Bioengineering secreted proteases converts divergent Rcr3 orthologs and paralogs into extracellular immune co-receptors

• DOI: 10.1101/2024.02.14.580413
• Publication Date: 2024-02-15T23:56:58Z
• Authors: Jiorgos Kourelis; Mariana Schuster; Fatih Demir; Oliver Mattinson; Sonja Krauter; Parvinderdeep S. ...; Ruby O’Grady; Samantha Royston; Ana Lucía Bravo-C...; Brian C. Mooney; Pitter F. Huesgen; Sophien Kamoun; Renier A. L. van ...
• Keywords: Nicotiana; 0301 basic medicine; 570; Bioengineering; 630; Fungal Proteins; 03 medical and health sciences; Solanum lycopersicum; Plant Immunity; Cladosporium; Research Article; Plant Proteins; Peptide Hydrolases; Plant Diseases; Signal Transduction

ABSTRACTSecreted immune proteases Rcr3 and Pip1 of tomato are both inhibited by Avr2 from the fungal plant pathogenCladosporium fulvumbut only Rcr3 act as a decoy co-receptor that detects Avr2 in the presence of the Cf-2 immune receptor. Here, we identified crucial residues from tomato Rcr3 required for Cf-2-mediated signalling and bioengineered various proteases to trigger Avr2/Cf-2 dependent immunity. Despite substantial divergences in Rcr3 orthologs from eggplant and tobacco, only minimal alterations were sufficient to trigger Avr2/Cf-2-triggered immune signalling. Tomato Pip1, by contrast, was bioengineered with 16 Rcr3-specific residues to initiate Avr2/Cf-2-triggered immune signalling. These residues cluster on one side next to the substrate binding groove, indicating a potential Cf-2 interaction site. Our findings also revealed that Rcr3 and Pip1 have distinct substrate preferences determined by two variant residues and that both are suboptimal for binding Avr2. This study advances our understanding of Avr2 perception and opens avenues to bioengineer proteases to broaden pathogen recognition in other crops.