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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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Organisation, Phylogenetic Analysis, and Subcellular Localisation of PDLP1a and Its HomologuesThe domain structure of PDLP1a is shown in (A). The signal peptide (SP; black box) and the TMD (grey box) were predicted using SignalP 3.0 and TMHMM 2.0, respectively. The TMD is followed by a short cytoplasmic tail. The DUF26 domain (Pfam PF01657) is shown in red. Protein homology searches using the PDLP1a amino acid sequence, followed by phylogenetic analysis (B), revealed the presence of two clades within the closely related PDLP1 family; the gene for PDLP1a is boxed. All of these genes, in addition to representatives of more distantly related DUF26 proteins, were cloned and expressed trangenically in Arabidopsis from the CaMV 35S promoter. Optical sections through epidermal (Ep) and spongy mesophyll (Me) tissues of transgenic Arabidopsis expressing representatives of the two families of PDLP1a homologs (At2g33330/clade 1 and At2g01660/clade 2) show punctate labelling at all areas of cell–cell contact (some are marked with arrows). 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; bright background fluorescence from stomata is also evident. The least-related 2xDUF26 with a TMD and a C-terminal kinase domain (encoded by At4g23140) also shows PM labelling.Bars indicate 10 μm .
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pbio-0060007-g003: Organisation, Phylogenetic Analysis, and Subcellular Localisation of PDLP1a and Its HomologuesThe domain structure of PDLP1a is shown in (A). The signal peptide (SP; black box) and the TMD (grey box) were predicted using SignalP 3.0 and TMHMM 2.0, respectively. The TMD is followed by a short cytoplasmic tail. The DUF26 domain (Pfam PF01657) is shown in red. Protein homology searches using the PDLP1a amino acid sequence, followed by phylogenetic analysis (B), revealed the presence of two clades within the closely related PDLP1 family; the gene for PDLP1a is boxed. All of these genes, in addition to representatives of more distantly related DUF26 proteins, were cloned and expressed trangenically in Arabidopsis from the CaMV 35S promoter. Optical sections through epidermal (Ep) and spongy mesophyll (Me) tissues of transgenic Arabidopsis expressing representatives of the two families of PDLP1a homologs (At2g33330/clade 1 and At2g01660/clade 2) show punctate labelling at all areas of cell–cell contact (some are marked with arrows). 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; bright background fluorescence from stomata is also evident. The least-related 2xDUF26 with a TMD and a C-terminal kinase domain (encoded by At4g23140) also shows PM labelling.Bars indicate 10 μm .

Mentions: Sequence analysis showed PDLP1a to have a domain structure (Figure 3A) conserved in a small family of plant-specific proteins, including representatives from Arabidopsis, rice, Medicago, and Phaseolus. 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. DUF26 domains have a conserved C-X8-C-X2-C motif, which is distinct from the Cys-rich regions found in S-locus glycoproteins [19]. The PDLP1 families in Arabidopsis and rice comprise two clades from eight members. 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). In addition, there are three other groups of DUF26-related proteins. 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 (Figure 3B).


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)

Organisation, Phylogenetic Analysis, and Subcellular Localisation of PDLP1a and Its HomologuesThe domain structure of PDLP1a is shown in (A). The signal peptide (SP; black box) and the TMD (grey box) were predicted using SignalP 3.0 and TMHMM 2.0, respectively. The TMD is followed by a short cytoplasmic tail. The DUF26 domain (Pfam PF01657) is shown in red. Protein homology searches using the PDLP1a amino acid sequence, followed by phylogenetic analysis (B), revealed the presence of two clades within the closely related PDLP1 family; the gene for PDLP1a is boxed. All of these genes, in addition to representatives of more distantly related DUF26 proteins, were cloned and expressed trangenically in Arabidopsis from the CaMV 35S promoter. Optical sections through epidermal (Ep) and spongy mesophyll (Me) tissues of transgenic Arabidopsis expressing representatives of the two families of PDLP1a homologs (At2g33330/clade 1 and At2g01660/clade 2) show punctate labelling at all areas of cell–cell contact (some are marked with arrows). 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; bright background fluorescence from stomata is also evident. The least-related 2xDUF26 with a TMD and a C-terminal kinase domain (encoded by At4g23140) also shows PM labelling.Bars indicate 10 μm .
© Copyright Policy
Related In: Results  -  Collection

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

pbio-0060007-g003: Organisation, Phylogenetic Analysis, and Subcellular Localisation of PDLP1a and Its HomologuesThe domain structure of PDLP1a is shown in (A). The signal peptide (SP; black box) and the TMD (grey box) were predicted using SignalP 3.0 and TMHMM 2.0, respectively. The TMD is followed by a short cytoplasmic tail. The DUF26 domain (Pfam PF01657) is shown in red. Protein homology searches using the PDLP1a amino acid sequence, followed by phylogenetic analysis (B), revealed the presence of two clades within the closely related PDLP1 family; the gene for PDLP1a is boxed. All of these genes, in addition to representatives of more distantly related DUF26 proteins, were cloned and expressed trangenically in Arabidopsis from the CaMV 35S promoter. Optical sections through epidermal (Ep) and spongy mesophyll (Me) tissues of transgenic Arabidopsis expressing representatives of the two families of PDLP1a homologs (At2g33330/clade 1 and At2g01660/clade 2) show punctate labelling at all areas of cell–cell contact (some are marked with arrows). 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; bright background fluorescence from stomata is also evident. The least-related 2xDUF26 with a TMD and a C-terminal kinase domain (encoded by At4g23140) also shows PM labelling.Bars indicate 10 μm .
Mentions: Sequence analysis showed PDLP1a to have a domain structure (Figure 3A) conserved in a small family of plant-specific proteins, including representatives from Arabidopsis, rice, Medicago, and Phaseolus. 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. DUF26 domains have a conserved C-X8-C-X2-C motif, which is distinct from the Cys-rich regions found in S-locus glycoproteins [19]. The PDLP1 families in Arabidopsis and rice comprise two clades from eight members. 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). In addition, there are three other groups of DUF26-related proteins. 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 (Figure 3B).

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