Regulatory roles of photoreceptors and non-coding RNA in Fusarium fujikuroi

The genus Fusarium comprises hundreds of species of pathogenic and saprophytic fungi, usually characterized by a complex secondary metabolism. This includes the production of carotenoids, terpenoid pigments that give a characteristic orange color to their colonies. All the biosynthetic genes have been identified, and two of them are grouped in a coregulated cluster. Light plays a major role in promoting their synthesis by activating their transcription, but their effects on fungal biology are much wider. Light is presumably perceived by F. fujikuroi by a battery of photoreceptors, including members of the White-Collar complex, cryptochromes and photolyases, rhodopsins, and phytochromes. The main photoresponses in fungi are normally dependent on the White-collar complex, and carotenoid biosynthesis in Fusarium mainly depends on it, with WcoA as the light-absorbing component. Another photoreceptor, the DASH cryptochrome CryD, also affects the response of carotenogenesis to light, although the available information suggests its participation in a post-transcriptional regulation. Carotenogenesis is downregulated by a protein of the RING Finger family, called CarS, whose mutation results in a carotenoid overproduction phenotype. The carS gene is preceded by a 4 kb sequence with no known genes, in which the presence of two putative genes for microRNA like precursors had previously been suggested. In this Thesis, a massive sequencing specific for small RNAs has been carried out in F. fujikuroi, which confirmed their occurrence in this fungus. The result was consistent with the existence of a functional RNA interference system, supported by the identification of all the predicted enzymatic components encoded in the genome. This system seems to play a role in the biology of the fungus, as suggests the association between some sRNAs and the sequences of some transposons, indicating their role in silencing of these mobile elements. Deletion of one of the components, the gene for a Dicer protein predictably involved in their generation, showed no phenotype under laboratory conditions, suggesting other roles of the produced sRNAs in processes related with other stages of its life cycle, such as pathogenesis or sexual reproduction. The global sRNA analysis failed to identify possible sRNA elements upstream of carS, contradicting the former hypothesis on the participation of microRNAS in carS regulation. However, a new 1.2 kb transcript, that was denominated carP, was identified in carS upstream region. Its sequence lacks relevant open reading frames and the few that exist do not coincide in the equivalent sequence of the close relative Fusarium oxysporum, indicating that the transcript is a long non-coding RNA (lncRNA). The results showed that carP is independently transcribed from carS, and its deletion produces an albino phenotype due to a drop in transcription of the structural car genes, probably as a result of the higher transcription of carS gene. This phenotype was only complemented by reintegration of the carP gene in the native locus, while its ectopic integration did not allow to recover carotenoid production in the recipient carP mutant, indicating a cis-acting regulatory mechanism for carP on carS expression. Global transcriptomic data showed that carP deletion affects the expression of many genes, most of them predictably through its effect on CarS. However, some of the differentially expressed genes are hardly affected by the carS mutation, pointing to specific regulatory effects of carP on other target genes. Global transcriptomic data after different illumination times revealed a diversity of kinetics patterns in mRNA accumulation in the wild strain. Photoinduced genes exhibited fast, intermediate and late responses, while only intermediate and late responses were found for light-repressed genes. A vast majority of these photoresponses were lost in the wcoA mutant, indicating that WcoA is the main photoreceptor responsible for light regulation in F. fujikuroi. Outstandingly, the wcoA mutation brought about massive changes in the transcriptome, affecting about 20% of the genes. Most of these effects were produced regardless of illumination, indicating that WcoA plays a more general light-independent regulatory role in F. fujikuroi. Outstandingly, many of the genes influenced by WcoA were related to secondary metabolism biosynthetic clusters, raising a biotechnological interest for this protein. Parallel analysis of the effect of light on a cryD deletion mutant revealed less severe transcriptomic effects. However, it resulted in changes in the degrees of photoinduction or photorepression of many genes, suggesting an accessory function of CryD in Fusarium photobiology. The summarized results constitute a significant contribution to the knowledge of the regulation of carotenogenesis in F. fujikuroi and its photobiology and provide further evidence on its molecular complexity

