Function Of P4 Protein Encoded By Pepper Vein Yellows Virus

Pepper vein yellows virus(PeVYV),a single-stranded RNA(+ssRNA)virus and a identified member belongs to genus Polerovirus(family Luteoviridae).The symptoms of vein-chlorosis,dwarf and abnormal leavies were appeared in infected plants.The severe yield loss would be resulted by this virus for decreasing the photosynthetic efficiency in infected plants.The one of critical steps of virus successful infecting host plant is viral cell-to-cell movement through plasmodesmata(also called short-distance movement)from infected cells to infect adjacent cells.The diameter of plasmodesmata was far smaller than that of virus particle,for overcoming the barrier of plasmodesmata in virus short-distance movement,the viruses generally encode movement proteins,to interact with plasmodesmata,guide viruses short-distance movement througth plasmodesmata.Pepper vein yellow virus encoding P4 protein,which predicted as its movement protein,but there was no experimental verifications untill now.So the mechanism of cell-to-cell movement of PeVYV remains ambiguously.In this study,PeVYV P4 gene was recombined into plant express vector using gateway recombinant method.The PeVYV P4 gene mutants(Transmember region deletion mutant ?P4127-129 and Ala replacement mutant ?P4 AAA 127-129)were also generated by overlap PCR combined with gateway recombinant method.All of constructed plant expressing vectors were transferred into Agrobacteria and infiltrated into Nicotiana benthamiana,the fluorescence was observed and photos were taken by confocal laser scanning microscope.Subcellular localization of P4 protein showed that P4 protein was localized on the cell membrane and could be localized in plasmodesmata,forming typical discontinuous fluorescent dots.Subcellular localization of P4 protein transmembrane domain of 127-129 aa mutants showed that 2 mutants(Ala replacement mutant and deletion mutant)could still locate on cell membrane,nevertheless,both mutant proteins also localize in cytoplasm,and the fluorescence dimensionality localized on cell membrane was decreased than P4 protein,indicated that the 127-129 aa of P4 protein was its transmember region,and transmembrane region of P4 protein may be contained the 117-138 aa,which was agreement with software "TMpred" predicted,but it needed further verification.Compared with YFP and PDL protein(Plasmadosmata localization marker protein),the fluorescence region of P4-YFP was extended bigger,indicated that P4 protein could drive YFP gene cell-to-cell movement.Taken together,the one of key functions of P4 protein of PeVYV was driving viral cell-to-cell movement through plasmdosmata.