Unveiling a Microexon Switch: Novel Regulation of the Activities of Sugar Assimilation and Plant-Cell-Wall-Degrading Xylanases and Cellulases by Xlr2 in Trichoderma virens.

Functional microexons have not previously been described in filamentous fungi. Here, we describe a novel mechanism of transcriptional regulation in Trichoderma requiring the inclusion of a microexon from the Xlr2 gene. In low-glucose environments, a long mRNA including the microexon encodes a protein with a GAL4-like DNA-binding domain (Xlr2-α), whereas in high-glucose environments, a short mRNA that is produced encodes a protein lacking this DNA-binding domain (Xlr2-ß). Interestingly, the protein isoforms differ in their impact on cellulase and xylanase activity. Deleting the Xlr2 gene reduced both xylanase and cellulase activity and growth on different carbon sources, such as carboxymethylcellulose, xylan, glucose, and arabinose. The overexpression of either Xlr2-α or Xlr2-ß in T. virens showed that the short isoform (Xlr2-ß) caused higher xylanase activity than the wild types or the long isoform (Xlr2-α). Conversely, cellulase activity did not increase when overexpressing Xlr2-ß but was increased with the overexpression of Xlr2-α. This is the first report of a novel transcriptional regulation mechanism of plant-cell-wall-degrading enzyme activity in T. virens. This involves the differential expression of a microexon from a gene encoding a transcriptional regulator.

Insights into Metabolic Changes Caused by the Trichoderma virens-Maize Root Interaction.

The interactions of crops with root-colonizing endophytic microorganisms are highly relevant to agriculture, because endophytes can modify plant resistance to pests and increase crop yields. We investigated the interactions between the host plant Zea mays and the endophytic fungus Trichoderma virens at 5 days postinoculation grown in a hydroponic system. Wild-type T. virens and two knockout mutants, with deletion of the genes tv2og1 or vir4 involved in specialized metabolism, were analyzed. Root colonization by the fungal mutants was lower than that by the wild type. All fungal genotypes suppressed root biomass. Metabolic fingerprinting of roots, mycelia, and fungal culture supernatants was performed using ultrahigh performance liquid chromatography coupled to diode array detection and quadrupole time-of-flight tandem mass spectrometry. The metabolic composition of T. virens-colonized roots differed profoundly from that of noncolonized roots, with the effects depending on the fungal genotype. In particular, the concentrations of several metabolites derived from the shikimate pathway, including an amino acid and several flavonoids, were modulated. The expression levels of some genes coding for enzymes involved in these pathways were affected if roots were colonized by the ∆vir4 genotype of T. virens. Furthermore, mycelia and fungal culture supernatants of the different T. virens genotypes showed distinct metabolomes. Our study highlights the fact that colonization by endophytic T. virens leads to far-reaching metabolic changes, partly related to two fungal genes. Both metabolites produced by the fungus and plant metabolites modulated by the interaction probably contribute to these metabolic patterns. The metabolic changes in plant tissues may be interlinked with systemic endophyte effects often observed in later plant developmental stages.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Yield and cold storage of Trichoderma conidia is influenced by substrate pH and storage temperature.

In this study we examined the influence of the ambient pH during morphogenesis on conidial yield of Trichoderma sp. "atroviride B" LU132 and T. hamatum LU593 and storage at low temperatures. The ambient pH of the growth media had a dramatic influence on the level of Trichoderma conidiation and this was dependent on the strain and growth media. On malt-extract agar, LU593 yield decreased with increasing pH (3-6), whereas yield increased with increasing pH for LU132. During solid substrate production the reverse was true for LU132 whereby yield decreased with increasing pH. The germination potential of the conidia decreased significantly over time in cold storage and the rate of decline was a factor of the strain, pH during morphogenesis, growth media, and storage temperature.

Identification of growth stage molecular markers in Trichoderma sp. 'atroviride type B' and their potential application in monitoring fungal growth and development in soil.

