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 \u3b3 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.Peer reviewed: YesNRC publication: Ye

A Conserved Amphipathic Ligand Binding Region Influences K‑Path-Dependent Activity of Cytochrome <i>c</i> Oxidase

A conserved, crystallographically defined bile acid binding site was originally identified in the membrane domain of mammalian and bacterial cytochrome <i>c</i> oxidase (C<i>c</i>O). 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 <i>Rhodobacter sphaeroides</i> C<i>c</i>O as well as altering the activity of wild-type and bovine enzymes. Dodecyl maltoside, Triton X100, C12E8, lysophophatidylcholine, and CHOBIMALT detergents further inhibit <i>Rs</i>C<i>c</i>O 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 C<i>c</i>O activity by altering the K-path-dependent electron transfer equilibrium between heme <i>a</i> and heme <i>a</i>₃. The high affinity and specificity of a number of compounds for this region, and its conservation and impact on C<i>c</i>O activity, support its physiological significance

Computational Prediction and <i>in Vitro</i> Analysis of Potential Physiological Ligands of the Bile Acid Binding Site in Cytochrome <i>c</i> Oxidase

A conserved bile acid site has been crystallographically defined in the membrane domain of mammalian and Rhodobacter sphaeroides cytochrome <i>c</i> oxidase (<i>Rs</i>C<i>c</i>O). Diverse amphipathic ligands were shown previously to bind to this site and affect the electron transfer equilibrium between heme <i>a</i> and <i>a</i>₃ 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: <i>ROCS</i> comparison of ligand shape and electrostatics, <i>SimSite3D</i> comparison of ligand binding site features, and <i>SLIDE</i> 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. <i>In vitro</i> oxygen consumption and ligand competition assays on <i>Rs</i>C<i>c</i>O wildtype and its Glu101Ala mutant support regulatory activity and specificity of some of these ligands. An ATP analog and GDP inhibit <i>Rs</i>C<i>c</i>O 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 <i>Rs</i>C<i>c</i>O inhibition in the presence of certain nucleotides is reminiscent of previously reported ATP inhibition of mammalian C<i>c</i>O, 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 <i>c</i> and appears to arrest C<i>c</i>O in a semioxidized state with some resemblance to the “resting” state of the enzyme

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), xray 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 \u3bcM). 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.Peer reviewed: YesNRC publication: Ye