Virus Genes

, Volume 53, Issue 2, pp 286–299 | Cite as

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

  • Chan-Hwan Park
  • Hye-Kyoung Ju
  • Jae-Yeong Han
  • Jong-Seo Park
  • Ik-Hyun Kim
  • Eun-Young Seo
  • Jung-Kyu Kim
  • John Hammond
  • Hyoun-Sub Lim


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 newly constructed binary vector pJY, which includes both the 35S and T7 promoters for versatile usage (agroinfiltration and in vitro RNA transcription) and a modified hepatitis delta virus 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; 49.85% to pepper mild mottle virus; 50.47% to tomato mosaic virus; 60.9% to zucchini green mottle mosaic virus; and 60.96% to kyuri green mottle mosaic virus, 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 nor 186 kDa RdRps of the three new isolates showed any detectable gene silencing suppressor function.


Cucumber green mottle mosaic virus (CGMMV) Full-length infectious cDNA clones Phylogenetic analysis Gene silencing suppressor function 



This research was supported by the Technology Commercialization Support Program (Project No. 113044–3), Ministry of Agriculture, Food and Rural Affairs.


  1. 1.
    D.B. Adams, C.W. Kuhn, seed transmission of peanut mottle virus in peanuts. Phytopathology. 67, 1126–1129 (1977)CrossRefGoogle Scholar
  2. 2.
    G.C. Ainsworth, Mosaic disease of cucumber. Ann. Appl. Biol. 22, 55–67 (1935)CrossRefGoogle Scholar
  3. 3.
    A. Ali, A. Hussain, M. Ahmad, Occurrence and molecular characterization of cucumber green mottle mosaic virus in cucurbit crops of KPK, Pakistan. Braz. J. Microbiol. 45, 1247–1253 (2015)CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Y. Antignus, M. Pearlsman, Rachel Ben-Yoseph, S. Cohen, Occurrence of a variant of cucumber green mottle mosaic virus in ISRAEL. Phytoparasitica 18, 50–56 (1990)CrossRefGoogle Scholar
  5. 5.
    T. Chujo, K. Ishibashi, S. Miyashita, M. Ishikawa, Functions of the 5′- and 3′-untranslated regions of tobamovirus RNA. Virus Res. 206, 82–89 (2015)CrossRefPubMedGoogle Scholar
  6. 6.
    N.C. Crowley, Studies on the seed transmission of plant virus diseases. Aust. J. Biol. Sci. 10, 449–464 (1957)CrossRefGoogle Scholar
  7. 7.
    T. Csorba, A. Bovi, T. Dalmay, J. Burgyán, The p122 subunit of tobacco mosaic virus replicase is a potent silencing suppressor and compromises both small interfering RNA- and MicroRNA-mediated pathways. J. Virol. 81, 11768–11780 (2007)CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    W.O. Dawson, D.L. Beck, D.A. Knorr, G.L. Grantham, cDNA cloning of the complete genome of tobacco mosaic virus and production of infectious transcripts. PNAS USA 83, 1832–1836 (1986)CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    L.L. Domier, H.A. Hobbs, N.K. McCoppin, C.R. Bowen, T.A. Steinlage, S. Chang, Y. Wang, G.L. Hartman, Multiple loci condition seed transmission of soybean mosaic virus (SMV) and SMV-Induced seed coat mottling in soybean. Phytopathology 10, 750–756 (2011)CrossRefGoogle Scholar
  10. 10.
    A. Ghanem, A. Kern, K.K. Conzeimann, Significantly improved rescue of rabies virus from cDNA plasmids. Eur. J. Cell Biol. 91(1), 10–16 (2012)CrossRefPubMedGoogle Scholar
  11. 11.
    M.M. Goodin, R.G. Dietzgen, D. Schichnes, S. Ruzin, A.O. Jackson, pGD vectors: versatile tools for the expression of green and red fluorescent protein fusions in agroinfiltrated plant leaves. Plant J. 31, 375–383 (2002)CrossRefPubMedGoogle Scholar
  12. 12.
