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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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The TMD of PDLP1a Is Sufficient to Target Foreign Proteins to Plasmodesmata(A and B) Chimeric constructs consisting of the signal peptide from PDLP1a, followed by the coding sequence for YFP (citrine) fused to the TMD and CT of PDLP1a (A), or the TMD alone (B), were constructed and expressed.(C and D) Upon plasmolysis of spongy mesophyll cells, the chimeric protein remained in the cell wall at the areas of cell–cell contact for both YFP:TMD+CT (C) and YFP:TMD (D) proteins. The former also showed accumulation of fluorescent bodies within the cytoplasm. Overlays of the YFP fluorescence and red chloroplast autofluorescence (bottom) images clearly show YFP labelling retained after plasmolysis at the points of cell-to-cell contact (arrows).Bars indicate 10 μm.
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pbio-0060007-g008: The TMD of PDLP1a Is Sufficient to Target Foreign Proteins to Plasmodesmata(A and B) Chimeric constructs consisting of the signal peptide from PDLP1a, followed by the coding sequence for YFP (citrine) fused to the TMD and CT of PDLP1a (A), or the TMD alone (B), were constructed and expressed.(C and D) Upon plasmolysis of spongy mesophyll cells, the chimeric protein remained in the cell wall at the areas of cell–cell contact for both YFP:TMD+CT (C) and YFP:TMD (D) proteins. The former also showed accumulation of fluorescent bodies within the cytoplasm. Overlays of the YFP fluorescence and red chloroplast autofluorescence (bottom) images clearly show YFP labelling retained after plasmolysis at the points of cell-to-cell contact (arrows).Bars indicate 10 μm.

Mentions: That PDLP1a was targeted to plasmodesmata in the absence of the C-terminal tail led us to ask whether the TMD alone would be sufficient. To assess this, two constructs were tested. The citrine variant of YFP (this has higher stability in the acidic extracellular environment) [29] was fused between the PDLP1a N-terminal signal peptide and the TMD plus C-terminal tail, or the TMD alone (Figure 8A and 8B). In both cases, the YFP open reading frame was flanked with flexible linker peptides [30], minimizing protein folding interference between the YFP and PDLP1a domains. Transgenic or transient expression of the latter fusion protein showed unique targeting to plasmodesmata, which was unaffected by plasmolysis (Figure 8C and 8D, arrows). For the former construct including the C-terminal tail, similar targeting was observed except that YFP fluorescence was also visible at discrete unidentified locations in the cytoplasm (Figure 7C). Therefore, our experiments have uniquely identified a short TMD sequence with the capacity to target proteins to plasmodesmata.


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)

The TMD of PDLP1a Is Sufficient to Target Foreign Proteins to Plasmodesmata(A and B) Chimeric constructs consisting of the signal peptide from PDLP1a, followed by the coding sequence for YFP (citrine) fused to the TMD and CT of PDLP1a (A), or the TMD alone (B), were constructed and expressed.(C and D) Upon plasmolysis of spongy mesophyll cells, the chimeric protein remained in the cell wall at the areas of cell–cell contact for both YFP:TMD+CT (C) and YFP:TMD (D) proteins. The former also showed accumulation of fluorescent bodies within the cytoplasm. Overlays of the YFP fluorescence and red chloroplast autofluorescence (bottom) images clearly show YFP labelling retained after plasmolysis at the points of cell-to-cell contact (arrows).Bars indicate 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

pbio-0060007-g008: The TMD of PDLP1a Is Sufficient to Target Foreign Proteins to Plasmodesmata(A and B) Chimeric constructs consisting of the signal peptide from PDLP1a, followed by the coding sequence for YFP (citrine) fused to the TMD and CT of PDLP1a (A), or the TMD alone (B), were constructed and expressed.(C and D) Upon plasmolysis of spongy mesophyll cells, the chimeric protein remained in the cell wall at the areas of cell–cell contact for both YFP:TMD+CT (C) and YFP:TMD (D) proteins. The former also showed accumulation of fluorescent bodies within the cytoplasm. Overlays of the YFP fluorescence and red chloroplast autofluorescence (bottom) images clearly show YFP labelling retained after plasmolysis at the points of cell-to-cell contact (arrows).Bars indicate 10 μm.
Mentions: That PDLP1a was targeted to plasmodesmata in the absence of the C-terminal tail led us to ask whether the TMD alone would be sufficient. To assess this, two constructs were tested. The citrine variant of YFP (this has higher stability in the acidic extracellular environment) [29] was fused between the PDLP1a N-terminal signal peptide and the TMD plus C-terminal tail, or the TMD alone (Figure 8A and 8B). In both cases, the YFP open reading frame was flanked with flexible linker peptides [30], minimizing protein folding interference between the YFP and PDLP1a domains. Transgenic or transient expression of the latter fusion protein showed unique targeting to plasmodesmata, which was unaffected by plasmolysis (Figure 8C and 8D, arrows). For the former construct including the C-terminal tail, similar targeting was observed except that YFP fluorescence was also visible at discrete unidentified locations in the cytoplasm (Figure 7C). Therefore, our experiments have uniquely identified a short TMD sequence with the capacity to target proteins to plasmodesmata.

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