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Research paper

An efficient transient gene expression system for protein subcellular localization assay and genome editing in citrus protoplasts

Wenhui Yanga, Jiaqin Rena, Wanrong Liua, Dan Liua, Kaidong Xiea, Fei Zhanga,b, Pengwei Wanga,b, Wenwu Guoa,b, Xiaomeng Wua( )
National Key Laboratory for Germplasm Innovation & Utilization of Horticultural Crops, College of Horticulture and Forestry Sciences, Huazhong Agricultural University, Wuhan, Hubei 430070, China
Hubei Hongshan Laboratory, Wuhan, Hubei 430070, China

Peer review under responsibility of Chinese Society of Horticultural Science (CSHS) and Institute of Vegetables and Flowers (IVF), Chinese Academy of Agricultural Sciences (CAAS)

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Abstract

Protoplast has been widely used in biotechnologies to circumvent the breeding obstacles in citrus, including long juvenility, polyembryony, and male/female sterility. The protoplast-based transient gene expression system is a powerful tool for gene functional characterization and CRISPR/Cas9 genome editing in higher plants, but it has not been widely used in citrus. In this study, the polyethylene glycol (PEG)-mediated method was optimized for citrus callus protoplast transfection, with an improved transfection efficiency of 68.4%. Consequently, the efficiency of protein subcellular localization assay was increased to 65.8%, through transient expression of the target gene in protoplasts that stably express the fluorescent organelle marker protein. The gene editing frequencies in citrus callus protoplasts reached 14.2% after transient expression of CRISPR/Cas9 constructs. We demonstrated that the intronic polycistronic tRNA-gRNA (inPTG) genome editing construct was functional in both the protoplast transient expression system and epicotyl stable transformation system in citrus. With this optimized protoplast transient expression system, we improved the efficiency of protein subcellular localization assay and developed the genome editing system in callus protoplasts, which provides an approach for prompt test of CRISPR vectors.

Keywords

Genome editingCitrusSubcellular localizationCallus protoplastTransient transfection

References

 

Bart, R., Chern, M., Park, C.J., Bartley, L., Ronald, P.C., 2006. A novel system for gene silencing using siRNAs in rice leaf and stem-derived protoplasts. Plant Methods, 2: 13.
Crossref Google Scholar
 

Burris, K.P., Dlugosz, E.M., Collins, A.G., Stewart, C.N., Lenaghan, S.C., 2016. Development of a rapid, low-cost protoplast transfection system for switchgrass (Panicum virgatum L.). Plant Cell Rep, 35: 693–704.
Crossref Google Scholar
 

Cai, X.D., Fu, J., Guo, W.W., 2017. Mitochondrial genome of callus protoplast has a role in mesophyll protoplast regeneration in citrus: evidence from transgenic GFP somatic homo-fusion. Hortic Plant J, 3: 177–182.
Crossref Google Scholar
 

Cao, H.B., Zhang, J.C., Xu, J.D., Ye, J.L., Yun, Z., Xu, Q., Xu, Juan, Deng, X.X., 2012. Comprehending crystalline β-carotene accumulation by comparing engineered cell models and the natural carotenoid-rich system of citrus. J Exp Bot, 63: 4403–4417.
Crossref Google Scholar
 

Chen, Y.T., Mao, W.W., Liu, T., Feng, Q.Q., Li, L., Li, B.B., 2020. Genome editing as a versatile tool to improve horticultural crop qualities. Hortic Plant J, 6: 372–384.
Crossref Google Scholar
 

Di, Y.H., Sun, X.J., Hu, Z., Jiang, Q.Y., Song, G.H., Zhang, B., Zhao, S.S., Zhang, H., 2019. Enhancing the CRISPR/Cas9 system based on multiple GmU6 promoters in soybean. Biochem Biophys Res Commun, 519: 819–823.
Crossref Google Scholar
 

