Discerning the relationship between geminiviral infection and vesicle trafficking using virus induced gene silencing

Tomato yellow leaf curl disease is one of the most important threats to tomato crops worldwide. One of its causal agents, Tomato yellow leaf curl Sardinian virus (TYLCSV) is a monopartite member of the genus Begomovirus from the family Geminiviridae. Due to the few proteins encoded by their viral genome, geminiviruses rely heavily on host cellular machineries and interact with a wide range of plant proteins to complete all processes required for infection, such as viral replication, movement and suppression or evasion of plant defence mechanisms. The identification of the host proteins involved in viral infection will be an important step towards the understanding of the mechanisms underlying this process. In our laboratory, transgenic Nicotiana benthamiana plants containing a green fluorescent protein (GFP) expression cassette flanked by two direct repeats of the intergenic region of TYLCSV have been constructed (2IR plants). When these plants are infected with TYLCSV, an overexpression of the reporter gene is observed in those cells where the virus is actively replicating. These plants have been used together with virus induced gene silencing (VIGS) in an effort to identify host genes involved in the infection process using a reverse genetics approach. Using this combined technique our group has identified two genes δ-COP and ARF 1, involved in retrograde vesicle trafficking, which are essential for the infectious process. We are currently assaying genes codifying proteins involved in different pathways of the vesicle trafficking system: Sar1b, γ subunit of AP1, Sec24, SYT1 and two that encode the heavy chain of triskelion proteins. Their effect over viral infection will be presented and discussedUniversidad de Málaga. Campues de Excelencia Internacional Andalucía Tech

Identifying the function of vesicle trafficking in geminiviral infection using virus induced gene silencing

Tomato yellow leaf curl Sardinian virus (TYLCSV) is one of the causal agent of the tomato yel-low leaf curl disease, one of the most important threats to tomato crops worldwide. TYLCSV is a monopartite member of the genus Begomovirus from the family Geminiviridae. To carry out a full infection, geminiviruses need to move inside the infected cell and from one cell to an-other for which they depend on diverse cellular factors. While cell-to-cell movement has been described to occur through plasmodesmata, the way in which geminiviruses move inside the host cells is yet unknown. The identification of the host proteins involved in viral infection will be an important step to-wards the understanding of the mechanisms underlying this process. In our laboratory, trans-genic Nicotianabenthamiana plants containing a green fluorescent protein (GFP) expression cassette flanked by two direct repeats of the intergenic region of TYLCSV have been construct-ed (2IR plants). When these plants are infected with TYLCSV, an overexpression of the reporter gene is observed in those cells where the virus replicates. These plants have been used to-gether with virus induced gene silencing (VIGS) in an effort to identify host genes involved in the infection process using a reverse genetics approach. Using this combined technique our group has identified two genes δ-COP and ARF 1, involved in retrograde vesicle trafficking, which are essential for the infectious process. We are current-ly assaying genes codifying proteins involved in different pathways of the vesicle trafficking system: Sar1b, γ subunit of AP1, Sec24, SYT1 and two that encode the heavy chain of triskelion proteins. Their effect over virus infection will be presented and discussed.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Geminivirus Rep Protein Interferes with the Plant DNA Methylation Machinery and Suppresses Transcriptional Gene Silencing

Viruses are masters at circumventing host defenses and manipulating the cellular environment for their own benefit. The replication of the largest known family of single-stranded DNA viruses, Geminiviridae, is impaired by DNA methylation but the fact that plants might use methylation as a defense against geminiviruses and the impact that viral genome methylation may have during the infection, remain controversial. We have found that geminiviruses reduce the expression of the plant maintenance DNA methyltransferases, MET1 and CMT3, in both, locally and systemically infected tissues. Furthermore, we demonstrated that the virus-mediated repression of these two maintenance DNA methyltransferases is widely spread among different geminivirus species and we have identified Rep as the geminiviral protein responsible for the repression of MET1 and CMT3. The presence of Rep, suppresses transcriptional gene silencing (TGS) of an Arabidopsis transgene and of host loci whose expression is strongly controlled by MET1. Bisulfite sequencing analyses showed that the expression of Rep caused a substantial reduction in the levels of DNA methylation at certain loci at CG sites. The biological relevance of these findings and the role of Rep as a TGS suppressor will be discussed.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Geminivirus C2 protein represses genes involved in sulphur assimilation and this effect can be counteracted by jasmonate treatment

