PAN Yuan, ZHOU Xiu-hong, Ll Shuai, FENG Ming-feng, SHl Man-ling, ZUO Deng-pan, JlANG Xizi, CHEN Jing, HU Ya-hui, ZHANG Xiang-xiang, JlANG Tong

1 Schoolof Plant Protection, Anhui Agriculturaluniversity, Hefei 230036, P.R.China

2 Biotechnology Center, Anhui Agriculturaluniversity, Hefei 230036, P.R.China

3 College of Life and Environmental Sciences, Hangzhou Normaluniversity, Hangzhou 310000, P.R.China

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.

Keywords: SVBV, IBs, intracellular transport, cytoskeleton, ER, P6 mutant

1. lntroduction

Strawberry vein banding virus (SVBV) is an important virus infecting strawberry (Fragaria spp.) (Stenger et al.1998). Since 1978, the European and Mediterranean Plant Protection Organization (EPPO) has listed SVBV as a quarantine pest. The virus has also been reported in Australia, America, Asia, Africa and Europe, as wellas in China, where it causes severe strawberry losses (Baker et al. 2014). SVBV is transmitted by aphids in a semipersistent 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 cauli flower 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 caulimovirusinfected 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. Wefirst cloned the gfp gene into pCAM2300 at the BamHI/SalI 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 KnpI/BamHI sites, respectively. The microfilament labeledfimbrin 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 KnpI/BamHI sites. Subsequently, we inserted the gfp gene into the 5´-terminus of ABD2 at the KnpI site. The microtubules marker protein (mCherry-MAP65-1) and ER marker protein(mCherry-HDEL) were kindly provided by Prof. Tao Xiaorong from Nanjing Agriculturaluniversity, China.

Seeds of N. benthamiana were germinated for 1 week at 22°C in a constant temperature incubator, and 2 weeks later the seedlings were transplanted and grown in a greenhouse under a 12 h light and 12 h dark cycle at 22°C with routine watering and fertilization. After 3-5 weeks when N. benthamiana reached the ten leaf stage, the upper emerged leaves were used for Agrobacterium infiltration.

2.2. Transient infiltration and DAPl staining

Agrobacterium transformed with SVBV ORFs or P6 mutants was diluted to OD=1.0 with MMA buffer (10 mmoL L-1MgCl2, 10 mmoL L-1MES (pH 5.6) and 100 μmoL L-1acetosyringone). After 2-3 h, N. benthamiana leaves were agroinfiltrated with Agrobacterium strains harboring the SVBV derivatives with ORFs or P6 mutants, respectively.For co-infiltrations with P1 and labeled P6 or P1 and the P6 mutant combinations, Agrobacterium strains were mixed at ratios of 3:5 respectively.

For visualization of nuclei, lower epidermal N. benthamiana leaf strips were excised on a microscopic slide, and then the unfold epidermis was soaked in 1 μg mL-1DAPI for 15 min with gently shaking. The stained epidermis was washed with water six times and visualized by confocal microscopy.

2.3. Confocal microscopy

Agroinfiltrated N. benthamiana leaf strips were mounted in water under a coverslip, and images were acquired on an OLYMPUS FluoView™ FV1000 Confocal Imaging System (Hamburg, Germany). RFP was excited at 525 nm and images were captured at 598 nm. GFP was excited at 488 nm and captured at 522 nm. For double labeling experiments, the green and red channels were imaged separately and then superimposed. For time-lapse microscopy, images were obtained every 6 or 10 s from a single optical plane. Images were processed using Adobe Photoshop Software.

2.4. RNA and protein extraction

Leaves co-infiltrated with P6 or P6 mutants and GFP were harvested at 3 days post infiltration (dpi). Total RNA was extracted from leaf tissue using Easy Pure Plant RNA Kit(Tiangen, China). Agarose gel electrophoresis was used to assess the RNA quality, and samples with minimal RNA degradation were used for Northern blot analysis. Proteins were extracted from leaf samples with 3 mL PBS extraction buffer (137 mmoL L-1NaCl, 2.7 mmoL L-1KCl, 10 mmoL L-1Na2HPO4, 2 mmoL L-1KH2PO4) per gram fresh leaf material.Insoluble materials were separated by centrifugation(10 min, 12 000×g, 4°C) and the lysate was collected for Western blot analysis.

