Una breve historia del fenómeno del silenciamiento del RNA. Contribuciones de la Virología a su descubrimiento

[ES] El descubrimiento del silenciamiento de RNA ha sido fruto del esfuerzo de muy diversos grupos entre los que hay que necesariamente destacar a los de A. Fire y C. Mello. No obstante, sin duda, la Virología y, en especial, la Virología de plantas, ha tenido una contribución muy meritoria en la elucidación de un apasionante mecanismo de regulación génica que la evolución ha ido moldeando en todos los seres vivosPallás Benet, V. (2013). Una breve historia del fenómeno del silenciamiento del RNA. Contribuciones de la Virología a su descubrimiento. Virología. 16(1):14-21. http://hdl.handle.net/10251/100986S142116

Polyvalent Detection of Members of the Genus Potyvirus by Molecular Hybridization Using a Genus-Probe

[EN] The use of a unique riboprobe named polyprobe, carrying partial sequences of different plant viruses or viroids fused in tandem, has permitted the polyvalent detection of up to 10 different pathogens by using a nonradioactive molecular hybridization procedure. In the present analysis, we have developed a unique polyprobe with the capacity to detect all members of the genus Potyvirus, which we have named genus-probe. To do this, we have exploited the capacity of the molecular hybridization assay to cross-hybridize with related sequences by reducing the hybridization temperature. We observed that sequences showing a percentage similarity of 68% or higher could be detected with the same probe by hybridizing at 50 to 55 degrees C, with a detection limit of picograms of viral RNA comparable to the specific individual probes. According to this, we developed several polyvalent polyprobes, containing three, five, or seven different 500-nucleotide fragments of a conserved region of the NIb gene. The polyprobe carrying seven different conserved regions was able to detect all the 32 potyviruses assayed in the present work with no signal in the healthy tissue, indicating the potential capacity of the polyprobe to detect all described, and probably uncharacterized, potyviruses being then considered as a genus-probe. The use of this technology in routine diagnosis not only for Potyvirus but also to other viral genera is discussed.We thank L. Corachan for her excellent technical assistance and I. Font and A. O. Alfaro, from the Mediterranean Agroforestal Institute at the Polytechnic University of Valencia, for provide part of the potyvirus-infected field samples. This work was supported by grant BIO2017-88321-R from the Spanish Direccion General de Investigacion Cientifica y Tecnica (DGICYT-MINECO).Sanchez Navarro, JA.; Cooper, C.; Pallás Benet, V. (2018). Polyvalent Detection of Members of the Genus Potyvirus by Molecular Hybridization Using a Genus-Probe. Phytopathology. 108(12):1522-1529. https://doi.org/10.1094/PHYTO-04-18-0146-RS152215291081

Phloem RNA-binding proteins as potential components of the long-distance RNA transport system

[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.This study has been supported by Grant B10201 1-25018 from the Spanish Granting Agency (Direccion General de Investigacion Cientifica) and from the PROMETEO Programme 2011/003 from the GeneralitalN'alenciana.Pallás Benet, V.; Gomez ., GG. (2013). Phloem RNA-binding proteins as potential components of the long-distance RNA transport system. Frontiers in Plant Science. 4(130):1-6. https://doi.org/10.3389/fpls.2013.00130S16413

Orchid fleck dichorhavirus movement protein shows RNA silencing suppressor activity.

