J Virol 87(8): 4313-4321

Tyrosine Phosphorylation of the Triple Gene Block Protein 3 Regulates Cell-to-Cell Movement and Protein Interactions of <em class="genus-species">Potato Mop-Top Virus</em>

INTRODUCTION

Plant viruses encode movement proteins (MPs) to facilitate intra- and intercellular movement of viral genomes to and through plasmodesmata by recruiting the host trafficking systems (1–5). Much research has focused on the mechanisms regulated by MPs, but less is known about regulation of MP activities. Protein phosphorylation causes a reversible posttranslational modification that plays a fundamental role in the regulation of many cellular processes in eukaryotic cells, including altering protein function, interactions, stability, or subcellular location (6). However, the regulation of plant virus movement by phosphorylation has been studied for only a few taxa.

Phosphorylation of MPs by cellular serine (Ser)/threonine (Thr) kinases can either enhance or inhibit virus movement, indicating the importance of phosphorylation in the virus infection cycle. For example, phosphorylation of the 30-kDa MP (30K MP) of Tobacco mosaic virus (TMV) (genus Tobamovirus) is needed for MP-mediated gating of plasmodesmata and mediation of viral cell-to-cell movement (7–9). In another member of the genus Tobamovirus (Tomato mosaic virus), phosphorylation enhances MP stability and controls its intracellular localization (10, 11). Phosphorylation of the coat protein of Potato virus A (PVA) (genus Potyvirus) by the host casein kinase II (CKII) in tobacco (Nicotiana tabacum) cells inhibits RNA binding by the coat protein as well as cell-to-cell movement of PVA (12, 13). In the bipartite viruses of the family Geminiviridae, the phosphorylated nuclear shuttle protein facilitates intracellular transport of viral DNA from the nucleus to the cytoplasm and promotes cell-to-cell movement of the virus in concert with MPs (14).

Genomes of many plant viruses with rod-shaped particles (family Virgaviridae and genus Benyvirus) or filamentous particles (family Alphaflexiviridae) contain three MP genes organized as a “triple gene block” (TGB) (15). However, little is known about phosphorylation of the three MPs. TGB protein 1 (TGBp1) of Potato virus X (genus Potexvirus) is phosphorylated in tobacco plants, but the functional role of the phosphorylation is not known (16).

Potato mop-top virus (PMTV) (genus Pomovirus, family Virgaviridae) is a TGB-containing virus that causes yield losses in potato production by inducing necrotic arcs in the infected tubers that preclude their marketability (17). PMTV has a tripartite, positive-sense, single-stranded RNA (ssRNA) genome consisting of RNA1 (6.0-kb), RNA2 (3.2-kb), and RNA3 (2.9-kb) segments (18, 19). RNA3 contains four open reading frames (ORFs) predicted to encode the TGB proteins TGBp1 (51K), TGBp2 (13K), and TGBp3 (21K) and an 8K cysteine-rich protein (19–21) (Fig. 1A). Coexpression of TGB proteins from plant expression vectors in the same cell has revealed their coordinated functions, suggesting a model in which TGBp3 and TGBp2 mediate the transport of a TGBp1-containing ribonucleoprotein complex to and through the plasmodesmata, whereas TGBp2 and TGBp3 are not transported to the adjacent cell (22). These roles of TGBp3 and TGBp2 are consistent with their ability to increase the size exclusion limit of plasmodesmata (23). Yeast two-hybrid assays have shown that TGBp3 and TGBp2 self-interact and interact with each other (24, 25), which is important for TGB functions, because disruption of the TGBp2-TGBp3 interaction inhibits the movement of Barley stripe mosaic virus (BSMV) (genus Hordeivirus) (26, 27). Previous studies suggested that TGB proteins must be expressed at suitable relative molarities for viral movement to occur. For example, the optimal relative expression ratio of TGBp2 and TGBp3 is 10:1 in BSMV and PMTV (25, 28), and TGBp2 and TGBp3 should be expressed at 10- and 100-fold-lower levels, respectively, than TGBp1 (27, 29, 30). Previous studies on PMTV TGBp3 have been done by producing the protein from a plant expression vector or heterologous virus (e.g., see references 23–25). Indeed, the TGBp3 proteins present in infected plant tissues are extremely difficult to detect due to a low titer (26, 29, 31, 32), and previous attempts to detect and localize PMTV TGBp3 in PMTV-infected plants have been unsuccessful (24). Hence, unequivocal evidence that TGBp3 of PMTV is expressed during virus infection remains to be obtained.

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Fig 1

Schematic representation of PMTV RNA3 and detection of PMTV TGBp3 in leaf tissues of N. benthamiana by Western blotting. (A) PMTV RNA3 with positions of the open reading frames (ORFs) for the triple gene block proteins (TGBp1 [51K], TGBp2 [13K], and TGBp3 [21K]) and the 8-kDa cysteine-rich protein (8K) depicted. Insertion sites for the green fluorescent protein (GFP) gene, expressed with two additional amino acids (GFP-Gly-Asn) linking GFP to TGBp1 and for the Myc epitope sequence expressed with a 3-amino-acid spacer (Glu-Phe-Gly-Myc) are shown. (B) Schematic representation of PMTV TGBp3 showing the amino acid motifs containing tyrosine residues 87 to 89 and 120 mutated to alanine in this study. The amino acid residues conserved among the hordei-like viruses (boldface type) and the putative tyrosine-based YXXΦ sorting motifs (underlined) are shown. Dashed lines indicate the end of the TGBp2 ORF and the beginning of the 8K ORF, which overlap the TGBp3 ORF. (C) Leaves agroinfiltrated with a 35S promoter-driven plant expression vector for TGBp3 overexpression (21K) and tested at 3 days postinfiltration by using the polyclonal antibodies (α-21K) raised against PMTV TGBp3. Mock leaves were infiltrated by using infiltration buffer alone. (D) Leaves systemically infected with PMTV_21K-Myc, in which TGBp3 was expressed as a fusion with a Myc tag and detected by using anti-Myc monoclonal antibody (α-myc) at 18 dpi. Mock inoculation was done with buffer only. The position of the 21K protein (arrowhead) is indicated in panels C and D.

TGBp3 in hordei-like viruses contains a conserved YQDLN motif in the central part of the protein (33). The ₈₉YQDLN motif in PMTV TGBp3 serves a critical role during infection of plants. When the motif is mutated to ₈₉GQDGN, TGBp3 is no longer targeted to plasmodesmata and is impaired in its ability to gate plasmodesmata open (23). Hence, tyrosine (Tyr) at position 89 appears to be crucial for viral cell-to-cell movement (25).

Little is known about phosphorylation of TGB proteins. It is also unclear whether tyrosine kinases participate in phosphorylation of MPs or other viral proteins in plants. Because the Tyr-containing motif in PMTV TGBp3 is important for viral movement, the aim of this study was to examine possible tyrosine phosphorylation of TGBp3 to gain further insight into the functions and regulation of TGBp3 activity in PMTV.

MATERIALS AND METHODS

Cloning and mutagenesis of DNA.

Plasmid pPMTV3 contains a full-length cDNA clone of PMTV RNA3 that can be used to generate RNA3 transcripts in vitro and generate infectious PMTV when coinoculated with the RNA1 and RNA2 transcripts into plants (34).

The putative phosphotyrosine sites in PMTV TGBp3 were predicted by using NetPhos 2 and Scansite. The NetPhos 2 algorithm is a neural network method with a false-positive prediction rate of 0 to 26% for tyrosine (35). Scansite predicts target motifs for different kinases using a positional selectivity matrix based on peptide library screening (36). Searches using Scansite applied a high level of stringency to identify the strongest motif matches.

