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

Date Published: April 18, 2019

Publisher: Public Library of Science

Author(s): Kun Li, Yanan Zhu, Wei Yan, Xin Deng, Yanmei Xiao, Liyang Song, Rongxiang Fang, Yantao Jia, Xiaoyan Tang, Wenbo Ma.


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.

Partial Text

Many Gram-negative bacterial pathogens rely on the T3SS for successful infection of their hosts [1]. The T3SS is encoded by a cluster of hrp/hrc genes that are essential for the induction of a hypersensitive response (HR) in resistant and nonhost plants and pathogenicity in susceptible plants [2]. The T3SS functions as a conduit to deliver an array of effector proteins into plant cells [3]. The effectors interfere with the host defense systems and contribute to bacterial pathogenicity [4]. Some effectors elicit HR and disease resistance in plants containing cognate disease resistance genes, and are therefore named avirulence proteins [5].

In the search for genes regulating the T3SS genes in Psph, we identified the mutant of the rhpC gene with diminished T3SS gene induction and reduced pathogenicity in the host plant. rhpC is downstream of rhpP which encodes a putative metalloprotease. RhpC is a cytoplasmic protein that interacts with RhpP and facilitates the translocation of RhpP to the periplasm. The expression of RhpP alone in Psph and E. coli inhibited bacterial growth, and the inhibition was abolished by mutation of the conserved protease activity sites, suggesting that RhpP alone is an active protease in the bacterial cytoplasm. Purified recombinant RhpP protein can degrade HrpL in vitro, further showing RhpP as an active protease. The ΔrhpC mutant exhibited a reduced level of HrpL protein, the direct regulator of the T3SS genes. The reduction in HrpL was consistent with the reduced T3SS gene induction in the ΔrhpC mutant. Interestingly, the ΔrhpC mutant did not show a reduction in the HrpS and HrpR protein levels, suggesting that RhpP has substrate specificity. Nonetheless, HrpL is unlikely to be the only substrate for RhpP in the Psph cytoplasm, because hrpL- mutant did not show any growth defect in KB and MM [13, 39]. In addition, RhpP probably degraded the E. coli proteins as well because expression of the RhpP-MBP recombinant protein in E. coli caused a severe growth defect, and the detrimental effect in E. coli required the protease active sites of RhpP. The reduced growth of the ΔrhpC mutant in the host plants was likely the result of reduced T3SS gene expression plus the reduced fitness of the bacterial cells.




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