Jasmonate-induced protein 60 (JIP60) is a ribosome-inactivating protein (RIP) from barley (Hordeum vulgare) and is involved in the plant immune response dependent on jasmonate hormones. Here, we demonstrate in Nicotiana benthamiana that transient expression of the N-terminal domain of JIP60, from which the inhibitor domain (amino acids 163-185) 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 Tyr 96, Glu 201, Arg 204, and Trp 234 in the presumptive active site of JIP60 are conserved in 815 plant RIPs in the Pfam database that were identified by HUMMR as containing a RIP domain. When these amino acid residues are individually mutated, the necrosis-inducing activity is completely abolished. We therefore propose that the role of these amino acids in JIP60 activity is to depurinate adenosine in ribosomes. This study provides insight into the catalytic mechanism of JIP60.

中文翻译：

---

JIP60 N 端核糖体失活结构域催化位点的作用模式。

茉莉酸诱导蛋白 60 (JIP60) 是一种来自大麦 (Hordeum vulgare) 的核糖体失活蛋白 (RIP)，参与依赖茉莉酸激素的植物免疫反应。在这里，我们在本塞姆氏烟草中证明，JIP60 N 端结构域的瞬时表达（其中抑制剂结构域（氨基酸 163-185）被去除）会引发细胞死亡，导致叶组织广泛坏死。我们使用 JIP60 的结构预测来鉴定活性位点中潜在的催化氨基酸，并通过诱变和在本塞姆氏烟草中表达诱导坏死的植物测定以及通过体外翻译失活测定来测试这些氨基酸。我们发现，JIP60 假定活性位点中的 Tyr 96、Glu 201、Arg 204 和 Trp 234 在 Pfam 数据库中的 815 个植物 RIP 中是保守的，这些 RIP 经 HUMMR 鉴定为包含 RIP 结构域。当这些氨基酸残基单独突变时，坏死诱导活性完全消失。因此，我们提出这些氨基酸在 JIP60 活性中的作用是使核糖体中的腺苷脱嘌呤。这项研究深入了解了 JIP60 的催化机制。