Characterization of a partially saturated and glycosylated apocarotenoid from wheat that is depleted upon leaf rust infection.

Recent semi-targeted metabolomics studies have highlighted a number of metabolites in wheat that associate with leaf rust resistance genes and/or rust infection. Here, we report the structural characterization of a novel glycosylated and partially saturated apocarotenoid, reminiscent of a reduced form of mycorradicin, (6E,8E,10E)-4,9-dimethyl-12-oxo-12-((3,4,5-trihydroxy-6-(2-hydroxyethoxy)tetrahydro-2H-pyran-2-yl)methoxy)-3-((3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)dodeca-6,8,10-trienoic acid, isolated from Triticum aestivum L. (Poaceae) variety 'Thatcher' (Tc) flag leaves. While its accumulation was not associated with any of Lr34, Lr67 or Lr22a resistance genes, infection of Tc with leaf rust was found to deplete it, consistent with the idea of this metabolite being a glycosylated-storage form of an apocarotenoid of possible relevance to plant defense. A comparative analysis of wheat transcriptomic changes shows modulation of terpenoid, carotenoid, UDP-glycosyltransferase and glycosylase -related gene expression profiles, consistent with anticipated biosynthesis and degradation mechanisms. However, details of the exact nature of the relevant pathways remain to be validated in the future. Together these findings highlight another example of the breadth of unique metabolites underlying plant host-fungal pathogen interactions.

Assay Development for Metal-Dependent Enzymes-Influence of Reaction Buffers on Activities and Kinetic Characteristics.

Buffers are often thought of as innocuous components of a reaction, with the sole task of maintaining the pH of a system. However, studies had shown that this is not always the case. Common buffers used in biochemical research, such as Tris (hydroxymethyl) aminomethane hydrochloride (Tris-HCl), can chelate metal ions and may thus affect the activity of metalloenzymes, which are enzymes that require metal ions for enhanced catalysis. To determine whether enzyme activity is influenced by buffer identity, the activity of three enzymes (BLC23O, Ro1,2-CTD, and trypsin) was comparatively characterized in N-2- hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), Tris-HCl, and sodium phosphate buffer. The pH and temperature optima of BLC23O, a Mn2+-dependent dioxygenase, were first identified, and then the metal ion dissociation constant (Kd) was determined in the three buffer systems. It was observed that BLC23O exhibited different Kd values depending on the buffer, with the lowest (1.49 ± 0.05 µM) recorded in HEPES under the optimal set of conditions (pH 7.6 and 32.5 °C). Likewise, the kinetic parameters obtained varied depending on the buffer, with HEPES (pH 7.6) yielding overall the greatest catalytic efficiency and turnover number (kcat = 0.45 ± 0.01 s-1; kcat/Km = 0.84 ± 0.02 mM-1 s-1). To corroborate findings, the characterization of Fe3+-dependent Ro1,2-CTD was performed, resulting in different kinetic constants depending on the buffer (Km (HEPES, Tris-HCl, and Na-phosphate) = 1.80, 6.93, and 3.64 µM; kcat(HEPES, Tris-HCl, and Na-phosphate) = 0.64, 1.14, and 1.01 s-1; kcat/Km(HEPES, Tris-HCl, and Na-phosphate)= 0.36, 0.17, and 0.28 µM-1 s-1). In order to determine whether buffer identity influenced the enzymatic activity of nonmetalloenzymes alike, the characterization of trypsin was also carried out. Contrary to the previous results, trypsin yielded comparable kinetic parameters independent of the buffer (Km (HEPES, Tris-HCl, and Na-Phosphate) = 3.14, 3.07, and 2.91 mM; kcat(HEPES, Tris-HCl, and Na-phosphate) = 1.51, 1.47, and 1.53 s-1; kcat/Km (HEPES, Tris-HCl, and Na-phosphate) = 0.48, 0.48, and 0.52 mM-1 s-1). These results showed that the activity of tested metalloenzymes was impacted by different buffers. While selected buffers did not influence the tested nonmetalloenzyme activity, other research had shown impacts of buffers on other enzyme activities. As a result, we suggest that buffer selection be optimized for any new enzymes such that the results from one lab to another can be accurately compared.

