小麦TaXI-Ⅳ互作蛋白的筛选及其与TaHRLI-4D的互作验证

doi:10.13560/j.cnki.biotech.bull.1985.2025-0993

摘要/Abstract

摘要：

目的小麦木聚糖酶抑制剂（Triticum aestivum XI, TaXI）TaXI-Ⅳ在小麦抵御赤霉病的过程中发挥重要作用，既能抑制禾谷镰刀菌木聚糖酶对细胞壁木聚糖的降解，又能抑制其引发的细胞坏死现象。筛选TaXI-Ⅳ互作蛋白，为研究TaXI-Ⅳ在小麦抵御禾谷镰刀菌侵染过程中的分子调控机制，以及发掘小麦中的抗病相关基因提供参考。方法通过对TaXI-Ⅳ蛋白进行生物信息学分析和亚细胞定位。用同源重组法构建TaXI-Ⅳ的诱饵载体，并对其进行功能检测和自激活检测。采用酵母双杂交技术筛选TaXI-Ⅳ的互作蛋白，并通过酵母回转试验、荧光素酶互补试验和双分子荧光互补试验进行验证。用RT-qPCR分析互作蛋白对应基因在禾谷镰刀菌侵染小麦后的表达情况，并构建转基因拟南芥，通过对拟南芥叶片注射禾谷镰刀菌，并分析其抗病性。结果 TaXI-Ⅳ亚细胞定位于细胞膜和（或）细胞壁。重组诱饵载体pBT3N-TaXI-Ⅳ在酵母细胞NMY51中无自激活现象，能够正常表达并翻译成具有功能的蛋白质，适合作为筛选cDNA文库的诱饵载体。采用分裂泛素膜系统酵母双杂交技术从小麦cDNA文库中共筛选获得23个TaXI-Ⅳ互作蛋白，涉及植物的转录调控、胁迫响应、生物防御反应、氧化应激响应等过程。TaXI-Ⅳ与TaHRLI-4D（参与超敏反应的HR样病变诱导蛋白）在酵母和植物体内均有互作。TaHRLI-4D受禾谷镰刀菌诱导表达，过表达TaHRLI-4D能够增强拟南芥对病原菌的抗性。结论筛选获得23个TaXI-Ⅳ阳性互作蛋白，其中，包括病程相关蛋白TaNLTP-4B（非特异性脂质转移蛋白）和活性氧代谢相关蛋白TaTrx-4A（硫氧还蛋白）。HR样病变诱导蛋白基因TaHRLI-4D参与小麦对禾谷镰刀菌侵染的防御反应。

关键词: 小麦, 木聚糖酶抑制剂TaXI-Ⅳ, 酵母双杂交, 双分子荧光互补试验, TaHRLI-4D

Abstract:

Objective The wheat xylanase inhibitor TaXI-Ⅳ plays an important role in wheat resistance to Fusarium head blight. It can inhibit both the degradation of cell wall xylan and the cell necrosis induced by Fusarium gramineum xylanase. Screening TaXI-Ⅳ-interacting proteins will provide insights into its molecular regulatory mechanisms during wheat defense against F. graminearum infection, as well as support the discovery of disease resistance-related genes in wheat. Method Bioinformatic analysis and subcellular localization were conducted for the TaXI-Ⅳ protein. The bait vector for TaXI-Ⅳ was constructed using homologous recombination and subsequently tested for functionality and self-activation. TaXI‑Ⅳ‑interacting proteins were screened by yeast two-hybrid (Y2H) assay and verified through yeast retransformation, luciferase complementation (LCA), and bimolecular fluorescence complementation (BiFC) assays. RT‑qPCR was performed to analyze the expressions of the corresponding interacting‑protein genes after F. graminearum infection. Furthermore, transgenic Arabidopsis thaliana lines were then generated and inoculated with F. graminearum via leaf injection to assess disease resistance. Result TaXI-Ⅳ was localized to the plasma membrane and/or cell wall. The recombinant bait vector pBT3N-TaXI-Ⅳ showed no auto-activation in the yeast strain NMY51 and produced functional protein, confirming its suitability for cDNA library screening. A total of 23 proteins that interacted with TaXI-Ⅳ were obtained by the split-ubiquitin membrane yeast two-hybrid system. These proteins were involved in diverse biological processes in plants, including transcriptional regulation, stress response, biotic defensive reaction, oxidative stress response. Notably, TaXI‑Ⅳ interacted with the hypersensitive response-like lesion-inducing protein TaHRLI‑4D, as demonstrated in both yeast and plant systems. The expression of TaHRLI-4D was induced by F. graminearum infection, and its overexpression in A. thaliana enhanced plant resistance to the pathogen. Conclusion This study screened 23 positive TaXI-Ⅳ-interacting proteins, including the pathogenesis-related protein TaNLTP-4B (non-specific lipid transfer protein) and the reactive oxygen metabolism-related protein TaTrx-4A (thioredoxin). It was further verified that the HR-like lesion inducing protein gene TaHRLI-4D participates in the wheat defense response against F. graminearum infection.

