The Study Of Relationship Between Cell Death Induced By P25 Protein Of Potato Virus X And NbALY916 Protein

Potato virus disease is one of the important factors restricting the potato industry and causing the quality of seed potatoes to be unqualified.Potato virus X(PVX)infects potatoes alone causing a mild symptom,but complexly infects with potato virus Y producing cells necrosis.P25 protein is a multifunctional protein encoded by PVX ORF2.It has nucleotide binding and RNA helicase activity.It is the largest movement protein of TGB and has the function of silencing suppressor.Studies have shown that accumulation of P25 protein is the main pathogenicity determinant responsible for systemic necrosis in PVX-Associated synergisms.P25 induces cell death by endoplasmic reticulum(ER)stress and the unfolded protein response(UPR),which ultimately lead to ER collapse.However,relevant host factors involved in the mechanism of P25-induced cell death have not yet been identified.In this study,we used a double 35 S promoter to construct a vector to express P25 transiently in N.Benthamian and found that the transient expression of P25 through high concentration of Agrobacterium can induce cell death.The cell death is related to the accumulation of H2O2.In the previous study,Co-immunoprecipitation(Co-IP)combined mass spectrometry(MS)technology was used to analyze and screen candidate proteins that interacted with P25.After strict screening conditions,we screened a relevant host factor Nb ALY916.We performed experiments of Bimolecular Fluorescence complementation(BIFC)and Co-IP to confirm the interaction between P25 and Nb ALY916.We discovered that Nb ALY916 is a multifunctional nuclear protein and has three homologous genes in N.Benthamian,namely ALY617,ALY1693,and ALY615.It was found that P25 specifically up-regulates the transcription level of Nb ALY916 by real-time quantitative PCR.When Nb ALY916 expression was reduced by Tobacco rattle virus(TRV)-based virus-induced gene silencing,plants had not been affected on growth and development.The silencing of Nb ALY916 increased the accumulation of P25 and reduced the extent of cell death caused by P25.We found that overexpression of Nb ALY916 triggered more extensive cell death when co-expressed with P25,even though accumulation of P25 was itself reduced by the increased expression of Nb ALY916.Nb ALY916 may degrade P25 through autophagy pathway and proteasome pathway.The expression level of Nb ALY916 was one of the factors which affects the cell death triggered by expression of P25,not related to P25 accumulation.Overexpression of Nb ALY916 could induce the accumulation of H2O2 and cell death.Hence,we hypothesized that P25 induces cell death by specifically up-regulates Nb ALY916.When we rub-inoculated PVX-GFP onto leaves of silent Nb ALY916 plants,no significant changes in fluorescent spots on the inoculated leaves were observed,the number of fluorescent spots on the system leaves increased significantly,the virus system infection rate was significantly accelerated,and the accumulation of CP of PVX-GFP increased.These results suggested that silencing of Nb ALY916 facilitated the systemic infection of PVX in N.Benthamian.In summary,this study identified a host protein Nb ALY916 and found that this protein is involved in P25-induced cell death.Silencing Nb ALY916 relieves P25-induced cell death and promotes systemic infection of PVX.These studies provide a theoretical basis for further understanding of the multifunctionality of P25,and also found that Nb ALY916 is involved in the resistance of plants to viruses.