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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 Utilises the Secretory Pathway for Delivery to PlasmodesmataN. benthamiana leaves were agro-infiltrated with a construct expressing 35S::PDLP1a:GFP (A to H) and either a construct expressing a Golgi marker (35S::ManI:RFP; [A to F]) or a construct expressing the Sar1 inhibitory mutant variant Sar1[H74L]:RFP that blocks ER to Golgi transport (G and H).(A–C) Cells expressing 35S::PDLP1a:GFP and 35S::ManI:RFP showed discrete punctate structures characteristic of plasmodesmata (arrowheads), and Golgi stacks, respectively. Inset, high magnification of the boxed region in (C) confirms the discrete nature of the two subcellular locations.(D–F) Treatment with BFA led to a severe reduction in number of cell wall–associated PDLP1a:GFP-labeled punctate structures ([D], compare to [A], arrowheads), and the near complete loss of individual Golgi stacks ([B], compare to [E]), and concomitantly in the formation of typical BFA compartments (asterisks) in which both PDLP1a:GFP and ManI:RFP colocalized.(G and H) Similar phenomenon was observed upon coexpression of PDLP1a:GFP and the GTP-restricted mutant Sar1[H74L]. In median section (G), PDLP1a:GFP formed large aggregates in the nuclear vicinity that resembled the BFA compartment. Fluorescence extended also all around the cells and within cytoplasmic strands (G). In the cortex of the same cells (H), PDLP1a:GFP was found to be located in a typical ER polygonal network that was better seen upon higher magnification (inset, 3× magnification of the boxed region in [H]). Coexpression of the Sar1[H74L] and PDLP1a constructs in the same cell was confirmed from their respective fluorescent tags.Scale bars indicate 5 μm.
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pbio-0060007-g006: PDLP1a Utilises the Secretory Pathway for Delivery to PlasmodesmataN. benthamiana leaves were agro-infiltrated with a construct expressing 35S::PDLP1a:GFP (A to H) and either a construct expressing a Golgi marker (35S::ManI:RFP; [A to F]) or a construct expressing the Sar1 inhibitory mutant variant Sar1[H74L]:RFP that blocks ER to Golgi transport (G and H).(A–C) Cells expressing 35S::PDLP1a:GFP and 35S::ManI:RFP showed discrete punctate structures characteristic of plasmodesmata (arrowheads), and Golgi stacks, respectively. Inset, high magnification of the boxed region in (C) confirms the discrete nature of the two subcellular locations.(D–F) Treatment with BFA led to a severe reduction in number of cell wall–associated PDLP1a:GFP-labeled punctate structures ([D], compare to [A], arrowheads), and the near complete loss of individual Golgi stacks ([B], compare to [E]), and concomitantly in the formation of typical BFA compartments (asterisks) in which both PDLP1a:GFP and ManI:RFP colocalized.(G and H) Similar phenomenon was observed upon coexpression of PDLP1a:GFP and the GTP-restricted mutant Sar1[H74L]. In median section (G), PDLP1a:GFP formed large aggregates in the nuclear vicinity that resembled the BFA compartment. Fluorescence extended also all around the cells and within cytoplasmic strands (G). In the cortex of the same cells (H), PDLP1a:GFP was found to be located in a typical ER polygonal network that was better seen upon higher magnification (inset, 3× magnification of the boxed region in [H]). Coexpression of the Sar1[H74L] and PDLP1a constructs in the same cell was confirmed from their respective fluorescent tags.Scale bars indicate 5 μm.