Several members of the genus Trichoderma are biocontrol agents of soil-borne fungal plant pathogens. The effectiveness of biocontrol agents depends heavily on how they perform in the complex field environment. Therefore, the ability to monitor and track Trichoderma within the environment is essential to understanding biocontrol efficacy. The objectives of this work were to: (a) identify key genes involved in Trichoderma sp. 'atroviride type B' morphogenesis; (b) develop a robust RNA isolation method from soil; and (c) develop molecular marker assays for characterizing morphogenesis whilst in the soil environment. Four cDNA libraries corresponding to conidia, germination, vegetative growth and conidiogenesis were created, and the genes identified by sequencing. Stage specificity of the different genes was confirmed by either Northern blot or quantitative reverse-transcriptase PCR (qRT-PCR) analysis using RNA from the four stages. con10, a conidial-specific gene, was observed in conidia, as well as one gene also involved in subsequent stages of germination (L-lactate/malate dehydrogenase encoding gene). The germination stage revealed high expression rates of genes involved in amino acid and protein biosynthesis, while in the vegetative-growth stage, genes involved in differentiation, including the mitogen-activated protein kinase kinase similar to Kpp7 from Ustilago maydis and the orthologue to stuA from Aspergillus nidulans, were preferentially expressed. Genes involved in cell-wall synthesis were expressed during conidiogenesis. We standardized total RNA isolation from Trichoderma sp. 'atroviride type B' growing in soil and then examined the expression profiles of selected genes using qRT-PCR. The results suggested that the relative expression patterns were cyclic and not accumulative.

An injury-response mechanism conserved across kingdoms determines entry of the fungus Trichoderma atroviride into development.

A conserved injury-defense mechanism is present in plants and animals, in which the production of reactive oxygen species (ROS) and lipid metabolism are essential to the response. Here, we describe that in the filamentous fungus Trichoderma atroviride, injury results in the formation of asexual reproduction structures restricted to regenerating cells. High-throughput RNA-seq analyses of the response to injury in T. atroviride suggested an oxidative response and activation of calcium-signaling pathways, as well as the participation of lipid metabolism, in this phenomenon. Gene-replacement experiments demonstrated that injury triggers NADPH oxidase (Nox)-dependent ROS production and that Nox1 and NoxR are essential for asexual development in response to damage. We further provide evidence of H(2)O(2) and oxylipin production that, as in plants and animals, may act as signal molecules in response to injury in fungi, suggesting that the three kingdoms share a conserved defense-response mechanism.

Ecological functions of fungal sesquiterpenes in the food preference and fitness of soil Collembola.

Volatile organic compounds (VOCs) emitted by fungi play a key role in locating and selecting hosts for fungivorous arthropods. However, the ecological functions of many common VOC classes, such as sesquiterpenes, remain unknown. Mutants of Trichoderma virens, defective in the emission of most sesquiterpenes owing to the deletion of the terpene cyclase vir4, were used to evaluate the role of this compound class in the food preference and fitness of the soil Collembola Folsomia candida. Choice experiments with and without direct contact with fungal mycelium revealed that Collembola were preferentially attracted to Δvir4 mutants impaired in sesquiterpene synthesis compared to wild-type T. virens. Grazing by F. candida on the sesquiterpene deficient T. virens strain had no effect on Collembola survival, reproduction and growth compared to wild-type T. virens. The results suggest that sesquiterpenes play an important role in fungal defence as repellents, but not as deterrents or toxins, against fungivorous Collembola. Our research contributes to the understanding of ecological interactions between fungi and fungivorous arthropods, providing insights into the specific ecological functions of sesquiterpenes. The study has implications for chemical ecology and the dynamics of multitrophic interactions in soil ecosystems.

Uncovering the multifaceted properties of 6-pentyl-alpha-pyrone for control of plant pathogens.

Some volatile organic compounds (VOCs) produced by microorganisms have the ability to inhibit the growth and development of plant pathogens, induce the activation of plant defenses, and promote plant growth. Among them, 6-pentyl-alpha-pyrone (6-PP), a ketone produced by Trichoderma fungi, has emerged as a focal point of interest. 6-PP has been isolated and characterized from thirteen Trichoderma species and is the main VOC produced, often accounting for >50% of the total VOCs emitted. This review examines abiotic and biotic interactions regulating the production of 6-PP by Trichoderma, and the known effects of 6-PP on plant pathogens through direct and indirect mechanisms including induced systemic resistance. While there are many reports of 6-PP activity against plant pathogens, the vast majority have been from laboratory studies involving only 6-PP and the pathogen, rather than glasshouse or field studies including a host plant in the system. Biopesticides based on 6-PP may well provide an eco-friendly, sustainable management tool for future agricultural production. However, before this can happen, challenges including demonstrating disease control efficacy in the field, developing efficient delivery systems, and determining cost-effective application rates must be overcome before 6-PP's potential for pathogen control can be turned into reality.