[EN] To counteract RNA interference-mediated antiviral defence, virus genomes evolved to express proteins that inhibit this plant defence mechanism. Using six independent biological approaches, we show that orchid fleck dichorhavirus citrus strain (OFV-citrus) movement protein (MP) may act as a viral suppressor of RNA silencing (VSR). By using the alfalfa mosaic virus (AMV) RNA 3 expression vector, it was observed that the MP triggered necrosis response in transgenic tobacco leaves and increased the viral RNA (vRNA) accumulation. The use of the potato virus X (PVX) expression system revealed that the cis expression of MP increased both the severity of the PVX infection and the accumulation of PVX RNAs, further supporting that MP could act as an RNA silencing suppressor (RSS). From the analysis of the RSS-defective turnip crinkle virus (TCV), we do not find local RSS activity for MP, suggesting a link between MP suppressor activity and the prevention of systemic silencing. In the analysis of local suppressive activity using the GFP-based agroinfiltration assay in Nicotiana benthamiana (16 c line), we do not identify local RSS activity for the five OFV RNA1-encoded proteins. However, when evaluating the small interfering RNA (siRNA) accumulation, we find that the expression of MP significantly reduces the accumulation of GFP-derived siRNA. Finally, we examine whether the MP can prevent systemic silencing in 16c plants. Our findings show that MP inhibits the long-distance spread of RNA silencing, but does not affect the short-distance spread. Together, our findings indicate that MP is part of OFV's counter-defence mechanism, acting mainly in the prevention of systemic long-distance silencing. This work presents the first report of a VSR for a member of the genus Dichorhavirus.This work was supported by grant PID2020-115571RB-100 from the Spanish MCIN/AEI/10.13039/501100011033 granting agency and Fondo Europeo de Desarrollo Regional (FEDER), and by the company INVESTIR IMOVEIS LDTA from Brasilia, Brazil.Oliveira Leastro, M.; Pallás Benet, V.; Sánchez-Navarro, JÁ. (2022). Orchid fleck dichorhavirus movement protein shows RNA silencing suppressor activity. Journal of General Virology. 103(11):1-14. https://doi.org/10.1099/jgv.0.0018051141031

A conserved motif in three viral movement proteins from different genera is required for host factor recruitment and cell-to-cell movement

[EN] Due to their minimal genomes, plant viruses are forced to hijack specific cellular pathways to ensure host colonization, a condition that most frequently involves physical interaction between viral and host proteins. Among putative viral interactors are the movement proteins, responsible for plasmodesma gating and genome binding during viral transport. Two of them, DGBp1 and DGBp2, are required for alpha-, beta- and gammacarmovirus cell-to-cell movement, but the number of DGBp-host interactors identified at present is limited. By using two different approaches, yeast two-hybrid and bimolecular fluorescence complementation assays, we found three Arabidopsis factors, eIF3g1, RPP3A and WRKY36, interacting with DGBp1s from each genus mentioned above. eIF3g1 and RPP3A are mainly involved in protein translation initiation and elongation phases, respectively, while WRKY36 belongs to WRKY transcription factor family, important regulators of many defence responses. These host proteins are not expected to be associated with viral movement, but knocking out WRKY36 or silencing either RPP3A or eIF3g1 negatively affected Arabidopsis infection by Turnip crinkle virus. A highly conserved FNF motif at DGBp1 C-terminus was required for protein-protein interaction and cell-to-cell movement, suggesting an important biological role.We thank Dr. Anne Simon and Dr. Steve A. Lommel for providing an infectious cDNA clone of the Turnip crinkle virus strain M (TCV-M) and PZP-TCV-sGFP plasmid, respectively. This work was funded by grant BIO2017-88321-R from the Spanish Agencia Estatal de Investigacion (AEI) and Fondo Europeo de Desarrollo Regional (FEDER). J.A.N. and M.S.-S. are the recipients of a postdoctoral contract and a PhD fellowship from the Ministerio de Ciencia, Innovacion y Universidades of Spain.Navarro Bohigues, JA.; Serra-Soriano, M.; Corachán Valencia, L.; Pallás Benet, V. (2020). 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Virology 273, 276–285 (2000).Martinez-Gil, L., Sauri, A., Vilar, M., Pallas, V. & Mingarro, I. Membrane insertion and topology of the p7B movement protein of Melon Necrotic Spot Virus (MNSV). Virology 367, 348–357 (2007).Sauri, A., Saksena, S., Salgado, J., Johnson, A. E. & Mingarro, I. Double-spanning plant viral movement protein integration into the endoplasmic reticulum membrane is signal recognition particle-dependent, translocon-mediated, and concerted. J. Biol. Chem. 280, 25907–25912 (2005).Genoves, A., Navarro, J. A. & Pallas, V. The intra- and intercellular movement of Melon necrotic spot virus (MNSV) depends on an active secretory pathway. Mol. Plant-Microbe Interact. 23, 263–272 (2010).Aparicio, F. & Pallás, V. The coat protein of Alfalfa mosaic virus interacts and interferes with the transcriptional activity of the bHLH transcription factor ILR3 promoting salicylic acid-dependent defence signalling response. Mol. Plant Pathol. 18(2), 173–186 (2017).Yang, Y. et al. 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Ribosomal protein P0 promotes Potato virus A infection and functions in viral translation together with VPg and eIF(iso)4E. J. Virol. 87, 4302–4312 (2013).Sato, H. et al. Measles virus N protein inhibits host translation by binding to eIF3-p40. J. Virol. 81, 11569–11576 (2007).Bhardwaj, U., Powell, P. & Goss, D. J. Eukaryotic initiation factor (eIF) 3 mediates barley yellow dwarf viral mRNA 3′–5′ UTR interactions and 40S ribosomal subunit binding to facilitate cap-independent translation. Nucleic Acids Res. 47, 6225–6235Park, H. S., Himmelbach, A., Browning, K. S., Hohn, T. & Ryabova, L. A. A plant viral ‘reinitiation’ factor interacts with the host translational machinery. Cell 106, 723–733 (2001).Thiébeauld, O., Pooggin, M. & Ryabova, L. Alternative translation strategies in plant viruses. Plant Viruses 1, 1–20 (2007).Bureau, M. et al. P6 protein of Cauliflower mosaic virus, a translation reinitiator, interacts with ribosomal protein L13 from Arabidopsis thaliana. J. Gen. 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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

