Distribution and identification of molecular interactions between tomato roots and bacterial biofilms

Conference (2012, September 05)

Some non pathogenic microorganisms evolving in the root micro-environment can trigger a positive effect on plant, increasing host defense against disease or/and directly inhibiting growth of pathogen in ... [more ▼]

Some non pathogenic microorganisms evolving in the root micro-environment can trigger a positive effect on plant, increasing host defense against disease or/and directly inhibiting growth of pathogen in soil (1). To initiate both phenomena leading to biocontrol activity, microorganisms use plant exudates to grow on roots and to produce in-situ active compounds. In Bacilli, cyclic lipopeptides of the surfactin, iturin and fengycin families represent important antibiotics involved in biocontrol (2). Recent studies in microbiology allowed a better understanding of plant microorganism interactions but few has been done at the molecular level. In this study, MALDI MS imaging has been used to study the nature of the secreted lipopeptide molecules, their relative quantity and their distribution in the root’s environment. Disinfected tomato seeds were first germinated at 28°C in sterile conditions for germination. Seedlings were then placed in Petri dish on ITO glass slide recovered with a thin layer of plant nutritive solution (Hoagland) containing 1,75% of agar and treated with freshly-grown cells of Bacillus amyloliquefaciens S499. Petri dishes were finally incubated vertically in phytotron at 28°C with a 16h photoperiod. Different root age / time of incubation were studied: 13 / 3; 13 / 7; 21 / 14 and 39 / 32. Control tomato root (without bacterial treatment) of the same ages were also analyzed (13 / 0; 21 / 0 and 42 / 0. For MALDI imaging experiments, the ITO slide was removed from the agar and dried in a dessiccator under vacuum. The matrix solution (α-cyano-hydroxycinnamic acid, 5mg/mL in ACN/0.2% TFA 70/30) was applied with an ImagePrep automated sprayer (Bruker Daltonics). An UltraFlex II TOF/TOF and a Solarix FT-ICR mass spectrometers were 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. The relative intensity of lipopeptides families varied with respect to the age of the root/biofilm system. In the 13/3 system, 3 homologues of surfactins were essentially detected (C13, C14 and C15), with very few iturins and fengycins. Their localizations were identical, whatever the considered homologue. Then the production of iturin and fengycin families increases in older systems (13/7 and 21/14) and a novel homologue of surfactin is detected (C12). Some variations in localizations within families may be observed (around the root or at the close vicinity of it in function of the considered homologue or alkali adduct). Then for the oldest system we studied, iturins and fengycins are not detected anymore and the localization of surfactins is less precise. In the 39/32 system, we also detected unknown compounds at 986.6, 1000.6, 1014.7 and 1028.7 m/z. The mass range of these compounds allied to the mass difference between two consecutive ion peaks let us think that these unknown compounds could be a new lipopeptide family. Investigations are in progress to identify these new secondary metabolites of Bacillus amyloliquefaciens. [less ▲]

Effect of environmental factors on the interaction plant-pathogen-Bacillus amyloliquefaciens S499

in Bulletin OILB/SROP = IOBC/WPRS Bulletin (2012), sous presse

The attachment of Bacillus amyloliquefaciens FZB42 to tomato roots is impaired in a mutant devoid of GalM and PBP4a.

Poster (2011, June 20)

In vivo experiments on tomato (Lycopersicon esculentum) roots have been performed: drench of 10-day hydroponic cultures with bacterial suspensions. A second identical treatment was applied one week later ... [more ▼]

In vivo experiments on tomato (Lycopersicon esculentum) roots have been performed: drench of 10-day hydroponic cultures with bacterial suspensions. A second identical treatment was applied one week later and after 30 days of culture, the plants were collected and the bacteria detached from a precise weight of roots were diluted, plated and counted. The mutant devoid of both GalM and PBP4a (encoded by the galM and dacC operon) is affected in its capacity to colonize the tomato roots. [less ▲]

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

Conference (2011, June 06)

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 ... [more ▼]

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. [less ▲]

The bacterial lipopeptide surfactin targets the lipid fraction of plasma membrane to trigger immune-related defense response in the host plant

in Cellular Microbiology (2011), 13

Differential roles of lipopeptides in plant host defenses and pathogen suppression.

in Phytopathology (2010), 100

Insights into the plant defense mechanisms induced by Bacillus lipopeptides.

Poster (2009, July)

Bacillus lipopeptides as MAMPs for non-pathogenic bacteria perception and defense responses elicitation in plant cells.

in Bulletin OILB/SROP = IOBC/WPRS Bulletin (2009), 43

Insights into the Defense-Related Events Occuring in Plant Cells Following Perception of Surfactin-Type Lipopeptide from Bacillus subtilis

in Molecular Plant-Microbe Interactions [=MPMI] (2009), 22(4), 456-468

Multiple strains of Bacillus subtilis were demonstrated to stimulate plant defense responses, and cyclic lipopeptides may be involved in the elicitation of this induced systemic resistance phenomenon ... [more ▼]

Multiple strains of Bacillus subtilis were demonstrated to stimulate plant defense responses, and cyclic lipopeptides may be involved in the elicitation of this induced systemic resistance phenomenon. Here, we further investigated molecular events underlying the interaction between wuch lipopeptides and plant cells. Addition of surfactin but not fengycin or iturin in the micromolar range to tobacco cell suspensions induced defense-related early events such as extracellular medium alkalinization coupled with ion fluxes and reactive oxygen species production. Surfactin also stimulated the defense enzymes phenylalanine ammonia lyase and lipoxygenase and modified the pattern of phenolics produced by the elicited cells. The occurence of these surfactin-elicited early events is closely related to Ca2+ influx and dynamic changes in protein phosphorylation but is not associed with any marked phytotoxicity or adverse effect on the integrity and growth potential of the treated tobacco cells. [less ▲]

Defence responses induced in tobacco cells by Bacillus lipopeptides acting as elicitors of systemic resistance in plants

Poster (2008, August)