Multiple Clonostachys rosea UDP-Glycosyltransferases Contribute to the Production of 15-Acetyl-Deoxynivalenol-3-O-Glycoside When Confronted with Fusarium graminearum.

Mycotoxins, derived from toxigenic fungi such as Fusarium, Aspergillus, and Penicillium species have impacted the human food chain for thousands of years. Deoxynivalenol (DON), is a tetracyclic sesquiterpenoid type B trichothecene mycotoxin predominantly produced by F. culmorum and F. graminearum during the infection of corn, wheat, oats, barley, and rice. Glycosylation of DON is a protective detoxification mechanism employed by plants. More recently, DON glycosylating activity has also been detected in fungal microparasitic (biocontrol) fungal organisms. Here we follow up on the reported conversion of 15-acetyl-DON (15-ADON) into 15-ADON-3-O-glycoside (15-ADON-3G) in Clonostachys rosea. Based on the hypothesis that the reaction is likely being carried out by a uridine diphosphate glycosyl transferase (UDP-GTase), we applied a protein structural comparison strategy, leveraging the availability of the crystal structure of rice Os70 to identify a subset of potential C. rosea UDP-GTases that might have activity against 15-ADON. Using CRISPR/Cas9 technology, we knocked out several of the selected UDP-GTases in the C. rosea strain ACM941. Evaluation of the impact of knockouts on the production of 15-ADON-3G in confrontation assays with F. graminearum revealed multiple UDP-GTase enzymes, each contributing partial activities. The relationship between these positive hits and other UDP-GTases in fungal and plant species is discussed.

Clonostachys rosea 'omics profiling: identification of putative metabolite-gene associations mediating its in vitro antagonism against Fusarium graminearum.

BACKGROUND: Clonostachys rosea is an established biocontrol agent. Selected strains have either mycoparasitic activity against known pathogens (e.g. Fusarium species) and/or plant growth promoting activity on various crops. Here we report outcomes from a comparative 'omics analysis leveraging a temporal variation in the in vitro antagonistic activities of C. rosea strains ACM941 and 88-710, toward understanding the molecular mechanisms underpinning mycoparasitism. RESULTS: Transcriptomic data highlighted specialized metabolism and membrane transport related genes as being significantly upregulated in ACM941 compared to 88-710 at a time point when the ACM941 strain had higher in vitro antagonistic activity than 88-710. In addition, high molecular weight specialized metabolites were differentially secreted by ACM941, with accumulation patterns of some metabolites matching the growth inhibition differences displayed by the exometabolites of the two strains. In an attempt to identify statistically relevant relationships between upregulated genes and differentially secreted metabolites, transcript and metabolomic abundance data were associated using IntLIM (Integration through Linear Modeling). Of several testable candidate associations, a putative C. rosea epidithiodiketopiperazine (ETP) gene cluster was identified as a prime candidate based on both co-regulation analysis and transcriptomic-metabolomic data association. CONCLUSIONS: Although remaining to be validated functionally, these results suggest that a data integration approach may be useful for identification of potential biomarkers underlying functional divergence in C. rosea strains.

Selective Quantification of Chemotropic Responses of Fusarium graminearum.

Chemotropism refers to the directional growth of a living organism toward a chemical stimulus. Molecular mechanisms underlying chemotropism of fungal pathogens have recently been enabled by advancements in biological chemotropic assays, with a particular focus on the roles of G-protein-coupled receptors and their plant-derived ligands in chemotropism. Here we describe in detail an assay that enables quantification of chemotropic responses of Fusarium graminearum, with variations recently reported for Fusarium oxysporum and Trichoderma atroviride.

Reduction of phenolics in faba bean meal using recombinantly produced and purified Bacillus ligniniphilus catechol 2,3-dioxygenase.