To produce the various constructs described below, pPMTV3 was subjected to PCR-based modification and site-directed mutagenesis using the high-fidelity Phusion DNA polymerase (Finnzymes, Espoo, Finland), as described previously (37). Tyr-to-alanine (Ala) substitutions were introduced into the residues Tyr₈₇, Tyr₈₈, and Tyr₈₉ (construct pPMTV3_21K87-89A) or Tyr₁₂₀ (pPMTV3_21K120A) (Fig. 1A and andB).B). Furthermore, all mutations were combined in the construct pPMTV3_{21K87-89A/120A} (Fig. 1B). The primers used to prepare the above-mentioned constructs and other constructs in this study are available upon request.

The sequence encoding the Myc epitope (EQKLISEEDL) was added to the 3′ end of TGBp3 (pPMTV3_21K-Myc) (Fig. 1A). To produce green fluorescent protein (GFP) fusion constructs of pPMTV3, an NcoI site was created at the 5′ end of the TGBp1 gene by using PCR-based mutagenesis. The GFP coding sequence was amplified from PVA-GFP (38) and subsequently inserted at the 5′ end of the 51K protein gene of wild-type (wt) or mutant pPMTV3 constructs (Fig. 1A) to obtain GFP-pPMTV3, GFP-pPMTV3_21K87-89A, GFP-pPMTV3_21K120A, and GFP-pPMTV3_{21K87-89A/120A}.

To express PMTV TGBp3 in fusion with glutathione S-transferase (GST) (GST-21K), the 21K protein gene was cloned into the pGEX6P-1 expression vector (Amersham Biosciences, Piscataway, NJ) to obtain construct 21KpGEX6P-1. For expression in plants under the Cauliflower mosaic virus 35S promoter, the 21K-Myc and 8K genes of PMTV were cloned into the binary vector pLH (39). Cloning was done according to standard molecular biology protocols (40). Construct p21K_MycpLH was used as the parental plasmid for site-directed mutagenesis to obtain the mutated genes for expression of 21K_87-89A, 21K_120A, and 21K_87-89A/120A proteins as Myc-tagged proteins from binary vectors in plants.

For yeast two-hybrid system (YTHS) analysis, the mutated TGBp3 gene sequences encoding the 21K_87-89A, 21K_120A, and 21K_87-89A/120A proteins were obtained by PCR from parental plasmids pPMTV3_21K87-89A, pPMTV3_21K120A, and pPMTV3_{21K87-89A/120A}, respectively, using Dynazyme II DNA polymerase (Finnzymes) according to manufacturer's instructions. The genes for the 13K and 21K proteins were amplified by PCR as described above, using the wt pPMTV3 plasmid as the template. The obtained PCR products were cloned into pGEM-T (Promega, Madison, WI), and plasmid DNA was then prepared and cleaved with appropriate restriction enzymes for subsequent cloning into the similarly cleaved pGADT7 and pGBKT7 vectors (Clontech, Mountain View, CA). All the constructs were verified by sequencing.

Expression and purification of recombinant proteins.

Escherichia coli strain BL21 was transformed with p21KpGEX6P-1 and grown to an optical density at 600 nm (OD₆₀₀) of 0.6 in Luria broth (Sigma-Aldrich, St. Louis, MO). Protein expression was induced by addition of isopropyl-β-d-1-thiogalactopyranoside (Promega) to a final concentration of 0.1 mM, and the cells were grown for 3 h at 30°C and collected by centrifugation (5,000 × g for 10 min). The protein was purified under native conditions on glutathione-Sepharose 4B (Amersham Biosciences) according to the manufacturer's protocols, with some modifications (41). Polyclonal antiserum against the 21K protein was produced by immunization of two rabbits with purified GST-21K. The IgG fraction was purified on protein A-Sepharose CL-4B (GE Healthcare, Waukesha, WI) according to the manufacturer's instructions.

In vitro and in vivo kinase assays and Western blotting.

Leaf tissue from full-grown leaves of 5-week-old Nicotiana benthamiana plants (see below) was homogenized in 20 mM HEPES-KOH buffer (pH 7.4) with a pestle and a mortar prechilled to 4°C. The leaf extract (total protein concentration, 10 μg/ml) as the source of plant kinase activity and 10× kinase buffer were mixed in a 10:1 ratio (final concentration of 20 mM HEPES-KOH [pH 7.4], 10 mM MgCl₂, 50 mM NaCl, 1 mM dithiothreitol, and 10 mM MnCl₂). Purified GST-21K protein (200 ng) was added to an aliquot (20 μl) of the solution and incubated in the presence of 10 μCi [γ-^{P]ATP (Amersham Biosciences) for 45 min at 37°C. The reaction was terminated by adding SDS-PAGE sample buffer, followed immediately by boiling for 5 min. The samples were analyzed by SDS-PAGE (12.5% [wt/vol] acrylamide) and subjected to autoradiography.}

In another experiment, 500 ng of purified GST-21K was incubated as described above in the presence of 1 mM unlabeled ATP. Subsequently, half of the probe was left untreated or treated with λ protein phosphatase (New England BioLabs, Ipswich, MA) according to the manufacturer's protocols. The samples were subjected to SDS-PAGE (12.5% [wt/vol] acrylamide) and Western blotting (see below).

In a third type of experiment, the Myc-tagged wt 21K protein (21K-Myc) and mutants were expressed in leaves of N. benthamiana by agroinfiltration (see below). Agroinfiltrated leaf tissue was sampled at 3 days postinfiltration, ground in SDS-PAGE sample buffer at a 1:3 ratio (wt/vol) at room temperature, and heated to 95°C for 5 min. Aliquots of 20 μl were analyzed by SDS-PAGE (12.5% [wt/vol] acrylamide), after which proteins were transferred onto a polyvinylidene fluoride membrane (Amersham Biosciences). Western blotting was done by using a specific anti-phosphotyrosine (anti-pY) (clone 4G-10 Platinum; Upstate, Millipore) or anti-Myc (clone 9E10; Santa Cruz Biotechnology, Santa Cruz, CA) monoclonal antibody (MAb) or polyclonal antibodies to the PMTV 21K protein (prepared in this study). Polyclonal rabbit anti-mouse immunoglobulins (IgG) conjugated with horseradish peroxidase (HRP) (Dako Denmark A/S, Glostrup, Denmark) and HRP-conjugated anti-rabbit donkey IgG (GE Healthcare, Little Chalfont, United Kingdom) were used as secondary antibodies. Each membrane was developed with the SuperSignal West Femto chemiluminescence detection system (Thermo Scientific, Rockford, IL).

YTHS assay.

Competent yeast cells were prepared and transformed by using Matchmaker GAL4 Two-Hybrid System 3 (Clontech) with Saccharomyces cerevisiae strain AH109 and a small transformation scale according to the manufacturer's instructions. Cotransformations were made with different combinations of pGADT7 activation domain (AD) and pGBKT7 DNA-binding domain (BD) constructs. Cotransformation of pGBKT7-53 (Clontech) with pGADT7-T (Clontech) was used as a positive interaction control, and cotransformation of pGBKT7-Lam (Clontech) with pGADT7-T was used as a negative interaction control. To select for cotransformants, the cells were plated onto supplement dropout medium lacking leucine and tryptophan (SD/−Leu/−Trp) and grown at 30°C for approximately 36 h. The colonies were then replated onto supplement dropout medium lacking adenine, histidine, leucine, and tryptophan (SD/−Ade/−His/−Leu/−Trp) to check for interactions between the two-hybrid proteins, as indicated by growth on the selection plate after a 1-week incubation at 30°C.

To test the strength of the interactions, yeast strain Y187 was cotransformed with the above-described constructs, and the pellet 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal) β-galactosidase (β-gal) assay was performed and analyzed as described previously (42). The intensity of the color, caused by cleavage of X-gal by β-gal activity, was determined with ImageJ software (43). These intensities were used to calculate β-gal activity relative to that of the positive control, which was arbitrarily set to 100%. Expression of the fusion proteins in yeast was verified by Western blot analysis using AD- and BD-specific MAbs (Clontech), as described previously (44).

Plant materials and growth conditions.

