Cloning, Expression, and Function Analysis Under Ralstonia solanacearum Stress of Pathogenesis-related Protein SlPR1b Gene in Tomato (Solanum lycopersicum)
CHEN Na1, LI Xiao-Peng1, LIU Jin-Fa2, SHAO Qin1,*
1 College of Life Science and Resources and Environment, Yichun University, Yichun 336000, China; 2 Management Committee of Modern Agricultural Science Park of Xinyu, Xinyu 338000, China
Abstract:Pathogenesis-related protein 1 (PR1) gene plays an important role in plant resistance to biotic and abiotic stresses. Based on the transcriptome data of Ralstonia solanacearum stress previously conducted by our research group, in this study, a PR1 gene, named SlPR1b (Solyc00g174340.2) was cloned from tomato (Solanum lycopersicum) by reverse transcription PCR (RT PCR) technology. qPCR was used to reveal SlPR1b gene tissue expression specificity and expression characteristic under the conditions including infection with Ralstonia solanacearum, and treatment with salicylic acid (SA) and methyl jasmonate acid (MeJA). SlPR1b gene virus induced gene silencing (VIGS) vector was constructed and then transformed into tomato resistance material to analyze the resistance ability of SlPR1b gene silenced tomato in R. solanacearum stress condition. The results showed that the full-length cDNA sequence of SlPR1b gene was 807 bp, its ORF was 480 bp, encoding 159 amino acids, including a conserved CAP-PR-1 domain (cd05381), belonging to the CAP superfamily. The predicted molecular weight of SlPR1b was 17 519.73 D, the isoelectric point was 8.86, and the protein was found to be a secreted protein with a transmembrane structure. Homologous sequence alignment and phylogenetic analysis indicated that SlPR1b was highly homologous with a S. pennellii SpPR4 protein, followed by S. tuberosum StPR1b protein. The results of tissue specific expression showed that SlPR1b gene expression level was the highest in tomato leaves. Furthermore, SlPR1b gene expression could be induced by R. solanacearum, SA, and MeJA. Silencing SlPR1b decreased plant resistance to bacterial wilt, these results suggested that SlPR1b played a positive role in tomato resistance to bacterial wilt. This study provided a reference for further exploring the role of SlPR1b gene in the response of tomato to bacterial wilt.
陈娜, 李晓鹏, 刘进法, 邵勤. 番茄病程相关蛋白SlPR1b基因的克隆、表达及其在青枯菌胁迫下的功能分析[J]. 农业生物技术学报, 2023, 31(12): 2477-2489.
CHEN Na, LI Xiao-Peng, LIU Jin-Fa, SHAO Qin. Cloning, Expression, and Function Analysis Under Ralstonia solanacearum Stress of Pathogenesis-related Protein SlPR1b Gene in Tomato (Solanum lycopersicum). 农业生物技术学报, 2023, 31(12): 2477-2489.
