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.

Chemo-Enzymatic Synthesis and Biological Assessment of p-Coumarate Fatty Esters: New Antifungal Agents for Potential Plant Protection.

One trend in agriculture is the replacement of classical pesticides with more ecofriendly solutions, such as elicitation, which is a promising approach consisting of stimulating the natural immune system of a plant to improve its resistance to pathogens. In this fashion, a library of p-coumaric-based compounds were synthesized in accordance with as many principles of green chemistry as possible. Then, these molecules were tested for (1) the direct inhibition of mycelium growth of two pathogens, Botrytis cinerea and Sclerotinia sclerotiorum, and (2) plasma membrane destabilization in Arabidopsis and rapeseed. Finally, the protective effect was evaluated on an Arabidopsis/B. cinerea pathosystem. Total inhibition of the growth of both fungi could be achieved, and significant ion leakage was observed using dihydroxylated fatty p-coumarate esters. A direct effect on plants was also recorded as a ca. three-fold reduction in the necrosis area.

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.

Phytostilbenes as agrochemicals: biosynthesis, bioactivity, metabolic engineering and biotechnology.

Covering: 1976 to 2020. Although constituting a limited chemical family, phytostilbenes represent an emblematic group of molecules among natural compounds. Ever since their discovery as antifungal compounds in plants and their ascribed role in human health and disease, phytostilbenes have never ceased to arouse interest for researchers, leading to a huge development of the literature in this field. Owing to this, the number of references to this class of compounds has reached the tens of thousands. The objective of this article is thus to offer an overview of the different aspects of these compounds through a large bibliography analysis of more than 500 articles. All the aspects regarding phytostilbenes will be covered including their chemistry and biochemistry, regulation of their biosynthesis, biological activities in plants, molecular engineering of stilbene pathways in plants and microbes as well as their biotechnological production by plant cell systems.

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.

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.

Large-scale proteomic analysis of the grapevine leaf apoplastic fluid reveals mainly stress-related proteins and cell wall modifying enzymes.

BACKGROUND: The extracellular space or apoplast forms a path through the whole plant and acts as an interface with the environment. The apoplast is composed of plant cell wall and space within which apoplastic fluid provides a means of delivering molecules and facilitates intercellular communications. However, the apoplastic fluid extraction from in planta systems remains challenging and this is particularly true for grapevine (Vitis vinifera L.), a worldwide-cultivated fruit plant. Large-scale proteomic analysis reveals the protein content of the grapevine leaf apoplastic fluid and the free interactive proteome map considerably facilitates the study of the grapevine proteome. RESULTS: To obtain a snapshot of the grapevine apoplastic fluid proteome, a vacuum-infiltration-centrifugation method was optimized to collect the apoplastic fluid from non-challenged grapevine leaves. Soluble apoplastic protein patterns were then compared to whole leaf soluble protein profiles by 2D-PAGE analyses. Subsequent MALDI-TOF/TOF mass spectrometry of tryptically digested protein spots was used to identify proteins. This large-scale proteomic analysis established a well-defined proteomic map of whole leaf and leaf apoplastic soluble proteins, with 223 and 177 analyzed spots, respectively. All data arising from proteomic, MS and MS/MS analyses were deposited in the public database world-2DPAGE. Prediction tools revealed a high proportion of (i) classical secreted proteins but also of non-classical secreted proteins namely Leaderless Secreted Proteins (LSPs) in the apoplastic protein content and (ii) proteins potentially involved in stress reactions and/or in cell wall metabolism. CONCLUSIONS: This approach provides free online interactive reference maps annotating a large number of soluble proteins of the whole leaf and the apoplastic fluid of grapevine leaf. To our knowledge, this is the first detailed proteome study of grapevine apoplastic fluid providing a comprehensive overview of the most abundant proteins present in the apoplast of grapevine leaf that could be further characterized in order to elucidate their physiological function.

Grapevine NAC1 transcription factor as a convergent node in developmental processes, abiotic stresses, and necrotrophic/biotrophic pathogen tolerance.