[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.We thank L. Corachan for her excellent technical assistance. This work was supported by grant BIO2014-54862-R from the Spanish Direccion General de Investigacion Cientifica y Tecnica (DGICYT), the Prometeo Program GV2014/010 from the Generalitat Valenciana, CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnologico), Capes (Conselho de Aperfeicoamento de Pessoal de Nivel Superior) and FAP-DF (Fundacao de Apoio a Pesquisa do Distrito Federal)Leastro, MO.; Pallás Benet, V.; Resende, RO.; Sanchez Navarro, JA. (2017). 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. Virus Research. 227:57-68. https://doi.org/10.1016/j.virusres.2016.09.023S576822

Rescue of a Cilevirus from infectious cDNA clones

[EN] Reverse genetics systems represent an important tool for studying the molecular and functional processes of viral infection. Citrus leprosis virus C (CiLV-C) (genus Cilevirus, family Kitaviridae) is the main pathogen responsible for the citrus leprosis (CL) disease in Latin America, one of the most economically important diseases of the citrus industry. Molecular studies of this pathosystem are limited due to the lack of infectious clones. Here, we report the construction and validation of a CiLV-C infectious cDNA clone based on an agroinfection system. The two viral RNA segments (RNA1 and RNA2) were assembled into two binary vectors (pJL89 and pLXAS). Agro-infiltrated Nicotiana benthamiana plants showed a response similar to that observed in the natural infection process with the formation of localized lesions restricted to the inoculated leaves. The virus recovered from the plant tissue infected with the infectious clones can be mechanically transmitted between N. benthamiana plants. Detection of CiLV-C subgenomic RNAs (sgRNAs) from agroinfiltrated and mechanically inoculated leaves further confirmed the infectivity of the clones. Finally, partial particle-purification preparations or sections of CiLV-C-infected tissue followed by transmission electron microscopy (TEM) analysis showed the formation of CiLV-C virions rescued by the infectious clone. The CiLV-C reverse genetic system now provides a powerful molecular tool to unravel the peculiarities of the CL pathosystem.This work was supported by grant PID2020-115571RB-100 from the Spanish MCIN/AEI/10.13039/501100011033 granting agency.Leastro, MO.; Kitajima, EW.; Pallás Benet, V.; Sanchez Navarro, JA. (2024). Rescue of a Cilevirus from infectious cDNA clones. Virus Research. 339. https://doi.org/10.1016/j.virusres.2023.19926433

