Characterization of begomoviruses sampled during severe epidemics in tomato cultivars carrying the Ty-1 gene

Tomato yellow leaf curl virus (TYLCV, genus Begomovirus, family Geminiviridae) is a major species that causes a tomato disease for which resistant tomato hybrids (mainly carriers of the Ty-1/Ty-3 gene) are being used widely. We have characterized begomoviruses severely affecting resistant tomato crops in Southeast Spain. Circular DNA was prepared from samples by rolling circle amplification, and sequenced by massive sequencing (2015) or cloning and Sanger sequencing (2016). Thus, 23 complete sequences were determined, all belonging to the TYLCV Israel strain (TYLCV-IL). Massive sequencing also revealed the absence of other geminiviral and beta-satellite sequences. A phylogenetic analysis showed that the Spanish isolates belonged to two groups, one related to early TYLCV-IL isolates in the area (Group 1), and another (Group 2) closely related to El Jadida (Morocco) isolates, suggesting a recent introduction. The most parsimonious evolutionary scenario suggested that the TYLCV isolates of Group 2 are back recombinant isolates derived from TYLCV-IS76, a recombinant virus currently predominating in Moroccan epidemics. Thus, an infectious Group 2 clone (TYLCV-Mu15) was constructed and used in in planta competition assays against TYLCV-IS76. TYLCV-Mu15 predominated in single infections, whereas TYLCV-IS76 did so in mixed infections, providing credibility to a scenario of co-occurrence of both types of isolates

Analysis of the melon (Cucumis melo) small RNAome by high-throughput pyrosequencing

Abstract Background Melon (Cucumis melo L.) is a commercially important fruit crop that is cultivated worldwide. The melon research community has recently benefited from the determination of a complete draft genome sequence and the development of associated genomic tools, which have allowed us to focus on small RNAs (sRNAs). These are short, non-coding RNAs 21-24 nucleotides in length with diverse physiological roles. In plants, they regulate gene expression and heterochromatin assembly, and control protection against virus infection. Much remains to be learned about the role of sRNAs in melon. Results We constructed 10 sRNA libraries from two stages of developing ovaries, fruits and photosynthetic cotyledons infected with viruses, and carried out high-throughput pyrosequencing. We catalogued and analysed the melon sRNAs, resulting in the identification of 26 known miRNA families (many conserved with other species), the prediction of 84 melon-specific miRNA candidates, the identification of trans-acting siRNAs, and the identification of chloroplast, mitochondrion and transposon-derived sRNAs. In silico analysis revealed more than 400 potential targets for the conserved and novel miRNAs. Conclusion We have discovered and analysed a large number of conserved and melon-specific sRNAs, including miRNAs and their potential target genes. This provides insight into the composition and function of the melon small RNAome, and paves the way towards an understanding of sRNA-mediated processes that regulate melon fruit development and melon-virus interactions.This work was supported by grants AGL2009-07552/AGR, BIO2006-13107 (Ministerio de Ciencia e Innovación, Spain) and MELONOMICS (Fundación Genoma España, Spain).Peer Reviewe

High-Throughput Sequencing of RNA Silencing-Associated Small RNAs in Olive (Olea europaea L.)

Small RNAs (sRNAs) of 20 to 25 nucleotides (nt) in length maintain genome integrity and control gene expression in a multitude of developmental and physiological processes. Despite RNA silencing has been primarily studied in model plants, the advent of high-throughput sequencing technologies has enabled profiling of the sRNA component of more than 40 plant species. Here, we used deep sequencing and molecular methods to report the first inventory of sRNAs in olive (Olea europaea L.). sRNA libraries prepared from juvenile and adult shoots revealed that the 24-nt class dominates the sRNA transcriptome and atypically accumulates to levels never seen in other plant species, suggesting an active role of heterochromatin silencing in the maintenance and integrity of its large genome. A total of 18 known miRNA families were identified in the libraries. Also, 5 other sRNAs derived from potential hairpin-like precursors remain as plausible miRNA candidates. RNA blots confirmed miRNA expression and suggested tissue- and/or developmental-specific expression patterns. Target mRNAs of conserved miRNAs were computationally predicted among the olive cDNA collection and experimentally validated through endonucleolytic cleavage assays. Finally, we use expression data to uncover genetic components of the miR156, miR172 and miR390/TAS3-derived trans-acting small interfering RNA (tasiRNA) regulatory nodes, suggesting that these interactive networks controlling developmental transitions are fully operational in olive

