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

Abstract

<i>Clavibacter michiganensis</i>, 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 <i>pat-1_Cm</i> 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 <i>Nicotiana tabacum</i>. Among orthologs and paralogs of <i>pat-1_Cm</i> , only <i>chp-7_Cs</i> from <i>Clavibacter sepedonicus</i>, a potato pathogen, successfully complemented <i>pat-1_Cm</i> function in pathogenicity in tomato, whereas all failed to complement <i>pat-1_Cm</i> function in HR induction in <i>N. tabacum</i>. 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 <i>C. michiganensis</i>. Ectopic expression of <i>pat-1_Cm</i> with an SP from tobacco secreted protein triggered HR in <i>N. tabacum</i>, but not in tomato, whereas a catalytic triad mutant failed to induce HR. Inoculation of the <i>pat-1_Cm</i> 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.