2.5. Northern blot analysis

Equalamounts of RNA (5 μg) mixed with an equal-volume of 2× loading buffer were loaded on gel lanes and separated in a 1.0% agarose gel. The RNA was transferred to a nylon membrane (Amersham, Little Chalfont, UK) in 20×SSC. GFP RNA probes were amplified by PCR with specific primers and then labeled with DIG (Labeling and Detection Starter Kit II, Roche, Switzerland). For labeling reactions, 1 μg of purified GFP DNA was mixed with 4 μL DIG-HIGH prime in a 20-μL reaction system at 37°C for 20 h. Prehybridization, hybridization, washing and detection of hybridizing bands were carried out according to the manufacturer’s protocolof Labeling and Detection Starter Kit II (Roche, Switzerland).

2.6. Western blot analysis

For GFP detection, 30 μL lysate was loaded into SDS-PAGE to separate total proteins and the proteins were transferred to a Pure Nitrocellulose Blotting Membrane (PALL) with Trans-Blot SD Semi-Dry Transfer Cell 170-3940 (Bio-Rad,USA) at 15 V for 15 min. GFP antibody of Boster Biological Technology was used at 1:5 000 dilution. The membranes were stained with BCIP/NBT.

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3. Results

3.1. The SVBV P6-GFP fusion protein forms lBs

IBs have been found in many viruses-infected cells and their components provide important clues about replication,movement and host defense (Novoa et al. 2005; Wileman 2006; Netherton et al. 2007; Moshe and Gorovits 2012). The P6 protein and icosahedral virus particles are the predominant IBs constituents in CaMV-infected cells (Baughman et al.1988). To determine which SVBV-encoded proteins form IBs, we fused GFP to the carboxy-terminus (C-terminus)of each SVBV ORF, and the corresponding constructs were transformed into Agrobacterium tumefaciens and agroinfiltrated into N. benthamiana leaves. Subsequently,the agroinfiltrated areas were examined by laser scanning confocal microscope at 3 dpi.

The SVBV-GFP proteins varied in their cytoplasmic and nuclear distribution. P1-GFP formed foci that were distributed along the cell boundary, which is similar to the localization of the CaMV movement protein (Linstead et al.1988) (Fig. 1, P1-GFP), whereas the SVBV P2-GFP protein formedfilaments 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 P2filament 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 wasfirst 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 to gether, 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.

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, brightfield photograph; right panel, brightfield overlay of GFP fluorescence. P1-GFP formed foci distributed along the cell boundary. P2-GFP formedfilaments 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.

3.3. P6 lBs traffic along microfilaments and colocalizes with P1 at the cell periphery

Fig. 2 P6 inclusion body (IB) accumulation in the nucleus, and colocalization with the cytoskeleton and endoplasmic reticulum(ER). Transient expression was performed in Nicotiana benthamiana leaves via Agrobacterium infiltration. A, P6 import into the nucleus. a, P6-GFP; b, DAPI stained nucleus;c, overlay of a and b. B, P6 IB and cytoskeleton colocalization.P6 IBs were marked with the red arrowheads. a, GFP labeled microfilaments; b, coexpression of P6-GFP with GFP-ABD2-GFP; c, P6-GFP; d, mCherry labeled microtubules, mCherry-MAP65-1; e, overlay of c and d. C, P6 IBs co-localized with ER. a, P6-GFP; b, mCherry labeled ER, mCherry-HDEL; c,overlay of a and b. Magnification bar for all images is 50 μm.

