Defective RNA particles derived from Tomato black ring virus genome interfere with the replication of parental virus

[EN] Tomato black ring virus (TBRV) is the only member of the Nepovirus genus that is known to form defective RNA particles (D RNAs) during replication. Here, de novo generation of D RNAs was observed during prolonged passages of TBRV isolates originated from Solanum lycopersicum and Lactuca sativa in Chenopodium quinoa plants. D RNAs of about 500 nt derived by a single deletion in the RNA1 molecule and contained a portion of the 5' untranslated region and viral replicase, and almost the entire 3' non coding region. Short regions of sequence complementarity were found at the 5' and 3' junction borders, which can facilitate formation of the D RNAs. Moreover, in this study we analyzed the effects of D RNAs on TBRV replication and symptoms development of infected plants. C. quinoa, S. lycopersicum, Nicotiana tabacum, and L. sativa were infected with the original TBRV isolates (TBRV-D RNA) and those containing additional D RNA particles (TBRV + D RNA). The viral accumulation in particular hosts was measured up to 28 days post inoculation by RT-qPCR. Statistical analyses revealed that D RNAs interfere with TBRV replication and thus should be referred to as defective interfering particles. The magnitude of the interference effect depends on the interplay between TBRV isolate and host species.This work was supported by the National Science Centre, Poland (grant number 2017/25/B/NZ9/01715); Ministry of Science and Higher Education, Poland (grant number IP2014 014973) (to B.H.-J.) and Spain Agencia Estatal de Investigacion-FEDER (grant number BFU2015-65037P) (to S.F.E.). The funding bodies were not involved into the design of the study, analysis, and interpretation of data in the manuscript.Hasiow, B.; Minicka, J.; Zarzynska, A.; Budzynska, D.; Elena Fito, SF. (2018). Defective RNA particles derived from Tomato black ring virus genome interfere with the replication of parental virus. Virus Research. 250:87-94. https://doi.org/10.1016/j.virusres.2018.04.010S879425

Strain-dependent mutational effects for Pepino mosaic virus in a natural host

[EN] Pepino mosaic virus (PepMV) is an emerging plant pathogen that infects tomatoes worldwide. Understanding the factors that influence its evolutionary success is essential for developing new control strategies that may be more robust against the evolution of new viral strains. One of these evolutionary factors is the distribution of mutational fitness effect (DMFE), that is, the fraction of mutations that are lethal, deleterious, neutral, and beneficial on a given viral strain and host species. The goal of this study was to characterize the DMFE of introduced nonsynonymous mutations on a mild isolate of PepMV from the Chilean 2 strain (PepMV-P22). Additionally, we also explored whether the fitness effect of a given mutation depends on the gene where it appears or on epistatic interactions with the genetic background. To address this latter possibility, a subset of mutations were also introduced in a mild isolate of the European strain (PepMV-P11) and the fitness of the resulting clones measured.This study was financially supported by grant 2011/01/D/NZ9/00279, from the Poland National Science Center, to B.H.J and by grants BFU2015-65037-P, from Spain Ministry of Economy and Competitiveness-FEDER, and PROMETEOII/2014/021, from Generalitat Valenciana, to S.F.E.Minicka, J.; Elena Fito, SF.; Borodynko-Filas, N.; Rubis, B.; Hasiów-Jaroszewska, B. (2017). Strain-dependent mutational effects for Pepino mosaic virus in a natural host. BMC Evolutionary Biology. 17:1-11. https://doi.org/10.1186/s12862-017-0920-4S11117Steinhauer DA, Domingo E, Holland JJ. Lack of evidence for proofreading mechanisms associated with an RNA virus polymerase. Gene. 1992;122:281–8.Sanjuán R, Nebot MR, Chirico N, Mansky LM, Belshaw R. Viral mutation rates. J Virol. 2010;84:9733–48.Domingo E. Viruses at the edge of adaptation. Virology. 2000;270:251–3.Chao L. Fitness of RNA virus decreased by muller ratchet. Nature. 1990;348:454–5.Duarte E, Clarke D, Moya A, Domingo E, Holland J. Rapid fitness losses in mammalian RNA virus clones due to Muller ratchet. Proc Natl Acad Sci U S A. 1992;89:6015–9.De la Iglesia F, Elena SF. Fitness declines in Tobacco etch virus upon serial bottleneck transfers. J Virol. 