Highly efficient construction of infectious viroid-derived clones

[EN] Background Viroid research generally relies on infectious cDNA clones that consist of dimers of the entire viroid sequence. At present, those dimers are generated by self-ligation of monomeric cDNA, a strategy that presents several disadvantages: (i) low efficiency, (ii) it is a non-oriented reaction requiring tedious screenings and (iii) additional steps are required for cloning into a binary vector for agroinfiltration or for in vitro RNA production. Results We have developed a novel strategy for simultaneous construction of a viroid dimeric cDNA and cloning into a multipurpose binary vector ready for agroinfiltration or in vitro transcription. The assembly is based on IIs restriction enzymes and positive selection and supposes a universal procedure for obtaining infectious clones of a viroid independently of its sequence, with a high efficiency. Thus, infectious clones of one viroid of each family were obtained and its infectivity was analyzed by molecular hybridization. Conclusion This is a zero-background strategy for direct cloning into a binary vector, optimized for the generation of infectious viroids. As a result, this methodology constitutes a powerful tool for viroid research and exemplifies the applicability of type IIs restriction enzymes and the lethal gene ccdB to design efficient and affordable direct cloning approaches of PCR products into binary vectors.This work was supported by the Spanish Ministry of Economy and Competitiveness (co-supported by FEDER) Grants BIO2017-88321-R (VP) and AGL2016-79825-R (GG). The funders had no role in the experiment design, data analysis, decision to publish, or preparation of the manuscript.Márquez-Molins, J.; Navarro Bohigues, JA.; Pallás Benet, V.; Gomez, GG. (2019). Highly efficient construction of infectious viroid-derived clones. Plant Methods. 15:1-8. https://doi.org/10.1186/s13007-019-0470-4S1815Flores R, Minoia S, Carbonell A, Gisel A, Delgado S, López-Carrasco A, et al. 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Extremely High Mutation Rate of a Hammerhead Viroid. Science (80-). 2009;323:1308–1308.Steger G, Riesner D. Viroid research and its significance for RNA technology and basic biochemistry. Nucleic Acids Res. 2018;46:10563–76.Gómez G, Torres H, Pallás V. Identification of translocatable RNA-binding phloem proteins from melon, potential components of the long-distance RNA transport system. Plant J. 2004;41:107–16.Takeda R, Petrov AI, Leontis NB, Ding B. A three-dimensional RNA motif in Potato spindle tuber viroid mediates trafficking from palisade mesophyll to spongy mesophyll in Nicotiana benthamiana. Plant Cell. 2011;23:258–72.Gómez G, Pallás V. Studies on subcellular compartmentalization of plant pathogenic noncoding RNAs give new insights into the intracellular RNA-traffic mechanisms. Plant Physiol. 2012;159:558–64.Wassenegger M, Heimes S, Riedel L, Sänger HL. RNA-directed de novo methylation of genomic sequences in plants. 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Small RNA derived from the virulence modulating region of the potato spindle tuber viroid silences callose synthase genes of tomato plants. Plant Cell. 2015;27:2178–94.Gibson DG, Young L, Chuang R-Y, Venter JC, Hutchison CA, Smith HO. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods. 2009;6:343–5.Engler C, Gruetzner R, Kandzia R, Marillonnet S. Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes. PLoS ONE. 2009;4:e5553.Carbonell A, Takeda A, Fahlgren N, Johnson SC, Cuperus JT, Carrington JC. New generation of artificial MicroRNA and synthetic trans-acting small interfering RNA vectors for efficient gene silencing in Arabidopsis. Plant Physiol. 2014;165:15–29.Genovés A, Navarro JA, Pallás V. Functional analysis of the five melon necrotic spot virus genome-encoded proteins. J Gen Virol. 2006;87:2371–80.Gómez G, Pallás V. 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¿PUEDEN LOS VIRUS DE PLANTAS SER PATOGÉNICOS EN HUMANOS?

