Unusual features of pomoviral RNA movement

This work is partially supported by the Scottish Government’s Rural and Environment Science and Analytical Services (RESAS) DivisionPotato mop-top pomovirus (PMTV) is one of a few viruses that can move systemically in plants in the absence of the capsid protein (CP). Pomoviruses encode the triple gene block genetic module of movement proteins (TGB 1, 2, and 3) and recent research suggests that PMTV RNA is transported either as ribonucleoprotein (RNP) complexes containing TGB1 or encapsidated in virions containing TGB1. Furthermore, there are different requirements for local or systemic (long-distance) movement. Research suggests that nucleolar passage of TGB1 may be important for the long-distance movement of both RNP and virions. Moreover, and uniquely, the long-distance movement of the CP-encoding RNA requires expression of both major and minor CP subunits and is inhibited when only the major CP sub unit is expressed. This paper reviews pomovirus research and presents a current model for RNA movement.Publisher PDFPeer reviewe

A case for a negative-strand coding sequence in a group of positive-sense RNA viruses.

Positive-sense single-stranded RNA viruses form the largest and most diverse group of eukaryote-infecting viruses. Their genomes comprise one or more segments of coding-sense RNA that function directly as messenger RNAs upon release into the cytoplasm of infected cells. Positive-sense RNA viruses are generally accepted to encode proteins solely on the positive strand. However, we previously identified a surprisingly long (∼1,000-codon) open reading frame (ORF) on the negative strand of some members of the family Narnaviridae which, together with RNA bacteriophages of the family Leviviridae, form a sister group to all other positive-sense RNA viruses. Here, we completed the genomes of three mosquito-associated narnaviruses, all of which have the long reverse-frame ORF. We systematically identified narnaviral sequences in public data sets from a wide range of sources, including arthropod, fungal, and plant transcriptomic data sets. Long reverse-frame ORFs are widespread in one clade of narnaviruses, where they frequently occupy >95 per cent of the genome. The reverse-frame ORFs correspond to a specific avoidance of CUA, UUA, and UCA codons (i.e. stop codon reverse complements) in the forward-frame RNA-dependent RNA polymerase ORF. However, absence of these codons cannot be explained by other factors such as inability to decode these codons or GC3 bias. Together with other analyses, we provide the strongest evidence yet of coding capacity on the negative strand of a positive-sense RNA virus. As these ORFs comprise some of the longest known overlapping genes, their study may be of broad relevance to understanding overlapping gene evolution and de novo origin of genes

Polycipiviridae: a proposed new family of polycistronic picorna-like RNA viruses

Solenopsis invicta virus 2 is a single-stranded positive-sense picorna-like RNA virus with an unusual genome structure. The monopartite genome of approximately 11 kb contains four open reading frames in its 5′ one third, three of which encode proteins with homology to picornavirus-like jelly-roll fold capsid proteins. These are followed by an intergenic region, and then a single long open reading frame that covers the 3′ two thirds of the genome. The polypeptide translation of this 3′ open reading frame contains motifs characteristic of picornavirus-like helicase, protease and RNA-dependent RNA polymerase domains. Inspection of public transcriptome shotgun assembly sequences revealed five related apparently nearly complete virus genomes isolated from ant species and one from a dipteran insect. By high-throughput sequencing and in silico assembly of RNA isolated from Solenopsis invicta and four other ant species, followed by targeted Sanger sequencing, we obtained nearly complete genomes for four further viruses in the group. Four further sequences were obtained from a recent large-scale invertebrate virus study. The 15 sequences are highly divergent (pairwise amino acid identities as low as 17% in the non-structural polyprotein), but possess the same overall polycistronic genome structure distinct from all other characterized picorna-like viruses. Consequently we propose the formation of a new virus family, Polycipiviridae, to classify this clade of arthropod-infecting polycistronic picorna-like viruses. We further propose that this family be divided into three genera: Chipolycivirus (2 species), Hupolycivirus (2 species), and Sopolycivirus (11 species), with members of the latter infecting ants in at least three different subfamilies.This work was supported by a Wellcome Trust grant [106207] and a European Research Council (ERC) European Union's Horizon 2020 research and innovation programme grant [646891] to A.E.F

Meiotic crossover reduction by virus-induced gene silencing enables the efficient generation of chromosome substitution lines and reverse breeding in Arabidopsis thaliana.

