Loss of a conserved disulfide bond defines Penetration2-related immune myrosinases in Brassicaceae

O_LIThis study investigated whether PEN2/BGLU26 has uniquely evolved as an indole glucosinolate-hydrolysing myrosinase required for Arabidopsis thaliana pre-invasive immunity, or whether related myrosinases can replace its function when targeted to the same subcellular context. C_LIO_LIPEN2-homologous and other selected myrosinases from A. thaliana and Brassica rapa were expressed in the pen2-2 mutant background using a PEN2-like targeting strategy. The resulting lines were assessed by gene expression, protein accumulation, metabolite analysis and pathogen resistance assays. In parallel, targeted mutagenesis, structural comparison and phylogenetic analysis were used to examine molecular and evolutionary features of PEN2-related myrosinases. C_LIO_LIAtBGLU27 and BrBABG.a, but not AtBGLU18, AtBGLU23 or AtBGLU28, partially restored indole glucosinolate hydrolysis and resistance to Colletotrichum tropicale in pen2-2. Unlike AtPEN2, both enzymes acted mainly constitutively and showed distinct substrate preferences. PEN2, BGLU27 and BABG proteins lacked conserved post-translational modification sites, including residues associated with a conserved disulfide bond. Restoring this disulfide bond in AtPEN2 abolished its activity. C_LIO_LIPEN2-related myrosinases form an evolutionarily distinct BGLU lineage associated with indole glucosinolate metabolism in Brassicales. Loss of the conserved disulfide bond appears to be required for PEN2 activity, whereas additional PEN2-specific regulatory features are needed for pathogen-triggered, rather than constitutive, glucosinolate metabolism. C_LI