The bacterium Pantoea stewartii uses two different type III secretion systems to colonize its plant host and insect vector.

Plant- and animal-pathogenic bacteria utilize phylogenetically distinct type III secretion systems (T3SS) that produce needle-like injectisomes or pili for the delivery of effector proteins into host cells. Pantoea stewartii subsp. stewartii (herein referred to as P. stewartii), the causative agent of Stewart's bacterial wilt and leaf blight of maize, carries phylogenetically distinct T3SSs. In addition to an Hrc-Hrp T3SS, known to be essential for maize pathogenesis, P. stewartii has a second T3SS (Pantoea secretion island 2 [PSI-2]) that is required for persistence in its flea beetle vector, Chaetocnema pulicaria (Melsh). PSI-2 belongs to the Inv-Mxi-Spa T3SS family, typically found in animal pathogens. Mutagenesis of the PSI-2 psaN gene, which encodes an ATPase essential for secretion of T3SS effectors by the injectisome, greatly reduces both the persistence of P. stewartii in flea beetle guts and the beetle's ability to transmit P. stewartii to maize. Ectopic expression of the psaN gene complements these phenotypes. In addition, the PSI-2 psaN gene is not required for P. stewartii pathogenesis of maize and is transcriptionally upregulated in insects compared to maize tissues. Thus, the Hrp and PSI-2 T3SSs play different roles in the life cycle of P. stewartii as it alternates between its insect vector and plant host.

Multiple activities of the plant pathogen type III effector proteins WtsE and AvrE require WxxxE motifs.

The broadly conserved AvrE-family of type III effectors from gram-negative plant-pathogenic bacteria includes important virulence factors, yet little is known about the mechanisms by which these effectors function inside plant cells to promote disease. We have identified two conserved motifs in AvrE-family effectors: a WxxxE motif and a putative C-terminal endoplasmic reticulum membrane retention/retrieval signal (ERMRS). The WxxxE and ERMRS motifs are both required for the virulence activities of WtsE and AvrE, which are major virulence factors of the corn pathogen Pantoea stewartii subsp. stewartii and the tomato or Arabidopsis pathogen Pseudomonas syringae pv. tomato, respectively. The WxxxE and the predicted ERMRS motifs are also required for other biological activities of WtsE, including elicitation of the hypersensitive response in nonhost plants and suppression of defense responses in Arabidopsis. A family of type III effectors from mammalian bacterial pathogens requires WxxxE and subcellular targeting motifs for virulence functions that involve their ability to mimic activated G-proteins. The conservation of related motifs and their necessity for the function of type III effectors from plant pathogens indicates that disturbing host pathways by mimicking activated host G-proteins may be a virulence mechanism employed by plant pathogens as well.

WtsE, an AvrE-family effector protein from Pantoea stewartii subsp. stewartii, causes disease-associated cell death in corn and requires a chaperone protein for stability.

The pathogenicity of Pantoea stewartii subsp. stewartii to sweet corn and maize requires a Hrp type III secretion system. In this study, we genetically and functionally characterized a disease-specific (Dsp) effector locus, composed of wtsE and wtsF, that is adjacent to the hrp gene cluster. WtsE, a member of the AvrE family of effector proteins, was essential for pathogenesis on corn and was complemented by DspA/E from Erwinia amylovora. An intact C-terminus of WtsE, which contained a putative endoplasmic reticulum membrane retention signal, was important for function of WtsE. Delivery of WtsE into sweet corn leaves by an Escherichia coli strain carrying the hrp cluster of Erwinia chrysanthemi caused water-soaking and necrosis. WtsE-induced cell death was not inhibited by cycloheximide treatment, unlike the hypersensitive response caused by a known Avr protein, AvrRxol. WtsF, the putative chaperone of WtsE, was not required for secretion of WtsE from P. stewartii, and the virulence of wtsF mutants was reduced only at low inoculum concentrations. However, WtsF was required for full accumulation of WtsE within the bacteria at low temperatures. In contrast, WtsF was needed for efficient delivery of WtsE from E. coli via the Erwinia chrysanthemi Hrp system.

A novel transcriptional autoregulatory loop enhances expression of the Pantoea stewartii subsp. stewartii Hrp type III secretion system.

The hrp type III secretion regulon of Pantoea stewartii is regulated by a cascade involving the HrpX/HrpY two-component system, the HrpS enhancer-binding protein and the HrpL alternate sigma factor. hrpXY is both constitutive and autoregulated; HrpY controls hrpS; and HrpS activates hrpL. These regulatory genes are arranged in the order hrpL, hrpXY and hrpS and constitute three operons. This study describes a novel autoregulatory loop involving HrpS. Genetic experiments using a chromosomal hrpS-lacZ fusion demonstrated that ectopic expression of HrpS increases hrpS transcription and that this effect is blocked by polar mutations in hrpXY and hrpL and by a nonpolar mutation in hrpY. RT-PCR and Northern blot analysis revealed a hrpL-hrpXY polycistronic mRNA. These results suggest that HrpS-mediated autoregulation is due to activation of hrpS by increased levels of HrpY resulting from read-through transcription of hrpXY from the hrpL promoter. This novel autoregulatory loop may serve to rapidly induce hrp genes during infection and to compensate for negative regulatory mechanisms that keep the regulon off in the insect vector.

