Study On The Interaction Between Apple Chlorotic Leaf Spot Virus Movement Protein And Pear Proteins And Biological Function

Apple chlorotic leaf spot virus(ACLSV)is one of the important viruses infecting some pome fruit trees such as pear(Pyrus spp.)and apple(Malus spp.),and causes chlorotic spots on the leaves of susceptible pear and apple trees.ACLSV is a typical species of the genus Trichovirus in the family Betaflexiviridae of the order Tymovirales.The genome of the virus is a positive-sense single-stranded RNA of 7.5-8.0 kb,containing 3 partially overlapping open reading frames,in which ORF2 encodes movement protein(MP)belonging to the “30K” superfamily.The identification of host proteins interacting with viruses can provide important information for understanding the biological characteristics and target of viruses.In this study,the pear proteins interacting with ACLSV MP were screened by yeast twohybrid method,and the interaction results were further verified and the interaction characteristics were analyzed.The main results were as follows.(1)Using pGBKT7-MP as the bait vector,39 pear proteins interacting with MP were preliminarily screened from the cDNA library of Hongxiangsu pear,mainly including proteins related to ubiquitination,phosphorylation,photosynthesis,glucose metabolism,purine metabolism,amino acid biosynthesis,biosynthesis of secondary metabolites,protein processing and catabolism,signal transduction and transcriptional regulation.According to the results of bioinformatics analysis of candidate proteins,12 pear proteins were selected and further analyzed by yeast two-hybrid,bimolecular fluorescence complementation and luciferase complementation assay.It was confirmed that BOI-related E3 ubiquitin-protein ligase 2(E3),probable protein phosphatase 2C 47(PP2C),cyclin-P3-1-like(Cyc),NADPdependent sorbitol-6-phosphate dehydrogenase(Nd S6PDH),thaumatin-like protein 1b(TLP),chorismate mutase 1(CM),chlorophyll a-b binding protein CP24A(CP24)and chlorophyll a-b binding protein 6(LHCB6)could interact with MP.The bimolecular fluorescence complementation showed that the interaction positions were all plasmodesmata.(2)The subcellular localization of MP and its interacting proteins were analyzed.The MP was localized in the plasmodesmata.E3 was localized in the nucleus and PD.PP2 C,Nd S6 PDH,Cyc and TLP were localized in the nucleus and cell membrane,and PP2 C and Nd S6 PDH were also localized in the endoplasmic reticulum.CM,CP24 and LHCB6 were located in chloroplast in an uneven granular form.Except for LHCB6,these pear proteins all co-localized with MP at plasmodesmata.At the same time,it was identified that the plasmodesmata localization signal of MP was located at 11-201 aa at the N-terminus,the key region where MP can interact with itself was 19-312 aa,and the key amino acid sequence of MP interacting with pear proteins CP24 and Cyc was 11-201 aa.(3)The biological functions of MP and these interacting proteins were preliminarily studied.It was found that MP interacted with proteins NbCP24,NbLHCB6 and NbCyc from Nicotiana benthamiana.Based on virus-induced gene silencing,the expression of genes NbLHCB6 and NbCyc in Nicotiana benthamiana were down-regulated without obvious phenotypic changes,while the down-regulated expression of gene NbCP24 caused chlorosis and mosaic in tobacco leaves.In summary,this study identified 8 pear proteins that interact with ACLSV MP,clarifying the subcellular sites and potential biological functions of the interacting proteins.