Instituto Universitario Mixto de Biología Molecular y Celular de Plantas

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How do plant viruses induce disease? Interactions and interference with host components
(Society for General Microbiology, 2011) Pallás Benet, Vicente; Garcia, J.A; Instituto Universitario Mixto de Biología Molecular y Celular de Plantas; Ministerio de Ciencia e Innovación; European Commission
[EN] Plant viruses are biotrophic pathogens that need living tissue for their multiplication and thus, in the infection-defence equilibrium, they do not normally cause plant death. In some instances virus infection may have no apparent pathological effect or may even provide a selective advantage to the host, but in many cases it causes the symptomatic phenotypes of disease. These pathological phenotypes are the result of interference and/or competition for a substantial amount of host resources, which can disrupt host physiology to cause disease. This interference/competition affects a number of genes, which seems to be greater the more severe the symptoms that they cause. Induced or repressed genes belong to a broad range of cellular processes, such as hormonal regulation, cell cycle control and endogenous transport of macromolecules, among others. In addition, recent evidence indicates the existence of interplay between plant development and antiviral defence processes, and that interference among the common points of their signalling pathways can trigger pathological manifestations. This review provides an update on the latest advances in understanding how viruses affect substantial cellular processes, and how plant antiviral defences contribute to pathological phenotypes. © 2011 SGM.
Identification and genomic characterization of a novel tobamovirus from prickly pear cactus
(Springer-Verlag, 2020-03) Salgado-Ortiz, Héctor; De La Torre-Almaraz, Rodolfo; Sanchez Navarro, Jesus Angel; Pallás Benet, Vicente; Instituto Universitario Mixto de Biología Molecular y Celular de Plantas; Agencia Estatal de Investigación; European Regional Development Fund; Universidad Nacional Autónoma de México
[EN] In this work, we describe the complete sequence and genome organization of a novel tobamovirus detected in a prickly pear plant (Opuntia sp.) by high-throughput sequencing, tentatively named "opuntia virus 2". The full genome of opuntia virus 2 is 6,453 nucleotides in length and contains four open reading frames (ORFs) coding for the two subunits of the RNA polymerase, the movement protein, and the coat protein, respectively. Phylogenetic analysis using the complete nucleotide sequence revealed that the virus belongs to the genus Tobamovirus (family Virgaviridae), showing the highest nucleotide sequence identity (49.8%) with cactus mild mottle virus (CMMoV), being indicating that it belongs in the Cactaceae subgroup of tobamoviruses.
Recent Advances on the Multiplex Molecular Detection of Plant Viruses and Viroids
(Frontiers Media SA, 2018) Pallás Benet, Vicente; Sanchez Navarro, Jesús Ángel; James, D.; Instituto Universitario Mixto de Biología Molecular y Celular de Plantas; Generalitat Valenciana; Agencia Estatal de Investigación
[EN] Plant viruses are still one of the main contributors to economic losses in agriculture. It has been estimated that plant viruses can cause as much as 50 billion euros loss worldwide, per year. This situation may be worsened by recent climate change events and the associated changes in disease epidemiology. Reliable and early detection methods are still one of the main and most effective actions to develop control strategies for plant viral diseases. During the last years, considerable progress has been made to develop tools with high specificity and low detection limits for use in the detection of these plant pathogens. Time and cost reductions have been some of the main objectives pursued during the last few years as these increase their feasibility for routine use. Among other strategies, these objectives can be achieved by the simultaneous detection and (or) identification of several viruses in a single assay. Nucleic acid-based detection techniques are especially suitable for this purpose. Polyvalent detection has allowed the detection of multiple plant viruses at the genus level. Multiplexing RT polymerase chain reaction (PCR) has been optimized for the simultaneous detection of more than 10 plant viruses/viroids. In this short review, we provide an update on the progress made during the last decade on techniques such as multiplex PCR, polyvalent PCR, non-isotopic molecular hybridization techniques, real-time PCR, and array technologies to allow simultaneous detection of multiple plant viruses. Also, the potential and benefits of the powerful new technique of deep sequencing/next-generation sequencing are described.
