Immune recognition of the secreted serine protease ChpG restricts the host range of Clavibacter michiganensis from eggplant varieties

Abstract

Bacterial wilt and canker caused by Clavibacter michiganensis (Cm) inflict considerable damage in tomato-growing regions around the world. Cm has a narrow host range and can cause disease in tomato but not in many eggplant varieties. The pathogenicity of Cm is dependent on secreted serine proteases, encoded by the chp/tomA pathogenicity island (PI), and the pCM2 plasmid. Screening combinations of PI deletion mutants and plasmid-cured strains found that Cm-mediated hypersensitive response (HR) in the Cm-resistant eggplant variety Black Queen is dependent on the chp/tomA PI. Singular reintroduction of PI-encoded serine proteases into Cm- increment PI identified that the HR is elicited by the protease ChpG. Eggplant leaves infiltrated with a chpG marker exchange mutant (Cm ohm chpG) did not display an HR, and infiltration of purified ChpG protein elicited immune responses in eggplant but not in Cm-susceptible tomato. Virulence assays found that while wild-type Cm and the Cm ohm chpG complemented strain were nonpathogenic on eggplant, Cm ohm chpG caused wilt and canker symptoms. Additionally, bacterial populations in Cm ohm chpG-inoculated eggplant stem tissues were c.1000-fold higher than wild-type and Cm ohm chpG-complemented Cm strains. Pathogenicity tests conducted in multiple Cm-resistance eggplant varieties demonstrated that immunity to Cm is dependent on ChpG in all tested varieties, indicating that ChpG-recognition is conserved in eggplant. ChpG-mediated avirulence interactions were disabled by alanine substitution of serine231 of the serine protease catalytic triad, suggesting that protease activity is required for immune recognition of ChpG. Our study identified ChpG as a novel avirulence protein that is recognized in resistant eggplant varieties and restricts the host range of Cm.