An Apoplastic Effector Pat-1(Cm) of the Gram-Positive Bacterium Clavibacter michiganensis Acts as Both a Pathogenicity Factor and an Immunity Elicitor in Plants

Clavibacter michiganensis, a Gram-positive plant-pathogenic bacterium, utilizes apoplastic effectors for disease development in host plants. Here, we determine the roles of Pat-1(Cm) (a putative serine protease) in pathogenicity and plant immunity. Pat-1(Cm) was found to be a genuine secreted protein, and the secreted mature form did not carry the first 33 amino acids predicted to be a signal peptide (SP). The pat-1(Cm) mutant impaired to cause wilting, but still caused canker symptom in tomato. Moreover, this mutant failed to trigger the hypersensitive response (HR) in a nonhost Nicotiana tabacum. Among orthologs and paralogs of pat-1(Cm), only chp-7(Cs) from Clavibacter sepedonicus, a potato pathogen, successfully complemented pat-1(Cm) function in pathogenicity in tomato, whereas all failed to complement pat-1(Cm) function in HR induction in N. tabacum. Based on the structural prediction, Pat-1(Cm) carried a catalytic triad for putative serine protease, and alanine substitution of any amino acids in the triad abolished both pathogenicity and HR-inducing activities of Pat-1(Cm) in C. michiganensis. Ectopic expression of pat-1(Cm) with an SP from tobacco secreted protein triggered HR in N. tabacum, but not in tomato, whereas a catalytic triad mutant failed to induce HR. Inoculation of the pat-1(Cm) mutant mixed with the mutant of another apoplastic effector CelA (cellulase) caused severe wilting in tomato, indicating that these two apoplastic effectors can functionally cooperate in pathogenicity. Overall, these results indicate that Pat-1(Cm) is a distinct secreted protein carrying a functional catalytic triad for serine protease and this enzymatic activity might be critical for both pathogenicity and HR-eliciting activities of Pat-1(Cm) in plants.