Estudio de las poblaciones de pequeños RNAs derivados de virus de plantas y su possible función como moléculas reguladoras en la interacción planta-virus

156 p.-47 fig.-13 tab.En los organismos eucariotas, el silenciamiento por RNA es un mecanismo de regulación que controla la expresión génica y la estabilidad genómica a través de la síntesis de pequeños RNAs (sRNAs) a partir de inductores de doble cadena de RNA (dsRNA). Estos sRNAs, de 20 a 25 nucleótidos (nts), se unen a complejos multienzimáticos y les confieren la facultad de reconocer y silenciar transcripcional o post-transcripcionalmente secuencias de DNA o RNA altamente complementarias. En el tejido vegetal infectado con virus, este proceso se activa de forma natural mediante la formación de dsRNAs de origen viral. Como resultado, la infección está inexorablemente unida a la síntesis de sRNAs virales (vsRNAs) cuyo potencial regulador se asocia no sólo con la función del silenciamiento por RNA como mecanismo de defensa antiviral sino también con fenómenos como la patogénesis o el rango de hospedador. En esta tesis doctoral hemos empleado distintas técnicas de secuenciación e hibridación molecular en el sistema modelo Virus del cascabeleo del tabaco (Tobacco rattle virus, TRV)-Arabidopsis thaliana y en otros sistemas planta-virus para inferir la composición y estructura de las poblaciones de vsRNAs que se forman en el tejido infectado. Hemos investigado los componentes genéticos y los requerimientos estructurales de la maquinaria de silenciamiento de RNA de la planta para producir vsRNAs. Finalmente hemos analizado el papel del silenciamiento como componente antiviral en la planta y su potencial para reprogramar el transcriptoma de la célula infectada a través de la interacción funcional entre vsRNAs y RNAs mensajeros (mRNAs). Nuestros resultados revelan que los vsRNAs forman poblaciones abundantes y diversas en su composición que comparten características comunes en todos los sistemas planta-virus investigados. Así los vsRNAs muestran un sesgo evidente en su tamaño (con mayoría de especies de 21 y 22 nts) y, con frecuencia, en su polaridad (predominio de secuencias positivas). Otra característica común es la ubicuidad de los vsRNAs en su origen a partir del genoma viral. Los vsRNAs se generan en cualquier posición a lo largo del RNA viral aunque su distribución es muy heterogénea alternándose regiones genómicas pobres en secuencias con otras más enriquecidas en vsRNAs en forma de picos de acumulación o de regiones más amplias con alta densidad de secuencias. Nuestro trabajo demuestra que las RNasas tipo III Dicer-like, DCL4, DCL3 y DCL2, procesan, solas o combinadas unas con otras, las dsRNAs virales para producir vsRNAs de ambas polaridades a lo largo de todo el genoma de TRV. No obstante, las tres enzimas DCL cortan sustratos virales de forma jerárquica (DCL4>DCL2>DCL3) produciendo vsRNAs de 21, 22 y 24 nts, respectivamente. Nuestros datos sugieren que estas enzimas actúan sobre dsRNAs inductores en forma de intermediarios replicativos o plegamientos imperfectos del RNA viral para generar vsRNAs primarios. Sin embargo, nuestros resultados confirman que la mayoría de los vsRNAs en el tejido infectado son secundarios y resultan de la actividad de DCL sobre dsRNAs que se originan por la actividad de proteínas RNA polimerasas dependientes de RNA de la planta (RDR). Estas enzimas amplificarían las respuestas iniciales de silenciamiento convirtiendo el RNA viral en dsRNAs.El análisis de las colecciones secuenciadas sugiere que los vsRNAs tienen afinidad por diversas proteínas ARGONAUTA (AGO) que actúan como efectoras de silenciamiento sobre moléculas diana de RNA o DNA. Esto indica que los vsRNA podrían mediar en distintas rutas reguladoras que controlasen la expresión de genes del virus y del huésped. Nuestros datos confirman el papel de distintos componentes de la maquinaria de silenciamiento en inmunidad antiviral puesto que la desactivación genética de DCL4 y DCL2 o de RDR1, RDR2 y RDR6 resulta en un fenotipo de híper-susceptibilidad a TRV. Este fenómeno no se asocia al procesamiento de dsRNAs virales por DCL sino muy probablemente a la actividad de los vsRNAs generados a partir de las dsRNAs. Empleando técnicas informáticas hemos identificado genes de arabidopsis con alto grado de complementariedad con vsRNAs y que podrían servir como posibles dianas de regulación negativa durante la infección viral.Beca de la Comunidad Autónoma de Madrid (CAM), proyectos GEN 2003-20222 y BIO 2006-13107 del Ministerio de Ciencia e Innovación y proyecto GR/SAL0831/2004 de la Comunidad Autónoma de MadridPeer reviewe