Plant breeding applications exploiting meiotic mutant phenotypes (like the increase or decrease of crossover (CO) recombination) have been proposed over the last years. As recessive meiotic mutations in breeding lines may affect fertility or have other pleiotropic effects, transient silencing techniques may be preferred. Reverse breeding is a breeding technique that would benefit from the transient downregulation of CO formation. The technique is essentially the opposite of plant hybridization: a method to extract parental lines from a hybrid. The method can also be used to efficiently generate chromosome substitution lines (CSLs). For successful reverse breeding, the two homologous chromosome sets of a heterozygous plant must be divided over two haploid complements, which can be achieved by the suppression of meiotic CO recombination and the subsequent production of doubled haploid plants. Here we show the feasibility of transiently reducing CO formation using virus-induced gene silencing (VIGS) by targeting the meiotic gene MSH5 in a wild-type heterozygote of Arabidopsis thaliana. The application of VIGS (rather than using lengthy stable transformation) generates transgene-free offspring with the desired genetic composition: we obtained parental lines from a wild-type heterozygous F1 in two generations. In addition, we obtained 20 (of the 32 possible) CSLs in one experiment. Our results demonstrate that meiosis can be modulated at will in A. thaliana to generate CSLs and parental lines rapidly for hybrid breeding. Furthermore, we illustrate how the modification of meiosis using VIGS can open routes to develop efficient plant breeding strategies

