Engineering CRISPR immune systems conferring GLRaV-3 resistance in grapevine

Abstract

Grapevine leafroll-associated virus 3 (GLRaV-3) is one of the causal agents of grapevine leafroll disease (GLD), which severely impacts grapevine production in most viticultural regions of the world. The development of virus-resistant plants is a desirable strategy for the efficient control of viral diseases. However, natural resistant resources have not been reported in the genus Vitis, and anti-GLRaV-3 research has been quite limited in grapevine. In this study, by expressing FnCas9 and LshCas13a, we established a highly effective transgenic construct screening system via an optimized Agrobacterium-mediated transient delivery system in grapevine plantlets. Our study indicated that CRISPR/FnCas9 and LshCas13a caused GLRaV-3 inhibition. Moreover, three vectors-pCR01-CP, pCR11-Hsp70h and pCR11-CP-exhibited the most robust inhibition efficiency compared to those targeting other sites and could be further engineered to generate GLRaV-3-resistant grapevine. In addition, the viral interference efficiency of FnCas9 was dependent on its RNA binding activity. The efficiency of virus inhibition was positively correlated with the level of Cas gene expression. Importantly, we demonstrated that LshCas13a had better interference efficiency against viruses than FnCas9. In summary, this study confirmed that these two RNA-targeting CRISPR mechanisms can confer immunity against viruses in grapevine, providing new avenues to control GLRaV-3 or other RNA viruses in fruit crops.

References

[1] Bacilieri R, Lacombe T, Cunff LL, et al. Genetic structure in cultivated grapevines is linked to geography and human selection. BMC Plant Biol. 2013; 13: 25.

[2] Tilman D, Balzer C, Hill J, et al. Global food demand and the sustainable intensification of agriculture. Proc Natl Acad Sci U S A. 2011; 108: 20260-4.

[3] Whitham SA, Yang C, Goodin MM . Global impact: elucidating plant responses to viral infection. Mol Plant-Microbe Interact. 2006; 19: 1207-15.

[4] Martelli GP . Directory of virus and virus-like diseases of the grapevine and their agents. J Plant Pathol. 2014; 96: 1-136.

[5] Naidu RA, Maree HJ, Burger JT . Grapevine leafroll disease and associated viruses: a unique pathosystem. Annu Rev Phytopathol. 2015; 53: 613-34.

[6] Sampol B, Bota J, Riera D, et al. Analysis of the virus-induced inhibition of photosynthesis in malmsey grapevines. New Phytol. 2003; 160: 403-12.

[7] Gutha LR, Casassa LF, Harbertson JF, et al. Modulation of flavonoid biosynthetic pathway genes and anthocyanins due to virus infection in grapevine (Vitis vinifera L.) leaves . BMC Plant Biol. 2010; 10: 187.

[8] Vega A, Gutierrez RA, Pena-Neira A, et al. Compatible GLRaV-3 viral infections affect berry ripening decreasing sugar accumulation and anthocyanin biosynthesis in Vitis vinifera . Plant Mol Biol. 2011; 77: 261-74.

[9] Moutinho-Pereira J, Correia C, Gonçalves B, et al. Impacts of leafroll-associated viruses (GLRaV-1 and -3) on the physiology of the Portuguese grapevine cultivar ‘Touriga Nacional’ growing under field conditions. Ann Appl Biol. 2012; 160: 237-49.

[10] Halldorson MM, Keller M . Grapevine leafroll disease alters leaf physiology but has little effect on plant cold hardiness. Planta. 2018; 248: 1201-11.

[11] Habili N, Nutter FW . Temporal and spatial analysis of grapevine leafroll-associated virus 3 in pinot noir grapevines in Australia. Plant Dis. 1997; 81: 625-8.

[12] Atallah SS, Gomez MI, Fuchs MF, et al. Economic impact of grapevine leafroll disease on Vitis vinifera cv. Cabernet franc in finger lakes vineyards of New York . Am J Enol Viticult. 2011; 63: 73-9.

[13] Maree HJ, Almeida RPP, Bester R, et al. Grapevine leafroll-associated virus 3. Front Microbiol. 2013; 4: 82.

[14] Panattoni A, D’Anna F, Triolo E . Antiviral activity of tiazofurin and mycophenolic acid against grapevine leafroll-associated virus 3 in Vitis vinifera explants. Antivir Res. 2007; 73: 206-11.

[15] Wang XY, Zhang CW, Huang WT, et al. Crude garlic extract significantly inhibits replication of grapevine viruses. Plant Pathol. 2020; 69: 149-58.

[16] Wang Q, Valkonen JP . Cryotherapy of shoot tips: novel pathogen eradication method. Trends Plant Sci. 2009; 14: 119-22.

[17] Tsai CW, Chau J, Fernandez L, et al. Transmission of grapevine leafroll-associated virus 3 by the vine mealybug (Planococcus ficus). Phytopathology. 2008; 98: 1093-8.

