Characterization And Function Analysis Of Soybean Mosaic Virus P3 Interactiors From Soybean

Soybean Mosaic Virus(SMV)is one of the most destructive viruses that affects seed quality and production of soybean.The SMV genome comprises a single ORF that producing a single polypeptide autoclaved by it s proteinase to get 11 mature proteins.P3 is the third protein have been identified as important for virus virulence on the soybean and host range.A split-ubiquitin yeast two-hybrid system was used to identify soybean proteins that interact with SMV virulence protein P3 and the roles of the identified proteins was uncovered during different pathogenesis affection including SMV.GmEFlb,one of the subunit of elongation factor complex,was identified in a proteomic analysis of SMV-infected plants.We used a previously described bean pod mottle virus(BPMV)-based vector to silence genes encoding the target proteins and analyzed the response to SMV in the various silenced lines.Results related to the functional characterization of two such proteins will be discussed.We showed that a soybean elongation factor 1 a(EF 1 a),elongation factor lb(EF1b)and a vacuolar ATPase(VATPase)are important for SMV virulence in susceptible soybean backgrounds.Details result as following:1.Yeast two hybrid cDNA library constructionFor high throughout analysis protein interaction,prey vector was modified to generate Gateway vector as pPR3-N-RlR2 containing attB sites.Three-open reading frame cDNA library was generated from NN1138-2 after SMV-SC15 infection by modified prey vector pPR3-N-R1R2 into gateway system.The capacity of library was 0.68×107.Randomly picked 32 clonies,PCR showed that inset is size range from 0.8 to 4.5 kb and recombination rate is 94%.High quality of soybean cDNA library was ready for yeast two hybrid screening.2.Screening for P3 interactors from Y2H library and interaction confirmationP3 was used as a bait to screening the library,and 120 positive clonies were selecteted for sequencing.The potential function of these proteins from plant were divided into 9 groups based on Blast online.18 proteins were used for one to one interaction test,and results showed that all these proteins have good ineraction with p3 in yeast.Soybean elongation factor 1A(GmEF1a)and vacuolar ATPase(GmVATPase)are selected for further analysis.Full length of this two genes were cloned.Firstly,interaction with P3 was showed in yeast,and then confirmd by BiFc and Co-IP.BiFc showed that this two proteins interact with P3 which derived three different strains:G5\G7\SC15 in both cytoplasm and nucleus respectively.Even P3 is one of the most variation protein among potyvirus 11 proteins.They might share a conserved domain which is essential for binding with GmEF1a and GmVATPase.As already known in other virus,EF1a interacts with virus RdRp,and NIb serves the RdRp function in SMV,my data showed that NIb interacts with EF la by Co-IP without P3.3.Candidant proteins proterty analysisDifferent component isolation showed that GmEFla,GmEFlb and GmVATPase were soluble protein,and P3 was menbrane protein.Subcelluar localization showed that GmEF1a localized at ER and little in nucleus,GmEF1b and P3 at both nucleus and ER.But NLS assay showed GmEF1a,GmEF1b and P3 contain NLS.Co-localization of GmEF1a and P3 showed that P3 promates GmEFla into nucleus.Co-expressed GmEF1b and P3 did not show any locate change as single loclization.Subcelluar localization showed that GmVATPase localized at ER along with multiple vacuolar,indacting the relationship with it's function.4.Silencing line construction based on BPMV VIGSGmEFla and from Y2H and GmEFlb from 2D were used for function analysis.180 bp(L63-G122)of GmEF1a,222 bp(L134-P208)of GmEF1b and 186bp(G9-T71)of GmVATPase were used to generate BPMV-RNA2 recombinant vectors.In vitro transcription of the recombinant vectors with RNA1 for rub inoculation.qRT-PCR showed all three genes were silenced individully with 42%silencing efficiency of GmEF1a-17g,68%silencing efficiency of GmEF1b-13g and 78%silencing efficiency of Gm VATPase-10g.5.Elongation factor family function anylyasisAfter