Chinese Journal OF Rice Science >
Orientation Improvement of Blast Resistance in Rice via CRISPR/Cas9 System
Received date: 2019-04-10
Revised date: 2019-05-24
Online published: 2019-07-10
【Objective】 CRISPR/Cas9 gene editing is an effective biotechnology for crop genetic improvement. This study aims to edit Pita, Pi21 and ERF922 genes for obtaining valuable and stable genetic materials with resistance to rice blast. 【Method】The target genes Pita, Pi21 and ERF922 were selected for constructing the pC1300-2×35S::Cas9-gPita-gPi21-gERF922 expression vector by CRISPR/Cas9 gene editing technology, and transformed by Agrobacterium-mediated method into long-grain japonica rice restore lines L1014. Stable genetic mutant lines were selected and their resistance to rice blast was identified. 【Result】 In the T0 transgenic lines, the mutation rates of Pita, Pi21 and ERF922 were 75%, 85% and 65%, respectively, and most of the mutant lines are biallelic mutations. The homozygous mutation lines without T-DNA components within the screened T1 were selected and transferred to T2 stably. Evaluation analysis of resistance to rice blast suggested that the mutant lines showed significantly higher resistance to M. oryzae compared with the wild type. The expression level of defense-related genes involved in signaling pathways of salicylic acid, jasmonic acid and ethylene metabolisms was up-regulated in the mutant lines compared to the wild type after inoculation of the physiological races of M. oryzae. Therefore, we hypothesized that the increased resistance of homozygous mutant lines to rice blast might be related to the activation of the response to M. oryzae. 【Conclusion】The homozygous mutant lines with stable inheritance and high blast resistance were obtained by CRISPR/Cas9 system, which would provide ideal materials for improving blast resistance in rice.
Key words： rice; CRISPR/Cas9; Pita; Pi21; ERF922; blast resistance
Peng XU, Hong WANG, Ranran TU, Qunen LIU, Weixun WU, Xiumin FU, Liyong CAO, Xihong SHEN . Orientation Improvement of Blast Resistance in Rice via CRISPR/Cas9 System[J]. Chinese Journal OF Rice Science, 2019 , 33(4) : 313 -322 . DOI: 10.16819/j.1001-7216.2019.9043
| [1] | Liu J, Wang X, Mitchell T, Hu Y, Liu X, Dai L, Wang G L.Recent progress and understanding of the molecular mechanisms of the rice-Magnaporthe oryzae interaction.Mol Plant Pathol, 2010, 11(3): 419-427. |
| [2] | Borrelli V, Brambilla V, Rogowsky P, Marocco A, Lanubile A.The enhancement of plant disease resistance using CRISPR/Cas9 technology. Front Plant Sci, 2018, 9: 1245. |
| [3] | Wilson R A, Talbot N J.Under pressure: investigating the biology of plant infection by Magnaporthe oryzae.Nat Rev Microbiol, 2009, 7(3): 185-195. |
| [4] | Xie K, Yang Y.RNA-guided genome editing in plants using a CRISPR-Cas system.Mol Plant, 2013, 6(6): 1975-1983. |
| [5] | Shan Q, Wang Y, Li J, Zhang Y, Chen K, Liang Z, Zhang K, Liu J, Xi J J, Qiu J L, Gao C.Targeted genome modification of crop plants using a CRISPR-Cas system.Nat Biotechnol, 2013, 31(8): 686-688. |
| [6] | Cong L, Ran F A, Cox D, Lin S, Barretto R, Habib N, Hsu P D, Wu X, Jiang W, Marraffini L A, Zhang F.Multiplex genome engineering using CRISPR/Cas systems.Science, 2013, 339(6121): 819-823 |
