title: Differential RNAi responses of Nicotiana benthamiana individuals transformed with a hairpin-inducing construct during Plum pox virus challenge.
abstract: Gene silencing and large-scale small RNA analysis can be used to develop RNA interference (RNAi)-based resistance strategies for Plum pox virus (PPV), a high impact disease of Prunus spp. In this study, a pPPViRNA hairpin-inducing vector harboring two silencing motif-rich regions of the PPV coat protein (CP) gene was evaluated in transgenic Nicotiana benthamiana (NB) plants. Wild-type NB plants infected with a chimeric PPV virus (PPV::GFP) exhibited affected leaves with mosaic chlorosis congruent to GFP fluorescence at 21 day post-inoculation; transgenic lines depicted a range of phenotypes from fully resistant to susceptible. ELISA values and GFP fluorescence intensities were used to select transgenic-resistant (TG-R) and transgenic-susceptible (TG-S) lines for further characterization of small interfering RNAs (siRNAs) by large-scale small RNA sequencing. In infected TG-S and untransformed (WT) plants, the observed siRNAs were nearly exclusively 21- and 22-nt siRNAs that targeted the whole PPV::GFP genome; 24-nt siRNAs were absent in these individuals. Challenged TG-R plants accumulated a full set of 21- to 24-nt siRNAs that were primarily associated with the selected motif-rich regions, indicating that a trans-acting siRNAs process prevented viral multiplication. BLAST analysis identified 13 common siRNA clusters targeting the CP gene. 21-nt siRNA sequences were associated with the 22-nt siRNAs and the scarce 23- and 24-nt molecules in TG-S plants and with most of the observed 22-, 23-, and 24-nt siRNAs in TG-R individuals. These results validate the use of a multi-hot spot silencing vector against PPV and elucidate the molecules by which hairpin-inducing vectors initiate RNAi in vivo.
authors: Biotechnology Laboratory, Instituto de Investigaciones Agropecuarias, La Platina Research Station, Avenida Santa Rosa 11610, La Pintana, 8831314, Santiago, Chile.
authors: Christian
authors: C
authors: Montes
authors: Álvaro
authors: Á
authors: Castro
authors: Paola
authors: P
authors: Barba
authors: Julia
authors: J
authors: Rubio
authors: Evelyn
authors: E
authors: Sánchez
authors: Denisse
authors: D
authors: Carvajal
authors: Carlos
authors: C
authors: Aguirre
authors: Eduardo
authors: E
authors: Tapia
authors: Paola
authors: P
authors: DelÍ Orto
authors: Veronique
authors: V
authors: Decroocq
authors: Humberto
authors: H
authors: Prieto
mesh_terms: D060467:Disease Resistance; D020869:Gene Expression Profiling; D020868:Gene Silencing; D054884:Host-Pathogen Interactions; D010935:Plant Diseases / Q000821:virology*; D030821:Plants, Genetically Modified; D017801:Plum Pox Virus / Q000254:growth & development*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000032:analysis / Q000235:genetics; D014026:Nicotiana / Q000821:virology*
chemical_list: D034741:RNA, Small Interfering