Biofilm-forming Ability of Bacillus thuringiensis Strains from Biopesticides on Polystyrene and their Attachment on Spinach.

Bacillus thuringiensis-based commercial products as a biopesticide have been used for more than 60 years in agriculture. However, as one of the species in B. cereus group, B. thuringiensis has been considered as an emerging hazard with the potential to cause food toxico-infections. The present study aimed to evaluate the biofilm-forming ability of B. thuringiensis biopesticide strains and their attachment on spinach, compared to foodborne B. cereus strains. Biofilm formations of tested strains were found to be strain-specific and affected by the nutrient conditions more than the incubation time. Nutrient starvation conditions generally reduced the biofilm formation of tested B. thuringiensis and B. cereus strains, particularly B. thuringiensis ABTS-1857 strain was found as the nonbiofilm former in starvation conditions. It is worth mentioning that B. thuringiensis SA-11 strain showed stronger biofilm-forming ability with more air-liquid interface biofilm than the other two B. thuringiensis biopesticide strains, but no such higher attachment of B. thuringiensis SA-11 to spinach was observed. These results indicate that B. thuringiensis SA-11 strain can enter the food processing lines by the attachment on spinach leaves, and it has the potential to form biofilms throughout the processing lines or the production environment when sufficient nutrients are available. However, more biofilm tests of B. thuringiensis biopesticide strains in the vegetable production chain should be performed. The dry formulation of commercial B. thuringiensis biopesticides enhanced their adhesion on spinach leaves, whereas the strength of adhesion was not improved by the formulation. In addition, 1-2 log reductions of spores after the intensive washing of spinach leaves in the lab were detected. However, the log reduction due to the actual washing done by the food processing companies in large-volume washing baths or by consumers at home would be limited and less than this lab simulation.

Breaking Cycles: Saponification-Enhanced NMR Fingerprint Matching for the Identification and Stereochemical Evaluation of Cyclic Lipodepsipeptides from Natural Sources.

We previously described NMR based fingerprint matching with peptide backbone resonances as a fast and reliable structural dereplication approach for Pseudomonas cyclic lipodepsipeptides (CLiPs). In combination with total synthesis of a small library of configurational CLiP congeners this also allows unambiguous determination of stereochemistry, facilitating structure-activity relationship studies and enabling three-dimensional structure determination. However, the on-resin macrocycle formation in the synthetic workflow brings considerable burden and limits universal applicability. This drawback is here removed altogether by also transforming the native CLiP into a linearized analogue by controlled saponification of the ester bond. This eliminates the need for macrocycle formation, limiting the synthesis effort to linear peptide analogues. NMR fingerprints of such linear peptide analogues display a sufficiently distinctive chemical shift fingerprint to act as effective discriminators. The approach is developed using viscosin group CLiPs and subsequently demonstrated on putisolvin, leading to a structural revision, and tanniamide from Pseudomonas ekonensis COR58, a newly isolated lipododecapeptide that defines a new group characterized by a ten-residue large macrocycle, the largest to date in the Pseudomonas CLiP portfolio. These examples demonstrate the effectiveness of the saponification- enhanced approach that broadens applicability of NMR fingerprint matching for the determination of the stereochemistry of CLiPs.

Does regulation hold the key to optimizing lipopeptide production in Pseudomonas for biotechnology?

Lipopeptides (LPs) produced by Pseudomonas spp. are specialized metabolites with diverse structures and functions, including powerful biosurfactant and antimicrobial properties. Despite their enormous potential in environmental and industrial biotechnology, low yield and high production cost limit their practical use. While genome mining and functional genomics have identified a multitude of LP biosynthetic gene clusters, the regulatory mechanisms underlying their biosynthesis remain poorly understood. We propose that regulation holds the key to unlocking LP production in Pseudomonas for biotechnology. In this review, we summarize the structure and function of Pseudomonas-derived LPs and describe the molecular basis for their biosynthesis and regulation. We examine the global and specific regulator-driven mechanisms controlling LP synthesis including the influence of environmental signals. Understanding LP regulation is key to modulating production of these valuable compounds, both quantitatively and qualitatively, for industrial and environmental biotechnology.

The phenylalanine ammonia-lyase inhibitor AIP induces rice defence against the root-knot nematode Meloidogyne graminicola.

