Molecular characterization of proteolytic cleavage sites of the <i>Pseudomonas syringae</i> effector AvrRpt2

During infection of Arabidopsis thaliana, the bacterium Pseudomonas syringae pv tomato delivers the effector protein AvrRpt2 into the plant cell cytosol. Within the plant cell, AvrRpt2 undergoes N-terminal processing and causes elimination of Arabidopsis RIN4. Previous work established that AvrRpt2 is a putative cysteine protease, and AvrRpt2 processing and RIN4 elimination require an intact predicted catalytic triad in that AvrRpt2. In this work, proteolytic events that depend on AvrRpt2 activity were characterized. The amino acid sequence surrounding the processing site of AvrRpt2 and two related sequences from RIN4 triggered Avr-Rpt2-dependent proteolytic cleavage of a synthetic substrate, demonstrating that these sequences are cleavage recognition sites for AvrRpt2 activity. Processing-deficient AvrRpt2 mutants were identified and shown to retain their ability to eliminate wild-type RIN4. Single amino acid substitutions were made in each of the two RIN4 cleavage sites, and mutation of both sites resulted in cleavage-resistant RIN4. Growth of Pseudomonas expressing AvrRpt2 was significantly higher than catalytically inactive mutants on Arabidopsis rin4/rps2 mutant plants, suggesting there are additional protein targets of AvrRpt2 that account for the virulence activity of this effector. Bioinformatics analysis identified putative Arabidopsis proteins containing sequences similar to the proteolytic cleavage sites conserved in AvrRpt2 and RIN4. Several of these proteins were eliminated in an AvrRpt2-dependent manner in a transient in planta expression system. These results identify amino acids important for AvrRpt2 substrate recognition and cleavage as well as demonstrate AvrRpt2 protease activity eliminates multiple Arabidopsis proteins in a transient expression system.