Screening of OsCRK5-interacted Proteins in Rice Using Yeast Two-hybrid System

doi:10.13560/j.cnki.biotech.bull.1985.2023-0090

Abstract

Abstract:

Rice（Oryza sativa L.）is an important food crop, research on the regulation mechanism of rice growth and development can lay a theoretical foundation for the improvement of rice varieties. Calcium dependent protein kinases（CDPKs）are important protein kinases in plants which participate in plant growth and development, as well as in response to environmental reactions. CRK5（CDPK related kinase 5）in rice is highly homologous to CDPK in protein sequence and structure. Yeast two-hybrid screening library technology was used to screen OsCRK5 interacting proteins related to plant drought resistance in order to explore the molecular mechanism of OsCRK5 participating in drought response in rice plants. Firstly, the 1-1 332 bp fragment of OsCRK5 was cloned into the plastmid pGBKT7, and the bait vector pGBKT7-OsCRK5 was obtained. After verification by sequencing, the bait vector was transformed into the yeast strain Y2H Gold.It was observed that the recombinant protein didn't demonstrate the toxic effects and self-activation in selective nutrition medium, and the expression of the recombinant protein was analyzed by Western blot. Furthermore, the interacting proteins of OsCRK5 were screened from the rice cDNA library and 77 positive clones were obtained. The functional prediction showed that the interacting proteins were involved in protein synthesis, degradation and storage process, transcriptional regulation, cell growth and division process, energy metabolism and cell metabolic processes. Finally, OsWR1 and OsDi19-1 related to plant drought stress response were selected from positive clones. The interaction between OsCRK5 and OsWR1 and OsDi19-1 was verified by yeast two-hybrid and biomolecular fluorescence complementation experiments. The results lay a foundation for the genetic improvement of drought tolerance in rice.

Key words: rice, OsCRK5, yeast two-hybrid system, cDNA library, drought stress

WANG Zi-ying, LONG Chen-jie, FAN Zhao-yu, ZHANG Lei. Screening of OsCRK5-interacted Proteins in Rice Using Yeast Two-hybrid System[J]. Biotechnology Bulletin, 2023, 39(9): 117-125.

