Mechanisms Of Helper Component Protease And Cylindrical Inclusion Protein In The Symptom Development Of Potato Virus Y

Potato virus Y（PVY）which is the representative of Potyvirus,encodes two open reading frame（ORF）cleaved into 11 mature proteins by its own three proteinase.The second mature protein helper component proteinase（HCpro）is a multifunctional protein involved in virus replication,RNA silencing suppression,aphid transmission and symptom development.The pinwheel inclusions formed by the cylindrical inclusion protein（CI）in host cells are one of the most important identification features of Potyviridae viruses.CI is involved in virus replication,cell-to-cell movement,symptom formation,etc.Both HCpro and CI are involved in potyviruses symptom development,but the mechanism is still unclear.Studies have shown that the three-dimensional structure of HCpro is critical for the recognition by the resistant gene,indicating a role of three-dimensional structure in potyviruses symptom development.Sequence analysis reveals a YxxxxLΦmotif which is a eIF4E potential binding motif at the C-terminus of PVY CI.To study the role of HCpro and CI during the virus symptoms formation,we inserted one tag at different positions of HCpro-encoding region based on the PVY^N605 infectious clone,analyzed the effect of inserting tag on HCpro three-dimensional structure and virus symptoms;and revealed the role of YxxxxLΦmotif in virus symptom development by site-directed mutagenesis.The main results are showed as follows:（1）Changes on HCpro three-dimensional structural correlated symptom of PVY.The IDAGGS tag was inserted into S1S2,I252V253 and L313V314 in HCpro of binary transient expression vector pCa-35S:HCpro.The obtained constructs were named HCpro and mutants HCproS₁-S₂,HCproI₂₅₂-V₂₅₃53 and HCproL₃₁₃-V₃₁₄,respectively.The HCpro encoding sequence from HCproS₁-S₂,HCproI₂₅₂-V₂₅₃53 and HCproL₃₁₃-V₃₁₄14 were cloned into PVY^N605 infectious clone by homologous recombination,resultant three PVY mutants named PVY-HCS₁-S₂,PVY-HCI₂₅₂-V₂₅₃,and PVY-HCL₃₁₃-V₃₁₄.Results showed that all the mutants can infect Nicotiana benthamiana plants systemically.At four days post agro-infiltration（dpi）,Green fluorescence was observed in the plants inoculated with mutant PVY-HCS₁-S₂.Compared to wild type PVY,plant inoculated with PVY-HCS₁-S₂ were seriously stunted at 25 dpi.Fluorescence was observed on plants inoculated with PVY-HCI₂₅₂-V₂₅₃53 at 25 dpi.Plants inoculated with PVY-HCL₃₁₃-V₃₁₄14 showed similar symptom to that of wild type PVY.（2）Insertion of IDAGGS tag affects HCpro RNA silencing suppression ability.To know the effect of tag insertion on HCpro RNA silencing suppression ability,the IDAGGS tag was inserted into S1S2,I252V253 and L313V314 in HCpro of binary transient expression vector pCa-35S:HCpro.Results showed that inserting IDAGGS into N-terminal and central region of HCpro can enhance and reduce HCpro RNA silencing suppression ability,respectively.In contrast,inserting IDAGGS into the C-terminal have no significant effect on HCpro RNA silencing suppression ability.These results reveal an important role of HCpro certain sequence or structure during HCpro RNA silencing suppression.（3）Mutations on YxxxxLΦmotif within PVY CI attenuate PVY cell-to-cell movement and virus symptoms.To know the role of conserved amino acid within the potential eIF4E binding motif,the amino acid residues Y and L in PVY CI YxxxxLΦwere mutated to Alanine（A）,and named PVY-CI_Y513AL518A.The PVY^N605 or mutant PVY-CI_Y513AL518A513AL518A was argoinifiltrated into N.benthamiana and N.tabacum.Results showed that,compared to PVY^N605,the virus RNA and CP accumulation of PVY-CI_Y513AL518A513AL518A were reduced.The virus spread ability was weaker than that of PVY^N605.（4）No enough evidence of interaction between PVY CI YxxxxLΦmotif and eIF4E was obtained.Studies have shown that motif YxxxxLΦmotif was conserved in eIF4E binding protein.To reveal the interaction between PVY CI and eIF4E,CI,eIF4E,eIF（iso）4E were cloned into yeast expression vectors,respectively.However,no evidence of interaction between CI YxxxxLΦmotif and eIF4E,eIF（iso）4E was found in yeast two-hybrid experiments.To study the interaction between CI and eIF4E（and eIF（iso）4E）,CI and mutated CI,eIF4E,eIF（iso）4E were cloned into BiFC vectors.Results showed that both CI and mutated CI can interact with eIF4E but not eIF（iso）4E.