Protecting-group free synthesis of glycoconjugates displaying dual fungicidal and plant defense-eliciting activities.

A straightforward synthesis of carbohydrate templated isoxazolidines is described, by reaction of unprotected glycosylhydroxylamines (operating as 1,3-dipoles) with methyl acrylate using microwave activation. Rhamno- and erythro-isoxazolidines are recognized by plant cells, resulting in a strong ROS-production as a plant immune response, and exert a high antifungal activity against Botrytis cinerea.

L-Rhamnose and Phenolic Esters-Based Monocatenar and Bolaform Amphiphiles: Eco-Compatible Synthesis and Determination of Their Antioxidant, Eliciting and Cytotoxic Properties.

Symmetrical and dissymmetrical bolaforms were prepared with good to high yields from unsaturated L-rhamnosides and phenolic esters (ferulic, phloretic, coumaric, sinapic and caffeic) using two eco-compatible synthetic strategies involving glycosylation, enzymatic synthesis and cross-metathesis under microwave activation. The plant-eliciting activity of these new compounds was investigated in Arabidopsis model plants. We found that the monocatenar rhamnosides and bolaforms activate the plant immune system with a response depending on the carbon chain length and the nature of the hydrophilic heads. Their respective antioxidant activities were also evaluated, as well as their cytotoxic properties on dermal cells for cosmetic uses. We showed that phenolic ester-based compounds present good antioxidant activities and that their cytotoxicity is low. These properties are also dependent on the carbon chains used.

Deciphering the role of plant plasma membrane lipids in response to invasion patterns: how could biology and biophysics help?

Plants have to constantly face pathogen attacks. To cope with diseases, they have to detect the invading pathogen as early as possible via the sensing of conserved motifs called invasion patterns. The first step of perception occurs at the plasma membrane. While many invasion patterns are perceived by specific proteinaceous immune receptors, several studies have highlighted the influence of the lipid composition and dynamics of the plasma membrane in the sensing of invasion patterns. In this review, we summarize current knowledge on how some microbial invasion patterns could interact with the lipids of the plasma membrane, leading to a plant immune response. Depending on the invasion pattern, different mechanisms are involved. This review outlines the potential of combining biological with biophysical approaches to decipher how plasma membrane lipids are involved in the perception of microbial invasion patterns.

Bacterial rhamnolipids and their 3-hydroxyalkanoate precursors activate Arabidopsis innate immunity through two independent mechanisms.

Plant innate immunity is activated upon perception of invasion pattern molecules by plant cell-surface immune receptors. Several bacteria of the genera Pseudomonas and Burkholderia produce rhamnolipids (RLs) from l-rhamnose and (R)-3-hydroxyalkanoate precursors (HAAs). RL and HAA secretion is required to modulate bacterial surface motility, biofilm development, and thus successful colonization of hosts. Here, we show that the lipidic secretome from the opportunistic pathogen Pseudomonas aeruginosa, mainly comprising RLs and HAAs, stimulates Arabidopsis immunity. We demonstrate that HAAs are sensed by the bulb-type lectin receptor kinase LIPOOLIGOSACCHARIDE-SPECIFIC REDUCED ELICITATION/S-DOMAIN-1-29 (LORE/SD1-29), which also mediates medium-chain 3-hydroxy fatty acid (mc-3-OH-FA) perception, in the plant Arabidopsis thaliana HAA sensing induces canonical immune signaling and local resistance to plant pathogenic Pseudomonas infection. By contrast, RLs trigger an atypical immune response and resistance to Pseudomonas infection independent of LORE. Thus, the glycosyl moieties of RLs, although abolishing sensing by LORE, do not impair their ability to trigger plant defense. Moreover, our results show that the immune response triggered by RLs is affected by the sphingolipid composition of the plasma membrane. In conclusion, RLs and their precursors released by bacteria can both be perceived by plants but through distinct mechanisms.

Total synthesis, isolation, surfactant properties, and biological evaluation of ananatosides and related macrodilactone-containing rhamnolipids.

