利用酵母双杂交系统筛选与大豆花叶病毒核内含体蛋白互作的大豆寄主因子

doi:10.3864/j.issn.0578-1752.2025.19.001

Abstract

Abstract:

【Objective】Soybean mosaic virus (SMV) is one of the most damaging viral diseases of soybean, which seriously affects soybean yield and quality. Identification of host proteins interacting with SMV nuclear inclusion proteins (NIa-Pro and NIb) using yeast two-hybrid library screening, aiming to establish a theoretical foundation and propose novel perspectives insights into the molecular mechanisms of SMV infection and soybean resistance.【Method】Firstly, the coding sequences of NIa-Pro and NIb were cloned from the SMV strain SMV-HN and recombined into the pGBKT7 vector to construct the bait plasmids, and then soybean proteins interacting with the two viral functional proteins were identified by yeast library screening. Secondly, the host gene GmOEP16 encoding Outer Envelope Pore Protein 16 (OEP16) was cloned, and the interactions of GmOEP16 with NIa-Pro and NIb were clarified by yeast two-hybrid (Y2H) and luciferase complementation assay (LCA). Quantitative real-time PCR (qRT-PCR) was used to analyse the expression pattern of GmOEP16 under SMV treatment and exogenous hormone induction. Finally, virus-induced gene silencing (VIGS) was used to validate the function of GmOEP16 gene in SMV disease response.【Result】pGBKT7-NIa-Pro and pGBKT7-NIb recombinant plasmids were successfully constructed, and 12 soybean host proteins were screened for interactions with NIa-Pro and NIb, respectively. The Y2H assay was further used to verify that NIa-Pro interacted with GmOEP16 and GmDEG5, and NIb interacted with GmOEP16, GmZC3H18 and GmAHP1. The LCA assay was further used to clarify that GmOEP16 interacted with both NIa-Pro and NIb. Expression analysis revealed that GmOEP16 was induced by SMV infection and responded rapidly to salicylic acid (SA) and abscisic acid (ABA) stimuli during early response. The VIGS assay showed that effectively silencing of GmOEP16 resulted in no obvious susceptibility phenotype in leaf tissues relative to the wild-type controls. Meanwhile, the expression of SMV-CP was significantly reduced in the GmOEP16-silenced plants, suggesting that the soybean resistance to SMV was enhanced. Collectively, these findings demonstrated that GmOEP16 could function as a negative regulator of SMV resistance in soybean.【Conclusion】The pGBKT7-NIa-Pro and pGBKT7-NIb bait vectors were successfully constructed, and each 12 soybean host proteins that respectively interacted with pGBKT7-NIa-Pro and pGBKT7-NIb were identified. Among them, GmOEP16 interacted with both NIa-Pro and NIb. GmOEP16 responded to SMV induction and negatively regulated SMV resistance, which promoted SMV infection on soybeans.

Key words: soybean mosaic virus, nuclear inclusion proteins, yeast library screening, outer envelope pore protein 16, virus-induced gene silencing

YU Zhe, ZHOU FangXue, LIU RunFa, TIAN YaQi, JIHAO MuHa, WANG YongXiang, FENG WenMi, MOU KeXin, JING Yan, LI HaiYan. Screening for Soybean Host Factors that Interact with Soybean Mosaic Virus Nuclear Inclusion Proteins Using the Yeast Two-Hybrid System[J].Scientia Agricultura Sinica, 2025, 58(19): 3799-3813.

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Figures/Tables 10

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Table 1

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Table 2

Fig. 3

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Table 3

Partial cis-acting elements in the promoter sequence of the GmOEP16"

元件 Element	序列 Sequences	作用 Function
HD-Zip 1	TAAT	参与叶绿体中层细胞分化的元素 Element involved in differentiation of the palisade mesophyll cells
ARE	ATTG	厌氧诱导所必需的顺式作用调控元件 Cis-acting regulatory element essential for the anaerobic induction
TCT-motif	TCTTAC	光敏元件的一部分 Part of a light responsive element
I-box	TATAATAAT	光敏元件的一部分 Part of a light responsive element
AE-box	AGAAACTT	光响应模块的一部分 Part of a module for light response
Gap-box	CAAATGAA(A/G)A	光敏元件的一部分 Part of a light responsive element
TATA-box	TATA(A/T)A(A/T)	转录起点-30附近的核心启动子元件 Core promoter element around -30 of transcription start
GATA-motif	AAGGATAAGG	光敏元件的一部分 Part of a light responsive element
TC-rich repeats	ATTCTCTAAC	参与防御和应激反应的顺式作用元件 Cis-acting element involved in defense and stress responsiveness
ABRE	CGCACGTGTC	参与脱落酸反应的顺式作用元件 Cis-acting element involved in the abscisic acid responsiveness
AT1-motif	AATTATTTTTTATT	光响应模块的一部分 Part of a light responsive module
TCA-element	CCATCTTTTT	参与水杨酸反应的顺式作用元件 Cis-acting element involved in salicylic acid responsiveness
GCN4_motif	TGAGTCA	参与胚乳表达的顺式调控元件 Cis-regulatory element involved in endosperm expression
MBS	CAACTG	涉及干旱诱导性的MYB结合位点 MYB binding site involved in drought-inducibility
Box 4	ATTAAT	参与光响应的保守DNA模块的一部分 Part of a conserved DNA module involved in light responsiveness
CAAT-box	CAAT	启动子和增强子区域的共同顺式作用元件 Common cis-acting element in promoter and enhancer regions
MRE	AACCTAA	参与光响应的MYB结合位点 MYB binding site involved in light responsiveness

