Mechanism of NtPLA2A-mediated Hormone Signaling Pathway in Enhancing Tobacco Resistance to Bacterial Wilt

doi:10.13560/j.cnki.biotech.bull.1985.2025-1168

Abstract

Abstract:

Objective To clone the NtPLA2A gene from tobacco (Nicotiana tabacum) and explore the molecular mechanism by which it mediates hormone signaling pathways to enhance tobacco resistance to bacterial wilt. Method This study cloned the NtPLA2A gene from tobacco, conducted bioinformatic analysis and expression profiling under Ralstonia solanacearum infection, and investigated the role of NtPLA2A in disease resistance using virus-induced gene silencing (VIGS) in N. benthamiana. Result The NtPLA2A coding sequence is 1 284 bp, encoding 428 amino acids. The protein has a molecular weight of 46.57 kD and a theoretical isoelectric point of 8.74, and is a stable hydrophilic protein. Bioinformatic analysis revealed that NtPLA2A contains a typical Patatin-like phospholipase domain and shares high amino acid sequence similarity with PLA proteins from other species. Promoter analysis indicated that the promoter region of NtPLA2A contains environmental factors such as light, low temperature, and hormones. Following R. solanacearum infection, NtPLA2A was significantly upregulated in both resistant and susceptible tobacco materials to varying degrees. Additionally, treatments with salicylic acid, jasmonic acid, and ethylene also induced its expression. VIGS results demonstrated that silencing NtPLA2A compromised resistance to R. solanacearum in N. benthamiana. Conclusion This study clarified the sequence, structure, and expression characteristics of tobacco NtPLA2A gene, which is induced by R. solanacearum and SA/JA/ET; its silencing significantly impairs N. benthamiana resistance to bacterial wilt.

Key words: tobacco, NtPLA2A gene, Ralstonia solanacearum, gene cloning, virus-induced gene silencing (VIGS)

ZHANG Zhong-wen, WANG Wei-min, XIAO Zhi-liang, SHI Jiu-chang, HAO Xian-wei, WANG Wei, XIE Peng-gang, XU Jia-dan, ZHAO Jiong-ping, YANG Ai-guo. Mechanism of NtPLA2A-mediated Hormone Signaling Pathway in Enhancing Tobacco Resistance to Bacterial Wilt[J]. Biotechnology Bulletin, 2026, 42(8): 207-216.

