Multifaceted Regulation of Translational Readthrough by RNA Replication Elements in a Tombusvirus

Translational readthrough of stop codons by ribosomes is a recoding event used by a variety of viruses, including plus-strand RNA tombusviruses. Translation of the viral RNA-dependent RNA polymerase (RdRp) in tombusviruses is mediated using this strategy and we have investigated this process using a variety of in vitro and in vivo approaches. Our results indicate that readthrough generating the RdRp requires a novel long-range RNA-RNA interaction, spanning a distance of ∼3.5 kb, which occurs between a large RNA stem-loop located 3'-proximal to the stop codon and an RNA replication structure termed RIV at the 3'-end of the viral genome. Interestingly, this long-distance RNA-RNA interaction is modulated by mutually-exclusive RNA structures in RIV that represent a type of RNA switch. Moreover, a different long-range RNA-RNA interaction that was previously shown to be necessary for viral RNA replicase assembly was also required for efficient readthrough production of the RdRp. Accordingly, multiple replication-associated RNA elements are involved in modulating the readthrough event in tombusviruses and we propose an integrated mechanistic model to describe how this regulatory network could be advantageous by (i) providing a quality control system for culling truncated viral genomes at an early stage in the replication process, (ii) mediating cis-preferential replication of viral genomes, and (iii) coordinating translational readthrough of the RdRp with viral genome replication. Based on comparative sequence analysis and experimental data, basic elements of this regulatory model extend to other members of Tombusviridae, as well as to viruses outside of this family

Global Analyses of Small Interfering RNAs Derived from Bamboo mosaic virus and Its Associated Satellite RNAs in Different Plants

Background: Satellite RNAs (satRNAs), virus parasites, are exclusively associated with plant virus infection and have attracted much interest over the last 3 decades. Upon virus infection, virus-specific small interfering RNAs (vsiRNAs) are produced by dicer-like (DCL) endoribonucleases for anti-viral defense. The composition of vsiRNAs has been studied extensively; however, studies of satRNA-derived siRNAs (satsiRNAs) or siRNA profiles after satRNA co-infection are limited. Here, we report on the small RNA profiles associated with infection with Bamboo mosaic virus (BaMV) and its two satellite RNAs (satBaMVs) in Nicotiana benthamiana and Arabidopsis thaliana. Methodology/Principal Findings: Leaves of N. benthamiana or A. thaliana inoculated with water, BaMV alone or coinoculated with interfering or noninterfering satBaMV were collected for RNA extraction, then large-scale Solexa sequencing. Up to about 20% of total siRNAs as BaMV-specific siRNAs were accumulated in highly susceptible N. benthamiana leaves inoculated with BaMV alone or co-inoculated with noninterfering satBaMV; however, only about 0.1% of vsiRNAs were produced in plants co-infected with interfering satBaMV. The abundant region of siRNA distribution along BaMV and satBaMV genomes differed by host but not by co-infection with satBaMV. Most of the BaMV and satBaMV siRNAs were 21 or 22 nt, of both (+) and (-) polarities; however, a higher proportion of 22-nt BaMV and satBaMV siRNAs were generated in N. benthamiana than in A. thaliana. Furthermore, the proportion of non-viral 24-nt siRNAs was greatly increased in N. benthamiana after virus infection. Conclusions/Significance: The overall composition of vsiRNAs and satsiRNAs in the infected plants reflect the combined action of virus, satRNA and different DCLs in host plants. Our findings suggest that the structure and/or sequence demands of various DCLs in different hosts may result in differential susceptibility to the same virus. DCL2 producing 24-nt siRNAs under biotic stresses may play a vital role in the antiviral mechanism in N. benthamiana

Relationship between Symptoms and Gene Expression Induced by the Infection of Three Strains of Rice dwarf virus

