Constraints to virus infection in Nicotiana benthamiana plants transformed with a potyvirus amplicon

<p>Abstract</p> <p>Background</p> <p>Plant genomes have been transformed with full-length cDNA copies of viral genomes, giving rise to what has been called 'amplicon' systems, trying to combine the genetic stability of transgenic plants with the elevated replication rate of plant viruses. However, amplicons' performance has been very variable regardless of the virus on which they are based. This has boosted further interest in understanding the underlying mechanisms that cause this behavior differences, and in developing strategies to control amplicon expression.</p> <p>Results</p> <p><it>Nicotiana benthamiana </it>plants were transformed with an amplicon consisting of a full-length cDNA of the potyvirus <it>Plum pox virus </it>(PPV) genome modified to include a GFP reporter gene. Amplicon expression exhibited a great variability among different transgenic lines and even among different plants of the same line. Plants of the line 10.6 initially developed without signs of amplicon expression, but at different times some of them started to display sporadic infection foci in leaves approaching maturity. The infection progressed systemically, but at later times the infected plants recovered and returned to an amplicon-inactive state. The failure to detect virus-specific siRNAs in 10.6 plants before amplicon induction and after recovery suggested that a strong amplicon-specific RNA silencing is not established in these plants. However, the coexpression of extra viral silencing suppressors caused some amplicon activation, suggesting that a low level of RNA silencing could be contributing to maintain amplicon repression in the 10.6 plants. The resistance mechanisms that prevent amplicon-derived virus infection were also active against exogenous PPV introduced by mechanical inoculation or grafting, but did not affect other viruses. Amplicon-derived PPV was able to spread into wild type scions grafted in 10.6 rootstocks that did not display signs of amplicon expression, suggesting that resistance has little effect on virus movement.</p> <p>Conclusions</p> <p>Our results suggest that amplicon-derived virus infection is limited in this particular transgenic line by a combination of factors, including the presumed low efficiency of the conversion from the transgene transcript to replicable viral RNA, and also by the activation of RNA silencing and other defensive responses of the plant, which are not completely neutralized by viral suppressors.</p

Host-Specific Involvement of the HC Protein in the Long-Distance Movement of Potyviruses

Plum pox virus (PPV) is a member of the Potyvirus genus that, in nature, infects trees of the Prunus genus. Although PPV infects systemically several species of the Nicotiana genus, such as N. clevelandii and N. benthamiana, and replicates in the inoculated leaves of N. tabacum, it is unable to infect systemically the last host. The long-distance movement defect of PPV was corrected in transgenic tobacco plants expressing the 5 -terminal region of the genome of tobacco etch virus (TEV), a potyvirus that infects systemically tobacco. The fact that PPV was unable to move to upper noninoculated leaves in tobacco plants transformed with the same TEV transgene, but with a mutation in the HC protein (HC-Pro)-coding sequences, identifies the multifunctional HC-Pro as the complementing factor, and strongly suggests that a defect in an HC-Pro activity is responsible for the long-distance movement defect of PPV in tobacco. Whereas PPV HC-Pro strongly intensifies the symptoms caused by potato virus X (PVX) in the PPV systemic hosts N. clevelandii and N. benthamiana, it has no apparent effect on PVX pathogenicity in tobacco, supporting the hypothesis that long-distance movement and pathogenicity enhancement are related activities of the potyviral HC proteins. The movement defect of PPV in tobacco could also be complemented by cucumber mosaic virus in a mixed infection, demonstrating that at least some components of the long-distance machinery of the potyviruses are not strictly virus specific. A general conclusion of this work is that the HC-Pro might be a relevant factor for controlling the host range of the potyvirusesCopyrighted by the American Society for Microbiology

Design and Validation of the Adaptation to Change Questionnaire: New Realities in Times of COVID-19

Emotional and cognitive-behavioral factors influence people’s adaptability to change. Based on this premise, the objective of this study was to develop, evaluate and validate the Adaptation to Change Questionnaire (ADAPTA-10) for identifying those who show poor adaptability to adverse situations, such as those caused by COVID-19. This study was carried out in a sample of 1160 adults and produced a 10-item instrument with good reliability and validity indices. It is an effective tool useful in research and in clinical practice. Calculation tables are provided for the general Spanish population and by sex to evaluate adaptability to change. The two-dimensional structure proposed in the original model was confirmed. This instrument will enable the needs for adaptation to the new reality associated with COVID-19 to be detected and also other situations in which the subject becomes immersed which demand adaptation strategies in the new situation lived in.S

Design and Validation of the Adaptation to Change Questionnaire: New Realities in Times of COVID-19

Emotional and cognitive-behavioral factors influence people’s adaptability to change. Based on this premise, the objective of this study was to develop, evaluate and validate the Adaptation to Change Questionnaire (ADAPTA-10) for identifying those who show poor adaptability to adverse situations, such as those caused by COVID-19. This study was carried out in a sample of 1160 adults and produced a 10-item instrument with good reliability and validity indices. It is an effective tool useful in research and in clinical practice. Calculation tables are provided for the general Spanish population and by sex to evaluate adaptability to change. The two-dimensional structure proposed in the original model was confirmed. This instrument will enable the needs for adaptation to the new reality associated with COVID-19 to be detected and also other situations in which the subject becomes immersed which demand adaptation strategies in the new situation lived in

Epidemiologic Features and Control Measures during Monkeypox Outbreak, Spain, June 2022