The phenylalanine ammonia-lyase (PAL) enzyme catalyses the conversion of l-phenylalanine to trans-cinnamic acid. This conversion is the first step in phenylpropanoid biosynthesis in plants. The phenylpropanoid pathway produces diverse plant metabolites that play essential roles in various processes, including structural support and defence. Previous studies have shown that mutation of the PAL genes enhances disease susceptibility. Here, we investigated the functions of the rice PAL genes using 2-aminoindan-2-phosphonic acid (AIP), a strong competitive inhibitor of PAL enzymes. We show that the application of AIP can significantly reduce the PAL activity of rice crude protein extracts in vitro. However, when AIP was applied to intact rice plants, it reduced infection of the root-knot nematode Meloidogyne graminicola. RNA-seq showed that AIP treatment resulted in a rapid but transient upregulation of defence-related genes in roots. Moreover, targeted metabolomics demonstrated higher levels of jasmonates and antimicrobial flavonoids and diterpenoids accumulating after AIP treatment. Furthermore, chemical inhibition of the jasmonate pathway abolished the effect of AIP on nematode infection. Our results show that disturbance of the phenylpropanoid pathway by the PAL inhibitor AIP induces defence in rice against M. graminicola by activating jasmonate-mediated defence.

Strigolactone deficiency induces jasmonate, sugar and flavonoid phytoalexin accumulation enhancing rice defense against the blast fungus Pyricularia oryzae.

Strigolactones (SLs) are carotenoid-derived phytohormones that regulate plant growth and development. While root-secreted SLs are well-known to facilitate plant symbiosis with beneficial microbes, the role of SLs in plant interactions with pathogenic microbes remains largely unexplored. Using genetic and biochemical approaches, we demonstrate a negative role of SLs in rice (Oryza sativa) defense against the blast fungus Pyricularia oryzae (syn. Magnaporthe oryzae). We found that SL biosynthesis and perception mutants, and wild-type (WT) plants after chemical inhibition of SLs, were less susceptible to P. oryzae. Strigolactone deficiency also resulted in a higher accumulation of jasmonates, soluble sugars and flavonoid phytoalexins in rice leaves. Likewise, in response to P. oryzae infection, SL signaling was downregulated, while jasmonate and sugar content increased markedly. The jar1 mutant unable to synthesize jasmonoyl-l-isoleucine, and the coi1-18 RNAi line perturbed in jasmonate signaling, both accumulated lower levels of sugars. However, when WT seedlings were sprayed with glucose or sucrose, jasmonate accumulation increased, suggesting a reciprocal positive interplay between jasmonates and sugars. Finally, we showed that functional jasmonate signaling is necessary for SL deficiency to induce rice defense against P. oryzae. We conclude that a reduction in rice SL content reduces P. oryzae susceptibility by activating jasmonate and sugar signaling pathways, and flavonoid phytoalexin accumulation.

Detection of Fusarium oxysporum f.sp. lactucae race 1 and 4 via race-specific real-time PCR and target enrichment.

Fusarium oxysporum f.sp. lactucae (Fol) causes a vascular disease in lettuce that results in significant yield losses. Race-specific and sensitive real-time PCR assays were developed for Fol races 1 and 4, which are prevalent in Europe. Using genotyping-by-sequencing, unique DNA loci specific to each race were identified and subsequently used for the design of primers and hydrolysis probes. Two assays per race were developed to ensure specificity. The two assays of each race could be run in duplex format, while still giving a sensitivity of 100 fg genomic DNA for all assays. Sample preparation methods were developed for plant tissue, soil, and surfaces, with an extra enrichment step when additional sensitivity was required. By controlling the incubation conditions during the enrichment step, the real-time PCR signal could be matched to the number of spore equivalents in the original sample. When enriching naturally infested soil, down to six conidiospore equivalents L-1 soil could be detected. As enrichment ensures sensitive detection and focuses on living Fol propagules, it facilitates the evaluation of control measures. The developed detection methods for soil and surfaces were applied to samples from commercial lettuce farms and confirmed the prevalence of Fol race 4 in Belgium. Monitoring of soil disinfestation events revealed that despite a dramatic decrease in quantity, the pathogen could still be detected either immediately after sheet steaming or after harvesting the first new crop. The detection method for plant tissue was successfully used to quantify Fol in lettuce inoculated with race 1, race 4 or a combination of both. Under the temperature conditions used, race 4 was more aggressive than race 1, as reflected in larger amounts of DNA of race 4 detected in the roots. These newly developed assays are a promising tool for epidemiological research as well as for the evaluation of control measures.