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

[EN] During virus infection, specific viral component-host factor interaction elicits the transcriptional reprogramming of diverse cellular pathways. Alfalfa mosaic virus (AMV) can establish a compatible interaction in tobacco and Arabidopsis hosts. We show that the coat protein (CP) of AMV interacts directly with transcription factor (TF) ILR3 of both species. ILR3 is a basic helix-loop-helix (bHLH) family member of TFs, previously proposed to participate in diverse metabolic pathways. ILR3 has been shown to regulate NEET in Arabidopsis, a critical protein in plant development, senescence, iron metabolism and reactive oxygen species (ROS) homeostasis. We show that the AMV CP-ILR3 interaction causes a fraction of this TF to relocate from the nucleus to the nucleolus. ROS, pathogenesis-related protein 1 (PR1) mRNAs, salicylic acid (SA) and jasmonic acid (JA) contents are increased in healthy Arabidopsis loss-of-function ILR3 mutant (ilr3.2) plants, which implicates ILR3 in the regulation of plant defence responses. In AMV-infected wild-type (wt) plants, NEET expression is reduced slightly, but is induced significantly in ilr3.2 mutant plants. Furthermore, the accumulation of SA and JA is induced in Arabidopsis wt-infected plants. AMV infection in ilr3.2 plants increases JA by over 10-fold, and SA is reduced significantly, indicating an antagonist crosstalk effect. The accumulation levels of viral RNAs are decreased significantly in ilr3.2 mutants, but the virus can still systemically invade the plant. The AMV CP-ILR3 interaction may down-regulate a host factor, NEET, leading to the activation of plant hormone responses to obtain a hormonal equilibrium state, where infection remains at a level that does not affect plant viability.F.A. was the recipient of a contract Ramon y Cajal (RYC-2010-06169) program of the Ministerio de Educacion, Cultura y Deporte of Spain. We thank L. Corachan for excellent technical assistance. This work was supported by Grants BIO2014-54862-R from the Spanish grant agency Direccion General de Investigacion Cientifica y Tecnica (DGICT) the Prometeo Program GV2015/010 from the Generalitat Valenciana and PAID-06-10-1496 from the Universitat Politecnica de Valencia (Spain).Aparicio Herrero, F.; Pallás Benet, V. (2017). 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. Molecular Plant Pathology. 18(2):173-186. https://doi.org/10.1111/mpp.12388S173186182Abbink, T. E. M., Peart, J. R., Mos, T. N. M., Baulcombe, D. C., Bol, J. F., & Linthorst, H. J. M. (2002). Silencing of a Gene Encoding a Protein Component of the Oxygen-Evolving Complex of Photosystem II Enhances Virus Replication in Plants. Virology, 295(2), 307-319. doi:10.1006/viro.2002.1332Alazem, M., & Lin, N. (2014). Roles of plant hormones in the regulation of host–virus interactions. Molecular Plant Pathology, 16(5), 529-540. doi:10.1111/mpp.12204Aparicio, F., Vilar, M., Perez-Payá, E., & Pallás, V. (2003). The coat protein of prunus necrotic ringspot virus specifically binds to and regulates the conformation of its genomic RNA. Virology, 313(1), 213-223. doi:10.1016/s0042-6822(03)00284-8Aparicio, F., Thomas, C. L., Lederer, C., Niu, Y., Wang, D., & Maule, A. J. (2005). Virus Induction of Heat Shock Protein 70 Reflects a General Response to Protein Accumulation in the Plant Cytosol. Plant Physiology, 138(1), 529-536. doi:10.1104/pp.104.058958Aparicio, F., Sánchez-Navarro, J. A., & Pallás, V. (2006). In vitro and in vivo mapping of the Prunus necrotic ringspot virus coat protein C-terminal dimerization domain by bimolecular fluorescence complementation. Journal of General Virology, 87(6), 1745-1750. doi:10.1099/vir.0.81696-0Balasubramaniam, M., Kim, B.-S., Hutchens-Williams, H. M., & Loesch-Fries, L. S. (2014). The Photosystem II Oxygen-Evolving Complex Protein PsbP Interacts With the Coat Protein of Alfalfa mosaic virus and Inhibits Virus Replication. Molecular Plant-Microbe Interactions®, 27(10), 1107-1118. doi:10.1094/mpmi-02-14-0035-rBhat, S., Folimonova, S. Y., Cole, A. B., Ballard, K. D., Lei, Z., Watson, B. S., … Nelson, R. S. (2012). Influence of Host Chloroplast Proteins on Tobacco mosaic virus Accumulation and Intercellular Movement. Plant Physiology, 161(1), 134-147. doi:10.1104/pp.112.207860Bol, J. F. (2005). Replication of Alfamo- and Ilarviruses: Role of the Coat Protein. Annual Review of Phytopathology, 43(1), 39-62. doi:10.1146/annurev.phyto.43.101804.120505Callaway, A., Giesman-Cookmeyer, D., Gillock, E. T., Sit, T. L., & Lommel, S. A. (2001). THEMULTIFUNCTIONALCAPSIDPROTEINS OFPLANTRNA VIRUSES. Annual Review of Phytopathology, 39(1), 419-460. doi:10.1146/annurev.phyto.39.1.419Collum, T. D., & Culver, J. N. (2016). The impact of phytohormones on virus infection and disease. Current Opinion in Virology, 17, 