Chimeric Virus Made from crTMV RNA and the Coat Protein of Potato Leafroll Virus is Targeted to the Nucleolus and Can Infect Nicotiana benthamiana Mechanically

Konstantin O Butenko; Inna A Chaban; Eugene V Skurat; Olga A Kondakova; Yuri F Drygin

doi:10.3390/ht9020011

Chimeric Virus Made from crTMV RNA and the Coat Protein of Potato Leafroll Virus is Targeted to the Nucleolus and Can Infect Nicotiana benthamiana Mechanically

High Throughput. 2020 Apr 26;9(2):11. doi: 10.3390/ht9020011.

Authors

Konstantin O Butenko¹, Inna A Chaban², Eugene V Skurat¹, Olga A Kondakova¹, Yuri F Drygin³

Affiliations

¹ Faculty of Biology, Lomonosov Moscow State University, Moscow 119992, Russian.
² All-Russian Research Institute of Agricultural Biotechnology, Russian Academy of Agricultural Sciences, Moscow 127422, Russian.
³ Belozersky Research Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119992, Russian.

Abstract

A genetically engineered chimeric virus crTMV-CP-PLRV composed of the crucifer-infecting tobacco mosaic virus (crTMV) RNA and the potato leafroll virus (PLRV) coat protein (CP) was obtained by agroinfiltration of Nicotiana benthamiana with the binary vector pCambia-crTMV-CP_PLRV. The significant levels of the chimeric virus enabled direct visualization of crTMV-CP-PLRV in the cell and to investigate the mechanism of the pathogenesis. Localization of the crTMV-CP-PLRV in plant cells was examined by immunoblot techniques, as well as light, and transmission electron microscopy. The chimera can transfer between vascular and nonvascular tissues. The chimeric virus inoculum is capable to infect N. benthamiana mechanically. The distinguishing feature of the chimeric virus, the RNA virus with the positive genome, was found to localize in the nucleolus. We also investigated the role of the N-terminal sequence of the PLRV P3 coat protein in the cellular localization of the virus. We believe that the gene of the PLRV CP can be substituted with genes from other challenging-to-study plant pathogens to produce other useful recombinant viruses.

Keywords: agroinoculation; binary vector; chimeric virus; electron microscopy; mechanical infection of plant; recombinant RNA; tissue blot-immunoassay.