Lauren Du Fall1, Peter Solomon1
1Australian National University, Canberra, ACT, Australia
First Published: 2nd Australasian Metabolomics Symposium and Workshop 2010
Stagonospora nodorum is a necrotrophic fungal pathogen of wheat causing over $100 million of yield losses in Australia annually. This fungus produces a number of proteins (effectors) required for disease of which ToxA was the first discovered. These effectors have unique protein sequences and the mechanism by which they cause cell death remains elusive. We are using a comprehensive metabolomics approach in order to investigate the biochemical response of wheat to these effectors. Presented here are results from the Gas Chromatography-Mass Spectrometry (GC-MS) component of this research.
Wheat cultivar BG261 was infiltrated 10 days old (Fig. 1) with purified effector (ToxA) or water. Six replicates of each treatment were collected at 4, 12, 24, 48 & 72 hrs. Frozenvtissue (~100mg) was homogenised and metabolites extracted at 4°C in 80/20 Methanol/Water. Extracts were derivatised online and analysed on a 5975C Gas Chromatograph coupled to a single quadrupole 7890A Mass Selective Detector. Samples were run using a 5.6°C/minute ramping method on a Varian Factor 4 capillary column. All data were analysed using AnalyzerPro® data mining software by SpectralWorks. This software was utilised to successfully deconvolute co-eluting peaks and automate quantification of metabolite abundances using an internal standard. AnalyzerPro® was used to rapidly batch process data and match deconvoluted spectra to the integrated NIST library and Golm Metabolome Database resulting in a high number of compound identifications. Results were exported from AnalyzerPro® and statistical analyses were carried out using JMP (SAS institute) and The Unscrambler (CAMO) to perform Principal Component Analysis, Hierarchical Cluster Analysis and Analysis of Variance.
Necrotic activity of ToxA following direct infiltration into the apoplastic space.
Figure 1 - The chlorotic reaction of wheat caused by ToxA can clearly by visualised at 48 and 72 hours post infiltration.
ToxA induces global metabolic changes in wheat
The major metabolic changes induced by ToxA are illustrated in Figure 2 (right). In total, 99 metabolites were determined to be present at significantly different abundance (p < 0.05) as a result of ToxA infiltration. A number are key intermediates in the Tricarboxylic Acid (TCA) cycle (Figure 3); this pathway is central to generating energy in a form usable to the cell. A number of amino acids synthesized from precursors generated by the TCA cycle were also significantly affected by ToxA treatment (Figure 3).
Figure 2. - HCA of metabolites affected by ToxA infiltration (p < 0.05). Abundance low to high = green < white < red.
Figure 3 - TCA cycle and amino acid metabolism. Metabolites significantly altered by ToxA treatment are displayed with graphs of their abundance over the 4 to 72 hour period sampled post infiltration. Metabolites outlined in black are those detected by GC-MS (split mode).
Preliminary metabolomic analysis of ToxA infiltrated wheat indicates significant changes in primary metabolism involving the TCA cycle and amino acid metabolism. A number of unknown compounds affected by ToxA infiltration are also likely to play interesting roles in the mechanism by which ToxA causes necrosis in wheat. Definitive compound identification will be carried out in such cases. Combining the results presented here describing effects of ToxA on primary metabolism with those on secondary metabolism (to be achieved using LC-Q-ToF-MS), will provide a strong understanding of the mechanism of ToxA at the cellular level. This approach which will also be applied to other Stagonospora nodorum effectors holds great potential for generating strategies to combat the extensive crop damage these effectors are capable of causing.
This research is supported by the Grains Research and Development Corporation and the Australian Research Council.