Editing of PCNA Gene by CRISPR/Cas9 Technology to Improve the Red Chili Resistance to Yellow Leaf Curl Disease

Devi Ayu Kurniawati, nFN Suharsono, Tri Joko Santoso


Yellow leaf curl disease caused by Pepper yellow leaf curl virus (PepYLCV), member of geminiviruses group, is responsible for heavy yield losses for chili pepper production. Resistant genes which can cause immunity to the disease have not been found in germplasm collection. The aim of the research was to edit proliferating cell nuclear antigen (PCNA) gene by using CRISPR/Cas9 technology for developing plant resistance against geminivirus in chili pepper. A CRISPR/Cas9 plasmid cassette construct harboring the guide RNA of PCNA gene was constructed by Golden Gate cloning strategy. The construct was then introduced into chili genome via in planta method using Agrobacterium tumefaciens EHA105. The transformed plants were bioassayed by virus inoculation and confirmed using PCR and DNA sequencing to identify a mutagenesis event in PCNA gene target. The results showed that CRISPR/Cas9 plasmid cassette harboring gRNA of PCNA gene was successfully constructed. In planta transformation using A. tumefaciens vector harboring CRISPR/Cas9-gRNA PCNA construct resulted in 307 and 193 transformed plants from chili var. Lingga and Ciko, respectively. Bioassay by using virus inoculation to the transformed plants obtained 6 and 14 lines of Lingga and Ciko, respectively, which were resistant to geminivirus (no symptom observed). The resistant lines of chili pepper var. Lingga and Ciko were mutated in PCNA gene with one base insertion or deletion mutation types. These results exhibit that the CRISPR/Cas9 genome editing can be used to induce mutant of PCNA gene in chili pepper. Further investigation is necessary to evaluate the selected chili lines resistant to PepYLCV infection.


Capsicum annuum L.;CRISPR/Cas9; yellow leaf curl disease; geminivirus

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Abdallah, N.A., Prakash, C.S. & McHughen, A.G. (2015) Genome editing for crop improvement: Challenges and opportunities. GM Crops & Food. [Online] 6 (4), 183–205. Tersedia pada: https://doi.org/10.1080/21645698.2015.1129937 [Diakses 29 September 2018].

Bagewadi, B., Chen, S., Lal, S.K., Choudhury, N.R. & Mukherjee, S.K. (2004) PCNA interacts with Indian mung bean yellow mosaic virus Rep and downregulates Rep activity. Journal of Virology. [Online] 78 (21), 11890–11903. Tersedia pada: https://doi.org/10.1128/JVI.78.21.11890-11903.2004 [Diakses 17 Oktober 2019].

Belhaj, K., Chaparro-Garcia, A., Kamoun, S. & Nekrasov, V. (2013) Plant genome editing made easy: Targeted mutagenesis in model and crop plants using the CRISPR/Cas system. Plant Methods. [Online] 9 (1), 1–10. Tersedia pada: https://doi.org/10.1186/1746-4811-9-39 [Diakses 17 Oktober 2019].

Bent, A.F. (2000) Arabidopsis in planta transformation. Uses, mechanisms, and prospects for transformation of other species. Plant Physiology. [Online] 124 (4), 1540–1547. Tersedia pada: https://doi.org/10.1104/pp.124.4.1540 [Diakses 17 Oktober 2019].

BPS (2019) Statistik Indonesia 2019. Hlm. 738. Jakarta, Badan Pusat Statistik.

Czosnek, H., Eybishtz, A., Sade, D., Gorovits, R., Sobol, I., Bejarano, E., Rosas-Diaz, T. & Lozano-Duran, R. (2013) Discovering host genes involved in the infection by the Tomato yellow leaf curl virus complex and in the establishment of resistance to the virus using Tobacco rattle virus-based post transcriptional gene silencing. Viruses. [Online] 5 (3), 998–1022. Tersedia pada: https://doi.org/10.3390/v5030998 [Diakses 5 November 2016].

