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A bacterial signal peptide is functional in plants and directs proteins to the secretory pathway.

Moeller L, Gan Q, Wang K - J. Exp. Bot. (2009)

Bottom Line: Maize kernel fractionation revealed that transgenic lines carrying BSP result in recombinant protein association with fibre and starch fractions.This is the first report providing evidence of the ability of a bacterial signal peptide to target proteins to the plant secretory pathway.The results provide important insights for further understanding the heterologous protein trafficking mechanisms and for developing effective strategies in molecular farming.

View Article: PubMed Central - PubMed

Affiliation: Iowa State University, Ames, IA 50011-1010, USA.

ABSTRACT
The Escherichia coli heat-labile enterotoxin B subunit (LT-B) has been used as a model antigen for the production of plant-derived high-valued proteins in maize. LT-B with its native signal peptide (BSP) has been shown to accumulate in starch granules of transgenic maize kernels. To elucidate the targeting properties of the bacterial LT-B protein and BSP in plant systems, the subcellular localization of visual marker green fluorescent protein (GFP) fused to LT-B and various combinations of signal peptides was examined in Arabidopsis protoplasts and transgenic maize. Biochemical analysis indicates that the LT-B::GFP fusion proteins can assemble and fold properly retaining both the antigenicity of LT-B and the fluorescing properties of GFP. Maize kernel fractionation revealed that transgenic lines carrying BSP result in recombinant protein association with fibre and starch fractions. Confocal microscopy analysis indicates that the fusion proteins accumulate in the endomembrane system of plant cells in a signal peptide-dependent fashion. This is the first report providing evidence of the ability of a bacterial signal peptide to target proteins to the plant secretory pathway. The results provide important insights for further understanding the heterologous protein trafficking mechanisms and for developing effective strategies in molecular farming.

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Confocal images of transiently and stably transformed Arabidopsis and maize cells expressing GFP or LT-B::GFP fusion proteins. Constructs A–E are described in Fig. 1. Transiently transformed Arabidopsis leaf (a, e, i, m, q) and root (b, f, j, n, r) protoplasts using the constitutive P35S promoter constructs were imaged 24–48 h after transformation. Stably transformed maize callus (c, g, k, o, s) also used the P35S promoter constructs. Fresh immature endosperm (12–26 d after pollination) from transgenic maize seed carrying the Pγzein promoter constructs were excised and imaged (d, h, l, p). Images are presented as merged green and red channels (presented in magenta color) for all samples. Green signal in all images corresponds to GFP. Red signal in leaf protoplasts is the autofluorescence of chlorophyll in chloroplasts. Red signal in root protoplasts corresponds to the expression of a VirD2::RFP construct, a nuclear marker. Red signal in maize callus and endosperm samples is propidium iodide used as a counter stain that labels nucleic acids. Organelle labelling: chloroplasts (cl), cytosol (cy), nucleus (nu), endoplasmic reticulum (er), vacuole (va), starch (st). Bars=10 μm.
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fig5: Confocal images of transiently and stably transformed Arabidopsis and maize cells expressing GFP or LT-B::GFP fusion proteins. Constructs A–E are described in Fig. 1. Transiently transformed Arabidopsis leaf (a, e, i, m, q) and root (b, f, j, n, r) protoplasts using the constitutive P35S promoter constructs were imaged 24–48 h after transformation. Stably transformed maize callus (c, g, k, o, s) also used the P35S promoter constructs. Fresh immature endosperm (12–26 d after pollination) from transgenic maize seed carrying the Pγzein promoter constructs were excised and imaged (d, h, l, p). Images are presented as merged green and red channels (presented in magenta color) for all samples. Green signal in all images corresponds to GFP. Red signal in leaf protoplasts is the autofluorescence of chlorophyll in chloroplasts. Red signal in root protoplasts corresponds to the expression of a VirD2::RFP construct, a nuclear marker. Red signal in maize callus and endosperm samples is propidium iodide used as a counter stain that labels nucleic acids. Organelle labelling: chloroplasts (cl), cytosol (cy), nucleus (nu), endoplasmic reticulum (er), vacuole (va), starch (st). Bars=10 μm.

Mentions: Confocal microscopy was used for localization studies in transiently transformed Arabidopsis protoplasts, and stably transformed maize callus and endosperm tissue using the constructs described in Fig. 1. The objective of such experiments was to establish the subcellular targeting properties of the bacterial signal peptide of LT-B and the LT-B protein itself. Figure 5 summarizes the results.