A novel lncRNA as a positive regulator of carotenoid biosynthesis in Fusarium

The fungi Fusarium oxysporum and Fusarium fujikuroi produce carotenoids, lipophilic terpenoid pigments of biotechnological interest, with xanthophyll neurosporaxanthin as the main end product. Their carotenoid biosynthesis is activated by light and negatively regulated by the RING-finger protein CarS. Global transcriptomic analysis identified in both species a putative 1-kb lncRNA that we call carP, referred to as Fo-carP and Ff-carP in each species, upstream to the gene carS and transcribed from the same DNA strand. Fo-carP and Ff-carP are poorly transcribed, but their RNA levels increase in carS mutants. The deletion of Fo-carP or Ff-carP in the respective species results in albino phenotypes, with strong reductions in mRNA levels of structural genes for carotenoid biosynthesis and higher mRNA content of the carS gene, which could explain the low accumulation of carotenoids. Upon alignment, Fo-carP and Ff-carP show 75-80% identity, with short insertions or deletions resulting in a lack of coincident ORFs. Moreover, none of the ORFs found in their sequences have indications of possible coding functions. We conclude that Fo-carP and Ff-carP are regulatory lncRNAs necessary for the active expression of the carotenoid genes in Fusarium through an unknown molecular mechanism, probably related to the control of carS function or expressio

Comparative transcriptomic analysis unveils interactions between the regulatory CarS protein and light response in Fusarium

Background The orange pigmentation of the agar cultures of many Fusarium species is due to the production of carotenoids, terpenoid pigments whose synthesis is stimulated by light. The genes of the carotenoid pathway and their regulation have been investigated in detail in Fusarium fujikuroi. In this and other Fusarium species, such as F. oxysporum, deep-pigmented mutants affected in the gene carS, which encodes a protein of the RING-finger family, overproduce carotenoids irrespective of light. The induction of carotenogenesis by light and its deregulation in carS mutants are achieved on the transcription of the structural genes of the pathway. We have carried out global RNA-seq transcriptomics analyses to investigate the relationship between the regulatory role of CarS and the control by light in these fungi. Results The absence of a functional carS gene or the illumination exert wide effects on the transcriptome of F. fujikuroi, with predominance of genes activated over repressed and a greater functional diversity in the case of genes induced by light. The number of the latter decreases drastically in a carS mutant (1.1% vs. 4.8% in the wild-type), indicating that the deregulation produced by the carS mutation affects the light response of many genes. Moreover, approximately 27% of the genes activated at least 2-fold by light or by the carS mutation are coincident, raising to 40% for an 8-fold activation threshold. As expected, the genes with the highest changes under both regulatory conditions include those involved in carotenoid metabolism. In addition, light and CarS strongly influence the expression of some genes associated with stress responses, including three genes with catalase domains, consistent with roles in the control of oxidative stress. The effects of the CarS mutation or light in the transcriptome of F. oxysporum were partially coincident with those of F. fujikuroi, indicating the conservation of the objectives of their regulatory mechanisms. Conclusions The CarS RING finger protein down-regulates many genes whose expression is up-regulated by light in wild strains of the two investigated Fusarium species, indicating a regulatory interplay between the mechanism of action of the CarS protein and the control by light.España, Ministerio de Economía y Competitividad, project BIO2015–69613-REspaña, Junta de Andalucía project CTS-6638 CTS-66

Efecto de un programa sobre las limitaciones funcionales observadas en el test de sentadilla overhead

INTRODUCCIÓN Y OBJETIVOS Los jugadores de fútbol presentan importantes déficits funcionales, lo cual predispone a sufrir numerosas lesiones importantes. Anteriormente la evaluación de los deportistas se basaba en la utilización de tests que casi exclusivamente analizaban parámetros condicionales, no obstante esto se considera incompleto en la actualidad. Así, surge la realización de la sentadilla overhead, encuadrado en una de las pruebas del Funcional Movement Screen, como un método importante a utilizar para evaluar las limitaciones funcionales de los deportistas. El objetivo del estudio es valorar las limitaciones funcionales de jugadores de fútbol de categoría juvenil a través del test de la sentadilla overhead y determinar los efectos que provoca un programa de intervención. MATERIAL Y MÉTODOS 63 participantes divididos en 3 grupos (GE, GE b y GC) fueron sometidos a la prueba de sentadilla overhead, realizando el GE un programa de intervención principal, el GE b un trabajo no tan específico ni completo como el GE, y el GC no hizo programa. RESULTADOS Y DISCUSIÓN Los resultados de la prueba muestran que los 3 grupos presentan importantes limitaciones funcionales. Los pocos estudios existentes que utilizan la sentadilla overhead para evaluar las limitaciones funcionales en jugadores de fútbol se correlacionan con los resultados, reflejando que la mayor parte muestran importantes limitaciones funcionales. CONCLUSIONES Los 3 grupos de la muestra evaluada presentan importantes limitaciones funcionales observadas a través del test de sentadilla overhead. Atendiendo al análisis global del Functional Movement Screen (FMS) un 74,6% realizaba una ejecución incorrecta del movimiento. El análisis analítico, en el plano frontal, un 33,3% de la muestra presentaba varo de rodilla, y un 7,9% valgo. En el plano lateral, un 63,5% presentaban los brazos neutros con la cabeza y un 19% caían hacia adelante. En el plano posterior, un 19% mostraban un movimiento asimétrico de caderas. La sentadilla overhead o overhead squat puede ser un método muy útil para establecer valores normativos en base a las limitaciones funcionales observadas en jugadores de fútbol de categoría juvenil y poder establecer programas de intervención específicos<br /

