Analysis of Subcellular Localization and Pathogenicity of Plum Bark Necrosis Stem-Pitting Associated Virus Protein P6

Infection of plum bark necrosis stem pitting associated virus (PBNSPaV) has been reported in many prunus species in several countries, causing signi�cant economic losses. The very small proteins encoded by plant viruses are often overlooked due to their short sequences and uncertain signi�cance. However, numerous studies have indicated that they might play important roles in the pathogenesis of virus infection. The role of small hydrophobic protein P6, encoded by the open reading frame 2 of PBNSPaV, has not been well explored. In this study, we ampli�ed the P6 fragment from a PBNSPaV isolate by RT-PCR using speci�c primers and found that it is 174 bp long and encodes a protein of approximately 6.3 kD with a transmembrane domain. Subcellular localization analysis of P6 proteins in tobacco leaves showed that P6 localizes in cytomembrane and nuclear membrane. To further clarify the pathogenicity of P6 proteins, a PVX-P6 expression vector was constructed by inserting the p6 fragment into a potato virus X (PVX)-based vector and transformed into Agrobacterium tumefaciens GV3101. In�ltration of N. benthamiana with the PVX vector-transformed A. tumefaciens led to slight mosaic symptoms at 14 days of post-inoculation. Meanwhile, in�ltration with the PVX-P6 vector-transformed A. tumefaciens resulted in no signi�cant symptoms. These results demonstrated that heterologous expression of P6 in N. benthamiana could not enhance the pathogenicity of PVX. Our study indicates that p6 may not be a potential pathogenic factor associate with the causing of symptoms, and mode of action of PBNSPaV-P6 protein remain to be further studied.