Archives of Virology

, Volume 164, Issue 1, pp 181–194 | Cite as

Molecular characterization and analysis of conserved potyviral motifs in bean common mosaic virus (BCMV) for RNAi-mediated protection

  • Elizabeth A. Worrall
  • Alice C. Hayward
  • Stephen J. Fletcher
  • Neena MitterEmail author
Original Article


Australian bean common mosaic virus (BCMV) isolates were sequenced, and the sequences were compared to global BCMV and bean common mosaic necrosis virus (BCMNV) sequences and analysed for conserved potyviral motifs to generate in planta RNA-interference (RNAi) resistance. Thirty-nine out of 40 previously reported potyvirus motifs were conserved among all 77 BCMV/BCMNV sequences. Two RNAi target regions were selected for dsRNA construct design, covering 920 bp of the nuclease inclusion b (NIb) protein and 461 bp of the coat protein (CP). In silico prediction of the effectiveness of these constructs for broad-spectrum defence against the 77 BCMV and BCMNV sequences was done via analysis of putative 21-nucleotide (nt) and 22-nt small-interfering RNAs (siRNAs) generated from the target regions. The effectiveness of both constructs for siRNA generation and BCMV RNAi-mediated resistance was validated in Nicotiana benthamiana transient assays.



The authors would like to thank Queensland Alliance of Agriculture and Food Innovation (QAAFI), Australia and the University of Queensland, Australia, for their support.


This study was funded by the Accelerated Partnership Grant, Queensland Government (2014000652), awarded to N.M. with Nufarm Australia Limited as the industry partner. E.A.W. PhD programme with N.M. is supported by a scholarship from the University of Queensland.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Data availability

The datasets generated and/or analysed during the current study are available in the NCBI GenBank repository, Only complete genomes of BCMV and BCMNV were used.

Supplementary material

705_2018_4065_MOESM1_ESM.doc (1.8 mb)
Supplementary material 1 (DOC 1792 kb)


