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.

Alternative pathways utilize or circumvent putrescine for biosynthesis of putrescine-containing rhizoferrin.

The siderophore rhizoferrin (N1,N4-dicitrylputrescine) is produced in fungi and bacteria to scavenge iron. Putrescine-producing bacterium Ralstonia pickettii synthesizes rhizoferrin and encodes a single nonribosomal peptide synthetase-independent siderophore (NIS) synthetase. From biosynthetic logic, we hypothesized that this single enzyme is sufficient for rhizoferrin biosynthesis. We confirmed this by expression of R. pickettii NIS synthetase in Escherichia coli, resulting in rhizoferrin production. This was further confirmed in vitro using the recombinant NIS synthetase, synthesizing rhizoferrin from putrescine and citrate. Heterologous expression of homologous lbtA from Legionella pneumophila, required for rhizoferrin biosynthesis in that species, produced siderophore activity in E. coli. Rhizoferrin is also synthesized by Francisella tularensis and Francisella novicida, but unlike R. pickettii or L. pneumophila, Francisella species lack putrescine biosynthetic pathways because of genomic decay. Francisella encodes a NIS synthetase FslA/FigA and an ornithine decarboxylase homolog FslC/FigC, required for rhizoferrin biosynthesis. Ornithine decarboxylase produces putrescine from ornithine, but we show here in vitro that FigA synthesizes N-citrylornithine, and FigC is an N-citrylornithine decarboxylase that together synthesize rhizoferrin without using putrescine. We co-expressed F. novicida figA and figC in E. coli and produced rhizoferrin. A 2.1 Å X-ray crystal structure of the FigC N-citrylornithine decarboxylase reveals how the larger substrate is accommodated and how active site residues have changed to recognize N-citrylornithine. FigC belongs to a new subfamily of alanine racemase-fold PLP-dependent decarboxylases that are not involved in polyamine biosynthesis. These data reveal a natural product biosynthetic workaround that evolved to bypass a missing precursor and re-establish it in the final structure.

Characterization of Triticum aestivum Abscisic Acid Receptors and a Possible Role for These in Mediating Fusairum Head Blight Susceptibility in Wheat.

Abscisic acid (ABA) is a well-characterized plant hormone, known to mediate developmental aspects as well as both abiotic and biotic stress responses. Notably, the exogenous application of ABA has recently been shown to increase susceptibility to the fungal pathogen Fusarium graminearum, the causative agent of Fusarium head blight (FHB) in wheat and other cereals. However roles and mechanisms associated with ABA's modulation of pathogen responses remain enigmatic. Here the identification of putative ABA receptors from available genomic databases for Triticum aestivum (bread wheat) and Brachypodium distachyon (a model cereal) are reported. A number of these were cloned for recombinant expression and their functionality as ABA receptors confirmed by in vitro assays against protein phosphatases Type 2Cs. Ligand selectivity profiling of one of the wheat receptors (Ta_PYL2DS_FL) highlighted unique activities compared to Arabidopsis AtPYL5. Mutagenic analysis showed Ta_PYL2DS_FL amino acid D180 as being a critical contributor to this selectivity. Subsequently, a virus induced gene silencing (VIGS) approach was used to knockdown wheat Ta_PYL4AS_A (and similar) in planta, yielding plants with increased early stage resistance to FHB progression and decreased mycotoxin accumulation. Together these results confirm the existence of a family of ABA receptors in wheat and Brachypodium and present insight into factors modulating receptor function at the molecular level. That knockdown of Ta_PYL4AS_A (and similar) leads to early stage FHB resistance highlights novel targets for investigation in the future development of disease resistant crops.

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.

Identification of an attenuated barley stripe mosaic virus for the virus-induced gene silencing of pathogenesis-related wheat genes.

