Edgardo U. Esquivel-Naranjo

Comparative genome sequence analysis underscores mycoparasitism as the ancestral life style of Trichoderma

BackgroundMycoparasitism, a lifestyle where one fungus is parasitic on another fungus, has special relevance when the prey is a plant pathogen, providing a strategy for biological control of pests for plant protection. Probably, the most studied biocontrol agents are species of the genus Hypocrea/Trichoderma.ResultsHere we report an analysis of the genome sequences of the two biocontrol species Trichoderma atroviride (teleomorph Hypocrea atroviridis) and Trichoderma virens (formerly Gliocladium virens, teleomorph Hypocrea virens), and a comparison with Trichoderma reesei (teleomorph Hypocrea jecorina). These three Trichoderma species display a remarkable conservation of gene order (78 to 96%), and a lack of active mobile elements probably due to repeat-induced point mutation. Several gene families are expanded in the two mycoparasitic species relative to T. reesei or other ascomycetes, and are overrepresented in non-syntenic genome regions. A phylogenetic analysis shows that T. reesei and T. virens are derived relative to T. atroviride. The mycoparasitism-specific genes thus arose in a common Trichoderma ancestor but were subsequently lost in T. reesei.ConclusionsThe data offer a better understanding of mycoparasitism, and thus enforce the development of improved biocontrol strains for efficient and environmentally friendly protection of plants.

Crucial factors of the light perception machinery and their impact on growth and cellulase gene transcription in Trichoderma reesei.

In Trichoderma reesei light stimulates transcription of cellulase genes and this regulation has been found to occur, at least in part, through the protein ENVOY. Here we analyzed the role of the BLR photoreceptor complex (BLR1/BLR2) in photoconidiation and the regulation of gene expression. Both responses were dependent on both BLR proteins. Analyses of Deltablr1, Deltablr2 and Deltaenv1 mutants showed that the BLR proteins regulate growth under illumination. Analysis of env1 mutant strains indicated that ENVOY allows the fungus to tolerate continuous exposure to light, damped the capacity of Trichoderma to perceive changes in light intensity, and suggested that it participates in a negative regulatory feedback. Its activity as repressor establishes a period of insensitivity to a second light treatment. Interestingly, the stimulation of cellulase gene expression by light was also modulated by both blr1 and blr2, indicating a key role of the BLR proteins in this pathway.

Trichoderma in the light of day - physiology and development.

In recent years, considerable progress has been made in the elucidation of photoresponses and the mechanisms responsible for their induction in species of the genus Trichoderma. Although an influence of light on these fungi had already been reported five decades ago, their response is not limited to photoconidiation. While early studies on the molecular level concentrated on signaling via the secondary messenger cAMP, a more comprehensive scheme is available today. The photoreceptor-orthologs BLR1 and BLR2 are known to mediate almost all known light responses in these fungi and another light-regulatory protein, ENVOY, is suggested to establish the connection between light response and nutrient signaling. As a central regulatory mechanism, this light signaling machinery impacts diverse downstream pathways including vegetative growth, reproduction, carbon and sulfur metabolism, response to oxidative stress and biosynthesis of peptaibols. These responses involve several signaling cascades, for example the heterotrimeric G-protein and MAP-kinase cascades, resulting in an integrated response to environmental conditions.

Trichoderma atroviride PHR1, a Fungal Photolyase Responsible for DNA Repair, Autoregulates Its Own Photoinduction