Molecular characterization, targeting and expression analysis of chloroplast and mitochondrion protein import components in Nicotiana benthamiana
(Frontiers Media SA, 2022-10-26) Sáiz-Bonilla, María; Martín Merchán, Andrea; Pallás Benet, Vicente; Navarro Bohigues, José Antonio; Instituto Universitario Mixto de Biología Molecular y Celular de Plantas; Agencia Estatal de Investigación; European Regional Development Fund; Ministerio de Ciencia, Innovación y Universidades
[EN] Improved bioinformatics tools for annotating gene function are becoming increasingly available, but such information must be considered theoretical until further experimental evidence proves it. In the work reported here, the genes for the main components of the translocons of the outer membrane of chloroplasts (Toc) and mitochondria (Tom), including preprotein receptors and protein-conducting channels of N. benthamiana, were identified. Sequence identity searches and phylogenetic relationships with functionally annotated sequences such as those of A. thaliana revealed that N. benthamiana orthologs mainly exist as recently duplicated loci. Only a Toc34 ortholog was found (NbToc34), while Toc159 receptor family was composed of four orthologs but somewhat different from those of A. thaliana. Except for NbToc90, the rest (NbToc120, NbToc159A and NbToc159B) had a molecular weight of about 150 kDa and an acidic domain similar in length. Only two orthologs of the Tom20 receptors, NbTom20-1 and NbTom20-2, were found. The number of the Toc and Tom receptor isoforms in N. benthamiana was comparable to that previously reported in tomato and what we found in BLAST searches in other species in the genera Nicotiana and Solanum. After cloning, the subcellular localization of N. benthamiana orthologs was studied, resulting to be identical to that of A. thaliana receptors. Phenotype analysis after silencing together with relative expression analysis in roots, stems and leaves revealed that, except for the Toc and Tom channel- forming components (NbToc75 and NbTom40) and NbToc34, functional redundancy could be observed either among Toc159 or mitochondrial receptors. Finally, heterodimer formation between NbToc34 and the NbToc159 family receptors was confirmed by two alternative techniques indicating that different Toc complexes could be assembled. Additional work needs to be addressed to know if this results in a functional specialization of each Toc complex.
Phloem RNA-binding proteins as potential components of the long-distance RNA transport system
(Frontiers Media, 2013-05-10) Pallás Benet, Vicente; Gomez ., Gustavo Germán; Instituto Universitario Mixto de Biología Molecular y Celular de Plantas; Ministerio de Ciencia e Innovación; Generalitat Valenciana
[EN] RNA-binding proteins (RBPs) govern a myriad of different essential processes in eukaryotic cells. Recent evidence reveals that apart from playing critical roles in RNA metabolism and RNA transport, RBPs perform a key function in plant adaptation to various environmental conditions. Long-distance RNA transport occurs in land plants through the phloem, a conducting tissue that integrates the wide range of signaling pathways required to regulate plant development and response to stress processes. The macromolecules in the phloem pathway vary greatly and include defense proteins, transcription factors, chaperones acting in long-distance trafficking, and RNAs (mRNAs, siRNAs, and miRNAs). How these RNA molecules translocate through the phloem is not well understood, but recent evidence indicates the presence of translocatable RBPs in the phloem, which act as potential components of long-distance RNA transport system. This review updates our knowledge on the characteristics and functions of RBPs present in the phloem.
