New multidrug transporter of Pseudomonas syringae pv. tomato involved in resistance to plant antimicrobials

Multidrug resistance efflux pumps protect bacterial cells against a wide spectrum of antimicrobial compounds. The number of published studies investigating multidrug resistance (MDR) systems in phytopathogenic bacteria is rather limited, particularly those focusing on plant-defense compounds as potential substrates of the efflux pumps. Most studies on Pseudomonas syringae MDRs are devoted to its major efflux pump, MexAB-OprM, which has a very dominant influence on bacterial antimicrobial resistance. Our work evidences the role of PSPTO_0820, a newly characterized MDR transporter of P. syringae pv. tomato DC3000 (PsPto), in the efflux of specific plant antimicrobials and its importance for the colonization of tomato plants. Orthologs of PSPTO_0820 are conserved within many Pseudomonas species that interact with plants. We also analyzed the role of PSPTO_4977, a TolC-like protein that could work as a potential outer membrane channel for this tripartite efflux system. Mutants in the genes encoding these two proteins were isolated and characterized. Since a number of studies using the tomato-PsPto pathosystem reported increased expression of plant phenylpropanoid biosynthetic genes upon bacterial infection, the responses of PSPTO_0820 and PSPTO_4977 mutants to plant antimicrobials from the phenylpropanoid family was evaluated. Both mutants were more susceptible to trans-cinnamic and chlorogenic acids and to the flavonoid (+)-catechin, when added to the culture medium. In addition, the expression level of both genes increased in the presence of (+)-catechin and, in the case of PSPTO_0820, also in response to trans-cinnamic acid. Moreover, PSPTO_0820 and PSPTO_4977 mutants were unable to colonize tomato at high population levels.

In short, the present work evidences the involvement of PSPTO_0820 and PSPTO_4977 proteins in the bacterial resistance to certain plant phenylpropanoids. The results suggest that chlorogenic, (+)-catechin, and trans-cinnamic acid are expelled by these systems, and both PsPto genes are important for the colonization of the tomato plant.