silencing GmEF1aand GmEF1b in soybean,plant show more resistance to both G5 and G7 on Essex background by morphology.And on Rsvl backgrond,silencing GmEF1a and GmEFlb does not give much necerosis as V control.qRT-PCR showed that SilEFla has less cell death marker gene r203j expression after SMV infection.Also Western blot and ELISA showed less virus in silencing lines on local and systemic leaves comparied with V.Based on the difference fold of virus,the amount of SMV reduced more than systemic.That means silencing EF1a and EF1b restrict SMV replication and cell-to-cell movement,but not affect long distance movement.Silencing GmEF1a and GmEFlb do not show any difference on BYMV level.We conclude that P3 help SMV'replication in soybean by assistant of EFla and EFlb,which faciliates SMV replication specificly.As silencing GmEFla on Harosoy,plant gave more bacterial after Psg.avrB infection and did not show any difference after Psg.Vir and Psg.avrC infection.Which means GmEF1a is important for Rpg2 mediate pathway,not requried for basal and Rpg3 pathway.Silencing GmEF1b did not change Psg.avrB,Psg.Vir and Psg.avrC,which means GmEF1b not involved in Psg pathways.There EF1a mutants in arabidopsis have been genotyped,T-DNA were inserted into three position of two chromosomes,RT-PCR showed that each target gene was knockout and not affect other two genes.Three mutant lines were used to test response to TCV and CMV.Western blot showed virus level did not change on local and distal leaves comparied with col-0.Also parequrat treatment did not give any morpholpgy difference with wildtype.And bacterial Pst.DC3000 and Pst.rpt2 infiltration did not diverse to wildtype.ER stress agents treatment data conserved.Which suggests that EFla not involed in pathogens response,or it may involved in,but single mutant is not enough.Other isoforms may confer it defect.6.Relationship between ER stress and virus replicationVirus infection usually cause ER stess and induce UPR related genes expression like BIP and NRP.Soybean with only SMV infection cause 8 times BIP and 10 times NRP induction.In V plant BIP was up regulated 10 times and NRP was upregulated 9 times at 7dpi SMV infection,while both BIP and NRP did not change much in the SilEF1a plant.Tunicamycin and DTT are agents that interfere with protein folding in ER and trigger ER stress.We treated the soybean by vacum with 10 ?M Tunicamycin DMSO as control and 4 mM DTT water as control.And SMV inoculaiton was taken after 1dpi ER stress inducer treatment.Both Western and ELISA showed more SMV after DTT and TM induction,while did not affect BYMV.We propose that SMV replication take place on ER,and EF1a and EFElb are essential for SMV survial in soybean.7.Chariacterization Vacuolar ATPase functionAfter silencing GmVATPase in sespectible soybean,plant show more resistance to both G5 and G7 on Essex background by morphology.And on Rsvl backgrond,silencing GmVATPase does not give much necerosis as V control.Also Western blot and ELISA showed less virus in silencing lines on local and systemic leaves comparied with V.Based on the difference fold of virus,the amount of SMV reduced more than systemic.That means silencing VATPase also restrict SMV replication and cell-to-cell movement,but not affect long distance movement.Since SilVATPase have high level expression of SA,we sespected VATPase function had relationship with SA pathway.SA spray was taken to SilVATPase line.SMV was rub inoculation after 2 days treatment.SMV accumulation was less detected by Western blot compaired with V control after AS spray.We concluded that GmVATPase was required for S A pathway.RNA-seq technology was used to detect the difference expression genes between SilVATPase and V after SMV infection.Data analysis showed that 64%genes were up-regulated within the different expression genes.Since methyltranseranse was upregulated in SilVATPase,we generated methyltranseranse silencing line and it's response to SMV was that after silencing methyltranseranse,more SMV accumulation on both inoculation and systemic leaves.Indicating it's positive regulated of SMV infection.