Pulse meal should be a valuable product in the animal feed industry based on its strong nutritional and protein profiles. However, it contains anti-nutritional compounds including phenolics (large and small molecular weight), which must be addressed to increase uptake by the industry. Microbial fermentation is currently used as a strategy to decrease larger molecular weight poly-phenolics, but results in the undesirable accumulation of small mono-phenolics. Here, we investigate cell-free biocatalytic reduction of phenolic content in faba bean (Vicia faba L.) meal. A representative phenolic ring-breaking catechol dioxygenase, Bacillus ligniniphilus L1 catechol 2,3-dioxygenase (BLC23O) was used in this proof-of concept based on its known stability and broad substrate specificity. Initially, large-scale fermentative recombinant production and purification of BLC23O was carried out, with functionality validated by in vitro kinetic analysis. When applied to faba bean meal, BLC23O yielded greatest reductions in phenolic content in a coarse air classified fraction (high carbohydrate), compared to either a fine fraction (high protein) or the original unfractionated meal. However, the upstream hydrolytic release of phenolics from higher molecular weight species (e.g. tannins, or complexes with proteins and carbohydrates) likely remains a rate limiting step, in the absence of other enzymes or microbial fermentation. Consistent with this, when applied to a selection of commercially available purified phenolic compounds, known to occur in faba bean, BLC23O was found to have high activity against monophenolic acids and little if any detectable activity against larger molecular weight compounds. Overall, this study highlights the potential viability of the biocatalytic processing of pulse meals, for optimization of their nutritional and economical value in the animal feed industry. Supplementary Information: The online version contains supplementary material available at 10.1186/s40643-023-00633-8.

A peroxidase-derived ligand that induces Fusarium graminearum Ste2 receptor-dependent chemotropism.

Introduction: The fungal G protein-coupled receptors Ste2 and Ste3 are vital in mediating directional hyphal growth of the agricultural pathogen Fusarium graminearum towards wheat plants. This chemotropism is induced by a catalytic product of peroxidases secreted by the wheat. Currently, the identity of this product, and the substrate it is generated from, are not known. Methods and results: We provide evidence that a peroxidase substrate is derived from F. graminearum conidia and report a simple method to extract and purify the FgSte2-activating ligand for analyses by mass spectrometry. The mass spectra arising from t he ligand extract are characteristic of a 400 Da carbohydrate moiety. Consistent with this type of molecule, glycosidase treatment of F. graminearum conidia prior to peroxidase treatment significantly reduced the amount of ligand extracted. Interestingly, availability of the peroxidase substrate appears to depend on the presence of both FgSte2 and FgSte3, as knockout of one or the other reduces the chemotropism-inducing effect of the extracts. Conclusions: While further characterization is necessary, identification of the F. graminearum-derived peroxidase substrate and the FgSte2-activating ligand will unearth deeper insights into the intricate mechanisms that underlie fungal pathogenesis in cereal crops, unveiling novel avenues for inhibitory interventions.

Pea and lentil 7S globulin crystal structures with comparative immunoglobulin epitope mapping.

Legumes represent an affordable high protein, nutrient dense food source. However, the vast majority of legume crops contain proteins that are known allergens for susceptible individuals. These include proteins from the 7S globulin family, which comprise a vast majority of seed storage proteins. Here, the crystal structures of 7S globulins from Pisum sativum L. (pea) and Lens culinaris Medicus (lentil) are presented for the first time, including pea vicillin and convicilin, and lentil vicilin. All three structures maintain the expected 7S globulin fold, with trimeric quaternary structure and monomers comprised of ß-barrel N- and C-modules. The potential impact of sequence differences on structure and packing in the different crystal space groups is noted, with potential relevance to packing upon seed deposition. Mapping on the obtained crystal structures highlights significant Ig epitope overlap between pea, lentil, peanut and soya bean and significant coverage of the entire seed storage protein, emphasizing the challenge in addressing food allergies. How recently developed biologicals might be refined to be more effective, or how these seed storage proteins might be modified in planta to be less immuno-reactive remain challenges for the future. With legumes representing an affordable, high protein, nutrient dense food source, this work will enable important research in the context of global food security and human health on an ongoing basis.