N. benthamiana plants were grown in a controlled growth room at 20°C with a 16-h photoperiod. Five-week-old plants were used in the experiments. For inoculations, the plants were moved to a growth chamber (16°C; 16-h photoperiod) and kept in the dark overnight prior to inoculations. Infiltrated and bombarded plants were kept in the growth room during the experiments. All plants were fertilized weekly with a 1% (wt/vol) solution of N-P-K (16:9:22) fertilizer (Yara, Espoo, Finland).

Agroinfiltration.

The binary vectors for expression of PMTV proteins were introduced by electroporation into Agrobacterium tumefaciens strain C58C1 containing Ti plasmid pGV3850 (45). The transformations were verified by PCR designed to target the pLH vector from both sides of the cloning site. For agroinfiltration, the transformed cells were grown overnight at 28°C with shaking. The cells were collected by centrifugation and resuspended in induction medium (10 mM MgCl₂, 10 mM morpholineethanesulfonic acid [MES], 200 μM acetosyringone) at a final OD₆₀₀ of 1.0 and induced at room temperature for 3 h. Two or three fully expanded leaves of N. benthamiana plants were used for agroinfiltration. After 3 days, the agroinfiltrated leaves were collected and analyzed.

Virus inoculation and detection.

RNA transcripts were synthesized from PMTV cDNA constructs as described previously (34). Linearized cDNA (2 μg) was used in each transcription reaction mixture. The fully expanded leaves of N. benthamiana plants were mechanically inoculated with RNAs in GKP buffer (46), consisting of 12.5 mM glycine, 7.5 mM K₂HPO₄ (pH 9.2), 0.25% (wt/vol) bentonite (Sigma-Aldrich), and 0.25% (wt/vol) celite (Fluka Chemie, Buchs, Switzerland). Inoculation was done with a mixture containing wt or mutant RNA1-RNA2-RNA3 (1:1:2). Control plants were mock inoculated with GKP buffer.

Inoculated and upper leaves were collected at 18 days postinoculation (dpi). Samples were tested for the presence of PMTV by a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), using a MAb specific to the PMTV coat protein (Science and Advice for Scottish Agriculture [SASA], Edinburgh, United Kingdom), as described previously (47), and/or reverse transcription-PCR (RT-PCR). In a parallel experiment, leaves were collected at 3, 7, 10, and 14 dpi, and the samples were analyzed by RT-PCR. For RT-PCR, RNA was extracted by using a TRIzol-like reagent (48). RNA samples were treated with RQ1 RNase-free DNase (Promega) at 37°C for 30 min. cDNA was synthesized with Moloney murine leukemia virus reverse transcriptase (Promega) according to the manufacturer's instructions. To initiate cDNA synthesis, random hexamers were used. RT-PCR was carried out by using Phusion DNA polymerase (Finnzymes) according to the manufacturer's instructions.

In another experiment, plants were coinoculated with wt RNA1 and RNA2 and wt or mutant GFP-RNA3. Infections were monitored daily up to 18 dpi by using a handheld UV light (B-100 AP; UVP, Upland, CA). To study the infection at the single-cell level, plants were inoculated by bombardment with gold particles coated with transcribed RNAs using a HandyGun as described previously (49). Each single bombardment contained 0.1 mg gold particles (1-μm diameter; Bio-Rad, Hercules, CA) coated with RNA transcribed from 50 ng pPMTV1, 50 ng pPMTV2, and 100 ng of wt or mutant pPMTV3 plasmids. Leaves were analyzed at different time points up to 7 dpi.

Confocal microscopy.

Leaf tissue from the edges of the bombardment site was fixed to a microscope slide by using coverslips and tape and mounted in water. Pictures were taken with a Leica TCS SP5II HCS A confocal microscope using an HC PL Apo 10×/0.4 objective. GFP was visualized by using argon laser excitation at 488 nm and an acquisition window of 500 to 552 nm. The LAS AF Lite software package (Leica Microsystems GmbH, Wetzlar, Germany) was used to construct the images from serial optical sections and for image processing.

Cloning and mutagenesis of DNA.

Expression and purification of recombinant proteins.

In vitro and in vivo kinase assays and Western blotting.

YTHS assay.

Plant materials and growth conditions.

Agroinfiltration.

Virus inoculation and detection.

Confocal microscopy.

RESULTS

Detection of TGBp3 in PMTV-infected plant tissues.

Extracts from N. benthamiana leaves infected with PMTV or agroinfiltrated for 35S promoter-driven TGBp3 expression were tested by Western blotting using polyclonal antibodies raised to PMTV TGBp3 (21K protein) in this study. The antibodies detected a protein with electrophoretic mobility corresponding to TGBp3 in the agroinfiltrated leaf tissues overexpressing TGBp3 (Fig. 1C). However, no protein band for TGBp3 was observed in the leaves infected with PMTV in repeated experiments (data not shown), most probably due to a low concentration of the protein and inadequate sensitivity of the antibodies. Therefore, the cDNA of PMTV RNA3 was engineered (designated PMTV_21K-Myc) to express TGBp3 with a C-terminal Myc tag sequence (Fig. 1A) and subsequently coinoculated with PMTV RNA1 and RNA2 into N. benthamiana leaves. The upper noninoculated leaves systemically infected with PMTV_21K-Myc were sampled at 18 dpi, and extracts were analyzed by Western blotting using anti-Myc antibody (α-myc). A protein with the expected electrophoretic mobility of TGBp3 was detected (Fig. 1D). No such signal was detected in the mock-inoculated control plant (Fig. 1D). The progeny viruses in the leaf samples infected with PMTV_21K-Myc were analyzed by RT-PCR and sequencing of the products, which revealed an intact Myc tag sequence downstream of the TGBp3 gene. These data showed that TGBp3 was expressed in PMTV-infected N. benthamiana plants.

PMTV TGBp3 is phosphorylated by plant tyrosine kinase activity.

TGBp3 was expressed in E. coli as an N-terminal GST fusion protein and purified to near homogeneity by affinity chromatography using glutathione-Sepharose (Fig. 2A). The purified protein (GST-21K) was assayed for phosphorylation in a reconstituted system in which GST-21K was incubated in a kinase buffer with freshly prepared leaf extracts of N. benthamiana in the presence of [γ-^{P]ATP. Analysis of the reaction by SDS-PAGE and autoradiography revealed a single radiolabeled band corresponding to the expected size of GST-21K (∼48 kDa), whereas no such signal was detected in the control reaction mixture, to which only GST was added (}Fig. 2B). Equal loading of GST-21K and GST in the samples was verified by Coomassie blue staining (Fig. 2B, bottom). The short exposure time resulting in detectable signals for the recombinant GST-21K protein added to the crude leaf extract was insufficient to reveal signals for endogenous tyrosine-phosphorylated host proteins, which were detected by Western blot analysis using anti-pY antibody (α-pY) (Fig. 2D and and33).

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Fig 2

In vitro and in vivo phosphorylation of PMTV TGBp3. (A) GST-tagged PMTV TGBp3 (GST-21K) (arrowhead) expressed in E. coli, purified, and analyzed by SDS-PAGE. The gel was stained with Coomassie blue. The molecular mass markers (M) and their sizes (at left, in kilodaltons) are indicated. (B) In vitro kinase assay of the GST-21K fusion protein and GST alone (control) by incubation in an N. benthamiana leaf extract diluted in kinase buffer in the presence of [γ-^{P]ATP. Samples were separated by SDS-PAGE (12.5% [wt/vol] acrylamide), stained with Coomassie blue (bottom), and analyzed by autoradiography (top). The positions of molecular mass markers (at left, in kilodaltons) are indicated. Filled and open arrowheads indicate the positions of GST-21K and GST, respectively. (C) Phosphorylation of GST-21K by plant protein kinase activity. GST-21K (0.5 μg) was incubated with leaf extracts from}N. benthamiana in the presence of unlabeled ATP. Subsequently, half of the sample was treated with λ protein phosphatase (+λ PPase), and half was left untreated (−λ PPase). Western blotting was done by using anti-phosphotyrosine MAb (α-pY). Equal loading of proteins was verified by Ponceau S staining. The position of the 50-kDa molecular marker is indicated. (D) Tyrosine phosphorylation of PMTV TGBp3 in leaves of N. benthamiana infected with PMTV_21K-Myc, as detected by Western blot analysis using anti-pY (tyrosine-phosphorylated, higher-molecular-weight host proteins were also detected by using anti-pY). Figure 1D shows detection of PMTV_21K-Myc in the same sample using anti-Myc. The positions of molecular mass markers (at right, in kilodaltons) and the position of the 21K protein (arrowhead) are indicated.

