Synthesis of Full-Length cDNA Infectious Clones of Soybean Mosaic Virus and Functional Identification of a Key Amino Acid in the Silencing Suppressor Hc-Pro.

Wenhua Bao,Xiaoyi Deng,Ting Yan,Hada Wuriyanghan

doi:10.3390/v12080886

Abstract

Soybean mosaic virus (SMV), which belongs to the Potyviridae, causes significant reductions in soybean yield and seed quality. In this study, both tag-free and reporter gene green fluorescent protein (GFP)-containing infectious clones for the SMV N1 strain were constructed by Gibson assembly and with the yeast homologous recombination system, respectively. Both infectious clones are suitable for agroinfiltration on the model host N. benthamiana and show strong infectivity for the natural host soybean and several other legume species. Both infectious clones were seed transmitted and caused typical virus symptoms on seeds and progeny plants. We used the SMV-GFP infectious clone to further investigate the role of key amino acids in the silencing suppressor helper component-proteinase (Hc-Pro). Among twelve amino acid substitution mutants, the co-expression of mutant 2—with an Asparagine→Leucine substitution at position 182 of the FRNK (Phe-Arg-Asn-Lys) motif—attenuated viral symptoms and alleviated the host growth retardation caused by SMV. Moreover, the Hc-Prom2 mutant showed stronger oligomerization than wild-type Hc-Pro. Taken together, the SMV infectious clones will be useful for studies of host–SMV interactions and functional gene characterization in soybeans and related legume species, especially in terms of seed transmission properties. Furthermore, the SMV-GFP infectious clone will also facilitate functional studies of both virus and host genes in an N. benthamiana transient expression system.

Highlights

The Potyviridae represents one of the largest families of plant viruses that cause severe yield losses in many major crops [1,2,3]
To build infections clones of Soybean mosaic virus (SMV), the SMV genome was split into four overlapping fragments: 1# (P1 + helper component-proteinase (Hc-Pro), 2455 bp), 2# (P3 + 6 kinase 1 (6K1) + cylindrical inclusion (CI) + 6 kinase 2 (6K2), 3282 bp), 3# (NIa-Vpg + nuclear inclusion a proteinase (NIa-Pro), 1323 bp) and #4 (NIb + coat protein (CP), 2659 bp) by RT-PCR amplification using the primers listed in Supplementary Table S1
The recombinant pLXB-SMV was transformed into A. tumefaciens GV3101 and agroinfiltrated on N. benthamiana seedlings followed by being rub-inoculated on susceptible soybeans

Summary

Introduction

The Potyviridae represents one of the largest families of plant viruses that cause severe yield losses in many major crops [1,2,3]. Soybean mosaic virus (SMV) is a monopartite positive-sense ssRNA virus in the Potyviridae with a genome of approximately 10 kb. SMV encodes 11 putative proteins, including potyvirus. 1 (P1), helper component-proteinase (Hc-Pro), potyvirus 3 (P3), N-terminal half of P3 fused to PIPO (P3N-PIPO), 6 kinase 1 (6K1), cylindrical inclusion (CI), 6 kinase 2 (6K2), Viral genome-linked Protein (VPg), nuclear inclusion a proteinase (NIa-Pro), nuclear inclusion b (NIb) and coat protein (CP) [4,5]. SMV causes obvious phenotypic changes including leaf discoloration, mosaic, curling and dwarfing, and it further leads to severe symptoms such as necrosis and seed mottle [6,7].

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Conclusion