Stereomeric Lipopeptides from a Single Non-Ribosomal Peptide Synthetase as an Additional Source of Structural and Functional Diversification in Pseudomonas Lipopeptide Biosynthesis.

In Pseudomonas lipopeptides, the D-configuration of amino acids is generated by dedicated, dual-function epimerization/condensation (E/C) domains. The increasing attention to stereochemistry in lipopeptide structure elucidation efforts has revealed multiple examples where epimerization does not occur, even though an E/C-type domain is present. While the origin of the idle epimerization in those E/C-domains remains elusive, epimerization activity has so far shown a binary profile: it is either 'on' (active) or 'off' (inactive). Here, we report the unprecedented observation of an E/C-domain that acts 'on and off', giving rise to the production of two diastereoisomeric lipopeptides by a single non-ribosomal peptide synthetase system. Using dereplication based on solid-phase peptide synthesis and NMR fingerprinting, we first show that the two cyclic lipopeptides produced by Pseudomonas entomophila COR5 correspond to entolysin A and B originally described for P. entomophila L48. Next, we prove that both are diastereoisomeric homologues differing only in the configuration of a single amino acid. This configurational variability is maintained in multiple Pseudomonas strains and typically occurs in a 3:2 ratio. Bioinformatic analysis reveals a possible correlation with the composition of the flanking sequence of the N-terminal secondary histidine motif characteristic for dual-function E/C-type domains. In permeabilization assays, using propidium iodide entolysin B has a higher antifungal activity compared to entolysin A against Botrytis cinerea and Pyricularia oryzae spores. The fact that configurational homologues are produced by the same NRPS system in a Pseudomonas strain adds a new level of structural and functional diversification to those already known from substrate flexibility during the recruitment of the amino acids and fatty acids and underscores the importance of complete stereochemical elucidation of non-ribosomal lipopeptide structures.

Molecular analysis of broad-spectrum induced resistance in rice by the green leaf volatile Z-3-hexenyl acetate.

Green leaf volatiles (GLVs), volatile organic compounds released by plants upon tissue damage, are key signaling molecules in plant immunity. The ability of exogenous GLV application to trigger an induced resistance (IR) phenotype against arthropod pests has been widely reported, but its effectiveness against plant pathogens is less well understood. In this study, we combined mRNA sequencing-based transcriptomics and phytohormone measurements with multispectral imaging-based precision phenotyping to gain insights into the molecular basis of Z-3-hexenyl acetate-induced resistance (Z-3-HAC-IR) in rice. Furthermore, we evaluated the efficacy of Z-3-HAC-IR against a panel of economically significant rice pathogens: Pyricularia oryzae, Rhizoctonia solani, Xanthomonas oryzae pv. oryzae, Cochliobolus miyabeanus, and Meloidogyne graminicola. Our data revealed rapid induction of jasmonate metabolism and systemic induction of plant immune responses upon Z-3-HAC exposure, as well as a transient allocation cost due to accelerated chlorophyll degradation and nutrient remobilization. Z-3-HAC-IR proved effective against all tested pathogens except for C. miyabeanus, including against the (hemi)biotrophs M. graminicola, X. oryzae pv. oryzae, and P. oryzae. The Z-3-HAC-IR phenotype was lost in the jasmonate (JA)-deficient hebiba mutant, which confirms the causal role of JA in Z-3-HAC-IR. Together, our results show that GLV exposure in rice induces broad-spectrum, JA-mediated disease resistance with limited allocation costs, and may thus be a promising alternative crop protection approach.

Overview and Management of the Most Common Eukaryotic Diseases of Flax (Linum usitatissimum).

Flax is an important crop cultivated for its seeds and fibers. It is widely grown in temperate regions, with an increase in cultivation areas for seed production (linseed) in the past 50 years and for fiber production (fiber flax) in the last decade. Among fiber-producing crops, fiber flax is the most valuable species. Linseed is the highest omega-3 oleaginous crop, and its consumption provides several benefits for animal and human health. However, flax production is impacted by various abiotic and biotic factors that affect yield and quality. Among biotic factors, eukaryotic diseases pose a significant threat to both seed production and fiber quality, which highlights the economic importance of controlling these diseases. This review focuses on the major eukaryotic diseases that affect flax in the field, describing the pathogens, their transmission modes and the associated plant symptoms. Moreover, this article aims to identify the challenges in disease management and provide future perspectives to overcome these biotic stresses in flax cultivation. By emphasizing the key diseases and their management, this review can aid in promoting sustainable and profitable flax production.

