Cloning,Expression,and Functional Analysis of WRKY Transcription Factor SlWRKY75 in Tomato

doi:10.7668/hbnxb.20194162

Abstract

Abstract:

WRKY transcription factor is a family of transcription factors unique to plants.Studies have demonstrated that WRKY transcription factor played an important role in plant growth and development and in plant response to biological and abiotic stress.In order to reveal the function of tomato WRKY gene,two inbred lines of tomato with high resistance to bacterial wilt Hm 2-2(R)and high susceptibility to bacterial wilt BY 1-2(S)were used as experimental materials based on the preliminary transcriptome data,and a WRKY transcription factor SlWRKY75 gene(Solyc05g015850.3)was cloned.The structure,expression pattern and function of the gene and its encoded protein were analyzed by bioinformatics analysis,multiple alignment of amino acid sequences,phylogenetic tree construction,Real-time Quantitative PCR(qRT-PCR)and virus induced gene silencing(VIGS).The results showed that the full length of the cDNA of this gene was 653 bp,its maximum open reading frame was 519 bp,encoding 172 amino acids,the relative molecular weight of the protein was 19.878 51 ku,the theoretical isoelectric point was 9.32.The protein belonged to the hydrophilic non-secreted protein,and there was no transmembrane structure.Meanwhile,the protein had a highly conserved WRKY domain and a CX₄CX₂₃HXH zinc finger motif,which belonged to the Class Ⅱ family.Phylogenetic tree analysis showed that SlWRKY75 was closely related to Solanum pennellii SpWRKY75 and clustered into a group with other Solanaceae,while it was far related to Hevea brasiliensis HbWRKY75 and Gossypium hirsutum GhWRKY75 and was in different branches in the phylogenetic tree.The results of qRT-PCR analysis showed that the expression of SlWRKY75 gene were tissue-specific and could be induced by Ralstonia solanacearum,salicylic acid and jasmonic acid.VIGS result showed that silencing SlWRKY75 reduced plant resistance to bacterial wilt,indicating that SlWRKY75 positively regulated tomato resistance to bacterial wilt.These results suggested that SlWRKY75 gene played an important role in regulating tomato resistance to bacterial wilt.

Key words: Tomato bacterial wilt, WRKY transcription factor, Gene cloning, Expression analysis, Virus induced gene silencing(VIGS)

CHEN Na, ZHAN Wenwen, LIU Xingyu, SHI Leixin, LI Ruonan, XIE Rong, QUE Zhiqun. Cloning,Expression,and Functional Analysis of WRKY Transcription Factor SlWRKY75 in Tomato[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(6): 1-10. doi: 10.7668/hbnxb.20194162.

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Figures/Tables 10

Fig.1 Tomato SlWRKY75 gene A:M.Marker DL2000;1.SlWRKY75 gene.B:M.Marker DL2000;5—7.Positive clone;1—4,8.Negative clone.

Fig.2 Sequences of SlWRKY75 cDNA and predicted ORF

Fig.3 Conservative domain of SlWRKY75

Fig.4 Hydrophilic analysis of SlWRKY75 protein

Fig.5 Signal peptide and transmembrane structure analysis of SlWRKY75 protein A.Signal peptide prediction of SlWRKY75 protein;B.Transmembrane structure prediction of SlWRKY75 protein.

Fig.6 Secondary structure and tertiary structure prediction of SlWRKY75 protein A.The secondary structure prediction of SlWRKY75 protein;B.The tertiary structure prediction of SlWRKY75 protein.

Fig.7 Multiple alignment of homologous protein for the SlWRKY75 in tomato and other species The box.The highly conserved WRKYGQK heptapeptide sequence;The line.The WRKY domain;The triangle.The zinc finger motif.

Fig.8 Phylogenetic tree of SlWRKY75 homologous protein

Fig.9 Expression analysis of tomato SlWRKY75 gene n=3,the different lowercase letters represent significant differences(P<0.05).The same as Fig.10.

