P1 of strawberry vein banding virus,a multilocalized protein,functions as a movement protein and interacts with the coat protein

RUl Peng-huan,WANG Zhan-qi,SHAN Wen-shu,XlA Wei-wei,ZHOU Xiu-hong,YANG Lian-lian,JlANG Lei,4,5,JlANG Tong,4,5

1 School of Plant Protection,Anhui Agricultural University,Hefei 230036,P.R.China

2 Key Laboratory of Vector Biology and Pathogen Control of Zhejiang Province,College of Life Sciences,Huzhou University,Huzhou 313000,P.R.China

3 Biotechnology Center,Anhui Agricultural University,Hefei 230036,P.R.China

4 Anhui Province Key Laboratory of Integrated Pest Management on Crops,Hefei 230036,P.R.China

5 Key Laboratory of Biology and Sustainable Management of Plant Diseases and Pests of Anhui Higher Education Institutes,Anhui Agricultural University,Hefei 230036,P.R.China

Abstract Although the complete nucleotide sequence of strawberry vein banding virus (SVBV) has been determined and bioinformatic analysis has revealed that the SVBV genome could encode seven proteins,the precise function of each protein is unclear.This study provided evidence that the P1 protein of SVBV (SVBV-P1) possesses the following features.Bioinformatic and subcellular localization analyses showed that SVBV-P1 is localized in the cytoplasm and cell walls of epidermal cells in Nicotiana benthamiana,and it forms inclusion bodies associated with microtubules and the endoplasmic reticulum.Dilution experiments demonstrated that SVBV-P1 could move from the original agro-infiltrated cells to adjacent cells in N.benthamiana leaves.Further trans-complementation experiments demonstrated that SVBV-P1 could facilitate the intercellular movement of a movement-deficient potato virus X mutant in N.benthamiana leaves.Finally,yeast twohybrid and bimolecular fluorescence complementation assays revealed that SVBV-P1 could interact with the SVBV coat protein,which is a major component of Caulimovirus virions.Results of the electrophoretic mobility shift assay indicated that SVBV-P1 lacks DNA-binding capability.In summary,the results suggest that SVBV-P1 is probably a movement protein of SVBV,providing new insights into the function of movement proteins of the Caulimovirus genus.

Keywords:strawberry vein banding virus,P1 protein,movement protein,coat protein,virus movement

1.lntroduction

Strawberry vein banding virus (SVBV) cause serious damages to strawberries.The virus is highly prevalent in many countries around the world,especially in North and South America,Europe,Africa,Australia,and Japan (Petrziket al.1998;Pattanaiket al.2004).In China,its distribution has expanded considerably in strawberry cultivation areas in the Jilin,Liaoning,Hebei,and Henan provinces (Ryabovaet al.2002;Fenget al.2016,2018).SVBV is mainly transmitted byChaetosiphonfragaefoliiin a semi-persistent manner or by grafting (Hullet al.1986;Mrázet al.1998;Dickisonet al.2017).When strawberry cultivars are infected with SVBV,the plants grow weaker,the number of creeping stems decrease,and the yield and quality decline significantly (Jiet al.2011;Chenet al.2016).

SVBV belongs to the genusCaulimovirusof the familyCaulimoviridae,and its isometric particle encapsidates a double-stranded DNA (dsDNA) (Petrziket al.1998;Chenet al.2000).The SVBV genome is approximately 7.8 kb in length,and its structure is very similar to that of the cauliflower mosaic virus (CaMV) (Citovskyet al.1991;Fenget al.2016).It contains seven open reading frames (ORFs)and encodes seven corresponding proteins of various sizes(Petrziket al.1998;Fenget al.2016).The protein encoded by ORF I may be related to cell-to-cell movement,while that encoded by ORF II may be involved in aphid infection.ORF III may encode a nonsequence-specific DNA-binding protein,ORF IV encodes a coat protein (CP),ORF V encodes a reverse transcriptase protein,ORF VI encodes a translation activator that functions as an RNA-silencing suppressor to control the symptomatic phenotype (Fenget al.2018),and ORF VII encodes an unknown protein.Among these seven proteins encoded by the SVBV genome,only the precise biological function of P6 encoded by ORF VI has been determined (Fenget al.2018).Thus,the biological functions of other proteins need to be elucidated in the future.