  1. 1.
    International Service for the Acquisition of Agri-Biotech Applications (ISAAA) (2018) Bean (Phaseolus vulgaris) GM EventsGoogle Scholar
  2. 2.
    Aleman-Verdaguer M-E, Goudou-Urbino C, Dubern J, Beachy RN, Fauquet C (1997) Analysis of the sequence diversity of the P1, HC, P3, NIb and CP genomic regions of several yam mosaic potyvirus isolates: implications for the intraspecies molecular diversity of potyviruses. J Gen Virol 78:1253–1264CrossRefPubMedGoogle Scholar
  3. 3.
    Anuradha C, Balasubramanian V, Selvarajan R (2015) Sequence motif comparison and homology modeling of helper component proteinase (HC–Pro) of banana bract mosaic virus.Google Scholar
  4. 4.
    Bartel D (2004) MicroRNAs: genomics, biogensis, mechanism and function. Cell 116:281–297CrossRefPubMedGoogle Scholar
  5. 5.
    Baulcombe D (2004) RNA silencing in plants. Nature 431:356–363CrossRefPubMedGoogle Scholar
  6. 6.
    Bhadramurthy V, Bhat A (2009) Biological and molecular characterization of Bean common mosaic virus associated with vanilla in India. Indian J Virol 20:70–77Google Scholar
  7. 7.
    Bonfim K, Faria JC, Nogueira EO, Mendes ÉA, Aragão FJ (2007) RNAi-mediated resistance to Bean golden mosaic virus in genetically engineered common bean (Phaseolus vulgaris). Mol Plant-microbe Interact 20:717–726CrossRefPubMedGoogle Scholar
  8. 8.
    Borges F, Martienssen RA (2015) The expanding world of small RNAs in plants. Nat Rev Mol Cell Biol 16:727CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Bravo E, Calvert LA, Morales FJ (2008) The complete nucleotide sequence of the genomic RNA of bean common mosaic virus strain nl4. Rev Acad Colomb Cienc Exactas 32:37–46Google Scholar
  10. 10.
    Brosnan C, Mitter N, Christie M, Smith N, Waterhouse P, Carroll B (2007) Nuclear gene silencing directs reception of long-distance mRNA silencing in Arabidopsis. Proc Natl Acad Sci 104:14741–14746CrossRefPubMedGoogle Scholar
  11. 11.
    Burand JP, Hunter WB (2013) RNAi: Future in insect management. J Invertebr Pathol 112:S68–S74CrossRefPubMedGoogle Scholar
  12. 12.
    Cheng G, Dong M, Xu Q, Peng L, Yang Z, Wei T, Xu J (2017) Dissecting the molecular mechanism of the subcellular localization and cell-to-cell movement of the sugarcane mosaic virus P3N-PIPO. Sci Rep 7:9868CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Damayanti T, Susilo D, Nurlaelah S, Sartiami D, Okuno T, Mise K (2008) First report of Bean common mosaic virus in yam bean [Pachyrhizus erosus (L.) Urban] in Indonesia. J Gen Plant Pathol 74:438–442CrossRefGoogle Scholar
  14. 14.
    Duan C-G, Wang C-H, Guo H-S (2012) Application of RNA silencing to plant disease resistance. Silence 3:5CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    El-Sawy MA, Mohamed HAE, Elsharkawy MM (2013) Serological and molecular characterisations of the Egyptian isolate of Bean common mosaic virus. Arch Phytopathol Plant Protect 47:1–13Google Scholar
  16. 16.
    Flores-Estévez N, Acosta-Gallegos J, Silva-Rosales L (2003) Bean common mosaic virus and Bean common mosaic necrosis virus in Mexico. Plant disease 87:21–25CrossRefPubMedGoogle Scholar
  17. 17.
    Gleave AP (1992) A versatile binary vector system with a T-DNA organisational structure conducive to efficient integration of cloned DNA into the plant genome. Plant Mol Biol 20:1203–1207CrossRefPubMedGoogle Scholar
  18. 18.
    Gong D, Wang J-H, Lin Z-S, Zhang S-Y, Zhang Y-L, Yu N-T, Xiong Z, Liu Z-X (2011) Genomic sequencing and analysis of Chilli ringspot virus, a novel potyvirus. Virus Genes 43:439–444CrossRefPubMedGoogle Scholar
  19. 19.
    Gordon KH, Waterhouse PM (2007) RNAi for insect-proof plants. Nat Biotechnol 25:1231–1232CrossRefPubMedGoogle Scholar
  20. 20.
    Guyatt K, Proll D, Menssen A, Davidson A (1996) The complete nucleotide sequence of bean yellow mosaic potyvirus RNA. Arch Virol 141:1231–1246CrossRefPubMedGoogle Scholar
  21. 21.
    Hamid A, Ahmad M, Padder B, Shah M, Saleem S, Sofi T, Mir A (2013) Pathogenic and coat protein characterization confirming the occurrence of Bean common mosaic virus on common bean (Phaseolus vulgaris) in Kashmir, India. Phytoparasitica 42:1–6Google Scholar
  22. 22.
    Ivanov K, Eskelin K, Lõhmus A, Mäkinen K (2014) Molecular and cellular mechanisms underlying potyvirus infection. J Gen Virol 95:1415–1429CrossRefPubMedGoogle Scholar
  23. 23.
    Ivanov KI, Mäkinen K (2012) Coat proteins, host factors and plant viral replication. Curr Opin Virol 2:712–718CrossRefPubMedGoogle Scholar
  24. 24.
    Kamenova I, Lohuis H, Peters D (2002) Loss of aphid transmissibility of plum pox virus isolates. Biotechnol Biotechnol Equip 16:48–54CrossRefGoogle Scholar
  25. 25.
