ACLSV外壳蛋白与梨两种E3泛素连接酶互作及亚细胞定位

doi:10.16420/j.issn.0513-353x.2023-0967

摘要/Abstract

摘要：

为深入了解苹果褪绿叶斑病毒（apple chlorotic leaf spot virus，ACLSV）与寄主植物的互作特性，构建了表达该病毒外壳蛋白（coat protein，CP）的诱饵载体pGBKT7-CP，采用酵母双杂交技术，从‘红香酥’梨cDNA文库中筛选出与ACLSV CP潜在互作的33种寄主因子。酵母双杂交和双分子荧光互补试验验证了CP与来自梨的两种RING型E3泛素连接酶（命名为MIEL和BOI）互作。亚细胞定位分析表明，CP与BOI具有相似的胞间连丝和细胞核分布特点，MIEL主要分布在内质网和细胞核。双分子荧光互补试验结果显示，CP与BOI互作不改变二者的亚细胞分布，而CP与MIEL互作信号分布与MIEL亚细胞定位相似。构建了这3种蛋白的截短突变体，发现含zf-CHY结构域（1 ~ 99 aa）的MIEL截短突变体可以与CP互作，而仅全长BOI可以与CP互作，CP截短突变体不能与这两种寄主蛋白互作。

关键词: 苹果褪绿叶斑病毒, 外壳蛋白, 酵母双杂交, E3泛素连接酶, 蛋白互作, 亚细胞定位

Abstract:

Apple chlorotic leaf spot virus（ACLSV）possesses a positive single stranded RNA and widely infects pear and other fruit trees. The coat protein（CP）of the virus functions in the viral pathogenesis. In order to gain a deep understanding of the interaction characteristics between the virus and its host plants，a yeast two hybrid（Y2H）assay was carried out by using pGBKT7-CP（expressing ACLSV CP）as a bait vector to screen pear proteins in a cDNA library of pear‘Hongxiangsu’，and 33 host factors potentially interacting with ACLSV CP were identified. Furthermore，the interaction relationships between CP and two RING type E3 ubiquitin ligases（named MIEL and BOI）from pear were confirmed by both Y2H and bimolecular fluorescence complementation（BiFC）assays. In BiFC assays，the interaction signal of CP with BOI located at plasmodesma and nucleus，which was similar to the subcellular locations of the two proteins. The interaction signal of CP with MIEL located at endoplasmic reticulum and nucleus，which was similar to the subcellular locations of MIEL. The truncated mutants of the three proteins were constructed. In Y2H assays，only MIEL mutant containing the zf CHY domain（1-99 aa）interacted with CP，and other MIEL mutants and all BOI mutants failed to interact with CP，and all CP mutants cannot interact with the two host proteins.

Key words: apple chlorotic leaf spot virus, coat protein, yeast two-hybrid, E3 ubiquitin ligase, protein-protein interaction, subcellular localization

邱辉, 朱德娟, 张永乐, 高玉洁, 李柳, 王国平, 洪霓. ACLSV外壳蛋白与梨两种E3泛素连接酶互作及亚细胞定位[J]. 园艺学报, 2024, 51(8): 1715-1727.

QIU Hui, ZHU Dejuan, ZHANG Yongle, GAO Yujie, LI Liu, WANG Guoping, HONG Ni. Interaction Between the Coat Protein of Apple Chlorotic Leaf Spot Virus and Two E3 Ubiquitin Ligases of Pear and Their Subcellular Localization[J]. Acta Horticulturae Sinica, 2024, 51(8): 1715-1727.

图/表 8

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分析方法 Assay	基因 Gene	引物（F/R，5′-3′） Primer
酵母双杂交	CP	GGAATTC CATATGATGGCAGCAGTTCTGAATCT/CCG GAATTCGATGCAAAGATCAGT
Y2H	MIEL	GGAATTC CATATGATGGAAGGCTCAGCCAATGAA/CCG GAATTCTTGAGGAAGAACCGGAGGGGC
	BOI	GGAATTC CATATGATGGCTCTTCCCCGGCACCAT/CCG GAATTCCACATATACCTCCATGCCGAT
亚细胞定位	CP	CTAG TCTAGAATGGCAGCAGTTCTGAATCT/ACGC GTCGACGATGCAAAGATCAGTTGAGAC
Localization	MIEL	CTAG TCTAGAATGGAAGGCTCAGCCAATGAA/ACGC GTCGACTTGAGGAAGAACCGGAGGGGC
	BOI	CTAG TCTAGAATGGCTCTTCCCCGGCACCAT/ACGC GTCGACCACATATACCTCCATGCCGAT