In order to cause a successful systemic infection,plant viruses must transfer their genomes,alone or together with viral proteins,from the replication sites to the plasmodesmata (PD),which facilitates movement into adjacent cells,and,ultimately,into the phloem to spread throughout the host plant (Lucas 2006;Fenget al.2013;Heinleinet al.2015).It has been shown that movement proteins (MPs),which are encoded by plant viruses themselves,could help viruses spread to non-infected cells by cell-to-cell movement (local movement) and longdistance movement (systemic movement) within host plant (Gillespieet al.2002;Margariaet al.2016;Yuanet al.2016;Pitzalis and Heinlein 2017).If the viruses lose the MPs,infection is restricted to the initial infection sites and the systemic infection is abolished (Holt and Beachy 1991;Kotlizkyet al.2001;Navarroet al.2020).Viral MPs frequently possess nonsequence-specific nucleic acid binding ability (Zhouet al.2019) and/or the ability to interact with viral proteins such as CP (Chowdhury and Savithri 2011) to potentiate the virus cell-to-cell and/or long-distance movements in plant hosts.Intracellular and intercellular movements of the viruses could be broadly classified into two types according to the mode of action of viral MPs (Gillespieet al.2002;Schoelzet al.2011;Margariaet al.2016;Hong and Ju 2017).The first type is represented by the MP of the tobacco mosaic virus (TMV),which could directly bind to viral RNA to form a virusspecific ribonucleoprotein complex (Dorokhovet al.1983;Sambadeet al.2008).The second type is represented by the MP of CaMV,which facilitates the transport of whole virions between cells by assembling hollow tubules within the PD (Gillespieet al.2002;Carluccioet al.2014).

This study demonstrated that the P1 protein of SVBV (SVBV-P1,GenBank accession no.CBK62754)is a multilocalized protein that is mainly located in the cytoplasm and cell walls of epidermal cells inN.benthamiana.Additionally,SVBV-P1 could form motile inclusion bodies associated with microtubules (MTs) and the endoplasmic reticulum (ER) in the cytoplasm and cell walls.Furthermore,this study investigated the ability of SVBV-P1 to complement the cell-to-cell movement of a movement-defective potato virus X (PVX) by using an agroinfiltration-mediatedtrans-complementation assay and found that a domain comprising 119-233 aa is necessary for the movement of SVBV-P1.In addition,it showed that SVBV-P1 is able to interact with SVBV-CP but fails to bind to SVBV DNA,thereby indicating the capacity of SVBV-P1 to recognize and transport SVBV virions.These results suggest that SVBV-P1 is an MP of SVBV and provides more insights into the MPs of theCaulimovirusgenus.

2.Materials and methods

2.1.Plant materials and growth conditions

In this study,wild-typeN.benthamianawas used.All experimental plants were grown in an insectfree greenhouse at 25°C with a 16-h/8-h (light/dark)photoperiod as described previously (Zhonget al.2017).

2.2.Subcellular localizations of SVBV-P1 and its mutants

To construct plasmids used for protein subcellular localization,ORF ofSVBV-P1(GenBank accession no.FN689701) and its mutants (SVBV-P1Δ30-55aa,SVBVP1Δ119-233aa,andSVBV-P1Δ283-328aa) were cloned by fusing them with the N-terminus of yellow fluorescent protein(YFP) under the control of a CaMV 35S (35S) promoter(Yaoet al.2014) to generate 35S:SVBV-P1-YFP,35S:SVBV-P1Δ30-55aa-YFP,35S:SVBV-P1Δ119-233aa-YFP,and 35S:SVBV-P1Δ283-328aa-YFP,respectively.The primers used for plasmid construction are listed in Appendix A.Constructs of mCherry-MAP65-1,mCherry-HDEL and PDLP5-CFP,which are used as markers for MTs (Lucas 2006),ER (Yaoet al.2014) and PD (Chaiet al.2020),respectively,were gifted by Prof.Tao Xiaorong (Nanjing Agricultural University,China) and Prof.Cheng Xiaofei(Northeast Agricultural University,China).The constructs were confirmed by sequencing and introduced into theAgrobacteriumstrain EHA105viaelectroporation.TheAgrobacteriumculture was adjusted to an optical density at 600 nm (OD600) of 1.0 with an infiltration buffer(10 mmol L-1MgCl2,10 mmol L-1MES (pH 5.6),and 100 μmol L-1acetosyringone) and infiltrated intoN.benthamianaleaves as described previously (Yanget al.2011).After 72 h,the infiltrated leaves ofN.benthamianawere examinedviaconfocal laser scanning microscopy,(Olympus,Japan).The experiments were repeated three times to confirm the results.

