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