Identification For Soybean Host Factors Interacting With P3n-pipo Protein Of Soybean Mosaic Virus

Soybean mosaic disease,caused by soybean mosaic virus(SMV),is one of the most broadly distributed viral diseases worldwide in soybean[Glycine max(L.)Merr.].It causes yield loss and seed quality deficiency seriously.It is pivotal to identify the genes that are effective resistance against Soybean mosaic virus for breeding resistant varieties.The researches of protein interaction between pathogen and host not only help us to understand pathogens infection mechanism,but also help us to dig some effective resistance genes to lay a foundation for cultivating a lasting resistant soybean varieties.SMV belong to the genus of Potyvirus,the SMV genome consists of a positive-sense single-stranded RNA,which includes a long open reading frame(ORF)and encoding a polyprotein that is cleaved into 11 mature proteins:P1,HC-Pro,P3,6K1,CI,6K2,NIa,NIb,CP,VPg,P3N-PIPO.As a novel protein of Potyvirus,there are fewer reports about P3N-PIPO’s function.Recent studies have shown that P3N-PIPO mainly associated with the intercellular movement of virus,mutations of P3N-PIPO in the SMV interference the viral intercellular movement,P3N-PIPO is essential to SMV to establish system infection.The realization of the function of viral proteins require the participation of host proteins,therefore this study used SMV-P3N-PIPO as bait to screening soybean factors interacting with that and further speculation P3N-PIPO’s function through the interactions with the host proteins.In additon,we chosed four soybean proteins:GmBSP、GmGOS 1-2、GmVAMP722、GmHB6 as candidate proteins and further studied the P3N-PIPO’s regions of interaction between SMV-P3N-PIPO and them.We also carried on preliminary analysis on the candidate genes by qRT-PCR under SMV infection.The main results were as follows:1.Identification of soybean proteins interact with SMV-P3N-PIPOUsing SMV 6067-1 P3N-PIPO as the bait to screening a cDNA library from soybean cultivars "Nannong 1136-2".Fifty-four proteins were identified,using soybean genome database Phytozome and NCBI database Blast analysis showed that these proteins are involve in Host defense,Transporting,Primary metabolism,Secondary metabolism,Energy metabolism,DNA binding,Signal transduction,Transcription and Membrane movement.Interaction network analysis for these proteins showed that these proteins involved in three distinct physiological pathways:Signal transduction pathways,cell vesicle transport pathway and chloroplast photosynthetic system pathway.The transporting-related protein own the major proportion(15%)in all of identified soybean proteins and interaction network analysis also showed they are involve in cell vesicle transport pathway,this is consistent to that host P3N-PIPO mediated viral intercellular movement.defense and transporting related proteins own the largest proportion in all.This is consistent with the reportorial function of P3N-PIPO.The host defense-related protein own the largest proportion(17%)in all of identified soybean proteins and interaction network analysis also showed some identified proteins are involve in signal transduction and chloroplast photosynthetic system pathway,These pathways are related to the symptom induced by SMV infection.These suggested that there is also a potential role in inhibiting the host defense mechanism for P3N-PIPO.2.Candidate genes cloning and bioinformatics analysisSelected 4 soybean factors which interact with SMV P3N-PIPO as candidate genes for further studying and amplified them from cDNA of Nannong1138-2 and sequenced.The analysis of bioinformatics analysis to them by Multiple sequence alignment,Phylogenetic tree,membrane structure domain prediction.Results showed that GmBSP belongs to Gluzincin superfamily and contains a basic secretory proteins(BSP)conservative domain.Amino acid sequence alignment showed GmBSP was highly homologous with the C-end of TMV resistance protein which was identified in soybean cultivar "Williams 82".GmBSP is not a transmembrane protein,there is no transmembrane region;GmGOS 1-2 doesn’t contain conservative domain and is closely related Glyma soja(GsGOS1-2).GmGOSl-2 is a transmembrane protein,there is a transmembrane region;GmVAMP722 is the most conservative protein and contains Login and R-SNARE conservative domain.GmVAMP722 is a transmembrane protein,there is a transmembrane region;GmHB6 is transcriptional factor which is only identified in plants and contains HD-ZIP domin.GmHB6 is not a transmembrane protein,there is no transmembrane region.3.The determined of interacting region between SMV-P3N-PIPO and candidate proteinsTo determine whether the P3N domain(N terminus of P3)or the PIPO domain of P3N-PIPO interacts with candidate genes by testing the interaction of P3N or PIPO with candidate genes using yeast two hybrid(Y2H)and Bimolecular fluorescence complementation(BiFC).Y2H and BiFC all showed that GmBSP and GmHB6 interacted only with the full length of SMV-P3N-PIPO,there was no interaction between them and P3N or PIPO alone;Y2H and BiFC all showed that GmGOS1-2 interacted with the full length of SMV-P3N-PIPO and PIPO alone,but did’t interact with P3N alone,thus the interacting region between SMV-P3N-PIPO and GmGOS1-2 is PIPO;Y2H showed that GmVAMP722 interacted with the full length of SMV-P3N-PIPO and PIPO alone,but did’t interact with P3N alone.However,BiFC showed that GmVAMP722 interacted only with the full length of SMV-P3N-PIPO,but didn’t interact with P3N or PIPO alone,the two results are not consistent,need to further study with more evidences.4.Expression patterns of candidate genes under SMV stressqRT-PCR were performed on RNA samples extracted from leaves of Nannong1138-2 seedling at different time after infected with SMV-6067-1 by rubbing method,with Phosphate Buffer Solution(PBS)as control.Results showed that all the candidate genes mRNA levels rising after SMV-infecting and control,but there is no obvious regularity between SMV-infecting and control in rubbed leaves before 24h.This indicated all the candicate genes were corresponding to mechanical wound.All the candidate genes mRNA levels showed obviously upregulated comparing with control at 24h post-inoculation,and GmBSP,GmVAMP722 and GmHB6 mRNA levels have been obviously upregulated comparing with control at 7d and 21d post-inoculation in the leaves upper rubbed leaves.These results suggested that GmBSP,GmVAMP722 and GmHB6 raised expression due to infect by SMV infection and need further to be studied as resistance to SMV related genes.