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.

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.

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.

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.

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.

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.

Transporter Gene-Mediated Typing for Detection and Genome Mining of Lipopeptide-Producing Pseudomonas.

Pseudomonas lipopeptides (LPs) are involved in diverse ecological functions and have biotechnological application potential associated with their antimicrobial and/or antiproliferative activities. They are synthesized by multimodular nonribosomal peptide synthetases which, together with transport and regulatory proteins, are encoded by large biosynthetic gene clusters (BGCs). These secondary metabolites are classified in distinct families based on the sequence and length of the oligopeptide and size of the macrocycle, if present. The phylogeny of PleB, the MacB-like transporter that is part of a dedicated ATP-dependent tripartite efflux system driving export of Pseudomonas LPs, revealed a strong correlation with LP chemical diversity. As each LP BGC carries its cognate pleB, PleB is suitable as a diagnostic sequence for genome mining, allowing assignment of the putative metabolite to a particular LP family. In addition, pleB proved to be a suitable target gene for an alternative PCR method for detecting LP-producing Pseudomonas sp. and did not rely on amplification of catalytic domains of the biosynthetic enzymes. Combined with amplicon sequencing, this approach enabled typing of Pseudomonas strains as potential producers of a LP belonging to one of the known LP families, underscoring its value for strain prioritization. This finding was validated by chemical characterization of known LPs from three different families secreted by novel producers isolated from the rice or maize rhizosphere, namely, the type strains of Pseudomonas fulva (putisolvin), Pseudomonas zeae (tensin), and Pseudomonas xantholysinigenes (xantholysin). In addition, a new member of the Bananamide family, prosekin, was discovered in the type strain of Pseudomonas prosekii, which is an Antarctic isolate. IMPORTANCE Pseudomonas spp. are ubiquitous bacteria able to thrive in a wide range of ecological niches, and lipopeptides often support their lifestyle but also their interaction with other micro- and macro-organisms. Therefore, the production of lipopeptides is widespread among Pseudomonas strains. Consequently, Pseudomonas lipopeptide research not only affects chemists and microbiologists but also touches a much broader audience, including biochemists, ecologists, and plant biologists. In this study, we present a reliable transporter gene-guided approach for the detection and/or typing of Pseudomonas lipopeptide producers. Indeed, it allows us to readily assess the lipopeptide diversity among sets of Pseudomonas isolates and differentiate strains likely to produce known lipopeptides from producers of potentially novel lipopeptides. This work provides a valuable tool that can also be integrated in a genome mining strategy and adapted for the typing of other specialized metabolites.

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.

Cyclic lipopeptide-producing Pseudomonas koreensis group strains dominate the cocoyam rhizosphere of a Pythium root rot suppressive soil contrasting with P. putida prominence in conducive soils.

Pseudomonas isolates from tropical environments have been underexplored and may form an untapped reservoir of interesting secondary metabolites. In this study, we compared Pseudomonas and cyclic lipopeptide (CLP) diversity in the rhizosphere of a cocoyam root rot disease (CRRD) suppressive soil in Boteva, Cameroon with those from four conducive soils in Cameroon and Nigeria. Compared with other soils, Boteva andosols were characterized by high silt, organic matter, nitrogen and calcium. Besides, the cocoyam rhizosphere at Boteva was characterized by strains belonging mainly to the P. koreensis and P. putida (sub)groups, with representations in the P. fluorescens, P. chlororaphis, P. jessenii and P. asplenii (sub)groups. In contrast, P. putida isolates were prominent in conducive soils. Regarding CLP diversity, Boteva was characterized by strains producing 11 different CLP types with cocoyamide A producers, belonging to the P. koreensis group, being the most abundant. However, putisolvin III-V producers were the most dominant in the rhizosphere of conducive soils in both Cameroon and Nigeria. Furthermore, we elucidated the chemical structure of putisolvin derivatives-putisolvin III-V, and described its biosynthetic gene cluster. We show that high Pseudomonas and metabolic diversity may be driven by microbial competition, which likely contributes to soil suppressiveness to CRRD.

Lipopeptide families at the interface between pathogenic and beneficial Pseudomonas-plant interactions.