    M.R. Hajimorad, A.L. Eggenberger, J.H. Hill, Evolution of soybean mosaic virus-G7 molecularly cloned genome in Rsv1-genotype soybean results in emergence of a mutant capable of evading Rsv1-mediated recognition. Virology 314, 497–509 (2001)CrossRefGoogle Scholar
  13. 13.
    C. Hemenway, J. Weiss, K. O’Connell, N.E. Tumer, Characterization of infectious transcripts from a potato virus X cDNA clone. Virology 175, 365–371 (1990)CrossRefPubMedGoogle Scholar
  14. 14.
    M. Ishikawa, T. Meshi, F. Motoyoshi, N. Takamatsu, Y. Okada, In vitro mutagenesis of the putative replicase genes of tobacco mosaic virus. Nucleic Acids Res. 14, 8291–8305 (1986)CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    E. Johansen, M.C. Edwards, R.O. Hampton, Seed transmission of viruses: current perspectives. Annu. Rev. Phytopathol. 32, 363–386 (1994)CrossRefGoogle Scholar
  16. 16.
    B.R. Junqueira, C. Nicolini, N. Lucinda, A.F. Orílio, T. Nagata, A simplified approach to construct infectious cDNA clones of a tobamovirus in a binary vector. J. Virol. Methods 198, 32–36 (2014)CrossRefPubMedGoogle Scholar
  17. 17.
    T. Kavanagh, M. Goulden, S. Santa Cruz, S. Chapman, I. Barker, D. Baulcombe, Molecular analysis of a resistance-breaking strain of potato virus X. Virology 189, 609–617 (1992)CrossRefPubMedGoogle Scholar
  18. 18.
    M. Kang, J.K. Seo, D. Song, H.S. Choi, K.H. Kim, Establishment of an Agrobacterium-mediated inoculation system for cucumber green mottle mosaic virus. Plant Pathol. J. 31, 433–437 (2015)CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    J.S. Kim, S.H. Lee, H.S. Choi, M.K. Kim, H.R. Kwak, J.S. Kim, M. Nam, J.D. Cho, I.S. Cho, G.S. Choi, 2007–2011 Characteristics of plant virus infections on crop samples submitted from agricultural places. Res. Plant Dis. 18, 277–289 (2012)CrossRefGoogle Scholar
  20. 20.
    J.S. Kim, S.H. Lee, H.S. Choi, G.S. Choi, J.D. Cho, B.N. Chung, Survey of viral diseases occurrence on major crops in 2007. Res. Plant Dis. 14, 1–9 (2008)CrossRefGoogle Scholar
  21. 21.
    J.S. Kim, S.H. Lee, H.S. Choi, M.K. Kim, H.R. Kwak, M. Nam, J.S. Kim, G.S. Choi, J.D. Cho, I.S. Cho, B.N. Chung, Occurrence of virus diseases on major crops in 2010. Res. Plant Dis. 17, 334–341 (2011)CrossRefGoogle Scholar
  22. 22.
    O.K. Kim, T. Mizutani, K.T. Natsuaki, K.W. Lee, K. Soe, First report and the genetic variability of cucumber green mottle mosaic virus occurring on bottle gourd in Myanmar. J. Phytopathol. 158, 572–575 (2010)CrossRefGoogle Scholar
  23. 23.
    S.M. Kim, J.M. Lee, K.O. Yim, M.H. Oh, J.W. Park, K.H. Kim, Nucleotide sequences of two Korean isolates of cucumber green mottle mosaic virus. Mol. Cells 16, 407–412 (2003)PubMedGoogle Scholar
  24. 24.
    Y. Komuro, Cucumber green mottle mosaic virus on cucumber and watermelon and melon necrotic spot virus on muskmelon. Jpn. Agric. Res. Q. 6, 41–45 (1971)Google Scholar
  25. 25.
    Y. Komuro, H. Tochihara, R. Fukatsu, Y. Nagai, S. Yoneyama, Cucumber green mottle mosaic virus (watermelon strain) in watermelon and its bearing on deterioration of watermelon fruit known as “Konnyaku” disease. Ann. Phytopathol. Soc. Jpn. 37, 34–42 (1971)CrossRefGoogle Scholar
  26. 26.
    S.J. Ko, Y.H. Lee, K.H. Cha, J.W. Park, H.G. Choi, Detection of CGMMV from commercial cucumber seed and resistance test of cultivars. Res. Plant Dis. 10, 154–158 (2004)CrossRefGoogle Scholar
  27. 27.
    Ko Sj, Y.H. Lee, T.S. Lee, K.Y. Yang, J.W. Park, H.S. Choi, Influence of CGMMV infection times on growth and quality of watermelon and cucumber. Res. Plant Dis. 10, 48–52 (2004)CrossRefGoogle Scholar
  28. 28.
