利用酵母双杂交系统筛选与柑橘黄化脉明病毒CP互作的寄主因子

doi:10.3864/j.issn.0578-1752.2023.10.006

Abstract

Abstract:

【Objective】Citrus yellow vein clearing virus (CYVCV) is one of the viruses mostly threatening the stable development of citrus industry in China, but its infection and pathogenic mechanism in citrus is still unclear. In this study, the coat protein (CP) of CYVCV was used as a bait to screen the Eureka lemon (Citrus limon Burm. f.) cDNA library, and the function of obtained host factors in the interaction between host and virus was analyzed by bioinformatics method.【Method】The total RNA of Eureka lemon leaves was extracted by the Trizol method, and then reversely transcribed to the First-Stand cDNA with SMART method, which was used as a template for obtaining ds cDNA through Long-Distance PCR. After homogenization, the ds cDNA fragments were ligated to pGADT7 plasmid vector by recombination junctions to construct the primary cDNA library of Eureka lemon. The recombinant plasmids were transfected into Escherichia coli DH10B to obtain the E. coli cDNA library of Eureka lemon, and its quality was identified. Simultaneously, the CP sequence of CYVCV was amplified by PCR and ligated into the yeast two-hybrid (Y2H) bait vector pGBKT7, and the plasmids of pGBKT7-CP and pGADT7 were co-transfected into yeast Y2H Gold. The positive yeast clones were grown on the plate of SD/-Trp, SD/-Leu-Trp, SD/-Leu-Trp/X-α-Gal and SD/-Leu-Trp-His medium, respectively, and then the growth situation of the yeast was tested to identify the toxicity of pGBKT7-CP on yeast Y2H Gold and the self-activating effect of pGBKT7-CP on the reporter gene of yeast was analyzed. Then the Y2H Gold containing bait vector pGBKT7-CP was transformed with the primary cDNA library of Eureka lemon, the co-transformed yeasts were coated on the plate of SD/-Leu-Trp, SD/-Leu-Trp-His/X-α-Gal and SD/-Leu-Trp-His-Ade/X-α-Gal medium in turn. Finally, the blue and well grown positive clones were selected. The plasmids of positive yeast clones were extracted and sequenced. The candidate genes were preliminarily compared in the GenBank, and the interacted protein factors were annotated and the protein’s biological functions were analyzed with gene ontology (GO) pathway of Uniprot online websites. According to the results of the analysis, candidate factors that may be involved in host disease resistance or symptom development were selected, their CDS full-length sequences were amplified and constructed into the target vector pGADT7 and then were verified with pGBKT7-CP by one for one in yeast, respectively.【Result】The titer of the Eureka lemon-E. coli cDNA library was 1.02×10⁸ cfu/mL, and it demonstrated that the cDNA library measured up to the experiment standard. The bait vector of pGBKT7-CP was constructed, which had no ability to activate the reporter gene and had no virulence to the yeast. The 41 positive clones were finally selected by using the SD/-Leu-Trp-His-Ade/X-α-Gal medium. After sequence similarity comparison, removing the repetitive sequences, the vector sequences and the frameshift sequences, the 32 host factors that interacted with CYVCV CP were screened. The GO pathway annotation results indicated that these host factors were involved in several chloroplast-related biological processes, including photosynthesis, metabolic process, carbohydrate metabolic process, response to light stimulus, etc. The molecular functions of the 32 host factors were multiple, including catalytic activity, hydrolase activity, transferase activity, protein binding, DNA-binding transcription factor activity, and translation factor activity, etc. Moreover, the cell components of the 32 host factors were involved in the cytoplasm, membrane, chloroplast, thylakoid, nucleolus, and golgi apparatus, etc. Validation of one-to-one yeast two-hybrid crosses of 14 important proteins selected from candidate host factors with CP showed that CP interacted with all 14 proteins.【Conclusion】The cDNA library of Eureka lemon was constructed successfully, and 32 host factors of Eureka lemon interacted with CYVCV CP were preliminarily screened. According to the results, the function of the important proteins was analyzed. It was presumed that CYVCV CP affects photosystem stability, thylakoid structure and chlorophyll synthesis by interacting with multiple chloroplast-related proteins such as oxygen-evolving enhancer protein 2 (PsbP), chlorophyll a-b binding protein 8 (Lhca3) and ribulose bisphosphate carboxylase small subunit (RbcS), resulting in reduced photosynthesis and damaged chloroplast morphology and function. The interaction of CP with these chloroplast-associated factors was separately verified one-to-one in yeast, which will provide a theoretical basis for revealing the molecular mechanism of CYVCV CP in viral pathogenicity.