BACKGROUND: Rice dwarf virus (RDV) is the causal agent of rice dwarf disease, which often results in severe yield losses of rice in East Asian countries. The disease symptoms are stunted growth, chlorotic specks on leaves, and delayed and incomplete panicle exsertion. Three RDV strains, O, D84, and S, were reported. RDV-S causes the most severe symptoms, whereas RDV-O causes the mildest. Twenty amino acid substitutions were found in 10 of 12 virus proteins among three RDV strains. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed the gene expression of rice in response to infection with the three RDV strains using a 60-mer oligonucleotide microarray to examine the relationship between symptom severity and gene responses. The number of differentially expressed genes (DEGs) upon the infection of RDV-O, -D84, and -S was 1985, 3782, and 6726, respectively, showing a correlation between the number of DEGs and symptom severity. Many DEGs were related to defense, stress response, and development and morphogenesis processes. For defense and stress response processes, gene silencing-related genes were activated by RDV infection and the degree of activation was similar among plants infected with the three RDV strains. Genes for hormone-regulated defense systems were also activated by RDV infection, and the degree of activation seemed to be correlated with the concentration of RDV in plants. Some development and morphogenesis processes were suppressed by RDV infection, but the degree of suppression was not correlated well with the RDV concentration. CONCLUSIONS/SIGNIFICANCE: Gene responses to RDV infection were regulated differently depending on the gene groups regulated and the strains infecting. It seems that symptom severity is associated with the degree of gene response in defense-related and development- and morphogenesis-related processes. The titer levels of RDV in plants and the amino acid substitutions in RDV proteins could be involved in regulating such gene responses

Analysis of the melon (Cucumis melo) small RNAome by high-throughput pyrosequencing

Abstract Background Melon (Cucumis melo L.) is a commercially important fruit crop that is cultivated worldwide. The melon research community has recently benefited from the determination of a complete draft genome sequence and the development of associated genomic tools, which have allowed us to focus on small RNAs (sRNAs). These are short, non-coding RNAs 21-24 nucleotides in length with diverse physiological roles. In plants, they regulate gene expression and heterochromatin assembly, and control protection against virus infection. Much remains to be learned about the role of sRNAs in melon. Results We constructed 10 sRNA libraries from two stages of developing ovaries, fruits and photosynthetic cotyledons infected with viruses, and carried out high-throughput pyrosequencing. We catalogued and analysed the melon sRNAs, resulting in the identification of 26 known miRNA families (many conserved with other species), the prediction of 84 melon-specific miRNA candidates, the identification of trans-acting siRNAs, and the identification of chloroplast, mitochondrion and transposon-derived sRNAs. In silico analysis revealed more than 400 potential targets for the conserved and novel miRNAs. Conclusion We have discovered and analysed a large number of conserved and melon-specific sRNAs, including miRNAs and their potential target genes. This provides insight into the composition and function of the melon small RNAome, and paves the way towards an understanding of sRNA-mediated processes that regulate melon fruit development and melon-virus interactions.This work was supported by grants AGL2009-07552/AGR, BIO2006-13107 (Ministerio de Ciencia e Innovación, Spain) and MELONOMICS (Fundación Genoma España, Spain).Peer Reviewe

Distinct Effects of p19 RNA Silencing Suppressor on Small RNA Mediated Pathways in Plants

RNA silencing is one of the main defense mechanisms employed by plants to fight viruses. In change, viruses have evolved silencing suppressor proteins to neutralize antiviral silencing. Since the endogenous and antiviral functions of RNA silencing pathway rely on common components, it was suggested that viral suppressors interfere with endogenous silencing pathway contributing to viral symptom development. In this work, we aimed to understand the effects of the tombusviral p19 suppressor on endogenous and antiviral silencing during genuine virus infection. We showed that ectopically expressed p19 sequesters endogenous small RNAs (sRNAs) in the absence, but not in the presence of virus infection. Our presented data question the generalized model in which the sequestration of endogenous sRNAs by the viral suppressor contributes to the viral symptom development. We further showed that p19 preferentially binds the perfectly paired ds-viral small interfering RNAs (vsiRNAs) but does not select based on their sequence or the type of the 5’ nucleotide. Finally, co-immunoprecipitation of sRNAs with AGO1 or AGO2 from virus-infected plants revealed that p19 specifically impairs vsiRNA loading into AGO1 but not AGO2. Our findings, coupled with the fact that p19-expressing wild type Cymbidium ringspot virus (CymRSV) overcomes the Nicotiana benthamiana silencing based defense killing the host, suggest that AGO1 is the main effector of antiviral silencing in this host-virus combination