Ding, D., Chen, K.Y., Chen, Y.D., Li, H., Xie, K.B., 2018. Engineering introns to express RNA guides for Cas9- and Cpf1-mediated multiplex genome editing. Mol Plant, 11: 542–552.
Crossref Google Scholar
 

Duan, Y.X., Guo, W.W., Meng, H.J., Tao, N.G., Li, D.D., Deng, X.X., 2007. High efficient transgenic plant regeneration from embryogenic calluses of Citrus sinensis. Biol Plant, 51: 212–216.
Crossref Google Scholar
 

Dutt, M., Mou, Z.L., Zhang, X.D., Tanwir, S.E., Grosser, J.W., 2020. Efficient CRISPR/Cas9 genome editing with citrus embryogenic cell cultures. BMC Biotechnol, 20: 58.
Crossref Google Scholar
 

Engler, C., Youles, M., Gruetzner, R., Ehnert, T.M., Werner, S., Jones, J.D.G., Patron, N.J., Marillonnet, S., 2014. A golden gate modular cloning toolbox for plants. ACS Synth Biol, 3: 839–843.
Crossref Google Scholar
 

Fleming, G.H., Olivares-Fuster, O., Del-Bosco, S.F., Grosser, J.W., 2000. An alternative method for the genetic transformation of sweet orange. Vitro Cell Dev Biol Plant, 36: 450.
Crossref Google Scholar
 

Fu, X.Z., Khan, E.U., Hu, S.S., Fan, Q.J., Liu, J.H., 2011. Overexpression of the betaine aldehyde dehydrogenase gene from Atriplex hortensis enhances salt tolerance in the transgenic trifoliate orange (Poncirus trifoliata L. Raf.). Environ Exp Bot, 74: 106–113.
Crossref Google Scholar
 

Gibson, D.G., Young, L., Chuang, R.Y., Venter, J.C., Hutchison, C.A., Smith, H.O., 2009. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods, 6: 343–345.
Crossref Google Scholar
 

Gong, J.L., Tian, Z., Qu, X.L., Meng, Q.N., Guan, Y.J., Liu, P., Chen, C.W., Deng, X.X., Guo, W.W., Cheng, Y.J., Wang, P.W., 2021. Illuminating the cells: transient transformation of citrus to study gene function and organelle activities related to fruit quality. Hortic Res, 8: 175.
Crossref Google Scholar
 

Gou, Y.J., Li, Y.L., Bi, P.P., Wang, D.J., Ma, Y.Y., Hu, Y., Zhou, H.C., Wen, Y.Q., Feng, J.Y., 2020. Optimization of the protoplast transient expression system for gene functional studies in strawberry (Fragaria vesca). Plant Cell Tissue Organ Cult, 141: 41–53.
Crossref Google Scholar
 

Grosser, J.W., Gmitter, F.G., 2011. Protoplast fusion for production of tetraploids and triploids: applications for scion and rootstock breeding in citrus. Plant Cell Tissue Organ Cult, 104: 343–357.
Crossref Google Scholar
 

Guo, W.W., Duan, Y.X., Olivares-Fuster, O., Wu, Z.C., Arias, C.R., Burns, J.K., Grosser, J.W., 2005. Protoplast transformation and regeneration of transgenic Valencia sweet orange plants containing a juice quality-related pectin methylesterase gene. Plant Cell Rep, 24: 482–486.
Crossref Google Scholar
 

Guo, W.W., Xiao, S.X., Deng, X.X., 2013. Somatic cybrid production via protoplast fusion for citrus improvement. Sci Hortic, 163: 20–26.
Crossref Google Scholar
 

Hao, Y., Zong, W.B., Zeng, D.C., Han, J.L., Chen, S.F., Tang, J.N., Zhao, Z., Li, X.J., Ma, K., Xie, X.R., Zhu, Q.L., Chen, Y.L., Zhao, X.C., Guo, J.X., Liu, Y.G., 2020. Shortened snRNA promoters for efficient CRISPR/Cas-based multiplex genome editing in monocot plants. Sci China Life Sci, 63: 933–935. (in Chinese)
Crossref Google Scholar
 