Geminiviruses are plant viruses that infect a broad range of crops and cause extensive losses worldwide, having an important economic impact. C2, a multifunctional pathogenicity factor encoded by geminiviruses, has been recently shown to suppress the responses to jasmonates in the host plant, which might at least partially explain its well-established role in pathogenicity. Sulphur is one of the essential macro-elements for plant life, and is considered to have a role in plant defence, in a phenomenon named sulphur-induced resistance (SIR) or sulphur-enhanced defence (SED). In this work, we show that geminivirus C2 protein represses the expression of genes involved in the sulphur assimilation pathway in Arabidopsis, but, interestingly, this effect can be neutralized by exogenous jasmonate treatment. These preliminary results may raise the idea that geminiviruses might be affecting sulphur metabolism, and maybe counteracting SIR/SED, through the manipulation of the jasmonate signalling pathway, which would define a novel strategy in plant-virus interactions and may unveil SIR/SED as an important player in the plant defence against viruses.Ministerio de Ciencia y Innovación/FEDER AGL2007-66062-C02-02/AGR AGL2010-22287-CO2European Regional Development Fund (ERDF) BIO2010-15201Junta de Andalucía BIO­27

Stranger in a strange land:the experiences of immigrant researchers

Continuing with our Q&A series discussing issues of diversity in STEM fields, Genome Biology spoke with three researchers on their experiences as immigrants.  International collaborations are key to advancing scientific research globally and often require mobility on the part of researchers. Migration of scientists enables the spread of ideas and skills around the world, giving researchers the opportunity to follow the best resources. Of course, migration adds a new set of challenges to the already monumental task of starting and running a lab. Genome Biology spoke to Sophien Kamoun, Rosa Lozano-Durán, and Luay Nakhleh about their personal experiences

Importance of vesicle trafficking in the establichsment of a geminiviral infection

Importance of vesicle trafficking in the establishment of a geminiviral infection P. CANA-QUIJADA1, T. ROSAS-DÍAZ2, LOZANO-DURÁN R. 2 AND E.R. BEJARANO1 1Dpto. Biología Celular, Genética y Fisiología. Área de Genética Instituto de Hortofruticultura Subtropical y Mediterránea “La Mayora” (IHSM-UMA-CSIC), Universidad de Málaga, Málaga, Spain E-mail: [email protected] 2. Shanghai Center for Plant Stress Biology (PSC), Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences, Shanghai 201602, China. INTRODUCTION Geminiviruses produces some of the most devastating diseases for agriculture worldwide. Geminiviral genomes encode only 5 to 7 proteins, forcing them to rely heavily on host cellular machineries and to interact with a high amount of host proteins in order to complete a full infection. The identification of the host proteins involved in viral infection will be an important step towards the understanding of the mechanisms underlying this process and develop new strategies to generate new sources of resistance. Previous efforts from our group have identified several genes involved in vesicle trafficking. OBJECTIVES The main aim of this work is to elucidate the role of the plant cell’s vesicle trafficking in a geminiviral infection. MATHERIALS AND METHODS Transgenic Nicotiana benthamiana plants containing a green fluorescent protein (GFP) expression cassette flanked by two direct repeats of the intergenic region of TYLCSV have been constructed (2IR plants). When these plants are infected with TYLCSV, an overexpression of the reporter gene is observed in those cells where the virus is actively replicating. 2IR plants were used in combination with virus induced gene silencing (VIGS), to identify vesicle trafficking genes involved in the infectious process. Viral replication was monitorized by GFP expression. Viral accumulation was determined using qPCR. RESULTS When silenced, four of the assessed genes reduced dramatically the viral amounts or completely abolished the infection. On the other hand, three of them had no significant effect over the infection and one of them seems to cause a slight increase in viral accumulation. CONCLUSIONS The vesicle trafficking machinery plays an essential role in geminivirus infection. Assays to determine whether the described effect is due to a lack of replication or movement of the virus inside the plant cells are in progress.Universidad de Málaga. Campues de Excelencia Internacional Andalucía Tech

Broad application of a simple and affordable protocol for isolating plant RNA

BACKGROUND: Standard molecular biological methods involve the analysis of gene expression in living organisms under diverse environmental and developmental conditions. One of the most direct approaches to quantify gene expression is the isolation of RNA. Most techniques used to quantify gene expression require the isolation of RNA, usually from a large number of samples. While most published protocols, including those for commercial reagents, are either labour intensive, use hazardous chemicals and/or are costly, a previously published protocol for RNA isolation in Arabidopsis thaliana yields high amounts of good quality RNA in a simple, safe and inexpensive manner. FINDINGS: We have tested this protocol in tomato and wheat leaves, as well as in Arabidopsis leaves, and compared the resulting RNA to that obtained using a commercial phenol-based reagent. Our results demonstrate that this protocol is applicable to other plant species, including monocots, and offers yield and purity at least comparable to those provided by commercial phenol-based reagents. CONCLUSIONS: Here, we show that this previously published RNA isolation protocol can be easily extended to other plant species without further modification. Due to its simplicity and the use of inexpensive reagents, this protocol is accessible and affordable and can be easily implemented to work on different plant species in laboratories worldwide

