The Reaction Between PaMsrB1/ Oxidized PRSV-NIa-Pro And Isolation Of PaMsrB1 Substrate Proteins

Papaya ringspot virus (PRSV), a member of the aphid-transmitted genus Potyvirus, is the most widespread and destructive virus disease affecting papaya cultivation worldwide. Typically,in compatible virus-host plant interactions, one of the earliest responses of plant cells to pathogens is the production of reactive oxygen species (ROS) and chloroplasts can be a source of oxidative stress during viral disease development.Plant methionine sulfoxide reductase B1 (PaMsrB 1) protects the photosynthetic apparatus from oxidative damage by scavenging ROS to repair Met-oxidized proteins. However, the chloroplast-localized papaya (Pa) MsrBl interacts with the virus-encoded PRSV nuclear inclusion protein a protease (NIa-Pro), disturbing import of PaMsrB 1 into chloroplasts. PRSV NIa-Pro contains 4 Met residues, one of which is predicted to be particularly prone to oxidation into Met sulfoxide (MetO). These findings led us to hypothesize that PaMsrB 1 catalyzes the reduction of oxidized PRSV NIa-Pro by ROS, representing a novel mechanism of PRSV towards the host defense. Additionally, tounderstand the anti-oxidative defense mechanism of PaMsrB 1 in chloroplasts during host-virus interaction, affinity chromatography and Nano LC-MS/MS were used to isolate and identify substrates of PaMsrB1.In this study, the main results were as follows:1.Expression, purification, and biological activity of recombinant PRSV NIa-ProThe gene encoding the PRSV NIa-Pro was cloned and expressed as a fusion protein MBP-NIa with maltose binding protein (MBP) fusion and a-His tag in Escherichia coli. The recombinant NIa-Pro were purified from the fusion protein by Ni2+-NTA agarose affinity chromatography, cleavage of the MBP from MBP-NIa and amylose resin affinity chromatography. The purified recombinant NIa-Pro exhibited the nonspecific double-stranded DNase activity and sequence-specific proteinase activity.2. Expression, purification, and biological activity of recombinant PaMsrB 1The gene encoding the PaMsrB 1 without the chloroplast transit peptide was cloned and expressed as a fusion protein MBP-NIa with maltose binding protein (MBP) fusion and a-His tag in Escherichia coli. The recombinant NIa-Pro were purified from the fusion protein by Ni2+-NTA agarose affinity chromatography, cleavage of the MBP from MBP-PaMsrBl and amylose resin affinity chromatography.The results of thin layer chromatography assay and HPLC analysis indicated that recombinant PaMsrB 1 had reductase activity that could reduce MetSO back to Met in vitro.3.Reduction of oxidized PRSV-NIa-Pro by PaMsrB 1After H2O2 treatment, PRSV-NIa-Pro migration during sodium dodecylsulfate-polyacrylamidegel electrophoresis was shifted compared with that of the reduced form. Subsequent incubation with PaMsrB1 partially restored the migration profile.4.Identification of PaMsrBl substrate proteins by affinity chromatography and LC-MS/MS.Papaya leaf proteins were applied to the Hi-Trap NHS-activated HP 1-ml affinity column coupled with the recombinant PaMsrB1. Three hundred and forty-one proteins from eluted proteins were identified by mass spectrometry. Among the 13 PaMsrBl-interacting proteins directly participate in photosynthetic processes, such as Chaperonin 60 subunit beta 1, Nitrite reductase,Chaperonin 60 subunit alpha 1,Ribulose bisphosphate carboxylase small chain 1A,50S Ribosomal protein L24,ATP synthase subunit beta,Glutamine synthetase,Linoleate 9S-lipoxygenase 5,Chlorophyll A-B binding protein 1,30S Ribosomal protein S3,Oxygen-evolving enhancer protein 1-1,Protein thylakoid formation 1, ABC transporter 1 family member 6.This study would lay the foundation of the anti-viral defense mechanism and resolve the problem of the important proteins associated with PaMsrBl protein involved in the theoretical of antioxidant regulation for the further research.