Strawberry vein banding virus P6 protein intracellular transport and an important domain identification

Abstract Strawberry vein banding virus (SVBV)-infected strawberry cells contain cytoplasmic inclusions with isometric particles. To identify the components of the inclusions, green fluorescent protein (GFP) was fused to the carboxy-terminus (C-terminus) of SVBV open reading frames, these constructs were separately transformed into Agrobacterium tumefaciens and infiltrated into Nicotiana benthamiana leaves. Results showed that the SVBV P6 protein assembled into prominent and amorphous inclusion bodies (IBs). To investigate P6 subcellular localization, P6-GFP was ectopically expressed in N. benthamiana leaves by agroinfiltration and then stained with 4’,6-diamidino-2-phenylindole (DAPI). We found the P6 protein accumulated in the nuclei and also formed cytoplasmic IBs with different sizes. To further determine the location of P6 IBs in the cytoplasm, and explore whether the P6 IBs move freely or depend on cytoskeleton and endoplasmic reticulum (ER), the microfilament marker protein (GFP-ABD2-GFP), microtubules marker protein (mCherry-MAP65-1) and ER marker protein (mCherry-HDEL) were separately coexpressed with P6-GFP and into N. benthamiana leaves by agroinfiltration, exhibiting that P6 IBs aligned with cytoskeleton and endoplasmic reticulum. Meanwhile, coinfiltration of P1 and P6 indicated the P6 colocalized with the P1 protein at periphery of cells. The P6 protein contains one C-terminal nuclear localization signal (NLS) region, a P6 protein mutant with a deleted NLS did not localize in the nucleus, did not form IBs, and was unable to facilitate exogenous GFP expression. These results demonstrate that the deleted NLS region is an important P6 domain required for biological functions. In summary, the mobile P6 IBs are associated with ER, microfilaments and microtubules and move along microfilaments to the SVBV P1 protein in the PD.