Lipopeptides (LPs) are a prominent class of molecules among the steadily growing spectrum of specialized metabolites retrieved from Pseudomonas, in particular soil-dwelling and plant-associated isolates. Among the multiple LP families, pioneering research focussed on phytotoxic and antimicrobial cyclic lipopeptides (CLPs) of the ubiquitous plant pathogen Pseudomonas syringae (syringomycin and syringopeptin). Their non-ribosomal peptide synthetases (NRPSs) are embedded in biosynthetic gene clusters (BGCs) that are tightly co-clustered on a pathogenicity island. Other members of the P. syringae group (Pseudomonas cichorii) and some species of the Pseudomonas asplenii group and Pseudomonas fluorescens complex have adopted these biosynthetic strategies to co-produce their own mycin and peptin variants, in some strains supplemented with an analogue of the P. syringae linear LP (LLP), syringafactin. This capacity is not confined to phytopathogens but also occurs in some biocontrol strains, which indicates that these LP families not solely function as general virulence factors. We address this issue by scrutinizing the structural diversity and bioactivities of LPs from the mycin, peptin, and factin families in a phylogenetic and evolutionary perspective. BGC functional organization (including associated regulatory and transport genes) and NRPS modular architectures in known and candidate LP producers were assessed by genome mining.

γ-Aminobutyric acid and related amino acids in plant immune responses: Emerging mechanisms of action.

The entanglement between primary metabolism regulation and stress responses is a puzzling and fascinating theme in plant sciences. Among the major metabolites found in plants, γ-aminobutyric acid (GABA) fulfils important roles in connecting C and N metabolic fluxes through the GABA shunt. Activation of GABA metabolism is known since long to occur in plant tissues following biotic stresses, where GABA appears to have substantially different modes of action towards different categories of pathogens and pests. While it can harm insects thanks to its inhibitory effect on the neuronal transmission, its capacity to modulate the hypersensitive response in attacked host cells was proven to be crucial for host defences in several pathosystems. In this review, we discuss how plants can employ GABA's versatility to effectively deal with all the major biotic stressors, and how GABA can shape plant immune responses against pathogens by modulating reactive oxygen species balance in invaded plant tissues. Finally, we discuss the connections between GABA and other stress-related amino acids such as BABA (ß-aminobutyric acid), glutamate and proline.

Canditate metabolites for ash dieback tolerance in Fraxinus excelsior.

Ash dieback, a forest epidemic caused by the invasive fungus Hymenoscyphus fraxineus, threatens ash trees throughout Europe. Within Fraxinus excelsior populations, a small proportion of genotypes show a low susceptibility to the pathogen. We compared the metabolomes from a cohort of low-susceptibility ash genotypes with a cohort of high-susceptibility ash genotypes. This revealed two significantly different chemotypes. A total of 64 candidate metabolites associated with reduced or increased susceptibility in the chemical families secoiridoids, coumarins, flavonoids, phenylethanoids, and lignans. Increased levels of two coumarins, fraxetin and esculetin, were strongly associated with reduced susceptibility to ash dieback. Both coumarins inhibited the growth of H. fraxineus in vitro when supplied at physiological concentrations, thereby validating their role as markers for low susceptibility to ash dieback. Similarly, fungal growth inhibition was observed when the methanolic bark extract of low-susceptibility ash genotypes was supplied. Our findings indicate the presence of constitutive chemical defense barriers against ash dieback in ash.

Syringopeptin Contributes to the Virulence of Pseudomonas fuscovaginae, Based on sypA Biosynthesis Mutant Analysis.

Pseudomonas fuscovaginae, first reported from Japan in 1976, is now present in many agroecological regions around the world; it causes sheath brown rot of rice and is reported as a pathogen of a broad range of hosts. The pathogen can infect rice plants at all stages of growth and is known to cause significant losses due to grain discoloration, poor spike emergence and panicle sterility. Limited information is available on the virulence and mechanisms of pathogenicity for P. fuscovaginae. To address this, an analysis of genomes was conducted, which identified the presence of a gene showing homology to one of the genes contributing to syringopeptin synthetase (sypA) of P. syringae pv. syringae. To study the potential role of this gene in the virulence and pathogenicity of P. fuscovaginae, a site-specific mutation was created. Following inoculation of seeds and plantlets of rice and wheat with P. fuscovaginae wild types and their respective mutants, we demonstrated that the mutation significantly reduced virulence. This was evident on rice and wheat inoculated with mutants causing a significantly higher number of roots, length of roots and seedling height compared with their respective wild types. Characteristic disease symptoms of necrotic lesions were significantly less in rice seedlings infected with bacterial suspensions of mutants indicating a reduction in virulence. Chromatography analysis of bacterial exudates showed suppression of synthesis of metabolites analogous to syringopeptin in the mutants. These data demonstrate that the protein encoded by this sypA homolog gene is a major virulence determinant of P. fuscovaginae.

Whole-Genome Deep Sequencing Reveals Host-Driven in-planta Evolution of Columnea Latent Viroid (CLVd) Quasi-Species Populations.

