Expression, Protein Purification And Optimization Of Crystalize Conditions For Plant Avirulance Gene AvrRpt2

Suppression of host defenses by pathogenic bacteria plays a vital role in theirpathogenesis, thus promoting their survival and replication. This can be exemplified byAvrRpt2-mediated suppression of gene-for-gene resistance. The Pseudomonas syringaeeffector protein AvrRpt2, a protein protease, can recognize and cleave the host proteinRIN4 when delivered by Typeâ…¢Secretion System (TTSS) into host plant cells.Degradation of RIN4 by AvrRpt2 results in inability of the resistance protein RPM1 todetect the other two Pseudomonas syringae effector proteins, AvrB and AvrRpml, whichwould otherwise trigger host disease resistance. Not surprisingly, to protect pathogen itselffrom the detrimental effect of AvrRpt2, it is synthesized as a zymogen and activated by itshost chaperone ROC1. Although AvrRpt2 is genetically and biochemically welldocumented, it is still remains unknown the mechanisms by which it is activated and itcatalyzes substrate reactions. To address these questions, the current study is aimed atdetermining the crystal structures of AvrRpt2, its complex with ROC1 as well as RIN4. Allthe three recombinant proteins were successfully purified to homogeneity suitable forcryatalliztion. To obtain the AvrRpt2-ROC1 complex, we purified the protein for thecatalytic residue mutation in AvrRpt2 mixed it with ROC1. However, the complex thusformed could not survive in gel-filtration assay, indicating that activation of AvrRpt2 byROC1 likely through their transient or weak interaction. Results from limited proteolysisand purification of various AvrRpt2 proteins showed that AvrRpt2 protein tends tocrystallize without any truncation. Although the crystals of AvrRpt2 are not large enoughfor data collection, successful crystallization of this protein will facilitate the structuralstudy in the future.