Rhamnolipids are a specific class of microbial surfactants, which hold great biotechnological and therapeutic potential. However, their exploitation at the industrial level is hampered because they are mainly produced by the opportunistic pathogen Pseudomonas aeruginosa. The non-human pathogenic bacterium Pantoea ananatis is an alternative producer of rhamnolipid-like metabolites containing glucose instead of rhamnose residues. Herein, we present the isolation, structural characterization, and total synthesis of ananatoside A, a 15-membered macrodilactone-containing glucolipid, and ananatoside B, its open-chain congener, from organic extracts of P. ananatis. Ananatoside A was synthesized through three alternative pathways involving either an intramolecular glycosylation, a chemical macrolactonization or a direct enzymatic transformation from ananatoside B. A series of diasteroisomerically pure (1→2), (1→3), and (1→4)-macrolactonized rhamnolipids were also synthesized through intramolecular glycosylation and their anomeric configurations as well as ring conformations were solved using molecular modeling in tandem with NMR studies. We show that ananatoside B is a more potent surfactant than its macrolide counterpart. We present evidence that macrolactonization of rhamnolipids enhances their cytotoxic and hemolytic potential, pointing towards a mechanism involving the formation of pores into the lipidic cell membrane. Lastly, we demonstrate that ananatoside A and ananatoside B as well as synthetic macrolactonized rhamnolipids can be perceived by the plant immune system, and that this sensing is more pronounced for a macrolide featuring a rhamnose moiety in its native 1 C 4 conformation. Altogether our results suggest that macrolactonization of glycolipids can dramatically interfere with their surfactant properties and biological activity.

Biosurfactants in Plant Protection Against Diseases: Rhamnolipids and Lipopeptides Case Study.

Biosurfactants are amphiphilic surface-active molecules that are produced by a variety of microorganisms including fungi and bacteria. Pseudomonas, Burkholderia, and Bacillus species are known to secrete rhamnolipids and lipopeptides that are used in a wide range of industrial applications. Recently, these compounds have been studied in a context of plant-microbe interactions. This mini-review describes the direct antimicrobial activities of these compounds against plant pathogens. We also provide the current knowledge on how rhamnolipids and lipopeptides stimulate the plant immune system leading to plant resistance to phytopathogens. Given their low toxicity, high biodegradability and ecological acceptance, we discuss the possible role of these biosurfactants as alternative strategies to reduce or even replace pesticide use in agriculture.

Synthetic Mono-Rhamnolipids Display Direct Antifungal Effects and Trigger an Innate Immune Response in Tomato against Botrytis Cinerea.

Natural rhamnolipids are potential biocontrol agents for plant protection against bacterial and fungal diseases. In this work, we synthetized new synthetic mono-rhamnolipids (smRLs) consisting in a rhamnose connected to a simple acyl chain and differing by the nature of the link and the length of the lipid tail. We then investigated the effects of these ether, ester, carbamate or succinate smRL derivatives on Botrytis cinerea development, symptoms spreading on tomato leaves and immune responses in tomato plants. Our results demonstrate that synthetic smRLs are able to trigger early and late immunity-related plant defense responses in tomato and increase plant resistance against B. cinerea in controlled conditions. Structure-function analysis showed that chain length of the lipidic part and type of acyl chain were critical to smRLs immune activity and to the extent of symptoms caused by the fungus on tomato leaves.

Semipurified Rhamnolipid Mixes Protect Brassica napus Against Leptosphaeria maculans Early Infections.

The rapeseed crop (Brassica napus) has to cope with fungal diseases that significantly impacts yields. In particular, the fungal pathogen Leptosphaeria maculans, the causal agent of blackleg disease (also named Phoma stem canker), is a worldwide issue to this crop. Considering environmental concerns, it is essential to propose alternative natural compounds for rapeseed crop protection to reduce chemical fungicide use. Here we report data showing the efficacy of semipurified rhamnolipid (RL) mixes from bacterial origin to protect rapeseed against L. maculans at early stages of infection in controlled conditions. In addition, we show that RL solutions have excellent adhesion properties when sprayed onto rapeseed leaves, without adding any adjuvant. We demonstrate that RL mixes display direct antimycelial properties against the pathogen and stimulate plant defense responses in rapeseed. Our results validate, a preventive action of low RL concentrations to protect rapeseed against L. maculans and a curative effect in specific conditions when applied after the inoculation of the pathogen spores. Semipurified RL mixes therefore appear to be real cost-effective compounds that could be used in fields as biocontrol products to fight L. maculans early infections of rapeseed.