ABSTRACT The photolyases, DNA repair enzymes that use visible and long-wavelength UV light to repair cyclobutane pyrimidine dimers (CPDs) created by short-wavelength UV, belong to the larger photolyase-cryptochrome gene family. Cryptochromes (UVA-blue light photoreceptors) lack repair activity, and sensory and regulatory roles have been defined for them in plants and animals. Evolutionary considerations indicate that cryptochromes diverged from CPD photolyases before the emergence of eukaryotes. In prokaryotes and lower eukaryotes, some photolyases might have photosensory functions. phr1 codes for a class I CPD photolyase in Trichoderma atroviride. phr1 is rapidly induced by blue and UVA light, and its photoinduction requires functional blue light regulator (BLR) proteins, which are White Collar homologs in Trichoderma. Here we show that deletion of phr1 abolished photoreactivation of UVC (200 to 280 nm)-inhibited spores and thus that PHR1 is the main component of the photorepair system. The 2-kb 5′ upstream region of phr1, with putative light-regulated elements, confers blue light regulation on a reporter gene. To assess phr1 photosensory function, fluence response curves of this light-regulated promoter were tested in null mutant (Δphr1) strains. Photoinduction of the phr1 promoter in Δphr1 strains was >5-fold more sensitive to light than that in the wild type, whereas in PHR1-overexpressing lines the sensitivity to light increased about 2-fold. Our data suggest that PHR1 may regulate its expression in a light-dependent manner, perhaps through negative modulation of the BLR proteins. This is the first evidence for a regulatory role of photolyase, a role usually attributed to cryptochromes.

The Genomes of Three Uneven Siblings: Footprints of the Lifestyles of Three Trichoderma Species

SUMMARY The genus Trichoderma contains fungi with high relevance for humans, with applications in enzyme production for plant cell wall degradation and use in biocontrol. Here, we provide a broad, comprehensive overview of the genomic content of these species for “hot topic” research aspects, including CAZymes, transport, transcription factors, and development, along with a detailed analysis and annotation of less-studied topics, such as signal transduction, genome integrity, chromatin, photobiology, or lipid, sulfur, and nitrogen metabolism in T. reesei, T. atroviride, and T. virens, and we open up new perspectives to those topics discussed previously. In total, we covered more than 2,000 of the predicted 9,000 to 11,000 genes of each Trichoderma species discussed, which is >20% of the respective gene content. Additionally, we considered available transcriptome data for the annotated genes. Highlights of our analyses include overall carbohydrate cleavage preferences due to the different genomic contents and regulation of the respective genes. We found light regulation of many sulfur metabolic genes. Additionally, a new Golgi 1,2-mannosidase likely involved in N-linked glycosylation was detected, as were indications for the ability of Trichoderma spp. to generate hybrid galactose-containing N-linked glycans. The genomic inventory of effector proteins revealed numerous compounds unique to Trichoderma, and these warrant further investigation. We found interesting expansions in the Trichoderma genus in several signaling pathways, such as G-protein-coupled receptors, RAS GTPases, and casein kinases. A particularly interesting feature absolutely unique to T. atroviride is the duplication of the alternative sulfur amino acid synthesis pathway.

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 H2O2 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.

The RNAi machinery regulates growth and development in the filamentous fungus Trichoderma atroviride

The RNAi machinery is generally involved in genome protection in filamentous fungi; however, the physiological role of RNAi has been poorly studied in fungal models. Here, we report that in the filamentous fungus Trichoderma atroviride, the products of the dcr2 and rdr3 genes control reproductive development, because mutations in these genes affect conidiation. In addition, Dcr1 together with Dcr2 control vegetative growth since Δdcr1, Δdcr2 and Δdcr1Δdcr2 present morphological alterations. Whole‐genome transcriptional analysis of WT, Δdcr1, Δdcr2 and Δdcr1Δdcr2 show that each Dicer controls different biological processes, such as development or metabolism, which could explain the lack of conidiation in the mutants. Finally, we observed sRNAs that are differentially expressed in the WT and Δdcr2. The expression of some of these sRNAs correlates with the expression of differential transcripts, suggesting that these mRNAs may contain the corresponding targets. Together these data show that in T. atroviride, the RNAi machinery plays a central role in endogenous processes such as development and fitness, beyond controlling genome protection against invasive nucleic acids as reported for other fungi.