Dieckman, L.M., Freudenthal, B.D. & Washington, M.T. (2012) PCNA structure and function: Insights from structures of PCNA complexes and post-translationally modified PCNA. Subcellular Biochemistry. [Online] 62, 281–299. Tersedia pada: https://doi.org/10.1007/978-94-007-4572-8_15 [Diakses 17 Oktober 2019].

Egelkrout, E.M., Robertson, D. & Hanley-Bowdoin, L. (2001) Proliferating cell nuclear antigen transcription is repressed through an E2F consensus element and activated by geminivirus infection in mature leaves. Plant Cell. [Online] 13 (6), 1437–1452. Tersedia pada: https://doi.org/10.1105/TPC.010004 [Diakses 17 Oktober 2019].

Engler, C. Kandzia, R. & Marillonnet, S. (2008) A one pot, one step, precision cloning method with high throughput capability. PLoS ONE. [Online] 3 (11), e3647. Tersedia pada: https://doi.org/10.1371/journal.pone.0003647 [Diakses 17 Oktober 2019].

Feng, Z., Zhang, B., Ding, W., Liu, X., Yang, D.L., Wei, P., Cao, F., Zhu, S., Zhang, F., Mao, Y. & Zhu, J.K. (2013) Efficient genome editing in plants using a CRISPR/Cas system. Cell Research. [Online] 23 (10), 1229–1232. Tersedia pada: https://doi.org/10.1038/cr.2013.114 [Diakses 21 Agustus 2015].

Ganefianti, D.W., Hidayat, S.H. & Syukur, M. (2017) Susceptible phase of chili pepper due to Yellow leaf curl begomovirus infection. International Journal on Advanced Science, Engineering and Information Technology. [Online] 7 (2), 594–601. Tersedia pada: http://dx.doi.org/10.18517/ijaseit.7.2.1872 [Diakses 17 Oktober 2019].

Gaswanto, R., Syukur, M., Hidayat, S.H. & Gunaeni, N. (2016) Identifikasi gejala dan kisaran inang enam isolat begomovirus cabai di Indonesia. Jurnal Hortikultura. [Online] 26 (2), 223–234. Tersedia pada: http://ejurnal.litbang.pertanian.go.id/index.php/jhort/article/view/5780 [Diakses 17 Oktober 2019].

Gunaeni, N. & Purwati, E. (2013) Uji ketahanan terhadap Tomato yellow leaf curl virus pada beberapa galur tomat. Jurnal Hortikultura. [Online] 23 (1), 65–71. Tersedia pada: http://ejurnal.litbang.pertanian.go.id/ index.php/jhort/article/view/3382 [Diakses 17 Oktober 2019].

Heigwer, F., Kerr, G. & Boutros, M. (2014) E-CRISP: Fast CRISPR target site identification. Nature Methods. [Online] 11, 122–123. Tersedia pada: https://doi.org/10.1038/nmeth.2812 [Diakses 17 Oktober 2019].

Jiang, W., Zhou, H., Bi, H., Fromm, M., Yang, B. & Weeks, D.P. (2013) Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice. Nucleic Acids Research. [Online] 41 (20), e188. Tersedia pada: https://doi.org/10.1093/nar/gkt780 [Diakses 17 Oktober 2019].

Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J.A. & Charpentier, E. (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. [Online] 337 (6096), 816–821. Tersedia pada: https://doi.org/10.1126/ science.1225829 [Diakses 19 Oktober 2020].

Low, L.Y., Yang, S.K., Kok, D.X.A., Abdullah, J.O., Tan, N.P. & Lai, K.S. (2018) Transgenic plants: Gene constructs, vector and transformation method. Dalam: Çelik, Ö. (ed.) New visions in plant science. [e-book] London, UK, IntechOpen. Tersedia pada: https:// www.intechopen.com/books/new-visions-in-plant-science/transgenic-plants-gene-constructs-vector-and-transformation-method [Diakses 17 Oktober 2020].

