致病疫霉效应蛋白Pi07555功能研究及其寄主靶标筛选

doi:10.13560/j.cnki.biotech.bull.1985.2026-0041

摘要/Abstract

摘要：

目的明确致病疫霉（Phytophthora infestans）效应蛋白Pi07555的生物学功能及其寄主靶标蛋白。方法利用SignalP 6.0网站和酵母转化酶分泌系统检测效应蛋白Pi07555信号肽的分泌活性；联合实时荧光定量PCR、农杆菌介导基因表达和离体叶片接种分析Pi07555基因毒性功能；通过酵母双杂交、荧光素酶互补成像实验及病毒诱导基因沉默技术鉴定Pi07555寄主靶标蛋白及其编码基因的功能。结果 Pi07555基因编码170个氨基酸，N-端信号肽具有分泌活性；Pi07555基因在致病疫霉侵染早期上调表达且定位在植物细胞核、细胞质和细胞膜中；瞬时表达Pi07555促进致病疫霉侵染但不抑制BAX、INF1介导的过敏性细胞死亡；通过酵母双杂交与荧光素酶互补成像实验证实马铃薯过敏性诱导反应蛋白1（StHIR1）与Pi07555发生互作；StHIR1基因在致病疫霉侵染48 h后上调表达，沉默其同源基因显著提高了本氏烟对致病疫霉的抗性。结论 Pi07555是一个定位于植物细胞核、细胞质和细胞膜，且具有分泌功能的毒性效应蛋白。

关键词: 马铃薯, 致病疫霉, 效应蛋白, Pi07555, 蛋白互作

Abstract:

Objective To elucidate the biological functions and host target proteins of the effector Pi07555 from Phytophthora infestans. Methods The secretory activity of the signal peptide of effector Pi07555 was verified using the SignalP 6.0 online server and the yeast invertase secretion system. The virulence function of the Pi07555 gene was analyzed via combined assays including quantitative real-time PCR (RT-qPCR), Agrobacterium-mediated transient gene expression, and in vitro leaf inoculation. The host target protein of Pi07555 and the function of its encoding gene were identified through yeast two-hybrid (Y2H) assay, luciferase complementation imaging (LCI) assay, and virus-induced gene silencing (VIGS) technology. Results The Pi07555 gene encodes 170 amino acids, and the N-terminal signal peptide has secretory activity. The Pi07555 gene is upregulated at the early stage of pathogenic Phytophthora infection and is localized to the plant nucleus, cytoplasm, and cell membrane. The transient expression of Pi07555 promotes pathogenic Phytophthora infection but does not inhibit BAX and INF1-mediated hypersensitive cell death. The yeast two-hybrid and luciferase complementation imaging assays demonstrated that potato hypersensitive-induced reaction 1 (StHIR1) interacts with Pi07555. The StHIR1 gene is upregulated 48 hours after pathogenic Phytophthora infection, and silencing its homolog significantly increased the resistance of Nicotiana benthamiana to pathogenic Phytophthora. Conclusion Pi07555 is a secreted virulence effector that localizes to the plant nucleus, cytoplasm, and plasma membrane.

Key words: potato, Phytophthora infestans, effector, Pi07555, protein interaction

王荟洁, 刀文静, 张贝妮, 付艳鸿, 黄娅楠, 王洪洋. 致病疫霉效应蛋白Pi07555功能研究及其寄主靶标筛选[J]. 生物技术通报, 2026, 42(6): 139-148.

WANG Hui-jie, DAO Wen-jing, ZHANG Bei-ni, FU Yan-hong, HUANG ya-nan, WANG Hong-yang. Functional Characterization of Effector Protein Pi07555 from Phytophthora infestans and Screening for Its Host Targets[J]. Biotechnology Bulletin, 2026, 42(6): 139-148.