Hu, B.W., Li, D.W., Liu, X., Qi, J.J., Gao, D.L., Zhao, S.Q., Huang, S.W., Sun, J.J., Yang, L., 2017. Engineering non-transgenic gynoecious cucumber using an improved transformation protocol and optimized CRISPR/Cas9 system. Mol Plant, 10: 1575–1578.
Crossref Google Scholar
 

Huang, X.E., Wang, Y.C., Xu, J., Wang, N., 2020. Development of multiplex genome editing toolkits for citrus with high efficacy in biallelic and homozygous mutations. Plant Mol Biol, 104: 297–307.
Crossref Google Scholar
 

Jia, H., Orbovic, V., Wang, N., 2019. CRISPR-LbCas12a-mediated modification of citrus. Plant Biotechnol J, 17: 1928–1937.
Crossref Google Scholar
 

Jia, H., Wang, N., 2014. Targeted genome editing of sweet orange using Cas9/sgRNA. PLoS One, 9: e93806.
Crossref Google Scholar
 

Jia, H., Wang, N., 2020. Generation of homozygous canker-resistant citrus in the T0 generation using CRISPR-SpCas9p. Plant Biotechnol J, 18: 1990–1992.
Crossref Google Scholar
 

Jia, H.H., Xu, Y.T., Yin, Z.P., Wu, X.M., Qing, M., Fan, Y.J., Song, X., Xie, K.D., Xie, Z.Z., Xu, Q., Deng, X.X., Guo, W.W., 2021. Transcriptomes and DNA methylomes in apomictic cells delineate nucellar embryogenesis initiation in citrus. DNA Res, dsab014.
Crossref Google Scholar
 

Jiang, Y.Y., Chai, Y.P., Lu, M.H., Han, X.L., Lin, Q.P., Zhang, Y., Zhang, Q., Zhou, Y., Wang, X.C., Gao, C.X., Chen, Q.J., 2020. Prime editing efficiently generates W542L and S621I double mutations in two ALS genes in maize. Genome Biol, 21: 257.
Crossref Google Scholar
 

Jin, S., Lin, Q.P., Luo, Y.F., Zhu, Z.X., Liu, G.W., Li, Y.J., Chen, K.L., Qiu, J.L., Gao, C.X., 2021. Genome-wide specificity of prime editors in plants. Nat Biotechnol, 39: 1292–1299.
Crossref Google Scholar
 

LeBlanc, C., Zhang, F., Mendez, J., Lozano, Y., Chatpar, K., Irish, V.F., Jacob, Y., 2018. Increased efficiency of targeted mutagenesis by CRISPR/Cas9 in plants using heat stress. Plant J, 93: 377–386.
Crossref Google Scholar
 

Li, W.T., Li, Z.X., Huang, J.Q., Xu, M.R., Zheng, Z., Deng, X.L., 2022. Characterization of Xanthomonas citri pv. citri from China based on spoligotyping. Hortic Plant J, 8: 727–736.
Crossref Google Scholar
 

Lin, C.S., Hsu, C.T., Yang, L.H., Lee, L.Y., Fu, J.Y., Cheng, Q.W., Wu, F.H., Hsiao, H.C.W., Zhang, Y.S., Zhang, R., Chang, W.J., Yu, C.T., Wang, W., Liao, L.J., Gelvin, S.B., Shih, M.C., 2018. Application of protoplast technology to CRISPR/Cas9 mutagenesis: from single-cell mutation detection to mutant plant regeneration. Plant Biotechnol J, 16: 1295–1310.
Crossref Google Scholar
 

Liu, H., Ding, Y.D., Zhou, Y.Q., Jin, W.Q., Xie, K.B., Chen, L.L., 2017. CRISPR-P 2.0: an improved CRISPR-Cas9 tool for genome editing in plants. Mol Plant, 10: 530–532.
Crossref Google Scholar
 