CaMV P6 is thought to be involved in the transport of virus particles, and CaMV P1 is also considered to be a movement protein (Baughman et al. 1988). P6 and P1 also interact in vitro as determined by yeast two-hybrid and pull-down experiments (Hapiak et al. 2008). In SVBV,laser scanning confocal microscope showed that at 3 dpi,SVBV P6 IBs co-localized with SVBV P1 at periphery of the cell (Fig. 3-B). Therefore, we speculate that P6 moves along the microfilament network to interact with P1 at the plasmodesmata (PD).

3.4. The P6 C-terminal 25 aa affect nuclear localization and inclusion body formation

To explore key amino acids responsible for P6 nuclear localization, we used a prediction program available at http://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi, this program identified two putative nuclear localization signal (NLS) regions present at 193-223 and 402-426 aa of the 514 aa P6 protein (Fig. 4-A). To validate this prediction, we constructed two P6 deletion mutants, M1(deletion 193-223 aa) and M2 (deletion 402-426 aa). The M1 and M2 mutants were fused to GFP and agroinfiltrated into N. benthamiana leaves, respectively. The results indicate that the P6 M1 mutant protein could assemble into amorphous IBs with different sizes, but these differed in their distribution. The larger IBs accumulated in, or adjacent to,the nucleus, and the smaller IBs were distributed throughout the cytoplasm (Fig. 4-B). However, the M2 mutant protein lacking the 25 aa C-terminal region was present only at the cell periphery, and there was no evidence for nuclear import or formation of cytoplasmic IBs (Fig. 4-C). We substituted the SV40 NLS into P6 for the 402-426 aa region to construct the chimeric mutant M5. The M5-GFP fluorescence concentrated in the nucleus, showing that the SV40 NLS could restore the M2 nuclear localization function(Fig. 4-C).

Analysis of the 402-426 aa P6 polypeptide revealed a polar region (16 of 25 aa) that consisted of one acidic and seven basic amino acids. Furthermore, a secondary structure prediction of the 25-aa polypeptide indicated that the region mainly contains α-helices, which is similar to the CaMV P6 key region required for IB formation (Lutz et al.2015).

To obtain further information about the function of the P6 25 residue peptide in IBs, we constructed two additional P6 mutants, M3 (1-402 aa) and M4 (1-426 aa). Visualization of infiltrated tissue showed that the GFP fluorescence of both the M3 and M4 mutants was distributed at the cell periphery without any IB formation, as is the case for the M2 mutant (Fig. 4-D). This result indicates that 402-426 aa is necessary but is not sufficient for P6 formation of IBs;hence, other P6 regions likely contribute to IB generation.

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.

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 offluorescence (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 tissues are involved in replication and assembly of virons.

Fig. 4 Fluorescence distribution of P6 mutants. A, schematic diagrams of P6-GFP and the P6 mutant derivatives. The blue and red regions in the top diagram represent the Strawberry vein banding virus (SVBV) P6 514 amino acid sequence. The single black lines identify deleted regions of the P6 mutants. The red regions designated a and b represent the nuclear location signals predicted by software. The purple region designated c represents the substituted SV40 nuclear location signal. The constructs were agroinfiltrated into Nicotiana benthamiana leaves and examined by fluorescence microscopy 3 days after infiltration. B, M1 inclusion body (IB) formation and nuclear accumulation. a, M1-GFP; b, brightfield; c, overlay of a and b; d, M1-GFP; e, DAPI stained nucleus; f, overlay of d and e. C, restoration of M2NLS nuclear localization by insertion of the SV40 nuclear localization signal. a, M2-GFP; b, brightfield; c, overlay of a and b; d, M5-GFP; e, brightfield; f, overlay of d and e; g, M5-GFP; h, DAPI stained nucleus; I, overlay of g and h. D, disruption of M3 and M4 nuclear import. a, M3-GFP; b, brightfield; c, overlay of a and b; d, M4-GFP; e, brightfield; f, overlay of d and e. Magnification bar equals 20 μm.