Figures/Tables 8

References 52

[1]	李琼, 姜雪. 水稻产量相关性状的遗传研究[J]. 安徽农业科学, 2022, 50(13): 10-13, 20.
	Li Q, Jiang X. Genetic studies on rice yield related traits[J]. J Anhui Agric Sci, 2022, 50(13): 10-13, 20.
[2]	李涵茂, 陆魁东, 戴平, 等. 不同品种双季晚稻产量结构对干旱胁迫的响应[J]. 气象与环境科学, 2019, 42(2): 27-34.
	Li HM, Lu KD, Dai P, et al. Research on yield structure of different varieties of double-cropping late rice response to drought stress[J]. Meteorol Environ Sci, 2019, 42(2): 27-34.
[3]	Asano T, Hayashi N, Kikuchi S, et al. CDPK-mediated abiotic stress signaling[J]. Plant Signal Behav, 2012, 7(7): 817-821. doi: 10.4161/psb.20351 pmid: 22751324
[4]	Boudsocq M, Sheen J. CDPKs in immune and stress signaling[J]. Trends Plant Sci, 2013, 18(1): 30-40. doi: 10.1016/j.tplants.2012.08.008 pmid: 22974587
[5]	Cheng SH, Willmann MR, Chen HC, et al. Calcium signaling through protein kinases. The Arabidopsis calcium-dependent protein kinase gene family[J]. Plant Physiol, 2002, 129(2): 469-485.
[6]	Asano T, Tanaka N, Yang G, et al. Genome-wide identification of the rice calcium-dependent protein kinase and its closely related kinase gene families: comprehensive analysis of the CDPKs gene family in rice[J]. Plant Cell Physiol, 2005, 46(2): 356-366. doi: 10.1093/pcp/pci035 pmid: 15695435
[7]	Atif RM, Shahid L, Waqas M, et al. Insights on calcium-dependent protein kinases(CPKs)signaling for abiotic stress tolerance in plants[J]. Int J Mol Sci, 2019, 20(21): 5298. doi: 10.3390/ijms20215298 URL
[8]	Li AL, Zhu YF, Tan XM, et al. Evolutionary and functional study of the CDPK gene family in wheat(Triticum aestivum L.)[J]. Plant Mol Biol. 2008, 66(4): 429-443. doi: 10.1007/s11103-007-9281-5 URL
[9]	Xu WW, Huang WC. Calcium-dependent protein kinases in phytohormone signaling pathways[J]. Int J Mol Sci, 2017, 18(11): 2436. doi: 10.3390/ijms18112436 URL
[10]	Yip Delormel T, Boudsocq M. Properties and functions of calcium-dependent protein kinases and their relatives in Arabidopsis thaliana[J]. New Phytol, 2019, 224(2): 585-604. doi: 10.1111/nph.16088 pmid: 31369160
[11]	Durian G, Sedaghatmehr M, Matallana-Ramirez LP, et al. Calcium-dependent protein kinase CPK1 controls cell death by in vivo phosphorylation of senescence master regulator ORE1[J]. Plant Cell, 2020, 32(5): 1610-1625. doi: 10.1105/tpc.19.00810 URL
[12]	Marques J, Matiolli CC, Abreu IA. Visualization of a curated Oryza sativa L. CDPKs protein-protein interaction network(CDPK-OsPPIN)[J]. MicroPubl Biol, 2022. DOI: 10.17912/micropub.biology.000513.
[13]	Zhang HF, Wei CH, Yang XZ, et al. Genome-wide identification and expression analysis of calcium-dependent protein kinase and its related kinase gene families in melon(Cucumis melo L.)[J]. PLoS One, 2017, 12(4): e0176352. doi: 10.1371/journal.pone.0176352 URL
[14]	Zuo R, Hu R, Chai G, et al. Genome-wide identification, classification, and expression analysis of CDPK and its closely related gene families in poplar(Populus trichocarpa)[J]. Mol Biol Rep, 2013, 40(3): 2645-2662. doi: 10.1007/s11033-012-2351-z URL
[15]	Cai H, Cheng J, Yan Y, et al. Genome-wide identification and expression analysis of calcium-dependent protein kinase and its closely related kinase genes in Capsicum annuum[J]. Front Plant Sci, 2015, 6: 737.
[16]	Wu P, Wang WL, Duan WK, et al. Comprehensive analysis of the CDPK-SnRK superfamily genes in Chinese cabbage and its evolutionary implications in plants[J]. Front Plant Sci, 2017, 8: 162. doi: 10.3389/fpls.2017.00162 pmid: 28239387
[17]	Wang JP, Xu YP, Munyampundu JP, et al. Calcium-dependent protein kinase(CDPK)and CDPK-related kinase(CRK)gene families in tomato: genome-wide identification and functional analyses in disease resistance[J]. Mol Genet Genomics, 2016, 291(2): 661-676. doi: 10.1007/s00438-015-1137-0 URL
[18]	Tao XC, Lu YT. Loss of AtCRK1 gene function in Arabidopsis thaliana decreases tolerance to salt[J]. J Plant Biol, 2013, 56(5): 306-314. doi: 10.1007/s12374-012-0352-z URL
[19]	Baba AI, Rigó G, Ayaydin F, et al. Functional analysis of the Arabidopsis thaliana CDPK-related kinase family: AtCRK1 regulates responses to continuous light[J]. Int J Mol Sci, 2018, 19(5): 1282. doi: 10.3390/ijms19051282 URL
[20]	Nemoto K, Ramadan A, Arimura GI, et al. Tyrosine phosphorylation of the GARU E3 ubiquitin ligase promotes gibberellin signalling by preventing GID1 degradation[J]. Nat Commun, 2017, 8(1): 1004. doi: 10.1038/s41467-017-01005-5 pmid: 29042542
[21]	Li RJ, Hua W, Lu YT. Arabidopsis cytosolic glutamine synthetase AtGLN1;1 is a potential substrate of AtCRK3 involved in leaf senescence[J]. Biochem Biophys Res Commun, 2006, 342(1): 119-126. doi: 10.1016/j.bbrc.2006.01.100 URL
[22]	Rigó G, Ayaydin F, Tietz O, et al. Inactivation of plasma membrane-localized CDPK-RELATED KINASE5 decelerates PIN2 exocytosis and root gravitropic response in Arabidopsis[J]. Plant Cell, 2013, 25(5): 1592-1608. doi: 10.1105/tpc.113.110452 URL
[23]	Yadav A, Garg T, Singh H, et al. Tissue-specific expression pattern of calcium-dependent protein kinases-related kinases(CRKs)in rice[J]. Plant Signal Behav, 2020, 15(11): 1809846. doi: 10.1080/15592324.2020.1809846 URL
[24]	Fields S, Song OK. A novel genetic system to detect protein-protein interactions[J]. Nature, 1989, 340(6230): 245-246. doi: 10.1038/340245a0
[25]	Chien CT, Bartel PL, Sternglanz R, et al. The two-hybrid system: a method to identify and clone genes for proteins that interact with a protein of interest[J]. Proc Natl Acad Sci USA, 1991, 88(21): 9578-9582. pmid: 1946372
[26]	de Folter S, Immink RGH. Yeast protein-protein interaction assays and screens[J]. Methods Mol Biol, 2011, 754: 145-165. doi: 10.1007/978-1-61779-154-3_8 pmid: 21720951