Liu, Q., Wang, C., Jiao, X.Z., Zhang, H.W., Song, L.L., Li, Y.X., Gao, C.X., Wang, K.J., 2019. Hi-TOM: a platform for high-throughput tracking of mutations induced by CRISPR/Cas systems. Sci China Life Sci, 62: 1–7.
Crossref Google Scholar
 

Long, J.M., Liu, C.Y., Feng, M.Q., Liu, Y., Wu, X.M., Guo, W.W., 2018a. miR156-SPL modules regulate induction of somatic embryogenesis in citrus callus. J Exp Bot, 69: 2979–2993.
Crossref Google Scholar
 

Long, L., Guo, D.D., Gao, W., Yang, W.W., Hou, L.P., Ma, X.N., Miao, Y.C., Botella, J.R., Song, C.P., 2018b. Optimization of CRISPR/Cas9 genome editing in cotton by improved sgRNA expression. Plant Methods, 14: 85.
Crossref Google Scholar
 

Ma, C.F., Liu, M.C., Li, Q.F., Si, J., Ren, X.S., Song, H.Y., 2019. Efficient BoPDS gene editing in cabbage by the CRISPR/Cas9 system. Hortic Plant J, 5: 164–169.
Crossref Google Scholar
 

Ming, M.L., Ren, Q.R., Pan, C.T., He, Y., Zhang, Y.X., Liu, S.S., Zhong, Z.H., Wang, J.H., Malzahn, A.A., Wu, J., Zheng, X.L., Zhang, Y., Qi, Y.P., 2020. CRISPR-Cas12b enables efficient plant genome engineering. Native Plants, 6: 202–208.
Crossref Google Scholar
 
Murashige, T., Tucker, D.H.P., 1969. Growth factors requirements of citrus tissue. In: Proceedings of the First International Citrus Symposium, 3. University of California, Riverside, CA, pp. 1155–1161.
 

Nelson, B.K., Cai, X., Nebenführ, A., 2007. A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants: fluorescent organelle markers. Plant J, 51: 1126–1136.
Crossref Google Scholar
 
Omar, A.A., Dutt, M., Gmitter, F.G., Grosser, J.W., 2016. Somatic embryogenesis: still a relevant technique in citrus improvement. In: Germana, M.A., Lambardi, M. (Eds.), In vitro embryogenesis in higher plants, methods in molecular biology. Springer, New York, pp. 289–327.
Crossref
 

Omar, A.A., Murata, M.M., El-Shamy, H.A., Graham, J.H., Grosser, J.W., 2018. Enhanced resistance to citrus canker in transgenic mandarin expressing Xa21 from rice. Transgen Res, 27: 179–191.
Crossref Google Scholar
 

Peng, A.H., Chen, S.C., Lei, T.G., Xu, L.Z., He, Y.R., Wu, L., Yao, L.X., Zou, X.P., 2017. Engineering canker-resistant plants through CRISPR/Cas9-targeted editing of the susceptibility gene CsLOB1 promoter in citrus. Plant Biotechnol J, 15: 1509–1519.
Crossref Google Scholar
 

Ren, C., Liu, X.J., Zhang, Z., Wang, Y., Duan, W., Li, S.H., Liang, Z.C., 2016. CRISPR/Cas9-mediated efficient targeted mutagenesis in Chardonnay (Vitis vinifera L.). Sci Rep, 6: 32289.
Crossref Google Scholar
 

Sparkes, I.A., Runions, J., Kearns, A., Hawes, C., 2006. Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants. Nat Protoc, 1: 2019–2025.
Crossref Google Scholar
 

Sun, X.J., Hu, Z., Chen, R., Jiang, Q.Y., Song, G.H., Zhang, H., Xi, Y.J., 2015. Targeted mutagenesis in soybean using the CRISPR-Cas9 system. Sci Rep, 5: 10342.
Crossref Google Scholar
 