    K. Kubota, S. Tsuda, A. Tamai, T. Meshi, Tomato mosaic virus replication protein suppresses virus-targeted posttranscriptional gene silencing. J. Virol. 77, 11016–11026 (2003)CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Y. Kurihara, N. Inaba, N. Kutsuna, A. Takeda, Y. Tagami, Y. Watanabe, Binding of tobamovirus replication protein with small RNA duplexes. J. Gen. Virol. 88, 2347–2352 (2007)CrossRefPubMedGoogle Scholar
  30. 30.
    J.X. Li, S.S. Liu, Q.S. Gu, Transmission efficiency of cucumber green mottle mosaic virus via seeds, soil, pruning and irrigation water. J. Phytopathol. 164, 300–309 (2016)CrossRefGoogle Scholar
  31. 31.
    H.S. Lim, A.M. Vaira, L.L. Domier, S.C. Lee, H.G. Kim, J. Hammond, Efficiency of VIGS and gene expression in a novel bipartite potexvirus vector delivery system as a function of strength of TGB1 silencing suppression. Virology 402(1), 149–163 (2010)CrossRefPubMedGoogle Scholar
  32. 32.
    M.J. Li, J.K. Kim, E.Y. Seo, S.M. Hong, E.I. Hwang, J.K. Moon, L.L. Domier, J. Hammond, Y.N. Youn, H.S. Lim, Sequence variability in HC-Pro genes of Korean soybean mosaic virus isolates is associated with differences in gene silencing suppression. Arch. Virol. 159, 1373–1383 (2014)CrossRefPubMedGoogle Scholar
  33. 33.
    R. Li, Y. Zheng, Z. Fei, K.S. Ling, First complete genome sequence of an emerging cucumber green mottle mosaic virus isolate in North America. Genome Announc. 3(3), pii: e00452-15 (2015). doi: 10.1128/genomeA.00452-15 Google Scholar
  34. 34.
    H.W. Liu, L.X. Luo, J.Q. Li, P.F. Liu, X.Y. Chen, J.J. Hao, Pollen and seed transmission of cucumber green mottle mosaic virus in cucumber. Plant Pathol. 63, 72–77 (2014). doi: 10.1111/ppa.12065 CrossRefGoogle Scholar
  35. 35.
    Y. Liu, Y. Wang, X. Wang, G. Zhou, Molecular characterization and distribution of cucumber green mottle mosaic virus in China. J. Phytopathol. 157, 393–399 (2009)CrossRefGoogle Scholar
  36. 36.
    S. Mandal, B. Mandal, Q. Mohd, R. Haq, A. Varma, Properties, diagnosis and management of cucumber green mottle mosaic virus. Plant Virus 2, 25–34 (2008)Google Scholar
  37. 37.
    T. Meshi, M. Ishikawa, F. Motoyoshi, K. Semba, Y. Okada, In vitro transcription of infectious RNAs from full-length cDNAs of tobacco mosaic virus. PNAS USA 83, 5043–5047 (1986)CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    G.I. Mink, Pollen and seed-transmitted viruses and viroids. Annu. Rev. Phytopathol. 31, 375–402 (1993)CrossRefPubMedGoogle Scholar
  39. 39.
    T.A.M. Osman, C.L. Hemenway, K.W. Buck, Role of the tRNA-like structure in tobacco mosaic virus minus-strand RNA synthesis by the viral RNA-dependent RNA polymerase in vitro. J. Virol. 74, 11671–11680 (2000)CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    S.J. Park, J.H. Lee, M. Nam, C.Y. Park, J.S. Kim, J.H. Lee, E.S. Jun, J.S. Lee, H.S. Choi, J.S. Kim, J.S. Moon, H.G. Kim, S.H. Lee, Virus disease incidences and transmission ecology of oriental melons in Seongju area. Res. Plant Dis. 17, 342–350 (2011)CrossRefGoogle Scholar
  41. 41.
    V. Reingold, O. Lachman, E. Blaosov, A. Dombrovsky, Seed disinfection treatments do not sufficiently eliminate the infectivity of cucumber green mottle mosaic virus (CGMMV) on cucurbit seed. Plant Pathol. 64, 245–255 (2015)CrossRefGoogle Scholar
  42. 42.
    H.B. Scholthof, Rapid delivery of foreign genes into plants by direct rub-inoculation with intact plasmid DNA of a tomato bushy stunt virus gene vector. J. Virol. 73(9), 7823–7829 (1999)PubMedPubMedCentralGoogle Scholar
  43. 43.
