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Proteomic analysis of Trichoderma atroviride reveals independent roles for transcription factors BLR-1 and BLR-2 in light and darkness.

ABSTRACT: The genus Trichoderma is one of the most widely used biological control agents of plant-pathogenic fungi. The main mechanism for survival and dispersal of Trichoderma is through the production of asexual spores (conidia). The transition from filamentous growth to conidiation can be triggered by light, nutrient deprivation, and mechanical damage of the mycelium. We conducted proteomic profiling analyses of Trichoderma atroviride after a blue light pulse. The use of two-dimensional electrophoresis (2-DE) and mass spectrometry (MS) analysis allowed us to identify 72 proteins whose expression was affected by blue light. Functional category analysis showed that the various proteins are involved in metabolism, cell rescue, and protein synthesis. We determined the relationship between mRNA levels of selected genes 30 min after a light pulse and protein expression levels at different times after the pulse and found this correlation to be very weak. The correlation was highest when protein and mRNA levels were compared for the same time point. The transcription factors BLR-1 and BLR-2 are vital to the photoconidiation process; here we demonstrate that both BLR proteins are active in darkness and affect several elements at both the transcript and protein levels. Unexpectedly, in darkness, downregulation of proteins prevailed in the Δblr-1 mutant, while upregulation of proteins predominated in the Δblr-2 mutant. Our data demonstrate that the BLR proteins play roles individually and as a complex.

SUBMITTER: Sanchez-Arreguin A

PROVIDER: S-EPMC3255938 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundMost 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.ResultsΔblu7 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.ConclusionsBlu7 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.

Project description:Fungal blue-light photoreceptors have been proposed as integrators of light and oxidative stress. However, additional elements participating in the integrative pathway remain to be identified. In Trichoderma atroviride, the blue-light regulator (BLR) proteins BLR-1 and -2 are known to regulate gene transcription, mycelial growth, and asexual development upon illumination, and recent global transcriptional analysis revealed that the histone deacetylase-encoding gene hda-2 is induced by light. Here, by assessing responses to stimuli in wild-type and Δhda-2 backgrounds, we evaluate the role of HDA-2 in the regulation of genes responsive to light and oxidative stress. Δhda-2 strains present reduced growth, misregulation of the con-1 gene, and absence of conidia in response to light and mechanical injury. We found that the expression of hda-2 is BLR-1 dependent and HDA-2 in turn is essential for the transcription of early and late light-responsive genes that include blr-1, indicating a regulatory feedback loop. When subjected to reactive oxygen species (ROS), Δhda-2 mutants display high sensitivity whereas Δblr strains exhibit the opposite phenotype. Consistently, in the presence of ROS, ROS-related genes show high transcription levels in wild-type and Δblr strains but misregulation in Δhda-2 mutants. Finally, chromatin immunoprecipitations of histone H3 acetylated at Lys9/Lys14 on cat-3 and gst-1 promoters display low accumulation of H3K9K14ac in Δblr and Δhda-2 strains, suggesting indirect regulation of ROS-related genes by HDA-2. Our results point to a mutual dependence between HDA-2 and BLR proteins and reveal the role of these proteins in an intricate gene regulation landscape in response to blue light and ROS. Trichoderma atroviride is a free-living fungus commonly found in soil or colonizing plant roots and is widely used as an agent in biocontrol as it parasitizes other fungi, stimulates plant growth, and induces the plant defense system. To survive in various environments, fungi constantly sense and respond to potentially threatening external factors, such as light. In particular, UV light can damage biomolecules by producing free-radical reactions, in most cases involving reactive oxygen species (ROS). In T. atroviride, conidiation is essential for its survival, which is induced by light and mechanical injury. Notably, conidia are typically used as the inoculum in the field during biocontrol. Therefore, understanding the linkages between responses to light and exposure to ROS in T. atroviride is of major basic and practical relevance. Here, the histone deacetylase-encoding gene hda-2 is induced by light and ROS, and its product regulates growth, conidiation, blue light perception, and oxidative stress responses.

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Proteomic analysis of Trichoderma atroviride reveals independent roles for transcription factors BLR-1 and BLR-2 in light and darkness.

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