Recognition of Elicitors in Grapevine: From MAMP and DAMP Perception to Induced Resistance.

In a context of a sustainable viticulture, the implementation of innovative eco-friendly strategies, such as elicitor-triggered immunity, requires a deep knowledge of the molecular mechanisms underlying grapevine defense activation, from pathogen perception to resistance induction. During plant-pathogen interaction, the first step of plant defense activation is ensured by the recognition of microbe-associated molecular patterns, which are elicitors directly derived from pathogenic or beneficial microbes. Vitis vinifera, like other plants, can perceive elicitors of different nature, including proteins, amphiphilic glycolipid, and lipopeptide molecules as well as polysaccharides, thanks to their cognate pattern recognition receptors, the discovery of which recently began in this plant species. Furthermore, damage-associated molecular patterns are another class of elicitors perceived by V. vinifera as an invader's hallmark. They are mainly polysaccharides derived from the plant cell wall and are generally released through the activity of cell wall-degrading enzymes secreted by microbes. Elicitor perception and subsequent activation of grapevine immunity end in some cases in efficient grapevine resistance against pathogens. Using complementary approaches, several molecular markers have been identified as hallmarks of this induced resistance stage. This review thus focuses on the recognition of elicitors by Vitis vinifera describing the molecular mechanisms triggered from the elicitor perception to the activation of immune responses. Finally, we discuss the fact that the link between elicitation and induced resistance is not so obvious and that the formulation of resistance inducers remains a key step before their application in vineyards.

Apoplastic invasion patterns triggering plant immunity: plasma membrane sensing at the frontline.

Plants are able to effectively cope with invading pathogens by activating an immune response based on the detection of invasion patterns (IPs) originating from the pathogen or released by the plant after infection. At a first level, this perception takes place at the plasma membrane through cell surface immune receptors and although the involvement of proteinaceous pattern recognition receptors (PRRs) is well established, increasing data are also pointing out the role of membrane lipids in the sensing of IPs. In this review, we discuss the evolution of various conceptual models describing plant immunity and present an overview of well-characterized IPs from different natures and origins. We summarize the current knowledge on how they are perceived by plants at the plasma membrane, highlighting the increasingly apparent diversity of sentinel-related systems in plants.

Bacterial medium-chain 3-hydroxy fatty acid metabolites trigger immunity in Arabidopsis plants.

In plants, cell-surface immune receptors sense molecular non-self-signatures. Lipid A of Gram-negative bacterial lipopolysaccharide is considered such a non-self-signature. The receptor kinase LIPOOLIGOSACCHARIDE-SPECIFIC REDUCED ELICITATION (LORE) mediates plant immune responses to Pseudomonas and Xanthomonas but not enterobacterial lipid A or lipopolysaccharide preparations. Here, we demonstrate that synthetic and bacterial lipopolysaccharide-copurified medium-chain 3-hydroxy fatty acid (mc-3-OH-FA) metabolites elicit LORE-dependent immunity. The mc-3-OH-FAs are sensed in a chain length- and hydroxylation-specific manner, with free (R)-3-hydroxydecanoic acid [(R)-3-OH-C10:0] representing the strongest immune elicitor. By contrast, bacterial compounds comprising mc-3-OH-acyl building blocks but devoid of free mc-3-OH-FAs-including lipid A or lipopolysaccharide, rhamnolipids, lipopeptides, and acyl-homoserine-lactones-do not trigger LORE-dependent responses. Hence, plants sense low-complexity bacterial metabolites to trigger immune responses.

Rhamnolipids From Pseudomonas aeruginosa Are Elicitors Triggering Brassica napus Protection Against Botrytis cinerea Without Physiological Disorders.