Columnea latent viroid (CLVd) is one of the most serious tomato diseases. In general, viroids have high mutation rates. This generates a population of variants (so-called quasi-species) that co-exist in their host and exhibit a huge level of genetic diversity. To study the population of CLVd in individual host plants, we used amplicon sequencing using specific CLVd primers linked with a sample-specific index sequence to amplify libraries. An infectious clone of a CLVd isolate Chaipayon-1 was inoculated on different solanaceous host plants. Six replicates of the amplicon sequencing results showed very high reproducibility. On average, we obtained 133,449 CLVd reads per PCR-replicate and 79 to 561 viroid sequence variants, depending on the plant species. We identified 19 major variants (>1.0% mean relative abundance) in which a total of 16 single-nucleotide polymorphisms (SNPs) and two single nucleotide insertions were observed. All major variants contained a combination of 4 to 6 SNPs. Secondary structure prediction clustered all major variants into a tomato/bolo maka group with four loops (I, II, IV and V), and a chili pepper group with four loops (I, III, IV and V) at the terminal right domain, compared to the CLVd Chaipayon-1 which consists of five loops (I, II, III, IV and V).

Importance of the C12 Carbon Chain in the Biological Activity of Rhamnolipids Conferring Protection in Wheat against Zymoseptoria tritici.

The hemibiotrophic fungus Zymoseptoria tritici, responsible for Septoria tritici blotch, is currently the most devastating foliar disease on wheat crops worldwide. Here, we explored, for the first time, the ability of rhamnolipids (RLs) to control this pathogen, using a total of 19 RLs, including a natural RL mixture produced by Pseudomonas aeruginosa and 18 bioinspired RLs synthesized using green chemistry, as well as two related compounds (lauric acid and dodecanol). These compounds were assessed for in vitro antifungal effect, in planta defence elicitation (peroxidase and catalase enzyme activities), and protection efficacy on the wheat-Z. tritici pathosystem. Interestingly, a structure-activity relationship analysis revealed that synthetic RLs with a 12 carbon fatty acid tail were the most effective for all examined biological activities. This highlights the importance of the C12 chain in the bioactivity of RLs, likely by acting on the plasma membranes of both wheat and Z. tritici cells. The efficacy of the most active compound Rh-Est-C12 was 20-fold lower in planta than in vitro; an optimization of the formulation is thus required to increase its effectiveness. No Z. tritici strain-dependent activity was scored for Rh-Est-C12 that exhibited similar antifungal activity levels towards strains differing in their resistance patterns to demethylation inhibitor fungicides, including multi-drug resistance strains. This study reports new insights into the use of bio-inspired RLs to control Z. tritici.

Fluorescent Pseudomonas and cyclic lipopeptide diversity in the rhizosphere of cocoyam (Xanthosoma sagittifolium).

Cocoyam (Xanthosoma sagittifolium (L.)), an important tuber crop in the tropics, is severely affected by the cocoyam root rot disease (CRRD) caused by Pythium myriotylum. The white cocoyam genotype is very susceptible while the red cocoyam has some field tolerance to CRRD. Fluorescent Pseudomonas isolates obtained from the rhizosphere of healthy red and white cocoyams from three different fields in Cameroon were taxonomically characterized. The cocoyam rhizosphere was enriched with P. fluorescens complex and P. putida isolates independent of the plant genotype. LC-MS and NMR analyses revealed that 50% of the Pseudomonas isolates produced cyclic lipopeptides (CLPs) including entolysin, lokisin, WLIP, putisolvin and xantholysin together with eight novel CLPs. In general, CLP types were linked to specific taxonomic groups within the fluorescent pseudomonads. Representative CLP-producing bacteria showed effective control against CRRD while purified CLPs caused hyphal branching or hyphal leakage in P. myriotylum. The structure of cocoyamide A, a CLP which is predominantly produced by P. koreensis group isolates within the P. fluorescens complex is described. Compared with the white cocoyam, the red cocoyam rhizosphere appeared to support a more diverse CLP spectrum. It remains to be investigated whether this contributes to the field tolerance displayed by the red cocoyam.

Sweet Immunity: Inulin Boosts Resistance of Lettuce (Lactuca sativa) against Grey Mold (Botrytis cinerea) in an Ethylene-Dependent Manner.

The concept of "Sweet Immunity" postulates that sugar metabolism and signaling influence plant immune networks. In this study, we tested the potential of commercially available inulin-type fructans to limit disease symptoms caused by Botrytis cinerea in lettuce. Spraying mature lettuce leaves, with inulin-type fructans derived from burdock or chicory was as effective in reducing grey mold disease symptoms caused by Botrytis cinerea as spraying with oligogalacturonides (OGs). OGs are well-known defense elicitors in several plant species. Spraying with inulin and OGs induced accumulation of hydrogen peroxide and levels further increased upon pathogen infection. Inulin and OGs were no longer able to limit Botrytis infection when plants were treated with the ethylene signaling inhibitor 1-methylcyclopropene (1-MCP), indicating that a functional ethylene signaling pathway is needed for the enhanced defense response. Soluble sugars accumulated in leaves primed with OGs, while 1-MCP treatment had an overall negative effect on the sucrose pool. Accumulation of γ-aminobutyric acid (GABA), a stress-associated non-proteinogenic amino acid and possible signaling compound, was observed in inulin-treated samples after infection and negatively affected by the 1-MCP treatment. We have demonstrated for the first time that commercially available inulin-type fructans and OGs can improve the defensive capacity of lettuce, an economically important species. We discuss our results in the context of a possible recognition of fructans as Damage or Microbe Associated Molecular Patterns.

