Chinese Journal OF Rice Science >
Cloning and Functional Verification of Rice-Blast Resistance Gene Pi-kf2(t) in Kangfeng B
Received date: 2024-04-30
Revised date: 2024-07-23
Online published: 2025-05-21
【Objective】Rice blast is a worldwide disease caused by filamentous ascomycete fungus Magnaporthe oryzae, which seriously affects the quality and yield of rice. At present, cultivating and planting disease-resistant varieties is the most economical and effective strategy to control rice blast, and the mining and utilization of blast-resistance genes is the key to disease-resistant rice breeding. In this study, Kangfeng B (KFB), an elite maintainer line of indica hybrid rice with broad-spectrum resistance derived from Sanming Academy of Agricultural Sciences was used as the test material, and the Pi-kf2(t) gene in which was used to conduct resistance spectrum analysis, cloning and functional verification.【Method】For resistance spectrum analysis, KFB (Pi-kf2(t)), 75-1-127 (Pi9), C101A51 (Pi2), IRBLzt-T (Piz-t), EBZ (Pi50) and GM-4 (Pigm) seedlings were inoculated with 100 rice blast isolates from different regions of China during 3-4 leaf stages; The expression analysis of two Pi-kf2(t) candidate genes, Pi-kf2(t)-NBS2 and Pi-kf2(t)-NBS4, were determined by semi-quantitative reverse-transcription PCR (RT-PCR) and quantitative real-time PCR (qRT-PCR); Pi-kf2(t) gene was cloned by homologous cloning strategy and a phylogenetic tree was constructed to investigate the possible relationships between Pi-kf2(t) and Pi9, Pi2, Piz-t, Pi50, and Pigm proteins in evolutionary history. Moreover, sequence and expression analyses revealed Pi-kf2(t)-NBS2 as the candidate Pi-kf2(t) gene, which was verified by transgenic complementation test.【Result】The resistance spectrum of Pi-kf2(t), Pi9, Pi2, Piz-t, Pi50 and Pigm were 93%, 90%, 91%, 78%, 95% and 96%, respectively. The candidate gene Pi-kf2(t)-NBS2 exhibited a high expression level in the leaves of KFB seedlings and was not induced by isolates of rice blast, which showed a constitutive expression, while Pi-kf2(t)-NBS4 exhibited an extremely poor expression. Multiple amino acid sequence alignment analysis revealed that there were 45, 31, 34, 2, and 2 amino acids differences between Pi-kf2(t)-NBS2 and Pi9, Pi2, Piz-t, Pi50, and Pigm proteins, respectively. Phylogenetic tree analysis showed that Pi-kf2(t)-NBS2 exhibited a closer genetic relationship and a higher homology with Pi50 and Pigm. Furthermore, genetic complementation test verified that the candidate gene Pi-kf2(t)-NBS2 was the Pi-kf2(t).【Conclusion】The rice blast-resistance gene Pi-kf2(t) is a novel member of the Pi2/Pi9 multigene family. This work provided a theoretical basis for further disease resistance mechanism study of Pi-kf2(t), and also provided an important germplasm resource for rice blast-resistance breeding.
Key words： rice; rice blast; Pi2/Pi9; resistance gene; NBS-LRR
WEI Xinyu, ZENG Yuehui, XIAO Changchun, HUANG Jianhong, RUAN Hongchun, YANG Wangxing, ZOU Wenguang, XU Xuming . Cloning and Functional Verification of Rice-Blast Resistance Gene Pi-kf2(t) in Kangfeng B[J]. Chinese Journal OF Rice Science, 2025 , 39(3) : 352 -364 . DOI: 10.16819/j.1001-7216.2025.240502
| [1] | Valent B, Chumley F G. Molecular genetic analysis of the rice blast fungus, Magnaporthe grisea[J]. Annual Review of Phytopathology, 1991, 29: 443-467. |
| [2] | Oliveira-Garcia E, Yan X, Oses-Ruiz M, de Paula S, Talbot N J. Effector-triggered susceptibility by the rice blast fungus Magnaporthe oryzae[J]. The New Phytologist, 2024, 241(3): 1007-1020. |
