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

doi:10.13560/j.cnki.biotech.bull.1985.2022-0972

摘要/Abstract

摘要：

植物质膜是响应侵染的第一道屏障，其受体蛋白是感知病原菌入侵的雷达，是植物免疫系统的重要组成部分。大豆疫霉（Phytophthora sojae）通过分泌效应子干扰寄主免疫反应以促进自身侵染定殖。构建大豆膜系统酵母双杂交cDNA文库，为进一步探究大豆疫霉效应子与寄主膜蛋白靶标互作机制奠定基础。以大豆疫霉无毒效应子PsAvr3a为诱饵蛋白筛选文库，初步获得198个候选寄主靶标。酵母一对一验证试验表明，有5个候选靶标蛋白与PsAvr3a互作，包括酪氨酸分解酶、囊泡转运蛋白、乙烯合成调控酶、细胞代谢结合蛋白、抗逆的渗透蛋白等。通过双分子荧光互补试验（bimolecular fluorescence complementation, BIFC）与荧光素酶互补试验（luciferase complementation assay, LCA）验证得出GmACO、GmSec61与PsAvr3a均存在互作。确定2个寄主大豆膜蛋白乙烯前体ACC氧化酶GmACO、三聚体易位子GmSec61是大豆疫霉无毒效应子PsAvr3a的互作靶标。

关键词: 大豆, 效应子, 大豆疫霉, 分裂泛素酵母双杂交, 膜蛋白, 无毒基因

Abstract:

The plant plasma membrane is the first barrier in response to pathogen infection, and the receptor proteins on it are the radar for sensing pathogen invasion, which is an important part of the plant immune system. Phytophthora sojae disrupts the host immune activities of plants by secreting effectors, promoting Phytophthora colonization and spread. The construction of soybean split-ubiquitin based membrane yeast two-hybridization cDNA library is significant for exploring the interaction mechanisms between effectors and host cell membrane protein targets. The avirulent effector PsAvr3a was used as a bait protein to screen the library, and 198 candidate host targets were preliminarily obtained. The yeast two-hybrid technique between the interested target proteins and PsAvr3a revealed that five of them interacted with the bait vector. The enzyme that regulates tyrosine breakdown, the vesicle transport protein, the ethylene biosynthesis regulatory enzyme, the cell metabolic binding protein, the stress-resistant osmotic protein, and others were among the prospective host targets. The interactions amony GmACO, GmSec61 and PsAvr3a were verified by Bimolecular Fluorescence Complementation（BIFC）and Luciferase Complementation Assay（LCA）. Therefore, two soybean membrane proteins, ethylene precursor ACC oxidase GmACO and trimeric transposon GmSec61, were identified as the host target of Phytophthora sojae avirulent effector PsAvr3a.

Key words: soybean, effector, Phytophthora sojae, split-ubiquitin based membrane yeast two-hybridization, membrane protein, avirulence gene

侯筱媛, 车郑郑, 李姮静, 杜崇玉, 胥倩, 王群青. 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 Xiao-yuan, CHE Zheng-zheng, LI Heng-jing, DU Chong-yu, XU Qian, WANG Qun-qing. Construction of the Soybean Membrane System cDNA Library and Interaction Proteins Screening for Effector PsAvr3a[J]. Biotechnology Bulletin, 2023, 39(4): 268-276.

图/表 9

图1 pBIN-GFP2-PsAvr3a的亚细胞定位 pBIN-GFP2-PsAvr3a与pCAMBIA1300-mCherry-PsAvh241在烟草中共表达定位，比例尺：20 μm

Fig. 1 Subcelluar localization of pBIN-GFP2-PsAvr3a The pBIN-GFP2-PsAvr3a fusion protein was coexpressed with pCAMBIA1300-mCherry-PsAvh241 in N. benthamiana through agroinfiltration. Scale bar: 20 μm

图2 自激活检测与功能验证结果

Fig. 2 Results of self-activation detection and functional verification A：pNubG-Fe65 + pTSU2-APP；B：pBT3-N-APP + pPR3-N；C：pBT3-N-PsAvr3a + pOst1-NubI；D：pBT3-N-PsAvr3a + pPR3-N。100、10-1、10-2为酵母菌液稀释倍数。下同100, 10-1, 10-2 are the dilution ratio of yeast solution. The same below

