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Specific targeting of a plasmodesmal protein affecting cell-to-cell communication.

Thomas CL, Bayer EM, Ritzenthaler C, Fernandez-Calvino L, Maule AJ - PLoS Biol. (2008)

Bottom Line: We focus our studies on the first identified type member (namely At5g43980, or PDLP1a) and show that, following its altered expression, it is effective in modulating cell-to-cell trafficking.These studies identify a new family of plasmodesmal proteins that affect cell-to-cell communication.They exhibit a mode of intracellular trafficking and targeting novel for plant biology and provide technological opportunities for targeting different proteins to plasmodesmata to aid in plasmodesmal characterisation.

View Article: PubMed Central - PubMed

Affiliation: John Innes Centre, Norwich Research Park, Colney, Norwich, United Kingdom.

ABSTRACT
Plasmodesmata provide the cytoplasmic conduits for cell-to-cell communication throughout plant tissues and participate in a diverse set of non-cell-autonomous functions. Despite their central role in growth and development and defence, resolving their modus operandi remains a major challenge in plant biology. Features of protein sequences and/or structure that determine protein targeting to plasmodesmata were previously unknown. We identify here a novel family of plasmodesmata-located proteins (called PDLP1) whose members have the features of type I membrane receptor-like proteins. We focus our studies on the first identified type member (namely At5g43980, or PDLP1a) and show that, following its altered expression, it is effective in modulating cell-to-cell trafficking. PDLP1a is targeted to plasmodesmata via the secretory pathway in a Brefeldin A-sensitive and COPII-dependent manner, and resides at plasmodesmata with its C-terminus in the cytoplasmic domain and its N-terminus in the apoplast. Using a deletion analysis, we show that the single transmembrane domain (TMD) of PDLP1a contains all the information necessary for intracellular targeting of this type I membrane protein to plasmodesmata, such that the TMD can be used to target heterologous proteins to this location. These studies identify a new family of plasmodesmal proteins that affect cell-to-cell communication. They exhibit a mode of intracellular trafficking and targeting novel for plant biology and provide technological opportunities for targeting different proteins to plasmodesmata to aid in plasmodesmal characterisation.

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PDLP1a Resides in PlasmodesmataFollowing 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. Bar indicates 5 μm.
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pbio-0060007-g002: PDLP1a Resides in PlasmodesmataFollowing 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. Bar indicates 5 μm.

Mentions: A survey of the subcellular targeting of cell wall–associated membrane proteins derived from highly purified Arabidopsis cell walls [17] 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. We refer to this newly identified protein 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). This pattern of fluorescence was most notable in leaf spongy mesophyll cells, where the punctate spots were present on adjoining walls, but absent from the nonadjoining walls (Figure 1E). Similar patterns of protein accumulation were observed following transient expression in the heterologous species N. benthamiana and onion (Figure S1). 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). Callose distribution was revealed by staining with the fluorescent dye aniline blue, and was especially informative in cases where patterns of both callose and PDLP1a distribution identified the characteristic organisation of groups of plasmodesmata in pit fields (Figure S2B). 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].


Specific targeting of a plasmodesmal protein affecting cell-to-cell communication.

Thomas CL, Bayer EM, Ritzenthaler C, Fernandez-Calvino L, Maule AJ - PLoS Biol. (2008)

PDLP1a Resides in PlasmodesmataFollowing 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. Bar indicates 5 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2211546&req=5

pbio-0060007-g002: PDLP1a Resides in PlasmodesmataFollowing 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. Bar indicates 5 μm.
Mentions: A survey of the subcellular targeting of cell wall–associated membrane proteins derived from highly purified Arabidopsis cell walls [17] 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. We refer to this newly identified protein 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). This pattern of fluorescence was most notable in leaf spongy mesophyll cells, where the punctate spots were present on adjoining walls, but absent from the nonadjoining walls (Figure 1E). Similar patterns of protein accumulation were observed following transient expression in the heterologous species N. benthamiana and onion (Figure S1). 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). Callose distribution was revealed by staining with the fluorescent dye aniline blue, and was especially informative in cases where patterns of both callose and PDLP1a distribution identified the characteristic organisation of groups of plasmodesmata in pit fields (Figure S2B). 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].

Bottom Line: We focus our studies on the first identified type member (namely At5g43980, or PDLP1a) and show that, following its altered expression, it is effective in modulating cell-to-cell trafficking.These studies identify a new family of plasmodesmal proteins that affect cell-to-cell communication.They exhibit a mode of intracellular trafficking and targeting novel for plant biology and provide technological opportunities for targeting different proteins to plasmodesmata to aid in plasmodesmal characterisation.

View Article: PubMed Central - PubMed

Affiliation: John Innes Centre, Norwich Research Park, Colney, Norwich, United Kingdom.

ABSTRACT
Plasmodesmata provide the cytoplasmic conduits for cell-to-cell communication throughout plant tissues and participate in a diverse set of non-cell-autonomous functions. Despite their central role in growth and development and defence, resolving their modus operandi remains a major challenge in plant biology. Features of protein sequences and/or structure that determine protein targeting to plasmodesmata were previously unknown. We identify here a novel family of plasmodesmata-located proteins (called PDLP1) whose members have the features of type I membrane receptor-like proteins. We focus our studies on the first identified type member (namely At5g43980, or PDLP1a) and show that, following its altered expression, it is effective in modulating cell-to-cell trafficking. PDLP1a is targeted to plasmodesmata via the secretory pathway in a Brefeldin A-sensitive and COPII-dependent manner, and resides at plasmodesmata with its C-terminus in the cytoplasmic domain and its N-terminus in the apoplast. Using a deletion analysis, we show that the single transmembrane domain (TMD) of PDLP1a contains all the information necessary for intracellular targeting of this type I membrane protein to plasmodesmata, such that the TMD can be used to target heterologous proteins to this location. These studies identify a new family of plasmodesmal proteins that affect cell-to-cell communication. They exhibit a mode of intracellular trafficking and targeting novel for plant biology and provide technological opportunities for targeting different proteins to plasmodesmata to aid in plasmodesmal characterisation.

Show MeSH