2.3.Movement complementation experiments

The ORFs ofSVBV-P1and its mutants (SVBV-P1Δ30-55aa,SVBV-P1Δ119-233aa,andSVBV-P1Δ283-328aa) were cloned into theBamHI-SalI sites of a pBin438 vector (Cuiet al.2005)to produce pBin438:SVBV-P1,pBin438:SVBV-P1Δ30-55aa,pBin438:SVBV-P1Δ119-233aaand pBin438:SVBV-P1Δ283-328aa,respectively,for plasmid construction.The primers used for plasmid construction are listed in Appendix A.The movement-defective PVX tagged with green fluorescent protein (GFP) (PVXΔP25-GFP) (Yanet al.2012) and construct P25,expressing the PVX p25 protein,was gifted by Prof.Fei Yan (Ningbo University,China).The constructs were confirmed through sequencing and introduced into theAgrobacteriumstrain EHA105viaelectroporation.TheAgrobacteriumculture containing PVXΔP25-GFP was adjusted to an OD600of 0.001 with the infiltration buffer andAgrobacteriumcultures carrying pBin438,pBin438:SVBV-P1,pBin438:SVBV-P1Δ30-55aa,pBin438:SVBV-P1Δ119-233aa,pBin438:SVBV-P1Δ283-328aa,or P25 were adjusted to an OD600of 1.0 with the infiltration buffer.The agro-infiltration ofN.benthamianaleaves was performed as described previously (Yanet al.2012).After 72 h,the infiltrated leaves ofN.benthamianawere examinedviaconfocal laser scanning microscopy.The experiments were repeated three times to confirm the results.

2.4.Confocal laser scanning microscopy

Leaf discs obtained from the agroinfiltrated leaf area ofN.benthamianaleaves were dissected and mounted in water between two coverslips as described previously(Fenget al.2013).The slides were viewed using an Olympus Fluoview FV1000 Confocal Microscope(Germany) coupled with the Fluoview Confocal Software(FV10-ASW v1.7;Olympus).CFP fluorescence was excited at 405 nm,and the emission was captured at 405-450 nm;YFP or GFP fluorescence was excited at 488 nm,and the emission was captured at 497-520 nm;mCherry was excited at 561 nm,and the emission was captured at 585-615 nm.

2.5.Electrophoretic mobility shift assay (EMSA)

To obtain the SVBV-P1 protein,the ORF ofSVBV-P1was amplifiedviaPCR and cloned into the pET-32a vector to generate the expression vector 32a:SVBV-P1 according to the manufacturer’s instructions (USA) and expressed as an N-terminal His6fusion protein inEscherichiacoli(BL21) cells as described previously (Xionget al.2008).The primers used for plasmid construction are listed in Appendix A.The His6-taged SVBV-P1 protein was purified using the His-Bind?Purification Kit (USA) according to the manufacturer’s instructions.The quality of the purified His6-taged SVBV-P1 protein was determinedviaSDS-PAGE coupled with Coomassie brilliant blue (CBB) staining or Western blot (WB)with an anti-His monoclonal antibody (Qiagen,Germany),and the immunoblot signal was visualized using a nitroblue tetrazolium/5-bromo-4-chloro-3-indolylphosphate (NBT/BCIP) liquid substrate system (Germany).

For gel retardation assays,a 210-bp dsDNA probe was amplified using PCR and labeled using a DIG DNA Labeling and Detection Kit (Roche,Switzerland)as described previously (McInerneyet al.2005).The resulting DIG-labeled dsDNA at different amounts was mixed with purified His6-tagged SVBV-P1 in 10 μL of binding buffer containing 45 mmol L-1Tris-HCl (pH 8.4)and 45 mmol L-1boric acid.The mixtures were incubated at 25°C for 10 min,separated through electrophoresis on a 1.5% agarose gel,and blotted on a nylon membrane(UK) using a semi-dry transfer device (Bio-Rad,USA)as described previously (Kunzeet al.2017).The DIG hybridization signals were detected using an anti-DIG monoclonal antibody (Roche,Switzerland) in accordance with the standard manufacturer’s instructions.Additionally,the DIG-labeled dsDNA was mixed with bovine serum albumin (BSA) to be used as a negative control during the assay.The experiments were repeated three times to confirm the results.