Figures/Tables 12

References 34

[1]	Wei YL, Cai ZC, Che ZY, et al. Effects of phospholipase C inhibition on the regulation of membrane lipid metabolism in maize leaves [J]. Front Plant Sci, 2025, 16: 1547477.
[2]	Campos ML. A novel role for a phospholipase D in plant immunity [J]. Plant Physiol, 2020, 183(1): 33-34.
[3]	Wang GL, Ryu S, Wang XM. Plant phospholipases: an overview [M]//Lipases and Phospholipases. Totowa, NJHumana Press2012: 123-137.
[4]	Hong YY, Pan XQ, Welti R, et al. Phospholipase Dα3 is involved in the hyperosmotic response in Arabidopsis [J]. Plant Cell, 2008, 20(3): 803-816.
[5]	Fan RY, Zhao F, Gong Z, et al. Insights into the mechanism of phospholipid hydrolysis by plant non-specific phospholipase C [J]. Nat Commun, 2023, 14: 194.
[6]	Hong YY, Zhao J, Guo L, et al. Plant phospholipases D and C and their diverse functions in stress responses [J]. Prog Lipid Res, 2016, 62: 55-74.
[7]	Gonorazky G, Guzzo MC, Abd-El-Haliem AM, et al. Silencing of the tomato phosphatidylinositol-phospholipase C2 (SlPLC2) reduces plant susceptibility to Botrytis cinerea [J]. Mol Plant Pathol, 2016, 17(9): 1354-1363.
[8]	Jang JH, Lee OR. Overexpression of ginseng patatin-related phospholipase pPLAIIIβ alters the polarity of cell growth and decreases lignin content in Arabidopsis [J]. J Ginseng Res, 2020, 44(2): 321-331.
[9]	Wu W, Li WX, Huang CH. Phospholipase A2, a nonnegligible enzyme superfamily in gastrointestinal diseases [J]. Biochimie, 2022, 194: 79-95.
[10]	Hirschberg HJHB, Simons JFA, Dekker N, et al. Cloning, expression, purification and characterization of patatin, a novel phospholipase A [J]. Eur J Biochem, 2001, 268(19): 5037-5044.
[11]	Rydel TJ, Williams JM, Krieger E, et al. The crystal structure, mutagenesis, and activity studies reveal that patatin is a lipid acyl hydrolase with a Ser-Asp catalytic dyad [J]. Biochemistry, 2003, 42(22): 6696-6708.
[12]	Tavernier E, Pugin A. Phospholipase activities associated with the tonoplast from Acer pseudoplatanus cells: identification of a phospholipase A1 activity [J]. BBA Biomembr, 1995, 1233(2): 118-122.
[13]	Singh A, Baranwal V, Shankar A, et al. Rice phospholipase a superfamily: organization, phylogenetic and expression analysis during abiotic stresses and development [J]. PLoS One, 2012, 7(2): e30947.
[14]	Li MY, Bahn SC, Guo L, et al. Patatin-related phospholipase pPLAIIIβ-induced changes in lipid metabolism alter cellulose content and cell elongation in Arabidopsis [J]. Plant Cell, 2011, 23(3): 1107-1123.
[15]	Kwak JS, Kwon DH, Song JT, et al. A mutation in the pPLA-IIα gene encoding PATATIN-RELATED PHOSPHOLIPASE a causes late flowering in Arabidopsis [J]. Biochem Biophys Res Commun, 2021, 582: 16-20.
[16]	Seo YS, Kim EY, Mang HG, et al. Heterologous expression, and biochemical and cellular characterization of CaPLA1 encoding a hot pepper phospholipase A1 homolog [J]. Plant J, 2008, 53(6): 895-908.
[17]	Liu CX, Li X, Meng DX, et al. A 4-bp insertion at ZmPLA1 encoding a putative phospholipase a generates haploid induction in maize [J]. Mol Plant, 2017, 10(3): 520-522.
[18]	Liu HY, Wang K, Jia ZM, et al. Efficient induction of haploid plants in wheat by editing of TaMTL using an optimized Agrobacterium-mediated CRISPR system [J]. J Exp Bot, 2020, 71(4): 1337-1349.
[19]	Scherer GFE, Ryu SB, Wang XM, et al. Patatin-related phospholipase A: nomenclature, subfamilies and functions in plants [J]. Trends Plant Sci, 2010, 15(12): 693-700.
[20]	Yang WY, Zheng Y, Bahn SC, et al. The patatin-containing phospholipase a pPLAIIα modulates oxylipin formation and water loss in Arabidopsis thaliana [J]. Mol Plant, 2012, 5(2): 452-460.
[21]	姜惠娜, 敬松, 李晗玉, 等. 西藏野生垂穗披碱草EnPLA1基因克隆与表达分析 [J]. 草地学报, 2021, 29(10): 2141-2148.
	Jiang HN, Jing S, Li HY, et al. Cloning and expression analysis of EnPLA1 gene in Tibetan wild Elymus nutans Griseb [J]. Acta Agrestia Sin, 2021, 29(10): 2141-2148.
[22]	Narusaka Y, Narusaka M, Seki M, et al. Expression profiles of Arabidopsis phospholipase a IIA gene in response to biotic and abiotic stresses [J]. Plant Cell Physiol, 2003, 44(11): 1246-1252.
[23]	李慧欣, 车致远, 王旭, 等. 玉米磷脂酶A基因家族鉴定及其在盐胁迫条件下的差异表达分析 [J]. 玉米科学, 2025, 33(1): 14-21.
	Li HX, Che ZY, Wang X, et al. Identification of maize phospholipase a gene family and analysis of its differential expression under salt stress [J]. J Maize Sci, 2025, 33(1): 14-21.
[24]	Yang WY, Devaiah SP, Pan XQ, et al. AtPLAI is an acyl hydrolase involved in basal jasmonic acid production and Arabidopsis resistance to Botrytis cinerea [J]. J Biol Chem, 2007, 282(25): 18116-18128.
[25]	Saddhe A, Potock M. Comparative phylogenomic and structural analysis of canonical secretory PLA2 and novelPLA2-like family in plants [J]. Front Plant Sci,14(14), 1118670.
[26]	Zhang Y, Wang L, Chen X. Phospholipase A2 family members orchestrate plant immune responses through hormone signaling and second messenger production [J]. Nat Commun, 14(1), 1-12.
[27]	Hayward AC. Biology and epidemiology of bacterial wilt caused by Pseudomonas solanacearum [J]. Annu Rev Phytopathol, 1991, 29: 65-87.
[28]	Xiao ZL, Liu ZW, Zhang HF, et al. Transcriptomics and virus-induced gene silencing identify defence-related genes during Ralstonia solanacearum infection in resistant and susceptible tobacco [J]. Genomics, 2024, 116(2): 110784.
[29]	Wicker E, Grassart L, Coranson-Beaudu R, et al. Ralstonia solanacearum strains from Martinique (French west Indies) exhibiting a new pathogenic potential [J]. Appl Environ Microbiol, 2007, 73(21): 6790-6801.
[30]	Jiang GF, Wei Z, Xu J, et al. Bacterial wilt in China: history, current status, and future perspectives [J]. Front Plant Sci, 2017, 8: 1549.
[31]	Wu W, Zou HS, Zheng HY, et al. Ralstonia solanacearum type III effector RipAF1 mediates plant resistance signaling by ADP-ribosylation of host FBN1 [J]. Hortic Res, 2024, 11(8): uhae162.
[32]	张林琳,宫瑞,崔彦玲,等.用VIGS分析SmWRKY30在茄子抗青枯病中的作用[J].中国农业科学, 58(3), 548-563.
	Zhang L, Gong R, Cu, Y, et al. Analysis of the role of SmWRKY30 in eggplant resistance against bacterial wilt using VIGS [J]. Sc Agric Sin, 2025, 58(3): 548-563.
[33]	周明,陈燕,李阳,等.植物激素信号交叉对话在抗病反应中的作用机制[J]. 植物生理学报,2023, 59 (4):721-732.
	Zhou M, Chen Y, Li Y, et al. Mechanism of hormone signal crosstalk in plant disease resistance response [J]. Plant Physiol J, 2023, 59(4), 721-732.
[34]	李明,张华,王伟,等. 乙烯与茉莉酸协同调控PLA基因表达增强番茄对青枯病抗性的分子机制[J]. 植物病理学报, 2024, 53(2): 210-225.
	Li M, Zhang H, Wang W, et al. Molecular mechanism of ethylene and jasmonate co-regulating PLA gene expression to enhance tomato resistance against bacterial wilt [J]. Acta Phytopathol Sin, 53(2), 210-225.

