Identification and Characterization of Plant-Interacting Targets of Tomato Spotted Wilt Virus Silencing Suppressor.

Ying Zhai,Prabu Gnanasekaran,Hanu R Pappu

doi:10.3390/pathogens10010027

Ying Zhai, Prabu Gnanasekaran + Show 1 more

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https://doi.org/10.3390/pathogens10010027

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Journal: Pathogens	Publication Date: Jan 1, 2021
Citations: 8	License type: CC BY 4.0

Affiliation: Washington State University

Abstract

Tomato spotted wilt virus (TSWV; species Tomato spotted wilt orthotospovirus) is an economically important plant virus that infects multiple horticultural crops on a global scale. TSWV encodes a non-structural protein NSs that acts as a suppressor of host RNA silencing machinery during infection. Despite extensive structural and functional analyses having been carried out on TSWV NSs, its protein-interacting targets in host plants are still largely unknown. Here, we systemically investigated NSs-interacting proteins in Nicotiana benthamiana via affinity purification and mass spectrometry (AP-MS) analysis. Forty-three TSWV NSs-interacting candidates were identified in N. benthamiana. Gene Ontology (GO) and protein–protein interaction (PPI) network analyses were carried out on their closest homologs in tobacco (Nicotiana tabacum), tomatoes (Solanum lycopersicum) and Arabidopsis (Arabidopsis thaliana). The results showed that NSs preferentially interacts with plant defense-related proteins such as calmodulin (CaM), importin, carbonic anhydrase and two heat shock proteins (HSPs): HSP70 and HSP90. As two major nodes in the PPI network, CaM and importin subunit α were selected for the further verification of their interactions with NSs via yeast two-hybrid (Y2H) screening. Our work suggests that the downstream signaling, transportation and/or metabolic pathways of host-NSs-interacting proteins may play critical roles in NSs-facilitated TSWV infection.

Highlights

Tomato spotted wilt virus (TSWV; species Tomato spotted wilt orthotospovirus) is the best known member in Orthotospovirus, which is the only genus with plant-infecting viruses in the family Tospoviridae [1]
TSWV NSs was fused with an mGFP5 tag at its C-terminal (NSs-GFP) and was transiently expressed in N. benthamiana leaves at the four-leaf stage
To identify the host proteins that interact with TSWV NSs, overlapping candidates were selected from two independent AP-mass spectrometry (MS) replicates

Summary

Introduction

Tomato spotted wilt virus (TSWV; species Tomato spotted wilt orthotospovirus) is the best known member in Orthotospovirus, which is the only genus with plant-infecting viruses in the family Tospoviridae [1]. The M RNA encodes a non-structural movement protein NSm, and the precursor of two structural glycoproteins GN and GC. A nucleocapsid protein (N) and another non-structural protein (NSs) are encoded by the S RNA [7]. Both the M and S RNAs are organized in an ambisense manner [8]. The three genomic RNAs of TSWV and the N protein form ribonucleoproteins encapsulated by the glycoprotein (GN and GC) envelope. TSWV infects plants via the thrips vector in the field [9]

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