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Fig 3

Tyrosine phosphorylation of PMTV TGBp3 expressed in leaves of N. benthamiana by agroninfiltration. Detection of Myc-tagged PMTV TGBp3 (21K-myc) and Myc-tagged 21K mutants was carried out by Western blot analysis using anti-pY (top) and anti-Myc (bottom). Positions of molecular mass markers are shown at the right. The position of 21K-Myc is indicated with an arrowhead. Lanes 1 and 2, leaf tissue agroinfiltrated for expression of the PMTV 8K protein (8K) (lane 1) and leaf tissue mock infiltrated with infiltration buffer alone (lane 2) as negative controls; lane 6, leaf tissue infiltrated for expression of 21K-Myc as a positive control; lane 3, TGBp3 with tyrosine residues at positions 87 to 89 and 120 substituted for Ala (21K_87-89A/120A); lane 4, TGBp3 with the tyrosine residue at position 120 substituted for alanine (21K_120A); lane 5, TGBp3 with tyrosine residues at positions 87 to 89 substituted for alanine (21K_87-89A). Anti-pY and anti-Myc also detected host proteins with molecular weights higher than that of 21K-Myc.

Subsequently, purified GST-21K was incubated with leaf extracts, as described above, but in the presence of unlabeled ATP. The sample was subsequently divided into two aliquots, one of which was treated with λ protein phosphatase that removes phosphate groups from serine, threonine, and tyrosine residues. Western blot analysis of the proteins using α-pY revealed a band corresponding to GST-21K only in the untreated aliquot of the sample (Fig. 2C); no protein band was detected in the aliquot treated with λ protein phosphatase (Fig. 2C). Equal loading of GST-21K in the samples was verified by Ponceau S staining (Fig. 2C). These results from two types of experiments, each done three times, provided evidence that TGBp3 of PMTV was phosphorylated on tyrosine by plant kinases in vitro.

The leaf samples from PMTV_21K-Myc-infected and mock-inoculated plants tested for the presence of TGBp3 with α-myc (Fig. 1D) were also tested with α-pY to examine whether TGBp3 was phosphorylated in vivo. For this purpose, all samples were loaded in duplicate for analysis by SDS-PAGE. The membrane was cut into two halves, each containing a similar set of samples. Development of one-half of the membrane with α-pY (Fig. 2D) and the other half with α-myc (Fig. 1D) revealed a protein band at the same position corresponding to 21 kDa. No such protein band was detected in the leaves of noninfected control plants (Fig. 1D and andD).D). This experiment was repeated four times independently, with similar results. These data indicated that TGBp3 was phosphorylated on tyrosine by plant kinase activity in the PMTV-infected plants of N. benthamiana.

Furthermore, leaves of N. benthamiana were agroinfiltrated to express Myc-tagged TGBp3 (21K-Myc). The extracts from the infiltrated leaf tissues were subjected to Western blotting using α-pY. Results revealed a tyrosine-phosphorylated protein with an electrophoretic mobility corresponding to the 21K protein (Fig. 3, top, lane 6). No such protein band was detected in the mock-infiltrated control leaves (Fig. 3, top, lane 2). Western blot analysis of the same samples using α-myc confirmed that the protein band detected with α-pY corresponds to TGBp3 (21K protein) (Fig. 3, bottom, lane 6). α-pY detected many unknown tyrosine-phosphorylated host proteins (Fig. 3), and also, α-myc reacted with some host proteins with a considerably higher molecular weight than 21K-Myc (Fig. 3). The experiment was done three times independently, with similar results.

Phosphorylation sites in TGBp3 of PMTV.

NetPhos 2 and Scansite software prediction of the phosphorylation sites in PMTV TGBp3 suggested phosphorylation of the tyrosine residues at positions 89 and 120 within the YQDLN and QXXPF/Y motifs, respectively (Fig. 1B), which are conserved in the TGBp3 proteins of the hordei-like viruses (33). Therefore, in subsequent experiments, the TGBp3 gene (tagged with Myc) was mutated by using site-directed alanine substitutions at (i) Y_87–89 (21K_87-89A mutant), (ii) Y₁₂₀ (21K_120A mutant), and (iii) the combined substitutions (21K_87-89A/120A mutant). All three adjacent tyrosine residues 87 to 89 were replaced in order to exclude the possibility that loss of phosphorylation at residue 89 would be complemented by phosphorylation of Y₈₇ and/or Y₈₈. The wt and mutated TGBp3-Myc genes introduced into N. benthamiana leaves by agroinfiltration showed similar levels of protein expression, as detected by Western blot analysis with α-myc at 3 days postinfiltration (Fig. 3, bottom). The same samples were subjected to Western blot analysis using α-pY (Fig. 3, top). wt TGBp3 (21K-Myc) (Fig. 3, top, lane 6) and the 21K_87-89A (lane 5) and 21K_120A (lane 4) mutants were found to be phosphorylated on tyrosine. In contrast, no tyrosine phosphorylation of TGBp3 was detected in leaves expressing the 21K_87-89A/120A mutant (Fig. 3, top, lane 3), the PMTV 8K protein (control) (lane 1), or the mock-infiltrated leaves (lane 2), whereas tyrosine-phosphorylated host proteins were detected. These results from three independent experiments indicated that the tyrosine residue at position 120 and at least one of the tyrosine residues at positions 87 to 89 were phosphorylated.

Mutation of the phosphorylation sites in TGBp3 impairs PMTV virulence.

The tyrosine residues at positions 87 to 89 and 120 were also substituted for alanine in TGBp3 in the full-length cDNA of PMTV RNA3, which gives rise to PMTV infection when the in vitro transcripts are coinoculated into plants with the RNA1 and RNA2 transcripts produced from their respective plasmids (34). Plants of N. benthamiana were mechanically coinoculated with the PMTV RNA1 and RNA2 transcripts and those of either PMTV_21K or the mutated construct pPMTV21K_87-89A, pPMTV21K_120A, or pPMTV21K_87-89A/120A. In three independent experiments, systemic infection was detected by RT-PCR at 7 dpi in plants inoculated with wt PMTV (Fig. 4A). In contrast, no systemic infection was detected in plants in which the mutant transcripts were used, as tested by RT-PCR (Fig. 4A) and DAS-ELISA (data not shown) at 14 and 18 dpi, respectively. RT-PCR analysis of the inoculated leaves revealed readily detectable amplification products of the expected size in leaves infected with wt PMTV, whereas no products or only very faint bands were detected in the leaves inoculated with the mutant constructs (Fig. 4A and data not shown). These results indicated that the mutations introduced into the phosphorylation sites in TGBp3 significantly decreased the infectivity of PMTV.

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Fig 4

Effect of amino acid substitutions in TGBp3 on the infectivity of PMTV mutants. (A) Detection of PMTV infection in inoculated leaves (I) and upper noninoculated leaves (S) of N. benthamiana. Samples were analyzed by RT-PCR using primers specific to the TGBp3 sequence at 3, 7, 10, and 14 dpi. M, DNA molecular size markers, with the 1,000-bp and 500-bp markers indicated. (B) Confocal microscopy of N. benthamiana leaves inoculated with PMTV that expresses GFP fused to TGBp1. GFP-PMTV expresses wt TGBp3, whereas GFP-PMTV21K_87-89A expresses TGBp3 where tyrosine residues at position 87 to 89 were replaced with alanine. Images were captured at 3 dpi. Bars, 100 μm.