[27]	Bollier N, Gonzalez N, Chevalier C, et al. Zinc finger-homeodomain and mini zinc finger proteins are key players in plant growth and responses to environmental stresses[J]. J Exp Bot, 2022, 73(14): 4662-4673. doi: 10.1093/jxb/erac194 URL
[28]	Yao W, Beckwith SL, Zheng TN, et al. Assembly of the Arp5(actin-related protein)subunit involved in distinct INO80 chromatin remodeling activities[J]. J Biol Chem, 2015, 290(42): 25700-25709. doi: 10.1074/jbc.M115.674887 URL
[29]	Rodriguez Milla MA, Uno Y, Chang IF, et al. A novel yeast two-hybrid approach to identify CDPK substrates: characterization of the interaction between AtCPK11 and AtDi19, a nuclear zinc finger protein 1[J]. FEBS Lett, 2006, 580(3): 904-911. pmid: 16438971
[30]	Yang GY, Zhao T, Lu S, et al. T1121G point mutation in the mitochondrial gene COX1 suppresses a null mutation in ATP23 required for the assembly of yeast mitochondrial ATP synthase[J]. Int J Mol Sci, 2022, 23(4): 2327. doi: 10.3390/ijms23042327 URL
[31]	Thibivilliers S, Farmer A, Libault M. Biological and cellular functions of the microdomain-associated FWL/CNR protein family in plants[J]. Plants, 2020, 9(3): 377. doi: 10.3390/plants9030377 URL
[32]	Li Q, Deng ZY, Gong CY, et al. The rice eukaryotic translation initiation factor 3 subunit f(OseIF3f)is involved in microgametogenesis[J]. Front Plant Sci, 2016, 7: 532.
[33]	Calero G, Wilson KF, Ly T, et al. Structural basis of m7GpppG binding to the nuclear cap-binding protein complex[J]. Nat Struct Biol, 2002, 9(12): 912-917. doi: 10.1038/nsb874 pmid: 12434151
[34]	Schmidt R, Schippers JHM, Mieulet D, et al. MULTIPASS, a rice R2R3-type MYB transcription factor, regulates adaptive growth by integrating multiple hormonal pathways[J]. Plant J, 2013, 76(2): 258-273. doi: 10.1111/tpj.2013.76.issue-2 URL
[35]	Nakamura T, Yamaguchi Y, Sano H. Four rice genes encoding cysteine synthase: isolation and differential responses to sulfur, nitrogen and light[J]. Gene, 1999, 229(1/2): 155-161. doi: 10.1016/S0378-1119(99)00019-0 URL
[36]	Moriguchi K, Suzuki T, Ito Y, et al. Functional isolation of novel nuclear proteins showing a variety of subnuclear localizations[J]. Plant Cell, 2005, 17(2): 389-403. pmid: 15659629
[37]	Shan SH, Yin RP, Shi JY, et al. Bowman-birk major type trypsin inhibitor derived from foxtail millet bran attenuate atherosclerosis via remodeling gut microbiota in ApoE-/-mice[J]. J Agric Food Chem, 2022, 70(2): 507-519. doi: 10.1021/acs.jafc.1c05747 URL
[38]	Chou WL, Chung YL, Fang JC, et al. Novel interaction between CCR4 and CAF1 in rice CCR4-NOT deadenylase complex[J]. Plant Mol Biol, 2017, 93(1): 79-96. doi: 10.1007/s11103-016-0548-6 URL
[39]	Tang WQ, Kim TW, Oses-Prieto JA, et al. BSKs mediate signal transduction from the receptor kinase BRI1 in Arabidopsis[J]. Science, 2008, 321(5888): 557-560. doi: 10.1126/science.1156973 URL
[40]	Jantasuriyarat C, Gowda M, Haller K, et al. Large-scale identification of expressed sequence tags involved in rice and rice blast fungus interaction[J]. Plant Physiol, 2005, 138(1): 105-115. pmid: 15888683
[41]	Ogawa-Ohnishi M, Matsubayashi Y. Identification of three potent hydroxyproline O-galactosyltransferases in Arabidopsis[J]. Plant J, 2015, 81(5): 736-746. doi: 10.1111/tpj.2015.81.issue-5 URL
[42]	Zhou CL, Lin QB, Lan J, et al. WRKY transcription factor OsWRKY29 represses seed dormancy in rice by weakening abscisic acid response[J]. Front Plant Sci, 2020, 11: 691. doi: 10.3389/fpls.2020.00691 URL
[43]	Tang JQ, Mei EY, He ML, et al. Functions of OsWRKY24, OsWRKY70 and OsWRKY53 in regulating grain size in rice[J]. Planta, 2022, 255(4): 1-8. doi: 10.1007/s00425-021-03785-z
[44]	Zhao SS, Hong W, Wu JG, et al. A viral protein promotes host SAMS1 activity and ethylene production for the benefit of virus infection[J]. eLife, 2017, 6: 27529.
[45]	Wang YH, Wan LY, Zhang LX, et al. An ethylene response factor OsWR1 responsive to drought stress transcriptionally activates wax synthesis related genes and increases wax production in rice[J]. Plant Mol Biol, 2012, 78(3): 275-288. doi: 10.1007/s11103-011-9861-2 pmid: 22130861
[46]	Rodriguez Milla MA, Townsend J, Chang IF, et al. The Arabidopsis AtDi19 gene family encodes a novel type of Cys2/His2 zinc-finger protein implicated in ABA-independent dehydration, high-salinity stress and light signaling pathways[J]. Plant Mol Biol, 2006, 61(1): 13-30. doi: 10.1007/s11103-005-5798-7 URL
[47]	Su L, Yang J, Li DD, et al. Dynamic genome-wide association analysis and identification of candidate genes involved in anaerobic germination tolerance in rice[J]. Rice, 2021, 14(1): 1. doi: 10.1186/s12284-020-00444-x pmid: 33409869
[48]	Liu WX, Zhang FC, Zhang WZ, et al. Arabidopsis Di19 functions as a transcription factor and modulates PR1, PR2, and PR5 expression in response to drought stress[J]. Mol Plant, 2013, 6(5): 1487-1502. doi: 10.1093/mp/sst031 URL
[49]	Qin LX, Li Y, Li DD, et al. Arabidopsis drought-induced protein Di19-3 participates in plant response to drought and high salinity stresses[J]. Plant Mol Biol, 2014, 86(6): 609-625. doi: 10.1007/s11103-014-0251-4 pmid: 25218132
[50]	Qin LX, Nie XY, Hu R, et al. Phosphorylation of serine residue modulates cotton Di19-1 and Di19-2 activities for responding to high salinity stress and abscisic acid signaling[J]. Sci Rep, 2016, 6: 20371. doi: 10.1038/srep20371
[51]	Guo YL, Tan YQ, Qu MH, et al. OsWR2 recruits OsHDA704 to regulate the deacetylation of H4K8ac in the promoter of OsABI5 in response to drought stress[J]. J Integr Plant Biol, 2023. DOI: 10.1111/jipb.13481.
[52]	Luban J, Goff SP. The yeast two-hybrid system for studying protein-protein interactions[J]. Curr Opin Biotechnol, 1995, 6(1): 59-64. doi: 10.1016/0958-1669(95)80010-7 URL

