Direct and Indirect Targeting of PP2A by Conserved Bacterial Type-III Effector Proteins.

Bacterial AvrE-family Type-III effector proteins (T3Es) contribute significantly to the virulence of plant-pathogenic species of Pseudomonas, Pantoea, Ralstonia, Erwinia, Dickeya and Pectobacterium, with hosts ranging from monocots to dicots. However, the mode of action of AvrE-family T3Es remains enigmatic, due in large part to their toxicity when expressed in plant or yeast cells. To search for targets of WtsE, an AvrE-family T3E from the maize pathogen Pantoea stewartii subsp.

Perturbation of maize phenylpropanoid metabolism by an AvrE family type III effector from Pantoea stewartii.

AvrE family type III effector proteins share the ability to suppress host defenses, induce disease-associated cell death, and promote bacterial growth. However, despite widespread contributions to numerous bacterial diseases in agriculturally important plants, the mode of action of these effectors remains largely unknown. WtsE is an AvrE family member required for the ability of Pantoea stewartii ssp.

Molecular differentiation of Pantoea stewartii subsp. indologenes from subspecies stewartii and identification of new isolates from maize seeds.

Aims: Assays to detect Pantoea stewartii from maize seeds should include differentiation of P. stewartii subsp. stewartii and P.

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.

Variations in the molecular masses of the capsular exopolysaccharides amylovoran, pyrifolan and stewartan.

Erwinia amylovora, causing fire blight of apple, pear and some ornamentals, Erwinia pyrifoliae, causing Asian pear blight, and Pantoea stewartii, causing Stewart's wilt of sweet maize, synthesize capsular extracellular polysaccharides (EPSs) with a high molecular mass. The EPSs are virulence factors and form viscous aggregates, which participate in clogging vessels of infected plants and causing wilting. The sizes of EPSs produced under different environmental growth conditions were determined by analysis with large pore HPLC columns.

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.

WtsE, an AvrE-family type III effector protein of Pantoea stewartii subsp. stewartii, causes cell death in non-host plants.

Pantoea stewartii subsp. stewartii (Pnss) causes Stewart's bacterial wilt of sweet corn and leaf blight of maize. The pathogenicity of Pnss depends on synthesis of extracellular polysaccharide and an Hrp type III secretion system.

A rapid bioassay for screening rhizosphere microorganisms for their ability to induce systemic resistance.

ABSTRACT We developed a rapid and miniaturized bioassay for screening large numbers of rhizosphere microorganisms for their ability to induce systemic resistance to bacterial leaf spot of radish caused by Xanthomonas campestris pv. armoraciae. In this bioassay, Pantoea agglomerans strain E278Ar controlled symptoms of disease as effectively as 2,6-dichloroisonicotinic acid when applied to the roots of seedlings produced in growth pouches in a soilless system.

Export of the HR eliciting protein, Harpin(Es), of the maize pathogen Erwinia stewartii is species-specific but is independent of the growth temperature.

The extra-cellular export of the HR-eliciting protein, Harpin(Es) of the maize pathogen Erwinia stewartii was studied to find out if the protein needs any species-specific signal for its export and to determine if the export of the protein to the medium is affected in any way by the growth temperature. Based upon the experimental evidence, it was proved that the protein (i.e.

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.

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.

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.

Quorum sensing in plant-pathogenic bacteria.

Quorum sensing (QS) allows bacteria to assess their local population density and/or physical confinement via the secretion and detection of small, diffusible signal molecules. This review describes how phytopathogenic bacteria have incorporated QS mechanisms into complex regulatory cascades that control genes for pathogenicity and colonization of host surfaces. Traits regulated by QS include the production of extracellular polysaccharides, degradative enzymes, antibiotics, siderophores, and pigments, as well as Hrp protein secretion, Ti plasmid transfer, motility, biofilm formation, and epiphytic fitness.

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.

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.

Biological activity of harpin produced by Pantoea stewartii subsp. stewartii.

