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Transient expression analysis of promoters of okra enation leaf curl virus in Nicotiana benthamiana, cotton and okra plants

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Abstract

Viral promoters can be used to drive heterologous gene expression in transgenic plants. As part of our quest to look for new promoters, we have explored, for the first time, the promoters of okra enation leaf curl virus (OELCuV), a begomovirus infecting okra (Abelmoschus esculentus). The Rep and CP promoters of OELCuV fused with the gfp reporter gene, were expressed transiently in the natural host okra and the laboratory host cotton and Nicotiana benthamiana. The expression levels of the promoters were quantified through confocal laser scanning microscopy and GFP assay in N. benthamiana and okra. The results indicated that the Rep promoter was more active than the CP promoter, whose activity was similar to that of CaMV 35S promoter. Additionally, the Rep and CP promoters showed increase of expression, probably due to transactivation, when assayed following inoculation of OELCuV and betasatellite DNAs in cotton plants. A moderate increase in promoter activity in N. benthamiana was also seen, when assayed following the inoculation of the heterologous begomovirus Sri Lankan cassava mosaic virus.

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References

  1. Porto MS, Pinheiro MPN, Batista VGL, dos Santos RC, de Albuquerque Melo Filho P, de Lima LM, (2014) Plant promoters: an approach of structure and function. Mol Biotechnol 56:38–49

    Article  CAS  PubMed  Google Scholar 

  2. Potenza C, Aleman L, Sengupta-Gopalan C (2004) Targeting transgene expression in research, agricultural, and environmental applications: promoters used in plant transformation. In Vitro Cell Dev Biol Plant 40:1–22

    Article  CAS  Google Scholar 

  3. Kummari D, Palakolanu SR, Kishor PBK, Bhatnagar-Mathur P, Singam P, Vadez V et al (2020) An update and perspectives on the use of promoters in plant genetic engineering. J Biosci 45:119. https://doi.org/10.1007/s12038-020-00087-6

    Article  CAS  PubMed  Google Scholar 

  4. Hernandez-Garcia CM, Finer JJ (2014) Identification and validation of promoters and cis-acting regulatory elements. Plant Sci 217:109–119

    Article  PubMed  Google Scholar 

  5. Zhan X, Haley A, Richardson K, Morris B (1991) Analysis of the potential promoter sequences of African cassava mosaic virus by transient expression of the β-glucuronidase gene. J Gen Virol 72:2849–2852

    Article  CAS  PubMed  Google Scholar 

  6. Cantú-Iris M, Pastor-Palacios G, Mauricio-Castillo JA, Bañuelos-Hernández B, Avalos-Calleros JA, Juárez-Reyes A et al (2019) Analysis of a new begomovirus unveils a composite element conserved in the CP gene promoters of several Geminiviridae genera: clues to comprehend the complex regulation of late genes. PLoS ONE 14:e0210485

    Article  PubMed  PubMed Central  Google Scholar 

  7. Ashraf MA, Shahid AA, Rao AQ, Bajwa KS, Husnain T (2014) Functional characterization of a bidirectional plant promoter from cotton leaf curl Burewala virus using an Agrobacterium-mediated transient assay. Viruses 6:223–242

    Article  PubMed  PubMed Central  Google Scholar 

  8. Khan ZA, Abdin MZ, Khan JA (2015) Functional characterization of a strong bi-directional constitutive plant promoter isolated from cotton leaf curl Burewala virus. PLoS ONE 10:e0121656

    Article  PubMed  PubMed Central  Google Scholar 

  9. Rasul F, Asad S, Zafar Y, Mansoor S (2014) Characterization of a strong constitutive promoter from cotton leaf curl Kokhran virus for high level gene expression in monocotyledonous and dicotyledonous plants. Int J Agric Biol 16:342–346

    CAS  Google Scholar 

  10. Xie Y, Liu Y, Meng M, Chen L, Zhu Z (2003) Isolation and identification of a super strong plant promoter from cotton leaf curl Multan virus. Plant Mol Biol 53:1–14

    Article  CAS  PubMed  Google Scholar 

  11. Mazithulela G, Sudhakar D, Heckel T, Mehlo L, Christou P, Davies JW et al (2000) The maize streak virus coat protein transcription unit exhibits tissue-specific expression in transgenic rice. Plant Sci 155:21–29