Prevalence, attachment ability and strength of the biological control agent Bacillus thuringiensis on tomato.

Bacillus thuringiensis (Bt) is commonly used as a biological control agent (BCA) to control insect pests in edible plant production and can as such be introduced into the food chain of fresh produce. Using standard food diagnostics Bt will be detected and reported as presumptive B. cereus. Tomato plants are often sprayed with Bt biopesticides for insect control, thus these Bt BCAs can also reach the tomato fruits and persist until consumption. In this study, vine tomatoes from the retail in Belgium (Flanders) were investigated for the occurrence and residual numbers of presumptive B. cereus and Bt. Of 109 tomato samples, 61 (56%) were tested positive for presumptive B. cereus. Of the presumptive B. cereus isolates (n = 213) recovered from these samples, 98% were identified as Bt by the production of parasporal crystals. Further quantitative real-time PCR assays on a subselection of Bt isolates (n = 61) showed that 95% of Bt isolates were indistinguishable from Bt biopesticide strains that are approved to be used on crops in the EU. Furthermore, the attachment strength of tested Bt biopesticide strains showed easier wash-off properties if using the commercial Bt granule formulation than the unformulated lab-cultured Bt or B. cereus spore suspensions.

The saponin bomb: a nucleolar-localized ß-glucosidase hydrolyzes triterpene saponins in Medicago truncatula.

Plants often protect themselves from their own bioactive defense metabolites by storing them in less active forms. Consequently, plants also need systems allowing correct spatiotemporal reactivation of such metabolites, for instance under pathogen or herbivore attack. Via co-expression analysis with public transcriptomes, we determined that the model legume Medicago truncatula has evolved a two-component system composed of a ß-glucosidase, denominated G1, and triterpene saponins, which are physically separated from each other in intact cells. G1 expression is root-specific, stress-inducible, and coregulated with that of the genes encoding the triterpene saponin biosynthetic enzymes. However, the G1 protein is stored in the nucleolus and is released and united with its typically vacuolar-stored substrates only upon tissue damage, partly mediated by the surfactant action of the saponins themselves. Subsequently, enzymatic removal of carbohydrate groups from the saponins creates a pool of metabolites with an increased broad-spectrum antimicrobial activity. The evolution of this defense system benefited from both the intrinsic condensation abilities of the enzyme and the bioactivity properties of its substrates. We dub this two-component system the saponin bomb, in analogy with the mustard oil and cyanide bombs, commonly used to describe the renowned ß-glucosidase-dependent defense systems for glucosinolates and cyanogenic glucosides.

Charting the Lipopeptidome of Nonpathogenic Pseudomonas.

