Construction of Yeast Library of Tomato Nucleus System and Screening of ToCV CP-interacting Proteins

doi:10.7668/hbnxb.20195549

Abstract

Abstract:

This study aims to identify potential proteins that interact with the coat protein(CP)of Tomato chlorosis virus (ToCV)from a cDNA library,to explore the infection mechanism of ToCV and the role of CP in the infection process.The research highlights the significant impact of Tomato chlorosis virus disease on tomato yield and quality during summer and late-autumn production.The Moneymaker tomato variety infected with ToCV was used as the experimental material.Using Gateway technology,a nuclear yeast cDNA library was constructed from ToCV-infected tomatoes,and a yeast two-hybrid bait vector,pGBKT7-CP(CP-BD),was developed.CP was employed as the bait protein to screen the nuclear yeast cDNA library,identifying hundreds of potential interacting proteins involved in various physiological processes.Further verification was performed using one-on-one yeast two-hybrid assays and NCBI BLAST analysis to confirm the proteins interacting with ToCV CP.The constructed nuclear yeast cDNA library had a primary capacity of 1.60×10⁷,with a 100% recombination rate and an average insert size exceeding 1 000 bp.The secondary library also achieved a capacity of 1.60×10⁷,with a 100% recombination rate and an average insert size greater than 1 000 bp,meeting the quality standards for subsequent yeast hybridization experiments.Proteins interacting with ToCV CP,identified through library screening,were categorized into cellular processes,biological regulation,and intracellular material transport.Notably,many of these proteins were associated with processes such as viral replication and transport,host cell infection,and the regulation of host cell metabolism and the cell cycle.Additionally,the identified proteins included those with functions such as protein binding,nucleic acid binding,and hydrolase activity.Among these,ribonucleases were the most abundant,playing a critical role in the viral infection process.Ultimately,30 proteins,including HSPs,DnaJ,and TCPs,were confirmed to interact with ToCV CP.These findings provide a strong foundation for further research into the infection mechanism of ToCV and the functional role of CP in the infection process.

Key words: Tomato chlorosis virus, Coat protein, Gateway technology, Yeast two-hybrid, Nuclear system

CLC Number:

S432.4侵(传)染性病害

WANG Lina, GAO Kang, KANG Chen, TIAN Zhejuan, LI Yadong, WANG Peng, LI Zhaowei, WU Zhiming. Construction of Yeast Library of Tomato Nucleus System and Screening of ToCV CP-interacting Proteins[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(3): 34-43. doi: 10.7668/hbnxb.20195549.

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http://www.hbnxb.net/EN/Y2025/V40/I3/34

Figures/Tables 8

Fig.1 Agarose gel electrophoresis of total RNA and mRNA as well as ds cDNA A.RNA extraction;B.Isolation and purification of mRNAs;C.Double-stranded cDNA synthesis;M.2 000 bp DNA Ladder Marker.

Fig.2 Detection of primary and secondary library quality of nuclear systems A.Primary library capacity identification;B.Primary library colony PCR identification;C.Secondary library capacity identification;D.PCR identification of secondary library colonies;M.2 000 bp DNA Marker;1—24.PCR products of positive clones.

Fig.3 PCR detection of CP-BD decoy vectors CP-BD.Target strip;M.2 000 bp DNA Marker.

Fig.4 Autoactivation detection of CP-BD decoy vectors CP-BD.Target gene;-.The negative control;+.The positive control.

