## 2 Results
### Root Metabolome Modulation  
The PLS-DA analysis of the _Phomis purpurea_ roots challenged with _Plutophthora_ _cinnamomi_ revealed a separation between the challenged and control samples for all the time points studied (Figure 4), representing a response modulation of the metabolism upon a pathogen attack. In the roots, the separation was not as evident as that in the leaf samples. For the different time points, components 1 and 2 accounted for over 30% of the sample variation (37.4%, 37%, and 33.6% for 6, 12, and 72 hpc, respectively), except for 24 hpc, which had a global explanation of 26.3%. The maximum separation obtained for both components was at 48 hpc with 48.5%. For both the leaf and root tissues, 48 hpc was the time point with the highest metabolic modulation between the control and challenged samples.  
Figure 4: Partial least squares discriminant analysis (PLS-DA) components 1 and 2 score plots of _Phomis purpurea_ roots metabolite profiles at **(A)** 6, **(B)** 12, **(C)** 24, **(D)** 48, and **(E)** 72 hpc with _Plutophthora_ _cinnamomi_. In the score plots, the ellipse represents the Hotelling T2 with 95% confidence interval. The red ellipses represent the controls and the green ellipses the challenged samples. Five biological replicates were performed per analysis.  
In the roots, a total of 34 ions putatively annotated presented significant modulation (\(p\leq 0.05\)) between the challenged and control samples. The predicted root-associated metabolites were from the lipids (44.12%), phytochemical compounds (23.53%), alkaloids (8.82%), and carbohydrates (2.94%) classes (Figure 5). A percentage (20.59%) of the putative metabolites were not assigned to any functional class but were found to occur in the plant kingdom. Complete lists of the putative metabolites for each of the time points analyzed are given in Supplementary Table S4.  
After the first 48 h of interaction, a positive modulation of most metabolites was observed in the challenged samples compared to the controls (Figure 6).  
Figure 5: Metabolic secondary classes’ annotation of _Pilomis purpurea_ roots for the 6, 12, 24, 48, and 72 hpc with _Phytopthora cinnamomi_ according to the KEGG and LipidMaps classification.  
Figure 6: Heatmap of differently accumulated ions in each metabolic class from _Pilomis purpurea_ roots challenged at 6, 12, 48, and 72 hpc with _Phytopthora cinnamomi_ (no metabolites were assigned for the 24 hpc). Each column indicates the time point (6, 12, 48, and 72 hpc). Each row represents a feature, i.e., ion putatively identified as a metabolite in _Pilomis purpurea_ roots challenged with _Phytopthora cinnamomi_. Coloring indicates differential accumulation between challenged and control samples: green indicates low accumulation (fold change < 1), light red indicates significant accumulation (fold change between 2 and 3), and red indicates high accumulation (fold change > 4).  
At the first hours of interaction (6 and 12 hpc), this modulation was associated with alkaloids (e.g., \(m/z\) 262.9, \(m/z\) 174.1, and \(m/z\) 246.0) and other phytochemical compounds (e.g., \(m/z\) 365.0, \(m/z\) 345.1, \(m/z\) 353.0, and \(m/z\) 423.0). Other ions presented a positive modulation but no annotation was available in the databases. At 48 and 72 h, the number of positively modulated putatively identified metabolites was reduced in the challenged samples compared to the controls. For these time points, some ions (\(m/z\) 461.1, \(m/z\) 230.0, \(m/z\) 231.0, \(m/z\) 387.1, \(m/z\) 127.0, and \(m/z\) 353.2) were putatively annotated as lipids and others (\(m/z\) 225.0, \(m/z\) 193.0, \(m/z\) 462.1, and \(m/z\) 425.1) as phytochemical compounds.