A major source of pseudomonad-specialized metabolites is the nonribosomal peptide synthetases (NRPSs) assembling siderophores and lipopeptides. Cyclic lipopeptides (CLPs) of the Mycin and Peptin families are frequently associated with, but not restricted to, phytopathogenic species. We conducted an in silico analysis of the NRPSs encoded by lipopeptide biosynthetic gene clusters in nonpathogenic Pseudomonas genomes, covering 13 chemically diversified families. This global assessment of lipopeptide production capacity revealed it to be confined to the Pseudomonas fluorescens lineage, with most strains synthesizing a single type of CLP. Whereas certain lipopeptide families are specific for a taxonomic subgroup, others are found in distant groups. NRPS activation domain-guided peptide predictions enabled reliable family assignments, including identification of novel members. Focusing on the two most abundant lipopeptide families (Viscosin and Amphisin), a portion of their uncharted diversity was mapped, including characterization of two novel Amphisin family members (nepenthesin and oakridgin). Using NMR fingerprint matching, known Viscosin-family lipopeptides were identified in 15 (type) species spread across different taxonomic groups. A bifurcate genomic organization predominates among Viscosin-family producers and typifies Xantholysin-, Entolysin-, and Poaeamide-family producers but most families feature a single NRPS gene cluster embedded between cognate regulator and transporter genes. The strong correlation observed between NRPS system phylogeny and rpoD-based taxonomic affiliation indicates that much of the structural diversity is linked to speciation, providing few indications of horizontal gene transfer. The grouping of most NRPS systems in four superfamilies based on activation domain homology suggests extensive module dynamics driven by domain deletions, duplications, and exchanges. IMPORTANCE Pseudomonas species are prominent producers of lipopeptides that support proliferation in a multitude of environments and foster varied lifestyles. By genome mining of biosynthetic gene clusters (BGCs) with lipopeptide-specific organization, we mapped the global Pseudomonas lipopeptidome and linked its staggering diversity to taxonomy of the producers, belonging to different groups within the major Pseudomonas fluorescens lineage. Activation domain phylogeny of newly mined lipopeptide synthetases combined with previously characterized enzymes enabled assignment of predicted BGC products to specific lipopeptide families. In addition, novel peptide sequences were detected, showing the value of substrate specificity analysis for prioritization of BGCs for further characterization. NMR fingerprint matching proved an excellent tool to unequivocally identify multiple lipopeptides bioinformatically assigned to the Viscosin family, by far the most abundant one in Pseudomonas and with stereochemistry of all its current members elucidated. In-depth analysis of activation domains provided insight into mechanisms driving lipopeptide structural diversification.

Plant-associated Bacillus mobilizes its secondary metabolites upon perception of the siderophore pyochelin produced by a Pseudomonas competitor.

Bacillus velezensis is considered as model species for plant-associated bacilli providing benefits to its host such as protection against phytopathogens. This is mainly due to the potential to secrete a wide range of secondary metabolites with specific and complementary bioactivities. This metabolite arsenal has been quite well defined genetically and chemically but much remains to be explored regarding how it is expressed under natural conditions and notably how it can be modulated upon interspecies interactions in the competitive rhizosphere niche. Here, we show that B. velezensis can mobilize a substantial part of its metabolome upon the perception of Pseudomonas, as a soil-dwelling competitor. This metabolite response reflects a multimodal defensive strategy as it includes polyketides and the bacteriocin amylocyclicin, with broad antibiotic activity, as well as surfactin lipopeptides, contributing to biofilm formation and enhanced motility. Furthermore, we identified the secondary Pseudomonas siderophore pyochelin as an info-chemical, which triggers this response via a mechanism independent of iron stress. We hypothesize that B. velezensis relies on such chelator sensing to accurately identify competitors, illustrating a new facet of siderophore-mediated interactions beyond the concept of competition for iron and siderophore piracy. This phenomenon may thus represent a new component of the microbial conversations driving the behavior of members of the rhizosphere community.

Versatile role of Pseudomonas fuscovaginae cyclic lipopeptides in plant and microbial interactions.

Pseudomonas fuscovaginae is the most prominent bacterial sheath rot pathogen, causing sheath brown rot disease in rice. This disease occurs worldwide and it is characterized by typical necrotic lesions on the sheath, as well as a reduction in the number of emitted panicles and filled grains. P. fuscovaginae has been shown to produce syringotoxin and fuscopeptin cyclic lipopeptides (CLPs), which have been linked to pathogenicity. In this study, we investigated the role of P. fuscovaginae UPB0736 CLPs in plant pathogenicity, antifungal activity and swarming motility. To do so, we sequenced the strain to obtain a single-contig genome and we constructed deletion mutants in the biosynthetic gene clusters responsible for the synthesis of CLPs. We show that UPB0736 produces a third CLP of 13 amino acids, now named asplenin, and we link this CLP with the swarming activity of the strain. We could then show that syringotoxin is particularly active against Rhizoctonia solani in vitro. By testing the mutants in planta we investigated the role of both fuscopeptin and syringotoxin in causing sheath rot lesions. We proved that the presence of these two CLPs considerably affected the number of emitted panicles, although their number was still significantly affected in the mutants deficient in both fuscopeptin and syringotoxin. These results reveal the importance of CLPs in P. fuscovaginae pathogenicity, but also suggest that other pathogenicity factors may be involved.

Improvement of fitness and biocontrol properties of Pseudomonas putida via an extracellular heme peroxidase.

