Complete nucleotide sequences and construction of full-length infectious cDNA clones of Cucumber green mottle virus (CGMMV) in a versatile newly developed binary vector including both 35S and T7 promoters

Authors: PARK, CHAN-HWAN - Chungnam National University; JU, HYE-KYOUNG - Chungnam National University; HAN, JAE-YEONG - Chungnam National University; PARK, JONG-SEO - Chungnam National University; KIM, IK-HYUN - Chungnam National University; SEO, EUN-YOUNG - Chungnam National University; KIM, JUNG-KYU - Chungnam National University; Hammond, John; LIM, HYOUN-SUB - Chungnam National University

Submitted to: Virus Genes
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/24/2016
Publication Date: 12/2/2016
Citation: Park, C., Ju, H., Han, J., Park, J., Kim, I., Seo, E., Kim, J., Hammond, J., Lim, H. 2016. Complete nucleotide sequences and construction of full-length infectious cDNA clones of Cucumber green mottle virus (CGMMV) in a versatile newly developed binary vector including both 35S and T7 promoters. Virus Genes. 53:286-299.

Interpretive Summary: Plant virus infections are a significant constraint on crop productivity; seed-transmitted viruses are among the most damaging, because they are introduced into the crop from the very early stages of plant growth and have the potential to spread further during the growing season. Cucumber green mottle mosaic virus (CGMMV) has caused major crop losses in Korea, and has recently been introduced into the United States in contaminated seed; treatment of seed to inactivate the virus has generally proven ineffective. Korean isolates of CGMMV from watermelon, melon, and oriental melon were sequenced, and infectious clones produced and characterized. Differences in nucleotide sequence and symptom severity between these isolates will be useful to screen germplasm and breeding lines for possible virus resistance, and to identify possible differences in seed transmissibility. Identification of viral determinants of seed transmission and disease severity will contribute towards disease management and increased crop productivity.

Technical Abstract: Seed-transmitted viruses have caused significant damage to watermelon crops in Korea in recent years, with Cucumber green mottle mosaic virus (CGMMV) infection widespread as a result of infected seed lots. To determine the likely origin of CGMMV infection, we collected CGMMV isolates from watermelon and melon fields and generated full-length infectious cDNA clones. The full-length cDNAs were cloned into a newly-constructed binary vector pJY, which includes the 35S and T7 promoters for versatile usage (Agroinfiltration and in vitro RNA transcription) and a modified Hepatitis delta virus (HDV) ribozyme sequence to precisely cleave RNA transcripts at the 3’ end of the tobamovirus genome. Three CGMMV isolates (OMpj, Wpj, and Mpj) were separately evaluated for infectivity in Nicotiana benthamiana, demonstrated by either Agroinfiltration or inoculation with in vitro RNA transcripts. CGMMV nucleotide identities to other tobamoviruses were calculated from pairwise alignments using DNAMAN. CGMMV identities were 49.89% to Tobacco mosaic virus (TMV); 49.85% to Pepper mild mottle virus (PMMoV); 50.47% to Tomato mosaic virus (ToMV); 60.9% to Zucchini green mottle mosaic virus (ZGMMV); and 60.96% to Kyuri green mottle mosaic virus (KGMMV), confirming that CGMMV is a distinct species most similar to other cucurbit-infecting tobamoviruses. We further performed phylogenetic analysis to determine relationships of our new Korean CGMMV isolates to previously characterized isolates from Canada, China, India, Israel, Japan, Korea, Russia, Spain and Taiwan available from NCBI. Analysis of CGMMV amino acid sequences showed three major clades, broadly typified as ‘Russian’, ‘Israeli’, and ‘Asian’ groups. All of our new Korean isolates fell within the ‘Asian’ clade. Neither the 128 kDa nor 186 kDa RdRps of the three new isolates showed any detectable gene silencing suppressor function.