JIA-2018-09

2036 PAN Yuan et al. Journal of Integrative Agriculture 2018, 17(9): 2031–2041 Fig. 3 P6 inclusion body (IB) trafficking along microfilaments and colocalization with P1. A, P6 IB trafficking along GFP-ABD2-GFP- labeled microfilaments over 48 s. The positions of the motile IBs at each time point are marked with white arrows. B, colocalization of P6 IBs and P1 along the cell boundary, IBs located along the cell boundary are marked with white arrows. a, P1-GFP; b, P6- RFP; c, overlay of a and b. Arrows identify co-localization positions. Magnification bar equals 50 μm. A B 0 s a a b c b c d e 12 s 24 s 36 s 48 s 3.5. The P6 25 aa C-terminal region is required for facilitating expression of exogenous gene gfp To explore the biological function of P6 derivatives, a GFP plasmid containing a 35S promoter for GFP expression was coinfiltrated with P6 or the M1 and M2 protein mutants into N . benthamiana leaves. At 3 dpi, the intense fluorescence of N . benthamiana leaves agroinfiltrated with P6 and the M1 mutant was consistent with the TBSV P19 control, indicative of strong suppression of host silencing (Fig. 5-A). However, leaves agroinfiltrated with M2 exhibited very weak fluorescence similar to agroinfiltrations with the empty vector plasmid, showing that the P6 25 aa C-terminal region is required for facilitating expression of exogenous gene gfp . Analysis of Northern and Western blotting also showed that the RNA and protein levels of GFP corresponded with the fluorescence in the respective N . benthamiana leaf infiltrations (Fig. 5-B and C). To further determine whether the SV40 NLS region could restore the ability for facilitating exogenous expression of M2, the M5 mutant containing the SV40 NLS and GFP plasmids were agroinfiltrated into N . benthamiana leaves, and the fluorescent intensity evaluated. However, leaves infiltrated with M5 and M2 exhibited the same low levels of fluorescence (Appendix B). These results show that the NLS of SV40 could not restore the ability for facilitating exogenous expression of M2, despite functioning to import M2 into the nucleus. 4. Discussion In this study, we have evaluated several properties of the SVBV P6 protein. Our results show that SVBV P6 protein is imported into the nucleus and forms mobile IBs that are associated with ER, microfilaments and microtubules and that the IBs move along microfilaments to the SVBV P1 protein in the PD. The P6 protein contains one NLS region and that the C-terminal NLS is not only a key region required for IB formation, but also is required for the P6 facilitating exogenous gene expression. Our research also has implications for IB transport and interactions with the P1 protein that may affect virus movement and pathogenesis mechanisms. IBs had been found in many virus-infected plant tissues. The members of Potyviridae family share a unique feature in production of pinwheel-shaped IBs in the cytoplasm (Otulak and Garbaczewska 2012). These pinwheel IBs are composed of the potyvirus-encoded cylindrical inclusion (CI) helicase protein, which is believed to be involved in virus replication and long-distance transport (Sorel et al . 2014). TMV-infected cells contain a large amount of crystalline IBs that function as replication complexes and move to adjacent cells through PD to accomplish cell-to-cell movement (Kawakami et al . 2004; Liu et al . 2005). The closely related CaMV IBs are responsible for virus replication and assembly in infected cells (Angel et al . 2013). We identified the subcellular localization of proteins encoded by SVBV by ectopic expression in N . benthamiana leaves. The major result of our experiments showed that the P6 protein can be distinguished from other SVBV proteins by assembling into amorphous variable sized IBs that are similar to those formed by CaMV P6. It is pertinent to note that IBs formed by P6 is an important component of IBs in SVBV-infected tissue. Therefore, we assumed that the IBs in SVBV-infected