Key words: wheat, xylanase inhibitor TaXI-Ⅳ, yeast two-hybrid (Y2H), BiFC, TaHRLI-4D

刘洋, 白裔凡, 陈微, 周晶晶, 蔡华, 司红起. 小麦TaXI-Ⅳ互作蛋白的筛选及其与TaHRLI-4D的互作验证[J]. 生物技术通报, 2026, 42(9): 286-296.

LIU Yang, BAI Yi-fan, CHEN Wei, ZHOU Jing-jing, CAI Hua, SI Hong-qi. Screening for TaXI-Ⅳ Interacting Proteins in Wheat and Validation of Its Interaction with TaHRLI-4D[J]. Biotechnology Bulletin, 2026, 42(9): 286-296.

图/表 8

参考文献 34

[1]	万育民, 乔伟. 浅析小麦赤霉病的发病原因与防治措施 [J]. 种子科技, 2012, 30(6): 45-46.
	Wan YM, Qiao W. Analysis on the causes and control measures of wheat scab [J]. Seed Sci Technol, 2012, 30(6): 45-46.
[2]	夏腾飞, 熊子君. 小麦赤霉病及其防治策略概况与展望 [J]. 分子植物育种, 2021, 19(19): 6460-6467.
	Xia TF, Xiong ZJ. Overview and perspective on Fusarium head blight in wheat and its control strategies [J]. Mol Plant Breed, 2021, 19(19): 6460-6467.
[3]	高德荣, 胡文静, 张勇, 等. 小麦抗赤霉病遗传育种研究进展及思考 [J]. 长江大学学报: 自科版, 2021, 18(5): 66-77.
	Gao DR, Hu WJ, Zhang Y, et al. Research progress and thinking on genetics and breeding of resistance to Fusarium head blight in wheat [J]. J Yangtze Univ Nat Sci Ed, 2021, 18(5): 66-77.
[4]	Tanaka S, Kahmann R. Cell wall-associated effectors of plant-colonizing fungi [J]. Mycologia, 2021, 113(2): 247-260.
[5]	Kikot GE, Hours RA, Alconada TM. Contribution of cell wall degrading enzymes to pathogenesis of Fusarium graminearum: a review [J]. J Basic Microbiol, 2009, 49(3): 231-241.
[6]	Collins T, Gerday C, Feller G. Xylanases, xylanase families and extremophilic xylanases [J]. FEMS Microbiol Rev, 2005, 29(1): 3-23.
[7]	Beliën T, Van Campenhout S, Van Acker M, et al. Cloning and characterization of two endoxylanases from the cereal phytopathogen Fusarium graminearum and their inhibition profile against endoxylanase inhibitors from wheat [J]. Biochem Biophys Res Commun, 2005, 327(2): 407-414.
[8]	Sella L, Gazzetti K, Faoro F, et al. A Fusarium graminearum xylanase expressed during wheat infection is a necrotizing factor but is not essential for virulence [J]. Plant Physiol Biochem, 2013, 64: 1-10.
[9]	Tundo S, Moscetti I, Faoro F, et al. Fusarium graminearum produces different xylanases causing host cell death that is prevented by the xylanase inhibitors XIP-I and TAXI-Ⅲ in wheat [J]. Plant Sci, 2015, 240: 161-169.
[10]	Dornez E, Croes E, Gebruers K, et al. Accumulated evidence substantiates a role for three classes of wheat xylanase inhibitors in plant defense [J]. Crit Rev Plant Sci, 2010, 29(4): 244-264.
[11]	Debyser W, Derdelinckx G, Delcour JA. Arabinoxylan solubilization and inhibition of the barley malt xylanolytic system by wheat during mashing with wheat wholemeal adjunct: evidence for a new class of enzyme inhibitors in wheat [J]. J Am Soc Brew Chem, 1997, 55(4): 153-156.