NAHG increases transient transformation of arabidopsis leaves by agrobacterium

Agrobacterium tumefaciens-mediated transient transformation has demonstrated to be an invaluable tool in plant molecular biology studies. However, low efficiency and inconsistency of this method in Arabidopsis has forced the implementation of Nicotiana benthamiana as a surrogate system, limiting applicability. One of the main reasons to underlie the recalcitrance of Arabidopsis to Agrobacterium-mediated transformation is the activation of plant immune responses upon perception of the bacteria. Perception of bacterial pathogen-associated molecular patterns (PAMPs), including EF-Tu, leads to activation of PAMP-triggered immunity (PTI). Activation of PTI ultimately induces salicylic acid (SA) accumulation, which in turn shuts down expression of the vir genes, potentially interfering with the transfer of the T-DNA, and therefore T-DNA gene expression (Yuan et al., 2007; Anand et al., 2008). However, previous results suggested that besides depletion of SA other hormone-mediated defence responses, including jasmonic acid (JA), might be responsible for the low efficiency of transient transformation in Arabidopsis (Tsuda et al., 2012). In this work, we evaluate the efficiency of Agrobacterium-mediated transient transformation in Arabidopsis genotypes affected in JA perception or signalling (coi1, jin1), or with low SA or JA content (sid2, NahG, aos). The results show that impairment of JA signalling reduces or does not affect transient expression in mature leaves, but expression of the NahG transgene dramatically improves this process. We demonstrate that Arabidopsis NahG plants can be efficiently used for transient expression-based functional assays routinely performed in N. benthamiana, such as determination of subcellular localization of GFP-fused proteins or analysis of protein-protein interactions by Bimolecular Fluorescent Complementation.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Percepciones sobre frontera colombo venezolana en el informativo Oriente Noticias del Canal de Televisión Regional TRO

This article publishes the results of a research project that aimed to analyze the perception from the audiovisual journalistic discourse on the Colombian-Venezuelan border in the Oriente Noticias news that is broadcast by the Regional Television Channel TRO, with coverage in the departments Santander and Norte de Santander, in eastern Colombia. Discourse analysis and semiotic analysis of the audiovisual text were applied in the methodological component of the analysis, applied in an observation window of forty-five (45) days. The study's findings showed that a contradiction appears in the journalistic articles of the Oriente Noticias news program: its images and graphic codes point to a notion of linearity -the camera and the infographics emphasize the bridges, alcabalas and demarcations that They divide the two countries-, while the linguistic and sound codes point towards a notion of zonality –the journalist's voice and sound story reiterates the notion of brotherhood, mutuality, spaces and customs that are shared between Colombia and Venezuela.  Este artículo presenta los resultados de un proyecto de investigación que tuvo como propósito analizar la percepción acerca del discurso periodístico audiovisual sobre frontera colombo venezolana en el informativo Oriente Noticias que se emite por el Canal de Televisión Regional TRO, con cobertura en los departamentos de Santander y Norte de Santander, en el oriente de Colombia. En el componente metodológico se utilizó el análisis del discurso y el análisis semiótico del texto audiovisual, aplicados a una ventana de observación de cuarenta y cinco (45) días. Los hallazgos del estudio evidenciaron que en las notas periodísticas del noticiero Oriente Noticias aparece una contradicción en su discurso periodístico audiovisual: las imágenes y códigos gráficos apuntan hacia una noción de linealidad –la cámara y las infografías enfatizan en los puentes, alcabalas y demarcaciones que dividen a los dos países-, mientras que los códigos lingüísticos y sonoros apuntan hacia una noción de zonalidad –la voz y el relato sonoro del periodista reiteran la noción de hermandad, de mutualidad, de espacios y costumbres que se comparten entre Colombia y Venezuela

Diseño de escenarios de aprendizaje basados en la indagación con soporte tecnológico. Estrategias de co-diseño entre docentes e investigadores.

En el marco del proyecto Design2Learn se presenta el proceso de codiseño docentes e investigadores de escenarios de aprendizaje basados en la indagación con soporte tecnológico. Se detallan los principios de diseño. Los resultados incluyen los retos identificados por profesores de distintas disciplinas, así como la secuencia seguida para el proceso de co-diseño y creación de escenarios de aprendizaje basado en el aprendizaje indagativo y potenciados por tecnología en la formación universitaria