A remarkable synergistic effect at the transcriptomic level in peach fruits doubly infected by Prunus necrotic ringspot virus and Peach latent mosaic viroid

[EN] Background: Microarray profiling is a powerful technique to investigate expression changes of large amounts of genes in response to specific environmental conditions. The majority of the studies investigating gene expression changes in virus-infected plants are limited to interactions between a virus and a model host plant, which usually is Arabidopsis thaliana or Nicotiana benthamiana. In the present work, we performed microarray profiling to explore changes in the expression profile of field-grown Prunus persica (peach) originating from Chile upon single and double infection with Prunus necrotic ringspot virus (PNRSV) and Peach latent mosaic viroid (PLMVd), worldwide natural pathogens of peach trees. Results: Upon single PLMVd or PNRSV infection, the number of statistically significant gene expression changes was relatively low. By contrast, doubly-infected fruits presented a high number of differentially regulated genes. Among these, down-regulated genes were prevalent. Functional categorization of the gene expression changes upon double PLMVd and PNRSV infection revealed protein modification and degradation as the functional category with the highest percentage of repressed genes whereas induced genes encoded mainly proteins related to phosphate, C-compound and carbohydrate metabolism and also protein modification. Overrepresentation analysis upon double infection with PLMVd and PNRSV revealed specific functional categories over- and underrepresented among the repressed genes indicating active counter-defense mechanisms of the pathogens during infection. Conclusions: Our results identify a novel synergistic effect of PLMVd and PNRSV on the transcriptome of peach fruits. We demonstrate that mixed infections, which occur frequently in field conditions, result in a more complex transcriptional response than that observed in single infections. Thus, our data demonstrate for the first time that the simultaneous infection of a viroid and a plant virus synergistically affect the host transcriptome in infected peach fruits. These field studies can help to fully understand plant-pathogen interactions and to develop appropriate crop protection strategies.We thank Drs M.A. Perez-Amador y J. Gadea for helping in the result analysis. This work was supported by grant BIO2011-25018 from the Spanish granting agency Direccion General de Investigacion Cientifica for the transcriptomic analyses and from the grant 2009CL0020 from the bilateral project INIA-Chile/CSIC-Spain for the phytosanitary evaluation. MC Herranz was the recipient of a contract from the Juan de la Cierva program of the Ministerio de Educacion y Ciencia of Spain.Herranz Gordo, MDC.; Niehl, A.; Rosales, M.; Fiore, N.; Zamorano, A.; Granell Richart, A.; Pallás Benet, V. (2013). A remarkable synergistic effect at the transcriptomic level in peach fruits doubly infected by Prunus necrotic ringspot virus and Peach latent mosaic viroid. Virology Journal. 10:11-15. https://doi.org/10.1186/1743-422X-10-164S111510Pallas V, Garcia JA: How do plant viruses induce disease? Interactions and interference with host components. J Gen Virol 2011, 92: 2691-2705.Whitham SA, Yang C, Goodin MM: Global impact: elucidating plant responses to viral infection. Mol Plant Microbe Interact 2006, 19: 1207-1215.Havelda Z, Varallyay E, Valoczi A, Burgyan J: Plant virus infection-induced persistent host gene downregulation in systemically infected leaves. Plant J 2008, 55: 278-288.Aranda M, Maule A: Virus-induced host gene shutoff in animals and plants. Virology 1998, 243: 261-267.Whitham SA, Quan S, Chang HS, Cooper B, Estes B, Zhu T, Wang X, Hou YM: Diverse RNA viruses