    Article  CAS  PubMed  Google Scholar 

  12. Shivaprasad PV, Akbergenov R, Trinks D, Rajeswaran R, Veluthambi K, Hohn T et al (2005) Promoters, transcripts, and regulatory proteins of mungbean yellow mosaic geminivirus. J Virol 79:8149–8163

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Sunitha S, Mahajan N, Veluthambi K (2012) The TrAP/REn monodirectional promoter of mungbean yellow mosaic geminivirus (MYMV) displays root-specific expression in transgenic tobacco. Plant Cell Tissue Organ Cult 109:535–545

    Article  CAS  Google Scholar 

  14. Usharani KS, Periasamy M, Malathi VG (2006) Studies on the activity of a bidirectional promoter of mungbean yellow mosaic India virus by agroinfiltration. Virus Res 119:154–162

    Article  CAS  PubMed  Google Scholar 

  15. Brough CL, Sunter G, Gardiner WE, Bisaro DM (1992) Kinetics of tomato golden mosaic virus DNA replication and coat protein promoter activity in Nicotiana tabacum protoplasts. Virology 187:1–9

    Article  CAS  PubMed  Google Scholar 

  16. Dry I, Krake L, Mullineaux P, Rezaian A (2000) Regulation of tomato leaf curl viral gene expression in host tissues. Mol Plant Microbe Interact 13:529–537

    Article  CAS  PubMed  Google Scholar 

  17. Hofer JM, Dekker EL, Reynolds HV, Woolston CJ, Cox BS, Mullineaux PM (1992) Coordinate regulation of replication and virion sense gene expression in wheat dwarf virus. Plant Cell 4:213–223

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Roumagnac P, Lett J-M, Fiallo-Olivé E, Navas-Castillo J, Zerbini FM, Martin DP et al (2022) Establishment of five new genera in the family Geminiviridae: Citlodavirus, Maldovirus, Mulcrilevirus, Opunvirus, and Topilevirus. Arch Virol 167:695–710

    Article  CAS  PubMed  Google Scholar 

  19. Varsani A, Roumagnac P, Fuchs M, Navas-Castillo J, Moriones E, Idris A et al (2017) Capulavirus and Grablovirus: two new genera in the family Geminiviridae. Arch Virol 162:1819–1831

    Article  CAS  PubMed  Google Scholar 

  20. Zerbini FM, Briddon RW, Idris A, Martin DP, Moriones E, Navas-Castillo J et al (2017) ICTV virus taxonomy profile: Geminiviridae. J Gen Virol 98:131

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Fiallo-Olivé E, Lett J-M, Martin DP, Roumagnac P, Varsani A, Zerbini FM et al (2021) ICTV virus taxonomy profile: Geminiviridae 2021. J Gen Virol 102:001696

    Article  PubMed  PubMed Central  Google Scholar 

  22. Eagle PA, Hanley-Bowdoin L (1997) cis elements that contribute to geminivirus transcriptional regulation and the efficiency of DNA replication. J Virol 71:6947–6955

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Fenoll C, Black DM, Howell SH (1988) The intergenic region of maize streak virus contains promoter elements involved in rightward transcription of the viral genome. EMBO J 7:1589–1596

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Morris-Krsinich BAM, Mullineaux PM, Donson J, Boulton MI, Markham PG, Short MN et al (1985) Bidirectional transcription of maize streak virus DNA and identification of the coat protein gene. Nucleic Acids Res 13:7237–7256

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Petty ITD, Coutts RHA, Buck KW (1988) Transcriptional mapping of the coat protein gene of tomato golden mosaic virus. J Gen Virol 69:1359–1365

    Article  CAS  Google Scholar 

  26. Ruiz-Medrano R, Guevara-Gonzalez RG, Argüello-Astorga GR, Monsalve-Fonnegra Z, Herrera-Estrella LR, Rivera-Bustamante RF (1999) Identification of a sequence element involved in AC2-mediated transactivation of the pepper huasteco virus coat protein gene. Virology 253:162–169

    Article  CAS  PubMed  Google Scholar 

  27. Borah BK, Zarreen F, Baruah G, Dasgupta I (2016) Insights into the control of geminiviral promoters. Virology 495:101–111

    Article  CAS  PubMed  Google Scholar 

  28. Hur J, Choi E, Buckley KJ, Lee S, Davis KR (2008) Identification of a promoter motif involved in Curtovirus sense-gene expression in transgenic Arabidopsis. Mol Cells 26:131–139

