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.

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.

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.

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.

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.

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.

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.

Abscisic Acid Analogues That Act as Universal or Selective Antagonists of Phytohormone Receptors.

The plant hormone abscisic acid (ABA) plays many important roles in controlling plant development and physiology, from flowering to senescence. ABA is now known to exert its effects through a family of soluble ABA receptors, which in Arabidopsis thaliana has 13 members divided into three clades. Homologues of these receptors are present in other plants, also in relatively large numbers. Investigation of the roles of each homologue in mediating the diverse physiological roles of ABA is hampered by this genetic redundancy. We report herein the in vitro screening of a targeted ABA-like analogue library and identification of novel antagonist hits, including the analogue PBI686 that had been developed previously as a probe for identifying ABA-binding proteins. Further in vitro characterization of PBI686 and development of second-generation leads yielded both receptor-selective and universal antagonist hits. In planta assays in different species have demonstrated that these antagonist leads can overcome various ABA-induced physiological changes. While the general antagonists open up a hitherto unexplored avenue for controlling plant growth through inhibition of ABA-regulated physiological processes, the receptor-selective antagonist can be developed into chemical probes to explore the physiological roles of individual receptors.

Probing allocrite preferences of 2 naturally occurring variants of the wheat LR34 ABC transporter.

For almost a century, the wheat Lr34 gene has conferred durable resistance against fungal rust diseases. While sequence homology predicts a putative ATP binding cassette transporter, the molecules that are transported (allocrites) by the encoded LR34 variants, and any associated mechanism of resistance, remain enigmatic. Here, the in vitro transport characteristics of 2 naturally occurring Lr34 variants (that differ in their ability to mediate disease resistance; Lr34sus and Lr34res) are investigated. Initially, a method to express and purify recombinant LR34Sus and LR34Res pseudo half-molecules from Saccharomyces cerevisiae, is described. Subsequently, a semi-targeted chlorophyll catabolite (CC) extraction from Lr34res-expressing wheat plants was performed based on previous reports highlighting increased accumulation of CCs in Lr34res-expressing flag leaves. Following partial biochemical characterization, this extract was applied to an LR34 in vitro proteoliposome transport assay. Mass spectroscopic analyses of transported metabolites revealed that LR34Sus imported a wheat metabolite of 618 Da and that the LR34Res transporter did not. While the identity of the LR34Sus transported metabolite remains to be confirmed and any allocrites of LR34Res remain to be detected, this work demonstrates that these variants have different allocrite preferences, a finding that may be relevant to the mechanism of disease resistance.

Evidence of a role for S. cerevisiae α-arrestin Art1 (Ldb19) in mating projection and zygote formations.

The discovery of arrestin-mediated biased signalling mechanisms for mammalian G-protein coupled receptors (GPCRs) has revolutionized the field over the last decade. Now, with the recent demonstration of a role for α-arrestins in internalization of the yeast pheromone GPCR, Ste2p, the possibility of arrestin-mediated alternate GPCR functionalities in yeast also follows. Here, the effects of knockout and complementation of yeast α-arrestin expression during mating are reported. Although minor effects on classical pheromone-related signalling are noted for a few arrestins, much stronger effects were observed downstream of cell cycle arrest, in particular linking Ldb19 (Art1) to mediation of zygote formation. Subsequent phenotypic observations linked this activity to more pronounced projection formation in an Art1 complemented noncuple α-arrestin knockout line, compared to the knockout-line alone, or either of the Art3 or Art6 complemented lines. Together with the observation of ligand-stimulated localization of Art-GFP to the mating projection, a possible role for this arrestin-like protein in projection formation is supported. While leaving the full mechanism of alternate Art1 functionality to be elucidated, together these findings implicate Art1 in selective regulation of mating events downstream of receptor internalization and cell cycle arrest, leading to schmoo, and ultimately zygote formation.

