Scientia Agricultura Sinica ›› 2024, Vol. 57 ›› Issue (15): 2986-2996.doi: 10.3864/j.issn.0578-1752.2024.15.006

• PLANT PROTECTION • Previous Articles     Next Articles

Screening and Verification of Pepper Host Factors Interacting with the 126 kDa Protein of Pepper Mild Mottle Virus by Yeast Two-Hybrid System

HE Yong(), FAN XiaoZhu, CHEN XinYue, DUAN ShuJing, HU TingTing, XIE RuXue, WANG YuQing, CHEN Jing()   

  1. School of Plant Protection, Anhui Agricultural University, Hefei 230036
  • Received:2024-04-05 Accepted:2024-05-24 Online:2024-08-05 Published:2024-08-05
  • Contact: CHEN Jing

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Abstract:

【Objective】Pepper mild mottle virus (PMMoV) is one of the major viruses that harm peppers worldwide. The 126 kDa protein is an important pathogenic factor encoded by PMMoV, but its pathogenic mechanism remains unclear. This study aims to screen for pepper host factors that interact with the 126 kDa protein, and to provide a theoretical basis for elucidating the pathogenic mechanism of PMMoV.【Method】Firstly, the bait vector pGBK-126 kDa was constructed using homologous recombination. Using pepper leaves as experimental materials, total RNA was extracted from pepper leaves using the Trizol method, and a pepper yeast cDNA library was prepared. Subsequently, the cDNA library was screened using pGBK-126 kDa, and the screening results were subjected to sequence alignment and bioinformatics functional analysis using NCBI and Uniprot. Based on the alignment and analysis results, host factors that may participate in plant disease resistance pathways were selected, and their full-length CDSs were cloned and constructed into the pGADT7 vector. Yeast two-hybrid (Y2H), BiFC, and LCI were used to further verify the interaction between 126 kDa and host factors. Finally, the role of transiently overexpressed host proteins during PMMoV infection was analyzed.【Result】High-quality pepper RNA was extracted without degradation. A high-quality yeast cDNA library was obtained, and the bait plasmid pGBK-126 kDa was successfully constructed. A total of 18 pepper host factors that interact with the 126 kDa protein were screened. Bioinformatics analysis revealed that these 18 host factors are widely involved in multiple pathways such as plant enzyme systems, regulation of material and energy metabolism, DNA-binding transcription, hormone synthesis, and defense responses. Among them, three host factors (LA2, PDHE1, BXL1) showed interactions with 126 kDa in one-to-one Y2H interaction verification, indicating the reliability of the initial screening results. The interaction between 126 kDa and BXL1 was further verified in vitro and in vivo using BiFC and LCI. Transient overexpression of BXL1 significantly inhibited PMMoV infection.【Conclusion】The pGBK-126 kDa bait plasmid was successfully constructed. Based on this plasmid, 18 interacting host factors were obtained when screening the yeast cDNA library, which are widely involved in multiple pathways of plant life activities. The screening results were verified to be reliable. Among them, BXL1 interacts with 126 kDa both in vitro and in vivo, and can inhibit PMMoV infection. The results can provide a good theoretical and material basis for further exploring of the infection mechanism of PMMoV.

Key words: pepper mild mottle virus (PMMoV), pepper (Capsicum annuum), yeast two-hybrid (Y2H), 126 kDa protein, pathogenic mechanism

Table 1

PCR amplification primers"