Cell-to-cell movement of PMTV is impaired by mutation of the phosphorylation sites in TGBp3.

Cell-to-cell movement was studied by introducing the mutated TGBp3 genes into an engineered cDNA of PMTV RNA3 that expressed GFP as an N-terminal fusion with TGBp1 (Fig. 1A). Development of virus infection in the inoculated leaves was monitored under a handheld UV lamp and by confocal microscopy. Lesions consisting of many epidermal cells became visible in leaves inoculated with wt GFP-PMTV at 3 dpi (Fig. 4B). In leaves inoculated with GFP-PMTV21K_87-89A, individual fluorescent epidermal cells were detected, indicating that the mutant virus was infectious but debilitated in cell-to-cell movement (Fig. 4B). RT-PCR analysis of the leaves and subsequent sequencing of the products confirmed that the virus had retained the introduced mutations. These results indicated that mutation of this site of TGBp3 was detrimental to cell-to-cell movement of PMTV. In contrast, no fluorescence was observed in leaves inoculated with the GFP-PMTV21K_120A or GFP-PMTV21K_87-89A/120A mutant virus at any time up to 18 dpi. The results obtained by confocal microscopy were consistently observed for a total of 12 leaves inoculated with each virus in three independent experiments.

Influence of TGBp3 phosphorylation sites on interactions with TGBp2.

Previous studies with yeast two-hybrid assays have shown that PMTV TGBp3 and TGBp2 interact (24, 25), and this interaction was suggested to be needed for viral movement (15). In the present study, the wt and mutated TGBp3 genes of PMTV were transferred into the yeast two-hybrid assay vectors for expression as a fusion protein with the activation domain (AD), whereas TGBp2 was cloned into the binding domain (BD) vector. The yeast was cotransformed with the BD-TGBp2-containing plasmid and one of the AD-TGBp3-containing plasmids. After selection on selective medium (Fig. 5A), cotransformants were transferred into a new selective medium allowing the growth of only those transformants in which the selective marker genes were activated by interaction of the pairs of proteins tested (Fig. 5B). Interactions were detected between TGBp2 and wt TGBp3 as well as between TGBp2 and the mutated TGBp3 proteins 21K_87-89A, 21K_120A, and 21K_87-89A/120A (Fig. 5B, lanes 1 to 4, respectively). None of the constructs activated reporter genes autonomously in yeast (Fig. 5B, lanes 5 to 9). Expression of the fusion proteins in yeast cells was confirmed by Western blot analysis (Fig. 5E).

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Fig 5

Yeast two-hybrid analysis of the interactions between TGBp2 (13K) and the mutated forms of TGBp3 (21K) of PMTV. (A) Cotransformation of the binding domain (BD) and activation domain (AD) vector plasmids is indicated as growth of the yeast on selective medium. The plasmids were selected by the lack of tryptophan (−Trp) and leucine (−Leu), respectively, in the culture medium. TGBp2 was expressed from the BD plasmid, and the TGBp3-based constructs were expressed from the AD plasmid in the combinations indicated above the panel. ni, no insert; + and −, positive and negative controls of the Clontech yeast two-hybrid system. (B) Growth of the cotransformed yeast strains on selective medium indicating interactions between the coexpressed PMTV proteins. The culture medium lacked adenine (−Ade), histidine (−His), leucine (−Leu), and tryptophan (−Trp). (C) An X-gal assay in a 96-well microtiter plate format (42) was used to estimate the strength of the protein-protein interactions, as detected by the intensity of the color resulting from the X-gal substrate processed by β-galactosidase activity in cotransformed yeast cells. Enhanced interaction between the test proteins resulted in enhanced expression of β-galactosidase and of color. (D) Quantification of the intensity of the blue color in the yeast cells observed in panel C by using ImageJ software. Columns indicate β-galactosidase activity relative to that of the positive control (+) of the yeast two-hybrid system provided by Clontech. Error bars indicate the standard deviation of the mean (n = 3). (E) Expression of the 13K-BD and 21K-AD fusion proteins in yeast cells, as tested by Western blot analysis using MAbs specific to the BD and AD domains, respectively. Ponceau S staining of the membrane was used as a protein loading control. Positions of molecular mass markers (at right, in kilodaltons) are indicated.

Quantitative differences in the strength of interactions were studied by cotransforming the plasmids in the above-mentioned combinations into another yeast strain that encoded β-galactosidase under a strong promoter, facilitating β-galactosidase measurements using a microplate assay (42). Although interactions between TGBp2 and wt TGBp3 or the 21K_120A protein (Fig. 5, lanes 1 and 3, respectively) were sufficient for growth of yeast on selective medium (Fig. 5B), the interactions were weak, as estimated by the β-galactosidase assay (Fig. 5C and andD).D). In contrast, the 21K_87-89A and 21K_87-89A/120A TGBp3 mutants (Fig. 5, lanes 2 and 4, respectively) showed an enhanced interaction with TGBp2, as suggested by a 10-fold-higher β-galactosidase activity than that observed with wt TGBp3 (Fig. 5C and andDD).

Detection of TGBp3 in PMTV-infected plant tissues.

PMTV TGBp3 is phosphorylated by plant tyrosine kinase activity.

Fig 2

Fig 3

Phosphorylation sites in TGBp3 of PMTV.

Mutation of the phosphorylation sites in TGBp3 impairs PMTV virulence.

Fig 4

Cell-to-cell movement of PMTV is impaired by mutation of the phosphorylation sites in TGBp3.

Influence of TGBp3 phosphorylation sites on interactions with TGBp2.

Fig 5

DISCUSSION

Plant viruses encode MPs for intra- and intercellular transport of viral ribonucleoprotein complexes. A few viral MPs are known to be phosphorylated by serine/threonine kinases, but a detailed understanding of how phosphorylation modulates their functions is limited (8–14, 50). Furthermore, it is unclear whether tyrosine kinases participate in phosphorylation of viral MPs. Viruses such as PMTV encode three movement-associated TGB proteins acting coordinately. TGBp3 plays an important role in intracellular viral movement by targeting the viral ribonucleoprotein complex to plasmodesmata for cell-to-cell movement (15). However, the relative abundance of TGBp3 is the lowest among the TGB proteins in infected cells, which makes it difficult to detect (27, 29, 30, 32, 51). Indeed, PMTV TGBp3 has previously been detected in infected leaf tissues only following overexpression from a heterologous viral vector (24). In the present study, however, PMTV TGBp3 was also detected in PMTV-infected plants. Furthermore, our study revealed that PMTV TGBp3 was phosphorylated by plant tyrosine kinase activity in infected plants.

The luminal motifs in the central part (₈₇YYYQDLN₉₃) and the C-proximal part (₁₁₆QEFPYGNI₁₂₃) of PMTV TGBp3 (25) were found to be sites of tyrosine phosphorylation. Substitution of both tyrosine residues Y_87–89 and Y₁₂₀ with alanine residues abolished phosphorylation of TGBp3, in contrast to the substitutions introduced at only one of the two sites, indicating that both sites were phosphorylated. These results also indicated that only these two sites were tyrosine phosphorylated in TGBp3. These mutations that abolished tyrosine phosphorylation of TGBp3 resulted in the loss of any discernible PMTV infectivity in N. benthamiana. In contrast, mutation of only Y_87–89 to alanine did not abolish infectivity but allowed high levels of virus multiplication in initially infected cells, as evidenced by GFP fluorescence following inoculation with GFP-PMTV21K_87-89A. However, infection with GFP-PMTV21K_87-89A was restricted to the initially infected cells. This result is in agreement with a previous study showing that substitution of Y₈₉ for glycine in TGBp3 of PMTV prevented targeting of TGBp3 to plasmodesmata, inhibited viral cell-to-cell movement, and resulted in infection of only individual cells (25). Our results also show that the defective cell-to-cell movement of PMTV was associated with alteration in the strength of the interactions between TGBp3 and TGBp2. Upon mutation of Y_87–89 to alanine, the interaction between TGBp3 and TGBp2 of PMTV was enhanced by an order of magnitude. In a previous study, substitution of Y₈₉ for glycine in TGBp3 did not appear to affect the interaction with TGBp2 (25), probably because the interaction was enhanced rather than inhibited, and no quantitative assay was used. Taken together, those previous studies are consistent with our findings in terms of the importance of interactions between TGBp3 and TGBp2 in the movement of PMTV (24, 25) and BSMV (26, 27), and our findings now suggest that tyrosine phosphorylation may be the mechanism regulating the interaction between TGBp3 and TGBp2.

