CRISPR/Cas9-mediated VvPR4b editing decreases downy mildew resistance in grapevine (Vitis vinifera L.)

Abstract

Downy mildew of grapevine (Vitis vinifera L.), caused by the oomycete pathogen Plasmopara viticola, is one of the most serious concerns for grape production worldwide. It has been widely reported that the pathogenesis-related 4 (PR4) protein plays important roles in plant resistance to diseases. However, little is known about the role of PR4 in the defense of grapevine against P. viticola. In this study, we engineered loss-of-function mutations in the VvPR4b gene from the cultivar “Thompson Seedless” using the CRISPR/Cas9 system and evaluated the consequences for downy mildew resistance. Sequencing results showed that deletions were the main type of mutation introduced and that no off-target events occurred. Infection assays using leaf discs showed that, compared to wild-type plants, the VvPR4b knockout lines had increased susceptibility to P. viticola. This was accompanied by reduced accumulation of reactive oxygen species around stomata. Measurement of the relative genomic abundance of P. viticola in VvPR4b knockout lines also demonstrated that the mutants had increased susceptibility to the pathogen. Our results confirm that VvPR4b plays an active role in the defense of grapevine against downy mildew.

References

[1] Bouquet, A., Torregrosa, L., Iocco, P. & Thomas, M. R. in Agrobacterium Protocols Volume 2 273-285 (Springer, 2006).

[2] Jürges, G., Kassemeyer, H. H., Dürrenberger, M., Düggelin, M. & Nick, P. The mode of interaction between Vitis and Plasmopara viticola Berk. & Curt. Ex de Bary depends on the host species. Plant Biol. 11, 886-898 (2009).

[3] Fröbel, S. & Zyprian, E. Colonization of Different Grapevine Tissues by Plasmopara viticola-A Histological Study. Front. Plant Sci. 10, https://doi.org/10.3389/fpls.2019.00951 (2019).

[4] Alexander, D. et al. Increased tolerance to two oomycete pathogens in transgenic tobacco expressing pathogenesis-related protein 1a. Proc. Natl Acad. Sci. USA. 90, 7327-7331 (1993).

[5] Datta, K. et al. Over-expression of the cloned rice thaumatin-like protein (PR-5) gene in transgenic rice plants enhances environmental friendly resistance to Rhizoctonia solani causing sheath blight disease. Theor. Appl. Genet. 98, 1138-1145 (1999).

[6] Melchers, L. S. et al. A new class of tobacco chitinases homologous to bacterial exo-chitinases displays antifungal activity. Plant J. 5, 469-480 (1994).

[7] Nookaraju, A. & Agrawal, D. C. Enhanced tolerance of transgenic grapevines expressing chitinase and β-1,3-glucanase genes to downy mildew. Plant Cell Tiss. Org. Cult. 111, 15-28 (2012).

[8] Menezes, S. P. et al. The pathogenesis-related protein PR-4b from Theobroma cacao presents RNase activity, Ca2+ and Mg2+ dependent-DNase activity and antifungal action on Moniliophthora perniciosa. BMC Plant Biol. 14, 161 (2014).

[9] Lu, H.-C. et al. Cloning and expression of pathogenesis-related protein 4 from jelly fig (Ficus awkeotsang Makino) achenes associated with ribonuclease, chitinase and anti-fungal activities. Plant Physiol. Biochem. 56, 1-13 (2012).

[10] Dai, L. et al. The Novel Gene VpPR4-1 from Vitis pseudoreticulata Increases Powdery Mildew Resistance in Transgenic Vitis vinifera L. Front. Plant Sci. 7, 695 (2016).

[11] Zhou, Q. et al. A circulatory system useful both for long-term somatic embryogenesis and genetic transformation in Vitis vinifera L. cv. Thompson Seedless. Plant Cell Tiss. Org. Cult. 118, 157-168 (2014).

[12] Dai, L. et al. Establishment of a picloram-induced somatic embryogenesis system in Vitis vinifera cv. chardonnay and genetic transformation of a stilbene synthase gene from wild-growing Vitis species. Plant Cell Tiss. Org. Cult. 121, 397-412 (2015).

[13] Xing, H.-L. et al. A CRISPR/Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biol. 14, 327 (2014).

