The Trichoderma atroviride putative transcription factor Blu7 controls light responsiveness and tolerance.

BACKGROUND: Most living organisms use sunlight as a source of energy and/or information about their environment. Consequently, they have developed mechanisms to sense light quality and quantity. In the fungus Trichoderma atroviride blue-light is perceived through the Blue Light Regulator Complex, which in turn up-regulates a set of genes (blu) and down-regulates another set (bld), triggering asexual reproduction. To gain insight into this process, we characterized the blu7 gene, which encodes a protein containing a C2H2 zinc finger domain. RESULTS: Δblu7 mutants show reduced conidiation at low light fluences, which is still clear even when exposed to saturating light. For the first time we show a genome wide survey of light regulated gene expression in T. atroviride, including RNA-seq analyses of the wild type and the Δblu7 strains after brief exposure to blue-light. Our data show a reduction in the number of induced genes and an increase in down-regulated genes in the mutant. Light activates stress responses and several metabolic processes in the wild type strain that are no longer activated in the mutant. In agreement with the misregulation of metabolic processes, continuous exposure to white light strongly inhibited growth of the ∆blu7 mutant, in a carbon source dependent fashion. RNA-seq analyses under constant white light using glucose as sole carbon source revealed that localization and transport process present the opposite regulation pattern in the ∆blu7 and wild type strains. Genes related to amino acid, sugar and general transporters were enriched in the induced genes in the mutant and the repressed genes of the wild type. Peptone supplemented in the media restored growth of the ∆blu7 mutant in constant light, suggesting a role of Blu7 in the regulation of nitrogen metabolism in the presence of light. CONCLUSIONS: Blu7 appears to regulate light sensitivity in terms of induction of conidiation, and to play a major role in supporting growth under continuous exposure to light. The diminished conidiation observed in ∆blu7 mutants is likely due to misregulation of the cAMP signaling pathway and ROS production, whereas their low tolerance to continuous exposure to light indicates that Blu7 is required for adaptation.

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.

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 Ca(2+) 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, Ca(2+), and ROS. Indeed, application of exogenous ATP and Ca(2+) 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, Ca(2+) 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.

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.

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.

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

BACKGROUND: Mycoparasitism, 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. RESULTS: Here 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. CONCLUSIONS: The data offer a better understanding of mycoparasitism, and thus enforce the development of improved biocontrol strains for efficient and environmentally friendly protection of plants.

Trichoderma atroviride PHR1, a fungal photolyase responsible for DNA repair, autoregulates its own photoinduction.

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 (Deltaphr1) strains. Photoinduction of the phr1 promoter in Deltaphr1 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.