Function Of The Tobacco CLC-Nt1 Protein In The Infection Process Of Potato Virus Y

Potato virus Y(PVY)seriously threatens solanaceae crops production,investigating the interaction between PVY and its host will provide theoretical basis for studying its pathogenesis and the disease control.Our previous work suggested a probable interaction between CLC-Nt1 protein of Nicotiana tabacum and the 6K2 protein of PVY.In this study,we wish to further reveal the function of CLC-Nt1in the infection process of PVY with multiple biochemical and molecular biological methods,here below are our main results:(1)By segmented clone and nonsense mutation,a PVY necrosis strain(PVY~N)infectious clone(PVY-S)was constructed.PVY-S can infect Nicotiana benthamiana,N.tabacum and Lycopersicon esculentum,showing similar virulence compared with that of the wild PVY virus.Foreign proteins,including GFP,Rosea1 or MtPsy2a were expressed in PVY-S respectively,resulting in convenient supervision of PVY infection progress and providing a useful tool for studying the pathogenesis of PVY.(2)By virus-induced gene silencing(VIGS),CRISPR/Cas9 and complementation assays,CLC-Nt1was demonstrated to be essential for plant growth and development.Expression pattern of CLC-Nt1 was determined by GUS staining,and the result showed that CLC-Nt1 was expressed in root,stem,leaf and flower.Subcellular localization experiment showed that CLC-Nt1 was primarily localized in ER and COP II vesicles.Using a pH sensitive fluorescent protein probe(pHluorin),CLC-Nt1 was proved to contribute to regulate ER luminal pH.Using secGFP(secreted GFP),CLC-Nt1 was proved to participate in the protein secretion pathway.(3)Yeast two hybrid assays,co-localization experiment and co-immunoprecipitation experiment were performed,and the results showed that CLC-Nt1 interacted with PVY 6K2 protein.Using the pHluorin,either PVY infection or expression of 6K2 was found to induce a rise in ER luminal pH.Loss function of CLC-Nt1 by virus-induced gene silencing(VIGS),CRISPR/Cas9 or using chemical inhibitor impaired PVY intracellular replication and systemic infection,indicating that CLC-Nt1 was an essential host factor for PVY infection.It was suggested from these results that PVY could regulate CLC-Nt1 through its 6K2 protein interacting with CLC-Nt1 to induce a raise in ER luminal pH;this raised ER luminal pH was supposed to be essential for PVY to collect component molecules for its intracellular replication and to facilitate its systematic infection.Our research demonstrated for the first time that plant CLC was able to regulate organelle luminal pH,and for the first time that the interaction between PVY virus and its host plant was shown from the view of cellular pH;this was thought of as a new insight for further studies on searching crop anti-virus drug.