Fig.10 SlWRKY75 gene positively regulates tomato resistance to bacterial wilt A.SlWRKY75 relative expression in tomato plants with SlWRKY75 silencing mediated by TRV;B.Phynotypic characterization of tomato leaf with SlWRKY75 silencing mediated by TRV.CK.Control group,trans-infected with water;TRV∷Empty.Control group,trans-infected with empty virus vector;TRV∷SlWRKY75.Experimental group,trans-infected with SlWRKY75 recombinant virus vector.

References 42

[1]	任敏华, 张静燕, 崔晓东, 陈荣华, 刘琼光. 赣南地区番茄青枯菌菌系多样性分析[J]. 华南农业大学学报, 2022, 43(1):67-76.doi:10.7671/j.issn.1001-411X.202101041.
	Ren M H, Zhang J Y, Cui X D, Chen R H, Liu Q G. Diversity of Ralstonia solanacearum strains from tomato in the south of Jiangxi Province[J]. Journal of South China Agricultural Universit, 2022, 43(1):67-76.
[2]	宫超, 黎振兴, 麦培婷, 孙保娟, 李植良, 李涛. 番茄青枯病抗性相关根际微生物的研究进展[J]. 广东农业科学, 2021, 48(9):51-61.doi:10.16768/j.issn.1004-874X.2021.09.006.
	Gong C, Li Z X, Mai P T, Sun B J, Li Z L, Li T. Research progress of rhizosphere microorganisms related to tomato bacterial wilt resistance[J]. Guangdong Agricultural Sciences, 2021, 48(9):51-61.
[3]	Wang L, Gao Y, Jiang N H, Yan J, Lin W P, Cai K Z. Silicon controls bacterial wilt disease in tomato plants and inhibits the virulence-related gene expression of Ralstonia solanacearum[J]. International Journal of Molecular Sciences, 2022, 23(13):6965.doi:10.3390/ijms23136965. URL
[4]	Shi J L, Shui D J, Su S W, Xiong Z L, Zai W S. Gene enrichment and co-expression analysis shed light on transcriptional responses to Ralstonia solanacearum in tomato[J]. BMC Genomics, 2023, 24(1):159.doi:10.1186/s12864-023-09237-0.
[5]	da Silva Xaviera A, de Almeida J C F, de Melo A G, Rousseau G M, Tremblay D M, de Rezende R R, Moineau S, Alfenas-Zerbini P. Characterization of CRISPR-Cas systems in the Ralstonia solanacearum species complex[J]. Molecular Plant Patholog, 2019, 20(2):223-239.doi:10.1111/mpp.12750.
[6]	佘小漫, 何自福. 作物青枯病研究进展[J]. 广东农业科学, 2020, 47(12):82-89.doi:10.16768/j.issn.1004-874X.2020.12.009.
	She X M, He Z F. Advances in studies on crop bacterial wilt caused by Ralstonia solanacearum[J]. Guangdong Agricultural Sciences, 2020, 47(12):82-89.
[7]	He M M, Shah Jahan M, Wang Y, Sun J, Shu S, Guo S R. Compost amendments based on vinegar residue promote tomato growth and suppress bacterial wilt caused by Ralstonia solanacearum[J]. Pathogens, 2020, 9(3):227.doi:10.3390/pathogens9030227. URL
[8]	Ulker B, Somssich I E. WRKY transcription factors:from DNA binding towards biological function[J]. Current Opinion in Plant Biolog, 2004, 7(5):491-498.doi:10.1016/j.pbi.2004.07.012.
[9]	Eulgem T, Rushton P J, Robatzek S, Somssich I E. The WRKY superfamily of plant transcription factors[J]. Trends in Plant Science, 2000, 5(5):199-206.doi:10.1016/s1360-1385(00)01600-9. pmid: 10785665
[10]	Rushton P J, Somssich I E, Ringler P, Shen Q J. WRKY transcription factors[J]. Trends in Plant Science, 2010, 15(5):247-258.doi:10.1016/j.tplants.2010.02.006. pmid: 20304701
