Characterization Of A Cysteine Protease From Phytolacca Americana And Its Association With Pokeweed Antiviral Protein

The plant apoplast, an essential extracellular space, contains diverse proteins crucial for plant defence. This study focuses on Phytolacca americana (American pokeweed), examining the interaction between the ribosome inactivating protein Pokeweed Antiviral Protein (PAP) and a putative papain-like cysteine protease, Phytolacca americana cysteine protease 1 (PaCP1). Bioinformatic predictions identified PaCP1 as a papain-like cysteine protease with conserved structural features, while enzymatic assays confirmed its functionality. Yeast-two hybrid assays validated the interaction between PAP and PaCP1, and localization studies indicated their extracellular co-localization in the apoplast. Enzymatic assays further demonstrated PaCP1's ability to cleave PAP, with mass spectrometry identifying the resulting degradation products. Differential expression studies under salicylic acid and Flg22 treatment revealed different patterns of expression for both PAP and PaCP1. This research enhances our understanding of PAP and its protein interactions as well as its potential role in the pokeweed apoplast.