Fusarium graminearum Ste3 G-Protein Coupled Receptor: A Mediator of Hyphal Chemotropism and Pathogenesis.

Fungal hyphal chemotropism has been shown to be a major contributor to host-pathogen interactions. Previous studies on Fusarium species have highlighted the involvement of the Ste2 G-protein-coupled receptor (GPCR) in mediating polarized hyphal growth toward host-released peroxidase. Here, the role of the opposite mating type GPCR, Ste3, is characterized with respect to Fusarium graminearum chemotropism and pathogenicity. Fgste3Δ deletion strains were found to be compromised in the chemotropic response toward peroxidase, development of lesions on germinating wheat, and infection of Arabidopsis thaliana leaves. In the absence of FgSte3 or FgSte2, F. graminearum cells exposed to peroxidase showed no phosphorylation of the cell-wall integrity, mitogen-activated protein kinase pathway component Mgv1. In addition, transcriptomic gene expression profiling yielded a list of genes involved in cellular reorganization, cell wall remodeling, and infection-mediated responses that were differentially modulated by peroxidase when FgSte3 was present. Deletion of FgSte3 yielded the downregulation of genes associated with mycotoxin biosynthesis and appressorium development, compared to the wild-type strain, both in the presence of peroxidase. Together, these findings contribute to our understanding of the mechanism underlying fungal chemotropism and pathogenesis while raising the novel hypothesis that FgSte2 and FgSte3 are interdependent on each other for the mediation of the redirection of hyphal growth in response to host-derived peroxidase. IMPORTANCE Fusarium head blight of wheat, caused by the filamentous fungus Fusarium graminearum, leads to devastating global food shortages and economic losses. Fungal hyphal chemotropism has been shown to be a major contributor to host-pathogen interactions. Here, the role of the opposite mating type GPCR, Ste3, is characterized with respect to F. graminearum chemotropism and pathogenicity. These findings contribute to our understanding of the mechanisms underlying fungal chemotropism and pathogenesis.

Wheat transcriptome profiling reveals abscisic and gibberellic acid treatments regulate early-stage phytohormone defense signaling, cell wall fortification, and metabolic switches following Fusarium graminearum-challenge.

BACKGROUND: Treatment of wheat with the phytohormones abscisic acid (ABA) and gibberellic acid (GA) has been shown to affect Fusarium head blight (FHB) disease severity. However, the molecular mechanisms underlying the elicited phenotypes remain unclear. Toward addressing this gap in our knowledge, global transcriptomic profiling was applied to the FHB-susceptible wheat cultivar 'Fielder' to map the regulatory responses effected upon treatment with ABA, an ABA receptor antagonist (AS6), or GA in the presence or absence of Fusarium graminearum (Fg) challenge. RESULTS: Spike treatments resulted in a total of 30,876 differentially expressed genes (DEGs) identified in 'Fielder' (26,004) and the Fg (4872) pathogen. Topology overlap and correlation analyses defined 9689 wheat DEGs as Fg-related across the treatments. Further enrichment analyses demonstrated that these included expression changes within 'Fielder' defense responses, cell structural metabolism, molecular transport, and membrane/lipid metabolism. Dysregulation of ABA and GA crosstalk arising from repression of 'Fielder' FUS3 was noted. As well, expression of a putative Fg ABA-biosynthetic cytochrome P450 was detected. The co-applied condition of Fg + ABA elicited further up-regulation of phytohormone biosynthesis, as well as SA and ET signaling pathways and cell wall/polyphenolic metabolism. In contrast, co-applied Fg + GA mainly suppressed phytohormone biosynthesis and signaling, while modulating primary and secondary metabolism and flowering. Unexpectedly, co-applied Fg + AS6 did not affect ABA biosynthesis or signaling, but rather elicited antagonistic responses tied to stress, phytohormone transport, and FHB disease-related genes. CONCLUSIONS: Observed exacerbation (misregulation) of classical defense mechanisms and cell wall fortifications upon ABA treatment are consistent with its ability to promote FHB severity and its proposed role as a fungal effector. In contrast, GA was found to modulate primary and secondary metabolism, suggesting a general metabolic shift underlying its reduction in FHB severity. While AS6 did not antagonize traditional ABA pathways, its impact on host defense and Fg responses imply potential for future investigation. Overall, by comparing these findings to those previously reported for four additional plant genotypes, an additive model of the wheat-Fg interaction is proposed in the context of phytohormone responses.