25-31. doi:10.1016/j.coviro.2015.11.003Culver, J. N., & Padmanabhan, M. S. (2007). Virus-Induced Disease: Altering Host Physiology One Interaction at a Time. Annual Review of Phytopathology, 45(1), 221-243. doi:10.1146/annurev.phyto.45.062806.094422Donze, T., Qu, F., Twigg, P., & Morris, T. J. (2014). Turnip crinkle virus coat protein inhibits the basal immune response to virus invasion in Arabidopsis by binding to the NAC transcription factor TIP. Virology, 449, 207-214. doi:10.1016/j.virol.2013.11.018Fryer, M. J., Ball, L., Oxborough, K., Karpinski, S., Mullineaux, P. M., & Baker, N. R. (2003). Control of Ascorbate Peroxidase 2 expression by hydrogen peroxide and leaf water status during excess light stress reveals a functional organisation of Arabidopsis leaves. The Plant Journal, 33(4), 691-705. doi:10.1046/j.1365-313x.2003.01656.xGarcía, J. A., & Pallás, V. (2015). Viral factors involved in plant pathogenesis. Current Opinion in Virology, 11, 21-30. doi:10.1016/j.coviro.2015.01.001Heim, M. A. (2003). The Basic Helix-Loop-Helix Transcription Factor Family in Plants: A Genome-Wide Study of Protein Structure and Functional Diversity. Molecular Biology and Evolution, 20(5), 735-747. doi:10.1093/molbev/msg088Herranz, M. C., Pallas, V., & Aparicio, F. (2012). Multifunctional Roles for the N-Terminal Basic Motif of Alfalfa mosaic virus Coat Protein: Nucleolar/Cytoplasmic Shuttling, Modulation of RNA-Binding Activity, and Virion Formation. Molecular Plant-Microbe Interactions®, 25(8), 1093-1103. doi:10.1094/mpmi-04-12-0079-rHuang, Z., Yeakley, J. M., Garcia, E. W., Holdridge, J. D., Fan, J.-B., & Whitham, S. A. (2005). Salicylic Acid-Dependent Expression of Host Genes in Compatible Arabidopsis-Virus Interactions. Plant Physiology, 137(3), 1147-1159. doi:10.1104/pp.104.056028Inaba, J., Kim, B. M., Shimura, H., & Masuta, C. (2011). Virus-Induced Necrosis Is a Consequence of Direct Protein-Protein Interaction between a Viral RNA-Silencing Suppressor and a Host Catalase. Plant Physiology, 156(4), 2026-2036. doi:10.1104/pp.111.180042Jiménez, I., López, L., Alamillo, J. M., Valli, A., & García, J. A. (2006). Identification of a Plum pox virus CI-Interacting Protein from Chloroplast That Has a Negative Effect in Virus Infection. Molecular Plant-Microbe Interactions®, 19(3), 350-358. doi:10.1094/mpmi-19-0350Kim, K.-C., Lai, Z., Fan, B., & Chen, Z. (2008). Arabidopsis WRKY38 and WRKY62 Transcription Factors Interact with Histone Deacetylase 19 in Basal Defense. The Plant Cell, 20(9), 2357-2371. doi:10.1105/tpc.107.055566Kim, S. A., Punshon, T., Lanzirotti, A., Li, L., Alonso, J. M., Ecker, J. R., … Guerinot, M. L. (2006). Localization of Iron in Arabidopsis Seed Requires the Vacuolar Membrane Transporter VIT1. Science, 314(5803), 1295-1298. doi:10.1126/science.1132563Liu, Z., Zhang, Z., Faris, J. D., Oliver, R. P., Syme, R., McDonald, M. C., … Friesen, T. L. (2012). The Cysteine Rich Necrotrophic Effector SnTox1 Produced by Stagonospora nodorum Triggers Susceptibility of Wheat Lines Harboring Snn1. PLoS Pathogens, 8(1), e1002467. doi:10.1371/journal.ppat.1002467Long, T. A., Tsukagoshi, H., Busch, W., Lahner, B., Salt, D. E., & Benfey, P. N. (2010). The bHLH Transcription Factor POPEYE Regulates Response to Iron Deficiency in Arabidopsis Roots. The Plant Cell, 22(7), 2219-2236. doi:10.1105/tpc.110.074096Lukhovitskaya, N. I., Solovieva, A. D., Boddeti, S. K., Thaduri, S., Solovyev, A. G., & Savenkov, E. I. (2013). An RNA Virus-Encoded Zinc-Finger Protein Acts as a Plant Transcription Factor and Induces a Regulator of Cell Size and Proliferation in Two Tobacco Species. The Plant Cell, 25(3), 960-973. doi:10.1105/tpc.112.106476Mandadi, K. K., & Scholthof, K.-B. G. (2013). Plant Immune Responses Against Viruses: How Does a Virus Cause Disease? The Plant Cell, 25(5), 1489-1505. doi:10.1105/tpc.113.111658Maule, A. J., Escaler, M., & Aranda, M. A. (2000). Programmed responses to virus replication in plants. Molecular Plant Pathology, 1(1), 9-15. doi:10.1046/j.1364-3703.2000.00002.xNechushtai, R., Conlan, A. R., Harir, Y., Song, L., Yogev, O., Eisenberg-Domovich, Y., … Mittler, R. (2012). Characterization of Arabidopsis NEET Reveals an Ancient Role for NEET Proteins in Iron Metabolism. The Plant Cell, 24(5), 2139-2154. doi:10.1105/tpc.112.097634Nelson, B. K., Cai, X., & Nebenführ, A. (2007). A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants. The Plant Journal, 51(6), 1126-1136. doi:10.1111/j.1365-313x.2007.03212.xNemeth, K., Salchert, K., Putnoky, P., Bhalerao, R., Koncz-Kalman, Z., Stankovic-Stangeland, B., … Koncz, C. (1998). Pleiotropic control of glucose and hormone responses by PRL1, a nuclear WD protein, in Arabidopsis. Genes & Development, 12(19), 3059-3073. doi:10.1101/gad.12.19.3059Ni, P., & Cheng Kao, C. (2013). Non-encapsidation activities of the capsid proteins of positive-strand RNA viruses. Virology, 446(1-2), 