网站（网址） Website （URL）	分析用途 Purpose of analysis
ProtParam （https：//web.expasy.org/protparam）	预测蛋白的理化性质
ProtScale（https：//web.expasy.org/protscale）	预测蛋白的亲疏水性
SOPMA（https：//npsa-prabi.ibcp.fr/cgibin/npsa_automat.pl page=npsa_sopm.html）	预测蛋白的二级结构
SWISS-MODEL（https：//swissmodel. expasy.org）	预测蛋白的三级结构
TMHMM-2.0（http：//www.csbio.sjtu.edu.cn/bioinf/MHMM/）	预测蛋白的跨膜结构域
Plant-mPLoc（http：//www.csbio.sjtu.edu.cn/bioinf/plantmulti）	预测蛋白的亚细胞定位
PlantCARE（http：//bioinformatics.psb.ugent.be/webtools/plantcare/html/）	预测基因启动子中的顺式作用元件

网站（网址） Website （URL）	分析用途 Purpose of analysis
ProtParam （https：//web.expasy.org/protparam）	预测蛋白的理化性质
ProtScale（https：//web.expasy.org/protscale）	预测蛋白的亲疏水性
SOPMA（https：//npsa-prabi.ibcp.fr/cgibin/npsa_automat.pl page=npsa_sopm.html）	预测蛋白的二级结构
SWISS-MODEL（https：//swissmodel. expasy.org）	预测蛋白的三级结构
TMHMM-2.0（http：//www.csbio.sjtu.edu.cn/bioinf/MHMM/）	预测蛋白的跨膜结构域
Plant-mPLoc（http：//www.csbio.sjtu.edu.cn/bioinf/plantmulti）	预测蛋白的亚细胞定位
PlantCARE（http：//bioinformatics.psb.ugent.be/webtools/plantcare/html/）	预测基因启动子中的顺式作用元件

