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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 Membrane Topology(A) shows a schematic presentation of the expression cassettes for PDLP1a C-terminal fusions with YN and YC of YFP. N. benthamiana were agro-infiltrated with agrobacteria containing 35S::PDLP1a:YN or 35S::PDLP1a:YC and the corresponding counterpart (pLH::YN, pLH::YC, or pLH::YN-ER), and 35S::HCPro.(B) Five days post-inoculation, fluorescence was analysed by confocal microscopy confirming YFP fluorescence reconstitution in cells coexpressing 35S::YN and 35S::YC, 35S::YN-ER and 35S::YC-ER, 35S::PDLP1a:YN and 35S::YC, and 35S:PDLP1a:YC and 35S::YN, but not in cells coexpressing 35S::YN-ER and 35S::YC or 35S::PDLP1a:YC and 35S::YN-ER.(C) Bimolecular fluorescence confirmed the predicted orientation of PDLP1a as a type I membrane protein. Bars = 20 μm
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pbio-0060007-g004: PDLP1a Membrane Topology(A) shows a schematic presentation of the expression cassettes for PDLP1a C-terminal fusions with YN and YC of YFP. N. benthamiana were agro-infiltrated with agrobacteria containing 35S::PDLP1a:YN or 35S::PDLP1a:YC and the corresponding counterpart (pLH::YN, pLH::YC, or pLH::YN-ER), and 35S::HCPro.(B) Five days post-inoculation, fluorescence was analysed by confocal microscopy confirming YFP fluorescence reconstitution in cells coexpressing 35S::YN and 35S::YC, 35S::YN-ER and 35S::YC-ER, 35S::PDLP1a:YN and 35S::YC, and 35S:PDLP1a:YC and 35S::YN, but not in cells coexpressing 35S::YN-ER and 35S::YC or 35S::PDLP1a:YC and 35S::YN-ER.(C) Bimolecular fluorescence confirmed the predicted orientation of PDLP1a as a type I membrane protein. Bars = 20 μm

Mentions: By analogy with the wider members of the 2xDUF26 class of proteins, including some kinases that have DUF26 domains located extracellularly and signal through a TMD to the cytoplasmic kinase module [20], we predicted that PDLP1a should be orientated with its short C-terminal tail in the cytoplasm and the 2xDUF26 domain in the apoplast. To confirm this prediction, we employed a bimolecular fluorescence complementation (BiFC) strategy in which half of yellow fluorescent protein (YFP) fused to the test protein was complemented with the corresponding half of YFP targeted to the cytoplasm [21]. Hence, when either half of YFP (YN or YC, Figure 4A) was fused to the C-terminal tail of PDLP1a and cotransiently expressed in N. benthamiana with the alternate unfused half-YFP, BiFC fluorescence in plasmodesmata was observed (Figure 4B). In contrast, when PDLP1a:YC was coexpressed with the alternate unfused half carrying signals for targeting to and retention in the ER (YN-ER; [21]), no BiFC was observed (Figure 4B), confirming our predictions that PDLP1a was orientated with the C-terminus in the cytoplasm (Figure 4C). Since PDLP1 is a type I membrane protein with a single TMD, the N-terminal portion of the protein is by default located facing the apoplast.


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 Membrane Topology(A) shows a schematic presentation of the expression cassettes for PDLP1a C-terminal fusions with YN and YC of YFP. N. benthamiana were agro-infiltrated with agrobacteria containing 35S::PDLP1a:YN or 35S::PDLP1a:YC and the corresponding counterpart (pLH::YN, pLH::YC, or pLH::YN-ER), and 35S::HCPro.(B) Five days post-inoculation, fluorescence was analysed by confocal microscopy confirming YFP fluorescence reconstitution in cells coexpressing 35S::YN and 35S::YC, 35S::YN-ER and 35S::YC-ER, 35S::PDLP1a:YN and 35S::YC, and 35S:PDLP1a:YC and 35S::YN, but not in cells coexpressing 35S::YN-ER and 35S::YC or 35S::PDLP1a:YC and 35S::YN-ER.(C) Bimolecular fluorescence confirmed the predicted orientation of PDLP1a as a type I membrane protein. Bars = 20 μm
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2211546&req=5

pbio-0060007-g004: PDLP1a Membrane Topology(A) shows a schematic presentation of the expression cassettes for PDLP1a C-terminal fusions with YN and YC of YFP. N. benthamiana were agro-infiltrated with agrobacteria containing 35S::PDLP1a:YN or 35S::PDLP1a:YC and the corresponding counterpart (pLH::YN, pLH::YC, or pLH::YN-ER), and 35S::HCPro.(B) Five days post-inoculation, fluorescence was analysed by confocal microscopy confirming YFP fluorescence reconstitution in cells coexpressing 35S::YN and 35S::YC, 35S::YN-ER and 35S::YC-ER, 35S::PDLP1a:YN and 35S::YC, and 35S:PDLP1a:YC and 35S::YN, but not in cells coexpressing 35S::YN-ER and 35S::YC or 35S::PDLP1a:YC and 35S::YN-ER.(C) Bimolecular fluorescence confirmed the predicted orientation of PDLP1a as a type I membrane protein. Bars = 20 μm
Mentions: By analogy with the wider members of the 2xDUF26 class of proteins, including some kinases that have DUF26 domains located extracellularly and signal through a TMD to the cytoplasmic kinase module [20], we predicted that PDLP1a should be orientated with its short C-terminal tail in the cytoplasm and the 2xDUF26 domain in the apoplast. To confirm this prediction, we employed a bimolecular fluorescence complementation (BiFC) strategy in which half of yellow fluorescent protein (YFP) fused to the test protein was complemented with the corresponding half of YFP targeted to the cytoplasm [21]. Hence, when either half of YFP (YN or YC, Figure 4A) was fused to the C-terminal tail of PDLP1a and cotransiently expressed in N. benthamiana with the alternate unfused half-YFP, BiFC fluorescence in plasmodesmata was observed (Figure 4B). In contrast, when PDLP1a:YC was coexpressed with the alternate unfused half carrying signals for targeting to and retention in the ER (YN-ER; [21]), no BiFC was observed (Figure 4B), confirming our predictions that PDLP1a was orientated with the C-terminus in the cytoplasm (Figure 4C). Since PDLP1 is a type I membrane protein with a single TMD, the N-terminal portion of the protein is by default located facing the apoplast.

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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