Deep-sequencing of plant viral small RNAs reveals effective and widespread targeting of viral genomes

12 páginas, 6 figuras, 1 tabla, 3 figuras suplementarias -- PAGS nros. 203-214Plant virus infection involves the production of viral small RNAs (vsRNAs) with the potential to associate with distinct Argonaute (AGO)-containing silencing complexes and mediate diverse silencing effects on RNA and chromatin. We used multiplexed, high-throughput pyrosequencing to profile populations of vsRNAs from plants infected with viruses from different genera. Sense and antisense vsRNAs of 20 to 24 nucleotides (nts) spread throughout the entire viral genomes in an overlapping configuration; virtually all genomic nucleotide positions were represented in the data set. We present evidence to suggest that every genomic position could be a putative cleavage site for vsRNA formation, although viral genomes contain specific regions that serve as preferential sources of vsRNA production. Hotspots for vsRNAs of 21-, 22-, and 24-nt usually coincide in the same genomic regions, indicating similar target affinities among Dicer-like (DCL) enzymes. In the light of our results, the overall contribution of perfectly base paired double-stranded RNA and imperfectly base paired structures within single-stranded RNA to vsRNA formation is discussed. Our census of vsRNAs extends the current view of the distribution and composition of vsRNAs in virus-infected plants, and contributes to a better understanding of vsRNA biogenesisThis wok was supported by PhD fellowships from the Comunidad de Madrid (to L.D.) and Ministerio de Educación y Ciencia (MEC, to D.G.I.) and by Grant BIO2006-13107 from the MEC, SpainPeer reviewe

Complete genome sequence of malva-associated soymovirus 1: a novel virus infecting common mallow

In this work, a novel viral genomic sequence with a gene organization typical of members of the genus Soymovirus was identified using high-throughput sequencing data from common mallow. This species is a vigorous wild weed native to the Mediterranean region, commonly found in borders and edges of cultivated fields, making it a suitable reservoir for plant pests and pathogens. Indeed, plant viruses belonging to different genera have been previously found infecting common malva. This new viral genome consists of a single molecule of circular double-stranded DNA of 8391 base pairs and contains eight open reading frames encoding polymerase, movement, coat, translational transactivator protein typical of caulimoviruses, and four hypothetical proteins. Phylogenetic and pairwise distance analyses showed its close relationship with soybean chlorotic mottle virus. Interestingly, a small intergenic region was detected between ORFs Ib and II. Based on the demarcation criteria of the genus Soymovirus, the new virus, provisionally named malva-associated soymovirus 1, could be a member of a new species Soymovirus masolus. To our knowledge, this is the first report of a soymovirus infecting common mallowFunding was provided by the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie Grant Agreement No 813542TPeer reviewe

