Expression of recombinant aequorin as an intracellular calcium reporter in the phytopathogenic fungus Phyllosticta ampelicida.
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.
Shaw
B D
BD
Department of Botany, University of Georgia, Athens, Georgia 30602, USA.
Kozlova
O
O
Read
N D
ND
Turgeon
B G
BG
Hoch
H C
HC
D000331:Aequorin / Q000096:biosynthesis* / Q000235:genetics; D002118:Calcium / Q000378:metabolism*; D008163:Luminescent Measurements; D003904:Mitosporic Fungi / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity; D015336:Molecular Probe Techniques; D011994:Recombinant Proteins / Q000096:biosynthesis*; D019076:Transgenes
D011994:Recombinant Proteins; D000331:Aequorin; D002118:Calcium