Functional Study Of NbHAD1 In Turnip Mosaic Virus Infection

Nicotiana benthamiana Haloacid dehalogenase 1(NbHADl)is a halogenacid dehalogenase hydrolase family protein.At present,reports on such family proteins are mostly characterized by their physiological and biochemical aspects,and there are few reports on its functional study against virus infection.To explore the function of NbHADl in virus infection,We have carried out a series of explorations.qRT-PCR method was used to analysis the expression level of NbHADl in Nicotiana benthamiana which was infected by virus.the Target P 1.1 serve predictive software was used to predict the transport peptide of NbHAD1,and its localization in the cell was analyzed by laser confocal microscopy.The NbHAD1 in Nicotiana benthamiana was silenced by TRV-mediated silencing technique,we injeced Turnip Mosaic Virus and other genus virus such as Potato Virus X which was a member of Potexvirus and Pepper Mild Mottle Virus which was a member of Tobamovirus to study the role of NbHAD1 in the process of virus infection.In order to explore whether the silenced NbHAD1 have an impact on virus RNA accumulation,PEG-mediated method was used to transfect the viral plasmid into protoplast that NbHAD1 was silenced.Finally,the interaction between NbHAD1 and viral proteins was verified by bimolecular fluorescence complementation,co-immunoprecipitation and yeast double hybridization experiments.After infecting TuMV,PVX and PMMoV,the expression level of NbHAD1 was reduced.NbHAD1 was located in the chloroplast,after removing its transit peptide,it located in the nucleus and cell membrane.When NbHAD1 was silenced,the resistance of Nicotiana benthamiana to TuMV was immune,the infection of Pepper Mild Mottle Virus and Potato X virus to Nicotinan benthamiana was also inhibited.The Nicotiana benthamiana that NbHADl was silenced inhibited the accumulation of TuMV RNA,and the transport of TuMV from injected leaves to systenatic leaves in Nicotiana benthamiana that NbHAD1 was silenced was also significantly inhibited.TuMV protein 6k2 interacted with NbHAD1.6k2 was a key membrane virus protein that caused endometrial rearrangement after TuMV infection,which induced the production of vesicles in the host,The vesicles will target chloroplast and lead to chloroplast aggregates and the formation of chloroplast-bound 6K2 elongated tubular structures,which have been showed the site of the replaction of potyvirus.We first discovered that the protein NbHAD1,which belongs to the family of halogenated dehalogenase hydrolase,plays a role in the process of virus infection.We proved that NbHAD1 is localized and interacts with TuMV protein 6k2 in chloroplast.Silencing NbHAD1 impairs the accumulaction of virus RNA.Therefore,we hypothesize that NbHAD1 that localized in chloroplast plays an important role in TuMV replication,and positively regulates TuMV infection through its interaction with 6k2 protein.