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Burgyan J.,CNR Institute of Plant virology | Burgyan J.,Agricultural Biotechnology Center | Havelda Z.,Agricultural Biotechnology Center
Trends in Plant Science | Year: 2011

The infection and replication of viruses in the host induce diverse mechanisms for combating viral infection. One of the best-studied antiviral defence mechanisms is based on RNA silencing. Consistently, several viral suppressors of RNA silencing (VSRs) have been identified from almost all plant virus genera, which are surprisingly diverse within and across kingdoms, exhibiting no obvious sequence similarities. VSRs efficiently inhibit host antiviral responses by interacting with the key components of cellular silencing machinery, often mimicking their normal cellular functions. Recent findings have revealed that the impact of VSRs on endogenous pathways is more complex and profound than had been estimated thus far. This review highlights the current understanding of and new insights into the mechanisms and functions of plant VSRs. © 2011 Elsevier Ltd.


Schuck J.,Martin Luther University of Halle Wittenberg | Gursinsky T.,Martin Luther University of Halle Wittenberg | Pantaleo V.,CNR Institute of Plant virology | Burgyan J.,Agricultural Biotechnology Center | Behrens S.-E.,Martin Luther University of Halle Wittenberg
Nucleic Acids Research | Year: 2013

AGO/RISC-mediated antiviral RNA silencing, an important component of the plant's immune response against RNA virus infections, was recapitulated in vitro. Cytoplasmic extracts of tobacco protoplasts were applied that supported Tombusvirus RNA replication, as well as the formation of RNA-induced silencing complexes (RISC) that could be functionally reconstituted with various plant ARGONAUTE (AGO) proteins. For example, when RISC containing AGO1, 2, 3 or 5 were programmed with exogenous siRNAs that specifically targeted the viral RNA, endonucleolytic cleavages occurred and viral replication was inhibited. Antiviral RNA silencing was disabled by the viral silencing suppressor p19 when this was present early during RISC formation. Notably, with replicating viral RNA, only (+)RNA molecules were accessible to RISC, whereas (-)RNA replication intermediates were not. The vulnerability of viral RNAs to RISC activity also depended on the RNA structure of the target sequence. This was most evident when we characterized viral siRNAs (vsiRNAs) that were particularly effective in silencing with AGO1-or AGO2/RISC. These vsiRNAs targeted similar sites, suggesting that accessible parts of the viral (+)RNA may be collectively attacked by different AGO/RISC. The in vitro system was, hence, established as a valuable tool to define and characterize individual molecular determinants of antiviral RNA silencing. © 2013 The Author(s).


Turina M.,CNR Institute of Plant virology | Tavella L.,University of Turin | Ciuffo M.,CNR Institute of Plant virology
Advances in Virus Research | Year: 2012

Tospoviruses are among the most serious threats to vegetable crops in the Mediterranean basin. Tospovirus introduction, spread, and the diseases these viruses cause have been traced by epidemiological case studies. Recent research has centered on the close relationship between tospoviruses and their arthropod vectors (species of the Thripidae family). Here, we review several specific features of tospovirus-thrips associations in the Mediterranean. Since the introduction of Frankliniella occidentalis in Europe, Tomato spotted wilt virus (TSWV) has become one of the limiting factors for vegetable crops such as tomato, pepper, and lettuce. An increasing problem is the emergence of TSWV resistance-breaking strains that overcome the resistance genes in pepper and tomato. F. occidentalis is also a vector of Impatiens necrotic spot virus, which was first observed in the Mediterranean basin in the 1980s. Its importance as a cause of vegetable crop diseases is limited to occasional incidence in pepper and tomato fields. A recent introduction is Iris yellow spot virus, transmitted by the onion thrips Thrips tabaci, in onion and leek crops. Control measures in vegetable crops specific to Mediterranean conditions were examined in the context of their epidemiological features and tospovirus species which could pose a future potential risk for vegetable crops in the Mediterranean were discussed. © 2012 Elsevier Inc.


