Skip to main content

Advertisement

SpringerLink
Log in

Towards developing resistance to chickpea chlorotic dwarf virus through CRISPR/Cas9-mediated gene editing using multiplexed gRNAs

  • Original Article
  • Published:
Journal of Plant Diseases and Protection Aims and scope Submit manuscript

Abstract

Mastreviruses are an emerging group of viruses transmitted by leafhoppers and infect both monocot and dicot plants. Chickpea chlorotic dwarf virus (CpCDV), a mastrevirus, is increasingly becoming important in many parts of the world. Various gRNA constructs targeting conserved genomic regions were used singly or in tandem (three constructs together) in CRISPR/Cas9-based approach for triggering resistance against CpCDV. Following inoculation with the infectious clone and each of the constructs, Nicotiana benthamiana was tested for the relative accumulation of the viral DNA through qPCR. Out of five uniplex and three multiplex, one multiplex construct consisting of three genomic regions (LIR, Rep and RepA) showed a significant decrease in viral titre when 2^-∆∆Ct was compared at 7 days after inoculation (dai), 14 dai and 21 dai. When this multiplex construct was agroinfiltrated into the tomato, the qPCR for the viral titre was 0.266 ± 0.06 at 7 dai and 0.069 ± 0.034 when compared with the CpCDV positive control. The expression of gRNA and Cas9 decreased from 7.08 ± 1.24 to 5.22 ± 0.36 and 111.2 ± 5.19 to 75.2 ± 5.26 from 4 to 7 dai, respectively. Positive control plants infiltrated with an infectious clone showed chlorotic leaves, thickened veins and stunted growth at 7 dai, whereas plants infiltrated with the multiplex construct showed mild leaf yellowing and thickened veins and ultimately recovered from the symptoms at 21 dai stage. CRISPR/Cas9-based multiplex of three sgRNAs significantly reduced the viral titre; however, other triplex constructs of sgRNA and individual sgRNA failed to produce consistent results. Our study shows the potential of Cas9-based genome editing technique to generate resistance to CpCDV.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Al Shihi AAM (2019) Geminivirus occurrence in Australia, China, Europe, and the Middle Eastern Countries. In: Geminiviruses. Springer. pp 65–83.

  • Ali Z, Abul-Faraj A, Li L, Ghosh N, Piatek M, Mahjoub A, Aouida M, Piatek A, Baltes NJ, Voytas DF (2015a) Efficient virus-mediated genome editing in plants using the CRISPR/Cas9 system. Mol Plant 8:1288–1291

    Article  CAS  PubMed  Google Scholar 

  • Ali Z, Abulfaraj A, Idris A, Ali S, Tashkandi M, Mahfouz MM (2015b) CRISPR/Cas9-mediated viral interference in plants. Genome Biol 16:1–11

    Article  CAS  Google Scholar 

  • Ali Z, Ali S, Tashkandi M, Zaidi SSA, Mahfouz MM (2016) CRISPR/Cas9-mediated immunity to geminiviruses: differential interference and evasion. Sci Rep 6:26912

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ammara U, Al-Sadi AM, Al-Shihi A, Amin I (2017) Real-time qPCR assay for the TYLCV titer in relation to symptoms-based disease severity scales. Int J Agric Biol 19:145–151

    Article  Google Scholar 

  • Aregger M, Borah BK, Seguin J, Rajeswaran R, Gubaeva EG, Zvereva AS, Windels D, Vazquez F, Blevins T, Farinelli L, Pooggin MM (2012) Primary and secondary siRNAs in geminivirus-induced gene silencing. PLoS Pathog 8:e1002941

    Article  PubMed  PubMed Central  Google Scholar 

  • Ascencio-Ibáñez JT, Bobay BG (2021) Conserved structural motif identified in peptides that bind to geminivirus replication protein rep. Biochemistry 60:2795–2809

    Article  PubMed  Google Scholar 

  • Avedi E, Kilalo C, Olubayo F, Macharia I, Adediji A, Ateka E, Machuka E, Mutuku JM (2020) Complete genome sequence of a new chickpea chlorotic dwarf virus strain isolated from tomato in Kenya, obtained from Illumina sequencing. Microbiol Resour Announc 9:e01344-e11319