NCBI号 Accession number of NCBI	基因名称 Gene name	基因注释 Gene annotation	参考文献 Reference
LOC4347054	ZHD9	锌指同源结构域蛋白9 Zinc-finger homeodomain protein 9-like	[27]
LOC107275681	ARP5	肌动蛋白相关蛋白5 Actin-related protein 5	[28]
LOC4339608	Di19-1	脱水诱导蛋白19 DEHYDRATION-INDUCED 19-like protein	[29]
LOC4336195	ATP23	线粒体内膜蛋白酶 Mitochondrial inner membrane protease	[30]
LOC4329772	OsFWL2	细胞数目调节因子2 Cell number regulator 2	[31]
LOC4337552	OseIF3f	真核翻译起始因子3 Eukaryotic translation initiation factor 3 subunit F	[32]
LOC4329963	OsCBP20	核帽结合蛋白亚基2 Nuclear cap-binding protein subunit 2-like	[33]
LOC4349349	OsEXPB3	类膨胀素B3 Expansin-B3-like	[34]
LOC4334101	rcs3	类半胱氨酸合成酶 Cysteine synthase-like	[35]
LOC4345839	OsMtATPd2	线粒体中d亚单位ATP合成酶 ATP synthase subunit d, mitochondrial	[36]
LOC4325989	BBTI8	胰蛋白酶抑制剂 Bowman-Birk type bran trypsin inhibitor	[37]
LOC4331740	OsCCR4b	碳分解代谢抑制蛋白4同源物1 Carbon catabolite repressor protein 4 homolog 1	[38]
LOC4349455	BSK2	丝氨酸/苏氨酸蛋白激酶 Serine/threonine-protein kinase	[39]
LOC4336306	OsHCT1	羟基肉桂酰转移酶 Hydroxycinnamoyltransferase 1-like	[40]
LOC4330573	BSH	染色体结构重塑蛋白 Chromatin structure-remodeling complex protein
LOC4340286	HPGT3	羟脯氨酸O-半乳糖基转移酶 Hydroxyproline O-galactosyltransferase	[41]
LOC9269857	OsWRKY29	WRKY转录因子29 Probable WRKY transcription factor 29	[42]
LOC4349610	OsWRKY70	WRKY转录因子70 Probable WRKY transcription factor 70	[43]
LOC4337733	OsSAMS1	类S-腺苷甲硫氨酸合酶1 S-adenosylmethionine synthase 1-like	[44]
LOC4339460	OsNAP1;2	核小体组装蛋白1;2 Nucleosome assembly protein 1;2-like
LOC4328653	OsWR1	乙烯应答转录因子WIN1 Ethylene-responsive transcription factor WIN1	[45]