[12]	Igawa T, Ochiai-Fukuda T, Takahashi-Ando N, et al. New TAXI-type xylanase inhibitor genes are inducible by pathogens and wounding in hexaploid wheat [J]. Plant Cell Physiol, 2004, 45(10): 1347-1360.
[13]	Dornez E, Croes E, Gebruers K, et al. 2-D DIGE reveals changes in wheat xylanase inhibitor protein families due to Fusarium graminearum ΔTri5 infection and grain development [J]. Proteomics, 2010, 10(12): 2303-2319.
[14]	Moscetti I, Faoro F, Moro S, et al. The xylanase inhibitor TAXI-Ⅲ counteracts the necrotic activity of a Fusarium graminearum xylanase in vitro and in durum wheat transgenic plants [J]. Mol Plant Pathol, 2015, 16(6): 583-592.
[15]	Moscetti I, Tundo S, Janni M, et al. Constitutive expression of the xylanase inhibitor TAXI-Ⅲ delays Fusarium head blight symptoms in durum wheat transgenic plants [J]. Mol Plant Microbe Interact, 2013, 26(12): 1464-1472.
[16]	Liu Y, Han NN, Wang S, et al. Genome-wide identification of Triticum aestivum xylanase inhibitor gene family and inhibitory effects of Ⅺ-2 subfamily proteins on Fusarium graminearum GH11 xylanase [J]. Front Plant Sci, 2021, 12: 665501.
[17]	张雨佳, 刘丽, 邹龙海, 等. 毛竹PheFT12a基因过表达对拟南芥开花及芽发育的影响 [J]. 核农学报, 2023, 37(11): 2142-2150.
	Zhang YJ, Liu L, Zou LH, et al. Effects of overexpression of the moso bamboo PheFT12a on flowering and bud development of Arabidopsis [J]. J Nucl Agric Sci, 2023, 37(11): 2142-2150.
[18]	刘蓉. 细胞自噬参与拟南芥对禾谷镰刀菌侵染响应的初步研究 [D]. 广州: 华南农业大学, 2020: 11.
	Liu R. Preliminary study on autophagy involved in the response of Arabidopsis thaliana to Fusarium graminearum infection [D]. Guangzhou: South China Agricultural University, 2020: 11.
[19]	Fields S, Song O. A novel genetic system to detect protein-protein interactions [J]. Nature, 1989, 340(6230): 245-246.
[20]	王子颖, 龙晨洁, 范兆宇, 等. 利用酵母双杂交系统筛选水稻中与OsCRK5互作蛋白 [J]. 生物技术通报, 2023, 39(9): 117-125.
	Wang ZY, Long CJ, Fan ZY, et al. Screening of OsCRK5-interacted proteins in rice using yeast two-hybrid system [J]. Biotechnol Bull, 2023, 39(9): 117-125.
[21]	谢瑞莹, 曾露桂, 姜超英, 等. 烟草NtMYB4a基因酵母双杂交诱饵载体的构建及互作蛋白的筛选 [J]. 核农学报, 2022, 36(7): 1318-1328.
	Xie RY, Zeng LG, Jiang CY, et al. Construction of yeast two-hybrid bait vector and identification of proteins interacting with NtMYB4a in tobacco [J]. J Nucl Agric Sci, 2022, 36(7): 1318-1328.
[22]	Johnsson N, Varshavsky A. Split ubiquitin as a sensor of protein interactions in vivo [J]. Proc Natl Acad Sci USA, 1994, 91(22): 10340-10344.
