title: Potato Virus Y HCPro Suppression of Antiviral Silencing in Nicotiana benthamiana Plants Correlates with Its Ability To BindIn Vivoto 21- and 22-Nucleotide Small RNAs of Viral Sequence.
abstract: We have investigated short and small RNAs (sRNAs) that were bound to a biologically active hexahistidine-taggedPotato virus Y(PVY) HCPro suppressor of silencing, expressed from a heterologous virus vector inNicotiana benthamianaplants, and purified under nondenaturing conditions. We found that RNAs in purified preparations were differentially enriched in 21-nucleotide (nt) and, to a much lesser extent, 22-nt sRNAs of viral sequences (viral sRNAs [vsRNAs]) compared to those found in a control plant protein background bound to nickel resin in the absence of HCPro or in a purified HCPro alanine substitution mutant (HCPro mutB) control that lacked suppressor-of-silencing activity. In both controls, sRNAs were composed almost entirely of molecules of plant sequence, indicating that the resin-bound protein background had no affinity for vsRNAs and also that HCPro mutB failed to bind to vsRNAs. Therefore, PVY HCPro suppressor activity correlated with its ability to bind to 21- and 22-nt vsRNAs. HCPro constituted at least 54% of the total protein content in purified preparations, and we were able to calculate its contribution to the 21- and the 22-nt pools of sRNAs present in the purified samples and its binding strength relative to the background. We also found that in the 21-nt vsRNAs of the HCPro preparation, 5'-terminal adenines were overrepresented relative to the controls, but this was not observed in vsRNAs of other sizes or of plant sequences.IMPORTANCEIt was previously shown that HCPro can bind to long RNAs and small RNAs (sRNAs)in vitroand, in the case ofTurnip mosaic virusHCPro, alsoin vivoin arabidopsis AGO2-deficient plants. Our data show that PVY HCPro bindsin vivoto sRNAs during infection in wild-typeNicotiana benthamianaplants when expressed from a heterologous virus vector. Using a suppression-of-silencing-deficient HCPro mutant that can accumulate in this host when expressed from a virus vector, we also show that sRNA binding correlates with silencing suppression activity. We demonstrate that HCPro binds at least to sRNAs with viral sequences of 21 nucleotides (nt) and, to a much lesser extent, of 22 nt, which were are also differentially enriched in 5'-end adenines relative to the purified controls. Together, our results support the physical binding of HCPro to vsRNAs of 21 and 22 nt as a means to interfere with antiviral silencing.
authors.affiliation: Departamento de Biología Medioambiental, Centro de Investigaciones Biológicas, CSIC, Madrid, Spain fjdts@cib.csic.es tomas.canto@cib.csic.es.
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Del Toro
authors.affiliation: Universidad Politécnica de Madrid, Centro de Biotecnología y Genómica de Plantas, Campus de Montegancedo, Madrid, Spain.
authors.forename: Livia
authors.initials: L
authors.lastname: Donaire
authors.affiliation: Departamento de Biología Medioambiental, Centro de Investigaciones Biológicas, CSIC, Madrid, Spain.
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Aguilar
authors.affiliation: National Institute of Horticultural & Herbal Science, Agricultural Research Center for Climate Change, Wanju, Republic of Korea.
authors.forename: Bong-Nam
authors.initials: BN
authors.lastname: Chung
authors.affiliation: Departamento de Biología Medioambiental, Centro de Investigaciones Biológicas, CSIC, Madrid, Spain.
authors.forename: Francisco
authors.initials: F
authors.lastname: Tenllado
authors.affiliation: Departamento de Biología Medioambiental, Centro de Investigaciones Biológicas, CSIC, Madrid, Spain fjdts@cib.csic.es tomas.canto@cib.csic.es.
authors.forename: Tomás
authors.initials: T
authors.lastname: Canto
mesh_terms: D020868:Gene Silencing; D005822:Genetic Vectors; D009711:Nucleotides; D010935:Plant Diseases / Q000821:virology; D017800:Potyvirus / Q000235:genetics; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014026:Nicotiana / Q000821:virology*; D014764:Viral Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D009711:Nucleotides; D012367:RNA, Viral; D014764:Viral Proteins
keywords: HCPro; antiviral silencing; potyvirus; sRNAs; suppressor of silencing