    Article  CAS  PubMed  Google Scholar 

  29. Sun R, Han J, Zheng L, Qu F (2020) The AC2 protein of a bipartite geminivirus stimulates the transcription of the BV1 gene through abscisic acid responsive promoter elements. Viruses 12:1403

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Hameed U, Zia-Ur-Rehman M, Herrmann H-W, Haider MS, Brown JK (2014) First report of Okra enation leaf curl virus and associated cotton leaf curl Multan betasatellite and cotton leaf curl Multan alphasatellite infecting cotton in Pakistan: a new member of the cotton leaf curl disease complex. Plant Dis 98:1447

    Article  CAS  PubMed  Google Scholar 

  31. Naresh M, Khan ZA, Kumar R, Kale SP, Patil VM, Rajput JC et al (2019) Occurrence and variability of begomoviruses associated with bhendi yellow vein mosaic and okra enation leaf curl diseases in south-western India. Virusdisease 30:511–525

    Article  PubMed  PubMed Central  Google Scholar 

  32. Emmanuel CJ, Manohara S, Shaw MW (2020) Molecular characterization of begomovirus–betasatellite–alphasatellite complex associated with okra enation leaf curl disease in Northern Sri Lanka. 3 Biotech 10:506

    Article  PubMed  PubMed Central  Google Scholar 

  33. Gupta K, Rishishwar R, Khan ZA, Dasgupta I (2022) Agrobacterium-mediated co-inoculation of okra plants with cloned okra enation leaf curl virus DNA and bhendi yellow vein mosaic beta-satellite DNA furthers Koch’s postulates for enation leaf curl disease. J Virol Methods 300:114413

    Article  CAS  PubMed  Google Scholar 

  34. Venkataravanappa V, Reddy CNL, Jalali S, Briddon RW, Reddy MK (2015) Molecular identification and biological characterisation of a begomovirus associated with okra enation leaf curl disease in India. Eur J Plant Pathol 141:217–235

    Article  CAS  Google Scholar 

  35. Saunders K, Salim N, Mali VR, Malathi VG, Briddon R, Markham PG et al (2002) Characterisation of Sri Lankan cassava mosaic virus and Indian cassava mosaic virus: evidence for acquisition of a DNA B component by a monopartite begomovirus. Virology 293:63–74

    Article  CAS  PubMed  Google Scholar 

  36. Holsters M, de Waele D, Depicker A, Messens E, van Montagu M, Schell J (1978) Transfection and transformation of Agrobacterium tumefaciens. Mol Gen Genet 163:181–187

    Article  CAS  PubMed  Google Scholar 

  37. Remans T, Schenk PM, Manners JM, Grof CPL, Elliott AR (1999) A protocol for the fluorometric quantification of mGFP5-ER and sGFP (S65T) in transgenic plants. Plant Mol Biol Report 17:385–395

    Article  CAS  Google Scholar 

  38. Mittal D, Borah BK, Dasgupta I (2008) Agroinfection of cloned Sri Lankan cassava mosaic virus DNA to Arabidopsis thaliana, Nicotiana tabacum and cassava. Arch Virol 153:2149–2155

    Article  CAS  PubMed  Google Scholar 

  39. Lescot M, Déhais P, Thijs G, Marchal K, Moreau Y, van de Peer Y et al (2002) PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res 30:325–327

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Rajeswaran R, Sunitha S, Shivaprasad PV, Pooggin MM, Hohn T, Veluthambi K (2007) The mungbean yellow mosaic begomovirus transcriptional activator protein transactivates the viral promoter-driven transgene and causes toxicity in transgenic tobacco plants. Mol Plant Microbe Interact 20:1545–1554

    Article  CAS  PubMed  Google Scholar 

  41. Khan ZA, Khan JA (2022) Geminivirus promoters: a breakthrough in transgenic research. Geminivirus: detection, diagnosis and management. Elsevier, Amsterdam, pp 357–366

    Book  Google Scholar 

  42. Sunter G, Bisaro DM (1991) Transactivation in a geminivirus: AL2 gene product is needed for coat protein expression. Virology 180:416–419

    Article  CAS  PubMed  Google Scholar 

  43. Csorba T, Kontra L, Burgyán J (2015) Viral silencing suppressors: Tools forged to fine-tune host-pathogen coexistence. Virology 479:85–103

    Article  PubMed  Google Scholar 

  44. Trinks D, Rajeswaran R, Shivaprasad PV, Akbergenov R, Oakeley EJ, Veluthambi K et al (2005) Suppression of RNA silencing by a geminivirus nuclear protein, AC2, correlates with transactivation of host genes. J Virol 79:2517–2527

