Molecular and Biological Characterization of an Isolate of Cucumber mosaic virus from Glycine soja by Generating its Infectious Full-genome cDNA Clones

Abstract

Molecular and biological characteristics of an isolate of Cucumber mosaic virus (CMV) from Glycine soja (wild soybean), named as CMV-209, was examined in this study. Comparison of nucleotide sequences and phylogenetic analyses of CMV-209 with the other CMV strains revealed that CMV-209 belonged to CMV subgroup I. However, CMV-209 showed some genetic distance from the CMV strains assigned to subgroup IA or subgroup IB. Infectious full-genome cDNA clones of CMV-209 were generated under the control of the Cauliflower mosaic virus 35S promoter. Infectivity of the CMV-209 clones was evaluated in Nicotiana benthamiana and various legume species. Our assays revealed that CMV-209 could systemically infect Glycine soja (wild soybean) and Pisum sativum (pea) as well as N. benthamiana, but not the other legume species.

Keywords: Cucumber mosaic virus; infectious clones; phylogenetic analyses; sequence analysis; wild soybean.

Fig. 1.

Schematic representation of the T-DNA vector, pSNU1, and CMV-209 cDNA constructs. The pSNU1 vector contains, in sequential order, a left border of T-DNA (LB), a double 35S promoter (P35Sx2), multiple cloning site (MCS), a cis-cleaving ribozyme sequence (Rz), a NOS terminator (NOSt), and a right border of T-DNA (RB). The constructs pCMV-209R1, pCMV-209R2 and pCMV-209R3 express RNA1, RNA2 and RNA2 of CMV strain 209, respectively. The restriction enzyme cleavage sites used to make the constructs are shown.

Fig. 2.

Infectivity of pCMV-209. Mock (A) represents healthy plant. pCMV-209 induced latent symptom in Nicotiana benthamiana (B) while pCMV-Fny caused typical symptoms including mosaic, stunting, or distortion of leaves (C). Photographs were taken at 13 days post-inoculation. (D) Detection of progeny viruses in N. benthamiana plants inoculated with CMV strains. Total RNAs, isolated from upper uninoculated leaves of each N. benthaminana plant harvested at 7 days post-agroinfiltration, were analyzed by RT-PCR. M indicates a 100 bp DNA ladder (Bioneer). Lanes 1 to 3 represent results from three RNA samples extracted from three N. benthaminana plants.

Fig. 3.

Host range of CMV-209. CMV-209 induced severe stunting (A), rosetting (B) in Pisum sativum, and necrosis local lesions in inoculated Vigna sinensis leaves (C). CMV-Fny did not infect Pisum sativum (A). Mock represents healthy plant. CMV-209 and CMVFny represnt plants infected by CMV-209 and CMV-Fny, respectively. Photographs were taken at 18 days (A, B) and 9 days (C) post-inoculation.

Fig. 4.

Phylogenic trees of cucumoviruses obtained from analysis in MEGA 6 for each genomic segment (panels A, B, and C for RNA1, 2, and 3, respectively). Numbers at nodes indicate the percent occurrence of nodes in 1000 bootstrap resampling. Bootstrap values lower than 70 are not indicated. Roman numerals indicate respective CMV subgroups.

Fig. 5.

Phylogenic trees of cucumoviruses obtained from analysis in MEGA 6 for each ORF amino acid sequences. Numbers at nodes indicate the percent occurrence of nodes in 1000 bootstrap resampling. Bootstrap values lower than 70 are not indicated. Roman numerals indicate respective CMV subgroups.