The current model of PMTV movement suggests that the putative viral movement complexes, which supposedly include TGBp2, TGBp3, and a ribonucleoprotein complex containing TGBp1 and viral RNA, associate with motile membrane compartments of the endoplasmic reticulum via the two transmembrane domains located at the N-proximal and C-terminal parts of TGBp3. The movement complex is targeted to plasmodesmata along the endoplasmic reticulum-actin network by TGBp3, and the ribonucleoprotein complex is passed through plasmodesmata to the adjacent cell. TGBp2 and TGBp3 gate plasmodesmata open but do not follow the ribonucleoprotein complex and are instead recycled via an endocytotic pathway (23, 25). The reversible nature of phosphorylation (6) may therefore provide the means for the proposed recycling and dynamic interaction between TGBp3 and TGBp2, allowing TGBp3 to participate in multiple intracellular viral transport events.

Substitution of Tyr₁₂₀ for alanine in TGBp3 resulted in an apparent loss of infectivity of PMTV in N. benthamiana. However, phosphorylation of TGBp3 was not lost by the mutation, owing to phosphorylation at the other site (Tyr_87–89). Also, no discernible effect on the TGBp3-TGBp2 interaction was associated with the Tyr₁₂₀Ala mutation, as tested by the yeast two-hybrid assay. The existence of two distant tyrosine phosphorylation sites in TGBp3, both important for the infection cycle of PMTV, suggests that they may be needed to achieve threshold levels of TGBp3 phosphorylation required for different MP functions. Tyr₈₉ and Tyr₁₂₀ lie within putative tyrosine-based sorting motifs of TGBp3 (YXXΦ, where Φ is a bulky hydrophobic amino acid). The sorting motif is important for protein endocytosis, which in some proteins is controlled by phosphorylation of the tyrosine residue of the motif (52–54). The motif ₈₉YQDL, located in the luminal central part of TGBp3, is conserved among the hordei-like viruses and is implicated in the endocytosis of TGBp2 and TGBp3 (23, 25). In contrast, the luminal motif ₁₂₀YGNI of PMTV TGBp3 (25) is less conserved among the other hordei-like viruses. The endocytotic pathways also play a role in the functions of MPs in other plant viruses, including the 30K MP of TMV and the MP of Cabbage leaf curl virus (genus Begomovirus) (55). However, the role of phosphorylation in the regulation of endocytosis of these MPs has not been reported.

The MPs of a few positive-sense ssRNA viruses are phosphorylated by serine- and threonine-specific protein kinases. Phosphorylation of the C-terminal Ser₂₅₈ or Ser₂₆₅ or of Thr₂₆₁ enhances the 30K MP-mediated cell-to-cell movement of TMV, whereas phosphorylation of multiple sites reduces or inhibits TMV movement (9). In the genus Potyvirus, coat protein is one of the proteins facilitating viral cell-to-cell and long-distance movement in plants. The CKII kinase of tobacco phosphorylates Thr₂₄₂ in the coat protein of PVA, which inhibits viral movement (13). TGBp1 of Potato virus X is phosphorylated on Ser₁₆₅ in tobacco, possibly by CKII, but the functional significance of phosphorylation is unknown (16). The results of our study expand the comprehension of phosphorylation-mediated regulation of viral MPs significantly by also implicating tyrosine phosphorylation in the process.

Our study links tyrosine kinases to virus infection in plants by showing that one of the TGB proteins, TGBp3, undergoes tyrosine phosphorylation, which is novel for TGB-containing viruses and plant virus proteins in general. This fundamental novel finding opens a new, unexploited arena to study interactions of viruses and plants and understand how viruses utilize host factors to advance infection. Tyrosine phosphorylation was previously considered to be rare in plants, but recent studies indicate otherwise. Up to 4.3% of all phosphorylation events of proteins in Arabidopsis thaliana may occur on tyrosine, which is a frequency comparable to that of tyrosine phosphorylation in animals (56, 57). Furthermore, over 75% of the tyrosine-phosphorylated proteins in Arabidopsis are phosphorylated at multiple sites (56). Considering how viruses depend on and utilize cellular factors during the infection cycle, tyrosine phosphorylation of MPs may be more common than previously thought. Proteins of mammalian viruses, including vaccinia, variola, and monkeypox viruses, are phosphorylated by tyrosine kinases, and kinase inhibitors can increase survival of the host by reducing viral load and dissemination of the virus to distal tissues (58, 59). It is conceivable that inhibition of tyrosine kinases required for phosphorylation of viral proteins could also have a role to play in combating viral infections in plants.

Department of Agricultural Sciences, University of Helsinki, Helsinki, Finland

^{Corresponding author.}

Address correspondence to Jari P. T. Valkonen, if.iknisleh@nenoklav.iraj.

O.S. and J.S. contributed equally.

Department of Agricultural Sciences, University of Helsinki, Helsinki, Finland

Address correspondence to Jari P. T. Valkonen, if.iknisleh@nenoklav.iraj.

O.S. and J.S. contributed equally.

Received 2012 Dec 8; Accepted 2013 Jan 25.

Abstract

Functions of viral proteins can be regulated through phosphorylation by serine/threonine kinases in plants, but little is known about the involvement of tyrosine kinases in plant virus infection. In this study, TGBp3, one of the three movement proteins encoded by a triple gene block (TGB) of Potato mop-top virus (PMTV), was detected for the first time in PMTV-infected plants and found to be tyrosine phosphorylated. Phosphorylation sites (Tyr_87–89 and Tyr₁₂₀) were located in two amino acid motifs conserved in the TGB-containing, rod-shaped plant viruses. Substitution of these tyrosine residues in both motifs was needed to abolish tyrosine phosphorylation of TGBp3. Substitution of Tyr_87–89 with alanine residues enhanced the interaction between TGBp3 and TGBp2 and inhibited cell-to-cell movement of PMTV. On the other hand, substitution of Tyr₁₂₀ with alanine resulted in no alteration in the interaction of TGBp3 with TGBp2, but the mutant virus was not infectious. The results suggest that tyrosine phosphorylation is a mechanism regulating the functions of plant virus movement proteins.

Abstract

ACKNOWLEDGMENTS

We thank Tuuli Haikonen for assistance in acquiring the confocal images.

We gratefully acknowledge financial support from the Ministry of Agriculture and Forestry (grant 1386/39/2005), the Academy of Finland (grants 1134759; and 1253126), and the Viikki Doctoral Programme in Molecular Biosciences (VGSB).