Mentions: Trafficking of proteins to the plasmodesmata has variously been shown to exploit (e.g., C1RGP [10]) or bypass (e.g., TMV MP; [23,24]) the secretory pathway. Chemical or protein inhibitors were used to investigate the role of the secretory pathway in PDLP1a trafficking to plasmodesmata. Brefeldin A (BFA), an inhibitor of specific ADP ribosylation factor (ARF) GTPase exchange factors (GEFs), arrests vesicle trafficking at various points along the secretory pathway [25], whereas Sar1[H74L], a GTPase-defective mutant of Sar1p, very specifically affects COPII-mediated ER-to-Golgi transport [26]. Both of these inhibitors provided a qualitative assessment of the roles of the ER and COPII pathways in PDLP1a targeting to plasmodesmata. Coexpression of PDLP1a:GFP and ManI:RFP, encoding a well-described secretory marker whose translocation to the Golgi apparatus depends on a functional secretory pathway [27], resulted in the identification of PDLP1a:GFP at plasmodesmata, and distinct and separate Golgi labelling with ManI:RFP (Figure 6A to 6C). Addition of BFA led to the complete loss of Golgi stacks and the concomitant formation of large ER–Golgi hybrid bodies/BFA compartment in which both PDLP1a:GFP and ManI:RFP accumulated (Figure 6D to 6F, stars). In addition to this location, PDLP1a:GFP could still occasionally be seen within plasmodesmata after treatment (compare Figure 6A and 6D, arrowheads). This was likely due to a (dose-dependent) incomplete inhibition of secretion or to pre-existing PDLP1a:GFP molecules within plasmodesmata before addition of BFA. Since BFA induces a range of effects on treated cells due to the inhibition of multiple Arf-GEFs [25], additional experiments were performed using a dominant-negative mutant of the Ras-like small GTPase, Sar1 (Sar1[H74L]), to specifically block ER export; the wild-type protein was used as the control. As expected, coexpression of wild-type Sar1:RFP with PDLP1a:GFP had no effect on PDLP1a:GFP steady-state accumulation within plasmodesmata (unpublished data). In contrast, coexpression with the GTPase-impaired mutant Sar1[H74L] led to the retention of PDLP1a:GFP in the ER as deduced from the perinuclear labelling (Figure 6G) and the visualisation of a typical polygonal network in cortical sections (Figure 6H). Similar to BFA treatment, incorporation of PDLP1a into plasmodesmata was not completely abolished in all cells. This could be as a consequence of partial inhibition of secretion following expression of Sar1[H74L], because inhibition of secretion is directly related to the steady-state accumulation of Sar1[H74L] [28], or prior accumulation of PDLP1a:GFP before Sar1[H74L] expression. Such variability is inherent in experiments using transient expression following agro-infiltration. Most importantly, however, induced retention of PDLP1a:GFP in the ER or in the BFA compartment was never observed under conditions where secretion was unperturbed. It is therefore concluded that PDLP1a utilises the secretory pathway for delivery 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)

PDLP1a Utilises the Secretory Pathway for Delivery to PlasmodesmataN. benthamiana leaves were agro-infiltrated with a construct expressing 35S::PDLP1a:GFP (A to H) and either a construct expressing a Golgi marker (35S::ManI:RFP; [A to F]) or a construct expressing the Sar1 inhibitory mutant variant Sar1[H74L]:RFP that blocks ER to Golgi transport (G and H).(A–C) Cells expressing 35S::PDLP1a:GFP and 35S::ManI:RFP showed discrete punctate structures characteristic of plasmodesmata (arrowheads), and Golgi stacks, respectively. Inset, high magnification of the boxed region in (C) confirms the discrete nature of the two subcellular locations.(D–F) Treatment with BFA led to a severe reduction in number of cell wall–associated PDLP1a:GFP-labeled punctate structures ([D], compare to [A], arrowheads), and the near complete loss of individual Golgi stacks ([B], compare to [E]), and concomitantly in the formation of typical BFA compartments (asterisks) in which both PDLP1a:GFP and ManI:RFP colocalized.(G and H) Similar phenomenon was observed upon coexpression of PDLP1a:GFP and the GTP-restricted mutant Sar1[H74L]. In median section (G), PDLP1a:GFP formed large aggregates in the nuclear vicinity that resembled the BFA compartment. Fluorescence extended also all around the cells and within cytoplasmic strands (G). In the cortex of the same cells (H), PDLP1a:GFP was found to be located in a typical ER polygonal network that was better seen upon higher magnification (inset, 3× magnification of the boxed region in [H]). Coexpression of the Sar1[H74L] and PDLP1a constructs in the same cell was confirmed from their respective fluorescent tags.Scale bars indicate 5 μm.