Pantoea stewartii subsp. stewartii causes Stewart's wilt of sweet corn. A hypersensitive response and pathogenicity (Hrp) secretion system is needed to produce water-soaking and wilting symptoms in corn and to cause a hypersensitive response (HR) in tobacco.

Genetic organization of the Pantoea stewartii subsp. stewartii hrp gene cluster and sequence analysis of the hrpA, hrpC, hrpN, and wtsE operons.

The hrp/wts gene cluster of Pantoea stewartii subsp. stewartii is required for pathogenicity on sweet corn and the ability to elicit a hypersensitive response (HR) in tobacco. Site-directed transposon mutagenesis and nucleotide sequencing were used to identify hrp/wts genes within the left 20 kb of this cluster.

Genetic organization of the hrp gene cluster and dspAE/BF operon in Erwinia herbicola pv. gypsophilae.

Erwinia herbicola pv. gypsophilae induces gall formation in gypsophila that is dependent on the existence of a pathogenicity plasmid (pPATHEhg). We previously demonstrated the presence of several hrp genes on this plasmid.

Identification of an RcsA/RcsB recognition motif in the promoters of exopolysaccharide biosynthetic operons from Erwinia amylovora and Pantoea stewartii subspecies stewartii.

The regulation of capsule synthesis (Rcs) regulatory network is responsible for the induction of exopolysaccharide biosynthesis in many enterobacterial species. We have previously shown that two transcriptional regulators, RcsA and RcsB, do bind as a heterodimer to the promoter of amsG, the first reading frame in the operon for amylovoran biosynthesis in the plant pathogenic bacterium Erwinia amylovora. We now identified a 23-base pair fragment from position -555 to -533 upstream of the translational start site of amsG as sufficient for the specific binding of the Rcs proteins.

Characterization of a gene locus from Erwinia amylovora with regulatory functions in exopolysaccharide synthesis of Erwinia spp.

In a genomic library of Erwinia amylovora, a locus has been identified that can suppress an Erwinia stewartii rcsA mutant. In addition, the locus induced a mucoid sticky phenotype of colonies in a wild-type strain of Erwinia stewartii and increased exopolysaccharide synthesis in several species of bacteria belonging to the genus Erwinia. An open reading frame was identified at this locus encoding a 225 amino acid protein that contained a helix-turn-helix motif typical of transcriptional regulators.

A negative regulator mediates quorum-sensing control of exopolysaccharide production in Pantoea stewartii subsp. stewartii.

Classical quorum-sensing (autoinduction) regulation, as exemplified by the lux system of Vibrio fischeri, requires N-acyl homoserine lactone (AHL) signals to stimulate cognate transcriptional activators for the cell density-dependent expression of specific target gene systems. For Pantoea stewartii subsp. stewartii, a bacterial pathogen of sweet corn and maize, the extracellular polysaccharide (EPS) stewartan is a major virulence factor, and its production is controlled by quorum sensing in a population density-dependent manner.

Bacterial polysaccharide synthesis and gene nomenclature.

Gene nomenclature for bacterial surface polysaccharides is complicated by the large number of structures and genes. We propose a scheme applicable to all species that distinguishes different classes of genes, provides a single name for all genes of a given function and greatly facilitates comparative studies.

Structure of stewartan, the capsular exopolysaccharide from the corn pathogen Erwinia stewartii.

Stewartan, the acidic exopolysaccharide of Erwinia stewartii, was purified from agar grown cells. To facilitate its structural analysis, chemical and enzymatic depolymerizations were carried out using hydrofluoric acid and E. amylovora phage phi-Ea1h, respectively.

Structure of amylovoran, the capsular exopolysaccharide from the fire blight pathogen Erwinia amylovora.

The acidic exopolysaccharide (EPS) of Erwinia amylovora, amylovoran, was purified from culture supernatants of bacteria in minimal medium and cleaved chemically either by treatment with trifluoracetic acid or hydrofluoric acid, and enzymatically by digestion with depolymerase from E. amylovora phage phi-Ealh. Structural characterization of the resulting oligosaccharides was performed by a combination of mass spectrometric and NMR [one- and two-dimensional (1D and 2D)] spectroscopic techniques.