123-132. doi:10.1016/j.virol.2013.07.023Olsen, A. N., Ernst, H. A., Leggio, L. L., & Skriver, K. (2005). NAC transcription factors: structurally distinct, functionally diverse. Trends in Plant Science, 10(2), 79-87. doi:10.1016/j.tplants.2004.12.010Paddock, M. L., Wiley, S. E., Axelrod, H. L., Cohen, A. E., Roy, M., Abresch, E. C., … Jennings, P. A. (2007). MitoNEET is a uniquely folded 2Fe 2S outer mitochondrial membrane protein stabilized by pioglitazone. Proceedings of the National Academy of Sciences, 104(36), 14342-14347. doi:10.1073/pnas.0707189104Pallas, V., & García, J. A. (2011). How do plant viruses induce disease? Interactions and interference with host components. Journal of General Virology, 92(12), 2691-2705. doi:10.1099/vir.0.034603-0Pallas, V., Aparicio, F., Herranz, M. C., Sanchez-Navarro, J. A., & Scott, S. W. (2013). The Molecular Biology of Ilarviruses. Advances in Virus Research, 139-181. doi:10.1016/b978-0-12-407698-3.00005-3Palukaitis, P., Groen, S. C., & Carr, J. P. (2013). The Rumsfeld paradox: some of the things we know that we don’t know about plant virus infection. Current Opinion in Plant Biology, 16(4), 513-519. doi:10.1016/j.pbi.2013.06.004Peng, X., Hu, Y., Tang, X., Zhou, P., Deng, X., Wang, H., & Guo, Z. (2012). Constitutive expression of rice WRKY30 gene increases the endogenous jasmonic acid accumulation, PR gene expression and resistance to fungal pathogens in rice. Planta, 236(5), 1485-1498. doi:10.1007/s00425-012-1698-7Pieterse, C. M. J., Van der Does, D., Zamioudis, C., Leon-Reyes, A., & Van Wees, S. C. M. (2012). Hormonal Modulation of Plant Immunity. Annual Review of Cell and Developmental Biology, 28(1), 489-521. doi:10.1146/annurev-cellbio-092910-154055Puranik, S., Sahu, P. P., Srivastava, P. S., & Prasad, M. (2012). NAC proteins: regulation and role in stress tolerance. Trends in Plant Science, 17(6), 369-381. doi:10.1016/j.tplants.2012.02.004Rampey, R. A., Woodward, A. W., Hobbs, B. N., Tierney, M. P., Lahner, B., Salt, D. E., & Bartel, B. (2006). An Arabidopsis Basic Helix-Loop-Helix Leucine Zipper Protein Modulates Metal Homeostasis and Auxin Conjugate Responsiveness. Genetics, 174(4), 1841-1857. doi:10.1534/genetics.106.061044Ren, T., Qu, F., & Morris, T. J. (2005). The nuclear localization of the Arabidopsis transcription factor TIP is blocked by its interaction with the coat protein of Turnip crinkle virus. Virology, 331(2), 316-324. doi:10.1016/j.virol.2004.10.039Rodrigo, G., Carrera, J., Ruiz-Ferrer, V., del Toro, F. J., Llave, C., Voinnet, O., & Elena, S. F. (2012). A Meta-Analysis Reveals the Commonalities and Differences in Arabidopsis thaliana Response to Different Viral Pathogens. PLoS ONE, 7(7), e40526. doi:10.1371/journal.pone.0040526Sanchez-Navarro, J., Miglino, R., Ragozzino, A., & Bol, J. F. (2001). Engineering of Alfalfa mosaic virus RNA 3 into an expression vector. Archives of Virology, 146(5), 923-939. doi:10.1007/s007050170125Sánchez-Navarro, J. A., Carmen Herranz, M., & Pallás, V. (2006). Cell-to-cell movement of Alfalfa mosaic virus can be mediated by the movement proteins of Ilar-, bromo-, cucumo-, tobamo- and comoviruses and does not require virion formation. Virology, 346(1), 66-73. doi:10.1016/j.virol.2005.10.024Selth, L. A., Dogra, S. C., Rasheed, M. S., Healy, H., Randles, J. W., & Rezaian, M. A. (2004). A NAC Domain Protein Interacts with Tomato leaf curl virus Replication Accessory Protein and Enhances Viral Replication. The Plant Cell, 17(1), 311-325. doi:10.1105/tpc.104.027235Seo, M., Jikumaru, Y., & Kamiya, Y. (2011). Profiling of Hormones and Related Metabolites in Seed Dormancy and Germination Studies. Methods in Molecular Biology, 99-111. doi:10.1007/978-1-61779-231-1_7Su, L.-W., Chang, S. H., Li, M.-Y., Huang, H.-Y., Jane, W.-N., & Yang, J.-Y. (2013). Purification and biochemical characterization of Arabidopsis At-NEET, an ancient iron-sulfur protein, reveals a conserved cleavage motif for subcellular localization. Plant Science, 213, 46-54. doi:10.1016/j.plantsci.2013.09.001Taschner, P. E. M., Van Der Kuyl, A. C., Neeleman, L., & Bol, J. F. (1991). Replication of an incomplete alfalfa mosaic virus genome in plants transformed with viral replicase genes. Virology, 181(2), 445-450. doi:10.1016/0042-6822(91)90876-dThompson, J. D., Higgins, D. G., & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22(22), 4673-4680. doi:10.1093/nar/22.22.4673Toledo-Ortiz, G., Huq, E., & Quail, P. H. (2003). The Arabidopsis Basic/Helix-Loop-Helix Transcription Factor Family. The Plant Cell, 15(8), 1749-1770. doi:10.1105/tpc.013839Torres, M. A. (2010). ROS in biotic interactions. Physiologia Plantarum, 138(4), 414-429. doi:10.1111/j.1399-3054.2009.01326.xUhrig, J. F., Canto, T., Marshall, D., & MacFarlane, S. A. (2004). 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Effect of RNA silencing suppression activity of chrysanthemum virus B p12 protein on small RNA species