Exogenous Abscisic Acid and Gibberellic Acid Elicit Opposing Effects on Fusarium graminearum Infection in Wheat.

Although the roles of salicylate (SA) and jasmonic acid (JA) have been well-characterized in Fusarium head blight (FHB)-infected cereals, the roles of other phytohormones remain more ambiguous. Here, the association between an array of phytohormones and FHB pathogenesis in wheat is investigated. Comprehensive profiling of endogenous hormones demonstrated altered cytokinin, gibberellic acid (GA), and JA metabolism in a FHB-resistant cultivar, whereas challenge by Fusarium graminearum increased abscisic acid (ABA), JA, and SA in both FHB-susceptible and -resistant cultivars. Subsequent investigation of ABA or GA coapplication with fungal challenge increased and decreased FHB spread, respectively. These phytohormones-induced effects may be attributed to alteration of the F. graminearum transcriptome because ABA promoted expression of early-infection genes, including hydrolases and cytoskeletal reorganization genes, while GA suppressed nitrogen metabolic gene expression. Neither ABA nor GA elicited significant effects on F. graminearum fungal growth or sporulation in axenic conditions, nor do these phytohormones affect trichothecene gene expression, deoxynivalenol mycotoxin accumulation, or SA/JA biosynthesis in F. graminearum-challenged wheat spikes. Finally, the combined application of GA and paclobutrazol, a Fusarium fungicide, provided additive effects on reducing FHB severity, highlighting the potential for combining fungicidal agents with select phytohormone-related treatments for management of FHB infection in wheat.

Abscisic Acid Acts as a Blocker of the Bitter Taste G Protein-Coupled Receptor T2R4.

Bitter taste receptors (T2Rs) belong to the G protein-coupled receptor superfamily. In humans, 25 T2Rs mediate bitter taste sensation. In addition to the oral cavity, T2Rs are expressed in many extraoral tissues, including the central nervous system, respiratory system, and reproductive system. To understand the mechanistic roles of the T2Rs in oral and extraoral tissues, novel blockers or antagonists are urgently needed. Recently, we elucidated the binding pocket of T2R4 for its agonist quinine, and an antagonist and inhibitory neurotransmitter, γ-aminobutyric acid. This structure-function information about T2R4 led us to screen the plant hormone abscisic acid (ABA), its precursor (xanthoxin), and catabolite phaseic acid for their ability to bind and activate or inhibit T2R4. Molecular docking studies followed by functional assays involving calcium imaging confirmed that ABA is an antagonist with an IC50 value of 34.4 ± 1.1 µM. However, ABA precursor xanthoxin acts as an agonist on T2R4. Interestingly, molecular model-guided site-directed mutagenesis suggests that the T2R4 residues involved in quinine binding are also predominantly involved in binding to the novel antagonist, ABA. The antagonist ability of ABA was tested using another T2R4 agonist, yohimbine. Our results suggest that ABA does not inhibit yohimbine-induced T2R4 activity. The discovery of natural bitter blockers has immense nutraceutical and physiological significance and will help in dissecting the T2R molecular pathways in various tissues.

Amino acid derivatives as bitter taste receptor (T2R) blockers.

