# A Novel Cysteine Protease from Phytolacca americana Cleaves Pokeweed Antiviral Protein Generating Bioactive Fragments

## TL;DR

A new plant protein from pokeweed cleaves another antiviral protein to create bioactive fragments that may boost plant defenses.

## Contribution

Discovery of a novel extracellular role for PAP through interaction with PaCP1, generating bioactive peptides.

## Key findings

- PaCP1 cleaves PAP at N- and C-termini to produce peptides that enhance MAPK phosphorylation in pokeweed leaves.
- PAP interacts specifically with the mature, active form of PaCP1, a C1A subfamily cysteine protease.
- The generated PAP fragments suggest a new role for PAP in stress signaling beyond ribosome inactivation.

## Abstract

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 Phytolacca americana (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 Phytolacca americana 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.