2.6.Yeast two-hybrid (Y2H) assay

For plasmid construction,the ORF ofSVBV-P1was cloned into theEcoRI-BamHI sites of the pGADT7 and pGBKT7 vectors (USA) to produce AD:SVBV-P1 and BD:SVBV-P1,respectively.In the same manner,the ORF ofSVBV-CPwas cloned into theEcoRI-BamHI sites of the pGADT7 and pGBKT7 vectors (USA) to produce AD:SVBV-CP and BD:SVBV-CP,respectively.The primers used for plasmid construction are listed in Appendix A.An analysis of the interactions was carried out as described previously (Konget al.2014;Meiet al.2018).Combinations of plasmids BD and AD:SVBV-CP,BD:SVBV-CP and AD,BD and AD:SVBV-P1,BD:SVBV-P1 and AD,or BD:SVBV-CP and AD:SVBV-P1 were cotransformed intoSaccharomycescerevisiaestrain Gold (USA)as described previously (Gietz and Schiestl 2007).AD-T and BD-Lam were co-transformed intoS.cerevisiaestrain Gold to be used as a negative control;AD-T and BD-P53 were co-transformed intoS.cerevisiaestrain Gold to serve as a positive control.All transformants were grown at 30°C for 72 h on an SD medium lacking Leu,Trp and His and containing 0.2 μg mL-1aureobasidin A (AbA).The experiments were repeated three times to confirm the results.

2.7.Bimolecular fluorescence complementation(BiFC) assay

To construct BiFC plasmids,the ORF ofSVBV-P1was cloned into theKpnI-BamHI sites of the pCV-nYFP and pCV-cYFP vectors (Luet al.2011) to generate nYFP:SVBV-P1 and cYFP:SVBV-P1,respectively.In the same manner,the ORF ofSVBV-CPwas cloned into theKpnI-BamHI sites of the pCV-nYFP and pCVcYFP vectors (Luet al.2011) to generate nYFP:SVBVCP and cYFP:SVBV-CP,respectively.The primers used for plasmid construction are listed in Appendix A.The constructs were confirmed through sequencing and introduced into theAgrobacteriumstrain EHA105viaelectroporation.The BiFC assay was then performed as described previously (Zhonget al.2017;Fuet al.2018).After 72 h,the infiltrated leaf discs ofN.benthamianawere examinedviaconfocal laser scanning microscopy.The experiments were repeated three times to confirm the results.

3.Results

3.1.SVBV-P1 is a multilocalized protein and form cytoplasmic inclusion bodies

To examine the subcellular localization of SVBV-P1,we predicted the subcellular localization using the protein localization prediction tools WoLF PSORT (Hortonet al.2007) and Cell-PLoc (Chou and Shen 2008).The results suggested that SVBV-P1 was probably localized in the cytoplasm and nucleus.To test the location of SVBV-P1,SVBV P1 was fused to the N-terminus of YFP.TheN.benthamianaleaves agroinfiltrated with 35S:YFP were used as controls.TheN.benthamianaepidermal cells transiently expressing YFP only (35S:YFP) displayed homogeneous YFP fluorescence throughout the cells(Fig.1-A).However,YFP fluorescence was observed in the cytoplasm,and punctate YFP fluorescence was observed in cell boundary inN.benthamianaepidermal cells expressing 35S:SVBV-P1-YFP (Fig.1-B),thereby indicating that SVBV-P1 may be localized in both compartments.To further realize whether SVBV-P1 is localized in cell walls,we performed a plasmolysis using a 10% NaCl treatment for 3 min.Surprisingly,compared with the 35S:YFP (Fig.1-C),most of the diffuse fluorescence was detected in the cell walls,leaving lower fluorescence in the cytoplasm (Fig.1-D),thereby indicating that SVBV-P1 is also localized in cell walls.Collectively,these results suggest that SVBV-P1 is probably localized in the cytoplasm and cell walls.

Interestingly,compared with 35S:YFP,which generated a diffuse YFP fluorescence that was uniformly distributed in the cytoplasm (Fig.1-A),inclusion bodies covered with YFP were observed at the edges of the epidermal cells in 35S:SVBV-P1-YFP-infiltratedN.benthamianaleaves(Fig.1-B).Previous studies have shown that inclusion bodies are frequently associated with MTs and ER (Lucas 2006;Cottonet al.2009;Harrieset al.2009).Thus,this study next determined whether SVBV-P1 is localized in the MTs and ER.As shown in Fig.1-E,the YFP fluorescence of SVBV-P1-YFP was superposed on RFP fluorescence of mCherry-MAP65-1,which is a marker protein for MTs (Lucaset al.2011),thereby indicating that SVBV-P1 is localized in MTs.Using a marker protein for ER (Lucas 2006),we found that SVBV-P1 was also localized in the ER of epidermal cells inN.benthamiana(Fig.1-F).In addition,SVBV-P1 was also localized in the PD of epidermal cells when co-infiltrated with PDLP5-CFP(Fig.1-G).Taken together,these results suggest that SVBV-P1 localized in the MTs,ER and PD.