    Kelly J, Afanador L, Haley S (1995) Pyramiding genes for resistance to Bean common mosaic virus. Euphytica 82:207–212CrossRefGoogle Scholar
  26. 26.
    Knierim D, Menzel W, Winter S (2017) Analysis of the complete genome sequence of euphorbia ringspot virus, an atypical member of the genus Potyvirus. Arch Virol 162:291–293CrossRefPubMedGoogle Scholar
  27. 27.
    Koch A, Biedenkopf D, Furch A, Weber L, Rossbach O, Abdellatef E, Linicus L, Johannsmeier J, Jelonek L, Goesmann A (2016) An RNAi-based control of Fusarium graminearum infections through spraying of long dsRNAs involves a plant passage and is controlled by the fungal silencing machinery. PLoS Pathog 12:e1005901CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Kwapata K, Nguyen T, Sticklen M (2012) Genetic transformation of common bean (Phaseolus vulgaris L.) with the gus color marker, the bar herbicide resistance, and the barley (Hordeum vulgare) HVA1 drought tolerance genes. Int J Agron 2012:1–8CrossRefGoogle Scholar
  29. 29.
    Li F, Xu D, Abad J, Li R (2012) Phylogenetic relationships of closely related potyviruses infecting sweet potato determined by genomic characterization of Sweet potato virus G and Sweet potato virus 2. Virus Genes 45:118–125CrossRefPubMedGoogle Scholar
  30. 30.
    Li Y, Jia A, Qiao Y, Xiang J, Zhang Y, Wang W (2017) Virome analysis of lily plants reveals a new potyvirus. Arch Virol 163:1–4Google Scholar
  31. 31.
    Li YQ, Liu ZP, Yang YS, Zhao B, Fan ZF, Wan P (2014) First report of Bean common mosaic virus infecting Azuki bean (Vigna angularis Ohwi & Ohashi) in China. Plant Disease 98:1017CrossRefPubMedGoogle Scholar
  32. 32.
    Liang WX, Song LM, Tian GZ, Li HF, Fan ZF (2006) The genomic sequence of Wisteria vein mosaic virus and its similarities with other potyviruses. Arch Virol 151:2311–2319CrossRefPubMedGoogle Scholar
  33. 33.
    Lilley C, Davies L, Urwin P (2012) RNA interference in plant parasitic nematodes: a summary of the current status. Parasitology 139:630–640CrossRefPubMedGoogle Scholar
  34. 34.
    Lin J-J (1995) Electrotransformation of Agrobacterium. 171–178Google Scholar
  35. 35.
    Lopez-Moya J, Wang R, Pirone T (1999) Context of the coat protein DAG motif affects potyvirus transmissibility by aphids. J Gen Virol 80:3281–3288CrossRefPubMedGoogle Scholar
  36. 36.
    Maillard P, Ciaudo C, Marchais A, Li Y, Jay F, Ding S, Voinnet O (2013) Antiviral RNA interference in mammalian cells. Science 342:235–238CrossRefPubMedGoogle Scholar
  37. 37.
    Mangrauthia SK, Jain R, Praveen S (2008) Sequence motifs comparisons establish a functional portrait of a multifunctional protein HC-Pro from papaya ringspot potyvirus. J Plant Biochem Biotechnol 17:201–204CrossRefGoogle Scholar
  38. 38.
    Mann KS, Johnson KN, Dietzgen RG (2015) Cytorhabdovirus phosphoprotein shows RNA silencing suppressor activity in plants, but not in insect cells. Virology 476:413–418CrossRefPubMedGoogle Scholar
  39. 39.
    Maoka T, Hataya T (2005) The complete nucleotide sequence and biotype variability of Papaya leaf distortion mosaic virus. Phytopathology 95:128–135CrossRefPubMedGoogle Scholar
  40. 40.
    Mishra R, Verma RK, Gaur RK (2015) Analysis of genome comparison of two Indian isolates of Cowpea aphid-borne mosaic virus from India. Virus Genes 51:306–309CrossRefPubMedGoogle Scholar
  41. 41.
    Mitter N, Worrall EA, Robinson KE, Li P, Jain RG, Taochy C, Fletcher SJ, Carroll BJ, Lu G, Xu ZP (2017) Clay nanosheets for topical delivery of RNAi for sustained protection against plant viruses. Nat Plants 3:16207CrossRefPubMedGoogle Scholar
  42. 42.
    Moghal S, Francki R (1976) Towards a system for the identification and classification of potyviruses: I. Serology and amino acid composition of six distinct viruses. Virology 73:350–362CrossRefPubMedGoogle Scholar
  43. 43.
    Moradi Z, Mehrvar M, Nazifi E, Zakiaghl M (2017) Iranian johnsongrass mosaic virus: the complete genome sequence, molecular and biological characterization, and comparison of coat protein gene sequences. Virus Genes 53:77–88CrossRefPubMedGoogle Scholar
  44. 44.
    Moura MF, Marubayashi JM, Mituti T, Gioria R, Kobori RF, Pavan MA, Krause-Sakate R (2012) Comparative analysis of coding region for the coat protein of PepYMV and PVY isolates collected in sweetpepper. Summa Phytopathol 38:93–96CrossRefGoogle Scholar
  45. 45.
    Mukeshimana G, Paneda A, Rodríguez-Suárez C, Ferreira JJ, Giraldez R, Kelly JD (2005) Markers linked to the bc-3 gene conditioning resistance to bean common mosaic potyviruses in common bean. Euphytica 144:291–299CrossRefGoogle Scholar
  46. 46.
    Pasev G, Kostova D, Sofkova S (2014) Identification of genes for resistance to Bean common mosaic virus and Bean common mosaic necrosis virus in snap bean (Phaseolus vulgaris L.) breeding lines ssing conventional and molecular methods. J Phytopathol 162:19–25CrossRefGoogle Scholar
