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

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.

Development and Evaluation of Stable Sugarcane Mosaic Virus Mild Mutants for Cross-Protection Against Infection by Severe Strain

“…In the molecular arms race, potyvirus has evolved an effective RNA silencing suppressor (HC-Pro) to counteract the RNA silencing mechanism in plants (Anandalakshmi et al, 1998;Ivanov et al, 2016;Valli et al, 2018). Helper component-proteinase is a major virulence determinant of potyviruses and an important candidate for screening attenuated mutants (Seo et al, 2011;Huang et al, 2019;Bao et al, 2020;Cheng et al, 2020). Single or multiple amino acid mutations in the conserved motifs of HC-Pro affected potyviral virulence (Desbiez et al, 2010;Kung et al, 2014;Tuo et al, 2020).…”

Home-made enzymatic premix and Illumina sequencing allow for one-step Gibson assembly and verification of virus infectious clones

“…A full-length TVMV cDNA clone, under the T7 RNA polymerase promoter ( Domier et al 1989 ), was used as a control during method validation ( Additional file 1: Table S1 ). The pLX series includes mini T-DNA binary vectors (~ 3.5 kb) that autonomously replicate in E. coli and Agrobacterium , have plasmid stabilizing features and duplicated left borders to reduce backbone transfer to plants; the pLX vectors have been used in the assembly of plant virus infectious clones for agro-infection ( Pasin et al 2017 , 2018 ; Bao et al 2020 ; Klenov and Hudak 2021 ). A pLX-based vector that includes the cauliflower mosaic virus (CaMV) 35S promoter and nopaline synthase (nos) terminator sequences was linearized by PCR using primers #1F/#1R ( Fig.…”

GsRSS3L, a Candidate Gene Underlying Soybean Resistance to Seedcoat Mottling Derived from Wild Soybean (Glycine soja Sieb. and Zucc)

“…Therefore, N. benthamiana combined with an Agrobacterium -mediated infectious cDNA clone of plant virus has been widely used in studies of plant–virus interactions [ 42 , 43 , 44 ]. However, the host range of SMV is narrow and most of the SMV strains only systematically infect leguminous plants [ 45 ]. According to the reported study by Gao et al, SC7 (a novel SMV strain) could systematically infect N. benthamiana from 21 SMV strains in China (SC1-SC21) and construct its infectious cDNA clone [ 41 ].…”