表1 部分比对检索互作蛋白

Table 1 Retrieved partial interacting proteins

编号No.	基因名称Gene name	功能预测Function forecasting
1	Glycine max protein transport protein Sec61 subunit beta-like	介导信号肽依赖性蛋白质转运到内质网 Mediating signal peptide-dependent protein transport to endoplasmic reticulum
2	Glycine soja ER membrane protein complex subunit 4-like	参与eIF2B 介导的翻译Participating in eIF2B-mediated translation
3	Glycine max bax inhibitor 1-like	凋亡抑制基因Apoptotic inhibitor gene
4	Glycine max peroxisomal membrane protein 11	过氧化物酶体膜蛋白Peroxisomal membrane protein
5	Glycine max 1-aminocyclopropane-1-carboxylate oxidase	参与乙烯生物合成Participating in ethylene biosynthesis
6	Glycine max zinc finger CCCH domain-containing protein 23	DNA结合蛋白DNA binding protein
7	Glycine soja RING-H2 finger protein ATL8-like	泛素蛋白连接酶Ubiquitin-protein ligase enzyme
8	Glycine max SNF1-related protein kinase regulatory subunit beta-1	参与种子内部碳水化合物代谢和储藏物质积累 Involving in carbohydrate metabolism and storage material accumulation in seeds
9	Glycine max 4-hydroxyphenylpyruvate dioxygenase	α-酮酸依赖性加氧酶，催化酪氨酸分解代谢 α-ketoacid-dependent oxygenase, catalyzing tyrosine catabolism
10	Glycine max wound-induced protein	创伤诱导蛋白Wound-induced protein
11	Glycine max formin-like protein 20	钙依赖性脂质结合域蛋白Calcium-dependent lipid-binding domain protein
12	Glycine max osmotin-like protein, acidic（OLPa）	具有抗逆功能的渗透蛋白The stress-resistant osmotic protein
13	Glycine max probable enoyl-CoA hydratase 1	烯酰辅酶A水合酶Enoyl-CoA hydratase

图3 酵母双杂交验证结果

Fig. 3 Results of yeast two-hybrid verification A: pPR3-N-GmOsmotin + pBT3-N-PsAvr3a; B: pPR3-N-GmSNF1 + pBT3-N-PsAvr3a; C: pPR3-N-GmHPPD + pBT3-N-PsAvr3a; D: pPR3-N-GmSec61 + pBT3-N-PsAvr3a; E: pPR3-N-GmACO + pBT3-N-PsAvr3a; F: pNubG-Fe65 + pTSU2-APP; G: pBT3-N-APP + pPR3-N

图4 利用BiFC方法在烟草细胞中验证蛋白互作双分子荧光互补PsAvr3a和GmACO、GmSec61在烟草叶片中表达，通过共聚焦显微镜观察YFP荧光信号，比例尺：20 μm

Fig. 4 Verifying protein interacting results in tobacco cells using the BiFC method BiFC of PsAvr3a and GmACO, GmSec61 in transiently transformed N. benthamiana leaves. The YFP fluorescent signal was observed using confocal microscopy. Scale bar: 20 μm

图5 利用荧光素酶互补在烟草中验证蛋白互作试验

Fig. 5 Verifying protein interacting results in tobacco using LCA A: p1300-35S-cLUC-PsAvr3a/p1300-35S-Nluc; B: p1300-35S-cLUC/p1300-35S-nLUC-GmACO; C: p1300-35S-cLUC /p1300-35S-nLUC-GmSec61; D: p1300-35S-cLUC-PsAvr3a/p1300-35S-nLUC-GmACO; E: p1300-35S-cLUC-PsAvr3a/p1300-35S-nLUC-GmSec61

图6 GmACO蛋白的TMHMM跨膜域预测

Fig. 6 Transmembrane domain prediction of GmACO using TMHMM

图7 GmSec61蛋白的TMHMM跨膜域预测

Fig. 7 Transmembrane domain prediction of GmSec61 using TMHMM

图8 GmACO、GmSec61的亚细胞定位农杆菌注射表达后GmACO、GmSec61和空载体的亚细胞定位，通过共聚焦显微镜观察GFP荧光信号，比例尺：20 μm

Fig. 8 Subcellular localization of GmACO and GmSec61 Subcellular localization of GmACO, GmSec61 or empty vector after agroinfiltration. The GFP signal was observed using confocal microscopy. Scale bar: 20 μm

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