elicit the expression of common sets of genes in susceptible Arabidopsis thaliana plants. Plant J 2003, 33: 271-283.Liu Y, Ren D, Pike S, Pallardy S, Gassmann W, Zhang S: Chloroplast-generated reactive oxygen species are involved in hypersensitive response-like cell death mediated by a mitogen-activated protein kinase cascade. Plant J 2007, 51: 941-954.Hadidi A, Barba M: Economic impact of pome and stone fruit viruses and viroids. In Virus and Virus Like Diseases of Pome and Stone Fruits. Edited by: Hadidi A, Barba M, Candresse T, Jelkmann W. St Paul, MN: American Phytopathological Society; 2011:1-8.Flores R, Delgado S, Rodio ME, Ambros S, Hernandez C, Serio FD: Peach latent mosaic viroid: not so latent. Mol Plant Pathol 2006, 7: 209-221.Pallas V, Aparicio F, Herranz MC, Amari K, Sanchez-Pina MA, Myrta A, Sanchez-Navarro JA: Ilarviruses of Prunus spp.: A continued concern for fruit trees. Phytopathology 2012,102(12):1108-1120.Rowland O, Jones JD: Unraveling regulatory networks in plant defense using microarrays. Genome Biol 2001,2(1):1001.1-1001.3.Trinks D, Rajeswaran R, Shivaprasad PV, Akbergenov R, Oakeley EJ, Veluthambi K, Hohn T, Pooggin MM: Suppression of RNA silencing by a geminivirus nuclear protein, AC2, correlates with transactivation of host genes. J Virol 2005, 79: 2517-2527.Senthil G, Liu H, Puram VG, Clark A, Stromberg A, Goodin MM: Specific and common changes in Nicotiana benthamiana gene expression in response to infection by enveloped viruses. J Gen Virol 2005, 86: 2615-2625.Marathe R, Guan Z, Anandalakshmi R, Zhao H, Dinesh-Kumar SP: Study of Arabidopsis thaliana resistome in response to cucumber mosaic virus infection using whole genome microarray. Plant Mol Biol 2004, 55: 501-520.Agudelo-Romero P, Carbonell P, de la Iglesia F, Carrera J, Rodrigo G, Jaramillo A, Perez-Amador MA, Elena SF: Changes in the gene expression profile of Arabidopsis thaliana after infection with Tobacco etch virus. Virol J 2008, 5: 92.Itaya A, Matsuda Y, Gonzales RA, Nelson RS, Ding B: Potato spindle tuber viroid strains of different pathogenicity induces and suppresses expression of common and unique genes in infected tomato. Mol Plant Microbe Interact 2002, 15: 990-999.Huang Z, Yeakley JM, Garcia EW, Holdridge JD, Fan JB, Whitham SA: Salicylic acid-dependent expression of host genes in compatible Arabidopsis-virus interactions. Plant Physiol 2005, 137: 1147-1159.Rizza S, Conesa A, Juarez J, Catara A, Navarro L, Duran-Vila N, Ancillo G: Microarray analysis of Etrog citron (Citrus medica L.) reveals changes in chloroplast, cell wall, peroxidase and symporter activities in response to viroid infection. Mol Plant Pathol 2012,13(8):852-864.Golem S, Culver JN: Tobacco mosaic virus induced alterations in the gene expression profile of Arabidopsis thaliana. Mol Plant Microbe Interact 2003, 16: 681-688.Dardick C: Comparative expression profiling of Nicotiana benthamiana leaves systemically infected with three fruit tree viruses. Mol Plant Microbe Interact 2007, 20: 1004-1017.Hull R: In Matthews’ Plant Virology. London: Edited by Academic Press; 2002.Gonzalez-Jara P, Tenllado F, Martinez-Garcia B, Atencio FA, Barajas D, Vargas M, Diaz-Ruiz J, Diaz-Ruiz JR: Host-dependent differences during synergistic infection by Potyviruses with potato virus X. Mol Plant Pathol 2004, 5: 29-35.Gonzalez-Jara P, Atencio FA, Martinez-Garcia B, Barajas D, Tenllado F, Diaz-Ruiz JR: A Single Amino Acid Mutation in the Plum pox virus Helper Component-Proteinase Gene Abolishes Both Synergistic and RNA Silencing Suppression Activities. Phytopathology 2005, 95: 894-901.Vance VB: Replication of potato virus X RNA is altered in coinfections with potato virus Y. Virology 1991, 182: 486-494.Garcia-Marcos A, Pacheco R, Martianez J, Gonzalez-Jara P, Diaz-Ruiz JR, Tenllado F: Transcriptional changes and