Genetic Characterization ofPseudomonas fluorescensSBW25rspGene Expression in the Phytosphere and In Vitro

Abstract

The plant-colonizing Pseudomonas fluorescens strain SBW25 harbors a gene cluster (rsp) whose products show similarity to type III protein secretion systems found in plant and animal pathogens. Here we report a detailed analysis of the expression and regulation of the P. fluorescens rsp pathway, both in the phytosphere and in vitro. A combination of chromosomally integrated transcriptional reporter fusions, overexpressed regulatory genes, and specific mutants reveal that promoters controlling expression of rsp are actively transcribed in the plant rhizosphere but not (with the exception of the rspC promoter) in the phyllosphere. In synthetic medium, regulatory (rspL and rspR) and structural (rspU, plus the putative effector ropE) genes are poorly expressed; the rspC promoter is subject to an additional level of regulatory control. Ectopic expression of regulatory genes in wild-type and mutant backgrounds showed that RspR controls transcription of the alternate sigma factor, rspL, and that RspL controls expression of gene clusters encoding structural genes. Mutation of rspV did not affect RspR-mediated expression of rspU. A search for additional regulators revealed two candidates-one with a role in the conversion of alanine to pyruvate-suggesting that expression of rsp is partly dependent upon the metabolic status of the cell. Mutations in rsp regulators resulted in a significant reduction in competitive colonization of the root tips of sugar beet seedlings but also caused a marked increase in the lag phase of laboratory-grown cultures, indicating that rsp regulatory genes play a more significant general role in the function of P. fluorescens SBW25 than previously appreciated.The plant rhizosphere is a complex environment comprising organic matter and a plethora of microbes, some of which can affect plant health. Pathogens, such as Pythium ultimum, can hinder plant growth, whereas strains of Pseudomonas fluorescens, including P. fluorescens SBW25, can promote plant growth. The mechanism of plant growth promotion involves a combination of competition, antagonism of pathogens via the production of antimicrobial compounds, and induction of systemic resistance (15,16,31,34).To understand the interaction between P. fluorescens SBW25 and plants, SBW25 genes specifically activated in the plant environment were identified via a promoter trapping strategy (11,36). Approximately 100 rhizosphere-induced (rhi) genes have been identified and categorized into six groups: nutrient acquisition, stress response, attachment and surface colonization, antibiotic production, secretion, and unknown. One rhi gene (rhi-18) is hrcC (redesignated rscC), whose product is a component of a type III protein secretion system (TTSS) termed Rsp (33, 36) (Fig. 1).The P. fluorescens SBW25 rsp cluster exhibits a high level of synteny to the group I TTSS clusters of Pseudomonas syringae pv. tomato DC3000 and Erwinia amylovora and is genetically most similar to the P. syringae cluster. The cluster is organized into three putative transcriptional units...

Evolutionary History of the phl Gene Cluster in the Plant-Associated Bacterium Pseudomonas fluorescens

“…Specifically for Pseudomonas, some success has been obtained using in vitro expression technology (21,27) and transcriptome analysis (37) to identify niche (plant)-specific or plant-induced regulatory genes. The data from the study reported here identify another strategy that may prove useful in dissecting intimate plant microbe interactions.…”

Modulation of Host Immunity by Beneficial Microbes

“…Interestingly, all three type III effectors were found to be capable of suppressing typical innate immune responses when ectopically expressed in N. benthamiana (Mavrodi et al 2011). Several mutations in components of the TTSS of P. fluorescens SBW25 have been shown to affect the competitive colonization capacity of the bacterium in the rhizosphere of sugar beet (Jackson et al 2005). However, it is worth noticing that all of these mutants displayed altered in vitro growth compared with the wild-type strain, suggesting a more general role of the TTSS in the physiology of P. fluorescens SBW25 (Jackson et al 2005).…”

“…Several mutations in components of the TTSS of P. fluorescens SBW25 have been shown to affect the competitive colonization capacity of the bacterium in the rhizosphere of sugar beet (Jackson et al 2005). However, it is worth noticing that all of these mutants displayed altered in vitro growth compared with the wild-type strain, suggesting a more general role of the TTSS in the physiology of P. fluorescens SBW25 (Jackson et al 2005). On the other hand, P. fluorescens Q8r1-96 mutants lacking functional TTSS are not altered in their rhizosphere competence (Mavrodi et al 2011).…”

Microbiology, Genomics, and Clinical Significance of the Pseudomonas fluorescens Species Complex, an Unappreciated Colonizer of Humans

“…The Hrp1 T3SS of P. fluorescens Pf29Arp, a strain known for its ability to reduce the severity of wheat take-all, shows activity during the colonization of wheat rhizospheres (46). The homologous Hrp1 T3SS in strain SBW25 is induced during sugar beet rhizosphere colonization (154) and can induce a hypersensitive response in tobacco (47,155). Interestingly, in addition to Hrp1 system effectors, SBW25 also contains the T3SS effector ExoY (156), which in P. aeruginosa targets the actin cytoskeleton of eukaryotic cells (157).…”