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Babu KSD, Manoharan P, Pandi G (2018) Computational studies on begomoviral AC2/C2 proteins. Bioinformation 14:294

    Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

Authors thank Science and Engineering Research Board (SERB), Department of Science and Technology (DST), Government of India, for funding this study under J. C. Bose Fellowship (SB/S2/JCB-057/2016). ZAK and KG acknowledge SERB (PDF/2016/003514) and DST (INSPIRE-2015/IF150761), respectively, for research fellowships under NPDF and INSPIRE. ID acknowledges Senior Scientist Fellowship (INSA/SP/SS/2022/479) of Indian National Science Academy, New Delhi.

Funding

Funds available under the J.C. Bose Fellowship (SB/S2/JCB-057/2016) awarded by Science and Engineering Research Board, Government of India to ID were utilized for this study. KG acknowledges DST-INSPIRE fellowship (2015/IF150761) by Department of Science and Technology, Government of India. ZAK acknowledges National Postdoctoral Fellowship (PDF/2016/003514) by the Science and Engineering Research Board, Department of Science and Technology, Government of India. ID acknowledges the support of Senior Scientist Fellowship (INSA/SP/SS/2022/479) awarded by the Indian National Science Academy, New Delhi.

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  1. Zainul A. Khan, and Kanika Gupta have contributed equally to the manuscript.

Authors and Affiliations

  1. Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi, 110021, India

    Zainul A. Khan, Kanika Gupta & Indranil Dasgupta

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Contributions

Zainul A. Khan, Kanika Gupta: Conceptualization, Methodology, Formal analysis, Writing—original draft, Visualization. Indranil Dasgupta: Conceptualization, Resources, Writing—review & editing, Supervision, Funding acquisition.

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Correspondence to Indranil Dasgupta.

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Supplementary Information

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11262_2024_2074_MOESM1_ESM.jpg

Supplementary Fig. S1. Visualization of GFP in agroinoculated okra plants under handheld UV lamp at 3 days post-infiltration: Leaves infiltrated with OELCuV pCP-GFP, OELCuV pRep-GFP and p35S-GFP showing GFP expression as green fluorescence around the infiltration zone. Mock (pGFP0029) infiltrated leaf was used as negative control. The site of the infiltration is indicated by an arrow (JPG 160 KB)

11262_2024_2074_MOESM2_ESM.jpg

Supplementary Fig. S2. Gel electrophoresis pattern showing the PCR amplification products of the coat protein (CP) gene of OELCuV (Lane 1-10) using CP-specific primers in cotton plants agroinoculated with OELCuV and BYVMB at 15 dpi (M: 1 kb ladder, -ve: PCR negative control) (JPG 48 KB)

11262_2024_2074_MOESM3_ESM.jpg

Supplementary Fig. S3. Visualization of GFP in agroinoculated cotton plants under handheld UV lamp at 3 days following agroinoculation with cloned DNAs of OELCuV and BYVMB. Leaves infiltrated with OELCuV pCP-GFP, OELCuV pRep-GFP and p35S-GFP showing GFP expression as green fluorescence around the infiltration zone. The mock (pGFP0029) infiltrated leaf was used as negative control. The site of the infiltration is indicated by an arrow (JPG 169 KB)

11262_2024_2074_MOESM4_ESM.jpg

Supplementary Fig. S4. Nicotiana benthamiana plants infiltrated with (A) SLCMV DNA-A and DNA-B, and (B) mock at 10 days post-infiltration. Arrows indicate typical leaf curling (JPG 98 KB)

11262_2024_2074_MOESM5_ESM.jpg

Supplementary Fig. S5. GFP quantification in transient expression assay in Nicotiana benthamiana leaves at 3 days post-infiltration to test the inducibility of OELCuV CP and Rep promoters upon SLCMV DNA-A and SLCMV DNA-B infection. The data represent average ± SD (n = 3) of each construct: p35S-GFP, pRep-GFP, pCP-GFP, and mock (pGFP0029). Error bars indicate SE (JPG 74 KB)

Supplementary File 6 (DOCX 17 KB)

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Khan, Z.A., Gupta, K. & Dasgupta, I. Transient expression analysis of promoters of okra enation leaf curl virus in Nicotiana benthamiana, cotton and okra plants. Virus Genes (2024). https://doi.org/10.1007/s11262-024-02074-7

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