[11]	Wu K L, Guo Z J, Wang H H, Li J. The WRKY family of transcription factors in rice and Arabidopsis and their origins[J]. DNA Research, 2005, 12(1):9-26.doi:10.1093/dnares/12.1.9. URL
[12]	Zhang Y J, Wang L J. The WRKY transcription factor superfamily:its origin in eukaryotes and expansion in plants[J]. BMC Evolutionary Biolog, 2005, 5:1.doi:10.1186/1471-2148-5-1.
[13]	Ishiguro S, Nakamura K. Characterization of a cDNA encoding a novel DNA-binding protein,SPF1,that recognizes SP8 sequences in the 5'upstream regions of genes coding for sporamin and beta-amylase from sweet potato[J]. Molecular & General Genetics, 1994, 244(6):563-571.doi:10.1007/bf00282746.
[14]	Ramamoorthy R, Jiang S Y, Kumar N, Venkatesh P N, Ramachandran S. A comprehensive transcriptional profiling of the WRKY gene family in rice under various abiotic and phytohormone treatments[J]. Plant and Cell Physiolog, 2008, 49(6):865-879.doi:10.1093/pcp/pcn061.
[15]	Huang S X, Gao Y F, Liu J K, Peng X L, Niu X L, Fei Z J, Cao S Q, Liu Y S. Genome-wide analysis of WRKY transcription factors in Solanum lycopersicum[J]. Molecular Genetics and Genomics, 2012, 287(6):495-513.doi:10.1007/s00438-012-0696-6. URL
[16]	Bencke-Malato M, Cabreira C, Wiebke-Strohm B, Bücker-Neto L, Mancini E, Osorio M B, Homrich M S, Turchetto-Zolet A C, De Carvalho M C, Stolf R, Weber R L, Westergaard G, Castagnaro A P, Abdelnoor R V, Marcelino-Guimarães F C, Margis-Pinheiro M, Bodanese-Zanettini M H. Genome-wide annotation of the soybean WRKY family and functional characterization of genes involved in response to Phakopsora pachyrhizi infection[J]. BMC Plant Biolog, 2014, 14:236.doi:10.1186/s12870-014-0236-0.
[17]	Ning P, Liu C C, Kang J Q, Lü J Y. Genome-wide analysis of WRKY transcription factors in wheat(Triticum aestivum L.) and differential expression under water deficit condition[J]. Peer J, 2017, 5:e3232.doi:10.7717/peerj.3232. URL
[18]	Zhang T, Tan D F, Zhang L, Zhang X Y, Han Z X. Phylogenetic analysis and drought-responsive expression profiles of the WRKY transcription factor family in maize[J]. Agri Gene, 2017, 3:99-108.doi:10.1016/j.aggene.2017.01.001. URL
[19]	岳茂兰, 江雷雨, 刘怡, 李栎, 刘勇强, 陈清, 林源秀, 汤浩茹. 草莓FaWRKY31的克隆、亚细胞定位及表达特性分析[J]. 园艺学报, 2019, 46(10):1947-1959.doi:10.16420/j.issn.0513-353x.2018-0904.
	Yue M L, Jiang L Y, Liu Y, Li Y, Liu Y Q, Chen Q, Lin Y X, Tang H R. Cloning,subcellular location and expression analysis of FaWRKY31 in Fragaria×ananassa[J]. Acta Horticulturae Sinica, 2019, 46(10):1947-1959.
[20]	Lei Y Y, Sun Y P, Wang B T, Yu S, Dai H Y, Li H, Zhang Z H, Zhang J X. Woodland strawberry WRKY71 acts as a promoter of flowering via a transcriptional regulatory cascade[J]. Horticulture Research, 2020, 7:137.doi:10.1038/s41438-020-00355-4.
[21]	Zhang J, Gu M, Liang R S H, Shi X Y, Chen L L, Hu X, Wang S C, Dai X L, Qu H Y, Li H H, Xu G H. OsWRKY21 and OsWRKY108 function redundantly to promote phosphate accumulation through maintaining the constitutive expression of OsPHT1;1 under phosphate-replete conditions[J]. The New Phytologist, 2021, 229(3):1598-1614.doi:10.1111/nph.16931. URL