Funder: Swedish University of Agricultural SciencesAbstract: Chrysanthemum virus B encodes a multifunctional p12 protein that acts as a transcriptional activator in the nucleus and as a suppressor of RNA silencing in the cytoplasm. Here, we investigated the impact of p12 on accumulation of major classes of small RNAs (sRNAs). The results show dramatic changes in the sRNA profiles characterised by an overall reduction in sRNA accumulation, changes in the pattern of size distribution of canonical siRNAs and in the ratio between sense and antisense strands, lower abundance of siRNAs with a U residue at the 5′-terminus, and changes in the expression of certain miRNAs, most of which were downregulated

Symptoms induced by transgenic expression of p23 from Citrus tristeza virus in phloem-associated cells of Mexican lime mimic virus infection without the aberrations accompanying constitutive expression

[EN] Citrus tristeza virus (CTV) is phloem restricted in natural citrus hosts. The 23-kDa protein (p23) encoded by the virus is an RNA silencing suppressor and a pathogenicity determinant. The expression of p23, or its N-terminal 157-amino-acid fragment comprising the zinc finger and flanking basic motifs, driven by the constitutive 35S promoter of cauliflower mosaic virus, induces CTV-like symptoms and other aberrations in transgenic citrus. To better define the role of p23 in CTV pathogenesis, we compared the phenotypes of Mexican lime transformed with p23-derived transgenes from the severe T36 and mild T317 CTV isolates under the control of the phloem-specific promoter from Commelina yellow mottle virus (CoYMV) or the 35S promoter. Expression of the constructs restricted to the phloem induced a phenotype resembling CTV-specific symptoms (vein clearing and necrosis, and stem pitting), but not the non-specific aberrations (such as mature leaf epinasty and yellow pinpoints, growth cessation and apical necrosis) observed when p23 was ectopically expressed. Furthermore, vein necrosis and stem pitting in Mexican lime appeared to be specifically associated with p23 from T36. Phloem-specific accumulation of the p23158-209(T36) fragment was sufficient to induce the same anomalies, indicating that the region comprising the N-terminal 157 amino acids of p23 is responsible (at least in part) for the vein clearing, stem pitting and, possibly, vein corking in this host.We thank Dr B. Ding for providing a construct with the phloem-specific promoter from CoYMV, and J. E. Peris, J. Juarez and M. T. Gorris for their excellent technical assistance. N.S. was supported by a PhD fellowship from the Instituto Valenciano de Investigaciones Agrarias (IVIA). This research was supported by grants AGL2009-08052, co-financed by Fondo Europeo de Desarrollo Regional-MICINN, and Prometeo/2008/121 from the Generalitat Valenciana.Soler, N.; Fagoaga García, CC.; López Del Rincón, C.; Moreno, P.; Navarro, L.; Flores Pedauye, R.; Peña Garcia, L. (2015). Symptoms induced by transgenic expression of p23 from Citrus tristeza virus in phloem-associated cells of Mexican lime mimic virus infection without the aberrations accompanying constitutive expression. Molecular Plant Pathology. 16(4):388-399. https://doi.org/10.1111/mpp.12188S38839916

