A GFP-based assay reveals a role for RHD3 in transport between the endoplasmic reticulum and Golgi apparatus

Authors

  • Huanquan Zheng,

    1. Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, UK,
    2. Research School of Biological & Molecular Sciences, Oxford Brookes University, Gipsy Lane, Oxford OX3 OBP, UK, and
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  • Ljerka Kunst,

    1. Department of Botany, University of British Columbia, 3529-6270 University Boulevard, Vancouver, BC, Canada V6T 1Z4
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  • Chris Hawes,

    1. Research School of Biological & Molecular Sciences, Oxford Brookes University, Gipsy Lane, Oxford OX3 OBP, UK, and
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  • Ian Moore

    Corresponding author
    1. Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, UK,
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For correspondence (fax +44 1865 275074; e-mail ian.moore@plants.ox.ac.uk).

Present address: Department of Botany, University of British Columbia, 3529-6270 University Boulevard, Vancouver, BC, Canada V6T 1Z4.

Summary

We describe the use of a secreted form of Aequoria victoria green fluorescent protein (secGFP) in a non-invasive live cell assay of membrane traffic in Arabidopsis thaliana. We show that in comparison to GFP-HDEL, which accumulates in the endoplasmic reticulum (ER), secGFP generates a weak fluorescence signal when transported to the apoplast. The fluorescence of secGFP in the apoplast can be increased by growth of seedlings on culture medium buffered at pH 8.1, suggesting that apoplastic pH is responsible, at least in part, for the low fluorescence intensity of seedlings expressing secGFP. Inhibition of secGFP transport between the ER and plasma membrane (PM), either by Brefeldin A (BFA) treatment or by genetic intervention results in increased intracellular secGFP accumulation accompanied by an increase in the secGFP fluorescence intensity. secGFP thus provides a valuable tool for forward and reverse genetic analysis of membrane traffic and endomembrane organisation in Arabidopsis. Using this assay for quantitative sublethal perturbation of secGFP transport, we identify a role for root hair defective 3 (RHD3) in transport of secreted and Golgi markers between the ER and the Golgi apparatus.

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