Fig.5 Detection of library transformation efficiency

Tab.1 Proteins interacting with ToCV CP screened from tomato cDNA library

候选蛋白 Putative protein	分子质量/ku Molecular weight	登录号 GenBank No.	基因号 Gene ID	长度/bp Length
热休克蛋白 Heat shock protein	53.495	XP_004239010	Solyc05g010670	464
热休克同源蛋白70 kDa Heat shock cognate 70 kDa protein	70.779	X54030	Solyc10g086410	645
胚胎特异性蛋白 Embryo-specific protein 3,(ATS3)	21.221	XP_004235174	Solyc03g093360	194
热休克蛋白90 Heat shock protein 90	81.008	AK224669	Solyc06g036290	705
J结构域蛋白 J domain-containing protein	30.600	XP_004241350	Solyc06g073950	261
肽酶A1家族 The peptidase A1 family	52.954	XP_004230031	Solyc01g096450	489
HSP20家族 HSP20 family	16.716	XP_004238455	Solyc04g082720	154
加氧酶 Pheophorbide a oxygenase	60.747	AF321984	Solyc11g066440	538
真核生物翻译起始因子2 Eukaryotic translation initiation factor 2	55.008	XP_004229250	Solyc01g066690	504
转录因子VIP1 Transcription factor VIP1	30.863	XP_004242016	Solyc06g060490	278
SNF7家族 The SNF7 family	24.378	XP_004239323	Solyc05g026050	219
50S核糖体蛋白L29 50S ribosomal protein L29	20.254	XP_004241462	Solyc06g072470	176
snRNP Sm蛋白 snRNP Sm proteins	10.713	XP_004241374	Solyc06g073700	99
GST超家族 The GST superfamily	26.032	XP_004229524	Solyc01g081250	224
60S核糖体蛋白 60S ribosomal protein	25.443	XP_004250254	Solyc11g012110	230
DNAj伴侣蛋白 Chaperone protein DNAj	44.260	XP_004253109	Solyc12g095800	395
40S核糖体蛋白 40S ribosomal protein	19.853	XP_004253050	Solyc12g096540	181
热休克同源蛋白80 Heat shock cognate protein 80	80.136	NP_001234439	Solyc07g065840	700
HSP60家族 The chaperonin(HSP60)family	62.992	XP_004228946	Solyc01g028810	600
SART-1家族蛋白DOT2 SART-1 family protein DOT2	56.438	XP_004250062	Solyc11g008470	500
ADF家族 ADF family	15.847	XP_004240356	Solyc06g005360	138
通用核糖体蛋白uS11家族 The universal ribosomal protein uS11 family	10.627	NP_001274895	Solyc02g070570	100
小核核糖核蛋白smd2 Small nuclear ribonucleoprotein Sm D2	12.561	XP_004247417	Solyc09g075130	110
60S核糖体蛋白L12 60S ribosomal protein L12	17.795	XP_004250933	Solyc11g069440	167
ATPase家族 ATPase family	75.948	XP_004243266	Solyc07g055320	706
信号识别粒子 Signal recognition particle	62.201	NP_001353332	Solyc09g009940	568
Actin家族 Belongs to the actin family	41.758	NP_001295376	Solyc03g078400	378
TCP家族转录因子 TCP family transcription factor	45.629	NP_001234572	Solyc06g069240	407
NmrA-like家族 NmrA-like family	44.890	XP_004231083	Solyc01g112060	407
苹果酸脱氢酶 Malate dehydrogenase	48.449	AJ849379	Solyc11g007990	443