oxidative stress associated with the synergistic interaction between Potato virus X and Potato virus Y and their relationship with symptom expression. Mol Plant Microbe Interact 2009, 22: 1431-1444.Postnikova OA, Nemchinov LG: Comparative analysis of microarray data in Arabidopsis transcriptome during compatible interactions with plant viruses. Virol J 2012, 9: 101.Zanchin A, Bonghi C, Casadoro G, Ramina A, Rascio N: Cell enlargement and cell separation during peach fruit development. International Journal of Plant Science 1994, 155: 49-56.Herranz MC, Sanchez-Navarro JA, Aparicio F, Pallas V: Simultaneous detection of six stone fruit viruses by non-isotopic molecular hybridization using a unique riboprobe or ‘polyprobe’. J Virol Methods 2005, 124: 49-55.Pallas V, Mas P, Sanchez-Navarro JA: Detection of plant RNA viruses by nonisotopic dot-blot hybridization. Methods Mol Biol 1998, 81: 461-468.Lilly ST, Drummond RS, Pearson MN, MacDiarmid RM: Identification and validation of reference genes for normalization of transcripts from virus-infected Arabidopsis thaliana. Mol Plant Microbe Interact 2011, 24: 294-304.Cosgrove JD: Expansive growth of plant cell walls. Plant Physiol Biochem 2000,38(1–2):109-124.Tessitori M, Maria G, Capasso C, Catara G, Rizza S, De Luca V, Catara A, Capasso A, Carginale V: Differential display analysis of gene expression in Etrog citron leaves infected by Citrus viroid III. Biochim Biophys Acta 2007, 1769: 228-235.Rizza S, Capasso C, Catara A, Capasso A, Conte E, Catara A Proceedings of the 17th Conference of the International Organization of Citrus Virologists-IOCV, pp. XVII. In Transcriptional response of Troyer citrange, sour orange and alemow rootstocks to Citrus viroid IIIb (CVd-IIIb) infection. Adana, Turkey: Conference of the International Organization of Citrus Virologists; 2010:142-149. http://www.ivia.es/iocv/Owens RA, Tech KB, Shao JY, Sano T, Baker CJ: Global analysis of tomato gene expression during Potato spindle tuber viroid infection reveals a complex array of changes affecting hormone signaling. Mol Plant Microbe Interact 2012, 25: 582-598.Ogundiwin EA, Marti C, Forment J, Pons C, Granell A, Gradziel TM, Peace CP, Crisosto CH: Development of ChillPeach genomic tools and identification of cold-responsive genes in peach fruit. Plant Mol Biol 2008, 68: 379-397.Sánchez-Navarro JA FA, Rowhani A, Pallás V: Comparative analysis of ELISA, nonradioactive molecular hybridization and PCR for the detection of Prunus necrotic ringspot virus in herbaceous and prunus host. Plant Pathol 1998, 47: 780-786.Astruc N, Marcos JF, Macquaire G, Candresse T, Pallas V: Studies on the diagnosis of hop stunt viroid in fruit trees: Identification of new hosts and application of a nucleic acid extraction procedure based on non-organic solvents. Eur J Plant Pathol 1996, 102: 837-846.Myrta A, Di Terlizzi B, Pallas V, Savino V: Viruses and viroids of apricot in the Mediterranean: incidence and biodiversity. Acta Horticulturae 2006, 701: 409-417.Bouzayen M, Latché A, Nath P, Pech JC: Mechanism of fruit ripening. In Plant Developmental Biology- Biotechnological Perspectives: Volume I Edited by: Pua EC, Darvey MR. 2010, 319-339. Chapter 16Trainotti L, Bonghi C, Ziliotto F, Zanin D, Rasori A, Casadoro G, Ramina A, T P: The use of microarray mPEACH 1.0 to investigate transcriptome changes during transition from pre-climateric to climacteric phase in peach fruit. Plant Sci 2006, 170: 606-613.Lombardo VA, Osorio S, Borsani J, Lauxmann MA, Bustamante CA, Budde CO, Andreo CS, Lara MV, Fernie AR MFD: Metabolic profiling during peach fruit development and ripening reveals the metabolic networks that underpin each developmental stage. Plant Physiol 2011,157(4):1696-1710.Manganaris GA RA, Bassi D, Geuna F, Ramina A, Tonutti P, Bonghi C: Comparative transcript