Proteomics and functional analyses of Arabidopsis nitrilases involved in the defense response to microbial pathogens

Abstract

Proteomics and functional analyses of the Arabidopsis - Pseudomonas syringae pv. tomato interactions reveal that Arabidopsis nitrilases are required for plant defense and R gene-mediated resistant responses to microbial pathogens. A high-throughput in planta proteome screen has identified Arabidopsis nitrilase 2 (AtNIT2), which was de novo-induced by Pseudomonas syringae pv. tomato (Pst) infection. The AtNIT2, AtNIT3, and AtNIT4 genes, but not AtNIT1, were distinctly induced in Arabidopsis leaves by Pst infection. Notably, avirulent Pst DC3000 (avrRpt2) infection led to significant induction of AtNIT2 and AtNIT4 in leaves. Pst DC3000 and Pst DC3000 (avrRpt2) significantly grew well in leaves of nitrilase transgenic (nit2i-2) and mutant (nit1-1 and nit3-1) lines compared to the wild-type leaves. In contrast, NIT2 overexpression in nit2 mutants led to significantly high growth of the two Pst strains in leaves. The nitrilase transgenic and mutant lines exhibited enhanced susceptibility to Hyaloperonospora arabidopsidis infection. The nit2 mutation enhanced Pst DC3000 (avrRpt2) growth in salicylic acid (SA)-deficient NahG transgenic and sid2 and npr1 mutant lines. Infection with Pst DC3000 or Pst DC3000 (avrRpt2) induced lower levels of indole-3-acetic acid (IAA) in nit2i and nit2i NahG plants than in wild-type plants, but did not alter the IAA level in NahG transgenic plants. This suggests that Arabidopsis nitrilase 2 is involved in IAA signaling of defense and R gene-mediated resistance responses to Pst infection. Quantification of SA in these transgenic and mutant plants demonstrates that Arabidopsis nitrilase 2 is not required for SA-mediated defense response to the virulent Pst DC3000 but regulates SA-mediated resistance to the avirulent Pst DC3000 (avrRpt2). These results collectively suggest that Arabidopsis nitrilase genes are involved in plant defense and R gene-mediated resistant responses to microbial pathogens.