We speculate that PCaP1 links a complex of viral proteins and genomic RNA to the plasma membrane by binding P3N-PIPO, enabling localization to the plasmodesmata and cell-to-cell movement. A c-myc tag was added to PCaP1 gene by PCR amplification from pGADT7-HF3 with primer pair HF3-1F/HF3-1R (Table S1). The size of the PIPO protein that accumulates in potyviral infections is consistent with it being the C‐terminal portion of a fusion with the N‐terminal region of P3; consequently, it was termed P3N‐PIPO (Fig 1B) 1. https://doi.org/10.1371/journal.ppat.1002639.g004. Here, we provide immunoblotting evidence that the protein PIPO is expressed as a trans-frame protein consisting of the amino-terminal half of P3 fused to PIPO (P3N-PIPO). Yes Accordingly, it will be interesting to determine the RNA structural features (proximal and/or distal to the slip sites) that modulate this process. However, viral RNA accumulated to high level in protoplasts from pcap1 plants indicating that PCaP1 is not required for TuMV RNA synthesis. Immunoblotting (IB). Proteins from crude extracts of N. benthamiana leaves (2 days post agroinfiltration) that co-expressed HA-P3N-PIPO and c-myc-PCaP1, or expressed HA-P3N-PIPO or c-myc-PCaP1 only were pulled-down using anti-HA (top panels) or anti-c-myc (bottom panels) antibodies, separated by 4–12% Novex Tris-Glycine PAGE, electroblotted onto PVDF membrane and probed with anti-HA or anti-c-myc antibody as indicated. TuMV-GFP infection was confirmed by immunodetection of expressed GFP. No dedicated MP has been identified but many viral proteins with other known functions have been reported to participate in potyvirus movement. (A) Images of epidermal cells expressing GFP alone or fused to P3N-PIPO (P3N-PIPO-GFP) at indicated hours post biolistic bombardment (hpb). Samples were from mock inoculated or TuMV-GFP infected wild-type (WT) or PCaP1 knockout (pcap1) plants. Equal loading of proteins was verified by similar levels of Coomassie staining of Rubisco protein (bottom panel). Immunodetection of the pipo-encoded protein in TuMV infected cells revealed a ∼25 kDa polypeptide, consistent with expression of pipo as a translational fusion with the N-terminus of P3 [11]. Pipo Finito is on Facebook. VIRION. Position of inserted GFP coding insertion is indicated by dashed lines. The leaves were sterilized in 30% ethanol for 1 min, followed by rinses in distilled water and incubated in 1–1.5% cellulase Onozuka R-10 (Yakult Pharamaceuticals, Japan) and 0.2–0.4% macerozyme R-10 (Yakult Honsha, Japan) in 0.4 M D-sorbitol, 20 mM KCl and 20 mM MES, pH 5.7 for 2 h at room temperature. Therefore, we tested potential of P3N-PIPO for cell-to-cell movement. Cover: Mouse pre-implantation embryo at hatching. In all cases where the resistance gene has been sequenced, it has proven to encode a translation initiation factor [44]. The occurrence of such events at defined times during an infection would also allow for temporal regulation. Although slippage may be a constitutive and unmodulated property of the polymerase, this activity could also be conferred or regulated though covalent modifications or interaction with cofactors. Thus, if pcap1 knockouts prove not to reduce yield in uninfected plants, knockouts or mutations in this gene could be used for resistance in crop plants. Total proteins from leaves were extracted at 2 days post agroinfiltration in 50 mM Tris-acetate, pH 7.5, 1 mM DTT, 20 µM PMSF, complete protease inhibitor cocktail (Roche) and 1% Triton X-100. [19] provided evidence that an RNP consisting of CI protein bound to CP which is bound to the viral RNA, possibly in the form of a virion, is required for cell-to-cell movement. Affiliation Local callose accumulation increases in the presence of Ca2+ [41], [42], which increases in a successful defense response to virus infection [43]. Coding sequences of P3N-PIPO, P3N, PIPO, P3, PCaP1 or G2A.PCaP1 were cloned in-frame with the gene encoding N-terminus (YN) or C-terminus (YC) of citrine in the binary vector pSP1823-YN or pSP1794-YC (kindly provided by Dr. S.P. Previous evidence suggested a requirement for pipo for efficient viral cell-to-cell movement. No, Is the Subject Area "Arabidopsis thaliana" applicable to this article? Above the split compose expression is as follows: HF3 (PCaP1), TuHC (TuMV HC-Pro), F (forward), R (Reverse), LP, RP and LB (primers used for genotyping). Mature proteins processed from the proteolytic cleavage of the large polyprotein