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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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Co-localization experiments in Arabidopsis root protoplasts. Protoplasts were co-transformed using the constructs presented in Fig. 1 and an ER marker protein fused to RFP, ER cherry (Nelson et al., 2007). (A) pLM01 (GFP control). (B) pLM02 (BSP-GFP). (C) pLM03, (BSP-LT-B::GFP). (D) pLM08 (ZSP-LT-B::GFP). (E) pLM09 (LT-B::GFP). Green channel corresponds to GFP signal. Red channels (presented in magenta color) corresponds to ER-cherry signal. Merged images are also presented. Organelle labelling: cytosol (cy), nucleus (nu), endoplasmic reticulum (er). Bars=10 μm.
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fig6: Co-localization experiments in Arabidopsis root protoplasts. Protoplasts were co-transformed using the constructs presented in Fig. 1 and an ER marker protein fused to RFP, ER cherry (Nelson et al., 2007). (A) pLM01 (GFP control). (B) pLM02 (BSP-GFP). (C) pLM03, (BSP-LT-B::GFP). (D) pLM08 (ZSP-LT-B::GFP). (E) pLM09 (LT-B::GFP). Green channel corresponds to GFP signal. Red channels (presented in magenta color) corresponds to ER-cherry signal. Merged images are also presented. Organelle labelling: cytosol (cy), nucleus (nu), endoplasmic reticulum (er). Bars=10 μm.

Mentions: Figure 6 shows the co-localization experiments for the confirmation of subcellular localization using a known ER marker (Nelson et al., 2007). This marker (ER cherry) contains a red fluorescent protein fused to the signal peptide of the Arabidopsis thaliana wall-associated kinase 2 (He et al., 1999) and an HDEL motif for retention in the ER (Munro and Pelham, 1987). Arabidopsis root protoplasts were co-transformed with the ER cherry marker construct and the various constructs listed in Fig. 1. The results show that the ER-targeted RFP signals co-localized with the GFP signals from the constructs that contained either BSP (Fig. 6B, C) or ZSP (Fig. 6D). By contrast, GFP signals from constructs carrying GFP alone (Fig. 6A) or LT-B::GFP fusion (Fig. 6E) with no signal peptide were constantly detected in separate subcellular compartments than ER-targeted RFP signal.


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)

Co-localization experiments in Arabidopsis root protoplasts. Protoplasts were co-transformed using the constructs presented in Fig. 1 and an ER marker protein fused to RFP, ER cherry (Nelson et al., 2007). (A) pLM01 (GFP control). (B) pLM02 (BSP-GFP). (C) pLM03, (BSP-LT-B::GFP). (D) pLM08 (ZSP-LT-B::GFP). (E) pLM09 (LT-B::GFP). Green channel corresponds to GFP signal. Red channels (presented in magenta color) corresponds to ER-cherry signal. Merged images are also presented. Organelle labelling: cytosol (cy), nucleus (nu), endoplasmic reticulum (er). Bars=10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig6: Co-localization experiments in Arabidopsis root protoplasts. Protoplasts were co-transformed using the constructs presented in Fig. 1 and an ER marker protein fused to RFP, ER cherry (Nelson et al., 2007). (A) pLM01 (GFP control). (B) pLM02 (BSP-GFP). (C) pLM03, (BSP-LT-B::GFP). (D) pLM08 (ZSP-LT-B::GFP). (E) pLM09 (LT-B::GFP). Green channel corresponds to GFP signal. Red channels (presented in magenta color) corresponds to ER-cherry signal. Merged images are also presented. Organelle labelling: cytosol (cy), nucleus (nu), endoplasmic reticulum (er). Bars=10 μm.
Mentions: Figure 6 shows the co-localization experiments for the confirmation of subcellular localization using a known ER marker (Nelson et al., 2007). This marker (ER cherry) contains a red fluorescent protein fused to the signal peptide of the Arabidopsis thaliana wall-associated kinase 2 (He et al., 1999) and an HDEL motif for retention in the ER (Munro and Pelham, 1987). Arabidopsis root protoplasts were co-transformed with the ER cherry marker construct and the various constructs listed in Fig. 1. The results show that the ER-targeted RFP signals co-localized with the GFP signals from the constructs that contained either BSP (Fig. 6B, C) or ZSP (Fig. 6D). By contrast, GFP signals from constructs carrying GFP alone (Fig. 6A) or LT-B::GFP fusion (Fig. 6E) with no signal peptide were constantly detected in separate subcellular compartments than ER-targeted RFP signal.

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