Molecular Mechanism Of VAP1 Involved In The Replication Of Potato Virus Y

Replication is a critical biological process involved in plant virus infection.During this process,plant viruses need to hijack specific host proteins for their replication.The process of potato virus Y replication occurs in the virus replication complex（VRC）induced by the second 6K protein（6K2）.In this study,we showed that the vesicular associated membrane protein-associated protein 1（VAP1）interacted with the 6K2 protein of potato virus Y（PVY）,and revealed the molecular mechanism of NbVAP1 involved in the infection of PVY.The main results are as follows:（1）NbVAP1 interacted with PVY 6K2 and involved in the replication of PVY.Y2H,Co-IP,LCI and Bi FC assays showed that NbVAP1 interacted with PVY6K2 in vivo and in vitro.Knock down of NbVAP1 using tobacco rattle virus（TRV）-based silencing system will promote PVY infection.The accumulation of PVY-GFP in N.benthamiana systemic leaves increased and severe symptoms was induced as compared to the control.Overexpression of NbVAP1 significantly reduced the accumulation of replication-defective mutant PVY^Repin N.benthamiana plants.（2）W41 and F42 of PVY 6K2 are key amino acids for the interaction between 6K2and VAP1,and affect the infection of PVY.The results of Y2H,LCI and Bi FC assays showed that the amino acids 36-45 of PVY 6K2 determine the interaction between 6K2 and VAP1.When the amino acids in the key interaction regions were mutated to alanine,the results of Y2H and LCI assays proved that two mutants,PVY 6K2^W41Aand PVY 6K2^F42A,could not interact with wild-type NbVAP1.Western blot detects the accumulation of GFP in system leaves of p Cam0390-PVY^N605,p Cam0390-PVY 6K2^W41Aand p Cam0390-PVY6K2^F42Ainoculated with N.benthamiana for 6 dpi.It was proved that the virus accumulation of mutants was lower than that of control.（3）F15 of NbVAP1 is key amino acid for the interaction between 6K2 and VAP1 and the regulation of PVY infection.The results of Y2H,LCI and Bi FC assays showed that the amino acids 11-20 of NbVAP1 determine the interaction between 6K2 and VAP1.Then the amino acids in the key regions of interaction were mutated to alanine,the results of Y2H and LCI assays proved that the mutant NbVAP1^F15Acould not interact with the wild-type PVY6K2.The result of Western blot showed that the accumulation of GFP in N.benthamiana plants overexpressing NbVAP1^F15Awas significantly higher than that overexpressing NbVAP1after inoculation with PVY-GFP.（4）NbSyp71 interacted with NbVAP1 and involved in the infection of PVY.The results of Y2H and LCI assays showed that NbVAP1 interacted with NbSyp71 in vivo and vitro.Knock down of NbSyp71 using TRV-based silencing system will inhibit PVY infection,and the accumulation level in the systemic leaves of N.benthamiana plants was low as compared to the control.（5）PVY 6K2 affects the interaction between NbVAP1 and NbSyp71.LCI results show that the interaction strength between NbVAP1 and NbSyp71 decreases when PVY 6K2exists.To sum up,after PVY successfully infects the host,PVY 6K2 can interact with NbVAP1to interfere the interaction between NbVAP1 and NbSyp71,thus releasing NbSyp71 for PVY infection.