are indicated in boxes. In TuMV‐infected cells, P3N‐PIPO is produced at an extremely low level, which nonetheless suffices for movement‐related functions, and correlates well with the minute levels of polymerase slippage observed 1. Plasmids pGBKT7-T-antigen, pGADT7-laminin C and pGADT7-murine p53 (Clontech) served as controls. Conceived and designed the experiments: PV WAM. 2), PCaP1-bound P3N-PIPO would move the virion through the plasmodesmata to the neighboring cell (Fig. However, these plants allowed normal infection by a tobamovirus. No, Is the Subject Area "Co-immunoprecipitation" applicable to this article? PIPO specific antibody recognized two polypeptides, a polypeptide that migrated as ∼28 kDa and a smaller unexpected polypeptide of ∼18 kDa (Fig. Thus, binding of HA-P3N-PIPO to c-myc-PCaP1 is evident from the immunodetection of both proteins that were captured as a complex with anti-HA antibody or anti-c-myc antibody. Here, we describe the interaction of a novel potyviral protein, called P3N-PIPO, with a previously unrecognized host protein that provides a key insight into the cell-to-cell movement process of the potyviruses. https://doi.org/10.1371/journal.ppat.1002639.g008. Interaction between P3N-PIPO and PCaP1 was verified in yeast transformants expressing P3N-PIPO bait in combination with either the rescued prey plasmid encoding PCaP1, empty prey vector, or prey plasmid encoding unrelated protein SV40 large T-antigen (Clontech). MPs exploit cellular pathways to regulate virus movement by interacting with host factors to change their specific intracellular localization in infected cells, [3], [6], [36], [37]. 5B, panels a–d). 1B). Competing interests: The authors have declared that no competing interests exist. The level of TuMV-GFP RNA accumulation, as measured by qRT-PCR was not significantly different in both plants at 3 days post inoculation (dpi) (Fig. Briefly, the library strain and Y2HGold strain harboring pGBKT7-P3N-PIPO were mated and plated on double dropout medium containing X- α-Gal (SD/−Leu/−Trp/+X-α-Gal) and incubated at 30°C for 4 days. 2A). The simplest explanation was that PIPO is expressed as a translational fusion with the N-terminus of P3 which would be a ∼25 kDa protein, we call P3N-PIPO. As negative controls, each of the fusion proteins was expressed alone or in pairwise combination with GUS-YC (Fig. Data are averages of three independent experiments, each consisting of five technical replicates and statistical significance was analyzed by the unpaired Student's t-test (P = 0.169). The nature of host factors involved in the intercellular trafficking of potyviruses is poorly understood. They are not selected or validated by us and can contain inappropriate terms or ideas. The spread of TuMV-GFP in the plant was observed by epifluorescence microscopy (Fig. Insertion of the T-DNA at an intron as a single copy was verified. An essential overlapping ORF, termed pipo, resides in an internal region of the main polyprotein ORF. The accession numbers for the PCaP1 orthologs are: Medicago truncatula; ACJ84038, Vitis vinifera; XP_002263090, Nicotiana tabacum; CAB91552, Glycine max; XP_003546380, Cicer arietinum; CAB61742, Ricinus communis; XP_002532713, Oryza sativa; NP_001046572, Zea mays; NP_001150000, Sorghum bicolor; XP_002453713, Picea sitchensis ABK21073. Only colonies co-transformed with P3N-PIPO and PCaP1 expression plasmids expressed α- galactosidase and appeared blue on low stringency medium SD/−Leu/−Trp/+X-α-Gal and could grow on high stringency selective medium SD/−Leu/−Trp/−Ade/−His, confirming the protein-protein interaction. It lacks a transmembrane domain and anchors to the plasma membrane via myristoylation of a glycine residue. Both proteins were detected in the plasma membrane and plasmodesmata. However, it is clear that P3N-PIPO-PCaP1 complexes are also present outside of the plasmodesmata. https://doi.org/10.1371/journal.ppat.1002639.g009. In summary, the interaction between the P3N-PIPO and PCaP1, identified first in the Y2H assay, was confirmed by co-IP and BiFC, which also demonstrated a direct physical interaction between the PIPO domain of P3N-PIPO and PCaP1 in planta. 