsible for extracellular siderophore biosynthesis) and ChAP1 (encoding a transcription factor regulating redox homeostasis) were studied. To identify members of the Sre1 regulon, expression of candidate iron and oxidative stress-related genes was assessed in wild-type (WT) and sre1 mutants using quantitative reverse-transcription polymerase chain reaction. In sre1 mutants, NPS6 and NPS2 genes, responsible for siderophore biosynthesis, were derepressed under iron replete conditions, whereas the high-affinity reductive iron uptake pathway associated gene, FTR1, was not, in contrast to outcomes with other well-studied fungal models. C. heterostrophus L-ornithine-N(5)- monooxygenase (SIDA2), ATP-binding cassette (ABC6), catalase (CAT1), and superoxide dismutase (SOD1) genes were also derepressed under iron-replete conditions in sre1 mutants. Chap1nps6 double mutants were more sensitive to oxidative stress than either Chap1 or nps6 single mutants, while Chap1sre1 double mutants showed a modest increase in resistance compared with single Chap1 mutants but were much more sensitive than sre1 mutants. These findings suggest that the NPS6 siderophore indirectly contributes to redox homeostasis via iron sequestration, while Sre1 misregulation may render cells more sensitive to oxidative stress. The double-mutant phenotypes are consistent with a model in which iron sequestration by NPS6 defends the pathogen against oxidative stress. C. heterostrophus sre1, nps6, Chap1, Chap1nps6, and Chap1sre1 mutants are all reduced in virulence toward the host, compared with the WT.

PacC and pH-dependent transcriptome of the mycotrophic fungus Trichoderma virens.

BACKGROUND: In fungi, environmental pH is an important signal for development, and successful host colonization depends on homeostasis. Surprisingly, little is known regarding the role of pH in fungal-fungal interactions. Species of Trichoderma grow as soil saprobes but many are primarily mycotrophic, using other fungi as hosts. Therefore, Trichoderma spp. are studied for their potential in biocontrol of plant diseases. Particularly in alkaline soil, pH is a critical limiting factor for these biofungicides, whose optimal growth pH is 4-6. Gaining an understanding of pH adaptability is an important step in broadening the activity spectrum of these economically important fungi. RESULTS: We studied the pH-responsive transcription factor PacC by gene knockout and by introduction of a constitutively active allele (pacCc). ΔpacC mutants exhibited reduced growth at alkaline pH, while pacCc strains grew poorly at acidic pH. In plate confrontation assays ΔpacC mutants showed decreased ability to compete with the plant pathogens Rhizoctonia solani and Sclerotium rolfsii. The pacCc strain exhibited an overgrowth of R. solani that was comparable to the wild type, but was unable to overgrow S. rolfsii. To identify genes whose expression is dependent on pH and pacC, we designed oligonucleotide microarrays from the transcript models of the T. virens genome, and compared the transcriptomes of wild type and mutant cultures exposed to high or low pH. Transcript levels from several functional classes were dependent on pacC, on pH, or on both. Furthermore, the expression of a set of pacC-dependent genes was increased in the constitutively-active pacCc strain, and was pH-independent in some, but not all cases. CONCLUSIONS: PacC is important for biocontrol-related antagonism of other fungi by T. virens. As much as 5% of the transcriptome is pH-dependent, and of these genes, some 25% depend on pacC. Secondary metabolite biosynthesis and ion transport are among the relevant gene classes. We suggest that ΔpacC mutants may have lost their full biocontrol potential due to their inability to adapt to alkaline pH, to perceive ambient pH, or both. The results raise the novel possibility of genetically manipulating Trichoderma in order to improve adaptability and biocontrol at alkaline pH.

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.

Structure-activity relationships delineate how the maize pathogen Cochliobolus heterostrophus uses aromatic compounds as signals and metabolites.

The necrotrophic maize pathogen Cochliobolus heterostrophus senses plant-derived phenolic compounds, which promote nuclear retention of the redox-sensitive transcription factor ChAP1 and alter gene expression. The intradiol dioxygenase gene CCHD1 is strongly upregulated by coumaric and caffeic acids. Plant phenolics are potential nutrients but some of them are damaging compounds that need to be detoxified. Using coumaric acid as an inducer (16 to 160 µM), we demonstrated the rapid and simultaneous upregulation of most of the ß-ketoadipate pathway genes in C. heterostrophus. A cchd1 deletion mutant provided genetic evidence that protocatechuic acid is an intermediate in catabolism of a wide range of aromatic acids. Aromatics catabolism was slowed for compounds showing toxicity, and this was strongly correlated with nuclear retention of GFP-ChAP1. The activity of a structure series of compounds showed complementary requirements for upregulation of CCHD1 and for ChAP1 nuclear retention. Thus, there is an inverse correlation between the ability to metabolize a compound and the stress response (ChAP1 nuclear retention) that it causes. The ability to metabolize phenolics and to respond to them as signals should be an advantage to plant pathogens and may explain the presence of at least two response pathways detecting these compounds.

Secondary metabolism in Trichoderma--a genomic perspective.