3'-(Phenyl alkynyl) analogs of abscisic acid: synthesis and biological activity of potent ABA antagonists.

We report here the synthesis and biological testing of 3'-(phenyl alkynyl) abscisic ABA analogs, a new class of potent ABA antagonists. These ABA analogs incorporate a rigid framework of eight carbon atoms attached at the 3'-carbon atom of ABA that prevents folding of the ABA analog-bound receptor required for ABA signalling. The two-step synthesis is based upon the optimized conversion of natural (S)-ABA to 3'-iodo ABA which can be coupled to phenyl acetylenes using Sonogashira conditions, or to styryl compounds through Suzuki chemistry. The parent 3'-(phenyl alkynyl) ABA analog 7 was obtained in 29% yield, 74% yield based on recovered starting material. In a lentil seed germination assay, compound 7 was found to have more potent activity than other known 3'-substituted ABA antagonists to date. In a structure activity study parasubstituted phenyl alkynyl analogs had comparable activity to the analog 7 while the 3'-styryl ABA 18 was only slightly less active. Analog 7 overcame ABA inhibition of germination and seedling growth in a wide range of mono and dicot plant species, including canola, lentil, soybean, rice, wheat, barley, cannabis and canary seed. 3'-(Phenyl alkynyl) ABA analogs have numerous potential practical agricultural applications including promoting ripening of crops, dormancy breaking of seeds and woody perennials, as well as promoting seed germination, and growth under stress conditions as demonstrated in this report.

Draft Genome Resources for Plant-Beneficial Fungi Clonostachys rosea Strains ACM941 and 88-710.

Clonostachys rosea strains ACM941 and 88-710 are beneficial microbes recognized for their plant disease control and growth promotion properties, respectively, when applied to economically important crops. In addition to their geographical and functional overlap, the two strains also share a high degree of genetic similarity. In an effort to identify the subtleties that underlie their strain-specific applications, their genomic sequence is reported here. The genome size of ACM941 was estimated to be 56.9 Mb, encoding 17,585 putative genes, while strain 88-710 was estimated to have a 55.5 Mb genome size, containing 17,188 predicted genes. Overall, ACM941 and 88-710 share >96% of their encoded genomes, such that their strain-specific characteristics are likely encoded in either the remaining variable 4% or differentially regulated shared genes or both. These genomic sequences form a foundation for future studies aimed at identifying the genomic and metabolic machinery driving their respective beneficial properties.

Barley "uzu" and Wheat "uzu-like" Brassinosteroid Receptor BRI1 Kinase Domain Variations Modify Phosphorylation Activity In Vitro.