Conformation and Dynamics of the Cyclic Lipopeptide Viscosinamide at the Water-Lipid Interface.

Cyclic lipodepsipeptides or CLiPs from Pseudomonas are secondary metabolites that mediate a wide range of biological functions for their producers, and display antimicrobial and anticancer activities. Direct interaction of CLiPs with the cellular membranes is presumed to be essential in causing these. To understand the processes involved at the molecular level, knowledge of the conformation and dynamics of CLiPs at the water-lipid interface is required to guide the interpretation of biophysical investigations in model membrane systems. We used NMR and molecular dynamics to study the conformation, location and orientation of the Pseudomonas CLiP viscosinamide in a water/dodecylphosphocholine solution. In the process, we demonstrate the strong added value of combining uniform, isotope-enriched viscosinamide and protein NMR methods. In particular, the use of techniques to determine backbone dihedral angles and detect and identify long-lived hydrogen bonds, establishes that the solution conformation previously determined in acetonitrile is maintained in water/dodecylphosphocholine solution. Paramagnetic relaxation enhancements pinpoint viscosinamide near the water-lipid interface, with its orientation dictated by the amphipathic distribution of hydrophobic and hydrophilic residues. Finally, the experimental observations are supported by molecular dynamics simulations. Thus a firm structural basis is now available for interpreting biophysical and bioactivity data relating to this class of compounds.

Pseudomonas sp. COW3 Produces New Bananamide-Type Cyclic Lipopeptides with Antimicrobial Activity against Pythium myriotylum and Pyricularia oryzae.

Pseudomonas species are metabolically robust, with capacity to produce secondary metabolites including cyclic lipopeptides (CLPs). Herein we conducted a chemical analysis of a crude CLP extract from the cocoyam rhizosphere-derived biocontrol strain Pseudomonas sp. COW3. We performed in silico analyses on its whole genome, and conducted in vitro antagonistic assay using the strain and purified CLPs. Via LC-MS and NMR, we elucidated the structures of four novel members of the bananamide group, named bananamides D-G. Besides variability in fatty acid length, bananamides D-G differ from previously described bananamides A-C and MD-0066 by the presence of a serine and aspartic acid at position 6 and 2, respectively. In addition, bananamide G has valine instead of isoleucine at position 8. Kendrick mass defect (KMD) allowed the assignment of molecular formulae to bananamides D and E. We unraveled a non-ribosomal peptide synthetase cluster banA, banB and banC which encodes the novel bananamide derivatives. Furthermore, COW3 displayed antagonistic activity and mycophagy against Pythium myriotylum, while it mainly showed mycophagy on Pyricularia oryzae. Purified bananamides D-G inhibited the growth of P. myriotylum and P. oryzae and caused hyphal distortion. Our study shows the complementarity of chemical analyses and genome mining in the discovery and elucidation of novel CLPs. In addition, structurally diverse bananamides differ in their antimicrobial activity.

Gibberellin antagonizes jasmonate-induced defense against Meloidogyne graminicola in rice.

Gibberellin (GA) regulates various plant growth and developmental processes, but its role in pathogen attack, and especially nematode-plant interactions, still remains to be elucidated. An in-depth characterization of the role of GA in nematode infection was conducted using mutant lines of rice, chemical inhibitors, and phytohormone measurements. Our results showed that GA influences rice-Meloidogyne graminicola interactions in a concentration-dependent manner. Foliar spray of plants with a low concentration of gibberellic acid enhanced nematode infection. Biosynthetic and signaling mutants confirmed the importance of gibberellin for rice susceptibility to M. graminicola infection. Our study also demonstrates that GA signaling suppresses jasmonate (JA)-mediated defense against M. graminicola, and likewise the JA-induced defense against M. graminicola requires SLENDER RICE1 (SLR1)-mediated repression of the GA pathway. In contrast to observations from other plant-pathogen interactions, GA plays a dominant role over JA in determining susceptibility to M. graminicola in rice. This GA-induced nematode susceptibility was largely independent of auxin biosynthesis, but relied on auxin transport. In conclusion, we showed that GA-JA antagonistic crosstalk is at the forefront of the interaction between rice and M. graminicola, and SLR1 plays a central role in the JA-mediated defense response in rice against this nematode.