Cohombrillo-associated virus: a novel virus infecting Ecballium elaterium plants

We determined the complete genome sequence of a new virus infecting Ecballium elaterium ('cohombrillo amargo') plants, a weed species common on the borders of cultivated fields in the Mediterranean region. The genome of this virus is composed of two molecules of monocistronic positive-sense RNA, 6,934 and 3,501 nucleotides in length, excluding their poly(A) tails. The highest amino acid sequence similarity (50 % identity) in the Pro-Pol core region encoded by RNA 1 was observed in the corresponding protein of strawberry latent ringspot virus. Based on pairwise comparisons and phylogenetic analysis, this virus, tentatively named "cohombrillo-associated virus" (CoAV), appears to be a member of a new species in the genus Stralarivirus (family Secoviridae), for which the name "Stralarivirus elaterii" is proposed. This new virus has different putative cleavage patterns from members of other species belonging to this genus.Peer reviewe

Silencing supresor activity of the Tobacco Rattle Virus-encoded 16-kDa protein and interference with endogenous small RNA-guided regulatory pathways

11 páginas, 6 figuras -- PAGS nros. 346-356Higher plants use RNA silencing as a defense mechanism against viral infections, but viruses may encode suppressor proteins that counteract these defenses. Several virus-encoded suppressors also exert an inhibitory effect on endogenous small RNA regulatory pathways. Here we characterized the Tobacco rattle virus-encoded 16-kDa (TRV-16K) protein as a suppressor that blocked local RNA silencing induced by single (s)- and double-stranded (ds) RNA, indicating that TRV-16K interfered with a step in the silencing pathway downstream of dsRNA formation. The suppressor activity of TRV-16K was severely compromised by moderate to high dosages of dsRNA inducer. When silencing was locally triggered by ssRNA or low levels of dsRNA, silencing suppression by TRV-16K was associated with reduced accumulation of silencing-related siRNAs. TRV-16K also prevented partially cell-to-cell movement and systemic propagation of silencing but not transitive amplification of RNA silencing. We showed that neither TRV nor TRV-16K caused a global deregulation of the microRNA-regulatory pathway in Arabidopsis, suggesting that interference with microRNA biology was not a prerequisite for TRV, and probably many other plant viruses, to develop systemic infections in plantsThis work was supported by Grants GEN2003-20222-C02-01 and BIO2006-13107 from the MEC (Spain) and GR/SAL/0821/2004 from the CAM (Spain)Peer reviewe

Imagen Corporal, Alimentación y Calidad de Vida. Tema 2 y 3: La percepción y la sensación

Material didáctico de la asignatura Imagen corporal, alimentación y calidad de vida de la Diplomatura en Nutrición Humana y Dietética

Virus infection and primary metabolism

58 p.-8 fig.1 tab. Llave, César et al.During compatible virus infections, plants respond by reprogramming gene expression and metabolite content. While gene expression studies are profuse, our knowledge of the metabolic changes that occur in the presence of the virus is limited. Here, we combine gene expression and metabolite profiling in Arabidopsis (Arabidopsis thaliana) infected with Tobacco rattle virus (TRV) in order to investigate the influence of primary metabolism on virus infection. Our results revealed that primary metabolism is reconfigured in many ways during TRV infection, as reflected by significant changes in the levels of sugars and amino acids. Multivariate data analysis revealed that these alterations were particularly conspicuous at the time points of maximal accumulation of TRV, although infection time was the dominant source of variance during the process. Furthermore, TRV caused changes in lipid and fatty acid composition in infected leaves. We found that several Arabidopsis mutants deficient in branched-chain amino acid catabolism or fatty acid metabolism possessed altered susceptibility to TRV. Finally, we showed that increments in the putrescine content in TRV-infected plants correlated with enhanced tolerance to freezing stress in TRV-infected plants and that impairment of putrescine biosynthesis promoted virus multiplication. Our results thus provide an interesting overview for a better understanding of the relationship between primary metabolism and virus infection.This work was supported by grants BIO2006-13107 and BIO2009-12004 from the Spanish Ministry of Science and Innovation (MICINN), and P09-AGR-4516 from the Andalusian Government (Spain). F.J.T. and I.B.H. were supported by graduate fellowships from MICINN. L.F.-C. and M.L.H. were the recipients of JAE-Doc contracts from Consejo Superior de Investigaciones Científicas (CSIC). S.O. was supported in part by grant from Ministerio de Ciencia e Innovación, Spain (Ramón and Cajal contract).Peer reviewe