Plasmodesmata are channels that cross the cell wall and establish symplastic continuity throughout most of the plant. Some small RNAs generated as part of the RNA silencing pathway can act non–cell-autonomousl. Plant virus pathogens, which are restricted to the symplast, must use plasmodesmata to invade neighbouring cells, and very recently it has been proposed that biotrophic fungi may exploit plasmodesmata during tissue invasion. From the identification of
proteins interacting with viral MPs, coimmunolocalisation studies applied to candidate proteins, and proteomics approaches, a number of proteins have been identified in association with plasmodesmata . The use of Arabidopsis thaliana suspension cultures is reported as a source of membrane proteins located in the cell wall to identify a novel family of proteins functioning within plasmodesmata to affect cell-to-cell communication, and identify the key principles by which specific subcellular
targeting of these proteins is achieved. Plasmodesmata-Located
protein 1 Targets Plasmodesmata in Diverse Plant Species A survey of the subcellular targeting of cell wall–associated membrane proteins derived from highly purified Arabidopsis cell walls was carried out. Translational N- and/or C-terminal fusions with green fluorescent protein (GFP) were expressed transiently using the cauliflower mosaic virus (CaMV) 35S promoter in Nicotiana benthamiana leaves and onion epidermal monolayers, and transgenically in Arabidopsis plants. From this survey, we identified the protein derived from At5g43980 as a plasmodesmal protein. This newly identified protein is referred as plasmodesmata-located protein 1a (PDLP1a). Critically, expression in
transgenic Arabidopsis using either the CaMV 35S promoter or the native promoter for At5g43980 showed that the fusion protein was located as punctate spots on the cell wall (Figure 1B to 1E) and that this fluorescence was retained on the wall after plasmolysis (Figure 1D and 1E). The pattern of localisation was most distinctive when PDLP1a was expressed from its own promoter, when the protein was targeted exclusively to plasmodesmata. (Figure S2A shows a confocal stack through pPDLP1a::PDLP1a:GFP–expressing Arabidopsis epidermal cells, and unique association with plasmodesmata.) Further evidence that these punctate sites were plasmodesmata was obtained by demonstrating colocalisation with callose (Figure 2A–2C) and with TMV MP:GFP (Figure 2D–2F). TMV MP is one of the best-characterised viral MPs, which shows strong targeting to plasmodesmata in newly infected cells and in transgenic
plants <4>. In transgenic plants expressing TMV MP, the protein characteristically is localised within complex plasmodesmata that are a feature of photosynthetic source tissues rather than in the simple plasmodesmata of sink tissues <18>. PDLP1a Resides in Plasmodesmata Following coexpression of PDLP1a:GFP (A and D) and TMV MP:RFP (E), or chemical staining of callose using aniline blue (B), colocation of PDLP1a with either marker (C and F) confirmed its localisation to plasmodesmata. Briefly, the proteins are predicted to be type I membrane proteins with molecular mass ranging from 30.2 to 35.3 kDa and comprising an N-terminal signal peptide, a large region containing two similar domains annotated as domains of unknown function 26 (DUF26), a single transmembrane domain (TMD), and a short C-terminal tail. C-terminal fusions with GFP were constructed for all eight PDLP1 proteins and analysed for their subcellular targeting after expression from the CaMV 35S promoter; all showed targeting to plasmodesmata (representative proteins from At2g01660 and At2g33330 are illustrated in Figure 3B). These include a group of proteins that contain a signal peptide for secretion, but lack the TMD domain (e.g., protein from At5g48540, which was secreted to form large, unresolved bodies in the apoplast), a second group where the TMD and short C-terminus are replaced with a glycosylphosphoinositol (GPI) anchor domain (e.g., protein from At1g63580, which was localized to the PM), and a third group of predicted receptor-like kinases (e.g., protein from At4g23140) where the short C-terminus is replaced with a serine/threonine kinase domain. In this last case, the protein was also targeted to the PM, although in this case, the distribution was less uniform than seen for the GPI-anchored protein from At1g63580. Optical sections through the epidermis of transgenic Arabidopsis expressing a 2xDUF26 protein lacking a TMD (encoded by At5g48540) shows fluorescent apoplastic bodies, whereas expression of a GPI-anchored 2xDUF26 relative (encoded by At1g63580) shows PM labelling; Transgenic plants expressing genes for either PDLP1a:GFP or haemaglutinin-tagged PDLP1a (PDLP1a:HA) showed a reduced-growth phenotype that correlated with transgene copy number, i.e., plants with homozygous single insertions were more dwarfed than heterozygous plants and this dwarfing phenotype positively correlated with protein accumulation, assessed using anti-HA antibody. To assess the importance of the C-terminal tail in directing PDLP1a to plasmodesmata, this region was deleted and the resulting C-terminal fusion to GFP tested for targeting to plasmodesmata. Transient and transgenic expression showed that the protein lacking the C-terminal tail was still targeted to plasmodesmata. Transgenic or transient expression of the latter fusion protein showed unique targeting to plasmodesmata, which was unaffected by plasmolysis. The experiments have uniquely identified a short TMD sequence with the capacity to target proteins to plasmodesmata.
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