Rhamnolipids (RLs) are amphiphilic molecules naturally produced by some bacteria with a large range of biological activities. Although some studies report their potential interest in plant protection, evaluation of their effects and efficiency on annual crops of worldwide agronomic interest is lacking. The main objective of this work was to investigate their elicitor and protective activities on rapeseed crop species while evaluating their physiological effects. Here we report that RLs from Pseudomonas aeruginosa secretome trigger an effective protection of Brassicanapus foliar tissues toward the fungus Botrytis cinerea involving the combination of plant defense activation and direct antimicrobial properties. We demonstrated their ability to activate canonical B.napus defense responses including reactive oxygen species production, expression of defense genes, along with callose deposits and stomatal closure as efficient physical protections. In addition, microscopic cell death observations and electrolyte leakage measurements indicated that RLs trigger a hypersensitive response-like defense in this plant. We also showed that foliar spray applications of RLs do not induce deleterious physiological consequences on plant growth or chlorophyll content and that RL protective properties are efficient on several grown cultivars of rapeseed. To our knowledge, this is the first report of RLs as an elicitor that suppresses fungal disease on tissues of an annual crop species under greenhouse conditions. Our results highlight the dual mode of action of these molecules exhibiting plant protection activation and antifungal activities and demonstrate their potential for crop cultures as environmental-friendly biocontrol solution.

Synthetic Rhamnolipid Bolaforms trigger an innate immune response in Arabidopsis thaliana.

Stimulation of plant innate immunity by natural and synthetic elicitors is a promising alternative to conventional pesticides for a more sustainable agriculture. Sugar-based bolaamphiphiles are known for their biocompatibility, biodegradability and low toxicity. In this work, we show that Synthetic Rhamnolipid Bolaforms (SRBs) that have been synthesized by green chemistry trigger Arabidopsis innate immunity. Using structure-function analysis, we demonstrate that SRBs, depending on the acyl chain length, differentially activate early and late immunity-related plant defense responses and provide local increase in resistance to plant pathogenic bacteria. Our biophysical data suggest that SRBs can interact with plant biomimetic plasma membrane and open the possibility of a lipid driven process for plant-triggered immunity by SRBs.

Draft Genome Sequence of Plant Growth-Promoting Burkholderia sp. Strain BE12, Isolated from the Rhizosphere of Maize.

Burkholderia sp. strain BE12, isolated from a French agricultural soil, possesses antifungal activity against a set of phytopathogenic fungi and has friendly interactions with grapevine. Here, we present the draft genome sequence of BE12, along with genes related to plant growth-promoting traits and siderophores that this strain contains, supporting its plant growth and antifungal activities.

Differential Interaction of Synthetic Glycolipids with Biomimetic Plasma Membrane Lipids Correlates with the Plant Biological Response.

Natural and synthetic amphiphilic molecules including lipopeptides, lipopolysaccharides, and glycolipids are able to induce defense mechanisms in plants. In the present work, the perception of two synthetic C14 rhamnolipids, namely, Alk-RL and Ac-RL, differing only at the level of the lipid tail terminal group have been investigated using biological and biophysical approaches. We showed that Alk-RL induces a stronger early signaling response in tobacco cell suspensions than does Ac-RL. The interactions of both synthetic RLs with simplified biomimetic membranes were further analyzed using experimental and in silico approaches. Our results indicate that the interactions of Alk-RL and Ac-RL with lipids were different in terms of insertion and molecular responses and were dependent on the lipid composition of model membranes. A more favorable insertion of Alk-RL than Ac-RL into lipid membranes is observed. Alk-RL forms more stable molecular assemblies than Ac-RL with phospholipids and sterols. At the molecular level, the presence of sterols tends to increase the RLs' interaction with lipid bilayers, with a fluidizing effect on the alkyl chains. Taken together, our findings suggest that the perception of these synthetic RLs at the membrane level could be related to a lipid-driven process depending on the organization of the membrane and the orientation of the RLs within the membrane and is correlated with the induction of early signaling responses in tobacco cells.

Modifications of Sphingolipid Content Affect Tolerance to Hemibiotrophic and Necrotrophic Pathogens by Modulating Plant Defense Responses in Arabidopsis.

