Molecular Mechanism Of PLASMA-MEMBRANE ASSOCIATED CATION-BINDING PROTEIN 1 Regulating The Infection Of Potato Virus Y And Tobacco Vein Banding Mosaic Virus

To setup a successful infection,plant viruses encoded movement protein(MP)need to interact with host protein to facilitate virus movement and systemic movement.Identifying these host proteins and understanding their role during virus infection can help us to find novel antiviral target and develop new strategy to control plant viruses.P3N-PIPO is one of striking protein encoded by potyviruses and involved in virus movement.However,host proteins interacted with P3N-PIPO is unclear.In this study,we found that potato membrane-cation-binding protein(PLASMA-MEMBRANE ASSOCIATED CATION-BINDING PROTEIN 1,PCaP1)could interact with P3 NPIPO.Silencing PCaP1 can enhance cell-to-cell movement of potato virus Y(PVY)and tobacco vein banding mosaic virus(TVBMV).We provided evidence that P3 NPIPO interact with PCaP1 based on Pull-Down and co-immunoprecipitation(Co-IP),and identified the essential region within PCaP1 responsible for the interaction with P3N-PIPO.The main results are as follows:(1)Potato StPCaP1 interacts with TVBMV P3N-PIPO,and this interaction is common in tomato and tobacco plant.Using TVBMV-P3N-PIPO as the bait protein,six potato proteins including PCaP1 interacted with TVBMV-P3N-PIPO were identified in potato cDNA libraries.The PVY-P3N-PIPO-and TVBMV-P3N-PIPO-encoding sequences were cloned into transient expression vector pCam35S-EGFP;StPCaP1-,NtPCaP1-,and SlPCaP1-encoding sequences were cloned into transient expression vector pCam-35S-Myc,respectively.Co-IP showed that TVBMV and PVY-encoded P3N-PIPO can interact with StPCaP1,NtPCaP1,and SlPCaP1,respectively,indicating PCaP1 is a common antiviral target against potyviruses.(2)PCaP1 is involved in cell-to-cell movement in two potyviruses.By replacing StPCaP1 with harpin StPCaP1,pCam-35S-harpin-StPCaP1-myc was obtained,and used to silencing PCaP1 gene in potato.Results showed that the cell-to-cell movement is reduced in PCaP1-silencing potato leaves,and the size of infection loci is also reduced.PVY need more time to move systemically and has lower virus accumulation in PCaP1 silenced potato plants.Virus-Induced Gene Silencing(VIGS)silencing PCaP1 in N.benthamiana plants obtained similar results,suggesting PCaP1 is involved in cell-to-cell movement of two potyviruses.(3)The central region of PCaP1 is the essential region for interacting with PCaP1,The Serine at position 108 is the critical site.Prokaryotic expressed TVBMV P3N-PIPO and StPCaP1 were used to study their interaction in vitro.We constructed different StPCaP1 truncated mutants and analyzed their interaction with P3N-PIPO.It showed the central region of StPCaP1 is essential for interacting with P3N-PIPO.Alanine scanning mutagenesis showed that Serine at position 108 is the critical site involved in interacting with P3N-PIPO.Mutating Serine at position 108 to Proline lost its interaction with P3N-PIPO,suggesting that this site can be the potential site for breeding antiviral plants.