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Rice stripe virus (RSV) is the type member of the genus Tenuivirus.The genome of RSV is composed of four negative-sense single-stranded RNA segments, designated RNA 1, 2, 3 and 4.RNA 1 is of negative polarity, encoding the putative RNA dependent RNA polymerase.The other three segments adopt an unusual ambisense coding strategies i.e.both the viral-sense RNA and viral complementary-sense RNA possess coding capacity.To determine the subcellular localizations of proteins of RSV (without RdRp), we constructed six ORFs of RSV (NS2, NSvc2, NS3, CP, NSvc4, and SP) in pEarleyGate 101 and pEarleyGate 104 vectors by Gateway technique to yield N-terminal or C-terminal YFP fusion proteins.The subcellular localization of six proteins of RSV fusion with YFP was observed by confocal microscopy at 2 days after agroinfiltration in the leaves of Nicotiana benthamiana, respectively.We found the NS2-YFP formed punctate structures at the periphery of cell or within nucleus in N.benthamiana leaves.Immunoelectron microscopy confirmed that NS2 of RSV distributed along cell wall in RSV-infected wheat after labeling by RSV NS2 antibodies.The membrane protein NSvc2-YFP formed distinct vesicles throughout the cytoplasm.NS3-YFP and SP-YFP were diffuse distributing in cytoplasm and nucleus.The transient expression of CP-YFP led to formation punctate structures around chloroplast.The NSvc4-YFP led to formation punctate structures targeting to plasmodesmata (PD), corresponding to its MP function.