The Screening And Functional Exploration Of The Interactive Host Factors In Papaya To Papaya Ringspot Virus NIa-Pro

Papaya ringspot virus (PRSV, genus Potyvirus, family Potyviridae) is considered to be the most destructive virus that occurs in papaya plantations across the globe. Potyviral nuclear inclusion protein a protease (NIa-Pro) is the main Potyvirus-encoded proteinase and involved in virus replication, host specificity determination and disease resistance, which should rely on multiple host-virus interactions. However, little is known about the host proteins that interact with the NIa-Pro protein and their role in virus infection. In this study, based on whole-genome sequencing and structural analysis of the PRSV Hainan isolate (PRSV-HN) and the construction of papaya cDNA library, we investigated the host factors interacting with PRSV NIa-Pro using a Sos recruitment two-hybrid system (SRS). In total,17positive clones were identified after two rounds of testing for galactose-dependent growth at37℃. Based on sequence alignment with the relevant homologous sequence from C. papaya, Arabidopsis thaliana, Gossypium arboreum, Ricinus communis, Populus trichocarpa, Platanus acerifolia, Oryza sativa and Populus tremula, the cDNA inserts of the17candidate plasmids encoded proteins were classified into six different host proteins as follows:FBPA1, MTLs, EIF3G, GTPBP, FK506BP and MsrB1. the full-length cDNA of the six genes were cloned by RT-PCR, which are designated PaFBPAl (GENBANK accession no. JN008888), PaMTLs (GENBANK accession no. JN008889), PaEIF3G (GENBANK accession no. JN008890), PaGTPBP (GENBANK accession no. JN008891), PaFK506BP (GENBANK accession no. JN008892) and PaMSRB1(GENBANK accession no. JF431992). Then, we confirmed that the full-length papaya proteins interact specifically with PRSV NIa-Pro in yeast and in plant cells using SRS and bimolecular fluorescent complementation assay, respectively.Based on bioinformatic analysis, we found PaFBPA1and PaMsrB1contain68and70amino acid residues of predicted chloroplast localization signal peptide, respectively. Then, we constructed plant transient expression vectors for subcellular localization of PaFBPA1, PaMsrB1and NIa-Pro by fusion with eGFP, respectively. The transient expression vectors were transformed into the papaya leaf mesophyll protoplasts, and the fluorescence was observed by confocal microscopy. The results indicated that NIa-Pro was localized in the cytoplasm of papaya leaf cells and both PaFBPA1and PaMsrB1were localized in the papaya leaf chloroplasts. We then confirmed that both the specific protein-protein interaction PaFBPA1-NIa-Pro and PaMsrBl-NIa-Pro were localized in the papaya leaf chloroplasts respect by bimolecular fluorescence complementation (BiFC).According to protein structure analysis, four deletion mutants of NIa-Pro, three deletion mutants and two point mutants of PaFBPA1and three deletion mutants and four point mutants of PaMsrB1, were designed, and subcloned into pSos or pMyr for SRS assays. The result indicated that the only C-domain (residues133-239) of NIaPro was the necessary domain for interaction with PaFBPA1and PaMsrB1. FBP-2domain (residues231-280) of PaFBPAl was the necessary domain for interaction with NIa-Pro, the mutants FBP (257VA) and FBP (260EA) of PaFBPA can lead to failure interac with NIa-Pro. And MsrB-2domain (residues112-175) of PaMsrBl was the necessary domain for interaction with NIa-Pro, the mutants MsrB(133TA) and MsrB(173HF) of PaMsrB1can lead to failure interac with NIa-Pro.NIa-Pro is the major proteinase of potyviruses. In this study, a preliminary analysis of the interaction between PaFBPA1or PaMsrB1and NIa-Pro whether based on digestion reaction was done. Prokaryotic expression of the GST fusion protein of NIa-Pro, and the fusion proteins of PaFBPA1and PaMsrB1, which contain both GST tag and His tag. Combine with the digestion analysis in vitro and the western blot assay, we confirmed that the PaFBPAl was digested by NIa-Pro in vitro, which was related to the NIa-Pro protease recognition sites in PaFBPAl. And the PaMsrB1was not digested by NIa-Pro in vitro.The results of qRT-PCR indicated that the expression level of PaFBPAl mRNA was down-regulated after10dpi, showed an increasing trend after12dpi, and decreased to a higher than basal expression levels in noninoculated (healthy) papaya plants after20dpi. Whereas the expression level of PaFBPA1mRNA was down-regulated after6dpi and peaked at8dpi, showed an obvious increasing trend from10dpi and peaked at12dpi, and decreased to a higher than basal expression levels in noninoculated (healthy) papaya plants after15dpi.One of the earliest responses of plant cells to pathogens is the production of reactivate oxygen species (ROS). In this study, we analyzed the expression levels of ROS at various times post-inoculation with PRSV by ELISA. The result indicated that the expression level of ROS showed an increasing trend from4dpi, peaked at10dpi and decreased to a higher than basal expression levels in noninoculated (healthy) papaya plants after15dpi. Therefore, PRSV infection resulted in the accumulation of ROS in papaya plants. MsrBl is an ubiquitous enzyme that catalyze the reduction of the chloroplast proteins oxidized ROS. Because of the interacton between PaMsrB1and NIa-Pro was localized in the papaya leaf chloroplasts, we propose the interaction may interfere with the PaMsrB1scavenging of ROS caused by PRSV infection, and involved in metabolism of chloroplast in papaya. Fructose-1,6-bisphosphate (FBP) aldolase is a key enzyme in Calvin cycle and can regulate the rate of photosynthesis in higher-plant chloroplast. The interaction between PaFBPAl and NIa-Pro was localized in the papaya leaf chloroplasts and based on digestion reaction, we propose the interaction may interfere with the role of PaFBPAl played in photosynthesis in host cells, lead to the digestion of PaFBPAl, and then lead to the formation of the symptoms in papaya chloroplasts. which provide some new clues in further evaluating the detailed mechanisms of PRSV infection and to control PRSV.