Sho1 and Msb2-related proteins regulate appressorium development in the smut fungus Ustilago maydis.

The dimorphic fungus Ustilago maydis switches from budding to hyphal growth on the plant surface. In response to hydrophobicity and hydroxy fatty acids, U. maydis develops infection structures called appressoria. Here, we report that, unlike in Saccharomyces cerevisiae and other fungi where Sho1 (synthetic high osmolarity sensitive) and Msb2 (multicopy suppressor of a budding defect) regulate stress responses and pseudohyphal growth, Sho1 and Msb2-like proteins play a key role during appressorium differentiation in U. maydis. Sho1 was identified through a two-hybrid screen as an interaction partner of the mitogen-activated protein (MAP) kinase Kpp6. Epistasis analysis revealed that sho1 and msb2 act upstream of the MAP kinases kpp2 and kpp6. Furthermore, Sho1 was shown to destabilize Kpp6 through direct interaction with the unique N-terminal domain in Kpp6, indicating a role of Sho1 in fine-tuning Kpp6 activity. Morphological differentiation in response to a hydrophobic surface was strongly attenuated in sho1 msb2 mutants, while hydroxy fatty acid-induced differentiation was unaffected. These data suggest that Sho1 and the transmembrane mucin Msb2 are involved in plant surface sensing in U. maydis.

Fungistatic Activity Mediated by Volatile Organic Compounds Is Isolate-Dependent in Trichoderma sp. "atroviride B".

Trichoderma spp. produce multiple bioactive volatile organic compounds (VOCs). While the bioactivity of VOCs from different Trichoderma species is well documented, information on intraspecific variation is limited. The fungistatic activity of VOCs emitted by 59 Trichoderma sp. "atroviride B" isolates against the pathogen Rhizoctonia solani was investigated. Eight isolates representing the two extremes of bioactivity against R. solani were also assessed against Alternaria radicina, Fusarium oxysporum f. sp. lycopersici and Sclerotinia sclerotiorum. VOCs profiles of these eight isolates were analyzed using gas chromatography-mass spectrometry (GC-MS) to identify a correlation between specific VOCs and bioactivity, and 11 VOCs were evaluated for bioactivity against the pathogens. Bioactivity against R. solani varied among the fifty-nine isolates, with five being strongly antagonistic. All eight selected isolates inhibited the growth of all four pathogens, with bioactivity being lowest against F. oxysporum f. sp. lycopersici. In total, 32 VOCs were detected, with individual isolates producing between 19 and 28 VOCs. There was a significant direct correlation between VOC number/quantity and bioactivity against R. solani. 6-pentyl-α-pyrone was the most abundant VOC produced, but 15 other VOCs were also correlated with bioactivity. All 11 VOCs tested inhibited R. solani growth, some by >50%. Some of the VOCs also inhibited the growth of the other pathogens by >50%. This study demonstrates significant intraspecific differences in VOC profiles and fungistatic activity supporting the existence of biological diversity within Trichoderma isolates from the same species, a factor in many cases ignored during the development of biological control agents.

Dual role of endophytic entomopathogenic fungi: induce plant growth and control tomato leafminer Phthorimaea absoluta.