The HrpX/HrpY two-component system activates hrpS expression, the first step in the regulatory cascade controlling the Hrp regulon in Pantoea stewartii subsp. stewartii.

A regulatory cascade activating hrp/hrc type III secretion and effector genes was delineated in Pantoea stewartii subsp. stewartii, a bacterial pathogen of corn. Four hrp regulatory genes were characterized: hrpX and hrpY encode the sensor kinase and response regulator, respectively, of a two-component signal transduction system; hrpS encodes an NtrC-like transcriptional enhancer; and hrpL encodes an alternative sigma factor. Epistasis analysis, expression studies using gene fusions, and genetic reconstruction of each step in Escherichia coli were used to delineate the following pathway: HrpY activates hrpS and also positively autoregulates the hrpXY operon. In turn, HrpS is required for full activation of the sigma54-dependent hrpL promoter. Finally, HrpL controls expression of all known hrp and wts genes. In vitro, hrpS and all downstream hrp genes were regulated by pH and salt concentration. Mutants with in-frame deletions in hrpX were still partially virulent on corn but were unable to sense the chemical or metabolic signals that induce hrp genes in vitro. Site-directed mutagenesis of HrpY indicated that aspartate 57 is the probable phosphorylation site and that it is needed for activity. These findings suggest that both HrpX and an alternate mechanism are involved in the activation of HrpY in planta.

Identification of Pantoea stewartii subsp. stewartii by PCR and Strain Differentiation by PFGE.

Stewart's bacterial wilt and leaf blight of sweet corn and maize is caused by Pantoea stewartii subsp. stewartii. This bacterium can be seed transmitted at a low frequency, so it is subject to quarantine restrictions by many countries. To develop a polymerase chain reaction assay for the identification of this pathogen from field samples and for use in seed health tests, four primer pairs were tested. These were selected from the sequences of hrpS, cpsDE, and the 16S rRNA intergenic transcribed spacer (ITS) region. Under optimal reaction conditions, about 20 and 200 cells of P. stewartii could be detected in pure cultures and leaf lesions, respectively. Other plant-associated enteric bacteria (e.g., P. agglomerans pv. herbicola, P. ananas, Erwinia amylovora, and E. carotovora) either did not produce amplicons or they were not the correct size for P. stewartii. To test further for possible false positives, 29 yellow-pigmented bacteria, mainly other Pantoea spp., were isolated from lesions on old corn leaves and assayed with the ITS primer sets. Except for weak, variable reactions with three P. ananas strains, the bacteria did not test positive. Pulsed field gel electrophoresis (PFGE) was evaluated as an additional test to confirm the identity of P. stewartii. After digestion with SpeI and XbaI, P. stewartii strains could be easily distinguished from related Erwinia and Pantoea spp. and each other.

Molecular characterization of Pantoea stewartii subsp. stewartii HrpY, a conserved response regulator of the Hrp type III secretion system, and its interaction with the hrpS promoter.

Pantoea stewartii subsp. stewartii is a bacterial pathogen of corn. Its pathogenicity depends on the translocation of effector proteins into host cells by the Hrp type III secretion system. We previously showed by genetic analysis that the HrpX sensor kinase and the HrpY response regulator are at the head of a complex cascade of regulators controlling hrp/hrc secretion and wts effector genes. This cascade also includes the HrpS response regulator and the HrpL alternative sigma factor. These regulators are shared among many important plant pathogens in the genera Pantoea, Erwinia, and Pseudomonas. In this study, we dissect the regulatory elements in the hrpS promoter region, using genetic and biochemical approaches, and show how it integrates various environmental signals, only some of which are dependent on phosphorylation of HrpY. Primer extension located the transcriptional start site of hrpS at a sigma70 promoter 601 bp upstream of the open reading frame. Electrophoretic mobility shift assays and DNase I footprinting analysis demonstrated that HrpY binds to conserved regulatory elements immediately adjacent to this promoter, and its binding affinity was increased by phosphorylation at D57. A consensus sequence for the two direct repeats bound by HrpY is proposed. Deletion analysis of the promoter region revealed that both the HrpY binding site and additional sequences farther upstream, including a putative integration host factor binding site, are required for hrpS expression. This finding suggests that other unknown regulatory proteins may act cooperatively with HrpY.