Tomato fruit as a model for tissue-specific gene silencing in crop plants

Abstract

Use of CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated 9)- mediated genome editing has proliferated for use in numerous plant species to modify gene function and expression, usually in the context of either transient or stably inherited genetic alternations. While extremely useful in many applications, modification of some loci yields outcomes detrimental to further experimental evaluation or viability of the target organism. Expression of Cas9 under a promoter conferring gene knockouts in a tissue-specific subset of genomes has been demonstrated in insect and animal models, and recently in Arabidopsis. We developed an in planta GFP (green fluorescent protein) assay system to demonstrate fruit-specific gene editing in tomato using a phosphoenolpyruvate carboxylase 2 gene promoter. We then targeted a SET-domain containing polycomb protein, SlEZ2, previously shown to yield pleiotropic phenotypes when targeted via³⁵S-driven RNA interference and we were able to characterize fruit phenotypes absent additional developmental perturbations. Tissue-specific gene editing will have applications in assessing function of essential genes otherwise difficult to study via germline modifications and will provide routes to edited genomes in tissues that could not otherwise be recovered when their germline modification perturbs their normal development.

References

[1] Barrangou, R. et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science 315, 1709-1712 (2007).

[2] Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816-821 (2012).

[3] Adli, M. The CRISPR tool kit for genome editing and beyond. Nat. Commun. 9, 1911 (2018).

[4] Barrangou, R. & Doudna, J. A. Applications of CRISPR technologies in research and beyond. Nat. Biotechnol. 34, 933-941 (2016).

[5] Demirci, Y., Zhang, B. & Unver, T. CRISPR/Cas9: an RNA-guided highly precise synthetic tool for plant genome editing. J. Cell Physiol. 233, 1844-1859 (2018).

[6] Dahan-Meir, T. et al. Efficient in planta gene targeting in tomato using geminiviral replicons and the CRISPR/Cas9 system. Plant J. 95, 5-16 (2018).

[7] Li, J. et al. Gene replacements and insertions in rice by intron targeting using CRISPR-Cas9. Nat. Plants 2, 16139 (2016).

[8] Piatek, A. et al. RNA-guided transcriptional regulation in planta via synthetic dCas9-based transcription factors. Plant Biotechnol. J. 13, 578-589 (2015).

[9] Kang, B.-C. et al. Precision genome engineering through adenine base editing in plants. Nat. Plants 4, 427-431 (2018).

[10] Decaestecker, W. et al. CRISPR-TSKO: a technique for efficient mutagenesis in specific cell types, tissues, or organs in arabidopsis. Plant Cell https://doi.org/10.1105/tpc.19.00454 (2019).

[11] Liang, Y. et al. A screening method to identify efficient sgRNAs in Arabidopsis, used in conjunction with cell-specific lignin reduction. Biotechnol. Biofuels 12, 130 (2019).

[12] Lloyd, J. P., Seddon, A. E., Moghe, G. D., Simenc, M. C. & Shiu, S. H. Characteristics of plant essential genes allow for within- and between-species prediction of lethal mutant phenotypes. Plant Cell 27, 2133-2147 (2015).

[13] Kohler, C. & Hennig, L. Regulation of cell identity by plant Polycomb and trithorax group proteins. Curr. Opin. Genet Dev. 20, 541-547 (2010).

[14] Pu, L. & Sung, Z. R. PcG and trxG in plants-friends or foes. Trends Genet. 31, 252-262 (2015).

[15] Xiao, J. & Wagner, D. Polycomb repression in the regulation of growth and development in Arabidopsis. Curr. Opin. Plant Biol. 23, 15-24 (2015).

[16] Boureau, L. et al. A CURLY LEAF homologue controls both vegetative and reproductive development of tomato plants. Plant Mol. Biol. 90, 485-501 (2016).

[17] Bucher, E., Kong, J., Teyssier, E. & Gallusci, P. in Advances in Botanical Research, Vol. 88 (eds Mirouze, M., Bucher, E. & Gallusci, P. ) 327-360 (Academic Press, 2018).

[18] Fernandez, A. I. et al. Flexible tools for gene expression and silencing in tomato. Plant Physiol. 151, 1729-1740 (2009).

[19] Jacobs, T. B., Zhang, N., Patel, D. & Martin, G. B. Generation of a collection of mutant tomato lines using pooled CRISPR libraries. Plant Physiol. 174, 2023-2037 (2017).

[20] Liu, D. D., Dong, Q. L., Fang, M. J., Chen, K. Q. & Hao, Y. J. Ectopic expression of an apple apomixis-related gene MhFIE induces co-suppression and results in abnormal vegetative and reproductive development in tomato. J. Plant Physiol. 169, 1866-1873 (2012).

[21] Chakrabarti, M. et al. A cytochrome P450 regulates a domestication trait in cultivated tomato. Proc. Natl Acad. Sci. USA 110, 17125-17130 (2013).

[22] Gouthu, S. & Deluc, L. G. Timing of ripening initiation in grape berries and its relationship to seed content and pericarp auxin levels. BMC Plant Biol. 15, 46 (2015).

[23] Tanksley, S. D. The genetic, developmental, and molecular bases of fruit size and shape variation in tomato. Plant Cell 16, S181-S189 (2004).

[24] Nitsch, J. P. The physiology of fruit growth. Annu. Rev. Plant Physiol. 4, 199-236 (1953).

[25] Gillaspy, G., Ben-David, H. & Gruissem, W. Fruits: a developmental perspective. Plant Cell 5, 1439-1451 (1993).

[26] Azzi, L. et al. Fruit growth-related genes in tomato. J. Exp. Bot. 66, 1075-1086 (2015).

[27] Zhong, S. et al. Single-base resolution methylomes of tomato fruit development reveal epigenome modifications associated with ripening. Nat. Biotechnol. 31, 154-159 (2013).

[28] Jacobs, T. B., LaFayette, P. R., Schmitz, R. J. & Parrott, W. A. Targeted genome modifications in soybean with CRISPR/Cas9. BMC Biotechnol. 15, 16 (2015).

[29] Floss, D. S., Levy, J. G., Levesque-Tremblay, V., Pumplin, N. & Harrison, M. J. DELLA proteins regulate arbuscule formation in arbuscular mycorrhizal symbiosis. Proc. Natl Acad. Sci. USA 110, E5025-E5034 (2013).

[30] Van Eck, J., Keen, P. & Tjahjadi, M. in Transgenic Plants: Methods and Protocols (eds. Kumar, V., Barone, P. & Smith, M.) 225-234 (Springer, New York, 2019).