BACKGROUND: Entomopathogenic fungi (EPF) are multifunctional microorganisms acting not only as biopesticides against insect pests but also as endophytes which regulate plant growth. The tomato leafminer, Phthorimaea absoluta (Tuta absoluta) is a devastating invasive pest of tomatoes worldwide. However, effective alternatives are needed for a sustainable management of this invasive pest. In this study, the functional effects of five EPF isolates Metarhizium flavoviride, M. anisopliae, M. rileyi, Cordyceps fumosorosea and Beauveria bassiana were evaluated on tomato growth promotion and pest protection against P. absoluta. RESULTS: When directly sprayed with conidia, P. absoluta larvae showed high cumulative mortality of 100% to M. anisopliae under 1 × 108 conidia/mL, whereas M. flavoviride, B. bassiana, C. fumosorosea and M. rileyi caused cumulative mortality of 92.65%, 92.62%, 92.16% and 68.95%, respectively. Moreover, all five EPF isolates can successfully colonize tomato plants, whilst the colonization rate for each EPF depends on the inoculation method used. The most efficient inoculation method for M. flavoviride and M. rileyi was root dipping, for M. anisopliae and C. fumosorosea it was coating seed, and for B. bassiana it was foliage spraying. The highest plant colonization was obtained by M. flavoviride. Meanwhile, all these isolates promoted tomato plant growth upon inoculation. Furthermore, endophytic colonization of plants by the five EPF negatively affected the performance of P. absoluta, among them M. anisopliae and C. fumosorosea showed strong negative effects on the performance of P. absoluta. CONCLUSION: Our results highlight the potential of incorporating entomopathogenic fungi as endophytes in integrated pest management practices to protect tomatoes against P. absoluta. © 2023 Society of Chemical Industry.

Osmotic Stress Responses, Cell Wall Integrity, and Conidiation Are Regulated by a Histidine Kinase Sensor in Trichoderma atroviride.

Trichoderma atroviride responds to various environmental stressors through the mitogen-activated protein kinase (MAPK) Tmk3 and MAPK-kinase Pbs2 signaling pathways. In fungi, orthologues to Tmk3 are regulated by a histidine kinase (HK) sensor. However, the role of T. atroviride HKs remains unknown. In this regard, the function of the T. atroviride HK Nik1 was analyzed in response to stressors regulated by Tmk3. The growth of the Δnik1 mutant strains was compromised under hyperosmotic stress; mycelia were less resistant to lysing enzymes than the WT strain, while conidia of Δnik1 were more sensitive to Congo red; however, ∆pbs2 and ∆tmk3 strains showed a more drastic defect in cell wall stability. Light-regulated blu1 and grg2 gene expression was induced upon an osmotic shock through Pbs2-Tmk3 but was independent of Nik1. The encoding chitin synthases chs1 and chs2 genes were downregulated after an osmotic shock in the WT, but chs1 and chs3 expression were enhanced in ∆nik1, ∆pbs2, and ∆tmk3. The vegetative growth and conidiation by light decreased in ∆nik1, although Nik1 was unrequired to activate the light-responsive genes by Tmk3. Altogether, Nik1 regulates responses related to the Pbs2-Tmk3 pathway and suggests the participation of additional HKs to respond to stress.

Insights from the genome of the biotrophic fungal plant pathogen Ustilago maydis.

Ustilago maydis is a ubiquitous pathogen of maize and a well-established model organism for the study of plant-microbe interactions. This basidiomycete fungus does not use aggressive virulence strategies to kill its host. U. maydis belongs to the group of biotrophic parasites (the smuts) that depend on living tissue for proliferation and development. Here we report the genome sequence for a member of this economically important group of biotrophic fungi. The 20.5-million-base U. maydis genome assembly contains 6,902 predicted protein-encoding genes and lacks pathogenicity signatures found in the genomes of aggressive pathogenic fungi, for example a battery of cell-wall-degrading enzymes. However, we detected unexpected genomic features responsible for the pathogenicity of this organism. Specifically, we found 12 clusters of genes encoding small secreted proteins with unknown function. A significant fraction of these genes exists in small gene families. Expression analysis showed that most of the genes contained in these clusters are regulated together and induced in infected tissue. Deletion of individual clusters altered the virulence of U. maydis in five cases, ranging from a complete lack of symptoms to hypervirulence. Despite years of research into the mechanism of pathogenicity in U. maydis, no 'true' virulence factors had been previously identified. Thus, the discovery of the secreted protein gene clusters and the functional demonstration of their decisive role in the infection process illuminate previously unknown mechanisms of pathogenicity operating in biotrophic fungi. Genomic analysis is, similarly, likely to open up new avenues for the discovery of virulence determinants in other pathogens.

