The potato mop-top virus TGB2 protein and viral RNA associate with chloroplasts and viral infection induces inclusions in the plastids.

Graham H Cowan,Angelika Ziegler,Lesley Torrance,Eugene I Savenkov,Sean N Chapman,Alison G Roberts

doi:10.3389/fpls.2012.00290

Abstract

The potato mop-top virus (PMTV) triple gene block 2 (TGB2) movement proteins fused to monomeric red fluorescent protein (mRFP-TGB2) was expressed under the control of the PMTV subgenomic promoter from a PMTV vector. The subcellular localizations and interactions of mRFP-TGB2 were investigated using confocal imaging [confocal laser-scanning microscope, (CLSM)] and biochemical analysis. The results revealed associations with membranes of the endoplasmic reticulum (ER), mobile granules, small round structures (1–2 μm in diameter), and chloroplasts. Expression of mRFP-TGB2 in epidermal cells enabled cell-to-cell movement of a TGB2 defective PMTV reporter clone, indicating that the mRFP-TGB2 fusion protein was functional and required for cell-to-cell movement. Protein-lipid interaction assays revealed an association between TGB2 and lipids present in chloroplasts, consistent with microscopical observations where the plastid envelope was labeled later in infection. To further investigate the association of PMTV infection with chloroplasts, ultrastructural studies of thin sections of PMTV-infected potato and Nicotiana benthamiana leaves by electron microscopy revealed abnormal chloroplasts with cytoplasmic inclusions and terminal projections. Viral coat protein (CP), genomic RNA and fluorescently-labeled TGB2 were detected in plastid preparations isolated from the infected leaves, and viral RNA was localized to chloroplasts in infected tissues. The results reveal a novel association of TGB2 and vRNA with chloroplasts, and suggest viral replication is associated with chloroplast membranes, and that TGB2 plays a novel role in targeting the virus to chloroplasts.

Highlights

Plant viral genomes are relatively small and most comprise positive-sense, single-stranded RNAs that encode a few multifunctional proteins
To investigate whether the localizations we observed were influenced by over-expression from the 35S promoter, the mRFP-triple gene block 2 (TGB2) fusion protein was expressed from a potato mop-top virus (PMTV) vector
It was previously shown that PMTV mRFP-TGB2 and green fluorescent protein (GFP)-TGB3 when expressed from a 35S promoter co-localized in cellular membranes and mobile granules, utilizing the endoplasmic reticulum (ER)-actin network www.frontiersin.org to facilitate movement to the cell periphery and PD

Summary

Introduction

Plant viral genomes are relatively small and most comprise positive-sense, single-stranded RNAs that encode a few multifunctional proteins. Targeting of viral replication machinery to particular compartments by virusencoded, non-structural proteins that associate with specific organellar membranes, can lead to the formation of membranebound, cytosolic viral replication complexes (VRCs). For those viruses, such as potato mop-top virus (PMTV), where the coat protein (CP) is not required for cell-to-cell or systemic movement (Torrance et al, 2009), different membranes may be involved in replication and movement of the viral ribonucleoprotein (vRNP) complex and virions. It is becoming apparent that some movement proteins are multi-functional, playing additional roles in virus replication, counter defense and pathogenicity, as well as in viral genome transport (Scholthof, 2005; Lucas, 2006; Torrance et al, 2006)

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Conclusion