RESUMO
Optogenetic switches allow light-controlled gene expression with reversible and spatiotemporal resolution. In Saccharomyces cerevisiae, optogenetic tools hold great potential for a variety of metabolic engineering and biotechnology applications. In this work, we report on the modular optimization of the fungal light-oxygen-voltage (FUN-LOV) system, an optogenetic switch based on photoreceptors from the fungus Neurospora crassa. We also describe new switch variants obtained by replacing the Gal4 DNA-binding domain (DBD) of FUN-LOV with nine different DBDs from yeast transcription factors of the zinc cluster family. Among the tested modules, the variant carrying the Hap1p DBD, which we call "HAP-LOV", displayed higher levels of luciferase expression upon induction compared to FUN-LOV. Further, the combination of the Hap1p DBD with either p65 or VP16 activation domains also resulted in higher levels of reporter expression compared to the original switch. Finally, we assessed the effects of the plasmid copy number and promoter strength controlling the expression of the FUN-LOV and HAP-LOV components, and observed that when low-copy plasmids and strong promoters were used, a stronger response was achieved in both systems. Altogether, we describe a new set of blue-light optogenetic switches carrying different protein modules, which expands the available suite of optogenetic tools in yeast and can additionally be applied to other systems.
Assuntos
Proteínas Fúngicas , Microrganismos Geneticamente Modificados , Neurospora crassa/genética , Optogenética , Fotorreceptores Microbianos , Saccharomyces cerevisiae , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/metabolismo , Neurospora crassa/metabolismo , Fotorreceptores Microbianos/biossíntese , Fotorreceptores Microbianos/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
We have described that formation of basidiocarps by Ustilago maydis requires illumination. In the current research, we have proceeded to analyze what kind of light receptors are involved in this phenomenon. Accordingly, we investigated whether the homologues of the White Collar (WC), and the phytochrome (PHY) genes played a role in this process. Mutants deficient in either one of the three U. maydis WC homologue genes (WCO1a, WCO1b, WCO2), or the phytochrome-encoding the PHY gene were obtained. Phenotypic analysis of the mutants showed that ∆wco1a mutants formed similar numbers of basidiocarps than wild-type strain, whereas ∆wco1b mutants were severely affected in basidiocarp formation when illuminated with white, blue or red light. ∆wco2 and ∆phy1 mutants did not form basidiocarps under any illumination condition. These data indicate that Wco1a is the main blue light receptor, and Wco1b may operate as a secondary blue light receptor; Phy1 is the red light receptor, and Wco2 the transcription factor that controls the photo stimulation of the genes involved in the formation of fruiting bodies. It is suggested that effectiveness of the light receptors depends on the whole structure of the complex, possibly, because their association is necessary to maintain their functional structure.
Assuntos
Carpóforos/fisiologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fotorreceptores Microbianos/genética , Fotorreceptores Microbianos/metabolismo , Ustilago/fisiologia , Carpóforos/efeitos da radiação , Ustilago/genética , Ustilago/efeitos da radiaçãoRESUMO
Light is increasingly recognized as an efficient means of controlling diverse biological processes with high spatiotemporal resolution. Optogenetic switches are molecular devices for regulating light-controlled gene expression, protein localization, signal transduction and protein-protein interactions. Such molecular components have been mainly developed through the use of photoreceptors, which upon light stimulation undergo conformational changes passing to an active state. The current repertoires of optogenetic switches include red, blue and UV-B light photoreceptors and have been implemented in a broad spectrum of biological platforms. In this review, we revisit different optogenetic switches that have been used in diverse biological platforms, with emphasis on those used for light-controlled gene expression in the budding yeast Saccharomyces cerevisiae. The implementation of these switches overcomes the use of traditional chemical inducers, allowing precise control of gene expression at lower costs, without leaving chemical traces, and positively impacting the production of high-value metabolites and heterologous proteins. Additionally, we highlight the potential of utilizing this technology beyond laboratory strains, by optimizing it for use in yeasts tamed for industrial processes. Finally, we discuss how fungal photoreceptors could serve as a source of biological parts for the development of novel optogenetic switches with improved characteristics. Although optogenetic tools have had a strong impact on basic research, their use in applied sciences is still undervalued. Therefore, the invitation for the future is to utilize this technology in biotechnological and industrial settings.