Importin-α-mediated nucleolar localization of potato mop-top virus TRIPLE GENE BLOCK1 (TGB1) protein facilitates virus systemic movement, whereas TGB1 self-interaction is required for cell-to-cell movement in Nicotiana benthamiana

This work was supported by the Swedish Research Council Formas (to E.I.S.), the Carl Tryggers Foundation (to E.I.S. and N.I.L.), the Swedish Institute (to N.I.L.), and the Rural and Environmental Science and Analytical Services Division strategic research fund of the Scottish Government (to L.T., J.T., and G.H.C.).Recently, it has become evident that nucleolar passage of movement proteins occurs commonly in a number of plant RNA viruses that replicate in the cytoplasm. Systemic movement of Potato mop-top virus (PMTV) involves two viral transport forms represented by a complex of viral RNA and TRIPLE GENE BLOCK1 (TGB1) movement protein and by polar virions that contain the minor coat protein and TGB1 attached to one extremity. The integrity of polar virions ensures the efficient movement of RNA-CP, which encodes the virus coat protein. Here, we report the involvement of nuclear transport receptors belonging to the importin-α family in nucleolar accumulation of the PMTV TGB1 protein and, subsequently, in the systemic movement of the virus. Virus-induced gene silencing of two importin-α paralogs in Nicotiana benthamiana resulted in significant reduction of TGB1 accumulation in the nucleus, decreasing the accumulation of the virus progeny in upper leaves and the loss of systemic movement of RNA-CP. PMTV TGB1 interacted with importin-α in N. benthamiana, which was detected by bimolecular fluorescence complementation in the nucleoplasm and nucleolus. The interaction was mediated by two nucleolar localization signals identified by bioinformatics and mutagenesis in the TGB1 amino-terminal domain. Our results showed that while TGB1 self-interaction is needed for cell-to-cell movement, importin-α-mediated nucleolar targeting of TGB1 is an essential step in establishing the efficient systemic infection of the entire plant. These results enabled the identification of two separate domains in TGB1: an internal domain required for TGB1 self-interaction and cell-to-cell movement and the amino-terminal domain required for importin-α interaction in plants, nucleolar targeting, and long-distance movement.Recently, it has become evident that nucleolar passage of movement proteins occurs commonly in a number of plant RNA viruses that replicate in the cytoplasm. Systemic movement of Potato mop-top virus (PMTV) involves two viral transport forms represented by a complex of viral RNA and TRIPLE GENE BLOCK1 (TGB1) movement protein and by polar virions that contain the minor coat protein and TGB1 attached to one extremity. The integrity of polar virions ensures the efficient movement of RNA-CP, which encodes the virus coat protein. Here, we report the involvement of nuclear transport receptors belonging to the importin-α family in nucleolar accumulation of the PMTV TGB1 protein and, subsequently, in the systemic movement of the virus. Virus-induced gene silencing of two importin-α paralogs in Nicotiana benthamiana resulted in significant reduction of TGB1 accumulation in the nucleus, decreasing the accumulation of the virus progeny in upper leaves and the loss of systemic movement of RNA-CP. PMTV TGB1 interacted with importin-α in N. benthamiana, which was detected by bimolecular fluorescence complementation in the nucleoplasm and nucleolus. The interaction was mediated by two nucleolar localization signals identified by bioinformatics and mutagenesis in the TGB1 amino-terminal domain. Our results showed that while TGB1 self-interaction is needed for cell-to-cell movement, importin-α-mediated nucleolar targeting of TGB1 is an essential step in establishing the efficient systemic infection of the entire plant. These results enabled the identification of two separate domains in TGB1: an internal domain required for TGB1 self-interaction and cell-to-cell movement and the amino-terminal domain required for importin-α interaction in plants, nucleolar targeting, and long-distance movement.Publisher PDFPeer reviewe