[1] 陈娜. 2016. 茄子SmNAC转录因子表达特性及功能鉴定分析[D]. 硕士学位论文, 华南农业大学, 导师: 曹必好, pp. 13-16. (Chen N.2016. The expression characteristic and functional identification analysis of SmNAC transcription factor in eggplant (Solanum melongena)[D]. Thesis for M.S., South China Agricultural University, Suppervisor: Cao B H, pp. 13-16.) [2] 衡友强, 游西龙, 王艳. 2020. 费尔干猪毛菜病程相关蛋白SfPR1a基因的异源表达增强了烟草对干旱、盐及叶斑病的抗性[J]. 作物学报, 46(04): 503-512. (Heng Y Q, You X L, Wang Y.2020. Pathogenesis-related protein gene SfPR1a from Salsola ferganica enhances the resistances to drought, salt and leaf spot disease in transgenic tobacco[J]. Acta Agronomica Sinica, 46(04): 503-512.) [3] 李乾玉, 王秀美, 倪珊珊, 等. 2021. 非洲菊PR-1基因的克隆与表达分析[J]. 热带作物学报, 42(07): 1851-1859. (Li Q Y, Wang X M, Ni S S, et al.2021. Cloning and expression analysis of PR-1 genes in Gerbera jamesonii[J]. Chinese Journal of Tropical Crops, 42(07): 1851-1859.) [4] 刘玉晴, 燕高伟, 张彤, 等. 2021. 超表达OsPR1A增强了Xa21介导的水稻对白叶枯病的抗性反应[J]. 中国农业科学, 54(23): 4933-4942. (Liu Y Q, Yan G W, Zhang T, et al.2021. Overexpression of OsPR1A enhanced Xa21-mediated resistance to rice bacterial blight[J]. Scientia Agricultural Sinica, 54(23): 4933-4942.) [5] 马立功, 张匀华, 孟庆林, 等. 2015. 向日葵病程相关蛋白HaPR1基因的克隆与功能[J]. 作物学报, 41(12): 1819-1827. (Ma L G, Zhang Y H, Meng Q L, et al.2015. Cloning and function analysis of pathogenesis related protein gene HaPR1 from sunflower (Helianthus annuus)[J]. Acta Agronomica Sinica, 41(12): 1819-1827.) [6] 牛吉山, 刘瑞, 郑磊. 2007. 小麦PR-1、PR-2、PR-5基因的白粉菌和水杨酸诱导表达分析及白粉病抗性研究[J]. 麦类作物学报, 27(6): 1132-1137. (Niu J S, Liu R, Zheng L.2007. Expression analysis of wheat PR-1, PR-2, PR-5 activated by Bgt and SA, and powdery mildew resistance[J]. Journal of Triticeae, 27(6): 1132-1137.) [7] 田丽梅. 2016. 番茄IMPα/β和LYK家族基因的鉴定、表达模式分析和功能研究[D]. 硕士学位论文, 浙江大学, 导师: 宋凤鸣, pp. 42-52. (Tian L M.2016. Genome-wide characterization, expression patterns and functional analysis of the IMPα/β and LYK gene families in tomato[D]. Thesis for M.S., Zhejiang University, Suppervisor: Song F M, pp. 42-52.) [8] 王静, 刘丽, 张志明, 等. 2012. 玉米病程相关蛋白1基因的克隆与表达分析[J]. 浙江大学学报(农业与生命科学版), 38(1): 35-42. (Wang J, Liu L, Zhang Z M, et al.2012. Cloning and expression analysis of pathogenesis-related protein 1 gene in maize[J]. Journal of Zhejiang University (Agricultural & Life Science), 38(1): 35-42.) [9] 王荣青, 杨悦俭, 周国治, 等. 2007. 番茄抗青枯病筛选方法及其在抗青枯病育种中的应用[J]. 浙江农业学报, 19(2): 89-92. (Wang R Q, Yang Y J, Zhou G Z, et al.2007. Screening methods of resistance to bacterial wilt caused by Pseudomonas solanacearum in tomato and its application in tomato breeding[J]. Acta Agriculturae Zhejiangensis, 19(2): 89-92.) [10] 杨德翠, 张玉喜, 郑国生. 2013. 牡丹病程相关蛋白1基因的克隆及表达分析[J]. 园艺学报, 40(8): 1583-1590. (Yang D C, Zhang Y X, Zheng G S.2013. Gene cloning and expression analysis of pathogenesis-related protein 1 of Paeonia suffruticosa[J]. Acta Horticulturae Sinica, 40(8): 1583-1590.) [11] 张应鹏, 陈虹均, 郑锂蕾, 等. 2020. 三七病程相关蛋白基因PnPR1的表达特性以及功能分析[J]. 西北植物学报, 40(12): 2000-2007. (Zhang Y P, Chen H J, Zheng L L, et al.2020. Expression patterns and functional analysis of pathogenesis-related protein PnPR1 gene of Panax notoginseng[J]. Acta Botanica Boreali-Occidentalia Sinica, 40(12): 2000-2007.) [12] Akbudak M A, Yildiz S, Filiz E.2020. Pathogenesis related protein-1 (PR-1) genes in tomato (Solanum lycopersicum L.): Bioinformatics analyses and expression profiles in response to drought stress[J]. Genomics, 112(6): 4089-4099. [13] AlHudaib K A, Alanazi N A, Ghorbel M, et al.2022. Isolation and characterization of a novel pathogenesis-related protein-1 gene (AvPR-1) with induced expression in Oat (Avena sativa L.) during abiotic and hormonal stresses[J]. Plants (Basel), 11(17): 1-18. [14] Ali S, Mir Z A, Bhat J A, et al.2018. Isolation and characterization of systemic acquired resistance marker gene PR1 and its promoter from Brassica juncea[J]. 