ACKNOWLEDGMENTS

Footnotes

Published ahead of print 30 January 2013

Footnotes

REFERENCES

References

1. Maule A. 2008 Plasmodesmata: structure, function and biogenesis. Curr. Opin. Plant Biol.11:680–686 [[PubMed][Google Scholar]
2. Lucas WJ, Ham BK, Kim JY. 2009 Plasmodesmata—bridging the gap between neighboring plant cells. Trends. Cell Biol.19:495–503 [[PubMed][Google Scholar]
3. Harries PA, Schoelz JE, Nelson R. 2010 Intracellular transport of viruses and their components: utilizing the cytoskeleton and membrane highways. Mol. Plant Microbe Interact.23:1381–1393 [[PubMed][Google Scholar]
4. Schoelz JE, Harries PA, Nelson RS. 2011 Intracellular transport of plant viruses: finding the door out of the cell. Mol. Plant4:813–831 [Google Scholar]
5. Ueki S, Citovsky V. 2011 To gate, or not to gate: regulatory mechanisms for intercellular protein transport and virus movement in plants. Mol. Plant4:782–793 [Google Scholar]
6. Bond AE, Row PE, Dudley E. 2011 Post-translational modification of proteins; methodologies and applications in plant sciences. Phytochemistry72:975–996 [[PubMed][Google Scholar]
7. Citovsky V, McLean BG, Zupan JR, Zambryski P. 1993 Phosphorylation of tobacco mosaic virus cell-to-cell movement protein by a developmentally regulated plant cell wall associated protein kinase. Genes Dev.7:904–910 [[PubMed][Google Scholar]
8. Waigmann E, Chen MH, Bachmaier R, Ghoshroy S, Citovsky V. 2000 Regulation of plasmodesmal transport by phosphorylation of tobacco mosaic virus cell-to-cell movement protein. EMBO J.19:4875–4884 [Google Scholar]
9. Trutnyeva K, Bachmaier R, Waigmann E. 2005 Mimicking carboxyterminal phosphorylation differentially effects subcellular distribution and cell-to-cell movement of Tobacco mosaic virus movement protein. Virology332:563–577 [[PubMed][Google Scholar]
10. Kawakami S, Padgett HS, Hosokawa D, Okada Y, Beachy RN, Watanabe Y. 1999 Phosphorylation and/or presence of serine 37 in the movement protein of tomato mosaic tobamovirus is essential for intracellular localization and stability in vivo. J. Virol.73:6831–6840 [Google Scholar]
11. Kawakami S, Hori K, Hosokawa D, Okada Y, Watanabe Y. 2003 Defective Tobamovirus movement protein lacking wild-type phosphorylation sites can be complemented by substitutions found in revertants. J. Virol.77:1452–1461 [Google Scholar]
12. Ivanov KI, Puustinen P, Merits A, Saarma M, Mäkinen K. 2001 Phosphorylation down-regulates the RNA binding function of the coat protein of Potato virus A. J. Biol. Chem.276:13530–13540 [[PubMed][Google Scholar]
13. Ivanov KI, Puustinen P, Gabrenaite R, Vihinen H, Rönnstrand L, Valmu L, Kalkkinen N, Mäkinen K. 2003 Phosphorylation of the potyvirus capsid protein by protein kinase CK2 and its relevance for virus infection. Plant Cell15:2124–2139 [Google Scholar]
14. Florentino LH, Santos AA, Fontenelle NR, Pinheiro GL, Zerbini FM, Baracat-Pereira MC, Fontes EP. 2006 A PERK-like receptor kinase interacts with the geminivirus nuclear shuttle protein and potentiates viral infection. J. Virol.80:6648–6656 [Google Scholar]
15. Verchot-Lubicz J, Torrance L, Solovyev AG, Morozov SY, Jackson AO, Gilmer D. 2010 Varied movement strategies employed by triple gene block-encoding viruses. Mol. Plant Microbe Interact.23:1231–1247 [[PubMed][Google Scholar]
16. Módena NA, Zelada AM, Conte F, Mentaberry A. 2008 Phosphorylation of the TGBp1 movement protein of Potato virus X by a Nicotiana tabacum CK2-like activity. Virus Res.137:16–23 [[PubMed][Google Scholar]
17. Santala J, Samuilova O, Hannukkala A, Latvala S, Kortemaa H, Beuch U, Kvarnheden A, Persson P, Topp K, Ørstad K, Spetz C, Nielsen SL, Kirk HG, Uth JG, Budziszewska M, Wieczorek P, Obrepalska-Steplowska A, Pospieszny H, Kryszczuk A, Sztangret-Wisniewska J, Yin Z, Chrzanowska M, Zimnoch-Guzowska E, Jackeviciene E, Taluntytė L, Pūpola N, Mihailova J, Lielmane I, Järvekülg L, Kotkas K, Rogozina E, Sozonov A, Tikhonovich I, Horn P, Broer I, Kuusiene S, Staniulis J, Adam G, Valkonen JPT. 2010 Detection, distribution and control of Potato mop-top virus, a soil-borne virus, in northern Europe. Ann. Appl. Biol.157:163–178 [PubMed][Google Scholar]
18. Savenkov EI, Sandgren M, Valkonen JPT. 1999 Complete sequence of RNA 1 and presence of tRNA-like structures in all RNAs of Potato mop-top pomovirus. J. Gen. Virol.80:2779–2784 [[PubMed][Google Scholar]
19. Sandgren M, Savenkov EI, Valkonen JPT. 2001 The readthrough region of Potato mop-top virus (PMTV) coat protein encoding RNA, the second largest RNA of PMTV genome, undergoes structural changes in naturally infected and experimentally inoculated plants. Arch. Virol.146:467–477 [[PubMed][Google Scholar]
20. Scott PK, Kashiwazaki S, Reavy B, Harrison BD. 1994 The nucleotide sequence of potato mop-top virus RNA 2: a novel type of genome organization for a furovirus. J. Gen. Virol.75:3561–3568 [[PubMed][Google Scholar]
21. Lukhovitskaya NI, Yelina NE, Zamyatnin AA, Jr, Schepetilnikov MV, Solovyev AG, Sandgren M, Morozov SY, Valkonen JPT, Savenkov EI. 2005 Expression, localization and effects on virulence of the cysteine-rich 8-kDa protein of Potato mop-top virus. J. Gen. Virol.86:2879–2889 [[PubMed][Google Scholar]
22. Zamyatnin AA, Jr, Solovyev AG, Savenkov EI, Germundsson A, Sandgren M, Valkonen JPT, Morozov SY. 2004 Transient coexpression of individual genes encoded by the triple gene block (TGB) of Potato mop-top virus reveals requirements for TGBp1 trafficking. Mol. Plant Microbe Interact.17:921–930 [[PubMed][Google Scholar]
23. Haupt S, Cowan GH, Ziegler A, Roberts AG, Oparka KJ, Torrance L. 2005 Two plant-viral movement proteins traffic in the endocytic recycling pathway. Plant Cell17:164–181 [Google Scholar]
24. Cowan GH, Lioliopoulou F, Ziegler A, Torrance L. 2002 Subcellular localization, protein interactions, and RNA binding of Potato mop-top virus triple gene block proteins. Virology298:106–115 [[PubMed][Google Scholar]
25. Tilsner J, Cowan GH, Roberts AG, Chapman SN, Ziegler A, Savenkov E, Torrance L. 2010 Plasmodesmal targeting and intercellular movement of potato mop-top pomovirus is mediated by a membrane anchored tyrosine-based motif on the luminal side of the endoplasmic reticulum and the C-terminal transmembrane domain in the TGB3 movement protein. Virology402:41–51 [[PubMed][Google Scholar]
26. Lim HS, Bragg JN, Ganesan U, Lawrence DM, Yu J, Isogai M, Hammond J, Jackson AO. 2008 Triple gene block protein interactions involved in movement of Barley stripe mosaic virus. J. Virol.82:4991–5006 [Google Scholar]
27. Lim HS, Bragg JN, Ganesan U, Ruzin S, Schichnes D, Lee MY, Vaira AM, Ryu KH, Hammond J, Jackson AO. 2009 Subcellular localization of the Barley stripe mosaic virus triple gene block proteins. J. Virol.83:9432–9448 [Google Scholar]