2007;81:4941–7.Elena SF, Carrasco P, Daròs JA, Sanjuán R. Mechanisms of genetic robustness in RNA viruses. EMBO Rep. 2006;7:168–73.Elena SF, Moya A. Rate of deleterious mutation and the distribution of its effects on fitness in Vesicular stomatitis virus. J Evol Biol. 1999;12:1078–88.Sanjuán R, Moya A, Elena SF. The distribution of fitness effects caused by single-nucleotide substitutions in an RNA virus. Proc Natl Acad Sci U S A. 2004;101:8396–401.Acevedo A, Brodsky L, Andino R. Mutational and fitness landscapes of an RNA virus revealed through population sequencing. Nature. 2014;505:686–90.Visher E, Whitefield SE, McCrone JT, Fitzsimmons W, Lauring AS. The mutational robustness of Influenza A virus. PLoS Pathog. 2016;12, e1005856.Carrasco P, de la Iglesia F, Elena SF. Distribution of fitness and virulence effects caused by single-nucleotide substitutions in Tobacco etch virus. J Virol. 2007;81:12979–84.Bernet GP, Elena SF. Distribution of mutational fitness effects and of epistasis in the 5′ untranslated region of a plant RNA virus. BMC Evol Biol. 2015;15:274.Domingo-Calap P, Cuevas JM, Sanjuán R. The fitness effects of random mutations in single-stranded DNA and RNA bacteriophages. PLoS Genet. 2009;5, e1000742.Peris JB, Davis P, Cuevas JM, Nebot MR, Sanjuán R. Distribution of fitness effects caused by single-nucleotide substitutions in bacteriophage f1. Genetics. 2010;185:603–9.Keightley PD, Ohnishi O. EMS-induced polygenic mutation rates for nine quantitative characters in Drosophila melanogaster. Genetics. 1998;148:753–66.Keightley PD, Davies EK, Peters AD, Shaw RG. Properties of ethylmethane sulfonate-induced mutations affecting life-history traits in Caenorhabditis elegans and inferences about bivariate distributions of mutation effects. Genetics. 2000;156:143–54.Koufopanou V, Lomas S, Tsai IJ, Burt A. Estimating the fitness effects of new mutations in the wild yeast Saccharomyces paradoxus. Genome Biol Evol. 2015;7:1887–95.Sanjuán R. Mutational fitness effects in RNA and single-stranded DNA viruses: common patterns revealed by site-directed mutagenesis. Philos Trans R Soc B. 2010;365:1975–82.Keightley PD, Lynch M. Toward a realistic model of mutations affecting fitness. Evolution. 2003;57:683–5.Orr HA. The distribution of fitness effects among beneficial mutations. Genetics. 2003;163:1519–26.Miralles R, Gerrish PJ, Moya A, Elena SF. Clonal interference and the evolution of RNA viruses. Science. 1999;285:1745–7.Escarmís C, Dávila M, Charpentier N, Bracho A, Moya A, Domingo E. Genetic lesions associated with Muller’s ratchet in an RNA virus. J Mol Biol. 1996;264:255–67.Phillips PC. Epistasis - the essential role of gene interactions in the structure and evolution of genetic systems. Nat Rev Genet. 2008;9:855–67.De Visser JAGM, Krug J. Empirical fitness landscapes and the predictability of evolution. Nat Rev Genet. 2014;15:480–90.Lalić J, Elena SF. Magnitude and sign epistasis among deleterious mutations in a positive-sense plant RNA virus. Heredity. 2012;109:71–7.Lalić J, Elena SF. The impact of higher-order epistasis in the within-host fitness of a positive-sense plant RNA virus. J Evol Biol. 2015;28:2236–47.Hillung J, Cuevas JM, Elena SF. Evaluating the within-host fitness effects of mutations fixed during virus adaptation to different ecotypes of a new host. Philos Trans R Soc B. 2015;370:20140292.Cervera H, Lalić J, Elena SF. Effect of host species on the topography of the fitness landscape for a plant RNA virus. J Virol. 2016;90:10160–9.Cervera H, Lalić J, Elena SF. Efficient escape from local optima in a highly rugged fitness landscape by evolving RNA virus populations. Proc R Soc B. 2016;283:20160984.Blystad DR, van der Vlugt R, Alfaro-Fernandez A, Cordoba MD, Bese G, Hristova D, Pospieszny H, Mehle N, Ravnikar M, Tomassoli L, Varveri C, Nielsen SL. Host range and symptomatology of Pepino mosaic virus strains occurring in Europe. Eur J Plant Pathol. 2015;143:43–56.Mumford RA, Metcalfe EJ. The partial sequencing of the genomic RNA of a UK isolate of Pepino mosaic virus and the comparison of the coat protein sequence with other isolates from Europe and Peru. Arch Virol. 2001;146:2455–60.Roggero P, Masenga V, Lenzi R, Coghe F, Ena S, Winter S. First report of Pepino mosaic virus in tomato in Italy. Plant Dis. 2001;3:8.Cotillon AC, Girard M, Ducouret S. Complete nucleotide sequence of the genomic RNA of a French isolate of Pepino mosaic virus (PepMV). Arch Virol. 2002;147:2231–8.Maroon-Lango CJ, Guaragna MA, Jordan RL, Hammond J, Bandla M, Marquardt SK. Two unique US isolates of Pepino mosaic virus from a limited source of pooled tomato tissue are distinct from a third (European-like) US isolate. Arch Virol. 2005;150:1187–201.Pagán I, Córdoba-Selles MD, Martínez-Priego L, Fraile A, Malpica JM, Jorda C, García-Arenal F. Genetic structure of the population of Pepino mosaic virus infecting tomato crops in Spain. Phytopathology. 