Pseudomonas syringae pathogenesis: Regulation of type III secretion and identification of a secreted effector

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Bretz, James Robert
Hutcheson, Steven W
The Pseudomonas syringae hrp pathogenicity island encodes a type III secretion system (TTSS) which is used to translocate effector proteins into host cells to facilitate pathogenesis. Expression of the hrp TTSS is controlled by the alternative sigma factor, HrpL, whose expression in turn is positively controlled by two truncated enhancer binding proteins, HrpR and HrpS. Although a number of environmental conditions are known to modulate hrp TTSS expression, such as stringent conditions and pathogenesis, the mechanism by which the activities of these transcriptional factors are modulated had not been established. Both HrpR and HrpS were shown to be required for full expression of hrpL. hrpRS were shown to be expressed as an operon and a promoter was identified 5' to hrpR. The hrpRS promoter and coding sequence were found to be conserved among P. syringae strains. The hrpRS operon was constitutively expressed under conditions in which the hrpL promoter was inactive, indicating the involvement of a negative regulatory factor. Transposome (Tnp) mutagenesis was used to identify Lon protease as a negative regulator of hrpL expression, suggesting an effect on HrpR and/or HrpS. HrpR was observed to be unstable in wild-type P. syringae strains grown in non-inductive media. The apparent half-life of HrpR increased more than 10-fold in the P. syringae lon::Tnp mutants or upon transfer to inductive (stringent) conditions. As a result, an interaction between factors involved in the stringent response, Lon protease, and hrp regulation was also investigated. The regulatory system described above was used to develop a HrpL-dependent promoter trap to identify effectors secreted by the Hrp TTSS. One of these potential effectors, HopPtoD2, was shown to encode a protein tyrosine phosphatase that was translocated into Arabidopsis thaliana cells via the hrp-encoded TTSS. A hopPtoD2 mutant exhibited strongly reduced virulence in Arabidopsis thaliana. Expression of hopPtoD2 delayed the development of several defense-associated responses in infected plants. These results indicate that HopPtoD2 is a translocated effector with protein tyrosine phosphatase activity that modulates plant defense responses.