Fig.1 Sublocalization of the P1 protein of strawberry vein banding virus (SVBV-P1).A,yellow fluorescent protein (YFP) localization in leaf epidermal cells of Nicotiana benthamiana at 72 h post-infiltration (hpi).B,SVBV-P1-YFP fusion protein localization in leaf epidermal cells of Nicotiana benthamiana at 72 hpi.Red arrows mark the position of selected individual inclusion bodies in the cytoplasm of N.benthamiana leaf epidermal cells.C and D,plasmolyzed N.benthamiana leaf epidermal cells transformed with YFP (C) and SVBV-P1-YFP fusion protein (D),respectively.E and F,co-localization of the SVBV-P1-YFP protein with microtubules(MTs) labeled by mCherry-MAP65-1 (E) and the endoplasmic reticulum (ER) labeled by mCherry-HDEL (F) in N.benthamiana leaf epidermal cells at 72 hpi,respectively.The merged image illustrates SVBV-P1-YFP inclusion bodies associated with both the MTs and cortical ER.G,co-localization of the SVBV-P1-YFP protein with plasmodesmata (PD) labeled by PDLP5-CFP in N.benthamiana leaf epidermal cells at 72 hpi.

3.2.SVBV-P1 moves between cells and complement movement-defective PVX

The different locations of SVBV-P1 led us to wonder whether it is an MP.To this end,this study initially investigated the ability of SVBV-P1 to transfer YFP from one cell to another inN.benthamianaleaves.Agrobacteriumcontaining 35S:YFP,35S:SVBV-P1-YFP infiltrated into leaves ofN.benthamiana.TheN.benthamianaleaves infiltrated with 35S:YFP was used as a negative control.The 35S:SVBV-P1-YFP fusion protein could move autonomously and spread to surrounding neighbor cells (Fig.2-A).On the contrary,inN.benthamianaleaves agro-infiltrated with 35S:YFP,green fluorescence could only be observed in the infiltrated cells but not in the surrounding neighbor cells (Fig.2-B).

Next,to clarify the movement function of SVBV-P1,we performed a complementation assay using movement-defective PVX as described previously(Xionget al.2008).Agrobacteriumcontaining pBin438:SVBV-P1,pBin438:PVX-P25,or an empty vector pBin438 was co-infiltrated withAgrobacteriumcontaining movement-defective PVX (PVXΔP25-GFP) intoN.benthamianaleaves at a dilution of OD600=0.001.The infiltrated leaves were photographed using a confocal laser scanning microscope,72 h post-infltration(hpi).TheN.benthamianaleaves co-infiltrated with the PVXΔP25-GFP and pBin438 was used as negative control.As expected,GFP fluorescence was only detected in originally infiltratedN.benthamianacells(Fig.2-C),thereby suggesting that pBin438 fails to complement the ability of PVXΔP25-GFP to move betweenN.benthamianacells.In contrast,inN.benthamianaleaves co-infiltrated with PVXΔP25-GFP and its MP pBin438:PVX-P25,patches of GFP fluorescence were observed not only in originally infiltrated cells but also in surrounding cells (Fig.2-D).Interestingly,a similar GFP fluorescence pattern was also observed inN.benthamianaleaves co-infiltrated with PVXΔP25-GFP and pBin438:SVBV-P1 (Fig.2-E),thereby indicating that SVBV-P1 is capable of complementing the function of movement-defective PVX,although its ability is slightly weaker than P25 as statistical analysis (Fig.2-F).

Fig.2 P1 protein of strawberry vein banding virus (SVBV-P1) can move between cells and complement movement-defective potato virus X (PVX).A and B,trafficking of yellow fluorescent protein (YFP) from one cell to another by SVBV-P1.The leaves of Nicotiana benthamiana were infiltrated with Agrobacterium carrying expression plasmids for SVBV-P1-YFP (A) or YFP (B).PVX-P25 and YFP were used as positive and negative controls,respectively.C-E,trans-complementation of movement-defective PVX (PVXΔP25-GFP) by SVBV-P1.The N.benthamiana leaves were infiltrated with Agrobacterium carrying transcription plasmids for PVXΔP25-GFP plus a binary plasmid (pBin438) to express PVX-P25 (D) or SVBV-P1 (E).The empty vector pBin438 (C) and PVX-P25 (D) was used as negative and positive control,respectively.F,statistical analyses of movement and complementation of P1.Error bars show the mean±standard deviation of three replicates.