基因 Gene	沉默序列 Silencing sequence （5'-3'）
NtPLA2A	GGTAGAACTTTTGTAGCTGCACTAACTTTATTAGTGACTCTTCATGTTCTACTACCTGTTATGGTTTCTGCTGCTACAAAAGGAAAGATAGTAACAGTTTTGAGCATAGATGGAGGTGGCATCAGAGGCATTATTCCTGGCACCCTTCTTGCTTTCCTTGAATCCAAGCTTCAGGACATAGATGGACCGAATGCAAGAATTGCAGACTATTTTGATGTTGTAGCTGGAACGAGCACAGGTGGACTAATAAGCACCATGCTCACAGCTCCAAACAAGGATAATCGCCCTTTATATGCA
NtPDS	ATGCTAACTAGCTGGAGACTCTTTCCTGCTTCGTTCACCAGGCCTAAGTACTGCCCAAGTGGATTTTATTGCAGGGCTGAGGATCAACTGAGTAGTATAAATGAACGTCAAAAGAAGAAGAAAGTGCTGATAGTAGGTTCAGGCTGGGCTGGCCTTGGAGCTGCTCACCATCTCTGCAAACAGGGCTTTGAGGTCGTTGTTCTTGAAGGTGGCTATGAATTTGGACCCAAAAATCAATCCCTAAGCCCTGACGATGTGGCTATTCGCGGTTTCTGGTATCCCTATCGAAATATATTTGAT

基因 Gene	沉默序列 Silencing sequence （5'-3'）
NtPLA2A	GGTAGAACTTTTGTAGCTGCACTAACTTTATTAGTGACTCTTCATGTTCTACTACCTGTTATGGTTTCTGCTGCTACAAAAGGAAAGATAGTAACAGTTTTGAGCATAGATGGAGGTGGCATCAGAGGCATTATTCCTGGCACCCTTCTTGCTTTCCTTGAATCCAAGCTTCAGGACATAGATGGACCGAATGCAAGAATTGCAGACTATTTTGATGTTGTAGCTGGAACGAGCACAGGTGGACTAATAAGCACCATGCTCACAGCTCCAAACAAGGATAATCGCCCTTTATATGCA
NtPDS	ATGCTAACTAGCTGGAGACTCTTTCCTGCTTCGTTCACCAGGCCTAAGTACTGCCCAAGTGGATTTTATTGCAGGGCTGAGGATCAACTGAGTAGTATAAATGAACGTCAAAAGAAGAAGAAAGTGCTGATAGTAGGTTCAGGCTGGGCTGGCCTTGGAGCTGCTCACCATCTCTGCAAACAGGGCTTTGAGGTCGTTGTTCTTGAAGGTGGCTATGAATTTGGACCCAAAAATCAATCCCTAAGCCCTGACGATGTGGCTATTCGCGGTTTCTGGTATCCCTATCGAAATATATTTGAT

顺式元件 Cis-element	序列 Sequence （5′-3′）	数量 Number	功能 Function
CAAT-box	CAAAT	24	启动子和增强子区域的常见顺式作用元件 Common cis-acting element in promoter and enhancer regions
GARE-motif	TCTGTTG	3	赤霉素响应元件 Gibberellin-responsive element
LTR	CCGAAA	2	参与低温响应的顺式作用元件 Cis-acting element involved in low-temperature responsiveness
TCT-motif	TCTTAC	2	光响应元件的组成部分 Components of a light responsive element
TATA-box	TACAAAA	47	转录起始点-30附近的核心启动子元件 Core promoter element around -30 of transcription start
TCA-element	CCATCTTTTT	1	参与水杨酸反应的顺式作用元件 Cis-acting element involved in salicylic acid responsiveness
TGACG-motif	TGACG	2	参与茉莉酸甲酯（MeJA）响应的顺式调控元件 Cis-acting regulatory element involved in the MeJA-responsiveness
O2-site	GATGATGTGG	1	参与玉米醇溶蛋白代谢调控的顺式调控元件 Cis-acting regulatory element involved in zein metabolism regulation
MRE	AACCTAA	1	参与光响应的MYB结合位点 MYB binding site involved in light responsiveness
chs-CMA2a	TCACTTGA	1	光响应元件 Components of a light responsive element
ATCT-motif	AATCTAATCC	1	光响应保守DNA模块的组成部分 Components of a conserved DNA module involved in light responsiveness
CGTCA-motif	CGTCA	1	参与茉莉酸甲酯响应的顺式作用调控元件 Cis-acting regulatory element involved in the MeJA-responsiveness
GT1-motif	GGTTAA	1	光响应元件 Light responsive element