[18] Saporta R, San Pedro T, Gisbert C . Attempts at grapevine (Vitis vinifera L.) breeding through genetic transformation: the main limiting factors . Vitis. 2016; 55: 173-86.

[19] Laimer M, Lemaire O, Herrbach E, et al. Resistance to viruses, phytoplasmas and their vectors in the grapevine in Europe: a review. J Plant Pathol. 2009; 91: 7-23.

[20] Shekhawat UKS, Ganapathi TR, Hadapad AB . Transgenic banana plants expressing small interfering RNAs targeted against viral replication initiation gene display high-level resistance to banana bunchy top virus infection. J Gen Virol. 2012; 93: 1804-13.

[21] Younis A, Siddique MI, Kim CK, et al. RNA interference (RNAi) induced gene silencing: a promising approach of hi-tech plant breeding. Int J Biol Sci. 2014; 10: 1150-8.

[22] Zhao Y, Yang X, Zhou G, et al. Engineering plant virus resistance: from RNA silencing to genome editing strategies. Plant Biotechnol J. 2020; 18: 328-36.

[23] Levy M, Edelbaum O, Sela I . Tobacco mosaic virus regulates the expression of its own resistance gene N. Plant Physiol. 2004; 135: 2392-7.

[24] Jones JD, Dangl JL . The plant immune system. Nature. 2006; 444: 323-9.

[25] Wiedenheft B, Sternberg SH, Doudna JA . RNA-guided genetic silencing systems in bacteria and archaea. Nature. 2012; 482: 331-8.

[26] Sorek R, Lawrence CM, Wiedenheft B . CRISPR-mediated adaptive immune systems in bacteria and archaea. Annu Rev Biochem. 2013; 82: 237-66.

[27] Cong L, Ran FA, Cox D, et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013; 339: 819-23.

[28] Ali Z, Abulfaraj A, Idris A, et al. CRISPR/Cas9-mediated viral interference in plants. Genome Biol. 2015; 16: 238.

[29] Baltes NJ, Hummel AW, Konecna E, et al. Conferring resistance to geminiviruses with the CRISPR-Cas prokaryotic immune system. Nat Plants. 2015; 1: 15145.

[30] Ji X, Zhang H, Zhang Y, et al. Establishing a CRISPR-Cas-like immune system conferring DNA virus resistance in plants. Nat Plants. 2015; 1: 15144.

[31] Zhang T, Zheng Q, Yi X, et al. Establishing RNA virus resistance in plants by harnessing CRISPR immune system. Plant Biotechnol J. 2018; 16: 1415-23.

[32] Aman R, Ali Z, Butt H, et al. RNA virus interference via CRISPR/Cas13a system in plants. Genome Biol. 2018; 19: 1.

[33] Pyott DE, Sheehan E, Molnar A . Engineering of CRISPR/Cas9-mediated potyvirus resistance in transgene-free Arabidopsis plants. Mol Plant Pathol. 2016; 17: 1276-88.

[34] Chandrasekaran J, Brumin M, Wolf D, et al. Development of broad virus resistance in non-transgenic cucumber using CRISPR/Cas9 technology. Mol Plant Pathol. 2016; 17: 1140-53.

[35] Mehta D, Stürchler A, Anjanappa RB, et al. Linking CRISPR-Cas9 interference in cassava to the evolution of editing-resistant geminiviruses. Genome Biol. 2019; 20: 80.

[36] Gauffier C, Lebaron C, Moretti A, et al. A TILLING approach to generate broad-spectrum resistance to potyviruses in tomato is hampered by eIF4E gene redundancy. Plant J. 2016; 85: 717-29.

[37] Abudayyeh OO, Gootenberg JS, Konermann S, et al. C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science. 2016; 353: aaf5573.

[38] Sampson TR, Saroj SD, Llewellyn AC, et al. CRISPR/Cas system mediates bacterial innate immune evasion and virulence. Nature. 2013; 497: 254-7.

[39] Price AA, Grakoui A, Weiss DS . Harnessing the prokaryotic adaptive immune system as a eukaryotic antiviral defense. Trends Microbiol. 2016; 24: 294-306.

[40] Ashraf MU, Salman HM, Khalid MF, et al. CRISPR-Cas13a mediated targeting of hepatitis C virus internal-ribosomal entry site (IRES) as an effective antiviral strategy. Biomed Pharmacother. 2021; 136: 111239.

[41] Zhang T, Zhao Y, Ye J, et al. Establishing CRISPR/Cas13a immune system conferring RNA virus resistance in both dicot and monocot plants. Plant Biotechnol J. 2019; 17: 1185-7.

[42] Dolja VV, Kreuze JF, Valkonen JP . Comparative and functional genomics of closteroviruses. Virus Res. 2006; 117: 38-51.

