Optimization of the protoplast transient expression system for gene functional studies in strawberry (Fragaria vesca)
- 63 Downloads
Polyethylene glycol (PEG)-mediated transient expression system in plant protoplasts has been widely used in a variety of plants for gene function characterization. However, such a system has not been developed for strawberry. In this study, we report a method for obtaining high quality and high yield protoplasts from strawberry leaves. In addition, we developed an efficient transient gene expression system based on the PEG-mediated method, and the system has been successfully applied to studies of protein expression, protein subcellular localization and protein–protein interaction in strawberry. Furthermore, the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) technology was used to enable targeted mutagenesis of the FvTCP17 gene in strawberry protoplasts. Taken together, our results demonstrate the potential of this highly efficient mesophyll protoplast system for transient gene expression and induction of CRISPR/Cas9-mediated genome editing in strawberry.
We report a highly efficient mesophyll protoplast system for transient gene expression and induction of CRISPR/Cas9‐mediated genome editing in strawberry.
KeywordsStrawberry Protoplast isolation Transient gene expression CRISPR/Cas9 FvTCP17
The present study was financially supported by Grants from the Opening Foundation of Key Laboratory of Fruit Breeding Technology, the Ministry of Agriculture and Rural Affairs of China (NYB201705-3), the Natural Science Foundation of Henan (162300410329), and the project for Agriculture Sci-Tech of Xi’an (20193020YF008NS008). We also thank Bruce Levine of University of Maryland for careful editing of this manuscript.
JYF conceived the research. YJG performed all treatments with assistance of YLL, PPB, DJW, YYM, and Y.H. YJG and YYM analyzed the data. YH provided assistance in constructing vectors for subcellular localization. YJG, JYF, HCZ and YQW contributed to the writing of the manuscript. All authors read and approved the final manuscript.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
- Bai Y, Han N, Wu J, Yang Y, Wang J, Zhu M, Bian H (2014) A transient gene expression system using barley protoplasts to evaluate microRNAs for post-transcriptional regulation of their target genes. Plant Cell Tissue Organ Cult 119(1):219. https://doi.org/10.1007/s11240-014-0527-z CrossRefGoogle Scholar
- Cui M-Y, Wei W, Gao K, Xie Y-G, Guo Y, Feng J-Y (2017) A rapid and efficient Agrobacterium-mediated transient gene expression system for strawberry leaves and the study of disease resistance proteins. Plant Cell Tissue Organ Cult 131(2):246. https://doi.org/10.1007/s11240-017-1279-3 CrossRefGoogle Scholar
- Edger PP, Poorten TJ, VanBuren R, Hardigan MA, Colle M, McKain MR, Smith RD, Teresi SJ, Nelson ADL, Wai CM, Alger EI, Bird KA, Yocca AE, Pumplin N, Ou S, Ben-Zvi G, Brodt A, Baruch K, Swale T, Shiue L, Acharya CB, Cole GS, Mower JP, Childs KL, Jiang N, Lyons E, Freeling M, Puzey JR, Knapp SJ (2019) Origin and evolution of the octoploid strawberry genome. Nat Genet 51(3):547. https://doi.org/10.1038/s41588-019-0356-4 CrossRefPubMedPubMedCentralGoogle Scholar
- Hu Y, Han Y, Wei W, Li Y, Zhang K, Gao Y, Zhao F, Feng J (2015) Identification, isolation, and expression analysis of heat shock transcription factors in the diploid woodland strawberry Fragaria vesca Front Plant Sci 6:736. https://doi.org/10.3389/fpls.2015.00736 CrossRefPubMedPubMedCentralGoogle Scholar
- Khidr YA, Flachowsky H, Haselmair-Gosch C, Thill J, Miosic S, Hanke MV, Stich K, Halbwirth H (2017) Evaluation of a MdMYB10/GFP43 fusion gene for its suitability to act as reporter gene in promoter studies in Fragaria vesca L. 'Rugen' Plant Cell Tissue Organ Cult 130(2):356. https://doi.org/10.1007/s11240-017-1229-0 CrossRefPubMedPubMedCentralGoogle Scholar
- Le C, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA (2015) Multiplex genome engineering using CRISPR/Cas systems. Science 1239(11):197Google Scholar
- Li JF, Norville JE, Aach J, McCormack M, Zhang D, Bush J, Church GM, Sheen J (2013) Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nat Biotechnol 31(8):691. https://doi.org/10.1038/nbt.2654 CrossRefPubMedPubMedCentralGoogle Scholar
- Lin CS, Hsu CT, Yang LH, Lee LY, Fu JY, Cheng QW, Wu FH, Hsiao HC, Zhang Y, Zhang R, Chang WJ, Yu CT, Wang W, Liao LJ, Gelvin SB, Shih MC (2018) Application of protoplast technology to CRISPR/Cas9 mutagenesis: from single-cell mutation detection to mutant plant regeneration. Plant Biotechnol J 16(7):1295–1310. https://doi.org/10.1111/pbi.12870 CrossRefPubMedPubMedCentralGoogle Scholar
- Marion J, Bach L, Bellec Y, Meyer C, Gissot L, Faure JD (2008) Systematic analysis of protein subcellular localization and interaction using high-throughput transient transformation of Arabidopsis seedlings. Plant J 56(1):169–179. https://doi.org/10.1111/j.1365-313X.2008.03596.x CrossRefPubMedGoogle Scholar
- Schaart JG, Dubos C, Romero De La Fuente I, van Houwelingen AM, de Vos RC, Jonker HH, Xu W, Routaboul JM, Lepiniec L, Bovy AG (2013) Identification and characterization of MYB-bHLH-WD40 regulatory complexes controlling proanthocyanidin biosynthesis in strawberry (Fragaria x ananassa) fruits. New Phytol 197(2):454–467. https://doi.org/10.1111/nph.12017 CrossRefPubMedGoogle Scholar
- Wei L, Mao W, Jia M, Xing S, Ali U, Zhao Y, Chen Y, Cao M, Dai Z, Zhang K, Dou Z, Jia W, Li B (2018) FaMYB44.2, a transcriptional repressor, negatively regulates sucrose accumulation in strawberry receptacles through interplay with FaMYB10. J Exp Bot 69(20):4805–4820. https://doi.org/10.1093/jxb/ery249 CrossRefPubMedPubMedCentralGoogle Scholar
- Yang JW, Fu JX, Li J, Cheng XL, Li F, Dong JF, Liu ZL, Zhuang CX (2014) A novel co-immunoprecipitation protocol based on protoplast transient gene expression for studying protein-protein interactions in rice. Plant Mol Biol Rep 32(1):153–161. https://doi.org/10.1007/s11105-013-0633-9 CrossRefGoogle Scholar
- Zhang Y, Su J, Duan S, Ao Y, Dai J, Liu J, Wang P, Li Y, Liu B, Feng D, Wang J, Wang H (2011) A highly efficient rice green tissue protoplast system for transient gene expression and studying light/chloroplast-related processes. Plant Methods 7(1):30. https://doi.org/10.1186/1746-4811-7-30 CrossRefPubMedPubMedCentralGoogle Scholar
- Zhao FL, Li YJ, Hu Y, Gao YR, Zang XW, Ding Q, Wang YJ, Wen YQ (2016) A highly efficient grapevine mesophyll protoplast system for transient gene expression and the study of disease resistance proteins. Plant Cell Tissue Organ Cult 125(1):43–57. https://doi.org/10.1007/s11240-015-0928-7 CrossRefGoogle Scholar