Transcription factors of the NAC family are known to be involved in various developmental processes and in response to environmental stresses. Whereas NAC genes have been widely studied in response to abiotic stresses, little is known about their role in response to biotic stresses, especially in crops. Here, the first characterization of a Vitis vinifera L. NAC member, named VvNAC1, and involved in organ development and defence towards pathogens is reported. Expression profile analysis of VvNAC1 showed that its expression is closely associated with later stages of leaf, flower, and berry development, suggesting a role in plant senescence. Moreover, VvNAC1 expression is stimulated in Botrytis cinerea- or microbe-associated molecular pattern (MAMP)-infected berries or leaves. Furthermore, cold, wounding, and defence-related hormones such as salicylic acid, methyl jasmonate, ethylene, and abscisic acid are all able to induce VvNAC1 expression in grapevine leaves. VvNAC1-overexpressing Arabidopsis plants exhibit enhanced tolerance to osmotic, salt, and cold stresses and to B. cinerea and Hyaloperonospora arabidopsidis pathogens. These plants present a modified pattern of defence gene markers (AtPR-1, AtPDF1.2, and AtVSP1) after stress application, suggesting that VvNAC1 is an important regulatory component of the plant signalling defence cascade. Collectively, these results provide evidence that VvNAC1 could represent a node of convergence regulating grapevine development and stress responses, including defence against necrotrophic and biotrophic pathogens.

Modulation of phytoalexin biosynthesis in engineered plants for disease resistance.

Phytoalexins are antimicrobial substances of low molecular weight produced by plants in response to infection or stress, which form part of their active defense mechanisms. Starting in the 1950's, research on phytoalexins has begun with biochemistry and bio-organic chemistry, resulting in the determination of their structure, their biological activity as well as mechanisms of their synthesis and their catabolism by microorganisms. Elucidation of the biosynthesis of numerous phytoalexins has permitted the use of molecular biology tools for the exploration of the genes encoding enzymes of their synthesis pathways and their regulators. Genetic manipulation of phytoalexins has been investigated to increase the disease resistance of plants. The first example of a disease resistance resulting from foreign phytoalexin expression in a novel plant has concerned a phytoalexin from grapevine which was transferred to tobacco. Transformations were then operated to investigate the potential of other phytoalexin biosynthetic genes to confer resistance to pathogens. Unexpectedly, engineering phytoalexins for disease resistance in plants seem to have been limited to exploiting only a few phytoalexin biosynthetic genes, especially those encoding stilbenes and some isoflavonoids. Research has rather focused on indirect approaches which allow modulation of the accumulation of phytoalexin employing transcriptional regulators or components of upstream regulatory pathways. Genetic approaches using gain- or less-of functions in phytoalexin engineering together with modulation of phytoalexin accumulation through molecular engineering of plant hormones and defense-related marker and elicitor genes have been reviewed.

Metabolic engineering of yeast and plants for the production of the biologically active hydroxystilbene, resveratrol.

Resveratrol, a stilbenic compound deriving from the phenyalanine/polymalonate route, being stilbene synthase the last and key enzyme of this pathway, recently has become the focus of a number of studies in medicine and plant physiology. Increased demand for this molecule for nutraceutical, cosmetic and possibly pharmaceutic uses, makes its production a necessity. In this context, the use of biotechnology through recombinant microorganisms and plants is particularly promising. Interesting results can indeed arise from the potential of genetically modified microorganisms as an alternative mechanism for producing resveratrol. Strategies used to tailoring yeast as they do not possess the genes that encode for the resveratrol pathway, will be described. On the other hand, most interest has centered in recent years, on STS gene transfer experiments from various origins to the genome of numerous plants. This work also presents a comprehensive review on plant molecular engineering with the STS gene, resulting in disease resistance against microorganisms and the enhancement of the antioxidant activities of several fruits in transgenic lines.

Protoplast: A Valuable Toolbox to Investigate Plant Stress Perception and Response.

Plants are constantly facing abiotic and biotic stresses. To continue to thrive in their environment, they have developed many sophisticated mechanisms to perceive these stresses and provide an appropriate response. There are many ways to study these stress signals in plant, and among them, protoplasts appear to provide a unique experimental system. As plant cells devoid of cell wall, protoplasts allow observations at the individual cell level. They also offer a prime access to the plasma membrane and an original view on the inside of the cell. In this regard, protoplasts are particularly useful to address essential biological questions regarding stress response, such as protein signaling, ion fluxes, ROS production, and plasma membrane dynamics. Here, the tools associated with protoplasts to comprehend plant stress signaling are overviewed and their potential to decipher plant defense mechanisms is discussed.

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.

Molecular engineering of resveratrol in plants.