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Baltes NJ, Hummel AW, Konecna E, Cegan R, Bruns AN, Bisaro DM, Voytas DF (2015) Conferring resistance to geminiviruses with the CRISPR–Cas prokaryotic immune system. Nature Plants 1:1–4

    Article  Google Scholar 

  • Berendsen H, Hess B, Lindahl E, Van Der Spoel D, Mark A, Groenhof G (2005) GROMACS: fast, flexible, and free. J Comput Chem 26:1701–1718

    Article  PubMed  Google Scholar 

  • Blum M, Chang H-Y, Chuguransky S, Grego T, Kandasaamy S, Mitchell A, Nuka G, Paysan-Lafosse T, Qureshi M, Raj S (2021) The InterPro protein families and domains database: 20 years on. Nucleic Acids Res 49:D344–D354

    Article  CAS  PubMed  Google Scholar 

  • Borrelli VM, Brambilla V, Rogowsky P, Marocco A, Lanubile A (2018) The enhancement of plant disease resistance using CRISPR/Cas9 technology. Front Plant Sci 9:1245

    Article  PubMed  PubMed Central  Google Scholar 

  • Carra JH, Privalov PL (1996) Thermodynamics of denaturation of staphylococcal nuclease mutants: an intermediate state in protein folding. FASEB J 10:67–74

    Article  CAS  PubMed  Google Scholar 

  • Chandrasekaran J, Brumin M, Wolf D, Leibman D, Klap C, Pearlsman M, Sherman A, Arazi T, Gal-On A (2016) Development of broad virus resistance in non-transgenic cucumber using CRISPR/Cas9 technology. Mol Plant Pathol 17:1140–1153

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Faal PG, Farsi M, Seifi A, Kakhki AM (2020) Virus-induced CRISPR-Cas9 system improved resistance against tomato yellow leaf curl virus. Mol Biol Rep 47:3369–3376

    Article  Google Scholar 

  • Gergen J, Coulon F, Creneguy A, Elain-Duret N, Gutierrez A, Pinkenburg O, Verhoeyen E, Anegon I, Nguyen TH, Halary FA (2018) Multiplex CRISPR/Cas9 system impairs HCMV replication by excising an essential viral gene. PLoS ONE 13:e0192602

    Article  PubMed  PubMed Central  Google Scholar 

  • Ghoshal B, Vong B, Picard CL, Feng S, Tam JM, Jacobsen SE (2020) A viral guide RNA delivery system for CRISPR-based transcriptional activation and heritable targeted DNA demethylation in Arabidopsis thaliana. PLoS Genet 16:e1008983

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gil-Humanes J, Wang Y, Liang Z, Shan Q, Ozuna CV, Sánchez-León S, Baltes NJ, Starker C, Barro F, Gao C, Voytas DF (2017) High-efficiency gene targeting in hexaploid wheat using DNA replicons and CRISPR/Cas9. Plant J 89:1251–1262

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hameed A, Iqbal Z, Asad S, Mansoor S (2014) Detection of multiple potato viruses in the field suggests synergistic interactions among potato viruses in Pakistan. Plant Pathol J 30:407

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hameed U, Zia-Ur-Rehman M, Ali S, Haider M, Brown J (2017) First report of chickpea chlorotic dwarf virus infecting cucumber in Pakistan. Plant Dis 101:848–848

    Article  Google Scholar 

  • Hameed U, Zia-Ur-Rehman M, Ali SA, Haider MS, Brown JK (2019) Invasion of previously unreported dicot plant hosts by chickpea chlorotic dwarf virus in Pakistan. VirusDisease 30:95–100

    Article  CAS  PubMed  Google Scholar 

  • Hassan MM, Zhang Y, Yuan G, De K, Chen J-G, Muchero W, Tuskan GA, Qi Y, Yang X (2021) Construct design for CRISPR/Cas-based genome editing in plants. Trends Plant Sci 26:1133–1152

    Article  CAS  PubMed  Google Scholar 

  • Iqbal Z, Sattar MN, Shafiq M (2016) CRISPR/Cas9: A tool to circumscribe cotton leaf curl disease. Front Plant Sci 7.