Key words: citrus yellow vein clearing virus (CYVCV), Eureka lemon, yeast two-hybrid, coat protein, host factor

BIN Yu, ZHANG Qi, WANG ChunQing, ZHAO XiaoChun, SONG Zhen, ZHOU ChangYong. Screening of the Host Factors Interacting with CP of Citrus Yellow Vein Clearing Virus by Yeast Two-Hybrid System[J].Scientia Agricultura Sinica, 2023, 56(10): 1881-1892.

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Figures/Tables 8

Table 1

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Table 2

Interacting candidate proteins obtained by Y2H screening of a lemon cDNA library using CYVCV CP as bait"

克隆号 Clone number	登录号 GenBank accession	蛋白注释ID Uniprot ID	寄主因子（蛋白名称缩写） Factor of host (Abbreviations of protein name)	与CP一对一酵母双杂交验证结果 Validation results for one-to-one Y2H with CP
1	XP_015383675.1	A0A067E581	可能的甲基转移酶20（PMT20）Probable methyltransferase 20	互作Interaction
2	XP_006487560.1	A0A067ESK0	转醛缩酶（TAL）Transaldolase	互作Interaction
3	XP_006488195.1	A0A067G0G4	L-抗坏血酸过氧化物酶（APX）L-ascorbate peroxidase	互作Interaction
4	XP_006494191.1	V4UB67	U11/U12小核糖核蛋白35 kDa类蛋白 U11/U12 small nuclear ribonucleoprotein 35 kDa protein-like	—
5	XP_006479566.1	V4TUF9	类真核翻译起始因子6-2（eIF6） Eukaryotic translation initiation factor 6-2-like	互作Interaction
6	NP_001306987.1	A0A067G915	WD40重复蛋白（WD40-repeat）WD40 repeat protein	互作Interaction
7	XP_006493749.1	A0A067F2B8	琥珀酸-半醛脱氢酶，线粒体亚型X2（SSADH） Succinate-semialdehyde dehydrogenase, mitochondrial isoform X2	—
8	XP_006480341.1	A0A067EMJ8	疏水蛋白LTI6B Hydrophobic protein LTI6B	—
9	XP_006469744.1	A0A067EA24	木葡聚糖内转葡糖基化酶蛋白6（XTH6） Xyloglucan endotransglucosylase/hydrolase protein 6	互作Interaction
10	XP_006466254.1	A0A067F028	Ras相关蛋白Rab11A Ras-related protein Rab11A	—
11	XP_006476755.1	A0A067FQW5	醛脱氢酶家族2成员B4，线粒体（ALDH2B4） Aldehyde dehydrogenase family 2 member B4, mitochondrial	—
12	XP_024949159.1	—	含有RAP结构域的蛋白质，叶绿体 RAP domain-containing protein, chloroplast	—
13	XP_006481758.1	A0A067FSV8	异质核糖核蛋白Q（HNRNPAQ） Heterogeneous nuclear ribonucleoprotein Q	—
14	XP_006493717.1	A0A067F2C0	类T复合蛋白1亚基 T-complex protein 1 subunit gamma-like	—
15	XP_006466732.1	A0A067GIJ6	氨基肽酶P1亚型X1 Aminopeptidase P1 isoform X1	—
16	XP_006488080.1	A0A067GCU3	类丝氨酸/苏氨酸蛋白磷酸酶6调节亚基3 Serine/threonine-protein phosphatase 6 regulatory subunit 3-like	—
17	XP_006473996.1	A0A067EWB7	可能的嘌呤渗透酶5 Probable purine permease 5	—
18	NP_001275850.1	Q8LPM6	蔗糖转运蛋白1（SUT）Sucrose transporter 1	—
19	XP_006490909.1	A0A067H1D4	转酮醇酶，叶绿体（TK）Transketolase, chloroplast	—
20	XP_006471243.2	A0A067DX06	光捕获叶绿素a-b结合蛋白3，叶绿体（Lhca3） Light-harvesting chlorophyll a-b binding protein 8, chloroplast	互作Interaction
21	XP_006477045.2	A0A067DAX7	类FCS锌指蛋白1（FLZ1）FCS-like zinc finger protein 1	互作Interaction
22	XP_006474598.1	A0A067GEE4	过氧化物还原酶Q，叶绿体（PrxQ）Peroxiredoxin Q, chloroplast	互作Interaction
23	XP_006492323.1	A0A067H7T2	通用应激蛋白PHOS34（USP PHOS34）Universal stress protein PHOS34	互作Interaction
24	XP_006490626.1	A0A067EZZ6	重金属相关异戊二烯化植物蛋白39（HIPP39） Heavy metal-associated isoprenylated plant protein 39	互作Interaction
25	XP_006482254.1	Q84LN2	核酮糖二磷酸羧化酶小亚基，叶绿体（RbcS） Ribulose bisphosphate carboxylase (RuBisCO) small subunit, chloroplast	互作Interaction
26	XP_006482548.1	A0A067G267	V型质子ATP酶亚基d2 V-type proton ATPase subunit d2	—
27	XP_006484494.1	A0A067G359	F盒/LRR重复蛋白4（FBXL4）F-box/LRR-repeat protein 4	互作Interaction
28	XP_006469469.1	A0A067D341	葡萄糖-6-磷酸1-脱氢酶，类叶绿体（G6PD） Glucose-6-phosphate 1-dehydrogenase, chloroplast-like	—
29	XP_006485001.1	A0A067DPL0	光系统II放氧强化蛋白2，叶绿体（PsbP） Photosystem II oxygen-evolving enhancer protein 2, chloroplast	互作Interaction
30	XP_006476226.1	—	未知蛋白Uncharacterized protein	—
31	XP_006491226.1	A0A067HEJ5	通用植物调节因子1亚型X2 Common plant regulatory factor 1 isoform X2	—
32	XP_006478862.1	A0A067E694	类溶质载体家族35成员F2 Solute carrier family 35 member F2-like	—