6). Symptoms were less attenuated in about 30% of the inoculated plants but ultimately they showed signs of recovery, unlike WT plants. Since its discovery, the activities of P3N‐PIPO have been intensely investigated. The interaction of P3N-PIPO with the membrane protein PCaP1 provides a key missing link in the model by which the potyvirus is localized to the plasmodesmata. In other instances, regulation could be meditated by the viral polymerase. However, this notion was dispelled when an additional essential viral ORF, PIPO, was discovered encoded in an alternative reading frame. In WT plants, at both time points, >90% of the infection foci were composed of more than a hundred cells. This is the first known example of involvement of PCaP1 in virus infection. 1B). Finally, pcap1 mutations or knockouts may provide a new strategy for breeding potyvirus resistance in crop plants. Falcarindiol (FaDOH) is a cytotoxic and anti-inflammatory polyacetylenic oxylipin found in food plants of the carrot family (Apiaceae). 4, top right panel), and anti-HA antibodies detected HA-P3N-PIPO among proteins pulled-down by anti-c-myc antibodies (Fig. To detect PCaP1, total proteins were extracted as described in [38] and solubilized in 1% Triton X-100. To determine if this putative P3N-PIPO protein is expressed as predicted, total protein from Arabidopsis leaves infected with GFP-tagged TuMV (TuMV-GFP) (Fig. To this end, we used the plasmodesmata callose binding protein PDCB1 fused to mCherry (provided by Dr. Andy Maule; John Innes Center, UK) [35] as a plasmodesmata marker in the BiFC assay (Fig. We constructed a gene that expresses a PIPO-GFP fusion in-frame with the N terminus of P3 to generate P3N-PIPO-GFP. (A) qRT-PCR quantification of TuMV-GFP RNA in inoculated leaves of indicated plant lines at 3 dpi. The fact that viral RNA accumulation was not reduced in protoplasts from PCaP1 knockout plants indicates that PCaP1 is not required for the viral RNA synthesis. At 48 hpb the number of cells in each cluster was counted, followed by statistical evaluation by the unpaired Student's t-test (P = 0.151). Calcium binding proteins have been found to interact with other, unrelated viral MPs. Two recent studies provide compelling evidence that potyviruses use a viral polymerase slippage mechanism for P3N‐PIPO production 12. Although polymerase slippage has been reported previously for hepatitis C virus 10, its biological relevance in infections remains to be established. In PCaP1 knockout mutants (pcap1) of Arabidopsis, accumulation of TuMV harboring a GFP gene (TuMV-GFP) was drastically reduced relative to the virus level in wild-type plants, only small localized spots of GFP were visible, and the plants showed few symptoms. BabyboiFinito Pipo (feat. 7C). Thus, PCaP1 is not required for efficient infection by the tobamovirus. Construct pSP862 (provided by Dr. S.P. Y2H screening and two independent plant-based assays, co-IP and BiFC, revealed that P3N-PIPO interacts with the host factor PCaP1. Together, these results suggest that PCaP1 represents a new type of plant protein required for efficient infection by potyviruses, and which may participate in intercellular trafficking of potyviruses. However the infection foci were many-fold smaller than in infected WT plants. Homozygous pcap1 and WT plants were inoculated with equal amounts of TuMV-GFP. Translation Spell check Synonyms Conjugation. It was long thought that their plus‐strand RNA genomes encode only a single large ORF that is translated into a polyprotein and subsequently cleaved into ten mature proteins (Fig 1A and B). The data are averages of four independent experiments and each consisting of at least eight replicates. Therefore, the work by Olspert et al 1 and Rodamilans et al 2 represents the first compelling evidence for this activity being functionally significant in plus‐strand RNA virus infections and lays the foundation for investigating both mechanistic details of the process and its prevalence in other viral systems. From the yeast cells that displayed a positive interaction, prey plasmids were rescued in E. coli and sequenced. However, PCaP1 protein level was unaffected by TuMV-GFP infection (Fig. Genomic DNA was extracted from leaves of Arabidopsis T-DNA insertion line SALK_022955 (The Arabidopsis Information Resource [45]) using DNeasy Plant Mini kit (Qiagen), and the T-DNA insertion at PCaP1 was detected using primer pair LB1.3/HF3-RP (Table S1). For qRT-PCR analysis, 30 ng of total RNA and gene-specific primers at a concentration of 10 pmol were used. For details, see the Article on p Statistical significance of difference in the size and number of the infection foci between WT and pcap1 was analyzed by the unpaired Student's t-test and the calculated P