28. Jackson AO, Lim HS, Bragg J, Ganesan U, Lee MY. 2009 Hordeivirus replication, movement, and pathogenesis. Annu. Rev. Phytopathol.47:385–422 [[PubMed][Google Scholar]
29. Donald RGK, Zhou H, Jackson AO. 1993 Serological analysis of barley stripe mosaic virus-encoded proteins in infected barley. Virology195:659–668 [[PubMed][Google Scholar]
30. Zhou H, Jackson AO. 1996 Expression of the barley stripe mosaic virus RNAβ “triple gene block.”Virology216:367–379 [[PubMed][Google Scholar]
31. Morozov SY, Solovyev AG. 2003 Triple gene block: modular design of a multifunctional machine for plant virus movement. J. Gen. Virol.84:1351–1366 [[PubMed][Google Scholar]
32. Shemyakina EA, Erokhina TN, Gorshkova EN, Schiemann J, Solovyev AG, Morozov SY. 2011 Formation of protein complexes containing plant virus movement protein TGBp3 is necessary for its intracellular trafficking. Biochimie93:742–748 [[PubMed][Google Scholar]
33. Solovyev AG, Savenkov EI, Agranovsky AA, Morozov SY. 1996 Comparison of the genomic cis-elements and coding regions in RNAβ components of the hordeiviruses barley stripe mosaic virus, lychnis ringspot virus and poa semilatent virus. Virology219:9–18 [[PubMed][Google Scholar]
34. Savenkov EI, Germundsson A, Zamyatnin AA, Jr, Sandgren M, Valkonen JPT. 2003 Potato mop-top virus: the coat protein-encoding RNA and the gene for cysteine-rich protein are dispensable for systemic virus movement in Nicotiana benthamiana. J. Gen. Virol.84:1001–1005 [[PubMed][Google Scholar]
35. Blom N, Gammeltoft S, Brunak S. 1999 Sequence and structure based prediction of eucaryotic protein phosphorylation sites. J. Mol. Biol.294:1351–1362 [[PubMed][Google Scholar]
36. Obenauer JC, Cantley LC, Yaffe MB. 2003 Scansite 2.0: proteome wide prediction of cell signaling interactions using short sequence motifs. Nucleic Acids Res.31:3635–3641 [Google Scholar]
37. Samuilova O, Krogerus C, Fabrichniy I, Hyypiä T. 2006 ATP hydrolysis and AMP kinase activities of nonstructural protein 2C of human parechovirus 1. J. Virol.80:1053–1058 [Google Scholar]
38. Kelloniemi J, Mäkinen K, Valkonen JPT. 2006 A potyvirus-based gene vector allows producing active human S-COMT and animal GFP, but not human sorcin, in vector-infected plants. Biochimie88:505–513 [[PubMed][Google Scholar]
39. Zamyatnin AA, Jr, Solovyev AG, Bozhkov PV, Valkonen JPT, Morozov SY, Savenkov EI. 2006 Assessment of the integral membrane protein topology in living cells. Plant J.46:145–154 [[PubMed][Google Scholar]
40. Sambrook J, Fritsch EF, Maniatis T. 1989 Molecular cloning: a laboratory manual, 2nd ed Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY [PubMed][Google Scholar]
41. Frangioni JV, Neel BG. 1993 Solubilization and purification of enzymatically active glutathione S-transferase (pGEX) fusion proteins. Anal. Biochem.210:179–187 [[PubMed][Google Scholar]
42. Möckli N, Auerbach D. 2004 Quantitative β-galactosidase assay suitable for high-throughput applications in the yeast two-hybrid system. Biotechniques36:872–876 [[PubMed][Google Scholar]
43. Abramoff MD, Magalhaes PJ, Ram SJ. 2004 Image processing with ImageJ. Biophotonics Int.11:36–42 [PubMed][Google Scholar]
44. Ala-Poikela M, Goytia E, Haikonen T, Rajamäki ML, Valkonen JPT. 2011 Helper component proteinase of the genus Potyvirus is an interaction partner of translation initiation factors elF(iso)4E and alF4E and contains a 4E binding motif. J. Virol.85:6784–6794 [Google Scholar]
45. Deblaere R, Bytebier B, De Greve H, Deboeck F, Schell J, Van Montagu M, Leemans J. 1985 Efficient octopine Ti plasmid-derived vectors for Agrobacterium-mediated gene transfer to plants. Nucleic Acids Res.13:4777–4788 [Google Scholar]
46. Petty IT, Hunter BG, Wei N, Jackson AO. 1989 Infectious barley stripe mosaic virus RNA transcribed in vitro from full-length genomic cDNA clones. Virology171:342–349 [[PubMed][Google Scholar]
47. Latvala-Kilby S, Aura JM, Pupola N, Hannukkala A, Valkonen JPT. 2009 Detection of Potato mop-top virus in potato tubers and sprouts: combinations of RNA2 and RNA3 variants and incidence of symptomless infections. Phytopathology99:519–531 [[PubMed][Google Scholar]
48. Caldo RA, Nettleton D, Wise RP. 2004 Interaction-dependent gene expression in Mla-specified response to barley powdery mildew. Plant Cell16:2514–2528 [Google Scholar]
49. Sikorskaite S, Vuorinen AL, Rajamäki ML, Nieminen A, Gaba V, Valkonen JPT. 2010 HandyGun: an improved custom-designed, non-vacuum gene gun suitable for virus inoculation. J. Virol. Methods165:320–324 [[PubMed][Google Scholar]
50. Sokolova M, Prüfer D, Tacke E, Rohde W. 1997 The potato leaf roll virus 17K movement protein is phosphorylated by a membrane-associated protein kinase from potato with biochemical features of protein kinase C. FEBS Lett.400:201–205 [[PubMed][Google Scholar]
51. Gorshkova EN, Erokhina TN, Stroganova TA, Yelina NE, Zamyatnin AA, Jr, Kalinina NO, Schiemann J, Solovyev AG, Morozov SY. 2003 Immunodetection and fluorescent microscopy of transgenically expressed hordeivirus TGBp3 movement protein reveals its association with endoplasmic reticulum elements in close proximity to plasmodesmata. J. Gen. Virol.84:985–994 [[PubMed][Google Scholar]
52. Shiratori T, Miyatake S, Ohno H, Nakaseko C, Isono K, Bonifacino JC, Saito T. 1997 Tyrosine phosphorylation controls internalization of CTLA-4 by regulating its interaction with clathrin-associated adaptor complex AP-2. Immunity6:583–589 [[PubMed][Google Scholar]
53. Nucifora PGP, Fox AP. 1999 Tyrosine phosphorylation regulates rapid endocytosis in adrenal chromaffin cells. J. Neurosci.19:9739–9746 [Google Scholar]
54. Schaefer AW, Kamei Y, Kamiguchi H, Wong EV, Rapoport I, Kirchhausen T, Beach CM, Landreht G, Lemmon SK, Lemmon V. 2002 L1 endocytosis is controlled by a phosphorylation-dephosphorylation cycle stimulated by outside-in signaling by L1. J. Cell Biol.157:1223–1232 [Google Scholar]
55. Lewis JD, Lazarowitz SG. 2010 Arabidopsis synaptotagmin SYTA regulates endocytosis and virus movement protein cell-to-cell transport. Proc. Natl. Acad. Sci. U. S. A.107:2491–2496 [Google Scholar]
56. Sugiyama N, Nakagami H, Mochida K, Daudi A, Tomita M, Shirasu K, Ishihama Y. 2008 Large-scale phosphorylation mapping reveals the extent of tyrosine phosphorylation in Arabidopsis. Mol. Syst. Biol.4:193–199 [Google Scholar]
57. De la Fuente van Bentem S, Hirt H. 2009 Protein tyrosine phosphorylation in plants: more abundant than expected?Trends Plant Sci.14:71–76 [[PubMed][Google Scholar]
58. Reeves PM, Smith SK, Olson VA, Thorne SH, Bornmann W, Damon IK, Kalman D. 2011 Variola and monkeypox viruses utilize conserved mechanisms of virion motility and release that depend on Abl and Src family tyrosine kinases. J. Virol.85:21–31 [Google Scholar]
59. Keating JA, Striker R. 2012 Phosphorylation events during viral infections provide potential therapeutic targets. Rev. Med. Virol.22:166–181 [Google Scholar]