BACKGROUND: Virus-induced gene silencing (VIGS) has become an emerging technology for the rapid, efficient functional genomic screening of monocot and dicot species. The barley stripe mosaic virus (BSMV) has been described as an effective VIGS vehicle for the evaluation of genes involved in wheat and barley phytopathogenesis; however, these studies have been obscured by BSMV-induced phenotypes and defense responses. The utility of BSMV VIGS may be improved using a BSMV genetic background which is more tolerable to the host plant especially upon secondary infection of highly aggressive, necrotrophic pathogens such as Fusarium graminearum. RESULTS: BSMV-induced VIGS in Triticum aestivum (bread wheat) cv. 'Fielder' was assessed for the study of wheat genes putatively related to Fusarium Head Blight (FHB), the necrotrophism of wheat and other cereals by F. graminearum. Due to the lack of 'Fielder' spike viability and increased accumulation of Fusarium-derived deoxynivalenol contamination upon co-infection of BSMV and FHB, an attenuated BSMV construct was generated by the addition of a glycine-rich, C-terminal peptide to the BSMV γ b protein. This attenuated BSMV effectively silenced target wheat genes while limiting disease severity, deoxynivalenol contamination, and yield loss upon Fusarium co-infection compared to the original BSMV construct. The attenuated BSMV-infected tissue exhibited reduced abscisic, jasmonic, and salicylic acid defense phytohormone accumulation upon secondary Fusarium infection. Finally, the attenuated BSMV was used to investigate the role of the salicylic acid-responsive pathogenesis-related 1 in response to FHB. CONCLUSIONS: The use of an attenuated BSMV may be advantageous in characterizing wheat genes involved in phytopathogenesis, including Fusarium necrotrophism, where minimal viral background effects on defense are required. Additionally, the attenuated BSMV elicits reduced defense hormone accumulation, suggesting that this genotype may have applications for the investigation of phytohormone-related signaling, developmental responses, and pathogen defense.

Identification of Interactions between Abscisic Acid and Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase.

Abscisic acid ((+)-ABA) is a phytohormone involved in the modulation of developmental processes and stress responses in plants. A chemical proteomics approach using an ABA mimetic probe was combined with in vitro assays, isothermal titration calorimetry (ITC), x-ray crystallography and in silico modelling to identify putative (+)-ABA binding-proteins in crude extracts of Arabidopsis thaliana. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was identified as a putative ABA-binding protein. Radiolabelled-binding assays yielded a Kd of 47 nM for (+)-ABA binding to spinach Rubisco, which was validated by ITC, and found to be similar to reported and experimentally derived values for the native ribulose-1,5-bisphosphate (RuBP) substrate. Functionally, (+)-ABA caused only weak inhibition of Rubisco catalytic activity (Ki of 2.1 mM), but more potent inhibition of Rubisco activation (Ki of ~ 130 µM). Comparative structural analysis of Rubisco in the presence of (+)-ABA with RuBP in the active site revealed only a putative low occupancy (+)-ABA binding site on the surface of the large subunit at a location distal from the active site. However, subtle distortions in electron density in the binding pocket and in silico docking support the possibility of a higher affinity (+)-ABA binding site in the RuBP binding pocket. Overall we conclude that (+)-ABA interacts with Rubisco. While the low occupancy (+)-ABA binding site and weak non-competitive inhibition of catalysis may not be relevant, the high affinity site may allow ABA to act as a negative effector of Rubisco activation.

Computational prediction and in vitro analysis of potential physiological ligands of the bile acid binding site in cytochrome c oxidase.

A conserved bile acid site has been crystallographically defined in the membrane domain of mammalian and Rhodobacter sphaeroides cytochrome c oxidase (RsCcO). Diverse amphipathic ligands were shown previously to bind to this site and affect the electron transfer equilibrium between heme a and a3 cofactors by blocking the K proton uptake path. Current studies identify physiologically relevant ligands for the bile acid site using a novel three-pronged computational approach: ROCS comparison of ligand shape and electrostatics, SimSite3D comparison of ligand binding site features, and SLIDE screening of potential ligands by docking. Identified candidate ligands include steroids, nicotinamides, flavins, nucleotides, retinoic acid, and thyroid hormones, which are predicted to make key protein contacts with the residues involved in bile acid binding. In vitro oxygen consumption and ligand competition assays on RsCcO wildtype and its Glu101Ala mutant support regulatory activity and specificity of some of these ligands. An ATP analog and GDP inhibit RsCcO under low substrate conditions, while fusidic acid, cholesteryl hemisuccinate, retinoic acid, and T3 thyroid hormone are more potent inhibitors under both high and low substrate conditions. The sigmoidal kinetics of RsCcO inhibition in the presence of certain nucleotides is reminiscent of previously reported ATP inhibition of mammalian CcO, suggesting regulation involving the conserved core subunits of both mammalian and bacterial oxidases. Ligand binding to the bile acid site is noncompetitive with respect to cytochrome c and appears to arrest CcO in a semioxidized state with some resemblance to the "resting" state of the enzyme.