Histidine kinase two-component response regulators Ssk1, Skn7 and Rim15 differentially control growth, developmental and volatile organic compounds emissions as stress responses in Trichoderma atroviride.

The Skn7, Ssk1 and Rim15 proteins are response regulators involved in osmotic, oxidative and nutritional stress in fungi. In order to verify the involvement of these genes in Trichoderma atroviride IMI206040's growth, conidiation, direct antagonism against plant pathogens (Rhizoctonia solani and Sclerotinia sclerotiorum), production of volatile organic compounds (VOCs) with fungistatic effect, and interaction with plants (growth promotion), single mutants were generated, and the phenotypic patterns were analysed in comparison to the wild-type (wt) strain. The mutants were submitted to osmotic, oxidative, membrane and cell wall stress conditions in vitro. The Δskn7 and Δrim15 mutants did not show either significant differences at morphological level, or marked decreases in mycelial growth and conidiation in relation to wt, whereas Δssk1 had altered phenotypes in most conditions tested. The plant-growth promotion of Arabidopsis thaliana seedlings induced by VOCs was not quantitatively modified by any of the mutants in relation to the wt strain, although possible differences in secondary root hairs was noticed for Δrim15. The fungistatic activity was significantly altered for Δssk1 and Δrim15. Overall, the Δssk1 strain showed remarkable morphological differences, with decrease in mycelial growth and conidiation, being also affected in the antagonistic capacity against plant pathogens. The impacts demonstrated by the deletion of ssk1 suggest this gene has a relevant participation in the signalling response to different stresses in T. atroviride and in the interactive metabolism with phytopathogens and plants. On the other hand, unlike other fungal models, Skn7 did not appear to have a critical participation in the above-mentioned processes; Rim15 seemed to confirm its involvement in modulating cellular responses to nutritional status, although with a possible cross-talk with other cellular processes. Our results suggest that Ssk1 likely plays a key regulatory role, not only in basic metabolisms of T. atroviride, but also in biocontrol-related characteristics.

Ustilago maydis as a Pathogen.

The Ustilago maydis-maize pathosystem has emerged as the current model for plant pathogenic basidiomycetes and as one of the few models for a true biotrophic interaction that persists throughout fungal development inside the host plant. This is based on the highly advanced genetic system for both the pathogen and its host, the ability to propagate U. maydis in axenic culture, and its unique capacity to induce prominent disease symptoms (tumors) on all aerial parts of maize within less than a week. The corn smut pathogen, though economically not threatening, will continue to serve as a model for related obligate biotrophic fungi such as the rusts, but also for closely related smut species that induce symptoms only in the flower organs of their hosts. In this review we describe the most prominent features of the U. maydis-maize pathosystem as well as genes and pathways most relevant to disease. We highlight recent developments that place this system at the forefront of understanding the function of secreted effectors in eukaryotic pathogens and describe the expected spin-offs for closely related species exploiting comparative genomics approaches.

Hap2 regulates the pheromone response transcription factor prf1 in Ustilago maydis.

In Ustilago maydis the pheromone signal is transmitted via a mitogen-activated protein kinase (MAP kinase) module to the transcription factor Prf1. Prf1 activates transcription of a and b mating type genes by binding to pheromone response elements (PREs) located in regulatory regions of these genes. Here we show that the CCAAT-box binding protein Hap2 from U. maydis regulates prf1 expression. Hap2 was initially identified as a potential interaction partner of the MAP kinase Kpp6 in yeast two-hybrid screens and was subsequently also shown to interact with the MAPK Kpp2. Deletion of hap2 in haploid cells abolished mating, resulting from a defect in pheromone-induced gene expression. Crosses of haploid hap2 deletion strains were completely impaired in pathogenicity. Constitutive expression of prf1 complemented the pheromone response defect in Δhap2 strains. Chromatin immunoprecipitation assays indicated that Hap2 binds directly to CCAAT motifs in the prf1 promoter. Point mutations in two putative MAPK phosphorylation sites in Hap2 attenuated the pheromone response. In a solopathogenic strain hap2 deletion affected filamentation and the mutants showed reduced pathogenicity symptoms. These data suggest that Hap2 is a novel regulator of prf1 with additional functions after cell fusion.