3 Biotech, 8(1): 2190-5738. [15] Almeida-Silva F, Venancio TM.2022. Pathogenesis-related protein 1 (PR-1) genes in soybean: Genome-wide identification, structural analysis and expression profiling under multiple biotic and abiotic stresses[J]. Gene, 809: 146013. [16] Brederode F T, Linthorst H J, Bol J F.1991. Differential induction of acquired resistance and PR gene expression in tobacco by virus infection, ethephon treatment, UV light and wounding[J]. Plant Molecular Biology, 17(6): 1117-1125. [17] Buch F, Pauchet Y, Rott M, et al.2014. Characterization and heterologous expression of a PR-1 protein from traps of the carnivorous plant Nepenthes mirabilis[J]. Phytochemistry, 100: 43-50. [18] Gamir J, Darwiche R, Van't Hof P, et al.2017. The sterol-binding activity of PATHOGENESIS-RELATED PROTEIN 1 reveals the mode of action of an antimicrobial protein[J]. The Plant Journal, 89(3): 502-509. [19] Ghorbel M, Zribi I, Missaoui K, et al.2021. Differential regulation of the durum wheat pathogenesis-related protein (PR1) by calmodulin TdCaM1.3 protein[J]. Molecular Biology Reports, 48(1): 347-362. [20] Gibbs G M, Roelants K, O'Bryan M K.2008. The CAP superfamily: Cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteins-roles in reproduction, cancer, and immune defense[J]. Endocrine Reviews, 29(7): 865-97. [21] Glazebrook J.2005. Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens[J]. Annual Review of Phytopathology, 43: 205-227. [22] Hamamouch N, Li C, Seo P J, et al.2011. Expression of Arabidopsis pathogenesis-related genes during nematode infection[J]. Molecular Plant Pathology, 12(4): 355-364. [23] Hou M, Xu W, Bai H, et al.2012. Characteristic expression of rice pathogenesis-related proteins in rice leaves during interactions with Xanthomonas oryzae pv. oryzae[J]. Plant Cell Reports, 31(5): 895-904. [24] Jo Y S, Park H B, Kim J Y, et al.2020. Menadione sodium bisulfite-protected tomato leaves against grey mould via antifungal activity and enhanced plant immunity[J]. The Plant Pathology Journal, 36(4): 335-345. [25] Li X Y, Zhang Y J, Zhang, W H, et al.2016. Expression profiles of pathogenesis-related gene, TaLr35PR1, as it relate to Lr35-mediated adult plant leaf rust resistance[J]. Plant Molecular Biology Reporter, 34: 1127-1135. [26] Li Y, Wang H, Zhang Y, et al.2018a. Can the world's favorite fruit, tomato, provide an effective biosynthetic chassis for high-value metabolites?[J] Plant Cell Reports, 37(10): 1443-1450. [27] Li Y Z, Muhammad T, Wang Y, et al.2018b. Salicylic acid collaborates with gene silencing to tomato defense against Tomato yellow leaf curl virus (TYLCV)[J]. Pakistan Journal of Botany, 50(5): 2041-2054. [28] Li Z T, Dhekney S A, Gray D J.2011. PR-1 gene family of grapevine: A uniquely duplicated PR-1 gene from a vitis interspecific hybrid confers high level resistance to bacterial disease in transgenic tobacco[J]. Plant Cell Reports, 30(1): 1-11. [29] Mitsuhara I, Iwai T, Seo S, et al.2008. Characteristic expression of twelve rice PR1 family