JIA-2018-09

2038 PAN Yuan et al. Journal of Integrative Agriculture 2018, 17(9): 2031–2041 P19 P6 M1 Vector M2 35S:GFP+ M P19 P6 M1 M2 Vector P19 A C B P6 M1 M2 Vector Fig. 5 Facilitating exogenous gene expression of P6 and P6 mutant proteins in Nicotiana benthamiana. A, GFP accumulation in leaf discs at 3 days post infiltration (dpi) after agroinfiltration with the 35S GFP plasmid and plasmid constructs designated above the plants. P19 is the tomato bushy stunt P19 suppressor protein. The red color is chlorophyll auto-fluorescence under UV and the green color represents the fluorescence from GFP. B, accumulation of GFP detected by Western blotting with GFP antiserum (upper panel). Total Rubisco protein stained by Coomassie brilliant blue was shown in low panel. C, accumulation of GFP mRNA detected by Northern blotting (upper panel). Gel electrophoresis of 28S RNA was shown in lower panel. both inhibit intracellular and intercellular movement of TMV (Liu et al . 2005). Therefore, these results demonstrate that microfilaments are associated with virus movement and infection. IBs of SVBV P6 moved along actin microfilaments, and co-localized with the SVBV P1 protein at the periphery of the cell. The SVBV P1-GFP is similar to the MPs of other viruses in that it forms foci distributed along the cell boundary. Sequence analysis showed that the SVBV P1 protein has several conserved domains similar to other plant virus MPs (Petrzik et al . 1998), suggesting that P1 may act as a MP. Therefore, we propose a model for SVBV intracellular movement in which IBs composed of P6 proteins facilitate virion trafficking along microfilaments to the cell boundary, where they interact with the P1 protein to facilitate intercellular transport of virons across modified PD. Several studies have shown that microtubules are involved in the transfer of plant viruses. In the early stages of TMV infection of N . benthamiana protoplasts, IBs are distributed in the cytoplasm or on the cell border at 29°C. However, the IBs were concentrated around the nucleus rather than being transported to the cell surface at 4°C, owing to microtubule destruction at low temperatures (Mas and Beachy 2000), suggesting that microtubules participate in the intracellular transport of TMV IBs. CaMV P6 IB associations can increase microtubule stability in the presence of disruptive chemical reagents (Harries et al . 2008). IBs of African swine fever virus (ASFV) disperse when cells are incubated with drugs that depolymerize microtubules (Christopher et al . 2007), suggesting the formation of IBs involves microtubule motors. We therefore suggest that SVBV P6 aligned with microtubules may be associated with IB formation, microtubule stability and virion transit. The plant secretory pathway is specialized for the synthesis, transport and modification of proteins and other bio-macromolecules, and is composed of a complex membrane network of organelles, including the ER, vesicles and Golgi network, etc. (Hanton et al . 2005). Moreover, recent studies indicate that the host secretory pathway can be hijacked by plant viruses for transport to PD and subsequent intercellular transport (Patarroyo et al . 2012). TMV IBs do not localize at the PD if the ER network is destroyed by the antagonist brefeldin A (BFA). The movement of 6K2 and the cell to cell movement of TuMV (Grangeon et al . 2012) and the PD movement of P3N- PIPO and CI proteins of other potyviruses were abolished in infected cells treated with BFA (Patarroyo et al . 2012). Considering that ER tubules traversed through the PD to neighboring cells and that P6 is also associated with the ER,