values are indicated. Mutations in the pipo coding region impeded the virus cell-to-cell movement, allowed virus accumulation in cells localized at the inoculation site, and rendered the virus noninfectious or nearly so in whole plants [11], [21], [22]. Recessive resistance has been applied widely against economically important Potyviridae in capsicum, lettuce, pea, wheat, barley and other crops and it is generally more durable than dominant resistance genes [44]. FaDOH has been shown to activate PPARγ and to increase the expression of the cholesterol transporter ABCA1 in cells, both of which play an important role in lipid m … It is known to colocalize to plasmodesmata, where it acts in conjunction with another potyviral protein, CI, to mediate cell‐to‐cell spread of the virus 5. In SPFMV, the same viral polymerase is responsible for both slippage events; therefore, differences in the RNA template are likely responsible for the observed disparity. By 12 dpi, the number of cells in the fluorescent cluster ranged from 15 to 20. For transient expression of HA-P3N-PIPO or c-myc-PCaP1 in planta, p35S::HA-P3N-PIPO or p35S::myc-P3N-PIPO was introduced into Agrobacterium tumefaciens GV3101 by electroporation, followed by induction and infiltration into youngest fully-expanded leaves of 3-week old N. benthamiana plants [46]. The viral RNA genome is transported to the plasmodesmata by a complex of viral proteins including a recently discovered protein, P3N-PIPO which is encoded in two reading frames. Using a yeast two-hybrid screen, co-immunoprecipitation assays, and bimolecular fluorescence complementation (BiFC) assays, we found that P3N-PIPO interacts with host protein PCaP1, a cation-binding protein that attaches to the plasma membrane via myristoylation. Data were statistically evaluated by the unpaired Student's t-test. [21] reported that synonymous mutations in the P3 cistron, but which altered the pipo ORF (which wasn't known at the time) of Wheat streak mosaic virus (WSMV) disrupted the movement of WSMV in plants. Yes Unlike in classical mechanics, quantum systems constantly fluctuate in their lowest energy state as described by the Heisenberg uncertainty principle. Systemic leaves turned yellow, plants were extremely stunted, and the inflorescence was strongly condensed by 30 dpi (Fig. Valentin, 25 ans, Grand fan de jeux vidéo, me lançant sur twitch et youtube, j'espère apporter du sourire et de la joie :) Ici Jeux, humour, découverte et musique sont aux rendez vous ! 1005. TuMV accumulation in each sample was normalized to the quantity of Actin8 that was amplified with primer pair Actin8qRT-F/Actin8qRT-R (Table S1). This lack of requirement for membrane binding may explain why the interaction was detected in the Y2H assay that does not detect interactions of integral membrane proteins. https://doi.org/10.1371/journal.ppat.1002639.g007. Amino acid sequence alignment shows that Arabidopsis PCaP1 shares up to 67% sequence identity with ortholog in dicots including potyvirus hosts and up to 54% with ortholog in monocot species (Fig. The Potyviridae comprise the largest and most important family of RNA plant viruses. [46]. Arabidopsis plants lacking PCaP1 allowed TuMV RNA replication but showed inefficient TuMV movement, reduced TuMV accumulation, and had greatly attenuated symptoms. Non-enveloped, flexuous, filamentous, of one (650-900 nm) or two (500-600 and 200-300 nm) lengths x 12-15 nm in diameter. The positions of GA. Download PDF of article text and main figures. Primer sequences used in this study. Proteins implicated in potyviral cell-to-cell movement include helper component-protease (HC-Pro) [12], coat protein (CP) [13], [14], [15], [16], [17], genome-linked protein (VPg) [18], and cylindrical inclusion protein (CI) [17], [19], [20]. Rabbit polyclonal anti-GFP antibodies (Life Technologies) were used at dilution of 1/1000. Solid circle indicates VPg at 5′ end; (A)n indicates poly(A) tail. At about 38 h post agroinfiltration, leaves were analyzed for BiFC or colocalization by confocal microscopy. We speculate that the virion-CI complex binds P3N-PIPO, via the CI protein. Statistical significance between WT and pcap1 was analyzed by the unpaired Student's t-test and calculated P values are indicated. P3N-PIPO of Turnip mosaic virus (TuMV) fused to GFP facilitates its own cell-to-cell movement. Total soluble proteins from leaves collected at 14 dpi were separated by 4–12% Novex Tris-glycine PAGE, blotted onto PVDF membrane, probed with anti-PCaP1 antibody or anti-GFP antibody and detected by ECL-Plus Western reagents. The cells in cyan, positive for a lineage marker newly identified in the gene-trap screen, will contribute to both the embryonic and the extra-embryonic tissues. Moreover, this model does not rule out roles for other host proteins in translocating the movement complex to and through the plasmodesmata. 