The grapevine phytoalexin resveratrol, the synthesis of which is achieved by stilbene synthase (STS), displays a wide range of biological effects. Most interest has centred, in recent years, on STS gene transfer experiments from grapevine to the genome of numerous plants. This work presents a comprehensive review on plant molecular engineering with the STS gene. Gene and promoter options are discussed, namely the different promoters used to drive the transgene, as well as the enhancer elements and/or heterologous promoters used to improve transcriptional activity in the transformed lines. Factors modifying transgene expression and epigenetic modifications, for instance transgene copy number, are also presented. Resveratrol synthesis in plants, together with that of its glucoside as a result of STS expression, is described, as is the incidence of these compounds on plant metabolism and development. The ectopic production of resveratrol can lead to broad-spectrum resistance against fungi in transgenic lines, and to the enhancement of the antioxidant activities of several fruits, highlighting the potential role of this compound in health promotion and plant disease control.

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.

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.

Engineering stilbene metabolic pathways in microbial cells.

Numerous in vitro and in vivo studies on biological activities of phytostilbenes have brought to the fore the remarkable properties of these compounds and their derivatives, making them a top storyline in natural product research fields. However, getting stilbenes in sufficient amounts for routine biological activity studies and make them available for pharmaceutical and/or nutraceutical industry applications, is hampered by the difficulty to source them through synthetic chemistry-based pathways or extraction from the native plants. Hence, microbial cell cultures have rapidly became potent workhorse factories for stilbene production. In this review, we present the combined efforts made during the past 15 years to engineer stilbene metabolic pathways in microbial cells, mainly the Saccharomyces cerevisiae baker yeast, the Escherichia coli and the Corynebacterium glutamicum bacteria. Rationalized approaches to the heterologous expression of the partial or the entire stilbene biosynthetic routes are presented to allow the identification and/or bypassing of the major bottlenecks in the endogenous microbial cell metabolism as well as potential regulations of the genes involved in these metabolic pathways. The contributions of bioinformatics to synthetic biology are developed to highlight their tremendous help in predicting which target genes are likely to be up-regulated or deleted for controlling the dynamics of precursor flows in the tailored microbial cells. Further insight is given to the metabolic engineering of microbial cells with "decorating" enzymes, such as methyl and glycosyltransferases or hydroxylases, which can act sequentially on the stilbene core structure. Altogether, the cellular optimization of stilbene biosynthetic pathways integrating more and more complex constructs up to twelve genetic modifications has led to stilbene titers ranging from hundreds of milligrams to the gram-scale yields from various carbon sources. Through this review, the microbial production of stilbenes is analyzed, stressing both the engineering dynamic regulation of biosynthetic pathways and the endogenous control of stilbene precursors.

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.

Vacuum Infiltration-Centrifugation Method for Apoplastic Protein Extraction in Grapevine.

The apoplastic fluid moving in the extracellular space external to the plasma membrane provides a means of delivering molecules and facilitates intercellular communications. However, the apoplastic fluid extraction from in planta systems remains challenging and this is particularly true for grapevine (Vitis vinifera L.), a worldwide-cultivated fruit plant. Here, we describe an optimized vacuum-infiltration-centrifugation method to extract soluble proteins from apoplastic fluid of grapevine leaves. This optimized method allows recovering of the grapevine apoplastic soluble proteins suitable for mono- and bi-dimensional gel electrophoresis for further proteomic analysis in order to elucidate their physiological functions.

Uncovering plant-pathogen crosstalk through apoplastic proteomic studies.

Plant pathogens have evolved by developing different strategies to infect their host, which in turn have elaborated immune responses to counter the pathogen invasion. The apoplast, including the cell wall and extracellular space outside the plasma membrane, is one of the first compartments where pathogen-host interaction occurs. The plant cell wall is composed of a complex network of polysaccharides polymers and glycoproteins and serves as a natural physical barrier against pathogen invasion. The apoplastic fluid, circulating through the cell wall and intercellular spaces, provides a means for delivering molecules and facilitating intercellular communications. Some plant-pathogen interactions lead to plant cell wall degradation allowing pathogens to penetrate into the cells. In turn, the plant immune system recognizes microbial- or damage-associated molecular patterns (MAMPs or DAMPs) and initiates a set of basal immune responses, including the strengthening of the plant cell wall. The establishment of defense requires the regulation of a wide variety of proteins that are involved at different levels, from receptor perception of the pathogen via signaling mechanisms to the strengthening of the cell wall or degradation of the pathogen itself. A fine regulation of apoplastic proteins is therefore essential for rapid and effective pathogen perception and for maintaining cell wall integrity. This review aims to provide insight into analyses using proteomic approaches of the apoplast to highlight the modulation of the apoplastic protein patterns during pathogen infection and to unravel the key players involved in plant-pathogen interaction.