图/表 9

表1 候选靶标基因测序比对结果

Table 1 Sequence alignment results of candidate target genes

Clone ID	Gene ID	Gene annotation
AD-12	XM_006364913.2	Solanum tuberosum transcription factor bHLH93-like， StbHLH93
AD-39	NM_001318700.1	Solanum tuberosum glucan endo-1，3-beta-glucosidase， StGLU
AD-51	XM_006367576.2	Solanum tuberosum 21 kD protein-like， St21kDa
AD-109	XM_006354735.2	Solanum tuberosum hypersensitive-induced response protein 1， StHIR1

图1 效应蛋白Pi07555的信号肽分泌功能验证阴性对照：pSUC2和YTK12菌株；阳性对照：pSUC2-PsAvr1b；实验组：pSUC2-Pi07555

Fig. 1 Validation of the signal peptide secretory function of effector Pi07555Negative control: pSUC2 and YTK12 strains; positive control: pSUC2-PsAvr1b; experimental group: pSUC2-Pi07555

图2 效应蛋白Pi07555促进病原菌定殖A：Pi07555在P. infestans侵染马铃薯‘合作88’过程中的动态表达分析；B：瞬时表达Pi07555后接种P. infestans 6 d 的本氏烟叶片表型；C：瞬时表达Pi07555后接种P. infestans 6 d的本氏烟病斑面积统计，**P0.01

Fig. 2 Effector Pi07555 promotes pathogen colonizationA: Expression dynamic of Pi07555 in potato ‘Cooperation-88’ during P. infestans infection; B: phenotypes of N. benthamiana leaves at 6 days post-inoculation (dpi) with P. infestans after transient expression of Pi07555; C: lesion areas of N. benthamiana leaves at 6 dpi with P. infestans after transient expression of Pi07555. ** P0.01

图3 效应蛋白Pi07555抑制细胞死亡分析A、B：Pi07555对INF1的免疫抑制功能分析；C、D：Pi07555对BAX的免疫抑制功能分析；阳性对照：INF1/BAX；阴性对照：INF1/BAX+GFP；实验组：Pi07555、INF1/BAX+Pi07555

Fig. 3 Analysis of cell death suppression by effector Pi07555A and B: Analysis of the immunosuppressive function of Pi07555 on INF1; C and D: Analysis of the immunosuppressive function of Pi07555 on BAX; positive control: INF1/BAX; negative control: INF1/BAX+GFP; experimental groups: Pi07555 alone, and INF1/BAX+Pi07555

图4 效应蛋白Pi07555的亚细胞定位及Western blot分析Merge：重叠场；GFP：绿色荧光场；Bright：明场；DAPI：蓝色荧光场；FM4-64：红色荧光场。标尺=50 μm。CBB staining代表考马斯亮蓝染色；*代表目标蛋白

Fig. 4 Subcellular localization and Western blot analysis of effector Pi07555Merge: Merged field; GFP: Green fluorescence field; Bright: Bright field; DAPI: Blue fluorescent field; FM4-64: Red fluorescent field. Scale bar=50 μm; CBB staining represents Coomassie brilliant blue staining; * represents the target protein

图5 Bait蛋白Pi07555毒性验证与自激活检测A：毒性验证；B：自激活验证

Fig. 5 Toxicity and autoactivation assays of the bait protein Pi07555A: Toxicity test; B: transcriptional autoactivation test

图6 效应蛋白Pi07555互作蛋白筛选

Fig. 6 Screening for interacting proteins of effector Pi07555

图7 荧光素酶互补成像实验验证候选蛋白互作

Fig. 7 Verification of candidate protein interactions by luciferase complementation imaging assay

图8 HIR1基因表达模式与功能分析A：StHIR1在P. infestans侵染马铃薯‘合作88’过程中的表达模式；B：NbHIR1的沉默效率检测；C：沉默NbHIR1后接种88069的本氏烟发病表型；D：沉默NbHIR1后接种88069的本氏烟病斑面积统计。*和**分别表示在P0.05和P0.01水平下存在显著差异

Fig. 8 Expression pattern and functional analysis of the HIR1 geneA: Expression pattern of StHIR1 in potato ‘Cooperation 88’ during P. infestans infection; B: silencing efficiency assay for NbHIR1; C: disease phenotypes of NbHIR1-silenced N. benthamiana after inoculation with strain 88069; D: quantification of lesion areas on NbHIR1-silenced N. benthamiana inoculated with strain 88069. * and ** indicate significant differences at P0.05 and P0.01, respectively

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