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.

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.

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.

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.