  • Ji X, Zhang H, Zhang Y, Wang Y, Gao C (2015) Establishing a CRISPR–Cas-like immune system conferring DNA virus resistance in plants. Nature Plants 1:1–4

    Article  Google Scholar 

  • Kanakala S, Kuria P (2019) Chickpea chlorotic dwarf virus: an emerging monopartite dicot infecting mastrevirus. Viruses 11:5

    Article  CAS  Google Scholar 

  • Kato Y, Tabata H, Sato K, Nakamura M, Saito I, Nakanishi T (2021) Adenovirus vectors expressing eight multiplex guide RNAs of CRISPR/Cas9 efficiently disrupted diverse hepatitis B virus gene derived from heterogeneous patient. Int J Mol Sci 22:10570

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Khan ZA, Kumar R, Dasgupta I (2022) CRISPR/Cas-Mediated Resistance against Viruses in Plants. Int J Mol Sci 23:2303

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kim D, Alptekin B, Budak H (2018) CRISPR/Cas9 genome editing in wheat. Funct Integ Genomic 18:31–41

    Article  CAS  Google Scholar 

  • Kis A, Hamar É, Tholt G, Bán R, Havelda Z (2019) Creating highly efficient resistance against wheat dwarf virus in barley by employing CRISPR/Cas9 system. Plant Biotechnol J 17:1004

    Article  PubMed  PubMed Central  Google Scholar 

  • Lim H, Jun S, Park M, Lim J, Jeong J, Lee JH, Bang D (2020) Multiplex generation, tracking, and functional screening of substitution mutants using a CRISPR/retron system. ACS Synth Biol 9:1003–1009

    Article  CAS  PubMed  Google Scholar 

  • Liu H, Soyars CL, Li J, Fei Q, He G, Peterson BA, Meyers BC, Nimchuk ZL, Wang X (2018) CRISPR/Cas9-mediated resistance to cauliflower mosaic virus. Plant Direct 2:e00047

    Article  PubMed  PubMed Central  Google Scholar 

  • Loriato VA, Martins LG, Euclydes NC, Reis PA, Duarte CE, Fontes EP (2020) Engineering resistance against geminiviruses: a review of suppressed natural defenses and the use of RNAi and the CRISPR/Cas system. Plant Sci 292:110410

    Article  CAS  PubMed  Google Scholar 

  • Lowder LG, Zhang D, Baltes NJ, Paul JW III, Tang X, Zheng X, Voytas DF, Hsieh T-F, Zhang Y, Qi Y (2015) A CRISPR/Cas9 toolbox for multiplexed plant genome editing and transcriptional regulation. Plant Physiol 169:971–985

    Article  PubMed  PubMed Central  Google Scholar 

  • Marwal A, Verma RK, Mishra M, Kumar R, Gaur R (2019) Mastreviruses in the African World: harbouring both monocot and dicot species. In: Geminiviruses. Springer. pp 85–102.

  • Mehta D, Stürchler A, Anjanappa RB, Zaidi SS-e-A, Hirsch-Hoffmann M, Gruissem W, Vanderschuren H, (2019) Linking CRISPR-Cas9 interference in cassava to the evolution of editing-resistant geminiviruses. Genome Biol 20:80

    Article  PubMed  PubMed Central  Google Scholar 

  • Meli M, Morra G, Colombo G (2020) Simple model of protein energetics to identify ab initio folding transitions from all-atom MD simulations of proteins. J Chem Theory Comput 16:5960–5971

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Mubarik MS, Wang X, Khan SH, Ahmad A, Khan Z, Amjid MW, Razzaq MK, Ali Z, Azhar MT (2021) Engineering broad-spectrum resistance to cotton leaf curl disease by CRISPR-Cas9 based multiplex editing in plants. GM Crops Food: 1–12.