The dual specificity phosphatase Rok1 negatively regulates mating and pathogenicity in Ustilago maydis.

In the phytopathogenic fungus Ustilago maydis a conserved mitogen-activated-protein-kinase (MAPK) module regulates sexual and pathogenic development. Kpp2 is the central MAPK of this module and is required for transcriptional and morphological responses to pheromone. Upon perception of the pheromone signal Kpp2 is phosphorylated by the MAPK kinase Fuz7. Here we demonstrate that the MAPK Kpp6, which has a partially redundant function with Kpp2, is also phosphorylated by Fuz7. We show that Rok1, a putative dual specificity phosphatase for MAPK signalling, controls the phosphorylation of Kpp2 as well as of Kpp6. rok1 mutants display increased filamentation and are enhanced in virulence. The enhanced virulence is caused by more efficient appressorium formation as well as plant invasion. Overexpression of rok1 reduced conjugation hyphae formation and strongly attenuated pathogenicity. This places Rok1 in a negative feedback loop regulating Kpp2 and Kpp6 activity upon pheromone stimulation and plant colonization.

Physical-chemical plant-derived signals induce differentiation in Ustilago maydis.

Ustilago maydis is able to initiate pathogenic development after fusion of two haploid cells with different mating type. On the maize leaf surface, the resulting dikaryon switches to filamentous growth, differentiates appressoria and penetrates the host. Here, we report on the plant signals required for filament formation and appressorium development in U. maydis. In vitro, hydroxy-fatty acids stimulate filament formation via the induction of pheromone genes and this signal can be bypassed by genetically activating the downstream MAP kinase module. Hydrophobicity also induces filaments and these resemble the dikaryotic filaments formed on the plant surface. With the help of a marker gene that is specifically expressed in the tip cell of those hyphae that have formed an appressorium, hydrophobicity is shown to be essential for appressorium development in vitro. Hydroxy-fatty acids or a cutin monomer mixture isolated from maize leaves have a stimulatory role when a hydrophobic surface is provided. Our results suggest that the early phase of communication between U. maydis and its host plant is governed by two different stimuli.

Reproduction without sex: conidiation in the filamentous fungus Trichoderma.

Trichoderma spp. have served as models for asexual reproduction in filamentous fungi for over 50 years. Physical stimuli, such as light exposure and mechanical injury to the mycelium, trigger conidiation; however, conidiogenesis itself is a holistic response determined by the cell's metabolic state, as influenced by the environment and endogenous biological rhythms. Key environmental parameters are the carbon and nitrogen status and the C : N ratio, the ambient pH and the level of calcium ions. Recent advances in our understanding of the molecular biology of this fungus have revealed a conserved mechanism of environmental perception through the White Collar orthologues BLR-1 and BLR-2. Also implicated in the molecular regulation are the PacC pathways and the conidial regulator VELVET. Signal transduction cascades which link environmental signals to physiological outputs have also been revealed.

TrichoGate: An Improved Vector System for a Large Scale of Functional Analysis of Trichoderma Genes.

Species of the genus Trichoderma are ubiquitous in the environment and are widely used in agriculture, as biopesticides, and in the industry for the production of plant cell wall-degrading enzymes. Trichoderma represents an important genus of endophytes, and several Trichoderma species have become excellent models for the study of fungal biology and plant-microbe interactions; moreover, are exceptional biotechnological factories for the production of bioactive molecules useful in agriculture and medicine. Next-generation sequencing technology coupled with systematic construction of recombinant DNA molecules provides powerful tools that contribute to the functional analysis of Trichoderma genetics, thus allowing for a better understanding of the underlying factors determining its biology. Here, we present the creation of diverse vectors containing (i) promoter-specific vectors for Trichoderma, (ii) gene deletions (using hygromycin phosphotransferase as selection marker), (iii) protein localization (mCherry and eGFP, which were codon-optimized for Trichoderma), (iv) gene complementation (neomycin phosphotransferase) and (v) overexpression of encoding gene proteins fused to fluorescent markers, by using the Golden Gate cloning technology. Furthermore, we present the design and implementation of a binary vector for Agrobacterium-mediated transformation in Trichoderma to increase the homologous recombination rate and the generation of a novel selection marker based on carboxin resistance.