Mode of action of the catalytic site in the N-terminal ribosome-inactivating domain of JIP60

Jasmonate-induced protein 60 (JIP60) is a ribosome-inactivating protein from barley (Hordeum vulgare) and is involved in the plant immune response dependent on the jasmonate hormones. Here, we demonstrate that transient expression in Nicotiana benthamiana of the N-terminal domain of JIP60, from which the inhibitor domain (amino acids 163 to 185) is removed, initiates cell death leading to extensive necrosis of leaf tissues. We used structure prediction of JIP60 to identify potential catalytic amino acids in the active site and tested these by mutagenesis and in planta assays of necrosis induction by expression in N. benthamiana, as well as through an in vitro translation-inactivation assay. We found that Tyrosine 96, Glutamate 202, Arginine 205, and Tryptophan 235 in the presumptive active site of JIP60 are conserved in 815 plant ribosome inactivating proteins (RIP) in the Pfam database identified using HMMER as containing a RIP domain. When these amino acid residues are individually mutated, the necrosis-inducing activity is completely abolished. We therefore propose a role for these amino acids in JIP60 activity (depurination of adenosine in ribosomes). This study provides insight into the catalytic mechanism of this protein.