2006;96:274–9.Ling KS. Molecular characterization of two Pepino mosaic virus variants from imported tomato seed reveals high levels of sequence identity between Chilean and US isolates. Virus Genes. 2007;34:1–8.Hanssen IM, Paeleman A, Wittemans L, Goen K, Lievens B, Bragard C, Vanachter A, Thomma B. Genetic characterization of Pepino mosaic virus isolates from Belgian greenhouse tomatoes reveals genetic recombination. Eur J Plant Pathol. 2008;121:131–46.Hasiów B, Borodynko N, Pospieszny H. Complete genomic RNA sequence of the Polish Pepino mosaic virus isolate belonging to the US2 strain. Virus Genes. 2008;36:209–14.Hanssen IM, Paeleman A, Vandewoestijne E, Van Bergen L, Bragard C, Lievens B, Vanacher ACRC, Thomma BPHJ. Pepino mosaic virus isolates and differential symptomatology in tomato. Plant Pathol. 2009;58:450–60.Moreno-Pérez MG, Pagán I, Aragón-Caballero L, Cáceres F, Fraile A, García-Arenal F. Ecological and genetic determinants of Pepino mosaic virus emergence. J Virol. 2014;88:3359–68.Ling K, Li R, Bledsoe M. Pepino mosaic virus genotype shift in North America and development of a loop-mediated isothermal amplification for rapid genotype identification. Virol J. 2013;10:117.Hasiów-Jaroszewska B, Paeleman A, Ortega-Parra N, Borodynko N, Minicka J, Czerwoniec A, Thomma BP, Hanssen IM. Ratio of mutated versus wild-type coat protein sequences in Pepino mosaic virus determines the nature and severity of yellowing symptoms on tomato plants. Mol Plant Pathol. 2013;14:923–33.Sempere RN, Gómez-Aix C, Ruiz-Ramon F, Gómez P, Hasiów-Jaroszewska B, Sánchez-Pina MA, Aranda MA. Pepino mosaic virus RNA-dependent RNA polymerase POL domain is a hypersensitive response-like elicitor shared by necrotic and mild isolates. Phytopathology. 2016;106:395–406.Hasiów-Jaroszewska B, Borodynko N, Jackowiak P, Figlerowicz M, Pospieszny H. Single mutation converts mild pathotype of the Pepino mosaic virus into necrotic one. Virus Res. 2011;159:57–61.Minicka J, Rymelska N, Elena SF, Czerwoniec A, Hasiów-Jaroszewska B. Molecular evolution of Pepino mosaic virus during long-term passaging in different hosts and its impact on virus virulence. Ann Appl Biol. 2015;166:389–401.Hasiów-Jaroszewska B, Jackowiak P, Borodynko N, Figlerowicz M, Pospieszny H. Quasispecies nature of Pepino mosaic virus and its evolutionary dynamics. Virus Genes. 2010;41:260–7.Eigen M, McCaskill J, Schuster P. Molecular quasi-species. J Phys Chem. 1988;92(24):6881–91.Schneider WL, Roossinck MJ. Evolutionarily related Sindbis-like plant viruses maintain different levels of population diversity in a common host. J Virol. 2000;74:3130–4.Legg JP, Thresh JM. Cassava mosaic virus disease in East Africa: a dynamic disease in a changing environment. Virus Res. 2000;71:135–49.Hasiów-Jaroszewska B, Borodynko N, Pospieszny H. Infectious RNA transcripts derived from cloned cDNA of a Pepino mosaic virus isolate. Arch Virol. 2009;154:853–6.Hasiów-Jaroszewska B, Komorowska B. A new method for detection and discrimination of Pepino mosaic virus isolates using high resolution melting analysis of the triple gene block 3. J Virol Methods. 2013;193:1–5.Poelwijk FJ, Tanase-Nicola S, Kiviet DJ, Tans SJ. Reciprocal sign epistasis is a necessary condition for multi-peaked fitness landscapes. J Theor Biol. 2011;272:141–4.Dean AM, Thornton JW. Mechanistic approaches to the study of evolution: the functional synthesis. Nat Rev Genet. 2007;8:675–88.Lalić J, Cuevas JM, Elena SF. Effect of host species on the distribution of mutational fitness effects for an RNA virus. PLoS Genet. 2011;7, e1002378.Vale PF, Choisy M, Froissart R, Sanjuán R, Gandon S. The distribution of mutational fitness effects of phage ϕX174 on different hosts. Evolution. 2012;66:3495–507.Hasiów-Jaroszewska B, Minicka J, Pospieszny H. Cross-protection between different pathotypes of Pepino mosaic virus representing chilean 2 genotype. Acta Sci Pol Hortoru. 2014;13:177–85.Minicka J, Otulak K, Garbaczewska G, Pospieszny H, Hasiów-Jaroszewska B. Ultrastructural insights into tomato infections caused by three different pathotypes of Pepino mosaic virus and immunolocalization of viral coat proteins. Micron. 2015;79:84–92.Gómez P, Sempere RN, Aranda MA, Elena SF. Phylodynamics of Pepino mosaic virus in Spain. Eur J Plant Pathol. 2012;134:445–9.Vignuzzi M, Stone JK, Arnold JJ, Cameron CE, Andino R. Quasispecies diversity determines pathogenesis through cooperative interactions in a viral population. Nature. 2006;439:344–8.Domingo E, Martíb V, Perales C, Grande-Pérez A, García-Arriaza Arias J. Viruses as quasispecies: biological implications. Curr Top Microbiol Immunol. 