Network Analysis Combining Proteomics and Metabolomics Reveals New Insights Into Early Responses of Eucalyptus grandis During Rust Infection

Eucalyptus rust is caused by the biotrophic fungus, <i>Austropuccinia psidii</i>, which affects commercial plantations of Eucalyptus, a major raw material for the pulp and paper industry in Brazil. In this manuscript we aimed to uncover the molecular mechanisms involved in rust resistance and susceptibility in <i>Eucalyptus grandis</i>. Epifluorescence microscopy was used to follow the fungus development inside the leaves of two contrasting half-sibling genotypes (rust-resistance and rust-susceptible), and also determine the comparative time-course of changes in metabolites and proteins in plants inoculated with rust. Within 24 h of complete fungal invasion, the analysis of 709 metabolomic features showed the suppression of many metabolites 6 h after inoculation (hai) in the rust-resistant genotype, with responses being induced after 12 hai. In contrast, the rust-susceptible genotype displayed more induced metabolites from 0 to 18 hai time-points, but a strong suppression occurred at 24 hai. Multivariate analyses of genotypes and time points were used to select 16 differential metabolites mostly classified as phenylpropanoid-related compounds. Applying the Weighted Gene Co-Expression Network Analysis (WGCNA), rust-resistant and rust-susceptible genotypes had, respectively, 871 and 852 proteins grouped into 5 and 6 modules, of which 5 and 4 of them were significantly correlated to the selected metabolites. Functional analyses revealed roles for photosynthesis and oxidative-dependent responses leading to temporal activity of metabolites and related enzymes after 12 hai in rust-resistance; while the initial over-accumulation of those molecules and suppression of supporting mechanisms at 12 hai caused a lack of progressive metabolite-enzyme responses after 12 hai in rust-susceptible genotype. This study provides some insights on how <i>E. grandis</i> plants are functionally modulated to integrate secondary metabolites and related enzymes from phenylpropanoid pathway and lead to temporal divergences of resistance and susceptibility responses to rust.