Cillo F.,CNR Institute of Plant virology | Palukaitis P.,Seoul Womens University
Advances in Virus Research | Year: 2014

Transgenic resistance to plant viruses is an important technology for control of plant virus infection, which has been demonstrated for many model systems, as well as for the most important plant viruses, in terms of the costs of crop losses to disease, and also for many other plant viruses infecting various fruits and vegetables. Different approaches have been used over the last 28 years to confer resistance, to ascertain whether particular genes or RNAs are more efficient at generating resistance, and to take advantage of advances in the biology of RNA interference to generate more efficient and environmentally safer, novel "resistance genes." The approaches used have been based on expression of various viral proteins (mostly capsid protein but also replicase proteins, movement proteins, and to a much lesser extent, other viral proteins), RNAs [sense RNAs (translatable or not), antisense RNAs, satellite RNAs, defective-interfering RNAs, hairpin RNAs, and artificial microRNAs], nonviral genes (nucleases, antiviral inhibitors, and plantibodies), and host-derived resistance genes (dominant resistance genes and recessive resistance genes), and various factors involved in host defense responses. This review examines the above range of approaches used, the viruses that were tested, and the host species that have been examined for resistance, in many cases describing differences in results that were obtained for various systems developed in the last 20 years. We hope this compilation of experiences will aid those who are seeking to use this technology to provide resistance in yet other crops, where nature has not provided such. © 2014 Elsevier Inc.


Margaria P.,CNR Institute of Plant virology | Palmano S.,CNR Institute of Plant virology
Proteomics | Year: 2011

Flavescence dorée is a serious phytoplasma disease affecting grapevine in several European countries. We studied the interaction of Flavescence dorée phytoplasma with its natural plant host by monitoring the effects of infection on the protein expression profile. Among the 576 analyzed spots, 33 proteins were differentially regulated in infected grapevines. Grouping into MIPS functional categories showed proteins involved in metabolism (21%), energy processes (9%), protein synthesis (3%), protein fate (18%), cellular transport and transport routes (6%), cell defense and virulence (42%). Among the differentially regulated proteins, we selected six targets (thaumatin I, thaumatin II, osmotin-like protein, plant basic secretory protein, AAA + Rubisco activase and proteasome α5 subunit) and we analyzed their expression by quantitative RT-PCR on samples collected in 2008 and 2009 in several vineyards in Piedmont region, Italy. There was a positive correlation between mRNA and protein expression for most of the genes in both the years. We discuss the involvement of these proteins in the specific response to phytoplasma infection. To our knowledge, this work is the first to investigate the response of the grapevine proteome to Flavescence dorée phytoplasma infection, and provides reference protein profiles for future comparative proteomic and genomic studies. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


Carluccio A.V.,CNR Institute of Plant virology | Zicca S.,CNR Institute of Plant virology | Stavolone L.,CNR Institute of Plant virology
Plant Physiology | Year: 2014

The transport of a viral genome from cell to cell is enabled by movement proteins (MPs) targeting the cell periphery to mediate the gating of plasmodesmata. Given their essential role in the development of viral infection, understanding the regulation of MPs is of great importance. Here, we show that cauliflower mosaic virus (CaMV) MP contains three tyrosine-based sorting signals that interact with an Arabidopsis (Arabidopsis thaliana) mA-adaptin subunit. Fluorophore-tagged MP is incorporated into vesicles labeled with the endocytic tracer N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide. The presence of at least one of the three endocytosis motifs is essential for internalization of the protein from the plasma membrane to early endosomes, for tubule formation, and for CaMV infection. In addition, we show that MP colocalizes in vesicles with the Rab GTPase AtRAB-F2b, which is resident in prevacuolar late endosomal compartments that deliver proteins to the vacuole for degradation. Altogether, these results demonstrate that CaMV MP traffics in the endocytic pathway and that virus viability depends on functional host endomembranes. © 2014 American Society of Plant Biologists. All rights reserved.


Pantaleo V.,CNR Institute of Plant virology
Advances in Experimental Medicine and Biology | Year: 2011

RNA silencing is described in plants and insects as a defence mechanism against foreign nucleic acids, such as invading viruses. The RNA silencing-based antiviral defence involves the production of virus-derived small interfering RNAs and their association to effector proteins, which together drive the sequence specific inactivation of viruses. The entire process of antiviral defence 'borrows' several plant factors involved in other specialized RNA silencing endogenous pathways. Different viruses use variable strategies to infect different host plants, which render the antiviral RNA silencing a complex phenomenon far to be completely clarified. This chapter reports current advances in understanding the main steps of the plant's RNA-silencing response to viral invasion and discusses some of the key questions still to be answered. © 2011 Landes Bioscience and Springer Science+Business Media, LLC.