Table 2

Fig. 5

Fig. 6

References 34

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引物名称Primer name	引物序列Primer sequence (5′-3′)
CP-Nde I-F	TGATCTCAGAGGAGGACCTGCATATGAGCTTCGACTACACTCACCC
CP-BamH I-R	TGCGGCCGCTGCAGGTCGACGGATCCTTAGATGTTGAAAGGGGTC
pGADT7-F	TAATACGACTCACTATAGGGCGAGCGCCGCCATG
pGADT7-R	TGCGGCCGCTGCAGGTCGACGGATCCTTAGATGTTGAAAGGGGTC
1-F	CCCGGGTGGGCATCGATACGGGATCCATGCAAGATGGGACAGTTCG
1-R	TATCTACGATTCATCTGCAGCTCGAGTCAACTAGAGGTTTGGTTAG
2-F	CCCGGGTGGGCATCGATACGGGATCCATGGCGACCATTTCGAAGCTTAC
2-R	TATCTACGATTCATCTGCAGCTCGAGTTACAGACTAACCAGCTTGAG
3-F	CCCGGGTGGGCATCGATACGGGATCCATGACGAAGAATTACCCCAC
3-R	TATCTACGATTCATCTGCAGCTCGAGTTAGGCTTCAGCAAATCCTAG
5-F	ACGACGTACCAGATTACGCTCATATGGCAACCAGACTTATGTTTGAG
5-R	ATCTGCAGCTCGAGCTCGATGGATCCTTAGACATAGCTGTCAATTAAAG
6-F	ACGACGTACCAGATTACGCTCATATGGAAAACTCAAGCCAAGAATC
6-R	ATCTGCAGCTCGAGCTCGATGGATCCTCAAACTTTCAAAAGTTGCATTTTG
9-F	ACGACGTACCAGATTACGCTCATATGGCTAAGTTTGATGATCTTTATC
9-R	ATCTGCAGCTCGAGCTCGATGGATCCCTAGTGGCGGTGGTGCACGC
20-F	ACGACGTACCAGATTACGCTCATATGGCAACCCAAGCTTTGGTTTC
20-R	TATCTACGATTCATCTGCAGCTCGAGTCAGTGAAACTTGAGGCTAG
21-F	ACGACGTACCAGATTACGCTCATATGGATTCTGGGGGTTCAAAGAG
21-R	ATCTGCAGCTCGAGCTCGATGGATCCTTAAGCAGCTGCAACAGTGC
22-F	ACGACGTACCAGATTACGCTCATATGGCTGCTTCACTGAATCTCCC
22-R	ATCTGCAGCTCGAGCTCGATGGATCCTCAACTGCTTTGAAGAAACTTC
23-F	ACGACGTACCAGATTACGCTCATATGAACACGAACGAGCGCAGAGTTG
23-R	ATCTGCAGCTCGAGCTCGATGGATCCTCAATTCTCCTCTGGATGCTTC
24-F	ACGACGTACCAGATTACGCTCATATGAAGAAAGCAGTGTTGAAACTTG
24-R	ATCTGCAGCTCGAGCTCGATGGATCCTCAGCAAATAACACAAGCATTAG
25-F	ACGACGTACCAGATTACGCTCATATGGCTTCCTCAATGATCTCATC
25-R	ATCTGCAGCTCGAGCTCGATGGATCCCTAGACGCCTGGTGGCTTGG
27-F	ACGACGTACCAGATTACGCTCATATGGGTTCCCTTTGCATAAACGAAG
27-R	ATCTGCAGCTCGAGCTCGATGGATCCTCAAAGTAACACATCTGGCTC
29-F	ACGACGTACCAGATTACGCTCATATGGCTTCAACTCAATGCTTCTTG
29-R	ATCTGCAGCTCGAGCTCGATGGATCCTCAAGCAACACTGAAAGAA

Screening of the Host Factors Interacting with CP of Citrus Yellow Vein Clearing Virus by Yeast Two-Hybrid System

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