A conserved amphipathic ligand binding region influences k-path-dependent activity of cytochrome C oxidase.

A conserved, crystallographically defined bile acid binding site was originally identified in the membrane domain of mammalian and bacterial cytochrome c oxidase (CcO). Current studies show other amphipathic molecules including detergents, fatty acids, steroids, and porphyrins bind to this site and affect the already 50% inhibited activity of the E101A mutant of Rhodobacter sphaeroides CcO as well as altering the activity of wild-type and bovine enzymes. Dodecyl maltoside, Triton X100, C12E8, lysophophatidylcholine, and CHOBIMALT detergents further inhibit RsCcO E101A, with lesser inhibition observed in wild-type. The detergent inhibition is overcome in the presence of micromolar concentrations of steroids and porphyrin analogues including deoxycholate, cholesteryl hemisuccinate, bilirubin, and protoporphyrin IX. In addition to alleviating detergent inhibition, amphipathic carboxylates including arachidonic, docosahexanoic, and phytanic acids stimulate the activity of E101A to wild-type levels by providing the missing carboxyl group. Computational modeling of dodecyl maltoside, bilirubin, and protoporphyrin IX into the conserved steroid site shows energetically favorable binding modes for these ligands and suggests that a groove at the interface of subunit I and II, including the entrance to the K-path and helix VIII of subunit I, mediates the observed competitive ligand interactions involving two overlapping sites. Spectral analysis indicates that ligand binding to this region affects CcO activity by altering the K-path-dependent electron transfer equilibrium between heme a and heme a(3). The high affinity and specificity of a number of compounds for this region, and its conservation and impact on CcO activity, support its physiological significance.

From static structure to living protein: computational analysis of cytochrome c oxidase main-chain flexibility.

Crystallographic structure and deuterium accessibility comparisons of CcO in different redox states have suggested conformational changes of mechanistic significance. To predict the intrinsic flexibility and low energy motions in CcO, this work has analyzed available high-resolution crystallographic structures with ProFlex and elNémo computational methods. The results identify flexible regions and potential conformational changes in CcO that correlate well with published structural and biochemical data and provide mechanistic insights. CcO is predicted to undergo rotational motions on the interior and exterior of the membrane, driven by transmembrane helical tilting and bending, coupled with rocking of the ß-sheet domain. Consequently, the proton K-pathway becomes sufficiently flexible for internal water molecules to alternately occupy upper and lower parts of the pathway, associated with conserved Thr-359 and Lys-362 residues. The D-pathway helices are found to be relatively rigid, with a highly flexible entrance region involving the subunit I C-terminus, potentially regulating the uptake of protons. Constriction and dilation of hydrophobic channels in RsCcO suggest regulation of the oxygen supply to the binuclear center. This analysis points to coupled conformational changes in CcO and their potential to influence both proton and oxygen access.

A conserved steroid binding site in cytochrome C oxidase.

Micromolar concentrations of the bile salt deoxycholate are shown to rescue the activity of an inactive mutant, E101A, in the K proton pathway of Rhodobacter sphaeroides cytochrome c oxidase. A crystal structure of the wild-type enzyme reveals, as predicted, deoxycholate bound with its carboxyl group at the entrance of the K path. Since cholate is a known potent inhibitor of bovine oxidase and is seen in a similar position in the bovine structure, the crystallographically defined, conserved steroid binding site could reveal a regulatory site for steroids or structurally related molecules that act on the essential K proton path.