BackgroundPuccinia striiformisf. sp.tritici(Pst) is an economically devasting disease that is prominent in cereal crops such as wheat (Triticum aestivum). The fungal pathogen can cause approximately 30–70% losses in crop productivity and yields.Psthas become difficult to manage due to its ease of transmission through wind dispersal over long distances, and intercontinental dispersal has been previously reported. The ease of transmission has resulted in further destruction because of new and more virulent strains infecting crops previously resistant to a different strain.ResultsIn this study, a liquid chromatography-mass spectrometry-based untargeted metabolomics approach, in combination with multivariate data analytical tools, was used to elucidate the mechanistic nature of the defence systems of aPst-resistant and a susceptible wheat cultivar infected withP. striiformis. We also investigated the time-dependant metabolic reconfiguration of infected plants over a four-week period. The untargeted metabolomic analysis revealed a time-course metabolic reprogramming involving phenylpropanoids (majority flavonoids), amino acids, lipids, benzoic acids, TCA cycle intermediates and benzoxazinoids responding toPstinfection. Interestingly, the results do not show a linear course for the decrease and increase (up-/down-regulation) of said classes of metabolites, but rather the up- or down-regulation of specific metabolites in response to the pathogen infection. The resistant Koonap cultivar had an abundance of phenolic compounds such as rutin, isoorintin-7-O-glucoside and luteolin-6-C-hexoside-O-hexoside. These compounds showed a decrease over time in control Koonap plants compared to an increase inPst-infected plants. These metabolites were down-regulated in the susceptible Gariep cultivar, which could serve as biomarkers for plant responses to biotic stress and resistance againstPst.ConclusionsOverall, an LC-MS-based metabolomics approach allowed for the metabolic profiling and analysis of the impact of plant-pathogen interactions on the overall plant metabolome and provided a real-time snapshot of the differential significant metabolic perturbations occurring in wheat plants responding to thePstpathogen. ThePst-resistant Koonap cultivar showed a rapid accumulation of defence metabolites in response to pathogen infection compared to the susceptible Gariep cultivar. These findings provide insight into the mechanistic biochemical nature of plant-microbe interactions and the prospects of metabolic engineering for improved plant tolerance and resistance to biotic stresses.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12870-023-04313-9.