Plant viruses encode movement proteins (MP) that localize to the PD and modify the PD structure to facilitate cell to cell movement of virons or nucleic acids (Rodriguez et al. 2014). Recently, it has been reported that other virus proteins can also participate in virus intracellular and intercellular movement. For example, the tomato yellow leaf curl virus (TYLCV) C4 protein is able to interact with PD to modify their diameter and promote the intercellular movement of the viral DNA (Rojas et al. 2001). In addition,the potyviral P3N-PIPO and CI encoded protein can form a complex that coordinates the formation of PD-associated structures that can facilitate the intercellular movement(Wei et al. 2010).

The cytoskeleton is also involved in transferring virus particles from replication sites to PD where they can be transported to adjacent cells. Ectopic expression of CaMV P6 in N. benthamiana leaves resulted in the formation of IBs that were capable of moving along actin microfilaments.When CaMV-infected Nicotiana edwardsonii leaves are treated with latrunculin B (Lat B), a chemicalagent used to destabilize microfilaments, local lesion formation is abbrograted, suggesting that microfilament functions are required for CaMV infection (Harries et al. 2008). The 126-kD protein encoded by TMV also can form IBs that traffic along actin microfilaments. TMV IBs form in Lat B-treated cells, but the drug suppresses intracellular movement of IBs. Virus-induced gene silencing of actin and Lat B can 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 P3NPIPO 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,it is possible that ER tubules are used directly by P6 IBs as a conduit for both intracellular and intercellular transports.

Viruses cause a large array of different disease symptoms in susceptible plants, and the plant evolves special mechanisms to resist virus invasion. RNA silencing is one of the main adaptive defense mechanisms against virus. However, viruses encode special proteins called viral silencing suppressors that interfere with many steps of host gene silencing pathways. These include the 2b protein encoded by cucumber mosaic virus (CMV), which interacts with argonaute 1 (AGO1) to prevent the assembly of the RNA-induced silencing complex (RISC) (Zhang et al.2006). The tombusvirus P19 protein exerts a different mechanism of activity by competing with AGO1 for virusinduced siRNA binding to prevent RISC targeting of viral RNA and cleavage of exogenous genes (Haas et al. 2008).Considering the function of CaMV P6, we postulated that SVBV P6 is a silencing suppressor protein that functions to interfere with the RISC pathway, and we demonstrated that the protein could enhance the ectopic GFP expression. We also showed that the function of SVBV P6 was completely destroyed by deleting the C-terminal 25 aa residues. We raise the hypothesis that the 25 aa is indispensable for SVBV infection, and this hypothesis is consistent with the studies showing that a CaMV mutant lacking the 25 aa C-terminal region fails to form inclusion bodies or infect turnips (Lutz et al. 2015).

5. Conclusion

SVBV ORF VI was expressed ectopically in N. benthamiana.P6 protein accumulated in the nuclei and formed amorphous,cytoplasmic IBs with different sizes. P6 IBs aligned with microtubules and the endoplasmic reticulum, and trafficked along microfilaments for co-localization with the P1 protein at periphery of cells, suggesting that P6 IBs might transport virons to P1 in the plasmodesmata via the cytoskeleton and host secretory pathways. In addition, P6 protein deleted NLS region did not localize in the nucleus, did not form IBs,and was unable to facilitate exogenous green fluorescent protein expression.

Acknowledgements

We gratefully acknowledge Prof. Tao Xiaorong (Nanjing Agriculturaluniversity, China) for providing the microtubules marker protein (mCherry-MAP65-1) and ER marker protein(mCherry-HDEL). This work was supported by the Fund of State Key Laboratory for Biology of Plant Diseases and Insect Pests (SKLOF201710), the National Natural Science Foundation of China (31671999, 31371915)and the Zhejiang Natural Science Foundation of China(LY17C140001).

Appendices associated with this paper can be available on http://www.ChinaAgriSci.com/V2/En/appendix.htm