Extracellular ATP activates MAPK and ROS signaling during injury response in the fungus Trichoderma atroviride

The response to mechanical damage is crucial for the survival of multicellular organisms, enabling their adaptation to hostile environments. Trichoderma atroviride, a filamentous fungus of great importance in the biological control of plant diseases, responds to mechanical damage by activating regenerative processes and asexual reproduction (conidiation). During this response, reactive oxygen species (ROS) are produced by the NADPH oxidase complex. To understand the underlying early signaling events, we evaluated molecules such as extracellular ATP (eATP) and Ca2+ that are known to trigger wound-induced responses in plants and animals. Concretely, we investigated the activation of mitogen-activated protein kinase (MAPK) pathways by eATP, Ca2+, and ROS. Indeed, application of exogenous ATP and Ca2+ triggered conidiation. Furthermore, eATP promoted the Nox1-dependent production of ROS and activated a MAPK pathway. Mutants in the MAPK-encoding genes tmk1 and tmk3 were affected in wound-induced conidiation, and phosphorylation of both Tmk1 and Tmk3 was triggered by eATP. We conclude that in this fungus, eATP acts as a damage-associated molecular pattern (DAMP). Our data indicate the existence of an eATP receptor and suggest that in fungi, eATP triggers pathways that converge to regulate asexual reproduction genes that are required for injury-induced conidiation. By contrast, Ca2+ is more likely to act as a downstream second messenger. The early steps of mechanical damage response in T. atroviride share conserved elements with those known from plants and animals.

A Trichoderma atroviride stress-activated MAPK pathway integrates stress and light signals.

Cells possess stress‐activated protein kinase (SAPK) signalling pathways, which are activated practically in response to any cellular insult, regulating responses for survival and adaptation to harmful environmental changes. To understand the function of SAPK pathways in T. atroviride, mutants lacking the MAPKK Pbs2 and the MAPK Tmk3 were analysed under several cellular stresses, and in their response to light. All mutants were highly sensitive to cellular insults such as osmotic and oxidative stress, cell wall damage, high temperature, cadmium, and UV irradiation. Under oxidative stress, the Tmk3 pathway showed specific roles during development, which in conidia are essential for tolerance to oxidant agents and appear to play a minor role in mycelia. The function of this pathway was more evident in Δpbs2 and Δtmk3 mutant strains when combining oxidative stress or cell wall damage with light. Light stimulates tolerance to osmotic stress through Tmk3 independently of the photoreceptor Blr1. Strikingly, photoconidiation and expression of blue light regulated genes was severally affected in Δtmk3 and Δpbs2 strains, indicating that this pathway regulates light responses. Furthermore, Tmk3 was rapidly phosphorylated upon light exposure. Thus, our data indicate that Tmk3 signalling cooperates with the Blr photoreceptor complex in the activation of gene expression.

The Trichoderma atroviride cryptochrome/photolyase genes regulate the expression of blr1-independent genes both in red and blue light.

Quantitative transcriptome analysis led to the identification of 331 transcripts regulated by white light. Evaluation of the response to white light in mutants affected in the previously characterized blue-light receptor Blr1, demonstrated the existence of both Blr1-dependent and independent responses. Functional categorization of the light responsive genes indicated the effect of light on regulation of various transcription factors, regulators of chromatin structure, signaling pathways, genes related to different kinds of stress, metabolism, redox adjustment, and cell cycle among others. In order to establish the participation of other photoreceptors, gene expression was validated in response to different wavelengths. Gene regulation by blue and red light suggests the involvement of several photoreceptors in integrating light signals of different wavelengths in Trichoderma atroviride. Functional analysis of potential blue light photoreceptors suggests that several perception systems for different wavelengths are involved in the response to light. Deletion of cry1, one of the potential photoreceptors, resulted in severe reduction in the photoreactivation capacity of the fungus, as well as a change in gene expression under blue and red light.