| [3] | 杨婕, 杨长登, 曾宇翔, 侯雨萱, 陈天晓, 梁燕. 水稻稻瘟病抗性基因挖掘与利用研究进展[J]. 中国水稻科学, 2024, 38(6): 5: 591-603. |
| Yang J, Yang C D, Zeng Y X, Hou Y X, Chen T X, Liang Y. Research progress on mining and utilization of rice blast resistance genes[J]. Chinese Journal of Rice Science, 2024, 38(6): 591-603. (in Chinese with English abstract) | |
| [4] | 殷得所, 夏明元, 李进波, 万丙良, 查中萍, 杜雪树, 戚华雄. 抗稻瘟病基因Pi9的STS连锁标记开发及在分子标记辅助育种中的应用[J]. 中国水稻科学, 2011, 25(1): 2: 25-30. |
| Yin D S, Xia M Y, Li J B, Wan B L, Zha Z P, Du X S, Qi H X. Development ofof STS marker linked to rice blast resistance gene Pi9 in marker-assisted selection breeding[J]. Chinese Journal of Rice Science, 2011, 25(1): 25-30. (in Chinese with English abstract) | |
| [5] | Zhu X Y, Chen S, Yang J Y, Zhou S C, Zeng L X, Han J L, Su J, Wang L, Pan Q H. The identification of Pi50(t), a new member of the rice blast resistance Pi2/Pi9 multigene family[J]. Theoretical and Applied Genetics, 2012, 124: 1295-1304. |
| [6] | Hulbert S H W, Webb C A S, Smith S M S, Sun Q. Resistance gene complexes: E: Evolution and utilization[J]. Annual Review of Phytopathology, 2001, 39:285-312. |
| [7] | 华丽霞, 汪文娟, 陈深, 汪聪颖, 曾烈先, 杨健源, 朱小源, 苏菁. 抗稻瘟病Pi2/9/z-t基因特异性分子标记的开发[J]. 中国水稻科学, 2015, 29(3): 3: 305-310. |
| Hua L X, Wang W J, Chen S, Wang C Y, Zeng L X, Yang J Y, Zhu X Y, Su J. Development of specific DNA markers for detecting the rice blast resistance gene alleles Pi2/9/z-t[J]. Chinese Journal of Rice Science, 2015, 29(3): 305-310. (in Chinese with English abstract) | |
| [8] | Deng Y W, Zhu X D, Shen Y, He Z H. Genetic characterization and fine mapping of the blast resistance locus Pigm(t) tightly linked to Pi2 and Pi9in a broad-spectrum resistant Chinese variety[J]. Theoretical and Applied Genetics, 2006, 113(4): 705-713. |
| [9] | Wang Z X, Yano M, Yamanouchi U, Iwamoto M, Monna L, Hayasaka H, Katayose Y, Sasaki T. The Pib gene for rice blast resistance belongs to the nucleotide binding and leucine-rich repeat class of plant disease resistance genes[J]. The Plant Journal, 1999, 19(1): 55-64. |
| [10] | Qu S L, Liu G Z, Zhou B, Bellizzi M Z, Zeng L D, Dai L H, Han B W, Wang G L. The broad-spectrum blast resistance gene Pi9 encodes a nucleotide-binding site-leucine-rich repeat protein and is a member of a multigene family in rice[J]. Genetics, 2006, 172(3): 1: 1901-1914. |
| [11] | Hassan B, Peng Y T, Li S, Yin X X, Chen C, Gulzar F, Zhou S X, Pu M, Ji Y P, Wang Y P, Zhao W S, Huang F, Peng Y L, Zhao Z X, Wang W M. Identification of the blast resistance genes in three elite restorer lines of hybrid rice[J]. Phytopathology Research, 2022, 4(1): 15. |
| [12] | Martin G B, Bogdanove A J, Sessa G. Understanding the functions of plant disease resistance proteins[J]. Annual Review of Plant Biology, 2003, 54: 23-61. |
| [13] | Belkhadir Y, Subramaniam R, Dangl J L. Plant disease resistance protein signaling: NBS-LRR proteins and their partners[J]. Current Opinion in Plant Biology, 2004, 7(4): 391-399. |
| [14] | Zhou B Q, Qu S H L, Liu G F D, Dolan M S, Sakai H L, Lu G D, Bellizzi M W, Wang G L. The eight amino-acid differences within three leucine-rich repeats between Pi2 and Piz-t resistance proteins determine the resistance specificity to Magnaporthe grisea[J]. Molecular Plant-Microbe Interactions, 2006, 19( 11):1216-1228. |
| [15] | Tamura M, Tachida H. Evolution of the number of LRRs in plant disease resistance genes[J]. Molecular Genetics and Genomics, 2011, 285(5): 393-402. |