The extracellular 373-kDa PehA heme peroxidase of Pseudomonas putida KT2440 has two enzymatic domains which depend on heme cofactor for their peroxidase activity. A null pehA mutant was generated to examine the impact of PehA in rhizosphere colonization competence and the induction of plant systemic resistance (ISR). This mutant was not markedly hampered in colonization efficiency. However, increase in pehA dosage enhanced colonization fitness about 30 fold in the root and 900 fold in the root apex. In vitro assays with purified His-tagged enzymatic domains of PehA indicated that heme-dependent peroxidase activity was required for the enhancement of root tip colonization. Evaluation of live/dead cells confirmed that overexpression of pehA had a positive effect on bacterial cell viability. Following root colonization of rice plants by KT2440 strain, the incidence of rice blast caused by Magnaporthe oryzae was reduced by 65% and the severity of this disease was also diminished in comparison to non-treated plants. An increase in the pehA dosage was also beneficial for the control of rice blast as compared with gene inactivation. The results suggest that PehA helps P. putida to cope with the plant-imposed oxidative stress leading to enhanced colonization ability and concomitant ISR-elicitation.

An Nuclear Magnetic Resonance Fingerprint Matching Approach for the Identification and Structural Re-Evaluation of Pseudomonas Lipopeptides.

Cyclic lipopeptides (CLiPs) are secondary metabolites secreted by a range of bacterial phyla. CLiPs from Pseudomonas in particular, display diverse structural variations in terms of the number of amino acid residues, macrocycle size, amino acid identity, and stereochemistry (e.g., d- versus l-amino acids). Reports detailing the discovery of novel or already characterized CLiPs from new sources appear regularly in literature. Increasingly, however, the lack of detailed characterization threatens to cause considerable confusion, especially if configurational heterogeneity is present for one or more amino acids. Using Pseudomonas CLiPs from the Bananamide, Orfamide, and Xantholysin groups as test cases, we demonstrate and validate that the combined 1H and 13C Nuclear Magnetic Resonance (NMR) chemical shifts of CLiPs constitute a spectral fingerprint that is sufficiently sensitive to differentiate between possible diastereomers of a particular sequence even when they only differ in a single d/l configuration. Rapid screening, involving simple matching of the NMR fingerprint of a newly isolated CLiP with that of a reference CLiP of known stereochemistry, can then be applied to resolve dead-ends in configurational characterization and avoid the much more cumbersome chemical characterization protocols. Even when the stereochemistry of a particular reference CLiP remains to be established, its spectral fingerprint allows to quickly verify whether a newly isolated CLiP is novel or already present in the reference collection. We show NMR fingerprinting leads to a simple approach for early on dereplication which should become more effective as more fingerprints are collected. To benefit research involving CLiPs, we have made a publicly available data repository accompanied by a 'knowledge base' at https://www.rhizoclip.be, where we present an overview of published NMR fingerprint data of characterized CLiPs, together with literature data on the originally determined structures. IMPORTANCE Pseudomonas CLiPs are ubiquitous specialized metabolites, impacting the producer's lifestyle and interactions with the (a)biotic environment. Consequently, they generate interest for agricultural and clinical applications. Establishing structure-activity relationships as a premise to their development is hindered because full structural characterization including stereochemical information requires labor-intensive analyses, without guarantee for success. Moreover, increasing use of superficial comparison with previously characterized CLiPs introduces or propagates erroneous attributions, clouding further scientific progress. We provide a generally applicable characterization methodology based on matching NMR spectral fingerprints of newly isolated CLiPs to natural and synthetic reference compounds with (un)known stereochemistry. In addition, NMR fingerprinting is shown to provide a suitable basis for structural dereplication. A publicly available reference compound repository promises to facilitate participation of the lipopeptide research community in structural assessment and dereplication of newly isolated CLiPs, which should also support further developments in genome mining for novel CLiPs.

Altering in vivo membrane sterol composition affects the activity of the cyclic lipopeptides tolaasin and sessilin against Pythium.