A bacterial signal peptide is functional in plants and directs proteins to the secretory pathway.

Moeller L, Gan Q, Wang K - J. Exp. Bot. (2009)

Confocal images of transiently and stably transformed Arabidopsis and maize cells expressing GFP or LT-B::GFP fusion proteins. Constructs A–E are described in Fig. 1. Transiently transformed Arabidopsis leaf (a, e, i, m, q) and root (b, f, j, n, r) protoplasts using the constitutive P35S promoter constructs were imaged 24–48 h after transformation. Stably transformed maize callus (c, g, k, o, s) also used the P35S promoter constructs. Fresh immature endosperm (12–26 d after pollination) from transgenic maize seed carrying the Pγzein promoter constructs were excised and imaged (d, h, l, p). Images are presented as merged green and red channels (presented in magenta color) for all samples. Green signal in all images corresponds to GFP. Red signal in leaf protoplasts is the autofluorescence of chlorophyll in chloroplasts. Red signal in root protoplasts corresponds to the expression of a VirD2::RFP construct, a nuclear marker. Red signal in maize callus and endosperm samples is propidium iodide used as a counter stain that labels nucleic acids. Organelle labelling: chloroplasts (cl), cytosol (cy), nucleus (nu), endoplasmic reticulum (er), vacuole (va), starch (st). Bars=10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2724687&req=5

fig5: Confocal images of transiently and stably transformed Arabidopsis and maize cells expressing GFP or LT-B::GFP fusion proteins. Constructs A–E are described in Fig. 1. Transiently transformed Arabidopsis leaf (a, e, i, m, q) and root (b, f, j, n, r) protoplasts using the constitutive P35S promoter constructs were imaged 24–48 h after transformation. Stably transformed maize callus (c, g, k, o, s) also used the P35S promoter constructs. Fresh immature endosperm (12–26 d after pollination) from transgenic maize seed carrying the Pγzein promoter constructs were excised and imaged (d, h, l, p). Images are presented as merged green and red channels (presented in magenta color) for all samples. Green signal in all images corresponds to GFP. Red signal in leaf protoplasts is the autofluorescence of chlorophyll in chloroplasts. Red signal in root protoplasts corresponds to the expression of a VirD2::RFP construct, a nuclear marker. Red signal in maize callus and endosperm samples is propidium iodide used as a counter stain that labels nucleic acids. Organelle labelling: chloroplasts (cl), cytosol (cy), nucleus (nu), endoplasmic reticulum (er), vacuole (va), starch (st). Bars=10 μm.
Mentions: Confocal microscopy was used for localization studies in transiently transformed Arabidopsis protoplasts, and stably transformed maize callus and endosperm tissue using the constructs described in Fig. 1. The objective of such experiments was to establish the subcellular targeting properties of the bacterial signal peptide of LT-B and the LT-B protein itself. Figure 5 summarizes the results.

Bottom Line: Maize kernel fractionation revealed that transgenic lines carrying BSP result in recombinant protein association with fibre and starch fractions.This is the first report providing evidence of the ability of a bacterial signal peptide to target proteins to the plant secretory pathway.The results provide important insights for further understanding the heterologous protein trafficking mechanisms and for developing effective strategies in molecular farming.

View Article: PubMed Central - PubMed

Affiliation: Iowa State University, Ames, IA 50011-1010, USA.

ABSTRACT
The Escherichia coli heat-labile enterotoxin B subunit (LT-B) has been used as a model antigen for the production of plant-derived high-valued proteins in maize. LT-B with its native signal peptide (BSP) has been shown to accumulate in starch granules of transgenic maize kernels. To elucidate the targeting properties of the bacterial LT-B protein and BSP in plant systems, the subcellular localization of visual marker green fluorescent protein (GFP) fused to LT-B and various combinations of signal peptides was examined in Arabidopsis protoplasts and transgenic maize. Biochemical analysis indicates that the LT-B::GFP fusion proteins can assemble and fold properly retaining both the antigenicity of LT-B and the fluorescing properties of GFP. Maize kernel fractionation revealed that transgenic lines carrying BSP result in recombinant protein association with fibre and starch fractions. Confocal microscopy analysis indicates that the fusion proteins accumulate in the endomembrane system of plant cells in a signal peptide-dependent fashion. This is the first report providing evidence of the ability of a bacterial signal peptide to target proteins to the plant secretory pathway. The results provide important insights for further understanding the heterologous protein trafficking mechanisms and for developing effective strategies in molecular farming.

Show MeSH
Related in: MedlinePlus