Pipo X11

[Lola Maroun]

Thesmall wire framing inside this futuristic-looking FALN pipo-hat gives it the aerodynamic shape of a swoop-skiers helmet, but none of its protective qualities. Covers the wearer's ears and eyebrows to bring down the drag coefficient.

The table pipo was created with a dualistic design approach: The reduced design language and solid appearance of this piece of furniture add tranquillity to offices and living spaces alike, while its large cross-sections belie its exceptional lightweight quality.

Because of its innovative structure in the panel core no reinforcing metal crossbars have to be used. The same applies to its remaining components. They, too, are almost exclusively made of wood-based materials. The table legs therefore consist of painted wooden tubes that can be anchored to the tabletop without the requirement of tools. They offer the possibility for invisible cable routing, while sockets and outlets are positioned in the tabletop above the table legs.

This table can be delivered in its individual parts and flat-packed, which reduces transport costs and cargo volumes to a minimum. pipo is available in numerous dimensions and surfaces and is very versatile in usage.

A small open reading frame (ORF), pipo, overlaps with the P3 coding region of the potyviral polyprotein ORF. Previous evidence suggested a requirement for pipo for efficient viral cell-to-cell movement. 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). P3N-PIPO of Turnip mosaic virus (TuMV) fused to GFP facilitates its own cell-to-cell movement. 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. BiFC revealed that it is the PIPO domain of P3N-PIPO that binds PCaP1 and that myristoylation of PCaP1 is unnecessary for interaction with P3N-PIPO. 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. In contrast, TuMV-GFP infection in wild-type Arabidopsis yielded large green fluorescent patches, and caused severe stunting. However, viral RNA accumulated to high level in protoplasts from pcap1 plants indicating that PCaP1 is not required for TuMV RNA synthesis. In contrast to TuMV, the tobamovirus Oilseed rape mosaic virus did not require PCaP1 to infect Arabidopsis plants. We conclude that potyviral P3N-PIPO interacts specifically with the host plasma membrane protein PCaP1 to participate in cell-to-cell movement. 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. The PCaP1 knockout may contribute to a new strategy for recessive resistance to potyviruses.

The Potyviridae is the largest and most economically important family of plant viruses. A key step in the life cycle of all plant viruses is transport of the viral genome through the plasmodesmata, highly regulated channels that connect cells. While the mechanisms of cell-to-cell movement of many plant viruses have been characterized, our understanding of Potyviridae movement is lacking. 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. The details of this localization process are unclear. Here, we identify a potential missing link that suggests how the potyviral movement complex may anchor to the plasma membrane including in the plasmodesmata. The host protein PCaP1, a divalent cation-binding plasma membrane protein, binds the P3N-PIPO protein of Turnip mosaic virus (TuMV). Both proteins were detected in the plasma membrane and plasmodesmata. Arabidopsis plants lacking PCaP1 allowed TuMV RNA replication but showed inefficient TuMV movement, reduced TuMV accumulation, and had greatly attenuated symptoms. However, these plants allowed normal infection by a tobamovirus. Thus, mutation of the PCaP1 gene may contribute to breeding potyvirus-resistant crops.

Copyright: 2012 Vijayapalani et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: The anti-P3 N-terminus antibody was funded by Science Foundation Ireland grant number 08/IN.1/B1889. This work was funded by USDA NIFA AFRI grant 2008-35319-19196. This journal paper of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA, Project No. 5176 was supported in part by Hatch Act and State of Iowa funds. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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]. Viral nucleic acid is too large to move through the plasmodesmata on its own, so viruses have evolved movement proteins (MPs) that interact with host proteins to modify the plasmodesmata and transport the viral genome from cell-to-cell [3], [4], [5], [6], [7]. Viruses have evolved diverse types of MPs such as the 30K-type MP of Tobacco mosaic virus (TMV) and related viruses [4], the triple gene block proteins of Potex-, Hordei- and other viruses [5], and the tubule-forming MPs of the Secoviridae, Bromoviridae and Caulimoviridae [8]. However the cell-to-cell movement mechanism of the largest family of plant viruses, the Potyviridae, falls into no previously known category, and is poorly understood. No dedicated MP has been identified but many viral proteins with other known functions have been reported to participate in potyvirus movement. 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.

The nature of host factors involved in the intercellular trafficking of potyviruses is poorly understood. A number of host proteins such as calmodulin and calmodulin-related protein [25], RING finger protein HIP1 [26], 20S proteasome and its four subunits [27], [28], chloroplast division-related factor NtMinD [29], chloroplast precursor of ferredoxin-5 [30] and calreticulin [12], have been reported to interact with HC-Pro of various potyviruses. However, these proteins may be important for functions of HC-Pro other than movement, such as suppression of host antiviral silencing, aphid transmission, or cleavage of the polyprotein. The CP of Potato virus Y (PVY) interacts with a subset of tobacco DnaJ-like proteins, NtCPIPs that act as important susceptibility factors during PVY infection that may be involved in virion assembly and/or movement [16]. Eukaryotic translation initiation factor eIF4E [31] and a cysteine-rich protein [18] have been identified as susceptibility factors supporting potyvirus movement.

To better understand the function of P3N-PIPO in potyvirus infection including cell-to-cell movement, we identified a host protein with which it interacts. We used a yeast two-hybrid screen to identify a hydrophilic plasma membrane-associated cation binding protein, PCaP1 that interacts with the P3N-PIPO of TuMV. The specificity of P3N-PIPO and PCaP1 interaction was validated in planta where P3N-PIPO was found to colocalize with PCaP1 in the plasma membrane and plasmodesmata. Virus accumulation, movement and disease symptoms were dramatically reduced in an Arabidopsis PCaP1 knockout. 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.

To identify the cellular proteins that interact with P3N-PIPO, a cDNA library from A. thaliana was screened, utilizing the GAL4-based yeast two-hybrid (Y2H) system with TuMV P3N-PIPO as bait. As a result of sequential screening steps, ten positive clones were isolated and sequenced. Out of the ten clones, five encoded a protein known as PCaP1 (TAIR accession AT4G20260; GenBank accession NM_118145). Since PCaP1 showed the strongest interaction with P3N-PIPO in the yeast cells compared to other interactors (data not shown), PCaP1 was chosen for further investigation.

PCaP1 is a hydrophilic cation binding protein (Mr 24.5 kDa, pI 4.6) associated with the plasma membrane [34]. It lacks a transmembrane domain and anchors to the plasma membrane via myristoylation of a glycine residue. The PCaP1 gene is present as a single copy in Arabidopsis. 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. S1). An ortholog was also detected in a gymnosperm but not in lower plants.

To determine whether P3N-PIPO and PCaP1 interact in planta, HA-tagged P3N-PIPO and c-myc-tagged PCaP1 were co-expressed transiently in N. benthamiana leaves and total protein was extracted and subjected to co-immunoprecipitation (co-IP) with either anti-HA or anti-c-myc antibodies. Proteins bound to the matrix were eluted and immunodetected with either anti-HA or anti-c-myc antibodies (Fig. 4). Anti-c-myc antibodies detected c-myc-PCaP1 among proteins pulled-down with anti-HA antibodies (Fig. 4, top right panel), and anti-HA antibodies detected HA-P3N-PIPO among proteins pulled-down by anti-c-myc antibodies (Fig. 4, bottom left panel), in both cases only when plants were co-infiltrated with agrobacterium harboring both the plasmids. 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. Expression and immunoprecipitation of HA-P3N-PIPO and c-myc-PCaP1 were confirmed by immunoblotting (Fig. 4, panels at top left and bottom right).

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