Impact of the Potential m(6) A Modification Sites at the 3 ' UTR of Alfalfa Mosaic Virus RNA3 in the Viral Infection
(MDPI AG, 2022-08) Alvarado-Marchena, Luis; Martínez-Pérez, Mireya; Úbeda, Jesús R.; Pallás Benet, Vicente; Aparicio Herrero, Frederic; Instituto Universitario Mixto de Biología Molecular y Celular de Plantas; Dpto. de Biotecnología; Escuela Técnica Superior de Ingeniería Agronómica y del Medio Natural; Agencia Estatal de Investigación; Ministerio de Ciencia Tecnología y Telecomunicaciones de Costa Rica
[EN] We have previously reported the presence of m(6)A in the AMV (Alfamovirus, Bromoviridae) genome. Interestingly, two of these putative m(6)A-sites are in hairpin (hp) structures in the 3'UTR of the viral RNA3. One site ((2012)AAACU(2016)) is in the loop of hpB, within the coat protein binding site 1 (CPB1), while the other ((1900)UGACC(1904)) is in the lower stem of hpE, a loop previously associated with AMV negative-strand RNA synthesis. In this work, we have performed in vivo experiments to assess the role of these two regions, containing the putative m(6)A-sites in the AMV cycle, by introducing compensatory point mutations to interfere with or abolish the m(6)A-tag of these sites. Our results suggest that the loop of hpB could be involved in viral replication/accumulation. Meanwhile, in the (1900)UGACC(1904) motif of the hpE, the maintenance of the adenosine residue and the lower stem hpE structure are necessary for in vivo plus-strand accumulation. These results extend our understanding of the requirements for hpE in the AMV infection cycle, indicating that both the residue identity and the base-pairing capacity in this structure are essential for viral accumulation.
The Molecular Biology of Ilarviruses
(Academic Press, 2013) Pallás Benet, Vicente; Aparicio Herrero, Frederic; Herranz Gordo, María Carmen; Sanchez Navarro, Jesus Angel; Scott, SW; Instituto Universitario Mixto de Biología Molecular y Celular de Plantas; Dpto. de Biotecnología; Escuela Técnica Superior de Ingeniería Agronómica y del Medio Natural
[EN] Ilarviruses were among the first 16 groups of plant viruses approved by ICTV. Like Alfalfa mosaic virus (AMV), bromoviruses, and cucumoviruses they are isometric viruses and possess a single-stranded, tripartite RNA genome. However, unlike these other three groups, ilarviruses were recognized as being recalcitrant subjects for research (their ready lability is reflected in the sigla used to create the group name) and were renowned as unpromising subjects for the production of antisera. However, it was recognized that they shared properties with AMV when the phenomenon of genome activation, in which the coat protein (CP) of the virus is required to be present to initiate infection, was demonstrated to cross group boundaries. The CP of AMV could activate the genome of an ilarvirus and vice versa. Development of the molecular information for ilarviruses lagged behind the knowledge available for the more extensively studied AMV, bromoviruses, and cucumoviruses. In the past 20 years, genomic data for most known ilarviruses have been developed facilitating their detection and allowing the factors involved in the molecular biology of the genus to be investigated. Much information has been obtained using Prunus necrotic ringspot virus and the more extensively studied AMV. A relationship between some ilarviruses and the cucumoviruses has been defined with the recognition that members of both genera encode a 2b protein involved in RNA silencing and long distance viral movement. Here, we present a review of the current knowledge of both the taxonomy and the molecular biology of this genus of agronomically and horticulturally important viruses.
Viral factors involved in plant pathogenesis
(Elsevier, 2015-04) Garcia, J.A; Pallás Benet, Vicente; Instituto Universitario Mixto de Biología Molecular y Celular de Plantas; Ministerio de Ciencia e Innovación; Ministerio de Economía y Competitividad
Plant viruses must usurp host factors/routes for their survival. Disturbances derived from this extreme dependence for host resources, together with physiological alterations associated with defensive responses, can cause, in some virus-host combinations, acute or chronic plant diseases. As the coding capacity of these biotrophic pathogens is limited, viral-encoded proteins must essentially be multifunctional proteins involved in very different steps of their life cycle, and are usually elicitors of defensive responses. Thus, most, if not all, viral-encoded proteins can act as pathogenicity determinants. Indeed, the viral proteins involved in the essential processes of their life cycle, such as replication, movement, encapsidation and transmission can be critical players of the pathogenesis process through direct or indirect interactions. This review updates our knowledge on how viral factors affect plant physiology and contribute to the development of symptomatology.