Cyclic lipopeptides (CLiPs) are secondary metabolites produced by a variety of bacteria. These compounds show a broad range of antimicrobial activities; therefore, they are studied for their potential applications in agriculture and medicine. It is generally assumed that the primary target of the CLiPs is the cellular membrane, where they can permeabilize the lipid bilayer. Model membrane systems are commonly used to investigate the effect of lipid composition on the permeabilizing activity of CLiPs, but these systems do not represent the full complexity of true biological membranes. Here, we introduce a novel method that uses sterol-auxotrophic oomycetes to investigate how the activity of membrane-active compounds is influenced by alterations in membrane sterol composition. More specifically, we investigated how ergosterol, cholesterol, beta-sitosterol and stigmasterol affect the activity of the structurally related Pseudomonas-derived CLiPs tolaasin and sessilin against the oomycete Pythium myriotylum. Both compounds were effective against oomycetes, although tolaasin was considerably more active. Interestingly, tolaasin and sessilin effects were similarly reduced by the presence of sterols, with cholesterol showing the highest reduction of activity.

Bioinspired Rhamnolipid Protects Wheat Against Zymoseptoria tritici Through Mainly Direct Antifungal Activity and Without Major Impact on Leaf Physiology.

Rhamnolipids (RLs), glycolipids biosynthesized by the Pseudomonas and Burkholderia genera, are known to display various activities against a wide range of pathogens. Most previous studies on RLs focused on their direct antimicrobial activity, while only a few reports described the mechanisms by which RLs induce resistance against phytopathogens and the related fitness cost on plant physiology. Here, we combined transcriptomic and metabolomic approaches to unravel the mechanisms underlying RL-induced resistance in wheat against the hemibiotrophic fungus Zymoseptoria tritici, a major pathogen of this crop. Investigations were carried out by treating wheat plants with a bioinspired synthetic mono-RL with a 12-carbon fatty acid tail, dodecanoyl α/ß-L-rhamnopyranoside (Rh-Est-C12), under both infectious and non-infectious conditions to examine its potential wheat defense-eliciting and priming bioactivities. Whereas, Rh-Est-C12 conferred to wheat a significant protection against Z. tritici (41% disease severity reduction), only a slight effect of this RL on wheat leaf gene expression and metabolite accumulation was observed. A subset of 24 differentially expressed genes (DEGs) and 11 differentially accumulated metabolites (DAMs) was scored in elicitation modalities 2, 5, and 15 days post-treatment (dpt), and 25 DEGs and 17 DAMs were recorded in priming modalities 5 and 15 dpt. Most changes were down-regulations, and only a few DEGs and DAMs associated with resistance to pathogens were identified. Nevertheless, a transient early regulation in gene expression was highlighted at 2 dpt (e.g., genes involved in signaling, transcription, translation, cell-wall structure, and function), suggesting a perception of the RL by the plant upon treatment. Further in vitro and in planta bioassays showed that Rh-Est-C12 displays a significant direct antimicrobial activity toward Z. tritici. Taken together, our results suggest that Rh-Est-C12 confers protection to wheat against Z. tritici through direct antifungal activity and, to a lesser extent, by induction of plant defenses without causing major alterations in plant metabolism. This study provides new insights into the modes of action of RLs on the wheat-Z. tritici pathosystem and highlights the potential interest in Rh-Est-C12, a low-fitness cost molecule, to control this pathogen.

Pseudomonas Lipopeptide-Mediated Biocontrol: Chemotaxonomy and Biological Activity.

Pseudomonas lipopeptides (Ps-LPs) play crucial roles in bacterial physiology, host-microbe interactions and plant disease control. Beneficial LP producers have mainly been isolated from the rhizosphere, phyllosphere and from bulk soils. Despite their wide geographic distribution and host range, emerging evidence suggests that LP-producing pseudomonads and their corresponding molecules display tight specificity and follow a phylogenetic distribution. About a decade ago, biocontrol LPs were mainly reported from the P. fluorescens group, but this has drastically advanced due to increased LP diversity research. On the one hand, the presence of a close-knit relationship between Pseudomonas taxonomy and the molecule produced may provide a startup toolbox for the delineation of unknown LPs into existing (or novel) LP groups. Furthermore, a taxonomy-molecule match may facilitate decisions regarding antimicrobial activity profiling and subsequent agricultural relevance of such LPs. In this review, we highlight and discuss the production of beneficial Ps-LPs by strains situated within unique taxonomic groups and the lineage-specificity and coevolution of this relationship. We also chronicle the antimicrobial activity demonstrated by these biomolecules in limited plant systems compared with multiple in vitro assays. Our review further stresses the need to systematically elucidate the roles of diverse Ps-LP groups in direct plant-pathogen interactions and in the enhancement of plant innate immunity.