A cyclic lipopeptide produced by an antagonistic bacterium relies on its tail and TRP-type Ca2+ channels to immobilize a green alga.

The antagonistic bacterium Pseudomonas protegens secretes the cyclic lipopeptide (CLiP) orfamide A, which triggers a Ca2+ signal causing rapid deflagellation of the microalga Chlamydomonas reinhardtii. We performed chemical synthesis of orfamide A variants and used an aequorin reporter line to measure their Ca2+ responses. Immobilization of algae was studied using a modulator and mutants of TRP-type channels. By investigating targeted synthetic orfamide A derivatives, we found that N-terminal amino acids of the linear part as well as the terminal fatty acid region are important for the specificity of the Ca2+ -signal causing deflagellation. Molecular editing indicates that at least two distinct Ca2+ -signaling pathways are triggered. One is involved in deflagellation (Thr3 change, fatty acid tail shortened by 4C), whereas the other still causes an increase in cytosolic Ca2+ in the algal cells, but does not cause substantial deflagellation (Leu1 change, fatty acid hydroxylation, fatty acid changes by 2C). Using mutants, we define four TRP-type channels that are involved in orfamide-A-signaling; only one (ADF1) responds additionally to low pH. These results suggest (i) that the linear part of the CLiP plays one major role in Ca2+ signaling and (ii) that orfamide A uses a network of algal TRP-type channels for deflagellation.