Audet Annabelle, Chivers Jennifer A, Hudak Katalin A
Department of Biology, York University, Toronto, ON M3J 1P3, Canada.
Plants (Basel). 2025 Aug 7;14(15):2441. doi: 10.3390/plants14152441.
The apoplast is often the first point of contact between plant cells and invading pathogens, serving as an important site for defense signaling. Pokeweed antiviral protein (PAP), a ribosome-inactivating protein from (pokeweed), is localized to the apoplast and is hypothesized to accompany a pathogen to the cytosol, where it would inactivate host ribosomes to prevent pathogen spread. However, it is not known whether PAP interacts with other proteins in the apoplast. In this study, we identified cysteine protease 1 (PaCP1), an extracellular cysteine protease, as a novel PAP interactor. Sequence and structural analyses classified PaCP1 as a member of the C1A subfamily of papain-like cysteine proteases. Immunoprecipitation, mass spectrometry, and yeast two-hybrid analysis showed that PAP specifically binds the mature, active form of PaCP1. Curiously, PaCP1 cleaves PAP at its N- and C-termini, generating peptides that enhance MAPK phosphorylation in pokeweed leaves, indicating their potential role in stress signaling. PaCP1 processing of PAP to generate bioactive peptides diversifies the function of a ribosome-inactivating protein beyond its canonical inhibition of translation. Our findings present a novel extracellular role for PAP and advance our understanding of how protein interactions in the apoplast contribute to plant immune responses.
质外体通常是植物细胞与入侵病原体接触的第一个位点，是防御信号传导的重要场所。商陆抗病毒蛋白（PAP）是一种来自商陆的核糖体失活蛋白，定位于质外体，据推测它会伴随病原体进入细胞质，在细胞质中它会使宿主核糖体失活以阻止病原体传播。然而，尚不清楚PAP是否与质外体中的其他蛋白质相互作用。在本研究中，我们鉴定了一种细胞外半胱氨酸蛋白酶——半胱氨酸蛋白酶1（PaCP1），它是一种新型的PAP相互作用蛋白。序列和结构分析将PaCP1归类为木瓜蛋白酶样半胱氨酸蛋白酶C1A亚家族的成员。免疫沉淀、质谱分析和酵母双杂交分析表明，PAP特异性结合成熟的、有活性的PaCP1形式。奇怪的是，PaCP1在PAP的N端和C端切割PAP，产生的肽可增强商陆叶片中丝裂原活化蛋白激酶（MAPK）的磷酸化，表明它们在胁迫信号传导中的潜在作用。PaCP1对PAP进行加工以产生生物活性肽，使核糖体失活蛋白的功能多样化，超越了其对翻译的经典抑制作用。我们的研究结果揭示了PAP一种新的细胞外作用，并加深了我们对质外体中的蛋白质相互作用如何促进植物免疫反应的理解。