2006;299:51–82.Jones RAC, Koenig R, Lesemann DE. Pepino mosaic virus, a new potexvirus from pepino (Solanum muricatum). Ann Appl Biol. 1980;94:61–8.Domingo E, Sheldon J, Perales C. Viral quasispecies evolution. Microbiol Mol Biol Rev. 2012;76:159–216.Lough TJ, Emerson SJ, Lucas WJ, Forster RLS. Trans-complementation of long-distance movement of White clover mosaic virus triple gene block (TGB) mutants: Phloem-associated movement of TGBp1. Virology. 2001;288:18–28.Pospieszny H, Hasiów B, Borodynko N. Characterization of two distinct Polish isolates of Pepino mosaic virus. Eur J Plant Pathol. 2008;122:443–5.Gómez P, Sempere RN, Elena SF, Aranda MA. Mixed infections of Pepino mosaic virus strains modulate the evolutionary dynamics of this emergent virus. J Virol. 2009;83:12378–87.Elena SF, Solé RV, Sardanyés J. Simple genomes, complex interactions: epistasis in RNA virus. Chaos. 2010;20:026106.Sanjuán R, Moya A, Elena SF. The contribution of epistasis to the architecture of fitness in an RNA virus. Proc Natl Acad Sci USA. 2004;101:15376–9.Elena SF. RNA virus genetic robustness: possible causes and some consequences. Curr Opin Virol. 2012;2:525–30.Stern A, Bianco S, Yeh MT, Wright CF, Butcher K, Tang C, Nielsen R, Andino R. Costs and benefits of mutational robustness in RNA viruses. Cell Rep. 2014;8:1–11.Elena SF, Lalić J. Plant RNA virus fitness predictability: contribution of genetic and environmental factors. Plant Pathol. 2013;62:10–8

The transcriptomics of an experimentally evolved plant-virus interaction

[EN] Models of plant-virus interaction assume that the ability of a virus to infect a host genotype depends on the matching between virulence and resistance genes. Recently, we evolved tobacco etch potyvirus (TEV) lineages on different ecotypes of Arabidopsis thaliana, and found that some ecotypes selected for specialist viruses whereas others selected for generalists. Here we sought to evaluate the transcriptomic basis of such relationships. We have characterized the transcriptomic responses of five ecotypes infected with the ancestral and evolved viruses. Genes and functional categories differentially expressed by plants infected with local TEV isolates were identified, showing heterogeneous responses among ecotypes, although significant parallelism existed among lineages evolved in the same ecotype. Although genes involved in immune responses were altered upon infection, other functional groups were also pervasively over-represented, suggesting that plant resistance genes were not the only drivers of viral adaptation. Finally, the transcriptomic consequences of infection with the generalist and specialist lineages were compared. Whilst the generalist induced very similar perturbations in the transcriptomes of the different ecotypes, the perturbations induced by the specialist were divergent. Plant defense mechanisms were activated when the infecting virus was specialist but they were down-regulated when infecting with generalist.We thank Francisca de la Iglesia and Paula Agudo for excellent technical assistance and our labmates for useful discussions and suggestions. This work was supported by grants BFU2012-30805 from the Spanish Ministry of Economy and Competitiveness (MINECO), PROMETEOII/2014/021 from Generalitat Valenciana and EvoEvo (ICT610427) from the European Commission 7th Framework Program to SFE, and grant PROMETEOII/2014/025 to JD. JMC was supported by a JAE-doc postdoctoral contract from CSIC. JH was recipient of a predoctoral contract from MINECO.Hillung, J.; García-García, F.; Dopazo, J.; Cuevas Torrijos, JM.; Elena Fito, SF. (2016). The transcriptomics of an experimentally evolved plant-virus interaction. Scientific Reports. 6:1-19. https://doi.org/10.1038/srep24901S1196Duffy, S., Shackelton, L. A. & Holmes, E. C. Rates of evolutionary change in viruses: patterns and determinants. Nat. Rev. Genet. 9, 267–276 (2008).Parrish, C. R. et al. Cross-species virus transmission and the emergence of new epidemic diseases. Microbiol. Mol. Biol. Rev. 72, 457–470 (2008).Holmes, E. C. The comparative genomics of viral emergence. Proc. Natl. Acad. Sci. USA 107, 1742–1746 (2010).Sanjuán, R., Nebot, M. R., Chirico, N., Mansky, L. M. & Belshaw, R. Viral mutation rates. J. Virol. 84, 9733–9748 (2010).Elena, S. F. et al. The evolutionary genetics of emerging plant RNA viruses. Mol. Plant-Microbe Interact. 24, 287–293 (2011).Holmes, E. C. The evolutionary genetics of emerging viruses. Annu. Rev. Ecol. Evol. Syst. 40, 353–372 (2009).Domingo, E. Mechanisms of viral emergence. Vet. Res. 41, 38 (2010).King, K. C. & Lively, C. M. Does genetic diversity limit disease spread in natural host populations? Heredity 109, 199–203 (2012).Kearney, C. M., Thomson, M. J. & Roland, K. E. Genome evolution of Tobacco mosaic virus populations during long-term passaging in a diverse range of hosts. Arch. Virol. 