Assuntos
Regulação Fúngica da Expressão Gênica , Luz , Optogenética , Saccharomyces cerevisiae/genética , Expressão Gênica , Microbiologia Industrial , Fotorreceptores Microbianos/genética , Transdução de Sinais/genética , Biologia Sintética/métodosRESUMO
Pathogens interaction with a host plant starts a set of immune responses that result in complex changes in gene expression and plant physiology. Light is an important modulator of plant defense response and recent studies have evidenced the novel influence of this environmental stimulus in the virulence of several bacterial pathogens. Xanthomonas citri subsp. citri is the bacterium responsible for citrus canker disease, which affects most citrus cultivars. The ability of this bacterium to colonize host plants is influenced by bacterial blue-light sensing through a LOV-domain protein and disease symptoms are considerably altered upon deletion of this protein. In this work we aimed to unravel the role of this photoreceptor during the bacterial counteraction of plant immune responses leading to citrus canker development. We performed a transcriptomic analysis in Citrus sinensis leaves inoculated with the wild type X. citri subsp. citri and with a mutant strain lacking the LOV protein by a cDNA microarray and evaluated the differentially regulated genes corresponding to specific biological processes. A down-regulation of photosynthesis-related genes (together with a corresponding decrease in photosynthesis rates) was observed upon bacterial infection, this effect being more pronounced in plants infected with the lov-mutant bacterial strain. Infection with this strain was also accompanied with the up-regulation of several secondary metabolism- and defense response-related genes. Moreover, we found that relevant plant physiological alterations triggered by pathogen attack such as cell wall fortification and tissue disruption were amplified during the lov-mutant strain infection. These results suggest the participation of the LOV-domain protein from X. citri subsp. citri in the bacterial counteraction of host plant defense response, contributing in this way to disease development.
Assuntos
Proteínas de Bactérias/genética , Citrus sinensis/imunologia , Regulação da Expressão Gênica de Plantas , Fotorreceptores Microbianos/genética , Doenças das Plantas/imunologia , Proteínas de Plantas/imunologia , Xanthomonas/patogenicidade , Proteínas de Bactérias/metabolismo , Citrus sinensis/genética , Citrus sinensis/microbiologia , Deleção de Genes , Perfilação da Expressão Gênica , Interações Hospedeiro-Patógeno , Evasão da Resposta Imune , Luz , Fotorreceptores Microbianos/metabolismo , Fotossíntese/fisiologia , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Imunidade Vegetal/genética , Proteínas de Plantas/genética , Estrutura Terciária de Proteína , Virulência , Xanthomonas/genéticaRESUMO
In Arabidopsis thaliana, light signals modulate the defences against bacteria. Here we show that light perceived by the LOV domain-regulated two-component system (Pst-Lov) of Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) modulates virulence against A. thaliana. Bioinformatic analysis and the existence of an episomal circular intermediate indicate that the locus encoding Pst-Lov is present in an active genomic island acquired by horizontal transfer. Strains mutated at Pst-Lov showed enhanced growth on minimal medium and in leaves of A. thaliana exposed to light, but not in leaves incubated in darkness or buried in the soil. Pst-Lov repressed the expression of principal and alternative sigma factor genes and their downstream targets linked to bacterial growth, virulence and quorum sensing, in a strictly light-dependent manner. We propose that the function of Pst-Lov is to distinguish between soil (dark) and leaf (light) environments, attenuating the damage caused to host tissues while releasing growth out of the host. Therefore, in addition to its direct actions via photosynthesis and plant sensory receptors, light may affect plants indirectly via the sensory receptors of bacterial pathogens.
