Fluorescent probes to study the perception of the elicitor surfactin at the plant plasma membrane

Induced systemic resistance triggered by beneficial bacteria is a promising solution to reduce the use of chemical pesticides in agriculture. These bacteria stimulate plant immunity through the production of small secondary metabolites, reducing the incidence of pathogen infection.
The lipopeptide surfactin (SRF) produced by plant beneficial bacilli is one of these metabolites stimulating plant immunity. Results have shown a sensing mechanism independent of protein pattern-recognition receptors commonly known for plant elicitors. As SRF can insert into the lipid fraction of plant plasma membrane (PM), its perception by plant was assumed to be a lipid-driven process but the detailed sensing mechanism is not yet understood. We hypothesize that SRF disturbs physical properties and consequently activates PM proteins sensitive to membrane mechanics.
Putative sensors of change in membrane mechanics are mechanosensitive (MS) ionic channels, which transduce change in membrane tension due to mechanical stimuli into ion fluxes. Among plant MS channels, the Mid1-Complementing Activity (MCA) channels and MscS-like (MSL) channels are two families reported in plant PM.
To test our hypothesis, we evaluated the disturbance of membrane mechanics by SRF through membrane fluidity measurements on root protoplasts of Arabidopsis thaliana stained with the fluorescent probe Laurdan. The results showed a decrease in membrane fluidity in presence of SRF. Then, analyses of cytosolic calcium variation using Fluo4 in root protoplasts from wild type plants, MCA- and MSL-depleted mutant showed a lower calcium influx under SRF treatment in the two mutants.
Altogether, our results are in accordance with an SRF eliciting mechanism triggered through a PM mechanical disturbance activating MS channels. To go deeper into details of the mechanism, visualisation of membrane domains with the proper fluorescent probes and their colocalization with MS channels is envisaged.