    D. Steinhauer, E. Domingo, J.J. Holland, Lack of evidence for proofreading mechanisms associated with an RNA virus polymerase. Gene 122, 281–288 (1992)CrossRefPubMedGoogle Scholar
  44. 44.
    N. Takamatsu, Y. Watanabe, T. Meshi, Y. Okada, Mutational analysis of the pseudoknot region in the 3′ noncoding region of tobacco mosaic virus RNA. J. Virol. 64, 3686–3693 (1990)PubMedPubMedCentralGoogle Scholar
  45. 45.
    P.G. Teoh, A.S. Ooi, S. AbuBakar, R.Y. Othman, Virus-specific read-through codon preference affects infectivity of chimeric cucumber green mottle mosaic viruses displaying a dengue virus epitope. J. Biomed. Biotechnol. (2009). doi: 10.1155/2009/781712 PubMedPubMedCentralGoogle Scholar
  46. 46.
    T.H. Turpen, A.M. Turpen, N. Weinzettl, M.H. Kumagai, W.O. Dawson, Transfection of whole plants from wounds inoculated with Agrobacterium tumefaciens containing cDNA of tobacco mosaic virus. J. Virol. Methods 42, 227–239 (1993)CrossRefPubMedGoogle Scholar
  47. 47.
    M. Ugaki, M. Tomiyama, T. Kakutani, S. Hidaka, T. Kiguchi, R. Nagata, T. Sato, F. Motoyoshi, M. Nishiguchi, The complete nucleotide sequence of cucumber green mottle mosaic virus (SH strain) genomic RNA. J. Gen. Virol. 72, 1487–1495 (1991)CrossRefPubMedGoogle Scholar
  48. 48.
    K. Vemana, R.K. Jain, New experimental hosts of tobacco streak virus and absence of true seed transmission in leguminous hosts. Indian J. Virol. 21, 117–127 (2010)CrossRefPubMedGoogle Scholar
  49. 49.
    D. Wang, A.J. Maule, A model for seed transmission of a plant virus: genetic and structural analyses of pea embryo invasion by Pea seed-borne mosaic virus. Plant Cell 6, 777–787 (1994)CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    T. Watanabe, A. Honda, A. Iwata, S. Ueda, T. Hibi, A. Ishihama, Isolation from tobacco mosaic virus-infected tobacco of a solubilized template-specific RNA-dependent RNA polymerase containing a 126K/183K protein heterodimer. J. Virol. 73, 2633–2640 (1999)PubMedPubMedCentralGoogle Scholar
  51. 51.
    J.Y. Yoon, G.S. Choi, S.K. Choi, J.S. Hong, J.K. Choi, W. Kim, G.P. Lee, K.H. Ryu, Molecular and biological; diversities of cucumber green mottle mosaic virus from cucurbitaceous crops in Korea. J. Phytopathol. 156, 408–412 (2008)CrossRefGoogle Scholar
  52. 52.
    J.Y. Yoon, B.E. Min, S.H. Choi, K.H. Ryu, Completion of nucleotide sequence and generation of highly infectious transcripts to cucurbits from full-length cDNA clone of kyuri green mottle mosaic virus. Arch. Virol. 146, 2085–2096 (2001)CrossRefPubMedGoogle Scholar
  53. 53.
    H. Zheng, C. Xiao, K. Han, J. Peng, L. Lin, Y. Lu, L. Xie, X. Wu, P. Xu, G. Li, J. Chen, F. Yan, Development of an agroinoculation system for full-length and GFP-tagged cDNA clone of cucumber green mottle mosaic virus. Arch. Virol. 160, 2867–2872 (2015)CrossRefPubMedGoogle Scholar
  54. 54.
    M. Zhong, X. Zhao, Y. Liu, Y. Wang, K. Cao, Completion sequence and cloning of the infectious cDNA of a chb isolate of cucumber green mottle mosaic virus. Acta Virol. 59, 49–56 (2015)CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Chan-Hwan Park
    • 1
  • Hye-Kyoung Ju
    • 1
  • Jae-Yeong Han
    • 1
  • Jong-Seo Park
    • 1
  • Ik-Hyun Kim
    • 1
  • Eun-Young Seo
    • 1
  • Jung-Kyu Kim
    • 1
  • John Hammond
    • 2
  • Hyoun-Sub Lim
    • 1
  1. 1.Department of Applied Biology, College of Agriculture and Life SciencesChungnam National UniversityDaejeonKorea
  2. 2.United States Department of Agriculture - Agricultural Research ServiceUnited States National Arboretum, Floral and Nursery Plants Research UnitBeltsvilleUSA

Personalised recommendations