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.

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Published in Journal of Virology, v. 87, no. 19, p. 10598-10611.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

The copyright holders have granted the permission for posting the article here.

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Funding Information

This research was supported in part by NSF competitive grant MCB-03, 16907, National Institutes of Health award RO3 AI059178-01, and U.S. Department of Agriculture competitive grant award 96-35303-3563 to A.O.J., NSF competitive grant IOS-07, 49519 to M.M.G., grant Y12C140049 from the Zhejiang Provincial Foundation for Natural Science of China to M.S., and China National Science Funds for Excellent Young Scientists grant 31222004 and Zhejiang Provincial Natural Science Foundation of China under grant no. LR12C14001 to Z.L.

Related Content

Erratum, Construction of a Sonchus Yellow Net Virus Minireplicon: a Step toward Reverse Genetic Analysis of Plant Negative-Strand RNA Viruses, published in Journal of Virology, v. 87, no. 23, p. 13081.

A correction to the original article: "Page 10600, column 2, last paragraph of Materials and Methods, line 4: 'JNO38404' should read 'JN038404.'"

A PDF of this Erratum is attached.

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J. Virol.-2013-Ganesan-13081.pdf (57 kB)
Erratum (v. 87, no. 19, p. 10598)