分析方法 Assay	基因 Gene	引物（F/R，5′-3′） Primer
酵母双杂交	CP	GGAATTC CATATGATGGCAGCAGTTCTGAATCT/CCG GAATTCGATGCAAAGATCAGT
Y2H	MIEL	GGAATTC CATATGATGGAAGGCTCAGCCAATGAA/CCG GAATTCTTGAGGAAGAACCGGAGGGGC
	BOI	GGAATTC CATATGATGGCTCTTCCCCGGCACCAT/CCG GAATTCCACATATACCTCCATGCCGAT
亚细胞定位	CP	CTAG TCTAGAATGGCAGCAGTTCTGAATCT/ACGC GTCGACGATGCAAAGATCAGTTGAGAC
Localization	MIEL	CTAG TCTAGAATGGAAGGCTCAGCCAATGAA/ACGC GTCGACTTGAGGAAGAACCGGAGGGGC
	BOI	CTAG TCTAGAATGGCTCTTCCCCGGCACCAT/ACGC GTCGACCACATATACCTCCATGCCGAT

候选梨蛋白 Candidate protein	登录号 Accession No.	一致性/% Identity	克隆数 Clone No.	大小/bp Size
BOI相关泛素蛋白连接酶2 Probable BOI-related E3 ubiquitin-protein ligase 2	XM_009361505.2	93.00	18	1 026
ATP依赖的Clp蛋白酶蛋白水解亚基6，叶绿体 ATP-dependent Clp protease proteolytic subunit 6，chloroplastic-like	XM_009351165.2	98.58	10	855
果糖二磷酸醛缩酶1，胞质型 Fructose-bisphosphate aldolase 1，cytoplasmic-like	XM_009354717.2	98.08	5	1 077
核酮糖二磷酸羧化酶/加氧酶激活酶，叶绿体Ribulose bisphosphate carboxylase/oxygenase activase，chloroplastic-like	XM_009345517.2	99.55	4	1 617
E3泛素蛋白连接酶MIEL1 E3 ubiquitin-protein ligase MIEL1	XM_018650219.1	98.93	4	804
假定E3泛素-蛋白连接酶UBR7 Putative E3 ubiquitin-protein ligase UBR7	XM_009360328.2	93.90	1	1 239
核酮糖二磷酸羧化酶/加氧酶激活酶，叶绿体 Ribulose bisphosphate carboxylase/oxygenase activase，chloroplastic-like	XM_009366078.2	99.89	4	1 419
天冬氨酸蛋白酶A1-like Aspartic proteinase A1-like	XM_009355023.1	94.50	4	1 533
类甜蛋白1a Thaumatin-like protein 1a	XM_009345430.2	98.29	2	747
LHCⅡ1型叶绿素ab结合蛋白 Chlorophyll a-b binding protein of LHCⅡ type 1	XM_018648424.1	96.50	1	795
叶绿素ab结合蛋白CP29.1 Chlorophyll a-b binding protein CP29.1	XM_009379505.2	91.96	1	861
克隆1118ab结合蛋白mRNA Clone 1118 a-b binding protein mRNA	KJ008964.1	98.41	1	816
半胱氨酸蛋白酶ATG4-like Cysteine protease ATG4-like	XM_009356387.2	97.54	1	1 464
半胱氨酸合成酶 Cysteine synthase	XM_009366277.2	92.62	1	978
F-box/kelch-repeat蛋白At1g67480 F-box/kelch-repeat protein At1g67480	XM_009374031.2	98.47	1	1 185
LEC14B同源物 LEC14B homolog	XM_009353905.2	97.34	1	1 425
金属烟酰胺转运蛋白YSL2 Metal-nicotianamine transporter YSL2	XM_018648503.1	97.22	1	1 998
甲基酯酶10 Methylesterase 10	XM_009368119.2	99.89	1	825
丝裂原活化蛋白激酶13 Mitogen-activated protein kinase 13	XM_009356240.2	98.04	1	1 104
磷脂酰肌醇4-激酶γ7 Phosphatidylinositol 4-kinase gamma 7	XM_009357275.2	97.77	1	1 926
可能无活性的紫色酸性磷酸酶27 Probable inactive purple acid phosphatase 27	XM_009370253.2	98.40	1	1 923
锌指转录因子STOP1 Protein SENSITIVE TO PROTON RHIZOTOXICITY 1-like	XM_018645092.1	99.55	1	972
假定tRNA（胞苷(32)/鸟苷(34)-2′-O）-甲基转移酶 Putative tRNA (cytidine(32)/guanosine(34)-2′-O)-methyltransferase	XM_009371927.2	99.25	1	951
醌氧化还原酶PIG3 Quinone oxidoreductase PIG3	XM_009379085.2	97.44	1	978
40S核糖体蛋白S3-3 40S ribosomal protein S3-3	XM_009364296.2	98.99	1	717
60S核糖体蛋白L7-2样 60S ribosomal protein L7-2-like	XM_009347721.2	99.14	1	732
富含丝氨酸/精氨酸的剪接因子RS31 Serine/arginine-rich splicing factor RS31	XM_009377158.2	97.58	1	777
茎特异性蛋白TSJT1 Stem-specific protein TSJT1	XM_009365918.2	87.93	1	711
通用应激蛋白PHOS32-like Universal stress protein PHOS32-like	XM_009343181.2	99.34	1	693
尿卟啉原-ⅢC-甲基化转移酶 Uroporphyrinogen-III C-methyltransferase-like	XM_009356405.2	99.16	1	1 140
未知蛋白LOC103938811 Uncharacterized LOC103938811	XM_009348858.2	95.47	1	489
未知蛋白LOC103952636 Uncharacterized LOC103952636	XM_009364273.2	97.54	1	1 170
未知蛋白LOC103958759 Uncharacterized LOC103958759	XM_009371077.2	97.74	1	606

