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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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Altered Expression of PDLP1 Changes Plasmodesmal TraffickingPDLP1 KO lines (A) or PDLP1a:HA overexpressing lines (B) were assessed for plasmodesmal trafficking potential by counting the number of GFP recipient cells surrounding primary bombardment sites. From three double KO lines, only At2g33330×At1g04520 and At5g43980×At2g33330 showed altered GFP diffusion. (Data for At2g33330×At1g04520 were obtained from two independent experiments and 96 bombardment sites each for wild-type (WT) and KO lines (p ≤ 0.05); data for At5g43980×At2g33330 were obtained from three independent experiments and 57 and 93 bombardment sites for WT and KO lines, respectively (p ≤ 0.01). (B) Transgenic Arabidopsis plants expressing 35S::PDLP1a:HA (or 35S::PDLP1a:GFP; unpublished data) exhibited a dwarf phenotype (right, upper picture) when compared with nontransformed (NT) plants. This dwarf phenotype showed a positive correlation with transgene copy number and protein accumulation, assessed by western analysis with anti-HA antibody (right, lower picture). Bombardment of the less-dwarfed hemizygous plants (Hemi; left) showed a dramatic reduction in GFP diffusion (data collected from two independent experiments and 70 and 114 bombardment sites for the NT and hemizygous plants, respectively; p ≤ 0.0001). Homo, homozygous.Bars indicate the standard error of the mean.
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pbio-0060007-g005: Altered Expression of PDLP1 Changes Plasmodesmal TraffickingPDLP1 KO lines (A) or PDLP1a:HA overexpressing lines (B) were assessed for plasmodesmal trafficking potential by counting the number of GFP recipient cells surrounding primary bombardment sites. From three double KO lines, only At2g33330×At1g04520 and At5g43980×At2g33330 showed altered GFP diffusion. (Data for At2g33330×At1g04520 were obtained from two independent experiments and 96 bombardment sites each for wild-type (WT) and KO lines (p ≤ 0.05); data for At5g43980×At2g33330 were obtained from three independent experiments and 57 and 93 bombardment sites for WT and KO lines, respectively (p ≤ 0.01). (B) Transgenic Arabidopsis plants expressing 35S::PDLP1a:HA (or 35S::PDLP1a:GFP; unpublished data) exhibited a dwarf phenotype (right, upper picture) when compared with nontransformed (NT) plants. This dwarf phenotype showed a positive correlation with transgene copy number and protein accumulation, assessed by western analysis with anti-HA antibody (right, lower picture). Bombardment of the less-dwarfed hemizygous plants (Hemi; left) showed a dramatic reduction in GFP diffusion (data collected from two independent experiments and 70 and 114 bombardment sites for the NT and hemizygous plants, respectively; p ≤ 0.0001). Homo, homozygous.Bars indicate the standard error of the mean.

Mentions: Insertional mutant lines (knock-out lines; KOs) for six of the eight PDLP1 genes are available from public collections. None of these lines showed any obvious growth or developmental phenotype. GFP diffusion in leaves of these lines was not significantly different from that in wild-type Arabidopsis leaves (unpublished data). However, scrutiny of the public microarray expression data (http://www.genevestigator.ethz.ch) showed that the tissue-specific pattern of expression of these genes differed widely with respect to expression in leaf tissues. To accommodate potential problems of functional redundancy within the family, various combinations of KOs were made by crossing. We concentrated on crosses between members of clade 1 (Figure 3B) that included examples of genes expressed relatively highly in leaf tissues. KO combinations of At5g43980 and At1g04520, At5g43980 and At2g33330, and At2g33330 and At1g04520 were tested in our GFP bombardment assay (Figure 5A). Whereas the homozygous progeny of At5g43980×At1g04520 showed no significant change in the spread of GFP (unpublished data), the progeny of the other crosses (At5g43980×At2g33330 and At2g33330×At1g04520) showed significantly increased trafficking ability (p ≤ 0.05 and ≤ 0.01, respectively).


