Functional Analysis And Sub-Localization Of Sugarcane Mosaic Virus P3N-PIPO Protein In Planta

Sugarcane mosaic disease is an important worldwide virus disease that seriously damages sugarcane production.Sugarcane mosaic disease causes heavy yield loss every year.Breeding anti-viral sugarcane varieties is the most cost-effective way to resist sugarcane mosaic disease.However,there is no enough effective anti-virus varieties available for sugarcane production.It is difficult to create new cultivars or germplasm combined disease-resistant,high-yield and high sugar content traits.In recent years,transgenic breeding of sugarcane anti-virus has been carried out gradually.However,the genome structure of SCMV is very simple and SCMV evolves very fast.It is difficult to gain a continuous antiviral cultivar by silencing the genes coded by SCMV.It is necessary to understand the biological functions of viral proteins and the molecular mechanisms based for the interaction between virus and host plant to develop new antiviral strategies.Plant viruses have to deploy host proteins to establish systemic infection.Genome editing or knocking down the host genes involving SCMV infection could confer the mutated sugarcane plants virus resistance.In this study,we cloned the SCMV-P3N-PIPO by infusion PCR and determined the subcellular localization of SCMV-P3N-PIPO.Then,we screen the cDNA library with SCMV-P3N-PIPO as bait.These studies can benefit the interaction mechanism between SCMV and sugarcane.Thus to improve the molecular model of potyviruses cell-to-cell movement.The main results are as follows:(1)The P3N-PIPO gene was cloned with the SCMV P3 crison as a template by infusion PCR.P3N-PIPO consists of 153 aa N-terminus of P3N and 82 aa of C-terminus of PIPO.SCMV P3N-PIPO can be localized to the plasmodesmata(PD),but mainly localized on the plasma membrane(PM).(2)In order to identify its PM localization signal,a series of deletion mutations were constructed to obtain truncated mutants P3N,PIPO,P3NPIPOT1,P3N-PIPOT2,PIPOT1,PIPOT2,and PIPOT3,and GFP was fused at their C-terminus,respectively.These constructs were transiently expressed in the leaves off.benthamiana individually.The results showed that the membrane localization of P3N-PIPOT1 was weakened,P3NPIPOT2 was localized to the PD.P3N,PIPOT1 and PIPOT2 had no clear subcellular localization.PIPOT3 showed clear PD localization,but they formed aggregates in the cytoplasm.It suggested that the 19 amino acids at the N-terminus of PIPO determine the PD localization of P3N-PIPO,while the PM localization of P3N-PIPO was determined by both P3N and PIPO,i.e.,the conformation of P3N-PIPO.(3)Yeast two-hybrid(Y2H)and bimolecular fluorescence complementation(BiFC)assays showed that ScPCaP1 interacts with P3NPIPO and P3N-PIPOT1,but not P3N-PIPOT2.It suggested that the 60 amino acids of the C-terminus of PIPO determine the interaction between P3N-PIPO and ScPCaP1.Latrunculin B treatment showed that P3N-PIPO localization to PD is independent of the myoblast actin system,and Brefeldin A treatment indicated that the intracellular movement of P3NPIPO requires an early secretory system.(4)Wild-type P3N-PIPO and mutated P3N-PIPO,namely P3N-PIPOT1 and P3N-PIPOT2 can recruit CI to PM or PD,and P3N-PIPO and P3NPIPOT2 can move between cells.These results indicated the abundant length polymorphism of P3N-PIPO,also the diversity of biological functions.(5)Through yeast two-hybrid library screening,we identified 16 host factors that interact with the SCMV-P3N-PIPO.These host factors involve host defense,transport,and photosynthesis.(5)Through yeast two-hybrid library screening,we identified 16 host factors that interact with the SCMV-P3N-PIPO.These host factors involve host defense,transport,and photosynthesis.