  • Nahid N, Amin I, Mansoor S, Rybicki E, Van Der Walt E, Briddon R (2008) Two dicot-infecting mastreviruses (family Geminiviridae) occur in Pakistan. Arch Virol 153:1441–1451

    Article  CAS  PubMed  Google Scholar 

  • Ophinni Y, Miki S, Hayashi Y, Kameoka M (2020) Multiplexed tat-targeting CRISPR-cas9 protects T cells from acute HIV-1 infection with inhibition of viral escape. Viruses 12:1223

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ouattara A, Tiendrébéogo F, Lefeuvre P, Hoareau M, Claverie S, Traoré EV, Barro N, Traoré O, Varsani A, Lett J-M (2017) New strains of chickpea chlorotic dwarf virus discovered on diseased papaya and tomato plants in Burkina Faso. Arch Virol 162:1791–1794

    Article  CAS  PubMed  Google Scholar 

  • Pikkemaat MG, Linssen ABM, Berendsen HJC, Janssen DB (2002) Molecular dynamics simulations as a tool for improving protein stability. Protein Eng Des Sel 15:185–192

    Article  CAS  Google Scholar 

  • Pramanik D, Shelake RM, Park J, Kim MJ, Hwang I, Park Y, Kim J-Y (2021) CRISPR/Cas9-mediated generation of pathogen-resistant tomato against tomato yellow leaf curl virus and powdery mildew. Int J Mol Sci 22:1878

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Pyott DE, Sheehan E, Molnar A (2016) Engineering of CRISPR/Cas9-mediated potyvirus resistance in transgene-free Arabidopsis plants. Mol Plant Pathol 17:1276–1288

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Roy A, Zhai Y, Ortiz J, Neff M, Mandal B, Mukherjee SK, Pappu HR (2019) Multiplexed editing of a begomovirus genome restricts escape mutant formation and disease development. PLoS ONE 14:e0223765

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Saleem RS, Siddiqui SN, Irshad S, Ashraf NM, Hamid A, Khan MAU, Khan MI, Micheal S (2022) Targeted gene sequencing of FYCO1 identified a novel mutation in a Pakistani family for autosomal recessive congenital cataract. Mol Genet Genomic Med 10:e1985

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sastry KS, Mandal B, Hammond J, Scott S, Briddon R, Sastry SK, Mandal B, Sano T, Hammond J (2019) Brassica carinata (Ethiopian Mustard). Encyclopedia of plant viruses and viroids.

  • Shahid MS, Sattar MN, Iqbal Z, Raza A, Al-Sadi AM (2021) Next-generation sequencing and the CRISPR-Cas nexus: a molecular plant virology perspective. Front Microbiol 11:609376

    Article  PubMed  PubMed Central  Google Scholar 

  • Shortle D (1996) The denatured state (the other half of the folding equation) and its role in protein stability. FASEB J 10:27–34

    Article  CAS  PubMed  Google Scholar 

  • Smith AW, Iglewski BH (1989) Transformation of Pseudomonas aeruginosa by electroporation. Nucleic Acids Res 17:10509

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Smith LM, Ladner JT, Hodara VL, Parodi LM, Harris RA, Callery JE, Lai Z, Zou Y, Raveedran M, Rogers J (2021) Multiplexed simian immunodeficiency virus-specific paired RNA-guided Cas9 nickases inactivate proviral DNA. J Virol 95:e00882-e1821

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tian B, Minero GAS, Fock J, Dufva M, Hansen MF (2020) CRISPR-Cas12a based internal negative control for nonspecific products of exponential rolling circle amplification. Nucleic Acids Res 48:e30–e30

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yang J, Yan R, Roy A, Xu D, Poisson J, Zhang Y (2015) The I-TASSER Suite: protein structure and function prediction. Nat Methods 12:7–8

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yang X, Wang Y, Guo W, Xie Y, Xie Q, Fan L, Zhou X (2011) Characterization of small interfering RNAs derived from the geminivirus/betasatellite complex using deep sequencing. PLoS ONE 6:e16928