144, 1513–1526 (1999).Tan, Z. et al. Mutations in Turnip mosaic virus genomes that have adapted to Raphanus sativus . J. Gen. Virol. 88, 501–510 (2005).Rico, P., Ivars, P., Elena, S. F. & Hernández, C. Insights into the selective pressures restricting Pelargonium flower break virus genome variability: evidence for host adaptation. J. Virol. 80, 8124–8132 (2006).Wallis, C. M. et al. Adaptation of Plum pox virus to a herbaceous host (Pisum sativum) following serial passages. J. Gen. Virol. 88, 2839–2845 (2007).Agudelo-Romero, P., de la Iglesia, F. & Elena, S. F. The pleiotropic cost of host-specialization in tobacco etch potyvirus. Infect. Genet. Evol. 8, 806–814 (2008).Bedhomme, S., Lafforgue, G. & Elena, S. F. Multihost experimental evolution of a plant RNA virus reveals local adaptation and host-specific mutations. Mol. Biol. Evol. 29, 1481–1492 (2012).García-Arenal, F. & Fraile A. Trade-offs in host range evolution of plant viruses. Plant Pathol. 62, S2–S9. (2013).Calvo, M., Malinowski, T. & García, J. A. Single amino acid changes in the 6K1-CI region can promote the alternative adaptation of Prunus- and Nicotiana- propagated Plum pox virus C isolates to either host. Mol. Plant-Microbe Interact. 27, 136–149 (2014).Cuevas, J. M., Willemsen, A., Hillung, J., Zwart, M. P. & Elena, S. F. Temporal dynamics of intra-host molecular evolution for a plant RNA virus. Mol. Biol. Evol. 32, 1132–1147 (2015).Minicka, J., Rymelska, N., Elena, S. F., Czerwoniec, A. & Hasiów-Jaroszewska, B. Molecular evolution of Pepino mosaic virus during long-term passaging in different hosts and its impact on virus virulence. Ann. Appl. Biol. 166, 389–401 (2015).Agudelo-Romero, P., Carbonell, P., Pérez-Amador, M. A. & Elena, S. F. Virus adaptation by manipulation of host's gene expression. PLos ONE 3, e2397 (2008).Weigel, D. Natural variation in arabidopsis: from molecular genetics to ecological genomics. Plant Physiol. 158, 2–22 (2012).Mahajan, S. K., Chisholm, S. T., Whitham, S. A. & Carrington, J. C. Identification and characterization of a locus (RTM1) that restricts long-distance movement of Tobacco etch virus in Arabidopsis thaliana . Plant J. 14, 177–186 (1998).Whitham, S. A., Yamamoto, M. L. & Carrington, J. C. Selectable viruses and altered susceptibility mutants in Arabidopsis thaliana . Proc. Natl. Acad. Sci. USA 96, 772–777 (1999).Whitham, S. A., Anderberg, R. J., Chisholm, S. T. & Carrington, J. C. Arabidopsis RTM2 gene is necessary for specific restriction of Tobacco etch virus and encodes an unusual small heat shock-like protein. Plant Cell 12, 569–582 (2000).Chisholm, S. T., Mahajan, S. K., Whitham, S. A., Yamamoto, M. L. & Carrington, J. C. Cloning of the Arabidopsis RTM1 gene, which controls restriction of long-distance movement of Tobacco etch virus . Proc. Natl. Acad. Sci. USA 97, 489–494 (2000).Chisholm, S. T., Parra, M. A., Anderberg, R. J. & Carrington, J. C. Arabidopsis RTM1 and RTM2 genes function in phloem to restrict long-distance movement of Tobacco etch virus . Plant Physiol. 127, 1667–1675 (2001).Cosson, P. et al. RTM3, which controls long-distance movement of potyviruses, is a member of a new plant gene family encoding a MEPRIN and TRAF homology domain-containing protein. Plant Physiol. 154, 222–232 (2010).Cosson, P., Sofer, L., Schurdi-Levraud, V. & Revers, F. A member of a new plant gene family encoding a MEPRIN and TRAF homology (MATH) domain-containing protein is involved in restriction of long distance movement of plant viruses. Plant Signal. Behav. 5, 1321–1323 (2010).Agudelo-Romero P. et al. Changes in gene expression profile of Arabidopsis thaliana after infection with Tobacco etch virus . Virol. J. 5, 92 (2008).Lalić, J., Agudelo-Romero, P., Carrasco, P. & Elena, S. F. Adaptation of tobacco etch potyvirus to a susceptible ecotype of Arabidopsis thaliana capacitates it for systemic infection of resistant ecotypes. Phil. Trans. R. Soc. B 65, 1997–2008 (2010).Hillung, J., Cuevas, J. M. & Elena, S. F. Transcript profiling of different Arabidopsis thaliana ecotypes in response to tobacco etch potyvirus infection. Front. Microbiol. 