  47. 47.
    Perotto MC, Pozzi EA, Celli MG, Luciani CE, Mitidieri MS, Conci VC (2018) Identification and characterization of a new potyvirus infecting cucurbits. Arch Virol 163:719–724CrossRefPubMedGoogle Scholar
  48. 48.
    Puli’uvea C, Khan S, Chang W-L, Valmonte G, Pearson MN, Higgins CM (2017) First complete genome sequence of vanilla mosaic strain of Dasheen mosaic virus isolated from the Cook Islands. Arch Virol 162:591–595CrossRefPubMedGoogle Scholar
  49. 49.
    Rajamäki M, Merits A, Rabenstein F, Andrejeva J, Paulin L, Kekarainen T, Kreuze J, Forster R, Valkonen J (1998) Biological, serological, and molecular differences among isolates of potato A potyvirus. Phytopathology 88:311–321CrossRefPubMedGoogle Scholar
  50. 50.
    Revers F, Garcia J (2015) Molecular biology of potyviruses. Adv Virus Res 92:101–199CrossRefPubMedGoogle Scholar
  51. 51.
    Rojas MR, Zerbini FM, Allison RF, Gilbertson RL, Lucas WJ (1997) Capsid protein and helper component-proteinase function as potyvirus cell-to-cell movement proteins. Virology 237:283–295CrossRefPubMedGoogle Scholar
  52. 52.
    Saqib M, Jones RAC, Cayford B, Jones MGK (2005) First report of Bean common mosaic virus in Western Australia. Plant Pathol 54:563CrossRefGoogle Scholar
  53. 53.
    Saqib M, Nouri S, Cayford B, Jones RAC, Jones MGK (2010) Genome sequences and phylogenetic placement of two isolates of Bean common mosaic virus from Macroptilium atropurpureum in north-west Australia. Aust Plant Pathol Soc 39:184–191CrossRefGoogle Scholar
  54. 54.
    Sastry KS (2013) Mechanism of seed transmission. Seed-borne plant virus diseases. Springer, Heidelberg, pp 85–100CrossRefGoogle Scholar
  55. 55.
    Sengooba TN, Spence NJ, Walkey DGA, Allen DJ, Femi Lana A (1997) The occurrence of Bean common mosaic necrosis virus in wild and forage legumes in Uganda. Plant Pathol 46:95–103CrossRefGoogle Scholar
  56. 56.
    Shapter FM, Waters DL Genome walking T cereal genomics, pp 133–146Google Scholar
  57. 57.
    Shukla DD, Frcnkel M, Ward CW (1991) Structure and function of the potyvirus genome with special reference to the coat protein coding region. Can J Plant Pathol 13:178–191CrossRefGoogle Scholar
  58. 58.
    Silbernagel MJ, Mink GI, Zhao RL, Zheng GY (2001) Phenotypic recombination between bean common mosaic and bean common mosaic necrosis potyviruses in vivo. Arch Virol 146:1007–1020CrossRefPubMedGoogle Scholar
  59. 59.
    Singh SP, Schwartz HF (2010) Breeding common bean for resistance to diseases: a review. Crop Sci 50:2199–2223CrossRefGoogle Scholar
  60. 60.
    Spence N, Walkey D (1995) Variation for pathogenicity among isolates of Bean common mosaic virus in Africa and a reinterpretation of the genetic relationship between cultivars of Phaseolus vulgaris and pathotypes of BCMV. Plant Pathol 44:527–546CrossRefGoogle Scholar
  61. 61.
    Urchqui-Inchima S, Haenni A-L, Bernardi F (2001) Potyvirus proteins: a wealth of functions. Virus Res 74:157–175CrossRefGoogle Scholar
  62. 62.
    Valli A, López-Moya JJ, García JA (2007) Recombination and gene duplication in the evolutionary diversification of P1 proteins in the family Potyviridae. J Gen Virol 88:1016–1028CrossRefPubMedGoogle Scholar
  63. 63.
    Velasco L, Salem N, Willemsen A, Lapidot M, Mansour AN, Rubio L, Galipienso L (2016) Genetic variation and evolutionary forces shaping Cucumber vein yellowing virus populations: risk of emergence of virulent isolates in Europe. Plant Pathol 65:847–856CrossRefGoogle Scholar
  64. 64.
    Verma P, Gupta U (2010) Immunological detection of Bean common mosaic virus in French bean (Phaseolus vulgaris L.) leaves. Indian j Microbiol 50:263–265CrossRefPubMedPubMedCentralGoogle Scholar
  65. 65.
    Worrall EA, Wamonje FO, Mukeshimana G, Harvey JJ, Carr JP, Mitter N (2015) Bean common mosaic virus and Bean common mosaic necrosis virus: relationships, biology, and prospects for control. Adv Virus Res 93:1–46CrossRefPubMedGoogle Scholar
  66. 66.
    Zheng L, Wayper PJ, Gibbs AJ, Fourment M, Rodoni BC, Gibbs MJ (2008) Accumulating variation at conserved sites in potyvirus genomes is driven by species discovery and affects degenerate primer design. PLoS One 3:e1586CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2018

Authors and Affiliations

  • Elizabeth A. Worrall
    • 1
  • Alice C. Hayward
    • 1
  • Stephen J. Fletcher
    • 1
  • Neena Mitter
    • 1
    Email author
  1. 1.Centre of Horticultural Science, Queensland Alliance for Agriculture and Food InnovationThe University of QueenslandBrisbaneAustralia

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