| [7] | Islam W.CRISPR-Cas9: An efficient tool for precise plant genome editing.Mol Cell Probes, 2018, 39: 47-52. |
| [8] | Vriend L E M, Prakash R, Chen C, Vanoli F, Cavallo F, Zhang Y, Jasin M, Krawczyk P M. Distinct genetic control of homologous recombination repair of Cas9-induced double-strand breaks, nicks and paired nicks.Nucleic Acids Res, 2016, 44(11): 5204-5217. |
| [9] | Mao Y, Zhang H, Xu N, Zhang B, Gou F, Zhu J K.Application of the CRISPR-Cas system for efficient genome engineering in plants.Mol Plant, 2013, 6(6): 2008-2011. |
| [10] | Shan Q, Wang Y, Li J, Gao C.Genome editing in rice and wheat using the CRISPR/Cas system.Nat Protoc, 2014, 9(10): 2395-2410. |
| [11] | Zhang J, Zhang H, Botella J R, Zhu J K.Generation of new glutinous rice by CRISPR/Cas9-targeted mutagenesis of the Waxy gene in elite rice varieties. J Integr Plant Biol, 2018, 60(5): 369-375. |
| [12] | Jiang W, Zhou H, Bi H, Fromm M, Yang B Weeks D P. Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice.Nucleic Acids Res, 2013, 41(20): e188. |
| [13] | Fauser F, Schiml S, Puchta H.Both CRISPR/Cas-based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana.Plant J, 2014, 79(2): 348-359. |
| [14] | Jiang W, Yang B Weeks D P. Efficient CRISPR/Cas9- mediated gene editing in Arabidopsis thaliana and inheritance of modified genes in the T2 and T3 generations.PLoS One, 2014, 9(6): e99225. |
| [15] | Wang Y, Cheng X, Shan Q, Zhang Y, Liu J, Gao C, Qiu J L.Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew.Nat Biotechnol, 2014, 32(9): 947-951. |
| [16] | Zhang Y, Bai Y, Wu G, Zou S, Chen Y, Gao C, Tang D.Simultaneous modification of three homologs of TaEDR1 by genome editing enhances powdery mildew resistance in wheat.Plant J, 2017, 91(4): 714-724. |
| [17] | Shen L, Hua Y, Fu Y, Li J, Liu Q, Jiao X, Xin G, Wang J, Wang X, Yan C, Wang K.Rapid generation of genetic diversity by multiplex CRISPR/Cas9 genome editing in rice.Sci China Life Sci, 2017, 60(5): 506-515. |
| [18] | Fei Y Y, Yang J, Wang F Q, Fan F J, Li W Q, Wang J, Xu Y, Zhu J Y, Zhong W G.Production of two elite glutinous rice varieties by editing Wx gene.Rice Sci, 2019, 26(2): 118-124. |
| [19] | Fukuoka S, Okuno K.QTL analysis and mapping of pi21, a recessive gene for field resistance to rice blast in Japanese upland rice.Theor Appl Genet, 2001, 103(2-3): 185-190. |
| [20] | Fukuoka S, Saka N, Koga H, Ono K, Shimizu T, Ebana K, Hayashi N, Takahashi A, Hirochika H, Okuno K, Yano M.Loss of function of a proline-containing protein confers durable disease resistance in rice. Science, 2009, 325(5943): 998-1001. |
| [21] | Liu D, Chen X, Liu J, Ye J, Guo Z.The rice ERF transcription factor OsERF922 negatively regulates resistance to Magnaporthe oryzae and salt tolerance.J Exp Bot, 2012, 63(10): 3899-3911. |
| [22] | Bryan G T, Wu K S, Farrall L, Jia Y, Hershey H P, McAdams S A, Faulk K N, Donaldson G K, Tarchini R, Valent B. A single amino acid difference distinguishes resistant and susceptible alleles of the rice blast resistance gene Pi-ta.Plant Cell, 2000, 12(11): 2033-2046. |
