JIA-2018-09

2032 PAN Yuan et al. Journal of Integrative Agriculture 2018, 17(9): 2031–2041 China, where it causes severe strawberry losses (Baker et al . 2014). SVBV is transmitted by aphids in a semi- persistent manner or by vegetative propogation. SVBV strawberry infections can elicit chlorotic leaf veins, reduce numbers of creeping stem growth, and interfere with fruit development (Vaskova et al . 2006). SVBV is a member of the genus Caulimovirus of the family Caulimoviridae with an approximately 7.8-kb circular double-stranded DNA that contains seven open reading frames (ORFs) whose positions are identical to those of corresponding cauliflower mosaic virus (CaMV) ORFs (Petrzik et al . 1998). Therefore, the CaMV-encoded proteins and their functions provide a good model for the roles of the SVBV-encoded proteins. In this regard, the CaMV ORF I encodes P1, a protein involved in virus intracellular movement and delivery of the virion within the host (Carluccio et al . 2014). ORF II produces the P2 protein that promotes aphid transmission (Bouchery et al . 1990), ORF III encodes a virion associated protein (Leclerc et al . 1998, 2001) that interacts with the P2 and P1 proteins to mediate aphid transmission and cell to cell spread of the virions, respectively (Leh et al . 2000; Stavolone et al . 2005). ORF IV encodes the major viral coat protein (Petrzik et al . 1998), and ORF V expresses P5, a reverse transcriptase protein that functions in viral DNA replication (Haas et al . 2002). The CaMV P6 ORF-encoded protein is the major viral protein found in infected plants and has multiple functions involved in virus replication (Schoelz et al . 1986). In contrast, the ORF VII product has no known function and has not been found in infected plants (Schoelz et al . 2016). Extensive analyses have shown that CaMV P6 is a multifunctional nucleocytoplasmic protein containing nuclear export and import domains (Haas et al . 2005; Haas et al . 2008). First, CaMV P6 is a translational transactivator that specifically regulates synthesis of other CaMV proteins encoded by the polycistronic 35S RNA and it interacts with the L18 protein from 60S ribosomal subunits (Leh et al . 2000). Second, the P6 protein is involved in elicitation of disease phenotypes, and P6 transgenic Arabidopsis plants display viral-like symptoms (Baughman et al . 1988). CaMV P6 is also a potent suppressor of host gene silencing suppressor (Love et al . 2007). A portion of the P6 protein is transported into the nucleus where it has been shown to interfere with production of small interfering RNAs (siRNAs) (Haas et al . 2008; Shivaprasad et al . 2008). When P6 transgenic Arabidopsis was crossed with GFP-silenced transgenic Arabidopsis , the progeny showed strong GFP fluorescence (Love et al . 2007). In addition, P6 interferes with disease resistance by acting as a pathogenicity effector that modifies the natriuretic peptide receptor 1 (NPR1), a key regulator of salicylic acid (SA)- and jasmonic acid (JA)- dependent signaling and inhibit SA-dependent defense responses (Love et al . 2012). In addition to the functions described above, CaMV P6 is also a component of the amorphous, electron dense cytoplasmic inclusion bodies (IBs) that are typically found in caulimovirus infected cells (Baughman et al . 1988; Angel et al . 2013). P6-containing IBs are likely “virion factories”, since they are primary sites for CaMV protein synthesis, genome replication and virion assembly (Rodriguez et al . 2014). SVBV-infected strawberry cells also contain cytoplasmic IBs with embedded 40–45 nm spherical virions that are similar to the structures produced in other caulimovirus- infected cells (Kitajima et al . 1973). Considering that the formation, composition and the biological functions of SVBV IBs have not been defined. Therefore, to provide more information about the roles of SVBV proteins during infection, we fused GFP to each SVBV ORF and transiently expressed each reporter protein in Nicotiana benthamiana . We show here that only the P6 fusion protein accumulated in the nucleus. P6 also formed IBs and P6-GFP associated with the cytoskeleton and the endoplasmic reticulum (ER), and trafficked along microfilaments to co-localize with the P1 protein at periphery of the cell. In addition, our data demonstrate that both IB formation and nuclear localization require a NLS located within the C-terminal 25 amino acids (aa) of P6. We utilized the Simian virus 40 (SV40) nuclear localization sequence (NLS) region to substitute for the 25 aa of P6. This chimeric P6 protein was imported into the nucleus, but lacked the ability to form cytoplasmic IBs. The results from co-infiltration of GFP with P6 or its mutants provides evidence that the 25 aa region is required for P6 facilitating of exogenous GFP expression. 2. Materials and methods 2.1. Plasmids and plants The binary vector pCAM2300 was used for all constructs involving in SVBV ORFs and P6 mutants (M1, M2, M3, M4 and M5). To generate plasmids for transiently expressing fusion proteins of each DNA fragment with gfp , the corresponding regions were amplified by PCR with specific primers using the SVBV infectious clone as a template. We first cloned the gfp gene into pCAM2300 at the Bam HI/ Sal I sites to produce a recombinant plasmid pCAM-GFP, then the PCR products of the ORFs or P6 mutants were cloned into pCAM-GFP at the Knp I/ Bam HI sites, respectively. The microfilament labeled fimbrin actin-binding domain 2 ( ABD2 ) spans a region between 1 050–2 061 nt of the Arabidopsis fimbrin1 gene. We used special primers to amplify ABD2 and cloned it into the pCAM-GFP plasmid at the Knp I/ Bam HI sites. Subsequently, we inserted the gfp gene into