Two components of the rhpPC operon coordinately regulate the type III secretion system and bacterial fitness in Pseudomonas savastanoi pv. phaseolicola

Many plant bacterial pathogens including Pseudomonas species, utilize the type III secretion system (T3SS) to deliver effector proteins into plant cells. Genes encoding the T3SS and its effectors are repressed in nutrient-rich media but are rapidly induced after the bacteria enter a plant or are transferred into nutrient-deficient media. To understand how the T3SS genes are regulated, we screened for P. savastanoi pv. phaseolicola (Psph) mutants displaying diminished induction of avrPto-luc, a reporter for the T3SS genes, in Arabidopsis. A mutant carrying transposon insertion into a gene coding for a small functional unknown protein, designated as rhpC, was identified that poorly induced avrPto-luc in plants and in minimal medium (MM). Interestingly, rhpC is located immediately downstream of a putative metalloprotease gene named rhpP, and the two genes are organized in an operon rhpPC; but rhpP and rhpC displayed different RNA expression patterns in nutrient-rich King's B medium (KB) and MM. Deletion of the whole rhpPC locus did not significantly affect the avrPto-luc induction, implying coordinated actions of rhpP and rhpC in regulating the T3SS genes. Further analysis showed that RhpC was a cytoplasmic protein that interacted with RhpP and targeted RhpP to the periplasm. In the absence of RhpC, RhpP was localized in the cytoplasm and caused a reduction of HrpL, a key regulator of the T3SS genes, and also reduced the fitness of Psph. Expression of RhpP alone in E. coli inhibited the bacterial growth. The detrimental effect of RhpP on the fitness of Psph and E. coli required metalloprotease active sites, and was repressed when RhpC was co-expressed with RhpP. The coordination between rhpP and rhpC in tuning the T3SS gene expression and cell fitness reveals a novel regulatory mechanism for bacterial pathogenesis. The function of RhpP in the periplasm remains to be studied. Author summary The induction of the type III secretion system (T3SS) is of great importance to the pathogenesis of bacterial pathogens in host plants. Pseudomonas savastanoi pv. phaseolicola (Psph) causes halo blight disease on beans. We discovered that the bicistronic genes in the rhpPC locus of Psph act coordinately to regulate the T3SS gene expression, bacterial fitness, and pathogenicity. rhpP encodes a metalloprotease that can degrade the key T3SS regulator protein HrpL and reduce bacterial fitness. rhpC encodes a chaperone that inhibits the RhpP activity and mediates translocation of RhpP to the periplasm. The level of rhpP RNA is high in KB but decreases in MM, but the rhpC RNA is low in KB but increases in MM. The elevated rhpC/rhpP transcript ratio in MM plus the inhibition of RhpC on RhpP activity in cytoplasm provide double insurance that warrants high induction of the T3SS genes in MM and bacterial fitness. The coordination between rhpP and rhpC reveals a new mechanism regulating bacterial pathogenicity, and may provide an important target for controlling bacterial pathogens.