Brassinosteroid insensitive1 (BRI1), a leucine-rich repeat receptor kinase, is responsible for the perception of the brassinosteroid (BR) phytohormone in plants. While recent evidence has implicated a naturally occurring Hordeum vulgare V. (barley) HvBRI1 kinase domain (KD) variant (H857R; "uzu" variation) in increased fungal disease resistance, the impact of the variation on receptor function and thus the mechanism by which disease resistance might be imparted remain enigmatic. Here, the functional implications of the uzu variation as well as the effects of newly identified naturally occurring Triticum aestivum L. (wheat) TaBRI1-KD variants are investigated. Recombinantly produced KDs of wild-type (WT) and uzu HvBRI1 were assessed for phosphorylation activity in vitro, yielding WT KM and VMAX values similar to those of other reports, but the uzu variation delayed saturation and reduced turnover levels. In silico modeling of the H857R variation showed it to be surface-exposed and distal from the catalytic site. Further evaluation of three naturally occurring wheat TaBRI1 variants, A907T, A970V, and G1019R (barley numbering) identified in the A, B, and D subgenomic genes, respectively, highlighted a significant loss of activity for A907T. A907T is located on the same surface as the H857R variation and a negative regulatory phosphorylation site (T982) in Arabidopsis thaliana BRI1. A fourth variation, T1031A (barley numbering), unique to both subgenomic A proteins and localized to the BKI1 binding site, also decreased activity. The outcomes are discussed with respect to the predicted structural contexts of the variations and their implications with respect to mechanisms of action.

A phenylpropanoid diglyceride associates with the leaf rust resistance Lr34res gene in wheat.

The gene Lr34res is one of the most long-lasting sources of quantitative fungal resistance in wheat. It is shown to be effective against leaf, stem, and stripe rusts, as well as powdery mildew and spot blotch. Recent biochemical characterizations of the encoded ABC transporter have outlined a number of allocrites, including phospholipids and abscisic acid, consistent with the established general promiscuity of ABC transporters, but ultimately leaving its mechanism of rust resistance unclear. Working with flag leaves of Triticum aestivum L. variety 'Thatcher' (Tc) and a near-isogenic line of 'Thatcher' into which the Lr34res allele was introgressed (Tc+Lr34res; RL6058), a comparative semi-targeted metabolomics analysis of flavonoid-rich extracts revealed virtually identical profiles with the exception of one metabolite accumulating in Tc+Lr34res, which was not present at comparable levels in Tc. Structural characterization of the purified metabolite revealed a phenylpropanoid diglyceride structure, 1-O-p-coumaroyl-3-O-feruloylglycerol (CFG). Additional profiling of CFG across a collection of near-isogenic lines and representative Lr34 haplotypes highlighted a broad association between the presence of Lr34res and elevated accumulations of CFG. Depletion of CFG upon infection, juxtaposed to its relatively lower anti-fungal activity, suggests CFG may serve as a storage form of the more potent anti-microbial hydroxycinnamic acids that are accessed during defense responses. Altogether these findings suggest a role for the encoded LR34res ABC transporter in modifying the accumulation of CFG, leading to increased accumulation of anti-fungal metabolites, essentially priming the wheat plant for defense.

Ste2 receptor-mediated chemotropism of Fusarium graminearum contributes to its pathogenicity against wheat.

Fusarium Head Blight of wheat, caused by the filamentous fungus Fusarium graminearum, leads to devastating global food shortages and economic losses. While many studies have addressed the responses of both wheat and F. graminearum during their interaction, the possibility of fungal chemotropic sensing enabling pathogenicity remains unexplored. Based on recent findings linking the pheromone-sensing G-protein-coupled receptor Ste2 to host-directed chemotropism in Fusarium oxysporum, we investigated the role of the Ste2 receptor and its downstream signaling pathways in mediating chemotropism of F. graminearum. Interestingly, a chemotropic response of growing hyphae towards catalytically active Triticum aestivum 'Roblin' cultivar secreted peroxidases was detected, with deletion of STE2 in F. graminearum leading to loss of the observed response. At the same time, deletion of STE2 significantly decreased infection on germinating wheat coleoptiles, highlighting an association between Ste2, chemotropism and infection by F. graminearum. Further characterization revealed that the peroxidase-directed chemotropism is associated with stimulation of the fungal cell wall integrity mitogen-activated protein kinase signaling cascade. Altogether, this study demonstrates conservation of Ste2-mediated chemotropism by Fusarium species, and its important role in mediating pathogenicity.

Transcriptomic and Exometabolomic Profiling Reveals Antagonistic and Defensive Modes of Clonostachys rosea Action Against Fusarium graminearum.