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yin K, Han T, Liu G, Chen T, Wang Y, Yu AYL, Liu Y (2015) A geminivirus-based guide RNA delivery system for CRISPR/Cas9 mediated plant genome editing. Sci Rep 5:1–10

    Article  Google Scholar 

  • Yin K, Han T, Xie K, Zhao J, Song J, Liu Y (2019) Engineer complete resistance to Cotton Leaf Curl Multan virus by the CRISPR/Cas9 system in Nicotiana benthamiana. Phytopathology Res 1:9

    Article  Google Scholar 

  • Yu Y, Wang X, Sun H, Liang Q, Wang W, Zhang C, Bian X, Cao Q, Li Q, Xie Y (2020) Improving CRISPR-Cas-mediated RNA targeting and gene editing using SPLCV replicon-based expression vectors in Nicotiana benthamiana. Plant Biotechnol J 18:1993

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zaagueri T, Mnari-Hattab M, Moussaoui N, Accotto G, Noris E, Marian D, Vaira A (2019) Chickpea chlorotic dwarf virus infecting tomato crop in Tunisia. Eur J Plant Pathol 154:1159–1164

    Article  CAS  Google Scholar 

  • Zaidi SSA, Mansoor S, Ali Z, Tashkandi M, Mahfouz MM (2016) Engineering plants for geminivirus resistance with CRISPR/Cas9 system. Trends Plant Sci 21:279–281

    Article  CAS  PubMed  Google Scholar 

  • Zhao H, Tan Z, Wen X, Wang Y (2017) An improved syringe agroinfiltration protocol to enhance transformation efficiency by combinative use of 5-azacytidine, ascorbate acid and tween-20. Plants 6:9

    Article  PubMed  PubMed Central  Google Scholar 

  • Zhou J, Deng K, Cheng Y, Zhong Z, Tian L, Tang X, Tang A, Zheng X, Zhang T, Qi Y (2017) CRISPR-Cas9 based genome editing reveals new insights into microRNA function and regulation in rice. Front Plant Sci 8:1598

    Article  PubMed  PubMed Central  Google Scholar 

  • Zia-Ur-Rehman M, Hameed U, Ali C, Haider M, Brown JK (2017) First report of chickpea chlorotic dwarf virus infecting okra in Pakistan. Plant Dis 101:1336–1336

    Article  Google Scholar 

  • Zia-Ur-Rehman M, Hameed U, Herrmann H-W, Iqbal M, Haider M, Brown JK (2015) First report of Chickpea chlorotic dwarf virus infecting tomato crops in Pakistan. Plant Dis 99:1287–1287

    Article  Google Scholar 

Download references

Acknowledgements

This study was part of the PhD dissertation research of MA and was funded in part by the Higher Education Commission of Pakistan and the University of the Punjab.

Author information

Authors and Affiliations

  1. Department of Plant Pathology, University of the Punjab, Lahore, Pakistan

    Muniba Abid Munir Malik & Muhammad Saleem Haider

  2. Department of Plant Pathology, Washington State University, Pullman, WA, USA

    Muniba Abid Munir Malik, Ying Zhai & Hanu R. Pappu

  3. Department of Biochemistry and Biotechnology, University of Gujrat, Gujrat, Pakistan

    Muhammad Azmat Ullah Khan

Authors
  1. Muniba Abid Munir Malik
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Muhammad Saleem Haider
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ying Zhai
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Muhammad Azmat Ullah Khan
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Hanu R. Pappu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Muhammad Saleem Haider or Hanu R. Pappu.

Ethics declarations

Conflict of interest

Authors declare no competing or conflict of interest while publishing this study.

Ethics approval

Not applicable.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 4647 KB)

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Malik, M.A.M., Haider, M.S., Zhai, Y. et al. Towards developing resistance to chickpea chlorotic dwarf virus through CRISPR/Cas9-mediated gene editing using multiplexed gRNAs. J Plant Dis Prot 130, 23–33 (2023). https://doi.org/10.1007/s41348-022-00677-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s41348-022-00677-6

Keywords

Search

Navigation