3.3.ldentification of the domains is necessary for the movement of SVBV-P1

In order to analyze the domains necessary for the movement of SVBV-P1,this study assessed three deletion mutants based on the functional domains predicted by Pfam (http://pfam.xfam.org/) and ExPASy (https://www.expasy.org/) (Fig.3-A).The function of a protein is often linked to its subcellular localization (Itzhaket al.2016).Therefore,this study examined the subcellular localization of the deletion mutants of SVBV-P1 as described above.Deletion mutants SVBV-P1Δ30-55aaand SVBV-P1Δ283-328aa(Fig.3-C and E) were localized in the cell boundary and displayed punctate fluorescence with a pattern similar to that of the wild-type SVBV-P1(Fig.3-B),while the fluorescence of SVBV-P1Δ119-233aa(Fig.3-D) was homogeneous along the cell boundary.These results suggest that the 119-233 aa domain may play a vital role in the subcellular localization of SVBV-P1.To further clarify whether the 119-233 aa domain is involved in the movement of SVBV-P1,this study performed a complementation assay using movement-defective PVX as described above.TheN.benthamianaleaves co-infiltrated with PVXΔP25-GFP,and pBin438 and pBin438:PVX-P25 were used as negative and positive controls,respectively (Fig.3-F and G).Similar to the positive control,N.benthamianaleaves co-infiltrated with PVXΔP25-GFP and either pBin438:SVBV-P1Δ30-55aaor pBin438:SVBV-P1Δ283-328aashowed GFP fluorescence in several cells around the originally infiltrated cell (Fig.3-H and J),thereby indicating that the deletion mutants SVBV-P1Δ30-55aaand SVBV-P1Δ283-328aahave the capability to complement the movement-defective PVXΔP25.In contrast,inN.benthamianaleaves co-infiltrated with PVXΔP25-GFP and pBin438:SVBV-P1Δ119-233aa,GFP fluorescence was only observed in the originally infiltrated cells (Fig.3-I),thereby indicating that the deletion mutant SVBVP1Δ119-233aais not able to complement the movementdefective PVX.SVBV-P1Δ30-55aahad the highest movement and complementation efficiency compared to SVBV-P1Δ119-233aaand SVBV-P1Δ283-328aaas statistical analysis (Fig.3-K).Western blot showed that all the truncated P1 proteins were successfully expressed in the complementation assay (Fig.3-L).Taken together,these results suggest that the 119-233 aa domain plays a key role in SVBV-P1 as a MP.

Fig.3 Identification of the domains necessary for the movement of P1 protein of strawberry vein banding virus (SVBV-P1).A,schematic representation of the full-length and truncated mutants of SVBV-P1.Different boxes represent different domains.B-E,subcellular localization of the full-length SVBV-P1 (B),and the truncated mutants SVBV-P1Δ30-55aa (C),SVBV-P1Δ119-233aa (D),or SVBV-P1Δ283-328aa (E) in leaf epidermal cells of Nicotiana benthamiana at 72 h post-infiltration (hpi).F-J,trans-complementation of movement-defective PVX (PVXΔP25-GFP) by the truncated mutants of SVBV-P1SVBV-P1.The N.benthamiana leaves were infiltrated with Agrobacterium carrying transcription plasmids for PVXΔP25-GFP plus a binary plasmid (pBin438) to express PVX-P25(G),SVBV-P1Δ30-55aa (H),SVBV-P1Δ119-233aa (I) or SVBV-P1Δ283-328aa (J).The empty vector pBin438 (F) and PVX-P25 (G) were used as negative and positive controls,respectively.K,statistical analyses of movement and complementation of truncated P1.Error bars show the mean±standard deviation of three replicates.L,Western blot (WB) with an anti-P1 monoclonal antibody.