Assuntos
Ilhas Genômicas , Luz , Fotorreceptores Microbianos/genética , Folhas de Planta/microbiologia , Pseudomonas syringae/patogenicidade , Virulência , Arabidopsis/microbiologia , Arabidopsis/efeitos da radiação , Regulação Bacteriana da Expressão Gênica , Transferência Genética Horizontal , Fases de Leitura Aberta , Óperon , Fotorreceptores Microbianos/efeitos da radiação , Doenças das Plantas/microbiologia , Folhas de Planta/efeitos da radiação , Pseudomonas syringae/genética , Percepção de Quorum , Fator sigma/metabolismoRESUMO
Organisms are exposed to a tough environment, where acute daily challenges, like light, can strongly affect several aspects of an individual's physiology, including pathogenesis. While several fungal models have been widely employed to understand the physiological and molecular events associated with light perception, various other agricultural-relevant fungi still remain, in terms of their responsiveness to light, in the dark. The fungus Botrytis cinerea is an aggressive pathogen able to cause disease on a wide range of plant species. Natural B. cinerea isolates exhibit a high degree of diversity in their predominant mode of reproduction. Thus, the majority of naturally occurring strains are known to reproduce asexually via conidia and sclerotia, and sexually via apothecia. Studies from the 1970's reported on specific developmental responses to treatments with near-UV, blue, red and far-red light. To unravel the signaling machinery triggering development--and possibly also connected with virulence--we initiated the functional characterization of the transcription factor/photoreceptor BcWCL1 and its partner BcWCL2, that form the White Collar Complex (WCC) in B. cinerea. Using mutants either abolished in or exhibiting enhanced WCC signaling (overexpression of both bcwcl1 and bcwcl2), we demonstrate that the WCC is an integral part of the mentioned machinery by mediating transcriptional responses to white light and the inhibition of conidiation in response to this stimulus. Furthermore, the WCC is required for coping with excessive light, oxidative stress and also to achieve full virulence. Although several transcriptional responses are abolished in the absence of bcwcl1, the expression of some genes is still light induced and a distinct conidiation pattern in response to daily light oscillations is enhanced, revealing a complex underlying photobiology. Though overlaps with well-studied fungal systems exist, the light-associated machinery of B. cinerea appears more complex than those of Neurospora crassa and Aspergillus nidulans.
Assuntos
Botrytis/patogenicidade , Botrytis/efeitos da radiação , Luz/efeitos adversos , Complexos Multiproteicos/metabolismo , Fotorreceptores Microbianos/metabolismo , Transdução de Sinais/genética , Fatores de Transcrição/metabolismo , 3,3'-Diaminobenzidina , Northern Blotting , Botrytis/genética , Botrytis/crescimento & desenvolvimento , Clonagem Molecular , Complexos Multiproteicos/genética , Fotorreceptores Microbianos/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genética , Azul Tripano , Virulência , Vitis/microbiologiaRESUMO
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.
Assuntos
Celulase/genética , Proteínas Fúngicas/genética , Regulação Enzimológica da Expressão Gênica/efeitos da radiação , Fotorreceptores Microbianos/metabolismo , Trichoderma/crescimento & desenvolvimento , Trichoderma/efeitos da radiação , Celulase/metabolismo , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica/efeitos da radiação , Luz , Fotorreceptores Microbianos/genética , Esporos Fúngicos/genética , Esporos Fúngicos/crescimento & desenvolvimento , Esporos Fúngicos/metabolismo , Esporos Fúngicos/efeitos da radiação , Trichoderma/genética , Trichoderma/metabolismoRESUMO
In fungi, phototropism, the induction of carotenogenesis and reproductive structures, and resetting of the circadian rhythm are controlled by blue light. Trichoderma atroviride, a fungus used in biological control, sporulates in a synchronized manner following a brief pulse of blue light. Due to its apparent simplicity, this response was chosen for pursuing photoreceptor isolation. Two genes were cloned, blue-light regulators 1 and 2 (blr-1 and blr-2), similar to the Neurospora crassa white-collar 1 and 2, respectively. The BLR-1 protein has all the characteristics of a blue-light photoreceptor, whereas the structure of the deduced BLR-2 protein suggests that it interacts with BLR-1 through PAS domains to form a complex. Disruption of the corresponding genes demonstrated that they are essential for blue-light-induced conidiation. blr-1 and blr-2 were also shown to be essential for the light-induced expression of the photolyase-encoding gene (phr-1). Mechanical injury of mycelia was found to trigger conidiation of T. atroviride, a response not described previously. This response was not altered in the mutants. A novel effect of both red and blue light on mycelial growth was found involving another light receptor, which is compensated by the BLR proteins.