Multiple strains of Bacillus spp. were demonstrated to stimulate plant defence responses. However, very little is known about the nature of molecular determinants secreted by these Gram-positive bacteria that are responsible for the elicitation of the induced systemic resistance (ISR) phenomenon. This study shows that the lipopeptides surfactins and fengycins may be involved in this elicitation process. In bean, pure fengycins and surfactins provided a significant ISR-mediated protective effect on bean plants, similar to the one induced by living cells of the producing strain S499. Moreover, experiments conducted on bean and tomato plants showed that overexpression of both surfactin and fengycin biosynthetic genes in the naturally poor producer Bacillus subtilis strain 168 was associated with a significant increase in the potential of the derivatives to induce resistance. In tomato cells, key enzymes of the lipoxygenase pathway appeared to be activated in resistant plants following induction by lipopeptide overproducers. To our knowledge, such lipopeptides constitute a novel class of compounds from non-pathogenic bacteria that can be perceived by plant cells as signals to initiate defence mechanisms.

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 such 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 occurrence of these surfactin-elicited early events is closely related to Ca(2+) influx and dynamic changes in protein phosphorylation but is not associated with any marked phytotoxicity or adverse effect on the integrity and growth potential of the treated tobacco cells. Reduced activity of some homologues also indicates that surfactin perception is dictated by structural clues in both the acyl moiety and cyclic peptide part. Our results suggest that these molecules could interact without irreversible pore formation but in a way sufficient to induce disturbance or transient channeling in the plasma membrane that can, in turn, activate a biochemical cascade of molecular events leading to defensive responses. The present study sheds new light not only on defense-related events induced following recognition of amphiphilic lipopeptides from Bacillus spp. but also more globally on the way elicitors from beneficial bacteria can be perceived by host plant cells.

SummaryThe lipopeptide surfactin secreted by plantbeneficial bacilli has crucial biological functions among which the ability to stimulate immunerelated responses in host tissues. This phenomenon is important for biological control of plant diseases but its molecular basis is still poorly understood. In this work, we used various approaches to study the mechanism governing the perception of this biosurfactant at the plant cell surface. Combining data on oxidative burst induction in tobacco cells, structure/activity relationship, competitive inhibition, insertion kinetics within plant membranes and thermodynamic determination of binding parameters on model membranes globally indicates that surfactin perception relies on a lipid-driven process at the plasma membrane level. Such a sensor role of the lipid bilayer is quite uncommon considering that plant basal immunity is usually triggered upon recognition of microbial molecular patterns by high-affinity proteic receptors.

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Results presented in this paper describe the ability of Bacillus subtilis strain M4 to reduce disease incidence caused by Colletotrichum lagenarium and Pythium aphanidermatum on cucumber and tomato, respectively. Disease protection in both pathosystems was most probably due to induction of resistance in the host plant since experiments were designed in order to avoid any direct contact between the biocontrol agent and the pathogen. Pre-inoculation with strain M4 thus sensitised both plants to react more efficiently to subsequent pathogen infection. In cucumber, the use of endospores provided a disease control level similar to that obtained with vegetative cells. In contrast, a mixture of lipopeptides from the surfactin, iturin and fengycin families showed no resistance-inducing potential. Interestingly, treatment with strain M4 was also associated with significant changes in gene transcription in the host plant as revealed by cDNA-AFLP analyses. Several AFLP fragments corresponded to genes not expressed in control plants and specifically induced by the Bacillus treatment. In support to the macroscopic protective effect, this differential accumulation of mRNA also illustrates the plant reaction following perception of strain M4, and constitutes one of the very first examples of defence-associated modifications at the transcriptional level elicited by a non-pathogenic bacterium in a host plant.

Some soil Bacilli living in association with plant roots can protect their host from infection by pathogenic microbes and are therefore being developed as biological agents to control plant diseases. The plant-protective activity of these bacteria has been correlated with the potential to secrete a wide array of antibiotic compounds upon growth as planktonic cells in isolated cultures under laboratory conditions. However, in situ expression of these antibiotics in the rhizosphere where bacterial cells naturally colonize root tissues is still poorly understood. In this work, we used matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) to examine spatiotemporal changes in the secreted antibiome of Bacillus amyloliquefaciens developing as biofilms on roots. Nonribosomal lipopeptides such as the plant immunity elicitor surfactin or the highly fungitoxic iturins and fengycins were readily produced albeit in different time frames and quantities in the surrounding medium. Interestingly, tandem mass spectrometry (MS/MS) experiments performed directly from the gelified culture medium also allowed us to identify a new variant of surfactins released at later time points. However, no other bioactive compounds such as polyketides were detected at any time, strongly suggesting that the antibiome expressed in planta by B. amyloliquefaciens does not reflect the vast genetic arsenal devoted to the formation of such compounds. This first dynamic study reveals the power of MALDI MSI as tool to identify and map antibiotics synthesized by root-associated bacteria and, more generally, to investigate plant-microbe interactions at the molecular level.

Some plant-associated bacteria such as Bacillus sp. can protect their host from pathogen ingress and this biocontrol activity correlates with their potential to form multiple antibiotics upon in vitro growth. However, our knowledge on antibiotic production by soil bacilli evolving on roots in natural conditions is still limited. In this work, antibiome imaging first revealed that the lipopeptide surfactin is the main bacterial ingredient produced in planta within the first hours of interaction with root tissues. We further demonstrated that surfactin synthesis is specifically stimulated upon perception of plant cell wall polymers such as xylan or arabinogalactan, leading to fast accumulation of micromolar amounts in the root environment. At such concentrations, the lipopeptide may not only favour the ecological fitness of the producing strain in term of root colonization, but also triggers systemic resistance in the host plant. This surfactin-induced immunity primes the plant to better resist further pathogen ingress, and involves only limited expression of defence-related molecular events and does not provoke seedling growth inhibition. By contrast with the strong response mounted upon perception of pathogens, this strongly attenuated defensive reaction induced by surfactin in plant tissues should help Bacillus to be tolerated as saprophytic partner by its host.

Root treatment of Phaseolus vulgaris with the nonpathogenic Pseudomonas putida BTP1 led to significant reduction of the disease caused by the pathogen Botrytis cinerea on leaves. The molecular determinant of P. putida BTP1 mainly responsible for the induced systemic resistance (ISR) was isolated from cell-free culture fluid after growth of the strain in the iron-poor casamino acid medium. Mass spectrometry analyses performed on both the bacterial product and synthetic analogues revealed a polyalkylated benzylamine structure, with the quaternary ammonium substituted by methyl, ethyl, and C13 aliphatic groups responsible for the relative hydrophobicity of the molecule. The specific involvement of the N-alkylated benzylamine derivative (NABD) in ISR elicitation was first evidenced by testing the purified compound that mimicked the protective effect afforded by crude supernatant samples. The evidence was supported by the loss of elicitor activity of mutants impaired in NABD biosynthesis. Our experiments also showed that other iron-regulated metabolites secreted by the strain are not involved in ISR stimulation. Thus, these results indicate a wider variety of Pseudomonas determinants for ISR than reported to date.