Miao, J., Guo, D., Zhang, J., Huang, Q., Qin, G., Zhang, X., Wan, J., Gu, H. & Qu, L.J. (2013) Targeted mutagenesis in rice using CRISPR-Cas system. Cell Research. [Online] 23 (10), 1233–1236. Tersedia pada: https://doi.org/10.1038/cr.2013.123 [Diakses 21 Agustus 2015].

Mishra, R. & Zhao, K. (2018) Genome editing technologies and their applications in crop improvement. Plant Biotechnology Reports. [Online] 12 (2), 57–68. Tersedia pada: https://doi.org/10.1007/s11816-018-0472-0 [Diakses 29 September 2018].

Naseri, G., Sohani, M.M., Pourmassalehgou, A. & Allahi, S. (2012) In planta transformation of rice (Oryza sativa) using thaumatin-like protein gene for enhancing resistance to sheath blight. African Journal of Biotechnology. [Online] 11 (31), 7885–7893. Tersedia pada: https://doi.org/10.5897/AJB11.3331 [Diakses 18 September 2020].

Pattanayak, V., Lin, S., Guilinger, J.P., Ma, E., Doudna, J.A. & Liu, D.R. (2013) High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nature Biotechnology. [Online] 31 (9), 839–843. Tersedia pada: https://doi.org/10.1038/nbt.2673 [Diakses 18 September 2020].

Peng, D. & Tarleton, R. (2015) EuPaGDT: A web tool tailored to design CRISPR guide RNAs for eukaryotic pathogens. Microbial Genomics. [Online] 1 (4). Tersedia pada: https://doi.org/10.1099/mgen.0.000033 [Diakses 7 Oktober 2019].

Sievers, F. & Higgins, D.G. (2014) Clustal Omega. Current Protocols in Bioinformatics. [Online] 48 (1), 3.13.1–3.13.16. Tersedia pada: https://doi.org/10.1002/0471250953.bi0313s48 [Diakses 7 Oktober 2019].

Wang, F., Wang, C., Liu, P., Lei, C., Hao, W., Gao, Y., Liu, Y.G. & Zhao, K. (2016) Enhanced rice blast resistance by CRISPR/Cas9-targeted mutagenesis of the ERF transcription factor gene OsERF922. PLoS ONE. [Online] 11 (4), 1–18. Tersedia pada: https://doi.org/10.1371/journal.pone.0154027 [Diakses 17 Oktober 2020].

Xu, R. & Li, Q.Q. (2008) Protocol: Streamline cloning of genes into binary vectors in Agrobacterium via the Gateway® TOPO vector system. Plant Methods. [Online] 4 (1), 1–7. Tersedia pada: https://doi.org/10.1186/1746-4811-4-4 [Diakses 12 Oktober 2019].

Yasmeen, A., Mirza, B., Inayatullah, S., Safdar, N., Jamil, M., Ali, S. & Choudhry, M.F. (2008) In planta transformation of tomato. Plant Molecular Biology Reporter. [Online] 27 (1), 20–28. Tersedia pada: https://doi.org/10.1007/s11105-008-0044-5 [Diakses 15 Oktober 2020].

Zhang, Y., Bai, Y., Wu, G., Zou, S. Chen, Y, Gao, C. & Tang, D. (2017) Simultaneous modification of three homoeologs of TaEDR1 by genome editing enhances powdery mildew resistance in wheat. Plant Journal. [Online] 91 (4), 714–724. Tersedia pada: https://doi.org/10.1111/tpj.13599 [Diakses 17 Oktober 2020].

Zhou, J., Peng, Z., Long, J., Sosso, D., Liu, B., Eom, J.S., Huang, S., Liu, S., Cruz, C.V., Frommer, W.B., White, F.F. & Yang, B. (2015) Gene targeting by the TAL effector PthXo2 reveals cryptic resistance gene for bacterial blight of rice. Plant Journal. [Online] 82 (4), 632–643. Tersedia pada: https://doi.org/10.1111/tpj.12838 [Diakses 17 Oktober 2020].

DOI: http://dx.doi.org/10.21082/jbio.v16n2.2020.p79-88


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