Spatiotemporal monitoring of the antibiome secreted by Bacillus biofilms on plant roots using MALDI mass spectrometry imaging.

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.

The bacterial lipopeptide surfactin targets the lipid fraction of the plant plasma membrane to trigger immune-related defence responses.

The lipopeptide surfactin secreted by plant-beneficial bacilli has crucial biological functions among which the ability to stimulate immune-related 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.

Isolation of an N-alkylated benzylamine derivative from Pseudomonas putida BTP1 as elicitor of induced systemic resistance in bean.

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.

Plant polysaccharides initiate underground crosstalk with bacilli by inducing synthesis of the immunogenic lipopeptide surfactin.

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.

Limited impact of abiotic stress on surfactin production in planta and on disease resistance induced by Bacillus amyloliquefaciens S499 in tomato and bean.

Understanding how temperature and water stress affect protocooperation between plants and beneficial rhizobacteria may enhance the efficacy of biocontrol agents in reducing plant diseases. However, little is known about the impact of these factors on biocontrol mechanisms and effectiveness, especially when provided by beneficial Bacillus spp. This work aimed to evaluate the influence of low/high temperature combined with a normal and reduced water regime on the interaction between Bacillus amyloliquefaciens strain S499 and plants, resulting in the induction of systemic resistance (ISR). A reduction in ISR level was observed when plants were subjected to stress before bacterization; however, root treatment with S499 prior to stress exposure attenuated this negative effect. Colonization of S499 during exposure to temperature/water stress allowed the three crops to conserve their overall ability to mount defense lines to a similar degree at all the temperatures tested. Further investigation revealed that relative production of surfactin by S499 was clearly enhanced at low temperature, making it possible to counter-balance the negative effect on traits associated with rhizosphere fitness (colonization, motility, and biofilm formation) observed in vitro in cold conditions. This work thus represents a first step in deciphering the effect of high/low temperatures and/or drought on key plant-microorganism interactions culminating in ISR.

Peptidomic comparison and characterization of the major components of the venom of the giant ant Dinoponera quadriceps collected in four different areas of Brazil.

Despite the noxious effects inflicted by Dinoponera ant's envenomation, the information about the biological properties and composition of their venom is still very limited. Ants from the genus Dinoponera are believed to be the world's largest living ants with a body length of 3cm. Their occurrence is restricted to tropical areas of South America. In this work, we study the venom of the giant Dinoponera quadriceps ant collected in 4 different regions of Brazil. By using a combination of complementary mass spectrometric approaches, we aim at: (i) characterizing the venom composition of these ants; (ii) establishing a comparative analysis of the venom from four geographically different regions in Brazil. This approach demonstrates that ant venom is a copious source of new compounds. Several peptides were identified and selected for "de novo sequencing". Since most of the new peptides showed similarities with antimicrobial peptides (AMPs), antimicrobial assays were performed with the purpose of evaluating their activity. In regard to the comparative study of the four regions, we observed not only major differences in the venom compositions, but also that the venoms collected in closest areas are more similar than the ones collected in distant regions. These observations seem to highlight an adaption of the ant venoms to the local environment. Concerning the biological assays, the peptides called Dq-3162 and Da-3177 showed a wide-ranging antimicrobial activity. The characterization of new AMPs with a broad spectrum of activity and different scaffolds may aid scientists to design new therapeutic agents and understand the mechanisms of those peptides to interact with microbial membranes. The results obtained betoken the biotechnological potential of ant's venom. BIOLOGICAL SIGNIFICANCE: For the first time this manuscript describes an extensive proteomics characterization of the D. quadriceps venom. In addition this study reports the variation in venom composition of primitive ants from 4 geographically different areas of Brazil. The results reveal the presence of ~335 compounds for each venom/area and inter-colony variations were observed. 16 new peptides were characterized and 2 of them were synthesized and biologically assayed. These findings highlight the considerable and still unexplored diversity of ant's venom which could be used as valuable research tools in different areas of knowledge.

Amino acids, iron, and growth rate as key factors influencing production of the Pseudomonas putida BTP1 benzylamine derivative involved in systemic resistance induction in different plants.

The biological control bacterium Pseudomonas putida BTP1 exerts its protective effect mostly by inducing an enhanced state of resistance in the host plant against pathogen attack [induced systemic resistance (ISR)]. We previously reported that a specific compound derived from benzylamine may be involved in the elicitation of the ISR phenomenon by this Pseudomonas strain. In this article, we provide further information about the N,N-dimethyl-N-tetradecyl-N-benzylammonium structure of this determinant for ISR and show that the benzylamine moiety may be important for perception of the molecule by root cells of different plant species. We also investigated some regulatory aspects of elicitor production with the global aim to better understand how in situ expression of these ISR elicitors can be modulated by physiological and environmental factors. The biosynthesis is clearly related to secondary metabolism, and chemostat experiments showed that the molecule is more efficiently produced at low cell growth rate. Interestingly, the presence of free amino acids in the environment is necessary for optimal production, and a specific positive effect of phenylalanine was evidenced in pulsed continuous cultures. The influence of other abiotic factors, such as mineral content, oxygen concentration, or pH, on elicitor production is also reported and discussed with respect to the specific conditions that the producing strain undergoes in the rhizosphere environment.

Surfactin and fengycin lipopeptides of Bacillus subtilis as elicitors of induced systemic resistance in plants.

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.

Bacillus subtilis M4 decreases plant susceptibility towards fungal pathogens by increasing host resistance associated with differential gene expression.

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.