3.4.SVBV-P1 lacks DNA-binding capability

It has been shown that some viral MPs could bind to their genomic DNA or RNA to transport them from one cell to another during infections (Hehnleet al.2004;Xionget al.2008;Genovéset al.2009).To test whether SVBV-P1 was able to bind to the genomic DNA of SVBV,this study expressed a His6-tagged SVBV-P1 inE.coli(BL21),purified it under native conditions (Fig.4-A and B),and determined its DNA-binding capability using an EMSA.As shown in Fig.4-C,no retarded electrophoretic mobility was detected under any SVBV-P1 concentration with a DIG-labeled 210-bp dsDNA probe.Moreover,the incubation of BSA with the dsDNA probe at comparable concentrations did not result in retarded mobility in the assay as expected.To ensure the accuracy of the test results,U2AF65-associated protein (UAP56) ofArabidopsisthaliana(Kammelet al.2013) incubated with the dsDNA probe was regarded as the positive control,as it could interact with dsDNA (Fig.4-C).These results indicate that SVBV-P1 is not able to interact with the DIGlabeled dsDNA probe and may not have DNA-binding properties.

Fig.4 Electrophoretic mobility shift assay (EMSA) for P1 protein of strawberry vein banding virus (SVBV-P1).A and B,His6-tagged SVBV-P1 protein detected via Coomassie brilliant blue (CBB) staining (A) and Western blot with an anti-His monoclonal antibody (α-His) (B).C,different concentrations of His6-tagged SVBV-P1 were incubated with a DIG-labeled dsDNA probe and analyzed on a native gel.The DIG-labeled dsDNA incubated with bovine serum albumin (BSA) was used as a negative control,and that incubated with UAP56 (U2AF65-associated protein) was used as a positive control.The experiment was repeated three times with similar results.

3.5.SVBV-P1 interacts with SVBV-CP

To examine whether SVBV-P1 interacts with the viral CP,the ORFs ofSVBV-P1andSVBV-CPwere separately cloned into the GAL4 activation domain vector pGADT7 to produce AD:SVBV-P1 and AD:SVBV-CP,and into the GAL4 DNA-binding domain in vector pGBKT7 to produce BD:SVBV-P1 and BD:SVBV-CP,respectively.TheS.cerevisiaecells co-transformed with plasmids AD-T and BD-P53,and AD-T and BD-Lam,were used as positive and negative controls,respectively.As shown in Fig.5-A,yeast transformants harboring AD:SVBV-P1 and BD:SVBV-CP could grow on SD/-His/-Leu/-Trp/AbA+plates,which was similar to the positive control(co-expression of AD-T and BD-P53).This result suggests that SVBV-P1 can interact with SVBV-CP in the Y2H system.Furthermore,to demonstrate theinvivointeraction between SVBV-P1 and SVBV-CP,a BiFC assay was performed using agro-infiltratedN.benthamianaleaves as described previously (Zhonget al.2017;Fuet al.2018).The co-expression of SVBV-P1 and SVBV-CP inN.benthamianaepidermal cells gave rise to a strong yellow fluorescence in the cytoplasm and cell walls after 72 hpi (Fig.5-B).Taken together,these results suggest that SVBV-P1 interacts with SVBV-CP in plant cells.

Fig.5 Interaction between SVBV-P1 and SVBV-CP.A,interaction between SVBV-P1 and SVBV-CP detected in the yeast twohybrid (Y2H) assay.Yeast strain Gold was co-transformed with the indicated plasmids shown on the top of the panel and grown on an SD/-His/-Leu/-Trp/AbA+ medium.AD-T and BD-Lam were co-transformed into Saccharomyces cerevisiae strain Gold to be used as a negative control (NC);AD-T and BD-P53 were co-transformed into S.cerevisiae strain Gold to serve as a positive control (PC).The experiment was repeated three times with similar results.B,a bimolecular fluorescence complementation(BiFC) analysis of the interaction between SVBV-P1 and SVBV-CP in the agroinfiltrated Nicotiana benthamiana leaves.After coinfiltration for 72 h,the leaf epidermal cells were photographed using a confocal laser scanning microscope.The experiment was repeated three times with similar results.

4.Discussion

In order to successfully infect host plants,plant viruses have to traverse the PD of neighboring cells,followed by long-distance movement through the phloem(Huanget al.2005;Lucas 2006;Xionget al.2008).To achieve intercellular movement between adjacent cells,plant viruses have evolved various strategies using MPs encoded by viral genomes (Gillespieet al.2002;Huanget al.2005;Xionget al.2008).Most studies onCaulimovirusgenus focused on the cell-to-cell movement of CaMV (Schoelzet al.2016);however,the intercellular movement of other viruses within this genus is less known.This study identified an MP of SVBV and investigated its possible role in trafficking virions.