The mycoparasite Clonostachys rosea ACM941 is under development as a biocontrol organism against Fusarium graminearum, the causative agent of Fusarium head blight in cereals. To identify molecular factors associated with this interaction, the transcriptomic and exometabolomic profiles of C. rosea and F. graminearum GZ3639 were compared during coculture. Prior to physical contact, the antagonistic activity of C. rosea correlated with a response heavily dominated by upregulation of polyketide synthase gene clusters, consistent with the detected accumulation of corresponding secondary metabolite products. Similarly, prior to contact, trichothecene gene clusters were upregulated in F. graminearum, while those responsible for fusarielin and fusarin biosynthesis were downregulated, correlating with an accumulation of trichothecene products in the interaction zone over time. A concomitant increase in 15-acetyl deoxynivalenol-3-glucoside in the interaction zone was also detected, with C. rosea established as the source of this detoxified mycotoxin. After hyphal contact, C. rosea was found to predominantly transcribe genes encoding cell wall-degradation enzymes, major facilitator superfamily sugar transporters, anion:cation symporters, as well as alternative carbon source utilization pathways, together indicative of a transition to necrotropism at this stage. F. graminearum notably activated the transcription of phosphate starvation pathway signature genes at this time. Overall, a number of signature molecular mechanisms likely contributing to antagonistic activity by C. rosea against F. graminearum, as well as its mycotoxin tolerance, are identified in this report, yielding several new testable hypotheses toward understanding the basis of C. rosea as a biocontrol agent for continued agronomic development and application.

A Universally Primed-Polymerase Chain Reaction (UP-PCR) Marker to Discriminate Clonostachys rosea ACM941 from Related Strains.

Clonostachys rosea strain ACM941 is an effective biocontrol agent against several crop diseases including Fusarium head blight. In anticipation of its increased relevance going forward, the development of a reliable DNA-based molecular marker to track it is essential. Universally primed-PCR (UP-PCR) has been used successfully to differentiate other C. rosea strains. Herein, the development of a UP-PCR marker for ACM941 is described. A combination of two primers (AS15 and L45) produced a ~450 bp fragment that was unique to ACM941 compared to other commercial biocontrol agents. Primers subsequently designed based on the obtained fragment also produced a similarly unique band from ACM941 alone. BLAST analysis of the amplified sequence did not yield any homologous sequence in available online databases or within the closely related C. rosea IK726 and CBS125111 strains' genomes. The specificity of this marker for ACM941 was validated against ten additional C. rosea strains isolated from Canada, with ACM941 producing the brightest band. Taken together, these results imply that the UP-PCR primers AS15 and L45 and the amplified fragment can be used to detect and monitor the ACM941 strain after its release into the environment.

The malate-activated ALMT12 anion channel in the grass Brachypodium distachyon is co-activated by Ca2+/calmodulin.

In plants, strict regulation of stomatal pores is critical for modulation of CO2 fixation and transpiration. Under certain abiotic and biotic stressors, pore closure is initiated through anionic flux, with calcium (Ca2+) playing a central role. The aluminum-activated malate transporter 12 (ALMT12) is a malate-activated, voltage-dependent member of the aluminum-activated malate transporter family that has been implicated in anionic flux from guard cells controlling the stomatal aperture. Herein, we report the characterization of the regulatory mechanisms mediating channel activities of an ALMT from the grass Brachypodium distachyon (BdALMT12) that has the highest sequence identity to Arabidopsis thaliana ALMT12. Electrophysiological studies in a heterologous cell system confirmed that this channel is malate- and voltage-dependent. However, this was shown to be true only in the presence of Ca2+ Although a general kinase inhibitor increased the current density of BdALMT12, a calmodulin (CaM) inhibitor reduced the Ca2+-dependent channel activation. We investigated the physiological relevance of the CaM-based regulation in planta, where stomatal closure, induced by exogenous Ca2+ ionophore and malate, was shown to be inhibited by exogenous application of a CaM inhibitor. Subsequent analyses revealed that the double substitutions R335A/R338A and R335A/K342A, within a predicted BdALMT12 CaM-binding domain (CBD), also decreased the channels' ability to activate. Using isothermal titration calorimetry and CBD-mimetic peptides, as well as CaM-agarose affinity pulldown of full-length recombinant BdALMT12, we confirmed the physical interaction between the CBD and CaM. Together, these findings support a co-regulatory mechanism of BdALMT12 activation by malate, and Ca2+/CaM, emphasizing that a complex regulatory network modulates BdALMT12 activity.

