Abstract: Little cherry virus 1（LChV-1）was as research subject in this study.The subcellular localization and pathogenicity of P3.5protein were studied by using molecular biology methods,in order to provide reference for the studying of the very small proteins encoded by plant viruses.The results showed that,1)P3.5 was encoded by ORF2of LChV-1,a small hydrophobic protein of unknown function.Primarily,P3.5 was cloned from an isolate of LChV-1from Tai′an,Shandong province using specific primers by RT-PCR,which was 96bp in length and encoded a protein of approximately 3.5kD with a transmembrane domain.2)Subcellular localization analysis by tobacco leaves showed that P3.5localized in cytomembrane and nuclear membrane.3)To further clarify the pathogenicity of P3.5,apotato virus X（PVX）-based vector was introduced and PVX-P3.5 was constructed.The PVX-P3.5was transformed into Agrobacterium tumefaciens GV3101and infiltrated into the N.benthamiana.Slight mosaic symptom was observed in the plants inoculated by PVX14days later.Meanwhile,when the plants inoculated by PVX-P3.5,the plants didn′t have visible symptoms.It demonstrated that heterologous expression of P3.5in N.benthamiana didn′t enhanced the pathogenicity of PVX,and not be related to the symptom-formation.