引物Primer 序列Sequence (5′-3′)
pGAD-LA2-F CGCTCATATGGCCATGGAGGCCAGTGAATTCatgggtggttgggctatagcg
pGAD-LA2-R GATTCATCTGCAGCTCGAGCTCGATGGATCCctattcccaaataccaacttcc
pGAD-BXL1-F CGCTCATATGGCCATGGAGGCCAGTGAATTCatgagtaaaaaatattccagt
pGAD-BXL1-R GATTCATCTGCAGCTCGAGCTCGATGGATCCctaaaaggaaaagtaggttagt
pGAD-PDHE1-F CGCTCATATGGCCATGGAGGCCAGTGAATTCatggctgctattatacaaggaa
pGAD-PDHE1-R GATTCATCTGCAGCTCGAGCTCGATGGATCCtcactggcaaagctgttcaaca
pGBK-126 kDa-F GAGGAGGACCTGCATATGGCCATGGAGGCCGAATTCatggcttacacacaacaagctacc
pGBK-126 kDa-R ATGCTAGTTATGCGGCCGCTGCAGGTCGACGGATCCctattgagtcgacacatcaactttg

Fig. 1

Symptoms of pepper infected with PMMoV"

Fig. 2

Electrophoresis patterns of total RNA isolated from pepper"

Fig. 3

Electrophoresis patterns of PCR amplification of 126 kDa gene"

Fig. 4

Growth status of yeast containing pGBK-126 kDa and pGBKT7 plasmids on SD/-Trp medium"

Fig. 5

Growth status of yeast co-transforming pGBK-126 kDa and pGADT7 plasmids on different SD dropout media"

Table 2

Eighteen host factors of pepper interacting with PMMoV 126 kDa protein"

GenBank登录号
GenBank accession		 基因注释
Gene annotation		 克隆数
Number of clone
PHT30078.1 AGO 1A蛋白Protein argonaute 1A 2
AVP43435.1 叶绿体诱导的β-淀粉酶1 Chloroplast induced beta amylase 1 1
KAF3682718.1 乙烯反应转录因子9 Ethylene-responsive transcription factor 9 1
XP_016562445.1 乙烯反应转录因子RAP2-12 Ethylene-responsive transcription factor RAP2-12 isoform X2 1
PHT62104.1 p-1, 3-葡聚糖苷酶Glucan endo-1, 3-beta-D-glucosidase 2
XP_006359610.1 预测的酰胺基磷酸核糖基转移酶，叶绿体Predicted amidophosphoribosyl transferase, chloroplastic-like 1
XP_009794582.1 预测的延长因子类似子-2 Predicted elongation factor 2-like 1
XP_016568807.1 可能的异天冬氨酰肽酶L-天冬酰胺酶2 Probable isoaspartyl peptidase L-asparaginase 2 1
XP_016569480.1 类ROH1蛋白Protein ROH1-like 2
PHU30596.1 推定的β-木糖苷酶2 Putative beta-D-xylosidase 2 1
KAF3627957.1 推定的木葡聚糖半乳糖基转移酶类KATAMARI 1 Putative xyloglucan galactosyltransferase KATAMARI 1-like 1
MCE0481271.1 丙酮酸脱氢酶E1 β亚基，叶绿体Pyruvate dehydrogenase E1 component subunit beta-3, chloroplastic 1
NP_001385273.1 RuBisCO小亚基，叶绿体Ribulose bisphosphate carboxylase small subunit, chloroplastic 1
KAF3647305.1 RuBisCO大亚基结合蛋白β亚基，叶绿体RuBisCO large subunit-binding protein subunit beta, chloroplastic 1
AFR41329.1 S-腺苷甲硫氨酸合成酶S-adenosylmethionine synthetase 3
PHT42653.1 mec-8和unc-52蛋白样蛋白2抑制因子Suppressor of mec-8 and unc-52 protein-like protein 2 1
PHU13958.1 伤诱导蛋白1 Wound-induced protein 1 3
TKY48728.1 WRKY转录因子42 WRKY transcription factor 42 1

Fig. 6

Annotation by gene ontology"

Fig. 7

Interaction between 126 kDa and LA2, PDHE1 and BXL1 in the yeast two-hybrid system"

Fig. 8

Interaction between 126 kDa and BXL1 in LCI experiment"

Fig. 9

Interaction between 126 kDa and BXL1 in BiFC experiment"

Fig. 10

The function of transient overexpression of BXL1 in PMMoV infection"

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