3, 229 (2012).Hillung, J., Cuevas, J. M. & Elena, S. F. Evaluating the within-host fitness effects of mutations fixed during virus adaptation to different ecotypes of a new host. Phil. Trans. R. Soc. B 370, 20140292 (2015).Hillung, J., Cuevas, J. M., Valverde, S. & Elena, S. F. Experimental evolution of an emerging plant virus in host genotypes that differ in their susceptibility to infection. Evolution 68, 2467–2480 (2014).Sartor, M. A., Leikauf, G. D. & Medvedovic, M. LRpath: a logistic regression approach for identifying enriched biological groups in gene expression data. Bioinformatics 25, 211–217 (2009).Montaner, D. & Dopazo, J. Multidimensional gene set analysis of genomic data. PLos ONE 5, e10348 (2010).Supek, F., Bosnjak, M., Skunca, N. & Smuc, T. REVIGO summarizes and visualizes long lists of gene ontology terms. PLos ONE 6, e21800 (2011).Grennan, A. K. Regulation of starch metabolism in Arabidopsis leaves. Plant Physiol. 142, 1343–1345 (2006).Johnson, P. R. & Ecker, J. R. The ethylene gas signal transduction pathway: a molecular perspective. Annu. Rev. Genet. 32, 227–254 (1998).Wang, K. L., Li, H. & Ecker, J. R. Ethylene biosynthesis and signaling networks. Plant Cell 14, S131–S151 (2002).Binns, D. et al. QuickGO: a web-based tool for gene ontology searching. Bioinformatics 25, 3045–3046 (2009).Stintzi, A., Weber, H., Reymond, P., Browse, J. & Farmer, E. E. Plant defense in the absence of jasmonic acid: the role of cyclopentenones. Proc. Natl. Acad. Sci. USA 98, 12837–12842 (2001).Luna, E. et al. Callose deposition: a multifaceted plant defense response. Mol. Plant-Microbe Interact. 24, 183–193 (2011).Ghoshroy, S., Freedman, K., Lartey, R. & Citovsky, V. Inhibition of plant viral systemic infection by non-toxic concentrations of cadmium. Plant J. 13, 591–602 (1998).Hayashi, N. et al. Nef of HIV-1 interacts directly with calcium-bound calmodulin. Protein Sci. 11, 529–537 (2002).Zacharias, D. A., Violin, J. D., Newton, A. C. & Tsien, R. Y. Partitioning of lipic-modified monomeric GFPs into membrane microdomains of live cells. Science 296, 913–916 (2002).Rojas, M. R. et al. Functional analysis of proteins involved in movement of the monopartite begomovirus, Tomato yellow leaf curl virus. Virology 291, 110–125 (2001).Padmanabhan, M. S., Goregaoker, S. P., Golem, S., Shiferaw, H. & Culver, J. N. Interaction of the Tobacco mosaic virus replicase protein with the Aux/IAA protein PAP1/IAA26 is associated with disease development. J. Virol. 79, 2549–2558 (2005).Lurin, C. et al. Genome-wide analysis of Arabidopsis pentatricopeptide repeat proteins reveals their essential role in organelle biogenesis. Plant Cell 16, 2089–2103 (2004).Takenaka, M., Verbitskiy, D., Zehrmann, A. & Brennicke, A. Reverse genetic screening identifies five E-class PPR proteins involved in RNA editing in mitochondria of Arabidopsis thaliana . J. Biol. Chem. 285, 27122–27129 (2010).Gillissen, B. et al. A new family of high-affinity transporters for adenine, cytosine, and purine derivatives in Arabidopsis . Plant Cell 12, 291–300 (2000).Li, S., Fu, Q., Chen, L., Huang, W. & Yu, D. Arabidopsis thaliana WRKY25, WRKY26, and WRKY33 coordinate induction of plant thermotolerance. Planta 233, 1237–1252 (2011).Divol, F. et al. Involvement of the xyloglucan endotransglycosylase/hydrolases encoded by celery XTH1 and Arabidopsis XTH33 in the phloem response to aphids. Plant Cell. Environ. 30, 187–201 (2007).Vissenberg, K., Fry, S. C., Pauly, M., Höfte, H. & Verbelen, J. P. XTH acts at the microfibril-matrix interface during cell elongation. J. Exp. Bot. 56, 673–683 (2005).Ham, B. K., Li, G., Kang, B. H., Zeng, F. & Lucas, W. J. Overexpression of Arabidopsis plasmodesmata germin-like proteins disrupts root growth and development. Plant Cell 24, 3630–3648 (2012).Bae, M. S., Cho, E. J., Choi, E. Y. & Park, O. K. Analysis of the Arabidopsis nuclear proteome and its response to cold stress. Plant J. 36, 652–663 (2003).Zargar, S. M. et al. Correlation analysis of proteins responsive to Zn, Mn, or Fe deficiency in Arabidopsis roots based on iTRAQ analysis. Plant Cell Rep. 34, 157–166 (2015).Kleffmann, T. et al. The Arabidopsis thaliana chloroplast proteome reveals pathway abundance and novel protein functions. Curr. Biol. 14, 354–362 (2004).Zybailov, B. et al. Sorting signals, N-terminal modifications and abundance of the chloroplast proteome. PLos ONE 3, e1994 (2008).Wu, P. et al. Phosphate starvation triggers distinct alterations of gene expression in Arabidopsis roots and leaves. Plant Physiol. 132, 1260–1271 (2003).Oh, S. A., Lee, S. Y., Chung, I. K., Lee, C. H. & Nam H. G. A senescence-associated gene of Arabidopsis thaliana is distinctively regulated during natural and artificially induced leaf senescence. Plant Mol. Biol. 30, 739–754 (1996).Schenk, P. M., Kazan, K., Rusu, A. G., Manners, J. M. & Maclean, D. J. The SEN1 gene of Arabidopsis is regulated by signals that link plant defence responses and senescence. Plant Physiol. Biochem. 43, 997–1005 (2005).Fernández-Calvino, L. et al. Activation of senescence-associated dark-inducible (DIN) genes during infection contributes to enhanced susceptibility to plant viruses. Mol. Plant Pathol. 