| [16] | Dodds P N L, Lawrence G J E, Ellis J G. Six amino acid changes confined to the leucine-rich repeat beta-strand/beta-turn motif determine the difference between the P and P2 rust resistance specificities in flax[J]. The Plant Cell, 2001, 13(1):163-178. |
| [17] | Wu J L, Fan Y Y, Li D B, Zheng K L, Leung H, Zhuang J Y. Genetic control of rice blast resistance in the durably resistant cultivar Gumei 2 against multiple isolates[J]. Theoretical and Applied Genetics, 2005, 111(1): 50-56. |
| [18] | Jeung J U, Kim B R, Cho Y C, Han S S, Moon H P, Lee Y T, Jena K K. A novel gene, Pi40(t), linked to the DNA markers derived from NBS-LRR motifs confers broad spectrum of blast resistance in rice[J]. Theoretical and Applied Genetics, 2007, 115(8): 1163-1177. |
| [19] | Jiang N, Li Z Q, Wu J, Wang Y, Wu L Q, Wang S H, Wang D, Wen T, Liang Y, Sun P Y, Liu J L, Dai L Y, Wang Z L, Wang C, Luo M Z, Liu X L, Wang G L. Molecular mapping of the Pi2/9 allelic genePi2-2 conferring broad-spectrum resistance to Magnaporthe oryzae in the rice cultivar Jefferson[J]. Rice, 2012, 5(1): 29. |
| [20] | Su J, Wang W J, Han J L, Chen S, Wang C Y, Zeng L X, Feng A Q, Yang J Y, Zhou B, Zhu X Y. Functional divergence of duplicated genes results in a novel blast resistance gene Pi50 at the Pi2/9 locus[J]. Theoretical and Applied Genetics, 2015, 128(11): 2213-2225. |
| [21] | Deng Y W, Zhai K R, Xie Z, Yang D Y, Zhu X D, Liu J Z, Wang X, Qin P, Yang Y Z, Zhang G M, Li Q, Zhang J F, Wu S Q, Milazzo J, Mao B Z, Wang E, Xie H A, Tharreau D, He Z H. Epigenetic regulation of antagonistic receptors confers rice blast resistance with yield balance[J]. Science, 2017, 355(6328): 962-965. |
| [22] | Xie Z Y, Yan B X S, Shou J Y T, Tang J W, Wang X Z, Zhai K R L, Liu J Y L, Li Q L, Luo M Z D, Deng Y W H, He Z H. A nucleotide-binding site-leucine-rich repeat receptor pair confers broad-spectrum disease resistance through physical association in rice[J]. Philosophical Transactions of the Royal Society of London Series B-Biological Sciences, 2019,374(1767): 20180308. |
| [23] | Lü Q M, Xu X, Shang J J, Jiang G H, Pang Z Q, Zhou Z Z, Wang J, Liu Y, Li T, Li X B, Xu J C, Cheng Z K, Zhao X F, Li S G, Zhu L H. Functional analysis of Pid3-A4, an ortholog of rice blast resistance gene Pid3 revealed by allele mining in common wild rice[J]. Phytopathology, 2013, 103: 594-599. |
| [24] | Zhou Y, Lei F, Wang Q, He W C, Yuan B, Yuan W Y. Identification of novel alleles of the rice blast-resistance gene Pi9 through sequence-based allele mining[J]. Rice, 2020, 13: 80. |
| [25] | Li L Y W, Wang L J, Jing J X L, Li Z Q L, Lin F H, Huang L F P, Pan Q H. The Pikm gene, c, conferring stable resistance to isolates of Magnaporthe oryzae,was finely mapped in a crossover-cold region on rice chromosome 11[J]. Molecular Breeding, 2007, 20:179-188. |
| [26] | Kovi B, Sakai T, Abe A, Kanzaki E, Terauchi R, Shimizu M. Isolation of Pikps, an allele of Pik, from the aus rice cultivar Shoni[J]. Genes and Genetic Systems, 2023, 97(5): 229-235. |
| [27] | 黄衍焱, 李燕, 王贺, 王文明. 水稻小种特异性抗稻瘟病基因的等位性变异研究进展[J]. 植物病理学报, 2023, 53(5):753-768. |
| Huang Y Y L, Li Y W, Wang H W, Wang W M, Allelic variation in the race-specific blast resistance genes in rice[J]. Acta Phytopathologica Sinica, 2023, 53(5):753-768. (in Chinese with English abstract) | |
| [28] | Rybka K M, Miyamoto M A, Ando I S, Saito A K, Kawasaki S. High resolution mapping of the indica-derived rice blast resistance genes:II. Pi-ta2 and Pi-ta and a consideration of their origin[J]. Molecular Plant-Microbe Interactions, 1997, 10(4):517-524. |