Impact of the White Collar Photoreceptor WcoA on the Fusarium fujikuroi Transcriptome

The proteins of the White Collar 1 family (WC) constitute a major class of flavin photoreceptors, widely distributed in fungi, that work in cooperation with a WC 2 protein forming a regulatory complex. The WC complex was investigated in great detail in Neurospora crassa, a model fungus in photobiology studies, where it controls all its major photoresponses. The fungus Fusarium fujikuroi, a model system in the production of secondary metabolites, contains a single WC-1 gene called wcoA. The best-known light response in this fungus is the photoinduction of the synthesis of carotenoids, terpenoid pigments with antioxidant properties. Loss of WcoA in F. fujikuroi results in a drastic reduction in the mRNA levels of the carotenoid genes, and a diversity of morphological and metabolic changes, including alterations in the synthesis of several secondary metabolites, suggesting a complex regulatory role. To investigate the function of WcoA, the transcriptome of F. fujikuroi was analyzed in the dark and after 15-, 60- or 240-min illumination in a wild strain and in a formerly investigated wcoA insertional mutant. Using a threshold of four-fold change in transcript levels, 298 genes were activated and 160 were repressed in the wild strain under at least one of the light exposures. Different response patterns were observed among them, with genes exhibiting either fast, intermediate, and slow photoinduction, or intermediate or slow repression. All the fast and intermediate photoresponses, and most of the slow ones, were lost in the wcoA mutant. However, the wcoA mutation altered the expression of a much larger number of genes irrespective of illumination, reaching at least 16% of the annotated genes in this fungus. Such genes include many related to secondary metabolism, as well as others related to photobiology and other cellular functions, including the production of hydrophobins. As judged by the massive transcriptomic changes exhibited by the wcoA mutant in the dark, the results point to WcoA as a master regulatory protein in F. fujikuroi, in addition to a central function as the photoreceptor responsible for most of the transcriptional responses to light in this fungus.España Ministerio de Economía y Competitividad, grant BIO2015-69613-RMinisterio de Ciencia e Innovación , MCI, Agencia Estatal de Investigación, AEI, and Fondo Europeo de Desarrollo Regional, FEDER, grant RTI2018-101902-B-I00)España Ministerio de Economía y Competitividad, grant BIO2015-71703-RED

Protein Activity of the Fusarium fujikuroi Rhodopsins CarO and OpsA and Their Relation to Fungus–Plant Interaction

Fungi possess diverse photosensory proteins that allow them to perceive different light wavelengths and to adapt to changing light conditions in their environment. The biological and physiological roles of the green light-sensing rhodopsins in fungi are not yet resolved. The rice plant pathogen Fusarium fujikuroi exhibits two different rhodopsins, CarO and OpsA. CarO was previously characterized as a light-driven proton pump. We further analyzed the pumping behavior of CarO by patch-clamp experiments. Our data show that CarO pumping activity is strongly augmented in the presence of the plant hormone indole-3-acetic acid and in sodium acetate, in a dose-dependent manner under slightly acidic conditions. By contrast, under these and other tested conditions, the Neurospora rhodopsin (NR)-like rhodopsin OpsA did not exhibit any pump activity. Basic local alignment search tool (BLAST) searches in the genomes of ascomycetes revealed the occurrence of rhodopsin-encoding genes mainly in phyto-associated or phytopathogenic fungi, suggesting a possible correlation of the presence of rhodopsins with fungal ecology. In accordance, rice plants infected with a CarO-deficient F. fujikuroi strain showed more severe bakanae symptoms than the reference strain, indicating a potential role of the CarO rhodopsin in the regulation of plant infection by this fungus.España Ministerio de Ciencia y tecnología BIO20010-15430Junta de Andalucía CTS-663