Previous studies have shown that viral MPs are frequently localized in different subcellular compartments in which they perform their functions (Genovéset al.2011;Qiaoet al.2018).In this study,our subcellular localization results showed that the SVBV-P1-YFP fusion protein was detected in different subcellular organelles,especially in areas adjacent to and within cell walls (Fig.1-A-D).The pattern of SVBV-P1 accumulation is similar to that observed previously for MPs of TMV,rice stripe virus(RSV),and cucumber mosaic virus (Itayaet al.1997;Sambadeet al.2008;Xionget al.2008).Our observation of SVBV-P1 in MTs,ER and PD using organellar markers and confocal laser scanning microscopy suggests that SVBV-P1 may travel along the cytoskeleton and/or ER network from viral replication sites to PD in cell walls.This multi-localization pattern indicates that SVBV-P1 could move among different subcellular organelles.Further dilution experiments demonstrated that SVBVP1-YFP was capable of accumulating in multicell clusters following its transient expression inN.benthamianaleaf epidermal cells (Fig.2-A and B).This accumulation pattern suggests that SVBV-P1 is capable of moving through the PD into adjacent cells inN.benthamiana.

The results of complementation experiments showed that SVBV-P1 was able to assist a movement-defective PVXΔP25to move inN.benthamianaleaves,probablyviatrafficking the mutant PVXΔP25to the sites adjacent to the PD and/or by enlarging the exclusion size of the PD.This result is in line with those of previous studies that showed that MPs encoded by several different viruses could complement the cell-to-cell movement of mutant viruses (Yanet al.2012).For instance,MPs of RSV and rice yellow stunt rhabdovirus are able to complement the movement of the movement-defective PVXΔP25inN.benthamiana(Huanget al.2005;Xionget al.2008).However,this study also found that the ability of SVBV-P1 to support the cell-to-cell movement of mutant PVXΔP25appeared to be less effective than that of P25,which is the MP of PVX (Fig.2-C-E),probably because SVBV-P1 is a heterologous protein for PVX.This phenomenon was also observed in the complementation assay of the MP of RSV (Xionget al.2008).Collectively,these results suggest that SVBV-P1 is a multilocalized protein that could complement movement-defective PVXΔP25.

It has been shown that MP-CP interactions of plant viruses are required for their effective intracellular and intercellular movements in host plants (Scholthof 2005;Lucas 2006).Using Y2H and BiFC,this study further demonstrated that SVBV-P1 was able to interact with SVBV-CP bothinvitroandinvivo(Fig.5).These results provide further evidence that SVBV-P1 is involved in virus transport if a model in which caulimoviruses move from cell to cell in the form of virions (Schoelzet al.2016,2017) is taken into account.CPs are major constituents of the virions;thus,this study hypothesized that a specific interaction of SVBV-P1 with SVBV-CP may be crucial for the intercellular movement of the SVBV virions in host plants.Consistent with our findings,it is very intriguing that viruses represented by theComovirusandClosterovirusgenera also require their MPs and CPs for both the cell-to-cell and systemic movements of the viral genomes (Gillespieet al.2002;Lucas 2006).Additionally,these viruses travel as virion particles,thus explaining the requirement of the CPs in virus movements,whose roles may be to facilitate MP activity or to protect the viral genomes.In addition,this study investigated the DNAbinding ability of SVBV-P1 using an EMSA.However,the EMSA experiment did not support the ability of SVBV-P1 to bind the dsDNA of the viral genome (Fig.5).Altogether,these results demonstrate that SVBV-P1 is able to interact with SVBV-CP and that this interaction may be essential for the intrarcellular and intercellular movements of SVBV.However,further research is required to investigate how SVBV-P1 participates in the transport of SVBV virions in infected plants.

5.Conclusion

The results demonstrate that SVBV-P1 is a multilocalized protein that has the capacity to complement movementdefective PVXΔP25.It interacted with SVBV-CP both in the Y2H and BiFC experiments.It is speculated that SVBV-P1 is an MP of SVBV and is involved in both the intracellular and intercellular movements of SVBV.Future research should explore how the interaction between host factors and P1 protein influences the transport of SVBV virions.

Acknowledgements

This work was supported by the grants from the National Natural Science Foundation of China (32072386 and 31801700),the Key Research and Development Project of Anhui Province,China (202004a06020013) and the Postdoctoral Science Fund of Anhui Province,China(2019B360).

Declaration of competing interest

The authors declare that they have no conflict of interest.

Appendixassociated with this paper is available on http://www.ChinaAgriSci.com/V2/En/appendix.htm