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Related In: Results  -  Collection

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pbio-0060007-g006: PDLP1a Utilises the Secretory Pathway for Delivery to PlasmodesmataN. benthamiana leaves were agro-infiltrated with a construct expressing 35S::PDLP1a:GFP (A to H) and either a construct expressing a Golgi marker (35S::ManI:RFP; [A to F]) or a construct expressing the Sar1 inhibitory mutant variant Sar1[H74L]:RFP that blocks ER to Golgi transport (G and H).(A–C) Cells expressing 35S::PDLP1a:GFP and 35S::ManI:RFP showed discrete punctate structures characteristic of plasmodesmata (arrowheads), and Golgi stacks, respectively. Inset, high magnification of the boxed region in (C) confirms the discrete nature of the two subcellular locations.(D–F) Treatment with BFA led to a severe reduction in number of cell wall–associated PDLP1a:GFP-labeled punctate structures ([D], compare to [A], arrowheads), and the near complete loss of individual Golgi stacks ([B], compare to [E]), and concomitantly in the formation of typical BFA compartments (asterisks) in which both PDLP1a:GFP and ManI:RFP colocalized.(G and H) Similar phenomenon was observed upon coexpression of PDLP1a:GFP and the GTP-restricted mutant Sar1[H74L]. In median section (G), PDLP1a:GFP formed large aggregates in the nuclear vicinity that resembled the BFA compartment. Fluorescence extended also all around the cells and within cytoplasmic strands (G). In the cortex of the same cells (H), PDLP1a:GFP was found to be located in a typical ER polygonal network that was better seen upon higher magnification (inset, 3× magnification of the boxed region in [H]). Coexpression of the Sar1[H74L] and PDLP1a constructs in the same cell was confirmed from their respective fluorescent tags.Scale bars indicate 5 μm.
Mentions: Trafficking of proteins to the plasmodesmata has variously been shown to exploit (e.g., C1RGP [10]) or bypass (e.g., TMV MP; [23,24]) the secretory pathway. Chemical or protein inhibitors were used to investigate the role of the secretory pathway in PDLP1a trafficking to plasmodesmata. Brefeldin A (BFA), an inhibitor of specific ADP ribosylation factor (ARF) GTPase exchange factors (GEFs), arrests vesicle trafficking at various points along the secretory pathway [25], whereas Sar1[H74L], a GTPase-defective mutant of Sar1p, very specifically affects COPII-mediated ER-to-Golgi transport [26]. Both of these inhibitors provided a qualitative assessment of the roles of the ER and COPII pathways in PDLP1a targeting to plasmodesmata. Coexpression of PDLP1a:GFP and ManI:RFP, encoding a well-described secretory marker whose translocation to the Golgi apparatus depends on a functional secretory pathway [27], resulted in the identification of PDLP1a:GFP at plasmodesmata, and distinct and separate Golgi labelling with ManI:RFP (Figure 6A to 6C). Addition of BFA led to the complete loss of Golgi stacks and the concomitant formation of large ER–Golgi hybrid bodies/BFA compartment in which both PDLP1a:GFP and ManI:RFP accumulated (Figure 6D to 6F, stars). In addition to this location, PDLP1a:GFP could still occasionally be seen within plasmodesmata after treatment (compare Figure 6A and 6D, arrowheads). This was likely due to a (dose-dependent) incomplete inhibition of secretion or to pre-existing PDLP1a:GFP molecules within plasmodesmata before addition of BFA. Since BFA induces a range of effects on treated cells due to the inhibition of multiple Arf-GEFs [25], additional experiments were performed using a dominant-negative mutant of the Ras-like small GTPase, Sar1 (Sar1[H74L]), to specifically block ER export; the wild-type protein was used as the control. As expected, coexpression of wild-type Sar1:RFP with PDLP1a:GFP had no effect on PDLP1a:GFP steady-state accumulation within plasmodesmata (unpublished data). In contrast, coexpression with the GTPase-impaired mutant Sar1[H74L] led to the retention of PDLP1a:GFP in the ER as deduced from the perinuclear labelling (Figure 6G) and the visualisation of a typical polygonal network in cortical sections (Figure 6H). Similar to BFA treatment, incorporation of PDLP1a into plasmodesmata was not completely abolished in all cells. This could be as a consequence of partial inhibition of secretion following expression of Sar1[H74L], because inhibition of secretion is directly related to the steady-state accumulation of Sar1[H74L] [28], or prior accumulation of PDLP1a:GFP before Sar1[H74L] expression. Such variability is inherent in experiments using transient expression following agro-infiltration. Most importantly, however, induced retention of PDLP1a:GFP in the ER or in the BFA compartment was never observed under conditions where secretion was unperturbed. It is therefore concluded that PDLP1a utilises the secretory pathway for delivery 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