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)

Altered Expression of PDLP1 Changes Plasmodesmal TraffickingPDLP1 KO lines (A) or PDLP1a:HA overexpressing lines (B) were assessed for plasmodesmal trafficking potential by counting the number of GFP recipient cells surrounding primary bombardment sites. From three double KO lines, only At2g33330×At1g04520 and At5g43980×At2g33330 showed altered GFP diffusion. (Data for At2g33330×At1g04520 were obtained from two independent experiments and 96 bombardment sites each for wild-type (WT) and KO lines (p ≤ 0.05); data for At5g43980×At2g33330 were obtained from three independent experiments and 57 and 93 bombardment sites for WT and KO lines, respectively (p ≤ 0.01). (B) Transgenic Arabidopsis plants expressing 35S::PDLP1a:HA (or 35S::PDLP1a:GFP; unpublished data) exhibited a dwarf phenotype (right, upper picture) when compared with nontransformed (NT) plants. This dwarf phenotype showed a positive correlation with transgene copy number and protein accumulation, assessed by western analysis with anti-HA antibody (right, lower picture). Bombardment of the less-dwarfed hemizygous plants (Hemi; left) showed a dramatic reduction in GFP diffusion (data collected from two independent experiments and 70 and 114 bombardment sites for the NT and hemizygous plants, respectively; p ≤ 0.0001). Homo, homozygous.Bars indicate the standard error of the mean.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2211546&req=5

pbio-0060007-g005: Altered Expression of PDLP1 Changes Plasmodesmal TraffickingPDLP1 KO lines (A) or PDLP1a:HA overexpressing lines (B) were assessed for plasmodesmal trafficking potential by counting the number of GFP recipient cells surrounding primary bombardment sites. From three double KO lines, only At2g33330×At1g04520 and At5g43980×At2g33330 showed altered GFP diffusion. (Data for At2g33330×At1g04520 were obtained from two independent experiments and 96 bombardment sites each for wild-type (WT) and KO lines (p ≤ 0.05); data for At5g43980×At2g33330 were obtained from three independent experiments and 57 and 93 bombardment sites for WT and KO lines, respectively (p ≤ 0.01). (B) Transgenic Arabidopsis plants expressing 35S::PDLP1a:HA (or 35S::PDLP1a:GFP; unpublished data) exhibited a dwarf phenotype (right, upper picture) when compared with nontransformed (NT) plants. This dwarf phenotype showed a positive correlation with transgene copy number and protein accumulation, assessed by western analysis with anti-HA antibody (right, lower picture). Bombardment of the less-dwarfed hemizygous plants (Hemi; left) showed a dramatic reduction in GFP diffusion (data collected from two independent experiments and 70 and 114 bombardment sites for the NT and hemizygous plants, respectively; p ≤ 0.0001). Homo, homozygous.Bars indicate the standard error of the mean.
Mentions: Insertional mutant lines (knock-out lines; KOs) for six of the eight PDLP1 genes are available from public collections. None of these lines showed any obvious growth or developmental phenotype. GFP diffusion in leaves of these lines was not significantly different from that in wild-type Arabidopsis leaves (unpublished data). However, scrutiny of the public microarray expression data (http://www.genevestigator.ethz.ch) showed that the tissue-specific pattern of expression of these genes differed widely with respect to expression in leaf tissues. To accommodate potential problems of functional redundancy within the family, various combinations of KOs were made by crossing. We concentrated on crosses between members of clade 1 (Figure 3B) that included examples of genes expressed relatively highly in leaf tissues. KO combinations of At5g43980 and At1g04520, At5g43980 and At2g33330, and At2g33330 and At1g04520 were tested in our GFP bombardment assay (Figure 5A). Whereas the homozygous progeny of At5g43980×At1g04520 showed no significant change in the spread of GFP (unpublished data), the progeny of the other crosses (At5g43980×At2g33330 and At2g33330×At1g04520) showed significantly increased trafficking ability (p ≤ 0.05 and ≤ 0.01, respectively).

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
Related in: MedlinePlus