Table 3

Fig. 6

Fig. 7

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1	Glyma.06G080100	1	肌动蛋白纤维包被蛋白——原肌球蛋白 Actin filament-coating protein tropomyosin
2	Glyma.02G247800	1	富丝氨酸/精氨酸剪接因子 Serine/arginine rich splicing factor
3	Glyma.11G072700	2	含FG-GAP重复蛋白 FG-GAP repeat-containing protein
4	Glyma.13G219400	3	叶绿体类蛋白酶5 Protease do-like 5, chloroplastic
5	Glyma.02G184800	5	外膜孔蛋白16-1，叶绿体 Outer envelope pore protein 16-1, chloroplastic
6	Glyma.05G225000	1	线粒体加工肽酶/加工增强肽酶 Mitochondrial processing peptidase/Processing enhancing peptidase
7	Glyma.13G162800	1	无机二磷酸酶/焦磷酸磷酸水解酶 Inorganic diphosphatase/Pyrophosphate phosphohydrolase
8	Glyma.07G034800	2	亚油酸13S-脂氧合酶/亚油酸9S-脂氧合酶/亚油酸9-脂氧合酶 Linoleate 13S-lipoxygenase/lipoxidase//linoleate 9s-lipoxygenase/linoleate 9-lipoxygenase
9	Glyma.06G118000	2	蓝藻和叶绿体NDH-1亚基M（NdhM） Cyanobacterial and plastid NDH-1 subunit M (NdhM)
10	Glyma.02G002000	1	甲基-6-植烷基-1,4-羟醌甲基转移酶，叶绿体 Methyl-6-phytyl-1,4-hydroquinone methyltransferase, chloroplastic
11	Glyma.09G010700	1	溶质运载家族成员13 Solute carrier family 13 member
12	Glyma.04G166700	1	肽脯氨酰顺反异构酶 Peptidyl-prolyl cis-trans isomerase

编号 Number	基因号 Gene identifier	克隆数量 No. of clones	基因描述 Gene description
1	Glyma.17G159000	1	生长因子蛋白相关 Growth factor protein-related
2	Glyma.15G275600	1	光系统Ⅱ5 kDa蛋白，叶绿体 PhotosystemⅡ 5 kDa protein, chloroplastic
3	Glyma.11G102300	2	含FG-GAP重复蛋白 FG-GAP repeat-containing protein
4	Glyma.18G002800	1	硒结合蛋白 Selenium-binding protein
5	Glyma.06G172000	1	肽酶Ⅰ锌金属蛋白酶（M18） AminopeptidaseⅠ zinc metalloprotease (M18)
6	Glyma.05G135300	1	液泡磷酸合成酶亚基f Vacuolar ATP synthase subunit f
7	Glyma.13G352100	2	60S核糖体蛋白L28 60S ribosomal protein L28
8	Glyma.05G216000	3	含锌指CCCH结构域的蛋白18 Zinc finger CCCH domain-containing protein 18
9	Glyma.11G140500	1	RNA聚合酶Ⅱ转录亚基37e相关介质 Mediator of rna polymeraseⅡ transcription subunit 37e-related
10	Glyma.09G008100	1	叶绿体相关质体脂质相关蛋白3 Plastid-lipid-associated protein 3, chloroplastic- related
11	Glyma.02G184800	4	外膜孔蛋白16-1，叶绿体 Outer envelope pore protein 16-1, chloroplastic
12	Glyma.15G099800	3	含组氨酸磷酸转移蛋白1 Histidine-containing phosphotransfer protein1

Screening for Soybean Host Factors that Interact with Soybean Mosaic Virus Nuclear Inclusion Proteins Using the Yeast Two-Hybrid System

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