Tang, T., Yu, X.W., Yang, H., Gao, Q., Ji, H.T., Wang, Y.X., Yan, G.B., Peng, Y., Luo, H.F., Liu, K.D., Li, X., Ma, C.Z., Kang, C.Y., Dai, C., 2018. Development and validation of an effective CRISPR/Cas9 vector for efficiently isolating positive transformants and transgene-free mutants in a wide range of plant species. Front Plant Sci, 9: 1533.
Crossref Google Scholar
 

Varkonyi-Gasic, E., Wang, T., Voogd, C., Jeon, S., Drummond, R.S.M., Gleave, A.P., Allan, A.C., 2019. Mutagenesis of kiwifruit CENTRORADIALIS-like genes transforms a climbing woody perennial with long juvenility and axillary flowering into a compact plant with rapid terminal flowering. Plant Biotechnol J, 17: 869–880.
Crossref Google Scholar
 

Wang, G.X., Zong, M., Liu, D., Wu, Y.G., Tian, S.W., Han, S., Guo, N., Duan, M.M., Miao, L.M., Liu, F., 2022. Efficient generation of targeted point mutations in the Brassica oleracea var. botrytis genome via a modified CRISPR/Cas9 system. Hortic Plant J, 8: 527–530.
Crossref Google Scholar
 

Wang, K., Gao, E.L., Liu, D., Wu, X.M., Wang, P.W., 2020. The ER network, peroxisomes and actin cytoskeleton exhibit dramatic alterations during somatic embryogenesis of cultured citrus cells. Plant Cell Tissue Organ Cult, 148: 259–270.
Crossref Google Scholar
 

Wang, R., Fang, Y.N., Wu, X.M., Qing, M., Li, C.C., Xie, K.D., Deng, X.X., Guo, W.W., 2020. The miR399-CsUBC24 module regulates reproductive development and male fertility in citrus. Plant Physiol, 183: 1681–1695.
Crossref Google Scholar
 

Wang, Y., Liu, X.J., Ren, C., Zhong, G.Y., Yang, L., Li, S.H., Liang, Z.C., 2016. Identification of genomic sites for CRISPR/Cas9-based genome editing in the Vitis vinifera genome. BMC Plant Biol, 16: 96.
Crossref Google Scholar
 

Woo, J.W., Kim, J., Kwon, S.I., Corvalán, C., Cho, S.W., Kim, H., Kim, S.G., Kim, S.T., Choe, S., Kim, J.S., 2015. DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins. Nat Biotechnol, 33: 1162–1164.
Crossref Google Scholar
 

Xiao, S.X., Biswas, M.K., Li, M.Y., Deng, X.X., Xu, Q., Guo, W.W., 2014. Production and molecular characterization of diploid and tetraploid somatic cybrid plants between male sterile Satsuma mandarin and seedy sweet orange cultivars. Plant Cell Tissue Organ Cult, 116: 81–88.
Crossref Google Scholar
 

Xie, K.B., Minkenberg, B., Yang, Y.N., 2015. Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system. Proc Natl Acad Sci USA, 112: 3570–3575.
Crossref Google Scholar
 

Xing, H.L., Dong, L., Wang, Z.P., Zhang, H.Y., Han, C.Y., Liu, B., Wang, X.C., Chen, Q.J., 2014. A CRISPR/Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biol, 14: 327.
Crossref Google Scholar
 

Yan, L.H., Wei, S.W., Wu, Y.R., Hu, R.L., Li, H.J., Yang, W.C., Xie, Q., 2015. High-efficiency genome editing in Arabidopsis using YAO promoter-driven CRISPR/Cas9 system. Mol Plant, 8: 1820–1823.
Crossref Google Scholar
 

Yang, F., Yang, Q.S., Gao, Y.H., Ma, Y.J., Xu, Y., Teng, Y.W., Bai, S.L., 2021. Establishment of dual-cut CRISPR/Cas9 gene editing system in pear calli. Acta Hortic Sin, 48: 873–882. (in Chinese)
Google Scholar
 

Yang, L.S., Guo, Y., Hu, Y., Wen, Y.Q., 2020. CRISPR/Cas9-mediated mutagenesis of VviEDR2 results in enhanced resistance to powdery mildew in grapevine (Vitis vinifera). Acta Hortic Sin, 47: 623–634. (in Chinese)
Google Scholar
 
Yang, W., 2016. Establishment and Application of Citrus Protoplast Transient Transformation System [M.D.]. Huazhong Agricultural University, Wuhan.
 