The functional analysis of distinct tospovirus movement proteins (NSM) reveals different capabilities in tubule formation, cell-to-cell and systemic virus movement among the tospovirus species
(Elsevier, 2017-01-02) Leastro, Mikhail O.; Pallás Benet, Vicente; Resende, Renato O.; Sanchez Navarro, Jesus Angel; Instituto Universitario Mixto de Biología Molecular y Celular de Plantas; Ministerio de Economía, Industria y Competitividad; Generalitat Valenciana; Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brasil
[EN] The lack of infectious tospovirus clones to address reverse genetic experiments has compromised the functional analysis of viral proteins. In the present study we have performed a functional analysis of the movement proteins (NSM) of four tospovirus species Bean necrotic mosaic virus (BeNMV), Chrysanthemum stem necrosis virus (CSNV), Tomato chlorotic spot virus (TCSV) and Tomato spotted wilt virus (TSWV), which differ biologically and molecularly, by using the Alfalfa mosaic virus (AMV) model system. All NSM proteins were competent to: i) support the cell-to-cell and systemic transport of AMV, ii) generate tubular structures on infected protoplast and iii) transport only virus particles. However, the NSM of BeNMV (one of the most phylogenetically distant species) was very inefficient to support the systemic transport. Deletion assays revealed that the C-terminal region of the BeNMV NSM, but not that of the CSNV, TCSV and TSWV NSM proteins, was dispensable for cell-to-cell transport, and that all the non-functional C-terminal NSM mutants were unable to generate tubular structures. Bimolecular fluorescence complementation analysis revealed that the C-terminus of the BeNMV NSM was not required for the interaction with the cognate nucleocapsid protein, showing a different protein organization when compared with other movement proteins of the `30K family¿. Overall, our results revealed clearly differences in functional aspects among movement proteins from divergent tospovirus species that have a distinct biological behavior.
DETECTION OF Prunus necrotic ringspot virus FROM PEACH (Prunus persica (L.) IN MEXICO AND MOLECULAR CHARACTERIZATION OF ITS COMPONENT RNA-3
(Colegio de Postgraduados. Institución de Enseñanza e Investigación en Ciencias Agrícolas. México, 2014) De la Torre-Almaraz, R; Sanchez Navarro, Jesus Angel; Pallás Benet, Vicente; Instituto Universitario Mixto de Biología Molecular y Celular de Plantas
During field observations from 2008 to 2012 in commercial peach orchards in the Estado de Mexico, states of Morelos and Puebla, leaf damage was observed in the form of yellow mottle, chlorotic rings, linear patterns and mosaic. A virus was transmitted mechanically with affected macerated peach leaves exhibiting yellow mottle to seedlings of several species of tobacco, resulting in whitish spots. In serological diagnoses (DAS-ELISA) and dot-bot type hybridization using riboprobes marked with digoxigenin for detection of six different viruses that infect peach, only the virus Prunus necrotic ringspot virus (PNRSV. Ilarvirus) was detected in the collected samples with symptoms. The electrophoretic analysis of viral ds-RNA obtained from foliage with symptoms showed three bands of 3.6, 2.5 and 1.8 Kbp, molecular weight corresponding to the PNRSV genome. In all of the localities infections by PNRSV were verified in peach plants by direct sequencing of RT-PCR final point products, using extracts of viral ds-RNA as molds and specific oligonucleotides that amplify a 455 pb fragment of the capsid protein gene of this virus. Identity of PNRSV was confirmed by cloning and determining the primary structure of the viral RNA-3 component, which contains the open reading frames corresponding to the movement protein (MP) and the capsid protein (CP). Comparison of nucleotide sequences and the corresponding sequences in amino acids with PNRSV sequences available in the Genbank database, showed 98% and 100%, similarity, respectively. Phylogenetic analyses grouped the three characterized isolates into groups PE5-III and PV32-I. Together, the data presented indicate that the variants of PNRSV in Mexico do not differ from other isolates previously reported, suggesting a probable common origin.