MALDI mass spectrometry imaging of secreted lipopeptides in a bacterial biofilm colonizing plant roots

During the aggression of a plant by a pathogen, different immune reactions may occur. "Induced Systemic Resistance” (ISR) is triggered by the specific interaction between plant and non-pathogenic microorganism. The first step (of three) consists in the perception by plant cells of elicitors produced by the inducing agents that initiates the phenomenon. One class of known elicitors is antibiotics including surfactin- and fengycin-type lipopeptides. Recent studies in biology, genetics or biochemistry allowed a better understanding of the interactions between plants and microorganism but few has been done at the molecular level. MALDI MS imaging has been used to study the nature of the secreted lipopeptides, their relative quantity and their distribution in the root’s environment. Disinfected tomato seeds were first incubated at 28°C in sterile conditions for germination. Germinated seeds were then treated with freshly-grown cells of Bacillus amyloliquefaciens S499 and placed in Petri dish on ITO glass slide recovered with a thin layer of plant nutritive solution (Hoagland) containing 1,75% of agar. Petri dishes were finally incubated vertically in phytotron during 10 days (28°C, photoperiod 16h).
For MALDI imaging experiments, the ITO slide was removed from the agar and dried in a dessiccator under vacuum. The matrix solution (9-aminoacridine) was applied with an ImagePrep automated sprayer (Bruker Daltonics). An UltraFlex II TOF/TOF mass spectrometer was used to record molecular cartographies.

The average mass spectra recorded around the tomato root (2-3 mm on both sides of the root) showed that lipopeptides were major compounds detected on the agar. Only the surfactins have been detected when working with the S499 strain. The most abundant surfactins were those with longer fatty acyl chain lengths, such as C14- and C15-homologues. Such a surfactin signature is interesting since homologues with the longest acyl chains are also the more active biologically. The distribution of surfactins showed a gradient representing the diffusion of the molecules during the root growth. The more the fatty acyl chain is long, the more the surfactin is detected near the root. Other compounds detected during the analysis showed a clear anti-colocalization with the surfactins.
Future work will be focused on the influence of the plant species (tobacco, salad, Arabidopsis thaliana) on the secretion of lipopeptides (type, concentration…) and the influence of the strain of Bacillus amyloliquefaciens regarding its ability to selectively produce specific lipopeptide families (overproducing or repressed mutants). This MS imaging technique thus appears to be a very powerful method to study in situ production of bioactive lipopeptides by bacteria developing on roots. This is crucial for a better understanding of the molecular dialogue governing perception of beneficial Bacillus strains by the host plant.
This study provides a first analysis over a long root section of lipopeptides secreted by a bacterial biofilm colonizing plant.