The Interaction Between TuMV VPg And NbCat1 In Virus Infection

Turnip mosaic virus(TuMV)is a Potyvirus of the family Potyviridae that causes damaging disease in many kinds of economically important cruciferous vegetable all over the world.Infected plants,especially the natural hosts,show symptoms such as chlorotic local lesions,mosaic,mottling,puckering or rugosity.VPg(Viral Protein genome-linked)is an important non-structural protein encoded by Potyvirus genome,which plays a key role in viral RNA replication and translation by covalently binding with the 5 ’end of viral genome or interacting with some host proteins.However,whether VPg is involved in symptom induction and development remains unclear.In this study,we demonstrated that VPg encoded by TuMV is involved in systemic necrosis and reactive oxygen species(ROS)generation in Nicotiana benthamiana.Furthermore,we showed that TuMV VPg could directly interact with a catalase 1(CAT1)N.benthamiana in nucleus to facilitate viral infection.The main results are as follows:(1)The ectopic expression of TuMV VPg using the PVX-based vector promotes systemic necrosis and reactive oxygen species(ROS)accumulation in N.benthamiana.The expression of recombinant TuMV VPg from the PVX-based vector was identified using RT-PCR Strep tag affinity chromatography and western blot.The ectopic expression of TuMV VPg caused severe systemic necrosis in N.benthamiana.Moreover,the overexpression of TuMV VPg resulted in accumulation of reactive oxygen species(ROS)using Hydrogen peroxide(H2O2)detection.These results demonstrated that VPg might be a pathogenicity determinant that plays key roles in symptom development of TuMV.(2)TuMV VPg alters the cellular location of NbCatl by their interactionNbCatl interacting with TuMV VPg was identified using yeast two-hybrid system and bimolecular fluorescence complementation(BiFC).BiFC analysis of two deletion mutants of TuMV VPg demonstrated that the C-terminal(residues 121 to 192)fragment of TuMV VPg were necessary for this interaction.Then we investigated whether the interaction between TuMV VPg and NbCatl alters the original localization of TuMV VPg or NbCatl.The result showed that NbCatl localized in the peroxisome was recruited to the nucleus by an interacting with TuMV VPg.(3)Silencing of NbCatl promotes TuMV infection which leads to accumulation of ROS and development of necrotic lesions in N.benthamiana.To further explore if NbCatl is required for TuMV infection,we used the Tobacco rattle virus(TRV)-based virus induced gene silencing(VIGS)system to silence NbCatl in N.benthamiana.Silencing of NbCatl was confrmed by qRT-PCR,and NbCat1-silenced plants in displayed Necrosis symptoms and detected the overproduction of ROS.Then NbCat1-silenced and control(TRV2:△ GFP/TRV1)plants were challenged with TuMV6K2-GFP//mCherry,as a result the former displayed more severe symptoms and stronger GFP green fluorescence in comparison to the latter.qRT-PCR confirmed that the viral genomic RNA levels of TuMV in both the upper new leaves of the NbCat1-silenced plants were significantly higher than those in the corresponding counterparts of the control plants.Taken together,these results suggest that interactions between TuMV VPg and NbCatl might play a crucial role in TuMV infection and pathogenicity.