Gambino G.,CNR Institute of Plant virology | Gribaudo I.,CNR Institute of Plant virology
Transgenic Research | Year: 2012

Genetic transformation has emerged as a powerful tool for genetic improvement of fruit trees hindered by their reproductive biology and their high levels of heterozygosity. For years, genetic engineering of fruit trees has focussed principally on enhancing disease resistance (against viruses, fungi, and bacteria), although there are few examples of field cultivation and commercial application of these transgenic plants. In addition, over the years much work has been performed to enhance abiotic stress tolerance, to induce modifications of plant growth and habit, to produce marker-free transgenic plants and to improve fruit quality by modification of genes that are crucially important in the production of specific plant components. Recently, with the release of several genome sequences, studies of functional genomics are becoming increasingly important: by modification (overexpression or silencing) of genes involved in the production of specific plant components is possible to uncover regulatory mechanisms associated with the biosynthesis and catabolism of metabolites in plants. This review focuses on the main advances, in recent years, in genetic transformation of the most important species of fruit trees, devoting particular attention to functional genomics approaches and possible future challenges of genetic engineering for these species in the post-genomic era. © 2012 Springer Science+Business Media B.V.


Margaria P.,CNR Institute of Plant virology | Abba S.,CNR Institute of Plant virology | Palmano S.,CNR Institute of Plant virology
BMC Genomics | Year: 2013

Background: Translational and post-translational protein modifications play a key role in the response of plants to pathogen infection. Among the latter, phosphorylation is critical in modulating protein structure, localization and interaction with other partners. In this work, we used a multiplex staining approach with 2D gels to study quantitative changes in the proteome and phosphoproteome of Flavescence dorée-affected and recovered 'Barbera' grapevines, compared to healthy plants.Results: We identified 48 proteins that differentially changed in abundance, phosphorylation, or both in response to Flavescence dorée phytoplasma infection. Most of them did not show any significant difference in recovered plants, which, by contrast, were characterized by changes in abundance, phosphorylation, or both for 17 proteins not detected in infected plants. Some enzymes involved in the antioxidant response that were up-regulated in infected plants, such as isocitrate dehydrogenase and glutathione S-transferase, returned to healthy-state levels in recovered plants. Others belonging to the same functional category were even down-regulated in recovered plants (oxidoreductase GLYR1 and ascorbate peroxidase). Our proteomic approach thus agreed with previously published biochemical and RT-qPCR data which reported down-regulation of scavenging enzymes and accumulation of H2O2 in recovered plants, possibly suggesting a role for this molecule in remission from infection. Fifteen differentially phosphorylated proteins (| ratio | > 2, p < 0.05) were identified in infected compared to healthy plants, including proteins involved in photosynthesis, response to stress and the antioxidant system. Many were not differentially phosphorylated in recovered compared to healthy plants, pointing to their specific role in responding to infection, followed by a return to a steady-state phosphorylation level after remission of symptoms. Gene ontology (GO) enrichment and statistical analysis showed that the general main category " response to stimulus" was over-represented in both infected and recovered plants but, in the latter, the specific child category " response to biotic stimulus" was no longer found, suggesting a return to steady-state levels for those proteins specifically required for defence against pathogens.Conclusions: Proteomic data were integrated into biological networks and their interactions were represented through a hypothetical model, showing the effects of protein modulation on primary metabolic ways and related secondary pathways. By following a multiplex-staining approach, we obtained new data on grapevine proteome pathways that specifically change at the phosphorylation level during phytoplasma infection and following recovery, focusing for the first time on phosphoproteome changes during pathogen infection in this host. © 2013 Margaria et al.; licensee BioMed Central Ltd.


Shimura H.,Hokkaido University | Pantaleo V.,CNR Institute of Plant virology
Biochimica et Biophysica Acta - Gene Regulatory Mechanisms | Year: 2011

RNA silencing in plants and insects can function as a defence mechanism against invading viruses. RNA silencing-based antiviral defence entails the production of virus-derived small interfering RNAs which guide specific antiviral effector complexes to inactivate viral genomes. As a response to this defence system, viruses have evolved viral suppressors of RNA silencing (VSRs) to overcome the host defence. VSRs can act on various steps of the different silencing pathways. Viral infection can have a profound impact on the host endogenous RNA silencing regulatory pathways; alterations of endogenous short RNA expression profile and gene expression are often associated with viral infections and their symptoms. Here we discuss our current understanding of the main steps of RNA-silencing responses to viral invasion in plants and the effects of VSRs on endogenous pathways. This article is part of a Special Issue entitled: MicroRNAs in viral gene regulation. © 2011 Elsevier B.V.

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