title: Construction of a Sonchus Yellow Net Virus minireplicon: a step toward reverse genetic analysis of plant negative-strand RNA viruses.
abstract: Reverse genetic analyses of negative-strand RNA (NSR) viruses have provided enormous advances in our understanding of animal viruses over the past 20 years, but technical difficulties have hampered application to plant NSR viruses. To develop a reverse genetic approach for analysis of plant NSR viruses, we have engineered Sonchus yellow net nucleorhabdovirus (SYNV) minireplicon (MR) reporter cassettes for Agrobacterium tumefaciens expression in Nicotiana benthamiana leaves. Fluorescent reporter genes substituted for the SYNV N and P protein open reading frames (ORFs) exhibited intense single-cell foci throughout regions of infiltrated leaves expressing the SYNV MR derivatives and the SYNV nucleocapsid (N), phosphoprotein (P), and polymerase (L) proteins. Genomic RNA and mRNA transcription was detected for reporter genes substituted for both the SYNV N and P ORFs. These activities required expression of the N, P, and L core proteins in trans and were enhanced by codelivery of viral suppressor proteins that interfere with host RNA silencing. As is the case with other members of the Mononegavirales, we detected polar expression of fluorescent proteins and chloramphenicol acetyltransferase substitutions for the N and P protein ORFs. We also demonstrated the utility of the SYNV MR system for functional analysis of SYNV core proteins in trans and the cis-acting leader and trailer sequence requirements for transcription and replication. This work provides a platform for construction of more complex SYNV reverse genetic derivatives and presents a general strategy for reverse genetic applications with other plant NSR viruses.
authors.affiliation: Department of Plant and Microbial Biology, University of California, Berkeley, California, USA.
authors.forename: Uma
authors.initials: U
authors.lastname: Ganesan
authors.forename: Jennifer N
authors.initials: JN
authors.lastname: Bragg
authors.forename: Min
authors.initials: M
authors.lastname: Deng
authors.forename: Sharon
authors.initials: S
authors.lastname: Marr
authors.forename: Mi Yeon
authors.initials: MY
authors.lastname: Lee
authors.forename: Shasha
authors.initials: S
authors.lastname: Qian
authors.forename: Manling
authors.initials: M
authors.lastname: Shi
authors.forename: Justin
authors.initials: J
authors.lastname: Kappel
authors.forename: Cole
authors.initials: C
authors.lastname: Peters
authors.forename: Yeon
authors.initials: Y
authors.lastname: Lee
authors.forename: Michael M
authors.initials: MM
authors.lastname: Goodin
authors.forename: Ralf G
authors.initials: RG
authors.lastname: Dietzgen
authors.forename: Zhenghe
authors.initials: Z
authors.lastname: Li
authors.forename: Andrew O
authors.initials: AO
authors.lastname: Jackson
mesh_terms: D018515:Plant Leaves / Q000378:metabolism / Q000821:virology; D010942:Plant Viruses / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D012328:RNA Viruses / Q000235:genetics* / Q000378:metabolism; D018749:RNA, Plant / Q000235:genetics; D012093:Replicon; D012209:Rhabdoviridae / Q000502:physiology*; D018353:Rhabdoviridae Infections / Q000235:genetics / Q000821:virology*; D031184:Sonchus; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism / Q000821:virology*; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D018749:RNA, Plant; D014764:Viral Proteins