17, 3–15 (2016).Vierstra, R. D. Proteolysis in plants: mechanisms and functions. Plant Mol. Biol. 32, 275–302 (1996).Bögre, L., Okrész, L., Henriques, R. & Anthony, R. G. Growth signalling pathways in Arabidopsis and the AGC protein kinases. Trends Plant Sci. 8, 424–431 (2003).An, L. et al. A zinc finger protein gene ZFP5 integrates phytohormone signalling to control root hair development in Arabidopsis . Plant J. 72, 474–490 (2012).Zhou, Z., An, L., Sun, L. & Gan, Y. ZFP5 encodes a functionally equivalent GIS protein to control trichome initiation. Plant Signal. Behav. 7, 28–30 (2012).Zhou, Z. et al. Zinc finger protein 5 is required for the control of trichome initiation by acting upstream of zinc finger protein 8 in Arabidopsis . Plant Physiol. 157, 673–682 (2011).Lee, D. J. et al. Genome-wide expression profiling of ARABIDOPSIS RESPONSE REGULATOR 7 (ARR7) overexpression in cytokinin response. Mol. Genet. Genomics 277, 115–137 (2007).Theologis, A. et al. Sequence and analysis of chromosome 1 of the plant Arabidopsis thaliana . Nature 408, 816–820 (2000).Heyndrickx, K. S. & Vandepoele, K. Systematic identification of functional plant modules through the integration of complementary data sources. Plant Physiol. 159, 884–901 (2012).Martinoia, E. et al. Multifunctionality of plant ABC transporter - more than just detoxifiers. Planta 214, 345–355 (2002).Kaneda, M. et al. ABC transporters coordinately expressed during lignification of Arabidopsis stems include a set of ABCBs associated with auxin transport. J. Exp. Bot. 62, 2063–2077 (2011).Alejandro, S. et al. AtABCG29 is a monolignol transporter involved in lignin biosynthesis. Curr. Biol. 22, 1207–1212 (2012).Riechmann, J. L. et al. Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes. Science 290, 2105–2110 (2000).Ohashi-Ito, K. & Bergmann, D. C. Regulation of the Arabidopsis root vascular initial population by LONESOME HIGHWAY . Development 134, 2959–2968 (2007).Averyanov, A. Oxidative burst and plant disease resistance. Front. Biosci. 1, 142–152 (2009).Flury, P., Klauser, D., Schulze, B., Boller, T. & Bartels, S. The anticipation of danger: microbe-associated molecular pattern perception enhances AtPep-triggered oxidative burst. Plant Physiol. 161, 2023–2035 (2013).Tanaka, K., Nguyen, C. T., Liang, Y., Cao, Y. & Stacey, G. Role of LysM receptors in chitin-triggered plant innate immunity. Plant Signal. Behav. 8, e22598 (2013).Nakamura, K. & Matsuoka, K. Protein targeting to the vacuole in plant cells. Plant Physiol. 101, 1–5 (1993).Elena, S. F., Agudelo-Romero, P. & Lalić, J. The evolution of viruses in multi-host fitness landscapes. Open Virol. J. 3, 1–6 (2009).Bolstad, B. M., Irizarry, R. A., Astrand, M. & Speed, T. P. A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinformatics 19, 185–193 (2003).Smyth, G. K. Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat. Appl. Genet. Mol. Biol. 3, 3 (2004).Benjamini, Y. & Hochberg, Y. Controlling the false discovery rate: A practical and powerful approach to multiple testing. J. R. Stat. Soc. B 57, 289–300 (1995).Benjamini,Y. & Yekutieli, D. The control of the false discovery rate in multiple testing under dependency. Ann. Statist. 29, 1165–1188 (2001).Sneath, P. & Sokal, R. Numerical Taxonomy. ( W.H. Freeman, 1973).D'Haeseler, P. How does gene expression clustering work? Nat. Biotech. 23, 1499–1501 (2005).Suzuki, R. & Shimodaira, H. Pvclust: an R package for assessing the uncertainty in hierarchical clustering. Bioinformatics 22, 1540–1542 (2006)

Ochrona krzyżowa pomiędzy różnymi patotypami wirusa mozaiki Pepino reprezentującymi genotyp chilijski 2

Viral cross-protection in plants is a phenomenon, where a mild virus isolate can protect plants against damage caused by a severe challenge isolate of the same virus. It has been used on a large scale in cases where no resistant plants are available. We examined differences in cross-protection between pathotypes of Pepino mosaic virus representing Chilean 2 genotype. The potential of a mild PepMV-P22 isolate to protect tomato against more aggressive challenge isolates causing yellowing and necrotic symptoms was established. The challenge isolates were PepMV-P5-IY (yellowing), PepMV-P19 (necrotic) and PepMV-P22 K67E (artificial necrotic mutant of PepMV-P22 which differ from PepMV-P22 only by a point mutation). Efficient cross-protection was obtained using mild PepMV-P22 against PepMV-P5-IY. After a challenge inoculation with PepMV-P19 or PepMV-P22 K67E symptoms severity were significantly reduced in comparison to non-protected plants; however, necrotic