genes in response to pathogen infection, wounding, and defense-related signal compounds (121/180)[J]. Molecular Genetics and Genomics, 279(4): 415-427. [30] Niderman T, Genetet I, Bruyère T, et al.1995. Pathogenesis-related PR-1 proteins are antifungal. Isolation and characterization of three 14-kilodalton proteins of tomato and of a basic PR-1 of tobacco with inhibitory activity against Phytophthora infestans[J]. Plant Physiology, 108(1): 17-27. [31] Pečenková T, Pleskot R, Žárský V.2017. Subcellular localization of Arabidopsis pathogenesis-related 1 (PR1) protein[J]. International Journal of Molecular Sciences, 18(4): 1-13. [32] Porcel R, Zamarreño Á M, García-Mina J M, et al.2014. Involvement of plant endogenous ABA in Bacillus megaterium PGPR activity in tomato plants[J]. BMC Plant Biology, 14: 36. [33] Punja, Z K.2001. Genetic engineering of plants to enhance resistance to fungal pathogensa review of progress and future prospects[J]. Canadian Journal of Plant Pathology, 23(3): 216-235. [34] Sarowar S, Kim Y J, Kim E N, et al.2005. Overexpression of a pepper basic pathogenesis-related protein 1 gene in tobacco plants enhances resistance to heavy metal and pathogen stresses[J]. Plant Cell Reports, 24(4): 216-224. [35] Sels J, Mathys J, De Coninck B M, et al.2008. Plant pathogenesis-related (PR) proteins: A focus on PR peptides[J]. Plant Physiology and Biochemistry, 46(11): 941-950. [36] Seo P J, Lee A K, Xiang F.2008. Molecular and functional profiling of Arabidopsis pathogenesis-related genes: Insights into their roles in salt response of seed germination[J]. Plant and Cell Physiology, 49(3): 334-344. [37] Shin S H, Pak J H, Kim M J, et al.2014. An acidic PATHOGENESIS-RELATED1 gene of Oryza grandiglumis is involved in disease resistance response against bacterial infection[J]. The Plant Pathology Journal, 30(2): 208-214. [38] van Loon L C, Rep M, Pieterse C M.2006. Significance of inducible defense-related proteins in infected plants[J]. Annual Review of Phytopathology, 44(1): 135-162. [39] van Loon L C, van Kammen A.1970. Polyacrylamide disc electrophoresis of the soluble leaf proteins from Nicotiana tabacum var. "Samsun" and "Samsun NN". II. Changes in protein constitution after infection with Tobacco mosaic virus[J]. Virology, 40(2): 190-211. [40] Xie X Z, Xue Y J, Zhou J J.2011. Phytochromes regulate SA and JA signaling pathways in rice and are required for developmentally controlled resistance to Magnaporthe grisea[J]. Molecular Plant, 4(4): 688-696. [41] Yang L, Ding W, Xu Y, et al.2016. New insights into the antibacterial activity of hydroxycoumarins against Ralstonia solanacearum[J]. Molecules, 21(4): 468. [42] Zaynab M, Peng J, Sharif Y, et al.2021. Expression profiling of pathogenesis-related protein-1 (PR-1) genes from Solanum tuberosum reveals its critical role in phytophthora infestans infection[J]. Microbial Pathogenesis, 161(Part B): 105290. [43] Zeiss D R, Mhlongo M I, Tugizimana F, et al.2019. Metabolomic profiling of the host response of tomato (Solanum lycopersicum) following infection by Ralstonia solanacearum[J]. International Journal of Molecular Sciences, 20(16): 1-22. [44] Zhang Y, Xu S, Ding P, et al.2010. Control of salicylic acid synthesis and systemic acquired resistance by two members of a plant-specific family of transcription factors[J]. Proceedings of the National Academy of Sciences of the USA, 107(42): 18220-18225.