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

Intelligent learning objects: an agent aproach to create interoperable learning objects

Reusing learning material is very important to design learning environments for real-life learning. The reusability of learning objects results from the product of three main features: modularity, discoverability and interoperability. We proposed learning objects built based on agent architectures, called Intelligent Learning Objects (ILO). This paper discusses how the ILO approach can be used to improve the interoperability among learning objects, learning menagement systems (LMS) and pedagogical agents.Education for the 21 st century - impact of ICT and Digital Resources ConferenceRed de Universidades con Carreras en Informática (RedUNCI

Improving interoperability among learning objects using FIPA agent communication framework

The reusability of learning material is based on three main features: modularity, discoverability and interoperability. Several researchers on Intelligent Learning Environments have proposed the use of architectures based on agent societies. Learning systems based on Multi-Agent architectures support the development of more interactive and adaptable systems and the Learning Objects approach gives reusability. We proposed an approach where learning objects are built based on agent architectures. This paper discusses how the Intelligent Learning Objects approach can be used to improve the interoperability between learning objects and pedagogical agents.Applications in Artificial Intelligence - AgentsRed de Universidades con Carreras en Informática (RedUNCI

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

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

Photometric Variability in the Ultracool Dwarf BRI 0021-0214: Possible Evidence for Dust Clouds

We report CCD photometric monitoring of the nonemission ultracool dwarf BRI 0021-0214 (M9.5) obtained during 10 nights in 1995 November and 4 nights in 1996 August, with CCD cameras at 1 m class telescopes on the observatories of the Canary Islands. We present differential photometry of BRI 0021-0214, and we report significant variability in the I-band light curve obtained in 1995. A periodogram analysis finds a strong peak at a period of 0.84 day. This modulation appears to be transient because it is present in the 1995 data but not in the 1996 data. We also find a possible period of 0.20 day, which appears to be present in both the 1995 and 1996 datasets. However, we do not find any periodicity close to the rotation period expected from the spectroscopic rotational broadening (< 0.14 day). BRI 0021-0214 is a very inactive object, with extremely low levels of Halpha and X-ray emission. Thus, it is unlikely that magnetically induced cool spots can account for the photometric variability. The photometric variability of BRI 0021-0214 could be explained by the presence of an active meteorology that leads to inhomogeneous clouds on the surface. The lack of photometric modulation at the expected rotational period suggests that the pattern of surface features may be more complicated than previously anticipated.Comment: Accepted for publication in ApJ. 26 pages, 13 figures include

Intelligent learning objects: an agent aproach to create interoperable learning objects

Reusing learning material is very important to design learning environments for real-life learning. The reusability of learning objects results from the product of three main features: modularity, discoverability and interoperability. We proposed learning objects built based on agent architectures, called Intelligent Learning Objects (ILO). This paper discusses how the ILO approach can be used to improve the interoperability among learning objects, learning menagement systems (LMS) and pedagogical agents.Education for the 21 st century - impact of ICT and Digital Resources ConferenceRed de Universidades con Carreras en Informática (RedUNCI

Improving interoperability among learning objects using FIPA agent communication framework

The reusability of learning material is based on three main features: modularity, discoverability and interoperability. Several researchers on Intelligent Learning Environments have proposed the use of architectures based on agent societies. Learning systems based on Multi-Agent architectures support the development of more interactive and adaptable systems and the Learning Objects approach gives reusability. We proposed an approach where learning objects are built based on agent architectures. This paper discusses how the Intelligent Learning Objects approach can be used to improve the interoperability between learning objects and pedagogical agents.Applications in Artificial Intelligence - AgentsRed de Universidades con Carreras en Informática (RedUNCI