[43] Pang X, Halaly T, Crane O, et al. Involvement of calcium signalling in dormancy release of grape buds. J Exp Bot. 2007; 58: 3249-62.

[44] Naidu R, Rowhani A, Fuchs M, et al. Grapevine leafroll: a complex viral disease affecting a high-value fruit crop. Plant Dis. 2014; 98: 1172-85.

[45] Kurth EG, Peremyslov VV, Prokhnevsky A, et al. Virus-derived gene expression and RNA interference vector for grapevine. J Virol. 2012; 86: 6002-9.

[46] Visser M, Stephan D, Jaynes JM, et al. A transient expression assay for the in planta efficacy screening of an antimicrobial peptide against grapevine bacterial pathogens. Lett Appl Microbiol. 2012; 54: 543-51.

[47] Jelly NS, Valat L, Walter B, et al. Transient expression assays in grapevine: a step towards genetic improvement. Plant Biotechnol J. 2014; 12: 1231-45.

[48] Santos-Rosa M, Poutaraud A, Merdinoglu D, et al. Development of a transient expression system in grapevine via agro-infiltration. Plant Cell Rep. 2008; 27: 1053-63.

[49] Xu W, Yu Y, Ding J, et al. Characterization of a novel stilbene synthase promoter involved in pathogen- and stress-inducible expression from Chinese wild Vitis pseudoreticulata . Planta. 2010; 231: 475-87.

[50] Le Henanff G, Heitz T, Mestre P, et al. Characterization of Vitis vinifera NPR1 homologs involved in the regulation of pathogenesis-related gene expression. BMC Plant Biol. 2009; 9: 54.

[51] Zottini M, Barizza E, Costa A, et al. Agroinfiltration of grapevine leaves for fast transient assays of gene expression and for long-term production of stable transformed cells. Plant Cell Rep. 2008; 27: 845-53.

[52] Zhan X, Zhang F, Zhong Z, et al. Generation of virus-resistant potato plants by RNA genome targeting. Plant Biotechnol J. 2019; 17: 1814-22.

[53] Acharya S, Mishra A, Paul D, et al. Francisella novicida Cas9 interrogates genomic DNA with very high specificity and can be used for mammalian genome editing . Proc Natl Acad Sci U S A. 2019; 116: 20959-68.

[54] Abbott TR, Dhamdhere G, Liu Y, et al. Development of CRISPR as an antiviral strategy to combat SARS-CoV-2 and influenza. Cell. 2020; 181: 865-876.e12.

[55] Freije CA, Myhrvold C, Boehm CK, et al. Programmable inhibition and detection of RNA viruses using Cas13. Mol Cell. 2019; 76: 826-837.e11.

[56] Yan WX, Chong S, Zhang H, et al. Cas13d is a compact RNA-targeting type VI CRISPR effector positively modulated by a WYL-domain-containing accessory protein. Mol Cell. 2018; 70: 327-339.e5.

[57] Abudayyeh OO, Gootenberg JS, Essletzbicheler P, et al. RNA targeting with CRISPR-Cas13. Nature. 2017; 550: 280-4.

[58] Cox DBT, Gootenberg JS, Abudayyeh OO, et al. RNA editing with CRISPR-Cas13. Science. 2017; 358: 1019-27.

[59] Mahas A, Aman R, Mahfouz M . CRISPR-Cas13d mediates robust RNA virus interference in plants. Genome Biol. 2019; 20: 263.

[60] Cui Z-H, Bi W-L, Hao XY, et al. Responses of in vitro-grown plantlets (Vitis vinifera) to grapevine leafroll-associated virus-3 and PEG-induced drought stress. Front Physiol. 2016; 7: 203.

[61] Cui ZH, Bi WL, Pan C, et al. Abiotic stress improves in vitro biological indexing of grapevine leafroll-associated virus-3 in red grapevine cultivars. Aust J Grape Wine Res. 2015; 21: 490-5.

[62] Cui ZH, Bi W-L, Hao X-Y, et al. Drought stress enhances up-regulation of anthocyanin biosynthesis in grapevine leafroll-associated virus 3-infected in vitro grapevine (Vitis vinifera) leaves. Plant Dis. 2017; 101: 1606-15.

[63] Wise AA, Liu Z, Binns AN . Three methods for the introduction of foreign DNA into Agrobacterium. In: Agrobacterium Protocols.Totowa: Humana Press, 2006, 43-54.

[64] Baltes NJ, Gil-Humanes J, Cermak T, et al. DNA replicons for plant genome engineering. Plant Cell. 2014; 26: 151-63.

[65] Osman F, Rowhani A . Application of a spotting sample preparation technique for the detection of pathogens in woody plants by RT-PCR and real-time PCR (TaqMan). J Virol Methods. 2006; 133: 130-6.

[66] Chen T, Peng J, Yin X, et al. Importin- αs are required for the nuclear localization and function of the Plasmopara viticola effector PvAVH53 . Hortic Res. 2021; 8: 46.