Expression of recombinant aequorin as an intracellular calcium reporter in the phytopathogenic fungus Phyllosticta ampelicida.

Abstract

Conidia of Phyllosticta ampelicida germinate only after they have made contact with a substratum. Previous work has shown that external free calcium must be available to the spore for germination to be initiated. Transgenic strains of P. ampelicida expressing apo-aequorin, a calcium-sensitive luminescent protein, were developed to monitor cytoplasmic free Ca(2+) ([Ca(2+)]c). Transformants were verified by PCR and Southern hybridization. Apo-aequorin production was quantified for each of 21 transformants. The transformant that emitted the most light per unit of protein was found to contain 0.59 mg apo-aequorin/g total protein. To ascertain the feasibility of aequorin-based [Ca(2+)]c quantification, [Ca(2+)]c changes were measured in mycelia during various physiologically perturbing treatments: exposure to high concentrations of external Ca(2+), hypoosmotic shock, and mechanical perturbation. This is the first report of a plant pathogenic fungus for which aequorin-based Ca(2+) measurement protocols have been developed.

• Authors: B D Shaw, O Kozlova, N D Read, B G Turgeon, H C Hoch
• Journal: Fungal genetics and biology : FG & B (Fungal Genet Biol) Vol. 34 Issue 3 Pg. 207-15 (Dec 2001) ISSN: 1087-1845 [Print] United States
• PMID: 11728158 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright 2001 Academic Press.

Chemical References

• Recombinant Proteins
• Aequorin
• Calcium

Topics

• Aequorin (biosynthesis, genetics)
• Calcium (metabolism)
• Luminescent Measurements
• Mitosporic Fungi (genetics, metabolism, pathogenicity)
• Molecular Probe Techniques
• Recombinant Proteins (biosynthesis)
• Transgenes