JIA-2018-09

2034 PAN Yuan et al. Journal of Integrative Agriculture 2018, 17(9): 2031–2041 localization of the CaMV movement protein (Linstead et al . 1988) (Fig. 1, P1-GFP), whereas the SVBV P2-GFP protein formed filaments and foci in cytoplasm (Fig. 1, P2-GFP). Previously, it was demonstrated that ectopically expressed CaMV P2 binds strongly in vitro to microtubules (Martiniere et al . 2009), so we presume that the SVBV P2 filament formation was the result of co-localization with microtubules. P3-GFP was completely distributed along the cell boundary (Fig. 1, P3-GFP). Both P4-GFP and P5-GFP produced cytoplasmic signals around the periphery of the cell (Fig. 1, P4-GFP and P5-GFP). The SVBV P6-GFP accumulation appeared to be similar to CaMV P6-GFP by producing a GFP signal that assembled into prominent amorphous cytoplasmic IBs and also was associated with the nucleus (Fig. 1, P6-GFP; Fig. 2). These results suggest that the P6 protein and resulting IBs are important components in SVBV-infected plant tissues. 3.2. P6 accumulates in the nucleus, cytoskeleton and endoplasmic reticulum To investigate P6 nuclear localization, we compared P6- GFP accumulation and DAPI nuclear staining. P6-GFP was first ectopically expressed in N . benthamiana leaves by agroinfiltration, and then the tissue was stained with DAPI and examined by confocal microscopy at 3 dpi. The P6-GFP fusion protein accumulated throughout the nucleus, where it co-localized with DAPI and also formed GFP-labeled IBs were visible in the cytoplasm (Fig. 2-A). To further determine the location of P6 IBs in the cytoplasm, P6-GFP and GFP-ABD2-GFP were co-infiltrated into N . benthamiana leaves. ABD2 is a marker protein that binds to a network of actin microfilaments (Wang et al . 2008). Although P6 and ABD2 were both labeled with GFP, we can easily distinguish the simple geometric P6 morphology from the irregular cytoplasmic microfilaments (Fig. 2-B, b). In addition, co-infiltration of P6-GFP with microtubules marker protein mCherry-MAP65-1, revealed that the P6 IBs aligned with microtubules in N . benthamiana leaves (Fig. 2-B, e). Plant viruses move from the infected cells through plasmodesmata to the healthy cells, the plant ER is coadjacent among cells via the desmotubules across the cell wall in the plasmodesma, forming a continuous network throughout the entire plant (Chen et al . 2012; Stefano et al . 2014). We investigated whether P6 IBs associated with the ER. Transient expression of P6-GFP- and ER-labeled protein by mCherry-HDEL fused to RFP by co-infiltrating N . benthamiana leaves clearly revealed a red web-like meshwork interspersed with green P6 IBs (Fig. 2-C, c). The chloroplast outer envelope protein (CHUP1) is an outer membrane protein that can promote chloroplast targeting to cell membranes (Oikawa et al . 2008; Angel et al . 2013). CaMV P6 was able to interact with CHUP1; therefore, we speculated that SVBV P6 might co-localize with chloroplasts. However, laser scanning confocal microscopy of agroinfiltrated leaves at 3 dpi failed to reveal colocalization of SVBV P6-GFP with the chloroplasts, and that P6 movement failed to correlate with that of the chloroplasts (Appendix A). Taken together, these data show that SVBV P6 is imported into the nucleus, and that P6 IBs co-localize with both the cytoskeleton and the ER. However, the P6 IBs appear not to be associated with the chloroplasts. Fluorescent P1-GFP P2-GFP P3-GFP P4-GFP P5-GFP P6-GFP Bright Merge Fig. 1 Subcellular localization of Strawberry vein banding virus (SVBV) proteins. Each open reading frame (ORF) was fused to GFP and constructs were agroinfiltrated into Nicotiana benthamiana leaves and examined by fluorescence microscopy after 3 days. Left panel, GFP fluorescence; middle panel, bright field photograph; right panel, bright field overlay of GFP fluorescence. P1-GFP formed foci distributed along the cell boundary. P2-GFP formed filaments or foci in the cytoplasm. P3-GFP accumulated entirely at the cell boundary. P4-GFP and P5-GFP produced a signal similar to free GFP. P6-GFP assembled into amorphous inclusion bodies (IBs). Magnification bar for all pictures is 20 μm.