Calcium imaging in filamentous fungi using recombinant chamaeleon FRET technology Calcium signalling plays a central role in signal transduction in filamentous fungi but we know very little about this important process in these organisms. My project involves developing the recombinant cameleon probe technology to image and measure calcium dynamics in cells of filamentous fungi (Cochliobolus, Aspergillus and Neurospora). The chamaeleon probe is a chimeric protein composed of CFP, calmodulin, a peptide linker and YFP. On binding to calcium CFP and YFP can undergo FRET and the amount of FRET is dependent on the calcium concentration. I will be determining the distribution of the chamaeleon probe in the cell, optimising its expression and optimising imaging and measurement protocols before performing experiments to analyse the role of calcium in relation to the following questions:

• Is there a tip-focused calcium gradient within growing hyphal tips (of Neurospora and Aspergillus) which is involved in regulating cell polarity in much the same way as it does in pollen tubes?
• Are there transient increases in calcium associated with spore germination and infection structure (in Cochliobolus) indicating a role for calcium in regualting these processes?