During June 2022, Spain was one of the countries most affected worldwide by a multicountry monkeypox outbreak with chains of transmission without identified links to disease-endemic countries. We provide epidemiologic features of cases reported in Spain and the coordinated measures taken to respond to this outbreak.S

Antiviral strategies in plants based on RNA silencing

One of the challenges being faced in the twenty-first century is the biological control of plant viral infections. Among the different strategies to combat virus infections, those based on pathogen-derived resistance (PDR) are probably the most powerful approaches to confer virus resistance in plants. The application of the PDR concept not only revealed the existence of a previously unknown sequence-specific RNA-degradation mechanism in plants, but has also helped to design antiviral strategies to engineer viral resistant plants in the last 25 years. In this article, we review the different platforms related to RNA silencing that have been developed during this time to obtain plants resistant to viruses and illustrate examples of current applications of RNA silencing to protect crop plants against viral diseases of agronomic relevance. This article is part of a Special Issue entitled: MicroRNAs in viral gene regulation. © 2011.The work in the author's laboratory is supported by grants BIO2010-18541 from Spanish MEC and KBBE-204429 from European Union.Peer Reviewe

Procesamiento de poliproteinas den el virus de la peste porcina africana|buna nueva estrategia de expresión génica en un virus con ADN

Tesis doctoral inédita leida en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular, 199

Rapid fluorescent reporter quantification by leaf disc analysis and its application in plant-virus studies

[Background] Fluorescent proteins are extraordinary tools for biology studies due to their versatility; they are used extensively to improve comprehension of plant-microbe interactions. The viral infection process can easily be tracked and imaged in a plant with fluorescent protein-tagged viruses. In plants, fluorescent protein genes are among the most commonly used reporters in transient RNA silencing and heterologous protein expression assays. Fluorescence intensity is used to quantify fluorescent protein accumulation by image analysis or spectroscopy of protein extracts; however, these methods might not be suitable for medium- to large-scale comparisons.[Results] We report that laser scanners, used routinely in proteomic studies, are suitable for quantitative imaging of plant leaves that express different fluorescent protein pairs. We developed a microtiter plate fluorescence spectroscopy method for direct quantitative comparison of fluorescent protein accumulation in intact leaf discs. We used this technique to measure a fluorescent reporter in a transient RNA silencing suppression assay, and also to monitor early amplification dynamics of a fluorescent protein-labeled potyvirus.[Conclusions] Laser scanners allow dual-color fluorescence imaging of leaf samples, which might not be acquired in standard stereomicroscope devices. Fluorescence microtiter plate analysis of intact leaf discs can be used for rapid, accurate quantitative comparison of fluorescent protein accumulation.FP is financed by a La Caixa PhD fellowship and acknowledges support from S. Pasin and L. Lievore. This work was funded by grants BIO2010-18541 and BIO2013-49053-R from the Spanish government. The publication fee was covered partially by the CSIC Open Access Publication Support Initiative through the Unit of Information Resources for Research (URICI).Peer reviewe

Plant virus RNA replication

Plant RNA (ribonucleic acid) viruses are obligate intracellular parasites with single-stranded (ss) or double-stranded RNA genome(s) generally encapsidated but rarely enveloped. For viruses with ssRNA genomes, the polarity of the infectious RNA (positive or negative) and the presence of one or more genomic RNA segments are the features that mostly determine the molecular mechanisms governing the replication process. RNA viruses cannot penetrate plant cell walls unaided and must enter the cellular cytoplasm through mechanically induced wounds or assisted by a biological vector. After desencapsidation, their genome remains in the cytoplasm where it is translated, replicated and encapsidated in a coupled manner. Replication occurs in large viral replication complexes (VRCs), tethered to modified membranes of cellular organelles and composed by the viral RNA templates and by viral and host proteins. Cis-acting elements located in viral RNA templates regulate the recognition by the virus-encoded RNA-dependent RNA polymerase and possibly contribute to VRC assembly and/or activation.Work in the authors’ laboratories is supported by grants BFU2012-36095 BIO2013-49053-R and Plant KBBE PCIN-2013-056 from the Ministerio de Economía y Competitividad (MINECO, Spain) and from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 655841.Peer Reviewe

The effect of intermittent fasting and water restriction on myocardial ischemia/reperfusion-induced arrhythmia in rats

WOS: 000321226500020Aim: To investigate the effect of intermittent fasting and water restriction on ischemia/reperfusion-induced arrhythmias. Materials and methods: Six minutes of ischemia followed by 6 min of reperfusion was produced by the ligation and then releasing of the left coronary artery. Intermittent fasting and water restriction were applied during 1 month for 12 h/day. The duration, type, and incidence of arrhythmias during reperfusion and the survival rate at the end of reperfusion were determined and compared. Results: The score of arrhythmia that was determined using the type and duration of arrhythmia did not show significant differences among groups. The arrhythmic period was significantly longer in animals subjected to 1 month of intermittent water and food restriction. The risk of ischemic zone was found to be significantly larger in the animals subjected to 1 month of normal feeding after 1 month of food and water restriction. Conclusion: Intermittent fasting and water restriction was not found to be effective in decreasing the arrhythmia that occurred during 6 min of myocardial reperfusion in rats. Since there is no other study compatible with the present study, further research is required on the effect of intermittent fasting and water restriction on ischemia/reperfusion-induced arrhythmia and on the risk of an ischemic zone.Abant Izzet Baysal University Research FundAbant Izzet Baysal University [2011.03.01.408]We thank the Abant Izzet Baysal University Research Fund for supporting this study (project number: 2011.03.01.408)