候选梨蛋白 Candidate protein	登录号 Accession No.	一致性/% Identity	克隆数 Clone No.	大小/bp Size
BOI相关泛素蛋白连接酶2 Probable BOI-related E3 ubiquitin-protein ligase 2	XM_009361505.2	93.00	18	1 026
ATP依赖的Clp蛋白酶蛋白水解亚基6，叶绿体 ATP-dependent Clp protease proteolytic subunit 6，chloroplastic-like	XM_009351165.2	98.58	10	855
果糖二磷酸醛缩酶1，胞质型 Fructose-bisphosphate aldolase 1，cytoplasmic-like	XM_009354717.2	98.08	5	1 077
核酮糖二磷酸羧化酶/加氧酶激活酶，叶绿体Ribulose bisphosphate carboxylase/oxygenase activase，chloroplastic-like	XM_009345517.2	99.55	4	1 617
E3泛素蛋白连接酶MIEL1 E3 ubiquitin-protein ligase MIEL1	XM_018650219.1	98.93	4	804
假定E3泛素-蛋白连接酶UBR7 Putative E3 ubiquitin-protein ligase UBR7	XM_009360328.2	93.90	1	1 239
核酮糖二磷酸羧化酶/加氧酶激活酶，叶绿体 Ribulose bisphosphate carboxylase/oxygenase activase，chloroplastic-like	XM_009366078.2	99.89	4	1 419
天冬氨酸蛋白酶A1-like Aspartic proteinase A1-like	XM_009355023.1	94.50	4	1 533
类甜蛋白1a Thaumatin-like protein 1a	XM_009345430.2	98.29	2	747
LHCⅡ1型叶绿素ab结合蛋白 Chlorophyll a-b binding protein of LHCⅡ type 1	XM_018648424.1	96.50	1	795
叶绿素ab结合蛋白CP29.1 Chlorophyll a-b binding protein CP29.1	XM_009379505.2	91.96	1	861
克隆1118ab结合蛋白mRNA Clone 1118 a-b binding protein mRNA	KJ008964.1	98.41	1	816
半胱氨酸蛋白酶ATG4-like Cysteine protease ATG4-like	XM_009356387.2	97.54	1	1 464
半胱氨酸合成酶 Cysteine synthase	XM_009366277.2	92.62	1	978
F-box/kelch-repeat蛋白At1g67480 F-box/kelch-repeat protein At1g67480	XM_009374031.2	98.47	1	1 185
LEC14B同源物 LEC14B homolog	XM_009353905.2	97.34	1	1 425
金属烟酰胺转运蛋白YSL2 Metal-nicotianamine transporter YSL2	XM_018648503.1	97.22	1	1 998
甲基酯酶10 Methylesterase 10	XM_009368119.2	99.89	1	825
丝裂原活化蛋白激酶13 Mitogen-activated protein kinase 13	XM_009356240.2	98.04	1	1 104
磷脂酰肌醇4-激酶γ7 Phosphatidylinositol 4-kinase gamma 7	XM_009357275.2	97.77	1	1 926
可能无活性的紫色酸性磷酸酶27 Probable inactive purple acid phosphatase 27	XM_009370253.2	98.40	1	1 923
锌指转录因子STOP1 Protein SENSITIVE TO PROTON RHIZOTOXICITY 1-like	XM_018645092.1	99.55	1	972
假定tRNA（胞苷(32)/鸟苷(34)-2′-O）-甲基转移酶 Putative tRNA (cytidine(32)/guanosine(34)-2′-O)-methyltransferase	XM_009371927.2	99.25	1	951
醌氧化还原酶PIG3 Quinone oxidoreductase PIG3	XM_009379085.2	97.44	1	978
40S核糖体蛋白S3-3 40S ribosomal protein S3-3	XM_009364296.2	98.99	1	717
60S核糖体蛋白L7-2样 60S ribosomal protein L7-2-like	XM_009347721.2	99.14	1	732
富含丝氨酸/精氨酸的剪接因子RS31 Serine/arginine-rich splicing factor RS31	XM_009377158.2	97.58	1	777
茎特异性蛋白TSJT1 Stem-specific protein TSJT1	XM_009365918.2	87.93	1	711
通用应激蛋白PHOS32-like Universal stress protein PHOS32-like	XM_009343181.2	99.34	1	693
尿卟啉原-ⅢC-甲基化转移酶 Uroporphyrinogen-III C-methyltransferase-like	XM_009356405.2	99.16	1	1 140
未知蛋白LOC103938811 Uncharacterized LOC103938811	XM_009348858.2	95.47	1	489
未知蛋白LOC103952636 Uncharacterized LOC103952636	XM_009364273.2	97.54	1	1 170
未知蛋白LOC103958759 Uncharacterized LOC103958759	XM_009371077.2	97.74	1	606