Sphingolipids are emerging as second messengers in programmed cell death and plant defense mechanisms. However, their role in plant defense is far from being understood, especially against necrotrophic pathogens. Sphingolipidomics and plant defense responses during pathogenic infection were evaluated in the mutant of long-chain base phosphate (LCB-P) lyase, encoded by the dihydrosphingosine-1-phosphate lyase1 (AtDPL1) gene and regulating long-chain base/LCB-P homeostasis. Atdpl1 mutants exhibit tolerance to the necrotrophic fungus Botrytis cinerea but susceptibility to the hemibiotrophic bacterium Pseudomonas syringae pv tomato (Pst). Here, a direct comparison of sphingolipid profiles in Arabidopsis (Arabidopsis thaliana) during infection with pathogens differing in lifestyles is described. In contrast to long-chain bases (dihydrosphingosine [d18:0] and 4,8-sphingadienine [d18:2]), hydroxyceramide and LCB-P (phytosphingosine-1-phosphate [t18:0-P] and 4-hydroxy-8-sphingenine-1-phosphate [t18:1-P]) levels are higher in Atdpl1-1 than in wild-type plants in response to B. cinerea. Following Pst infection, t18:0-P accumulates more strongly in Atdpl1-1 than in wild-type plants. Moreover, d18:0 and t18:0-P appear as key players in Pst- and B. cinerea-induced cell death and reactive oxygen species accumulation. Salicylic acid levels are similar in both types of plants, independent of the pathogen. In addition, salicylic acid-dependent gene expression is similar in both types of B. cinerea-infected plants but is repressed in Atdpl1-1 after treatment with Pst. Infection with both pathogens triggers higher jasmonic acid, jasmonoyl-isoleucine accumulation, and jasmonic acid-dependent gene expression in Atdpl1-1 mutants. Our results demonstrate that sphingolipids play an important role in plant defense, especially toward necrotrophic pathogens, and highlight a novel connection between the jasmonate signaling pathway, cell death, and sphingolipids.

Perception of pathogenic or beneficial bacteria and their evasion of host immunity: pattern recognition receptors in the frontline.

Plants are continuously monitoring the presence of microorganisms to establish an adapted response. Plants commonly use pattern recognition receptors (PRRs) to perceive microbe- or pathogen-associated molecular patterns (MAMPs/PAMPs) which are microorganism molecular signatures. Located at the plant plasma membrane, the PRRs are generally receptor-like kinases (RLKs) or receptor-like proteins (RLPs). MAMP detection will lead to the establishment of a plant defense program called MAMP-triggered immunity (MTI). In this review, we overview the RLKs and RLPs that assure early recognition and control of pathogenic or beneficial bacteria. We also highlight the crucial function of PRRs during plant-microbe interactions, with a special emphasis on the receptors of the bacterial flagellin and peptidoglycan. In addition, we discuss the multiple strategies used by bacteria to evade PRR-mediated recognition.

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.

Cyclic lipopeptides from Bacillus subtilis activate distinct patterns of defence responses in grapevine.

Non-self-recognition of microorganisms partly relies on the perception of microbe-associated molecular patterns (MAMPs) and leads to the activation of an innate immune response. Bacillus subtilis produces three main families of cyclic lipopeptides (LPs), namely surfactins, iturins and fengycins. Although LPs are involved in induced systemic resistance (ISR) activation, little is known about defence responses induced by these molecules and their involvement in local resistance to fungi. Here, we showed that purified surfactin, mycosubtilin (iturin family) and plipastatin (fengycin family) are perceived by grapevine plant cells. Although surfactin and mycosubtilin stimulated grapevine innate immune responses, they differentially activated early signalling pathways and defence gene expression. By contrast, plipastatin perception by grapevine cells only resulted in early signalling activation. Gene expression analysis suggested that mycosubtilin activated salicylic acid (SA) and jasmonic acid (JA) signalling pathways, whereas surfactin mainly induced an SA-regulated response. Although mycosubtilin and plipastatin displayed direct antifungal activity, only surfactin and mycosubtilin treatments resulted in a local long-lasting enhanced tolerance to the necrotrophic fungus Botrytis cinerea in grapevine leaves. Moreover, challenge with specific strains overproducing surfactin and mycosubtilin led to a slightly enhanced stimulation of the defence response compared with the LP-non-producing strain of B. subtilis. Altogether, our results provide the first comprehensive view of the involvement of LPs from B. subtilis in grapevine plant defence and local resistance against the necrotrophic pathogen Bo. cinerea. Moreover, this work is the first to highlight the ability of mycosubtilin to trigger an immune response in plants.

Deciphering the role of phytoalexins in plant-microorganism interactions and human health.

Phytoalexins are low molecular weight antimicrobial compounds that are produced by plants as a response to biotic and abiotic stresses. As such they take part in an intricate defense system which enables plants to control invading microorganisms. In this review we present the key features of this diverse group of molecules, namely their chemical structures, biosynthesis, regulatory mechanisms, biological activities, metabolism and molecular engineering.