Carotenoid Biosynthesis in Fusarium

Many fungi of the genus Fusarium stand out for the complexity of their secondary metabolism. Individual species may differ in their metabolic capacities, but they usually share the ability to synthesize carotenoids, a family of hydrophobic terpenoid pigments widely distributed in nature. Early studies on carotenoid biosynthesis in Fusarium aquaeductuum have been recently extended in Fusarium fujikuroi and Fusarium oxysporum, well-known biotechnological and phytopathogenic models, respectively. The major Fusarium carotenoid is neurosporaxanthin, a carboxylic xanthophyll synthesized from geranylgeranyl pyrophosphate through the activity of four enzymes, encoded by the genes carRA, carB, carT and carD. These fungi produce also minor amounts of β-carotene, which may be cleaved by the CarX oxygenase to produce retinal, the rhodopsin’s chromophore. The genes needed to produce retinal are organized in a gene cluster with a rhodopsin gene, while other carotenoid genes are not linked. In the investigated Fusarium species, the synthesis of carotenoids is induced by light through the transcriptional induction of the structural genes. In some species, deep-pigmented mutants with up-regulated expression of these genes are affected in the regulatory gene carS. The molecular mechanisms underlying the control by light and by the CarS protein are currently under investigation.Junta de Andalucía project CTS-6638España, Gobierno BIO2012-39716, BIO2015-69613-R, AGL2014-53195R BIO2015-71703-RED

Exploiting ambipolarity in graphene field-effect transistors for novel designs on high-frequency analog electronics

This work was funded by FEDER/Junta de Andalucía-Consejería de Transformación Económica, Industria, Conocimiento y Universidades through the Projects A-TIC-646-UGR20, B-RNM-375-UGR18 and P20_00633; by Junta de Andalucía-Consejería de Universidad, Investigación e Innovación under ENERGHENE Project No. P21_00149; and by MCIN/AEI/10.13039/501100011033 through the projects PID2020- 116518GB-I00 and PID2021-127840NB-I00 (MCIN/AEI/FEDER, UE). The authors also acknowledge the support by the European Union’s Horizon 2020 Framework Programme for Research and Innovation through the Project GrapheneCore3 under Grant Agreement No. 881603. F.P. acknowledges funding from PAIDI 2020 – European Social Fund Operational Programme 2014–2020 No. 20804. A.M. acknowledges the support of the MCIN/AEI/PTA grant, with reference PTA2020-018250-I. M.C.P. acknowledges the FPU program with reference FPU21/04904. A.P.-S. acknowledges the support from Ministerio de Ciencia, Innovación y Universidades under Grant Agreement No. FJC2020-046213-I. E.R.-G. acknowledges the support from IPN Contract No. SIP/20230362. Funding for open access charge: Universidad de Granada / CBUA.Exploiting ambipolar electrical conductivity based on graphene field-effect transistors has raised enormous interest for high-frequency (HF) analog electronics. Controlling the device polarity, by biasing the graphene transistor around the vertex of the V-shaped transfer curve, enables to redesign and highly simplify conventional analog circuits, and simultaneously to seek for multifunctionalities, especially in the HF domain. This study presents new insights for the design of different HF applications such as power amplifiers, mixers, frequency multipliers, phase shifters, and modulators that specifically leverage the inherent ambipolarity of graphene-based transistors.FEDER/Junta de Andalucía-Consejería de Transformación Económica, Industria, Conocimiento y Universidades through the Projects A-TIC-646-UGR20, B-RNM-375-UGR18 and P20_00633Junta de Andalucía-Consejería de Universidad, Investigación e Innovación under ENERGHENE Project No. P21_00149MCIN/AEI/10.13039/501100011033 through the projects PID2020- 116518GB-I00 and PID2021-127840NB-I00 (MCIN/AEI/FEDER, UE)European Union’s Horizon 2020 Framework Programme for Research and Innovation through the Project GrapheneCore3 under Grant Agreement No. 881603PAIDI 2020 – European Social Fund Operational Programme 2014–2020 No. 20804MCIN/AEI/PTA grant, with reference PTA2020-018250-IFPU21/04904Ministerio de Ciencia, Innovación y Universidades under Grant Agreement No. FJC2020-046213-IIPN Contract No. SIP/20230362Funding for open access charge: Universidad de Granada / CBU