[14] Wiedenheft, B., Sternberg, S. H. & Doudna, J. A. RNA-guided genetic silencing systems in bacteria and archaea. Nature 482, 331-338 (2012).

[15] Symington, L. S. & Gautier, J. Double-strand break end resection and repair pathway choice. Annu. Rev. Genet. 45, 247-271 (2011).

[16] Wang, X., Tu, M., Li, Z., Wang, Y. & Wang, X. Current Progress and Future Prospects for the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Genome Editing Technology in Fruit Tree Breeding. Crit. Rev. Plant Sci. 37, 233-258 (2018).

[17] Xie, K. & Yang, Y. RNA-guided genome editing in plants using a CRISPR-Cas system. Mol. Plant 6, 1975-1983 (2013).

[18] Ma, X. et al. A Robust CRISPR/Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants. Mol. Plant 8, 1274-1284 (2015).

[19] Liu, D., Hu, R., Palla, K. J., Tuskan, G. A. & Yang, X. Advances and perspectives on the use of CRISPR/Cas9 systems in plant genomics research. Curr. Opin. Plant Biol. 30, 70-77 (2016).

[20] Chen, L. et al. A method for the production and expedient screening of CRISPR/Cas9-mediated non-transgenic mutant plants. Hortic. Res. 5, 1-12 (2018).

[21] Saika, H., Mori, A., Endo, M. & Toki, S. Targeted deletion of rice retrotransposon Tos17 via CRISPR/Cas9. Plant Cell Rep. 38, 455-458 (2018).

[22] Malnoy, M. et al. DNA-Free Genetically Edited Grapevine and Apple Protoplast Using CRISPR/Cas9 Ribonucleoproteins. Front. Plant Sci. 7, 1904 (2016).

[23] Ren, C. et al. CRISPR/Cas9-mediated efficient targeted mutagenesis in Chardonnay (Vitis vinifera L.). Sci. Rep. 6, 32289, https://doi.org/10.1038/srep32289 (2016).

[24] Wang, X. et al. CRISPR/Cas9-mediated efficient targeted mutagenesis in grape in the first generation. Plant Biotechnol. J. 16, 844-855 (2018).

[25] Nakajima, I. et al. CRISPR/Cas9-mediated targeted mutagenesis in grape. PLoS ONE 12, e0177966 (2017).

[26] Ren, F. et al. Efficiency optimization of CRISPR/Cas9-mediated targeted mutagenesis in grape. Front. Plant Sci. 10, 612 (2019).

[27] Ren, C. et al. Knockout of VvCCD8 gene in grapevine affects shoot branching. BMC Plant Biol. 20, 1-8 (2020).

[28] Dhekney, S., Li, Z., Dutt, M. & Gray, D. Agrobacterium-mediated transformation of embryogenic cultures and plant regeneration in Vitis rotundifolia Michx. (muscadine grape). Plant Cell Rep. 27, 865-872 (2008).

[29] Carroll, D. Genome Engineering with Targetable Nucleases. Annu. Rev. Biochem. 83, 409-439 (2014).

[30] Jia, H. et al. Development of a CRISPR/Cas9-mediated gene-editing tool in Streptomyces rimosus. Microbiology 163, 1148-1155 (2017).

[31] Zhang, J.-H., Adikaram, P., Pandey, M., Genis, A. & Simonds, W. F. Optimization of genome editing through CRISPR-Cas9 engineering. Bioengineered 7, 166-174 (2016).

[32] Mao, Y. et al. Application of the CRISPR-Cas system for efficient genome engineering in plants. Mol. Plant 6, 2008-2011 (2013).

[33] Li, Z. T., Dhekney, S., Dutt, M. & Gray, D. An improved protocol for Agrobacterium-mediated transformation of grapevine (Vitis vinifera L.). Plant Cell Tiss. Org. Cult. 93, 311-321 (2008).

[34] Brooks, C., Nekrasov, V., Lippman, Z. B. & Van Eck, J. Efficient gene editing in tomato in the first generation using the clustered regularly interspaced short palindromic repeats/CRISPR-associated9 system. Plant Physiol. 166, 1292-1297 (2014).

