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

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

Isothermal titration calorimetry analysis of spinach Rubisco-ligand interactions.

<p>Raw ITC data of sequential injections of (A) RuBP (0.75 mM) and (B) (+)-ABA (5 mM) titrated against spinach Rubisco (39.42 μM). The processed fit is shown below each binding isotherm. The binding parameters are shown in the inset. The ligands were titrated until saturation was reached showing a specific binding interaction. The data were fitted using the ‘Independent Binding’ model in the NanoAnalyze software and the best fit is represented by the black line.</p

X-ray data collection and structure refinement statistics.

<p>Values in parenthesis are for the highest resolution shell.</p

ABA and related ABA analogs.

<p>Compounds are labeled accordingly, with (+)-PBI686 representing the photoactive, bioactive ABA-mimetic biotinylated probe used to pull-out putative ABA-binding proteins.</p

Co-crystallography of RuBP-bound pea Rubisco with ABA.

<p>A) L-Subunit B showing (+)-ABA (in yellow) bound to a surface cleft adjacent to Y100. While distal from the RuBP (in green) binding site (~ 30 Å away), this site is in closer proximity to the positively charged regulatory latch region comprised of residues R41, R134, K305, H310, R312 and involves residues that immediately precede and follow the 90–97 loop region implicated in binding to Rubisco activase. B) The omit F_o-F_c electron density maps represented by the green hatching at ~ 2 sigma, were calculated without ABA included in the model. (+)-ABA (in yellow with red oxygens) is shown, bound to the A L-subunit. The terminal carboxylate is shown anchored to R139; the ketone forms a short H-bond with Y85; and the hydroxyl group at the chiral center forms a H-bond with K356. The hydroxyl group also has a weak 3.3 Å interaction with a water that is part of an H-bond and ionic matrix involving the side chains of Y100, E88, R358 and Y363.</p

ABA binds to spinach Rubisco with high affinity.

<p>A) Saturation curve and Scatchard plot representing specific binding of [³H]-(±)-ABA, as a difference between total and non-specific binding signal. B) Competition Binding Assay. Displacement of 25 nM (±) [³H]ABA by non-radiolabeled (+) ABA (diamonds), (-) ABA (circles), PA (squares) and trans-(+) ABA (triangle) at the indicated concentrations.</p

Predicted ABA binding to the spinach Rubisco active site.

<p>A) The Rubisco structure was previously solved in the presence of its two 3PGA product molecules (tan sticks with red oxygen atoms and orange phosphorus atoms), which may be used to define the protein’s active site (PDB ID: 1AA1). Small molecule docking of (+)-ABA (cyan sticks with red oxygen atoms) into B) the open, non-activated; C) closed, non-activated (PDB ID: 1RCX); and D) open, activated Rubisco active site, demonstrated the potential for competitive inhibition in a variety of enzyme states. In both non-activated enzyme forms, (+)-ABA was docked to the same location as the 3PGA molecule proximal to Mg2+ (PDB ID: 1AA1, residue 3PG 447) and was stabilized by hydrogen bonding to basic and acid residues (gray sticks) in Rubisco loops. In the activated enzyme form, (+)-ABA was docked to the region between the two 3PGA molecules, making ionic interactions with the Mg2+ cofactor (green spheres).</p