[22]	Rosado D, Ackermann A, Spassibojko O, Rossi M, Pedmale U V. WRKY transcription factors and ethylene signaling modify root growth during the shade-avoidance response[J]. Plant Physiolog, 2022, 188(2):1294-1311.doi:10.1093/plphys/kiab493. URL
[23]	Wu M, Zhang K M, Xu Y Z, Wang L N, Liu H X, Qin Z L, Xiang Y. The Moso bamboo WRKY transcription factor,PheWRKY86,regulates drought tolerance in transgenic plants[J]. Plant Physiology and Biochemistr, 2022, 170:180-191.doi:10.1016/j.plaphy.2021.10.024.
[24]	Yang J, Wang Q N, Luo H L, He C Z, An B. HbWRKY40 plays an important role in the regulation of pathogen resistance in Hevea brasiliensis[J]. Plant Cell Reports, 2020, 39(8):1095-1107.doi:10.1007/s00299-020-02551-x. pmid: 32399673
[25]	李笑, 王金彦, 刘廷利, 张保龙, 杨旭. 野生茄托鲁巴姆StWRKY-1的克隆及功能分析[C]. 江苏省遗传学会, 2017.doi:10.26914/c.cnkihy.2017.004816.
	Li X, Wang J Y, Liu T L, Zhang X. Cloning and functional analysis of StWRKY-1 in Solanum torvum[C]. Jiangsu Provincial Genetic Society, 2017.
[26]	Jiang Y Y, Zheng W R, Li J, Liu P, Zhong K L, Jin P, Xu M Z, Yang J, Chen J P. NbWRKY40 positively regulates the response of Nicotiana benthamiana to tomato mosaic virus via salicylic acid signaling[J]. Frontiers in Plant Science, 2020, 11:603518.doi:10.3389/fpls.2020.603518. URL
[27]	Wang W J, Li T, Chen Q, Yao S X, Deng L L, Zeng K F. CsWRKY25 improves resistance of Citrus fruit to Penicillium digitatum via modulating reactive oxygen species production[J]. Frontiers in Plant Science, 12:818198.doi:10.3389/fpls.2021.818198. URL
[28]	Yin W C, Wang X H, Liu H, Wang Y, van Nocker S, Tu M X, Fang J H, Guo J Q, Li Z, Wang X P. Overexpression of VqWRKY31 enhances powdery mildew resistance in grapevine by promoting salicylic acid signaling and specific metabolite synthesis[J]. Horticulture Research, 2022, 9:uhab064.doi:10.1093/hr/uhab064. URL
[29]	Ahammed G J, Li X, Mao Q, Wan H J, Zhou G Z, Cheng Y. The SlWRKY81 transcription factor inhibits stomatal closure by attenuating nitric oxide accumulation in the guard cells of tomato under drought[J]. Physiologia Plantarum, 2021, 172(2):885-895.doi:10.1111/ppl.13243. URL
[30]	Ramos R N, Martin G B, Pombo M A, Rosli H G. WRKY22 and WRKY25 transcription factors are positive regulators of defense responses in Nicotiana benthamiana[J]. Plant Molecular Biolog, 2021, 105(1/2):65-82.doi:10.1007/s11103-020-01069-w.
[31]	Guo M Y, Yang F J, Liu C X, Zou J P, Qi Z Y, Fotopoulos V, Lu G, Yu J Q, Zhou J. A single-nucleotide polymorphism in WRKY33 promoter is associated with the cold sensitivity in cultivated tomato[J]. The New Phytologist, 2022, 236(3):989-1005.doi:10.1111/nph.18403. URL
[32]	Huang H, Zhao W C, Li C H, Qiao H, Song S S, Yang R, Sun L L, Ma J L, Ma X C, Wang S H. SlVQ15 interacts with jasmonate-ZIM domain proteins and SlWRKY31 to regulate defense response in tomato[J]. Plant Physiolog, 2022, 190(1):828-842.doi:10.1093/plphys/kiac275. URL