[35] Kortekamp, A. Expression analysis of defence-related genes in grapevine leaves after inoculation with a host and a non-host pathogen. Plant Physiol. Biochem. 44, 58-67 (2006).

[36] Kiefer, B., Riemann, M., Büche, C., Kassemeyer, H.-H. & Nick, P. The host guides morphogenesis and stomatal targeting in the grapevine pathogen Plasmopara viticola. Planta 215, 387-393 (2002).

[37] Kumar, Y. et al. Fusarium oxysporum mediates systems metabolic reprogramming of chickpea roots as revealed by a combination of proteomics and metabolomics. Plant Biotechnol. J. 14, 1589-1603 (2016).

[38] Hong, J. K., Hwang, I. S. & Hwang, B. K. Functional roles of the pepper leucine-rich repeat protein and its interactions with pathogenesis-related and hypersensitive-induced proteins in plant cell death and immunity. Planta 246, 351-364 (2017).

[39] Kim, Y. H., Park, S. C., Yun, B. W. & Kwak, S. S. Overexpressing sweetpotato peroxidase gene swpa4 affects nitric oxide production by activating the expression of reactive oxygen species- and nitric oxide-related genes in tobacco. Plant Physiol. Biochem. 120, 52-60 (2017).

[40] Vo, K. T. X. et al. OsWRKY67 Plays a Positive Role in Basal and XA21-Mediated Resistance in Rice. Front. Plant Sci. 8, https://doi.org/10.3389/fpls.2017.02220 (2018).

[41] Hong, Y. et al. The OsMPK15 Negatively Regulates Magnaporthe oryza and Xoo Disease Resistance via SA and JA Signaling Pathway in Rice. Front. Plant Sci. 10, https://doi.org/10.3389/fpls.2019.00752 (2019).

[42] Wu, G. et al. Activation of host defense mechanisms by elevated production of H2O2 in transgenic plants. Plant Physiol. 115, 427-435 (1997).

[43] Yu, Y.-H. et al. Grape (Vitis davidii) VdGATA2 functions as a transcription activator and enhances powdery mildew resistance via the active oxygen species pathway. Sci. Hortic. 267, 109327 (2020).

[44] Kortekamp, A. & Zyprian, E. v.a. Characterization of Plasmopara-Resistance in grapevine using in vitro plants. J. Plant Physiol. 160, 1393-1400 (2003).

[45] Wise, A. A., Liu, Z. & Binns, A. N. Three methods for the introduction of foreign DNA into Agrobacterium. in Agrobacterium protocols 43-54 (Springer, 2006).

[46] Su, H. et al. Overexpression of VpPR10.1 by an efficient transformation method enhances downy mildew resistance in V. vinifera. Plant Cell Rep. 37, 819-832 (2018).

[47] Ma, H. et al. Grapevine VpPR10.1 functions in resistance to Plasmopara viticola through triggering a cell death-like defence response by interacting with VpVDAC3. Plant Biotechnol. J. 16, 1488-1501 (2018).

[48] Wong, F. P. & Wilcox, W. F. Distribution of baseline sensitivities to azoxystrobin among isolates of Plasmopara viticola. Plant Dis. 84, 275-281 (2000).

[49] Liu, R. et al. Histological responses to downy mildew in resistant and susceptible grapevines. Protoplasma 252, 259-270 (2015).

[50] Yin, X. et al. Pathogen development and host responses to Plasmopara viticola in resistant and susceptible grapevines: an ultrastructural study. Hortic. Res. 4, 17033 (2017).

[51] Valsesia, G. et al. Development of a high-throughput method for quantification of Plasmopara viticola DNA in grapevine leaves by means of quantitative real-time polymerase chain reaction. Phytopathology 95, 672-678 (2005).

[52] Ioos, R., Laugustin, L., Rose, S., Tourvieille, J. & Tourvieille de Labrouhe, D. Development of a PCR test to detect the downy mildew causal agent Plasmopara halstedii in sunflower seeds. Plant Pathol. 56, 209-218 (2007).

[53] Trouvelot, S. et al. A β-1, 3 glucan sulfate induces resistance in grapevine against Plasmopara viticola through priming of defense responses, including HR-like cell death. Mol. Plant-Microbe Interact. 21, 232-243 (2008).