Ye, S.W., Chen, G., Kohnen, M.V., Wang, W.J., Cai, C.Y., Ding, W.S., Wu, C., Gu, L.F., Zheng, Y.S., Ma, X.Q., Lin, C.T., Zhu, Q., 2020. Robust CRISPR/Cas9 mediated genome editing and its application in manipulating plant height in the first generation of hexaploid Ma bamboo (Dendrocalamus latiflorus Munro). Plant Biotechnol J, 18: 1501–1503.
Crossref Google Scholar
 

Yoo, S.D., Cho, Y.H., Sheen, J., 2007. Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc, 2: 1565–1572.
Crossref Google Scholar
 

Zhang, F., LeBlanc, C., Irish, V.F., Jacob, Y., 2017. Rapid and efficient CRISPR/Cas9 gene editing in citrus using the YAO promoter. Plant Cell Rep, 36: 1883–1887.
Crossref Google Scholar
 

Zhang, F., Rossignol, P., Huang, T.B., Wang, Y.W., May, A., Dupont, C., Orbovic, V., Irish, V.F., 2020. Reprogramming of stem cell activity to convert thorns into branches. Curr Biol, 30: 2951–2961.
Crossref Google Scholar
 

Zhu, C.Q., Zheng, X.J., Huang, Y., Ye, J.L., Chen, P., Zhang, C.L., Zhao, F., Xie, Z.Z., Zhang, S.Q., Wang, N., Li, H., Wang, L., Tang, X.M., Chai, L.J., Xu, Q., Deng, X.X., 2019. Genome sequencing and CRISPR/Cas9 gene editing of an early flowering Mini-Citrus (Fortunella hindsii). Plant Biotechnol J, 17: 2199–2210.
Crossref Google Scholar
 

Zhu, F., Luo, T., Liu, C.Y., Wang, Y., Yang, H.B., Yang, W., Zheng, L., Xiao, X., Zhang, M.F., Xu, R.W., Xu, J.G., Zeng, Y.L., Xu, J., Xu, Q., Guo, W.W., Larkin, R.M., Deng, X.X., Cheng, Y.J., 2017. An R2R3-MYB transcription factor represses the transformation of α- and β-branch carotenoids by negatively regulating expression of CrBCH2 and CrNCED5 in flavedo of Citrus reticulata. New Phytol, 216: 178–192.
Crossref Google Scholar
 

Zou, X.P., Fan, D., Peng, A.H., He, Y.R., Xu, L.Z., Lei, T.G., Yao, L.X., Li, Q., Luo, K.M., Chen, S.C., 2019. CRISPR/Cas9-mediated editing of multiple sites in the citrus CsLOB1 promoter. Acta Hortic Sin, 46: 337–344. (in Chinese)
Google Scholar
Horticultural Plant Journal
Volume 9 Issue 3,
June 2023
Pages 425-436
DOI: 10.1016/j.hpj.2022.06.006
Cite this article:
Yang W, Ren J, Liu W, et al. An efficient transient gene expression system for protein subcellular localization assay and genome editing in citrus protoplasts. Horticultural Plant Journal, 2023, 9(3): 425-436. https://doi.org/10.1016/j.hpj.2022.06.006

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Received: 22 April 2022
Revised: 10 June 2022
Accepted: 17 June 2022
Published: 23 June 2022
© 2022 Chinese Society for Horticultural Science (CSHS) and Institute of Vegetables and Flowers (IVF), Chinese Academy of Agricultural Sciences (CAAS).

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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