profiling of apricot (Prunus armeniaca L.) fruit development and on-tree ripening. Tree Genet Genomes 2011, 7: 609-616.Uyemoto JK, Scott SW: Important diseases of Prunus caused by viruses and other graft-transmissible pathogens in California and South Carolina. Plant Dis 1992, 76: 5-11.Li J, Yang H, Peer WA, Richter G, Blakeslee J, Bandyopadhyay A, Titapiwantakun B, Undurraga S, Khodakovskaya M, Richards EL, et al.: Arabidopsis H+-PPase AVP1 regulates auxin-mediated organ development. Science 2005, 310: 121-125.Paponov IA, Paponov M, Teale W, Menges M, Chakrabortee S, Murray JA, Palme K: Comprehensive transcriptome analysis of auxin responses in Arabidopsis. Mol Plant 2008, 1: 321-337.Padmanabhan MS, Goregaoker SP, Golem S, Shiferaw H, Culver JN: Interaction of the tobacco mosaic virus replicase protein with the Aux/IAA protein PAP1/IAA26 is associated with disease development. J Virol 2005, 79: 2549-2558.Padmanabhan MS, Shiferaw H, Culver JN: The Tobacco mosaic virus replicase protein disrupts the localization and function of interacting Aux/IAA proteins. Mol Plant Microbe Interact 2006, 19: 864-873.Padmanabhan MS, Kramer SR, Wang X, Culver JN: Tobacco mosaic virus replicase-auxin/indole acetic acid protein interactions: reprogramming the auxin response pathway to enhance virus infection. J Virol 2008, 82: 2477-2485.Kuhn JM, Boisson-Dernier A, Dizon MB, Maktabi MH, Schroeder JI: The protein phosphatase AtPP2CA negatively regulates abscisic acid signal transduction in Arabidopsis, and effects of abh1 on AtPP2CA mRNA. Plant Physiol 2006, 140: 127-139.Whenham RJ, Fraser RSS, Brown LP, Payne JA: Tobacco-mosaic-virus-induced increase in abscisic-acid concentration in tobacco leaves: Intracellular location in light and dark-green areas, and relationship to symptom development. Planta 1986, 168: 592-598.Bari R, Jones JD: Role of plant hormones in plant defence responses. Plant Mol Biol 2009, 69: 473-488.Kotchoni SO, Kuhns C, Ditzer A, Kirch HH, Bartels D: Over-expression of different aldehyde dehydrogenase genes in Arabidopsis thaliana confers tolerance to abiotic stress and protects plants against lipid peroxidation and oxidative stress. Plant Cell Environ 2006, 29: 1033-1048.Mowla SB, Cuypers A, Driscoll SP, Kiddle G, Thomson J, Foyer CH, Theodoulou FL: Yeast complementation reveals a role for an Arabidopsis thaliana late embryogenesis abundant (LEA)-like protein in oxidative stress tolerance. Plant J 2006, 48: 743-756.Amari K, Diaz-Vivancos P, Pallas V, Sanchez-Pina MA, Hernandez JA: Oxidative stress induction by Prunus necrotic ringspot virus infection in apricot seeds. Physiol Plant 2007, 131: 302-310.Gilroy EM, Hein I, van der Hoorn R, Boevink PC, Venter E, McLellan H, Kaffarnik F, Hrubikova K, Shaw J, Holeva M, et al.: Involvement of cathepsin B in the plant disease resistance hypersensitive response. Plant J 2007, 52: 1-13.Kruger J, Thomas CM, Golstein C, Dixon MS, Smoker M, Tang S, Mulder L, Jones JD: A tomato cysteine protease required for Cf-2-dependent disease resistance and suppression of autonecrosis. Science 2002, 296: 744-747.Bernoux M, Timmers T, Jauneau A, Briere C, De Wit PJ, Marco Y, Deslandes L: RD19, an Arabidopsis cysteine protease required for RRS1-R-mediated resistance, is relocalized to the nucleus by the Ralstonia solanacearum PopP2 effector. Plant Cell 2008, 20: 2252-2264.Shabab M, Shindo T, Gu C, Kaschani F, Pansuriya T, Chintha R, Harzen A, Colby T, Kamoun S, van der Hoorn RA: Fungal effector protein AVR2 targets diversifying defense-related cys proteases of tomato. Plant Cell 2008, 20: 1169-1183.Van Esse HP, Van’t Klooster JW, Bolton MD, Yadeta KA, Van Baarlen P, Boeren S, Vervoort J, De Wit PJ, Thomma BP: The Cladosporium fulvum virulence protein Avr2 inhibits host proteases required for basal defense. Plant Cell 2008, 20: 