[33]	Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCT method[J]. Methods, 2001, 25(4):402-408.doi:10.1006/meth.2001.1262. pmid: 11846609
[34]	陈娜. 茄子SmNAC转录因子表达特性及功能鉴定分析[D]. 广州: 华南农业大学, 2016.doi:10.7666/d.D01037009.
	Chen N. Expression characteristics and functional identification of SmNAC transcription factor in eggplant[D]. Guangzhou: South China Agricultural University, 2016.
[35]	张婷, 柏杨, 亓希武, 于盱, 房海灵, 李莉, 刘冬梅, 梁呈元, 李维林. 薄荷MhWRKY57基因克隆及响应茉莉酸信号的表达分析[J]. 南京林业大学学报(自然科学版), 2022, 46(6):279-287.doi:10.12302/j.issn.1000-2006.202109036.
	Zhang T, Bai Y, Qi X W, Yu X, Fang H L, Li L, Liu D M, Liang C Y, Li W L. Cloning and expression analyses of MhWRKY57 response to jasmonic acid in Mentha canadensis[J]. Journal of Nanjing Forestry University (Natural Science Edition), 2022, 46(6):279-287.
[36]	张冬梅, 冯亚艳, 修志君, 杨春芳, 杜美娥, 李得宙, 张笑宇. 硅酸钠诱导的马铃薯抗黑痣病StWRKY11基因克隆及生物信息学分析[J]. 华北农学报, 2022, 37(5):194-203.doi:10.7668/hbnxb.20192773.
	Zhang D M, Feng Y Y, Xiu Z J, Yang C F, Du M E, Li D Z, Zhang X Y. Cloning and bioinformatics analysis of resistant gene StWRKY11 induced by sodium silicate in potato by Rhizoctonia solani[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(5):194-203.
[37]	尹梦莹, 蔚亚楠, 杜光辉, 桂敏, 黎志彬, 鲍锐, 龚亚菊, 吴丽艳. 蒜芥茄SsWRKY1基因克隆及黄萎病病原菌胁迫下表达分析[J]. 南方农业学报, 2022, 53(4):1000-1010.doi:10.3969/j.issn.2095-1191.2022.04.013.
	Yin M Y, Yu Y N, Du G H, Gui M, Li Z B, Bao R, Gong Y J, Wu L Y. Cloning and expression analysis of SsWRKY1 gene in Solanum sisymbriifolium under stress of verticillium wilt disease[J]. Journal of Southern Agriculture, 2022, 53(4):1000-1010.
[38]	Hong Y H, Cui J, Liu Z, Luan Y S. SpWRKY6 acts as a positive regulator during tomato resistance to Phytophthora infestans infection[J]. Biochemical and Biophysical Research Communications, 2018, 506(4):787-792.doi:10.1016/j.bbrc.2018.10.155. URL
[39]	Gao Y F, Liu J K, Yang F M, Zhang G Y, Wang D, Zhang L, Ou Y B, Yao Y A. The WRKY transcription factor WRKY8 promotes resistance to pathogen infection and mediates drought and salt stress tolerance in Solanum lycopersicum[J]. Physiologia Plantarum, 2020, 168(1):98-117.doi:10.1111/ppl.12978. URL
[40]	Gharsallah C, Gharsallah Chouchane S, Werghi S, Mehrez M, Fakhfakh H, Gorsane F. Tomato contrasting genotypes responses under combined salinity and viral stresses[J]. Physiology and Molecular Biology of Plants, 2020, 26(7):1411-1424.doi:10.1007/s12298-020-00835-w. pmid: 32647458
[41]	Shu P, Zhang S J, Li Y J, Wang X Y, Yao L, Sheng J P, Shen L. Over-expression of SlWRKY46 in tomato plants increases susceptibility to Botrytis cinerea by modulating ROS homeostasis and SA and JA signaling pathways[J]. Plant Physiology and Biochemistr, 2021, 166:1-9.doi:10.1016/j.plaphy.2021.05.021.
[42]	Negi N, Khurana P. A salicylic acid inducible mulberry WRKY transcription factor,MiWRKY53 is involved in plant defence response[J]. Plant Cell Reports, 2021, 40(11):2151-2171.doi:10.1007/s00299-021-02710-8.

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