[23]	张瑞芳, 张合红, 魏中艳, 等. 与南方水稻黑条矮缩病毒外壳蛋白P10互作的寄主因子筛选 [J]. 核农学报, 2021, 35(5): 1067-1073.
	Zhang RF, Zhang HH, Wei ZY, et al. Screening of host factors and interaction with coat protein P10 of southern rice black streak dwarf virus [J]. J Nucl Agric Sci, 2021, 35(5): 1067-1073.
[24]	侯筱媛, 车郑郑, 李姮静, 等. 大豆膜系统cDNA文库的构建及大豆疫霉效应子PsAvr3a互作蛋白的筛选 [J]. 生物技术通报, 2023, 39(4): 268-276.
	Hou XY, Che ZZ, Li HJ, et al. Construction of the soybean membrane system cDNA library and interaction proteins screening for effector PsAvr3a [J]. Biotechnol Bull, 2023, 39(4): 268-276.
[25]	Zhu F, Cao MY, Zhu PX, et al. Non-specific lipid transfer protein 1 enhances immunity against tobacco mosaic virus in Nicotiana benthamiana [J]. J Exp Bot, 2023, 74(17): 5236-5254.
[26]	de Oliveira Dias R, Dos Santos Machado L, Migliolo L, et al. Insights into animal and plant lectins with antimicrobial activities [J]. Molecules, 2015, 20(1): 519-541.
[27]	Song PW, Zhang LF, Wu LL, et al. A ricin B-like lectin protein physically interacts with TaPFT and is involved in resistance to Fusarium head blight in wheat [J]. Phytopathology, 2021, 111(12): 2309-2316.
[28]	Xu QL, Reed JC. Bax inhibitor-1, a mammalian apoptosis suppressor identified by functional screening in yeast [J]. Mol Cell, 1998, 1(3): 337-346.
[29]	Babaeizad V, Imani J, Kogel KH, et al. Over-expression of the cell death regulator BAX inhibitor-1 in barley confers reduced or enhanced susceptibility to distinct fungal pathogens [J]. Theor Appl Genet, 2009, 118(3): 455-463.
[30]	Lu PP, Yu TF, Zheng WJ, et al. The wheat bax inhibitor-1 protein interacts with an aquaporin TaPIP1 and enhances disease resistance in Arabidopsis [J]. Front Plant Sci, 2018, 9: 20.
[31]	Potters G, Horemans N, Jansen MAK. The cellular redox state in plant stress biology-A charging concept [J]. Plant Physiol Biochem, 2010, 48(5): 292-300.
[32]	闫宝琴, 陈燕桂, 史贤成, 等. 拟南芥AtTrx5增强植物菌核病的抗性研究 [J]. 中国油料作物学报, 2023, 45(6): 1109-1118.
	Yan BQ, Chen YG, Shi XC, et al. Arabidopsis thioredoxin gene (AtTrx5) enhances plant resistance to Sclerotinia [J]. Chin J Oil Crop Sci, 2023, 45(6): 1109-1118.
[33]	Park SR, Moon SJ, Shin DJ, et al. Isolation and characterization of rice OsHRL gene related to bacterial blight resistance [J]. Plant Pathol J, 2010, 26(4): 417-420.
[34]	Wu XY, Lai YC, Rao SF, et al. Genome-wide identification reveals that Nicotiana benthamiana hypersensitive response (HR)-like lesion inducing protein 4 (NbHRLI4) mediates cell death and salicylic acid-dependent defense responses to turnip mosaic virus [J]. Front Plant Sci, 2021, 12: 627315.