Study Of The Interaction Between PVY 6K2 And The Small Subunit Of RubisCO In Nicotiana Benthamiana

Potato virus Y(PVY)is a single-stranded,positive-sense RNA virus with a wide range of host.It can infect potatoes,peppers,tomatoes and other plants,which has the serious influence of quality and yield,resulting in huge economic losses.The study on the interaction between PVY and its host is helpful to understand the pathogenesis of PVY and provide some theoretical basis for controlling PVY disease.6K2 encoded by PVY induces the formation of replication vesicles that contain viral replication complexes(VRC)and essential for virus replication.In our previous work,the 6K2 protein of PVY was used as bait in the yeast-2-hybrid(Y2H)screenings system to identify proteins in Nicotiana benthamiana with a putative role in PVY infection.Totally,we obtained 13 interacting proteins of 6K2.In this study,we reported the NbRbcS protein,which is a small subunit of Ribulose 1,5-bisphosphate carboxylase/oxygenase(RubisCO)in N.benthamiana.This study aims to confirm the interaction between 6K2 and NbRbcS and reveals the main roles and related mechanisms of NbRbcS protein in PVY infection with multiple molecular biological,biochemical,cell biological and virus biological methods.The main results are as follows:(1)6K2 could interact with NbRbcS.In order to verify the results of yeast-2-hybrid screenings system,NbRbcS was cloned by homologous cloning technology,and phylogenetic analysis was performed.And the protein interaction was confirmed in vitro and in planta by Y2 H and co-immunoprecipitation(Co-IP).(2)PVY 6K2 could change NbRbcS subcellular localization and reduce the activity of RubisCO and the content of chlorophyll.To clarify the role of PVY infection in regulating NbRbcS,the real-time RT-PCR was used to verify that the mRNA level of NbRbcS was increased and then gradually decreased,which suggested that NbRbcS might participate in the immune defense response to PVY.And using RbcS-RFP fusion protein,it was observed by laser confocal microscopy that 6K2 protein could change the localization of NbRbcS protein from chloroplast to endoplasmic reticulum.By spectrophotometry,it was shown that both PVY infection and 6K2 protein expression alone could inhibit the activity of RubisCO enzyme and chlorophyll synthesis.(3)NbRbcS could inhit PVY infection.To further understand the influence of NbRbcS on PVY infection and replication,NbRbcS was slienced by virus-induced gene silencing(VIGS).It was found that NbRbcS-silencing in N.benthamiana produced the chlorosis and vein clearing phenotypes in agroinfiltrated leaves,like the symptoms caused by PVY in primary infection.It proposed a hypothesis that the vein clearing symptoms in primary infection might be related to the down-regulation and afunction of NbRbcS protein.After inoculating PVY,it was found that NbRbcS-silencing promoted PVY infection and replication,and transient overexpression of NbRbcS gene inhibited PVY infection.Taken together,we speculated that in PVY infection,6K2 inhibited the expression of NbRbcS and changed the location of NbRbcS via interaction to reduce RubisCO activity and the synthesis of chlorophyll,thus contributing to infection and vein clearing symptoms.These findings further improve the understanding of the role of 6K2 in PVY replication and infection and reveal the molecular mechanism underlying PVY disease symptom,providing theoretical basis for controlling PVY disease and the development of anti-virus drug in agriculture.