NCBI号 Accession number of NCBI	基因名称 Gene name	基因注释 Gene annotation	参考文献 Reference
LOC4347054	ZHD9	锌指同源结构域蛋白9 Zinc-finger homeodomain protein 9-like	[27]
LOC107275681	ARP5	肌动蛋白相关蛋白5 Actin-related protein 5	[28]
LOC4339608	Di19-1	脱水诱导蛋白19 DEHYDRATION-INDUCED 19-like protein	[29]
LOC4336195	ATP23	线粒体内膜蛋白酶 Mitochondrial inner membrane protease	[30]
LOC4329772	OsFWL2	细胞数目调节因子2 Cell number regulator 2	[31]
LOC4337552	OseIF3f	真核翻译起始因子3 Eukaryotic translation initiation factor 3 subunit F	[32]
LOC4329963	OsCBP20	核帽结合蛋白亚基2 Nuclear cap-binding protein subunit 2-like	[33]
LOC4349349	OsEXPB3	类膨胀素B3 Expansin-B3-like	[34]
LOC4334101	rcs3	类半胱氨酸合成酶 Cysteine synthase-like	[35]
LOC4345839	OsMtATPd2	线粒体中d亚单位ATP合成酶 ATP synthase subunit d, mitochondrial	[36]
LOC4325989	BBTI8	胰蛋白酶抑制剂 Bowman-Birk type bran trypsin inhibitor	[37]
LOC4331740	OsCCR4b	碳分解代谢抑制蛋白4同源物1 Carbon catabolite repressor protein 4 homolog 1	[38]
LOC4349455	BSK2	丝氨酸/苏氨酸蛋白激酶 Serine/threonine-protein kinase	[39]
LOC4336306	OsHCT1	羟基肉桂酰转移酶 Hydroxycinnamoyltransferase 1-like	[40]
LOC4330573	BSH	染色体结构重塑蛋白 Chromatin structure-remodeling complex protein
LOC4340286	HPGT3	羟脯氨酸O-半乳糖基转移酶 Hydroxyproline O-galactosyltransferase	[41]
LOC9269857	OsWRKY29	WRKY转录因子29 Probable WRKY transcription factor 29	[42]
LOC4349610	OsWRKY70	WRKY转录因子70 Probable WRKY transcription factor 70	[43]
LOC4337733	OsSAMS1	类S-腺苷甲硫氨酸合酶1 S-adenosylmethionine synthase 1-like	[44]
LOC4339460	OsNAP1;2	核小体组装蛋白1;2 Nucleosome assembly protein 1;2-like
LOC4328653	OsWR1	乙烯应答转录因子WIN1 Ethylene-responsive transcription factor WIN1	[45]