Retos de la democracia y de la participación ciudadana

El libro Retos de la democracia y participación ciudadana es producto de un conjunto de reflexiones que surgieron tras percibir que, pese a la importancia que la Constitución concedió a la democracia y al principio, valor y derecho fundamental de participación ciudadana, existen problemas conceptuales, retos y perspectivas que han influido en su desarrollo, así como también en los mecanismos legales de participación del pueblo en ejercicio de su soberanía y de la participación ciudadana en lo administrativo y en su proyección social. Así, esta valiosa contribución académica indaga las razones que llevaron al constitucionalismo colombiano a reforzar la democracia representativa, adoptando adicionalmente el modelo participativo de la democracia, por lo cual se estudian las condiciones necesarias para su exitosa implementación y se reflexiona sobre las dificultades y riesgos de su puesta en marcha. Para ello, se hace una profunda revisión de las garantías democráticas que ofrece la Constitución y la ley a los ciudadanos tanto en el Derecho Comparado como en el Derecho colombiano. En el espectro de esta investigación se analiza la conjunción de la democracia representativa y la democracia participativa y el significado en el modelo de Estado, sus principales retos y tendencias. En segundo lugar, se analiza la participación de los ciudadanos en lo administrativo como garantía de eficacia en las decisiones administrativas y se delimita de otros conceptos aledaños, precisando las condiciones requeridas para su implementación, cuyo vértice principal es la vigencia del Estado de derecho. Teniendo en cuenta los derechos fundamentales de las personas que son el eje principal de la democracia y de la participación. En el último capítulo, se estudia el papel del juez constitucional en un Estado democrático y social de derecho. Siguiendo estas grandes líneas temáticas, en el primer capítulo del libro consagrado a la delimitación conceptual de la democracia representativa, participativa y sus retos actuales, se hace un análisis doctrinario para situar los conceptos de democracia representativa y participativa. Luego, con fundamento en la doctrina más avezada sobre el tema, se hace referencia a las distintas clases de democracia. En este sentido, se realiza un análisis del concepto de democracia representativa en sus diversas concepciones. También, se estudia la democracia directa y semidirecta, así como los mecanismos de participación política del pueblo en ejercicio de su soberanía, teniendo presentes las disposiciones que en este sentido aparecen en la Constitución Política de 1991, así como la interpretación que la Corte Constitucional ha efectuado frente al tema. Se establecen además los aspectos más relevantes a través de los cuales se deduce la importancia actual de la democracia en el Estado-Nación en el ámbito internacional, lo cual permite identificar una relación inseparable entre la democracia y el derecho, porque los ciudadanos no producen las disposiciones jurídicas, sino que eligen a quienes están autorizados para producirlas. Por lo cual, en el Estado social de derecho la fuente principal de Derecho es la Constitución, seguida por la ley, aunque esta no tenga la misma fuerza que tenía en el Estado liberal de Derecho. Dentro de este marco conceptual, también se define que la democracia es cultura, es estilo de vida, caracterizado por el diálogo, la información, la comunicación, la fraternidad de los ciudadanos con la organización política. Es un lenguaje y un conjunto de actitudes, convicciones, creencias, tolerancias, responsabilidades y concepciones de la ciudadanía, de su civismo y solidaridad que penetra en el espectro de lo público y de lo privado

El Papel de las TIC en la transformación de la sociedad

Este libro ha sido concebido como un libro de texto, el cual se enfoca en la influencia que han tenido las TIC en la sociedad actual y el avance que han proporcionado en los diferentes campos del saber: campos que se tratan en distintos capítulos, como son, en la educación. La didáctica, la medicina, la inclusión social, la sociedad del conocimiento y las soluciones TIC que pueden darse a partir del uso del almacenamiento de información en la nube. Todos los elementos nombrados, han contribuido para que a través de los años impacten de alguna manera en el desarrollo de la humanidad, cambiando los estilos de vida, la forma de pensar, actuar y aprender; de ahí que el mundo entero se conecta en instante a través de los diferentes medios en donde han incursionado las Tecnologías de la Información y las Comunicaciones. Los cambios no se han dado solo a nivel educativo, sino que las empresas, hoy se han transformado en conglomerados empresariales que lideran la economía mundial y por medio de Internet y las redes sociales, el resto del mundo se entera de los grandes progresos de manera inmediata