[EN] Virus infections in plants are very frequent, including those affecting crops of major economic importance. Plant viruses are present in many foods that are part of our daily diet, especially in fruits and vegetables. Although there are some studies suggesting that barriers between plants and mammals could be surpassed by some viruses, there is no evidence that plant viruses are causative agents of any disease in humans or other mammals.[ES] Las infecciones de virus en plantas son muy frecuentes, incluyendo las que afectan a cultivos de gran impor tancia económica. Los virus de plantas están presentes en muchos alimentos que forman par te de nuestra dieta diaria, especialmente en frutas y verduras. Aunque existen algunos trabajos que sugieren que las barreras entre plantas y mamíferos podrían ser sobrepasadas por algunos virus, no existen evidencias de que los virus de plantas sean agentes causales de ninguna enfermedad en humanos u otros mamíferos.Fiallo-Olivé, E.; Pallás Benet, V.; Navas-Castillo, J. (2017). ¿PUEDEN LOS VIRUS DE PLANTAS SER PATOGÉNICOS EN HUMANOS?. Virología. 20(2):60-63. http://hdl.handle.net/10251/102628S606320

An Evolved 5 ' Untranslated Region of Alfalfa Mosaic Virus Allows the RNA Transport of Movement-Defective Variants

[EN] The results obtained in the present work could challenge the view of the role of the virus particle in the systemic transport of plant viruses. In this sense, we show that two different MPs are competent to systemically transport the AMV genome without the requirement of the virus particles, as reported for viruses lacking a CP (e.g., Umbravirus). Although the coat protein (CP) has a relevant role in the long-distance movement of alfalfa mosaic virus (AMV) and brome mosaic virus (BMV), its precise function is not fully understood. Previous results showed that a specific interaction between the C termini of the movement protein (MP) and the cognate CP is required for systemic transport. Thus, we have performed a compensatory evolution experiment using an AMV RNA3 derivative defective in long-distance transport that carries a BMV MP lacking the C-terminal 48 residues and unable to interact with the AMV CP. After several passages, five independent evolution lineages were able to move long distance. The analysis of the viral RNA of these lineages showed the presence of three different modifications located exclusively at the 5 ' untranslated region (5 ' UTR). The three evolved 5 ' UTR variants accumulated comparable levels of viral RNA and CP but reduced the accumulation of virus particles and the affinity between the 5 ' UTR and the AMV CP. In addition, the evolved 5 ' UTR increased cell-to-cell transport for both the AMV RNA3 carrying the BMV MP and that carrying the AMV MP. Finally, the evolved 5 ' UTRs allowed the systemic transport of an AMV RNA3 carrying a CP mutant defective in virus particles and increased the systemic transport of several AMV RNA3 derivatives carrying different viral MPs associated with the 30K superfamily. Altogether, our findings indicate that virus particles are not required for the systemic transport of AMV but also that BMV MP is competent for the short- and long-distance transport without the interaction with the CP. IMPORTANCE The results obtained in the present work could challenge the view of the role of the virus particle in the systemic transport of plant viruses. In this sense, we show that two different MPs are competent to systemically transport the AMV genome without the requirement of the virus particles, as reported for viruses lacking a CP (e.g., Umbravirus). The incapability of the viral MP to interact with the CP triggered virus variants that evolved to reduce the formation of virus particles, probably to increase the accessibility of the MP to the viral progeny. Our results point to the idea that virus particles would not be necessary for the viral systemic transport but would be necessary for vector virus transmission. This idea is reinforced by the observation that heterologous MPs also increased the systemic transport of the AMV constructs that have reduced encapsidation capabilities.We are grateful to Lorena Corachan for excellent technical support. This work was supported by grants PID2020-115571RB-I00 and PID2019-103998GB-I00 from the Spanish MCIN/AEI/10.13039/501100011033 granting agency-FEDER (V.P. and S.F.E., respectively) and PROMETEO/2015/010 and PROMETEO2019/012 from the Generalitat Valenciana (V.P. and S.F.E., respectively).Villar-Álvarez, D.; Pallás Benet, V.; Elena Fito, SF.; Sánchez-Navarro, JÁ. (2022). An Evolved 5 ' Untranslated Region of Alfalfa Mosaic Virus Allows the RNA Transport of Movement-Defective Variants. Journal of Virology. 96(22):1-17. https://doi.org/10.1128/jvi.00988-22117962