| [23] | Jia Y, Martin R.Identification of a new locus, Ptr(t), required for rice blast resistance gene Pi-ta-mediated resistance.Mol Plant Microbe Interact, 2008, 21(4): 396-403. |
| [24] | Tu Z, Yang W, Yan S, Guo X, Li X.CRISPR/Cas9: A powerful genetic engineering tool for establishing large animal models of neurodegenerative diseases.Mol Neurod, 2015, 10: 35. |
| [25] | Hruscha A, Krawitz P, Rechenberg A, Heinrich V, Hecht J, Haass C, Schmid B.Efficient CRISPR/Cas9 genome editing with low off-target effects in zebrafish.Development, 2013, 140(24): 4982-4987. |
| [26] | Cai Y, Chen L, Liu X, Guo C, Sun S, Wu C, Jiang B, Han T, Hou W.CRISPR/Cas9-mediated targeted mutagenesis of GmFT2a delays flowering time in soya bean.Plant Biotechnol J, 2018, 16(1): 176-185. |
| [27] | Yu Z, Chen Q, Chen W, Zhang X, Mei F, Zhang P, Zhao M, Wang X, Shi N, Jackson S, Hong Y.Multigene editing via CRISPR/Cas9 guided by a single-sgRNA seed in Arabidopsis.J Integr Plant Biol, 2018, 60(5): 376-381. |
| [28] | Jakočiu?nas T, Rajkumar AS, Zhang J, Arsovska D, Rodriguez A, Jendresen C B, Skjødt M L, Nielsen A T, Borodina I, Jensen M K, Keasling J D. CasEMBLR: Cas9-facilitated multiloci genomic integration of in vivo assembled DNA parts in Saccharomyces cerevisiae.ACS Synthetic Biol, 2015, 4(11): 1226-1234. |
| [29] | 沈兰. 基于CRISPR/Cas9的水稻多基因编辑及其在育种中的应用. 扬州: 扬州大学, 2017. |
| [29] | Shen L.CRISPR/Cas9-mediated multiples genome editing in rice (Oryza sativa) and the application in rice breeding. Yangzhou: Yangzhou University, 2017. (in Chinese with English abstract) |
| [30] | 王芳权, 范方军, 李文奇, 朱金燕, 王军. 利用CRISPR/Cas9技术敲除水稻Pi21基因的效率分析. 中国水稻科学, 2016, 30(5): 469-478. |
| [30] | Wang F Q, Fan F J, Li W Q, Zhu J Y, Wang J.Knock-out efficiency analysis of Pi21 gene using CRISPR/Cas9 in rice.Chin J Rice Sci, 2016, 30(5): 469-478. (in Chinese with English abstract) |
| [31] | 杨海河, 毕冬玲, 张玉, 邹小维, 高晓庆, 袁正杰, 曲海艳, 何海燕, 瞿绍洪. 基于CRISPR/Cas9技术的水稻pi21基因编辑材料的创制及稻瘟病抗性鉴定. 分子植物育种, 2017, 15(11): 4451-4465. |
| [31] | Yang H H, Bi D L, Zhang Y, Zou X W, Gao X Q, Yuan Z J, Qu H Y, He H Y, Qu S H.Generation of rice pi21 gene editing lines based on CRISPR/Cas9 technology and evaluation of their blast resistance.Mol Plant Breed, 2017, 15(11): 4451-4465. (in Chinese with English abstract) |
| [32] | Wang F, Wang C, Liu P, Lei C, Hao W, Gao Y, Liu Y, Zhao K.Enhanced rice blast resistance by CRISPR/Cas9-targeted mutagenesis of the ERF transcription factor gene OsERF922.Plos One, 2016, 11(4): e0154027. |
| [33] | Zhang Y, Zhao J, Li Y, Yuan Z, He H, Yang H, Qu H, Ma C, Qu S.Transcriptome analysis highlights defense and signaling pathways mediated by rice pi21 gene with partial resistance to Magnaporthe oryzae.Front Plant Sci, 2016, 7: 1834. |
| [34] | Wamaitha M J, Yamamoto R, Wong H L, Kawasaki T, Kawano Y, Shimamoto K. OsRap2.6 transcription factor contributes to rice innate immunity through its interaction with Receptor for Activated Kinase-C1(RACK1). Rice, 2012, 5: 35. |
| [35] | Zhao H, Wang X, Jia Y, Minkenberg B, Wheatley M, Fan J, Jia M H, Famoso A, Edwards J D, Wamishe Y, Valent B, Wang G, Yang Y.The rice blast resistance gene Ptr encodes an atypical protein required for broad-spectrum disease resistance.Nat Commun, 2018, 9(1): 2039. |
/
| 〈 |
|
〉 |