P-262 - Specific function of glutathione-S-transferases in indole glucosinolate metabolism and penetration resistance towards filamentous pathogens

In Arabidopsis thaliana the pre-invasive resistance towards filamentous pathogens involves bioactive metabolites derived from indole glucosinolates (IG). This metabolic pathway is initiated by PEN2 myrosinase, which hydrolyses IGs. Our former study revealed glutathione-S-transferase U13 (GSTU13) as another component of this pathway. This enzyme conjugates isothiocyanates, generated upon PEN2-mediated IG hydrolysis, with glutathione to form adducts that are processed to end products, including indol-3-ylmethyl amine (I3A) and raphanusamic acid (RA). In this study, we address molecular determinants of specific contribution of GSTU13 to the PEN2 pathway. To this end, we expressed selected AtGSTs in gstu13 mutant under the native GSTU13 promoter. To assure PEN2-pathway specific subcellular localization we fused GST sequences with the coding sequence of the unique PEN2 C-terminal tail (TA_PEN2), which anchor this protein in the mitochondrial membrane. We analyzed obtained transgenic lines for their capacity to restore accumulation of I3A and RA. Our results indicated that GSTU13-TA_PEN2 fusion protein restores I3A and RA accumulation in gstu13 plants confirming that association of GSTU13 with mitochondrial membrane supports its function in IG metabolism. Subsequent experiments with transgenic lines expressing different selected GST-TA_PEN2 constructs indicated that some, but not all, of the investigated AtGSTs are capable to replace GSTU13 during the immune response.