Structure and function of a lignostilbene-α,ß-dioxygenase orthologue from Pseudomonas brassicacearum.

BACKGROUND: Stilbene cleaving oxygenases (SCOs), also known as lignostilbene-α,ß-dioxygenases (LSDs) mediate the oxidative cleavage of the olefinic double bonds of lignin-derived intermediate phenolic stilbenes, yielding small modified benzaldehyde compounds. SCOs represent one branch of the larger carotenoid cleavage oxygenases family. Here, we describe the structural and functional characterization of an SCO-like enzyme from the soil-born, bio-control agent Pseudomonas brassicacearum. METHODS: In vitro and in vivo assays relying on visual inspection, spectrophotometric quantification, as well as liquid-chormatographic and mass spectrometric characterization were applied for functional evaluation of the enzyme. X-ray crystallographic analyses and in silico modeling were applied for structural investigations. RESULTS: In vitro assays demonstrated preferential cleavage of resveratrol, while in vivo analyses detected putative cleavage of the straight chain carotenoid, lycopene. A high-resolution structure containing the seven-bladed ß-propeller fold and conserved 4-His-Fe unit at the catalytic site, was obtained. Comparative structural alignments, as well as in silico modelling and docking, highlight potential molecular factors contributing to both the primary in vitro activity against resveratrol, as well as the putative subsidiary activities against carotenoids in vivo, for future validation. CONCLUSIONS: The findings reported here provide validation of the SCO structure, and highlight enigmatic points with respect to the potential effect of the enzyme's molecular environment on substrate specificities for future investigation.

Profiling of the Transcriptomic Responses of Clonostachys rosea Upon Treatment With Fusarium graminearum Secretome.

Clonostachys rosea strain ACM941 is a fungal bio-control agent patented against the causative agent of Fusarium Head Blight, Fusarium graminearum. Although the molecular details remain enigmatic, previous studies have suggested that C. rosea may secrete F. graminearum growth inhibitors. Further toward this, experiments described herein show that induction of C. rosea cultures by the addition of an aliquot of F. graminearum(Fg)-spent media (including macroconidia), yield C. rosea (Cr)-spent media that elicited higher anti-F. graminearum activity than either control or deoxynivalenol (DON)-induced Cr-spent media. To gain additional insight into the genetic and metabolic factors modulating this interaction, transcriptomic (RNAseq) profiles of C. rosea in response to DON and Fg-spent media treatment, were developed. This analysis revealed 24,112 C. rosea unigenes, of which 5,605 and 6,285 were differentially regulated by DON and F-spent media, respectively. More than half of these unigenes were up-regulated, with annotations, most notably in the Fg-spent media treatment data, suggesting enhancement of polyketide (PK) and non-ribosomal peptide (NRP) secondary metabolite precursor synthesis, and PK/NRP-like synthases. Four ABC transporters were also up-regulated in response to Fg-spent media. Further analysis showed that the PK and NRP-like synthases belong to three gene clusters that also include ABC transporters, and other genes known to tailor secondary metabolite biosynthesis. The RNAseq data was further validated using quantitative RT-qPCR. Taken together, these results show that C. rosea responds to the presence of Fg-spent media (and to a lesser extent, DON-alone) by up-regulating unique aspects of its secondary metabolism-related genetic repertoire. The identities and roles of C. rosea secondary metabolites produced by the targeted gene clusters are now under investigation.