5A, panel j). The blots were developed with anti-rabbit antibody (Thermo Scientific) or anti-mouse antibody (AMRESCO) conjugated to horse-radish peroxidase and detected using the ECL-Plus Western blotting reagents (GE Health Care). © Cover image by Laura Panavaite, EMBL. GUS-YC expression in the negative controls was confirmed by histochemical staining (Fig. 5A, panels c, f, i, l and o). PCaP1 is constitutively expressed in most organs of the plant, and its expression is increased by elicitors flagellin-oligopeptide, sorbitol or copper [38]. Mutant Turnip mosaic virus (TuMV) genomes containing modifications in their GA6 motifs designed to inhibit ribosome frameshifting were able to express P3N‐PIPO, spread from inoculation sites and systemically infect plants 1. Error bars represent standard deviations. 5A, panel m) indicating that myristoylation, and thus membrane binding, of PCaP1 is apparently not necessary for binding by P3N-PIPO. This anchors PCaP1 in the plasma membrane. P3N-PIPO and PCaP1 were expressed in planta with an N-terminal HA or c-myc tag, respectively. pathogens and how they interact with host organisms. With regard to disease, in WT plants the symptoms appeared first at 7 dpi and severe symptoms arose systemically in 100% of the inoculated plants. Accordingly, these in vivo results supported only the polymerase slippage mechanism for P3N‐PIPO expression. 9) because it relies on SYTA and ANK, for similar function(s) provided by PCaP1 to potyviruses. https://doi.org/10.1371/journal.ppat.1002639.g006. pcap1 plants did not differ phenotypically from the WT plants and had normal fecundity. Merged citrine signals and mCherry signals show colocalization of P3N-PIPO, PCaP1 and PDCB1-mCherry as orange spots (panel c). All fluorescent images are projections of optical sections. To avoid overexpression of recombinant proteins in both assays, Agrobacterium was used at A600 0.05. "Finito Pipo" entre Maeva Ghennam et Greg Yega ! These mutations do not alter the amino acid sequence of the overlapping P3 region of the polyprotein. 974 Followers, 1 Following, 0 Posts - See Instagram photos and videos from Finito pipo ♂️ (@samy_vdl) Whether the retained slippage events occur during synthesis of the minus‐strand RNA replicative intermediate or the plus‐strand progeny genome remains unclear. This property, along with being synthesized by polymerase transcription during infections and mediating the expression of a specific viral protein, is consistent with classifying P3N‐PIPO‐encoding mRNAs as a novel type of sg mRNA that, unlike typical sg mRNAs, is larger than the parental genome (albeit by a single nucleotide). Plant viruses that contain plus‐sensed single‐stranded RNA genomes are highly abundant in nature. PCaP1 is a hydrophilic cation binding protein (Mr 24.5 kDa, pI 4.6) associated with the plasma membrane [34]. Finito Pipo (Le bebew) is on Facebook. The calcium-modulated synaptotagmin, SYTA, interacts with the MPs of Cabbage leaf curl (CaLCuV) geminivirus and the unrelated TMV [37]. To spread beyond the initially infected cell, the genome of a plant virus must move through the plasmodesmata, which are narrow tunnels through the impervious cell wall that connect cytoplasm, endoplasmic reticulum and plasma membrane between adjoining cells [1], [2]. To identify P3N-PIPO interacting host proteins, an Arabidopsis (Col-0) cDNA library (Clontech) was screened using the Matchmaker Gold Yeast Two-Hybrid System (Clontech). Also, a conserved GAAAAAA (GA6) motif located at the beginning of PIPO was identified and proposed to mediate the frameshift event required for its expression (Fig 1A), either by a −1 ribosomal frameshifting process during translation of the polyprotein ORF or by gaining a single adenylate (+1A) through slippage of the viral RNA‐dependent RNA polymerase during viral genome replication 4. Regardless of the stage at which slippage occurs, the products generated are modified viral genomes that template the translation of a distinct protein.
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