Intracellular Localization, Interactions and Functions of Capsicum Chlorosis Virus Proteins.

Shirani M K Widana Gamage,Ralf G Dietzgen

doi:10.3389/fmicb.2017.00612

Shirani M K Widana Gamage, Ralf G Dietzgen

Open Access

https://doi.org/10.3389/fmicb.2017.00612

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Abstract

Tospoviruses are among the most devastating viruses of horticultural and field crops. Capsicum chlorosis virus (CaCV) has emerged as an important pathogen of capsicum and tomato in Australia and South-east Asia. Present knowledge about CaCV protein functions in host cells is lacking. We determined intracellular localization and interactions of CaCV proteins by live plant cell imaging to gain insight into the associations of viral proteins during infection. Proteins were transiently expressed as fusions to autofluorescent proteins in leaf epidermal cells of Nicotiana benthamiana and capsicum. All viral proteins localized at least partially in the cell periphery suggestive of cytoplasmic replication and assembly of CaCV. Nucleocapsid (N) and non-structural movement (NSm) proteins localized exclusively in the cell periphery, while non-structural suppressor of silencing (NSs) protein and Gc and Gn glycoproteins accumulated in both the cell periphery and the nucleus. Nuclear localization of CaCV Gn and NSs is unique among tospoviruses. We validated nuclear localization of NSs by immunofluorescence in protoplasts. Bimolecular fluorescence complementation showed self-interactions of CaCV N, NSs and NSm, and heterotypic interactions of N with NSs and Gn. All interactions occurred in the cytoplasm, except NSs self-interaction was exclusively nuclear. Interactions of a tospoviral NSs protein with itself and with N had not been reported previously. Functionally, CaCV NSs showed strong local and systemic RNA silencing suppressor activity and appears to delay short-distance spread of silencing signal. Cell-to-cell movement activity of NSm was demonstrated by trans-complementation of a movement-defective tobamovirus replicon. CaCV NSm localized at plasmodesmata and its transient expression led to the formation of tubular structures that protruded from protoplasts. The D155 residue in the 30K-like movement protein-specific LxD/N50-70G motif of NSm was critical for plasmodesmata localization and movement activity. Compared to other tospoviruses, CaCV proteins have both conserved and unique properties in terms of in planta localization, interactions and protein functions which will effect viral multiplication and movement in host plants.

Highlights

Capsicum chlorosis virus was first discovered in capsicum (Capsicum annuum L.) and tomato (Solanum lycopersicum L.) crops in Queensland, Australia in 1999 (McMichael et al, 2002)
M RNA contains two open reading frame (ORF) separated by an intergenic region (IGR), which code for non-structural movement (NSm) and precursor of glycoproteins (GP) (Kormelink et al, 1992)
We have experimentally demonstrated the functions of Capsicum chlorosis virus (CaCV) non-structural proteins NSm and non-structural suppressor of silencing (NSs) that are involved in cellto-cell movement and RNA silencing suppression, respectively

Summary

Introduction

Capsicum chlorosis virus was first discovered in capsicum (Capsicum annuum L.) and tomato (Solanum lycopersicum L.) crops in Queensland, Australia in 1999 (McMichael et al, 2002). CaCV was recorded in China, India, Taiwan, and Thailand from capsicum, groundnut and tomato crops and ornamental plants (Knierim et al, 2006; Chen C. et al, 2007; Chen K. et al, 2007; Kunkalikar et al, 2007). The genome of CaCV has the typical tospovirus genome structure consisting of three linear RNA segments with negative or ambisense coding polarity (Knierim et al, 2006; Zheng et al, 2011; Widana Gamage et al, 2015). M RNA contains two ORFs separated by an intergenic region (IGR), which code for non-structural movement (NSm) and precursor of glycoproteins (GP) (Kormelink et al, 1992). S RNA contains two ORFs separated by an IGR, which code for non-structural suppressor of silencing (NSs) and nucleocapsid (N) proteins (de Haan et al, 1990). Encapsidated tospovirus RNA genome segments with several copies of RdRP are collectively bound by a lipid envelope, which is decorated by Gn and Gc glycoprotein projections (van Poelwuk et al, 1993; Kikkert et al, 1999)

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