Trichoderma spp. are a rich source of secondary metabolites (SMs). The recent publication of the genome sequences of three Trichoderma spp. has revealed a vast repertoire of genes putatively involved in the biosynthesis of SMs, such as non-ribosomal peptides, polyketides, terpenoids and pyrones. Interestingly, the genomes of the mycoparasitic species Trichoderma virens and Trichoderma atroviride are enriched in secondary metabolism-related genes compared with the biomass-degrading Trichoderma reesei: 18 and 18 polyketide synthases compared with 11; 28 and 16 non-ribosomal peptide synthetases compared with 10, respectively. All three species produce a special class of non-ribosomally synthesized peptides known as peptaibols, containing non-proteinogenic amino acids (particularly α-aminoisobutyric acid). In common with other filamentous ascomycetes, Trichoderma spp. may require siderophores (also produced by non-ribosomal peptide synthetases) to grow in iron-poor conditions and to compete with their hosts for available iron. Two generalizations can be made about fungal SM genes: they are often found in clusters, and many are not expressed under standard laboratory conditions. This has made it difficult to identify the compounds. Trichoderma, in particular, interacts with other microbes in the soil and with plant roots in the rhizosphere. A detailed metabolomic-genomic study would eventually unravel the roles of many of these SMs in natural ecosystems. Novel genetic tools developed recently, combined with biological understanding of the function of SMs as toxins or signals, should lead to 'awakening' of these 'silent' clusters. Knowledge of the SM repertoire should precede application of Trichoderma strains for biocontrol: some metabolites could be toxic to plants and their consumers, and thus should be avoided. Others could be beneficial, antagonizing pathogens or inducing resistance in crop plants.

Trichoderma-plant-pathogen interactions: advances in genetics of biological control.

Trichoderma spp. are widely used in agriculture as biofungicides. Induction of plant defense and mycoparasitism (killing of one fungus by another) are considered to be the most important mechanisms of Trichoderma-mediated biological control. Understanding these mechanisms at the molecular level would help in developing strains with superior biocontrol properties. In this article, we review our current understanding of the genetics of interactions of Trichoderma with plants and plant pathogens.

Adhesion as a Focus in Trichoderma-Root Interactions.

Fungal spores, germlings, and mycelia adhere to substrates, including host tissues. The adhesive forces depend on the substrate and on the adhesins, the fungal cell surface proteins. Attachment is often a prerequisite for the invasion of the host, hence its importance. Adhesion visibly precedes colonization of root surfaces and outer cortex layers, but little is known about the molecular details. We propose that by starting from what is already known from other fungi, including yeast and other filamentous pathogens and symbionts, the mechanism and function of Trichoderma adhesion will become accessible. There is a sequence, and perhaps functional, homology to other rhizosphere-competent Sordariomycetes. Specifically, Verticillium dahliae is a soil-borne pathogen that establishes itself in the xylem and causes destructive wilt disease. Metarhizium species are best-known as insect pathogens with biocontrol potential, but they also colonize roots. Verticillium orthologs of the yeast Flo8 transcription factor, Som1, and several other relevant genes are already under study for their roles in adhesion. Metarhizium encodes relevant adhesins. Trichoderma virens encodes homologs of Som1, as well as adhesin candidates. These genes should provide exciting leads toward the first step in the establishment of beneficial interactions with roots in the rhizosphere.

Gliotoxin, an Immunosuppressive Fungal Metabolite, Primes Plant Immunity: Evidence from Trichoderma virens-Tomato Interaction.

Beneficial interaction of members of the fungal genus Trichoderma with plant roots primes the plant immune system, promoting systemic resistance to pathogen infection. Some strains of Trichoderma virens produce gliotoxin, a fungal epidithiodioxopiperazine (ETP)-type secondary metabolite that is toxic to animal cells. It induces apoptosis, prevents NF-κB activation via the inhibition of the proteasome, and has immunosuppressive properties. Gliotoxin is known to be involved in the antagonism of rhizosphere microorganisms. To investigate whether this metabolite has a role in the interaction of Trichoderma with plant roots, we compared gliotoxin-producing and nonproducing T. virens strains. Both colonize the root surface and outer layers, but they have differential effects on root growth and architecture. The responses of tomato plants to a pathogen challenge were followed at several levels: lesion development, levels of ethylene, and reactive oxygen species. The transcriptomic signature of the shoot tissue in response to root interaction with producing and nonproducing T. virens strains was monitored. Gliotoxin producers provided stronger protection against foliar pathogens, compared to nonproducing strains. This was reflected in the transcriptomic signature, which showed the induction of defense-related genes. Two markers of plant defense response, PR1 and Pti-5, were differentially induced in response to pure gliotoxin. Gliotoxin thus acts as a microbial signal, which the plant immune system recognizes, directly or indirectly, to promote a defense response. IMPORTANCE A single fungal metabolite induces far-reaching transcriptomic reprogramming in the plant, priming immune responses and defense, in contrast to its immunosuppressive effect on animal cells. While the negative effects of gliotoxin-producing Trichoderma strains on growth may be observed only under a particular set of laboratory conditions, gliotoxin-linked molecular patterns, including the potential for limited cell death, could strongly prime plant defense, even in mature soil-grown plants in which the same Trichoderma strain promotes growth.

Synthetic cells with self-activating optogenetic proteins communicate with natural cells.

Development of regulated cellular processes and signaling methods in synthetic cells is essential for their integration with living materials. Light is an attractive tool to achieve this, but the limited penetration depth into tissue of visible light restricts its usability for in-vivo applications. Here, we describe the design and implementation of bioluminescent intercellular and intracellular signaling mechanisms in synthetic cells, dismissing the need for an external light source. First, we engineer light generating SCs with an optimized lipid membrane and internal composition, to maximize luciferase expression levels and enable high-intensity emission. Next, we show these cells' capacity to trigger bioprocesses in natural cells by initiating asexual sporulation of dark-grown mycelial cells of the fungus Trichoderma atroviride. Finally, we demonstrate regulated transcription and membrane recruitment in synthetic cells using bioluminescent intracellular signaling with self-activating fusion proteins. These functionalities pave the way for deploying synthetic cells as embeddable microscale light sources that are capable of controlling engineered processes inside tissues.