| [29] | Liu X Q, Yang Q Z, Lin F, Hua L X, Wang C T, Wang L, Pan Q H. Identification and fine mapping of Pi39(t), a major gene conferring the broad-spectrum resistance to Magnaporthe oryzae[J]. Molecular Genetics and Genomics, 2007, 278(4): 403-410. |
| [30] | 吴俊, 刘雄伦, 戴良英, 王国梁. 水稻广谱抗稻瘟病基因研究进展[J]. 生命科学, 2007, 19(2):233-238. |
| Wu J, Liu X L, Dai L Y, Wang G L. Advances on the identification and characterization of broad-spectrum blast resistance genes in rice[J]. Chinese Bulletin of Life Science, 2007, 19(2): 233-238. (in Chinese with English abstract) | |
| [31] | Sallaud C, Lorieux M, Roumen E, Tharreau D, Berruyer R, Svestasrani P, Garsmeur O, Ghesquiere A, Notteghem J L. Identification of five new blast resistance genes in the highly blast-resistant rice variety IR64 using a QTL mapping strategy[J]. Theoretical and Applied Genetics, 2003, 106: 794-803. |
| [32] | Liu B, Zhang S H, Zhu X Y, Yang Q Y, Wu S Z, Mei M T, Mauleon R, Leach J, Mew T, Leung H. Candidate defense genes as predictors of quantitative blast resistance in rice[J]. Molecular Plant-Microbe Interactions, 2004, 17: 1146-1152. |
| [33] | 韦新宇, 许旭明, 张锐, 陈美莲, 马彬林, 邹文广, 杨旺兴, 卓伟, 王宗华, 梁康迳. 籼粳交新种质康丰A对稻瘟病抗性的遗传[J]. 植物遗传资源学报, 2014, 15(5):1133-1137. |
| Wei X Y, Xu X M, Zhang R, Chen M L, Ma B L, Zou W G, Yang W X, Zhuo W, Wang Z H, Liang K J. Inheritance of blast resistance in new germplasm Kangfeng A from indica-japonica crosses[J]. Journal of Plant Genetic Resources, 2014, 15(5):1133-1137. (in Chinese with English abstract) | |
| [34] | Wei X Y Z, Zeng Y H Z, Zhang R H, Huang J H Y, Yang W X Z, Zou W G X, Xu X M. Fine mapping and identification of the rice blast-resistance locus Pi-kf2(t) as a new member of the Pi2/Pi9 multigene family[J]. Molecular Breeding, 2019,39:108. |
| [35] | Bonman J M, Khush G S, N, Nelson R J. Breeding rice for resistance to pests[J]. Annual Review of Phytopathology, 1992, 30:507-528. |
| [36] | 韦新宇, 曾跃辉, 杨旺兴, 肖长春, 候新坡, 黄建鸿, 邹文广, 许旭明. 利用CRISPR-Cas9技术编辑Badh2基因创制优质香型籼稻三系不育系[J]. . 作物学报, 2023, 49(8):2144-2159. |
| Wei X Y, Zeng Y H, Yang W X, Xiao C C, Hou X P, Huang J H, Zou W G, Xu X M. Development of high-quality fragrant indica CMS line by editing Badh2 gene using CRISPR-Cas9 technology in rice (Oryza sativa L.)[J]. Acta Agronomica Sinica, 2023, 49(8):2144-2159. (in Chinese with English abstract) | |
| [37] | Zeng Y H, Wei X Y, Xiao C C, Zhang R, Huang J H, Xu X M. Fine mapping and identification of a novel albino gene OsAL50 that is required for chlorophyll biosynthesis and chloroplast development in rice (Oryza sativa L.)[J]. Plant Growth Regulation, 2024, 103(2):389-407. |
| [38] | 曾跃辉, 韦新宇, 黄建鸿, 肖长春, 张锐, 尚伟, 许旭明. 不同来源特种稻香味和黑色种皮基因的鉴定与遗传特性分析[J]. 植物遗传资源学报, 2021, 22(4):951-962. |
| Zeng Y H, Wei X Y, Huang J H, Xiao C C, Zhang R, Shang W, Xu X M. Identification and genetic analysis of the genes for fragrance and black pericarp in special rice from different regions[J]. Journal of Plant Genetic Resources, 2021, 22(4):951-962. (in Chinese with English abstract) | |
| [39] | 陈深, 苏菁, 华丽霞, 汪文娟, 汪聪颖, 杨健源, 曾烈先, 朱小源. 水稻恢复系华占抗稻瘟病遗传分析及基因鉴定[J]. 植物病理学报, 2015, 45(6):598-605. |
| Chen S, Su J, Hua L X, Wang W J, Wang C Y, Yang J Y, Zeng L X, Zhu X Y. Genetic analysis and gene identification of restorer line Huazhan against rice blast[J]. Acta Phytopathologica Sinica, 2015, 45(6):598-605. (in Chinese with English abstract) | |
| [40] | Ellis J, Dodds P, Pryor T. The generation of plant disease resistance gene specificities[J]. Trends in Plant Science, 2000, 5(9):373-379. |
/
| 〈 |
|
〉 |