In humans, the 25 bitter taste receptors (T2Rs) are activated by hundreds of structurally diverse bitter compounds. However, only five antagonists or bitter blockers are known. In this study, using molecular modeling guided site-directed mutagenesis, we elucidated the ligand-binding pocket of T2R4. We found seven amino acids located in the extracellular side of transmembrane 3 (TM3), TM4, extracellular loop 2 (ECL2), and ECL3 to be involved in T2R4 binding to its agonist quinine. ECL2 residues Asn-173 and Thr-174 are essential for quinine binding. Guided by a molecular model of T2R4, a number of amino acid derivatives were screened for their ability to bind to T2R4. These predictions were tested by calcium imaging assays that led to identification of γ-aminobutryic acid (GABA) and Nα,Nα-bis(carboxymethyl)-L-lysine (BCML) as competitive inhibitors of quinine-activated T2R4 with an IC50 of 3.2 ± 0.3 µM and 59 ± 18 nM, respectively. Interestingly, pharmacological characterization using a constitutively active mutant of T2R4 reveals that GABA acts as an antagonist, whereas BCML acts as an inverse agonist on T2R4. Site-directed mutagenesis confirms that the two novel bitter blockers share the same orthosteric site as the agonist quinine. The signature residues Ala-90 and Lys-270 play important roles in interacting with BCML and GABA, respectively. This is the first report to characterize a T2R endogenous antagonist and an inverse agonist. The novel bitter blockers will facilitate physiological studies focused on understanding the roles of T2Rs in extraoral tissues.

Molecular mechanisms in the activation of abscisic acid receptor PYR1.

The pyrabactin resistance 1 (PYR1)/PYR1-like (PYL)/regulatory component of abscisic acid (ABA) response (RCAR) proteins comprise a well characterized family of ABA receptors. Recent investigations have revealed two subsets of these receptors that, in the absence of ABA, either form inactive homodimers (PYR1 and PYLs 1-3) or mediate basal inhibition of downstream target type 2C protein phosphatases (PP2Cs; PYLs 4-10) respectively in vitro. Addition of ABA has been shown to release the apo-homodimers yielding ABA-bound monomeric holo-receptors that can interact with PP2Cs; highlighting a competitive-interaction process. Interaction selectivity has been shown to be mediated by subtle structural variations of primary sequence and ligand binding effects. Now, the dynamical contributions of ligand binding on interaction selectivity are investigated through extensive molecular dynamics (MD) simulations of apo and holo-PYR1 in monomeric and dimeric form as well as in complex with a PP2C, homology to ABA insensitive 1 (HAB1). Robust comparative interpretations were enabled by a novel essential collective dynamics approach. In agreement with recent experimental findings, our analysis indicates that ABA-bound PYR1 should efficiently bind to HAB1. However, both ABA-bound and ABA-extracted PYR1-HAB1 constructs have demonstrated notable similarities in their dynamics, suggesting that apo-PYR1 should also be able to make a substantial interaction with PP2Cs, albeit likely with slower complex formation kinetics. Further analysis indicates that both ABA-bound and ABA-free PYR1 in complex with HAB1 exhibit a higher intra-molecular structural stability and stronger inter-molecular dynamic correlations, in comparison with either holo- or apo-PYR1 dimers, supporting a model that includes apo-PYR1 in complex with HAB1. This possibility of a conditional functional apo-PYR1-PP2C complex was validated in vitro. These findings are generally consistent with the competitive-interaction model for PYR1 but highlight dynamical contributions of the PYR1 structure in mediating interaction selectivity suggesting added degrees of complexity in the regulation of the competitive-inhibition.

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.

Structure of Pisum sativum Rubisco with bound ribulose 1,5-bisphosphate.

The first structure of a ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from a pulse crop is reported. Rubisco was purified from Pisum sativum (garden pea) and diffraction-quality crystals were obtained by hanging-drop vapour diffusion in the presence of the substrate ribulose 1,5-bisphosphate. X-ray diffraction data were recorded to 2.20 Šresolution from a single crystal at the Canadian Light Source. The overall quaternary structure of non-activated P. sativum Rubisco highlights the conservation of the form I Rubisco hexadecameric complex. The electron density places the substrate in the active site at the interface of the large-subunit dimers. Lys201 in the active site is not carbamylated as expected for this non-activated structure. Some heterogeneity in the small-subunit sequence is noted, as well as possible variations in the conformation and contacts of ribulose 1,5-bisphosphate in the large-subunit active sites. Overall, the active-site conformation most closely correlates with the `closed' conformation observed in other substrate/inhibitor-bound Rubisco structures.

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.

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.

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.