1948-1963.Song J, Win J, Tian M, Schornack S, Kaschani F, Ilyas M, van der Hoorn RA, Kamoun S: Apoplastic effectors secreted by two unrelated eukaryotic plant pathogens target the tomato defense protease Rcr3. Proc Natl Acad Sci U S A 2009, 106: 1654-1659.Tian M, Win J, Song J, van der Hoorn R, van der Knaap E, Kamoun S: A Phytophthora infestans cystatin-like protein targets a novel tomato papain-like apoplastic protease. Plant Physiol 2007, 143: 364-377.Rooney H, Van’t Klooster J, Van der Hoorn R, Joosten M, Jones J: Cladosporium Avr2 inhibits tomato Rcr3 protease required for Cf-2-dependent disease resistance. Science 2005, 308: 1783-1786.Auger AJ: Tomato ringspot virus associated with brownline disease on prune trees in Chile. Acta Horticulturae 1989, 235: 197-204.Herrera G: Enfermedades causadas por virus en frutales en Chile. Santiago, Chile: Instituto de Investigación Agropecuaria; 2001. Boletín INIA N°52. 65pFiore N, Abou Ghanem-Sabanadzovic N, Infante R, Myrta A, Pallás V: Detection of Peach latent mosaic viroid in stone fruits from Chile. In Option Méditerranéennes, Sér. B/n°45 –Virus ad virus-like disease of stone fruits, with particular reference to the Mediterranean region Edited by: Myrta A, Di Terlizzi B, Savino V. 2003, 143-145.Torres H, Gómez G, Pallás V, Stamo B, Shalaby A, Aouane B, Gavriel I, Kominek P, Caglayan K, Sipahioglu M, et al.: Detection by tissue printing of stone fruit viroids, from europe, the mediterranean and north and south America. Acta Horticulturae 2004, 657: 379-383.Peiró A, Pallás V, Sánchez-Navarro JA: Simultaneous detection of eight viruses and two viroids affecting stone fruit trees by using a unique polyprobe. Eur J Plant Pathol 2012,132(4):469-475.Meisel L, Fonseca B, Gonzalez S, Baeza-Yates R, Cambiazo V, Campos R, Gonzalez M, Orellana A, Retamales J, Silva H: A rapid and efficient method for purifying high quality total RNA from peaches (Prunus persica) for functional genomics analyses. Biol Res 2005, 38: 83-88.Van Gelder RN, Von Zastrow ME, Yool A, Dement WC, Barchas JD JHE: Amplified RNA synthesized from limited quantities of heterogeneous cDNA. Proc Natl Acad Sci U S A 1990,87(5):1663-1667.Tusher VG, Tibshirani R, Chu G: Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci U S A 2001, 98: 5116-5121.Sanchez-Navarro JA, Canizares MC, Cano EA, Pallas V: Simultaneous detection of five carnation viruses by non-isotopic molecular hybridization. J Virol Methods 1999, 82: 167-175

Antiviral Silencing and Suppression of Gene Silencing in Plants

RNA silencing is an evolutionary conserved sequence-specific gene inactivation mechanism that contributes to the control of development, maintains heterochromatin, acts in stress responses, DNA repair and defends against invading nucleic acids like transposons and viruses. In plants RNA silencing functions as one of the main immune systems. RNA silencing process involves the small RNAs and trans factor components like Dicers, Argonautes and RNA-dependent RNA poly- merases. To deal with host antiviral silencing responses viruses evolved mecha- nisms to avoid or counteract this, most notably through expression of viral suppressors of RNA silencing. Due to the overlap between endogenous and antiviral silencing pathways while blocking antiviral pathways viruses also impact endogenous silencing processes. Here we provide an overview of antiviral silencing pathway, host factors implicated in it and the crosstalk between antiviral and endogenous branches of silencing. We summarize the current status of knowledge about the viral counter-defense strategies acting at various steps during virus infection in plants with the focus on representative, well studied silencing suppres- sor proteins. Finally we discuss future challenges of the antiviral silencing and counter-defense research field