symptoms appeared two months after coinfection. The real-time PCR analysis revealed that the level of accumulation of the necrotic isolate in tomato plants was even 5–7 times higher than that of PepMV-P22.Zjawisko ochrony krzyżowej (ang. cross-protection) zachodzi tylko pomiędzy izolatami pokrewnymi danego gatunku wirusa. Polega ono na celowym zakażeniu roślin bardzo łagodnym izolatem wirusa (izolat ochronny), aby chronić je przed innym, ostrym izolatem tego samego wirusa (izolat konkurencyjny). W prezentowanej pracy analizowano potencjał wykorzystania łagodnego izolatu (PepMV-P22) wirusa mozaiki pepino (Pepino mosaic virus, PepMV) do ochrony roślin pomidora przeciwko innym izolatom, powodującym zróżnicowane objawy na roślinach. Jako izolaty konkurencyjne wykorzystano: PepMV-P5-IY (żółtaczkowy), PepMV-P19 (nekrotyczny) oraz PepMV-P22 K67E (mutant nekrotyczny, różniący się jedynie pojedynczą mutacją od PepMV-P22). Zjawisko ochrony krzyżowej zachodziło efektywnie w przypadku wykorzystania PepMVP22 przeciwko PepMV-P5-IY. W przypadku PepMV-P19 oraz PepMV-P22 K67E ochrona krzyżowa została przełamana, jednakże symptomy były mniej intensywne i pojawiły się później niż u roślin, u których nie stosowano ochrony krzyżowej. Ponadto analiza realtime PCR wykazała, że akumulacja wirusa w przypadku nekrotycznych wariantów była około 5–7 razy większa w porównaniu z łagodnym izolatem wirusa

Rapid evolutionary dynamics of the Pepino mosaic virus - status and future perspectives

Pepino mosaic virus (PepMV) has emerged as an important pathogen of greenhouse tomato crops and is currently distributed worldwide. Population genetic studies have revealed a shift in the dominant PepMV genotype from European (EU) to Chilean 2 (CH2) in North America and several European countries. New genetic variants are constantly being created by mutation and recombination events. Single nucleotide substitutions in different parts of the genome were found to affect on development of symptoms resulting in new pathotypes and accumulation of viral RNA. The variability of the PepMV population has a great impact on designing specific diagnostic tools and developing efficient and durable strategies of disease control. In this paper we review the current knowledge about the PepMV population, the evolutionary dynamics of this highly infective virus, methods for its detection and plant protection strategies

Aphid‐borne viruses infecting cultivated watermelon and squash in Spain: Characterization of a variant of cucurbit aphid‐borne yellows virus (CABYV)

Aphid-borne viruses are responsible for major cucurbit diseases and hamper the sustainability of crop production. Systematic monitoring can reveal the occurrence and distribution of these viruses, in addition to unadvertised viruses, facilitating the control of diseases. For three consecutive (2018–2020) seasons, the presence of aphid-borne viruses was monitored from a total of 292 samples of watermelon and squash plants that showed yellowing symptoms in three major cucurbit-producing areas (Castilla La-Mancha, Alicante, and Murcia) in Spain. We observed that cucurbit aphid-borne yellows virus (CABYV) was the most common virus found (29%) in the plants from both crops. Likewise, except for squash samples from Castilla La-Mancha and Alicante, watermelon mosaic virus (WMV) was also found (23%) with a relatively high frequency. Furthermore, we observed the exacerbation of bright yellowing symptoms in watermelon plants that was often accompanied by considerable fruit abortion. CABYV was the only causative agent for this new yellowing disease, and two infectious cDNA clones (one from watermelon, CABYV-LP63, and another from melon, CABYV-MEC12.1) were constructed to further compare and characterize this CABYV disease. Based on the full-length genome, both isolates were grouped phylogenetically together within the Mediterranean clade. However, the Koch's postulates tests were only successfully completed for the LP63 isolate, which also showed several amino acid changes and two potential recombination events, as compared to MEC12.1. Remarkably, the LP63 isolate caused more severe symptoms and showed higher RNA accumulation than MEC12.1 in five cucurbit plant species. These results suggest that a novel CABYV variant that causes severe yellowing symptoms may be causing outbreaks in cucurbit cropsM.P.R. was supported by funding of the Ministry of sciences, innovation and universities (MICINN, Spain) within a PhD programme grant (PRE2018-083915). This work was supported by the Spanish research grant; AGL2017-89550-R from the Agencia Estatal de Investigación (AEI) and FEDER (EU) fundsPeer reviewe