Tab.1 Proteins interacting with ToCV CP screened from tomato cDNA library

候选蛋白 Putative protein	分子质量/ku Molecular weight	登录号 GenBank No.	基因号 Gene ID	长度/bp Length
热休克蛋白 Heat shock protein	53.495	XP_004239010	Solyc05g010670	464
热休克同源蛋白70 kDa Heat shock cognate 70 kDa protein	70.779	X54030	Solyc10g086410	645
胚胎特异性蛋白 Embryo-specific protein 3,(ATS3)	21.221	XP_004235174	Solyc03g093360	194
热休克蛋白90 Heat shock protein 90	81.008	AK224669	Solyc06g036290	705
J结构域蛋白 J domain-containing protein	30.600	XP_004241350	Solyc06g073950	261
肽酶A1家族 The peptidase A1 family	52.954	XP_004230031	Solyc01g096450	489
HSP20家族 HSP20 family	16.716	XP_004238455	Solyc04g082720	154
加氧酶 Pheophorbide a oxygenase	60.747	AF321984	Solyc11g066440	538
真核生物翻译起始因子2 Eukaryotic translation initiation factor 2	55.008	XP_004229250	Solyc01g066690	504
转录因子VIP1 Transcription factor VIP1	30.863	XP_004242016	Solyc06g060490	278
SNF7家族 The SNF7 family	24.378	XP_004239323	Solyc05g026050	219
50S核糖体蛋白L29 50S ribosomal protein L29	20.254	XP_004241462	Solyc06g072470	176
snRNP Sm蛋白 snRNP Sm proteins	10.713	XP_004241374	Solyc06g073700	99
GST超家族 The GST superfamily	26.032	XP_004229524	Solyc01g081250	224
60S核糖体蛋白 60S ribosomal protein	25.443	XP_004250254	Solyc11g012110	230
DNAj伴侣蛋白 Chaperone protein DNAj	44.260	XP_004253109	Solyc12g095800	395
40S核糖体蛋白 40S ribosomal protein	19.853	XP_004253050	Solyc12g096540	181
热休克同源蛋白80 Heat shock cognate protein 80	80.136	NP_001234439	Solyc07g065840	700
HSP60家族 The chaperonin(HSP60)family	62.992	XP_004228946	Solyc01g028810	600
SART-1家族蛋白DOT2 SART-1 family protein DOT2	56.438	XP_004250062	Solyc11g008470	500
ADF家族 ADF family	15.847	XP_004240356	Solyc06g005360	138
通用核糖体蛋白uS11家族 The universal ribosomal protein uS11 family	10.627	NP_001274895	Solyc02g070570	100
小核核糖核蛋白smd2 Small nuclear ribonucleoprotein Sm D2	12.561	XP_004247417	Solyc09g075130	110
60S核糖体蛋白L12 60S ribosomal protein L12	17.795	XP_004250933	Solyc11g069440	167
ATPase家族 ATPase family	75.948	XP_004243266	Solyc07g055320	706
信号识别粒子 Signal recognition particle	62.201	NP_001353332	Solyc09g009940	568
Actin家族 Belongs to the actin family	41.758	NP_001295376	Solyc03g078400	378
TCP家族转录因子 TCP family transcription factor	45.629	NP_001234572	Solyc06g069240	407
NmrA-like家族 NmrA-like family	44.890	XP_004231083	Solyc01g112060	407
苹果酸脱氢酶 Malate dehydrogenase	48.449	AJ849379	Solyc11g007990	443

Fig.6 Identification of interacting proteins of ToCV CP A.Growth of the 30 candidate proteins on SD/-Trp/-Leu/-His plates;B.Growth of the 30 candidate proteins on SD/-Trp/-Leu/-His/-Ade plates;-.The negative control;+.The positive control.

Fig.7 GO annotation analysis 1.Intracellular protein transport;2.Cellular component assembly;3.Regulation of cell differentiation;4.Nuclear transport;5.Macromolecular complex assembly;6.Nucleocytoplasmic transport;7.Ribonucleoprotein complex assembly;8.Vacuolar transport;9.Ribonucleoprotein complex subunit organization;10.Protein localization to organization;11.Cellular localization;12.Protein transport;13.Transcription,DNA-templated;14.Regulation of multicellular organismal process;15.Phosphorylation;16.Biosynthetic process;17.Response to osmotic stress;18.Response to acid chemical;19.Rransmembrane transport;20.Regulation of cellular proten in metabolic process;21.Response to hormone;22.Regulation of RNA metabolic process;23.Positive regulation of gene expression;24.System development;25.Regulaton of cellular biosynthetic process;26.Vacuolar part;27.Vesicle;28.Intracellular non-membrane-bounded organelle;29.Non-membrane-bounded organelle;30.Bounding membrane of organelle;31.Nuclear part;32.Intracellular organelle;33.Intracellular;34.Golgi apparatus;35.Chloroplast;36.Organelle membrane;37.Plasma membrane;38.Envelope;39.Cell junction;40.Nucleoplasm;41.Organelle envelope;42.Membrane;43.Symplast;44.Plasmodesma;45.Cell-cell junction;46.Plastid;47.Membrane part;48.Endoplasmic reticulum;49.mRNA binding;50.Poly(A)RNA binding;51.Kinase binding;52.Protein kinase binding;53.Enzyme binding;54.RNA binding;55.Protein binding;56.Macromolecular complex binding;57.Nucleic acid binding;58.Heterocyclic compound binding;59.Hydrolase activity;60.Small molecule binding;61.Catalytic activity;62.Organic cyclic compound binding;63.Binding;64.Ion binding.

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