Pantoea stewartii subsp. stewartii: lessons learned from a xylem-dwelling pathogen of sweet corn

Pantoea stewartii subsp. stewartii is a Gram-negative enteric bacterium that primarily infects sweet corn. Studies of this bacterium have provided useful insight into how xylem-dwelling bacteria establish themselves and incite disease in their hosts. Pantoea stewartii subsp. stewartii is a remarkable bacterial system for laboratory studies because of its relative ease of propagation and genetic manipulation, and the fact that it appears to employ a minimal number of pathogenicity mechanisms. In addition, P. stewartii subsp. stewartii produces copious amounts of its quorum sensing (QS) signal, acyl-homoserine lactone (AHL), making it an excellent organism for studying QS-controlled gene regulation in a plant-pathogenic bacterium. In fact, P. stewartii subsp. stewartii has become the microbial paradigm for QS control of gene expression by both repression and activation via a QS regulator that binds DNA in the absence and dissociates in the presence of the signal ligand. Moreover, P. stewartii subsp. stewartii is a member of the Enterobacteriaceae, and lessons learned from its interaction with plants may be extrapolated to other plant-associated enterics, such as Erwinia, Dickeya and Pectobacterium spp., or enteric human pathogens associated with plants, such as Escherichia coli and Salmonella spp. TAXONOMY: Bacteria; Gammaproteobacteria; family Enterobacteriaceae; genus Pantoea; species stewartii (Mergaert et al., 1993). MICROBIOLOGICAL PROPERTIES: Gram-negative, motile, yellow pigmented, mucoid, facultative anaerobe. HOST RANGE: Pantoea stewartii subsp. stewartii (Smith, 1898) Dye causes Stewart's wilt of corn (Zea mays). Early-maturing sweet corn varieties and some elite inbred maize lines are particularly susceptible. DISEASE SYMPTOMS: There are two major phases of Stewart's wilt disease: (i) wilt and (ii) leaf blight. The wilt phase occurs when young seedlings are infected with P. stewartii subsp. stewartii (Fig. 1A). Water-soaked lesions first appear on the young expanding leaves and, later, seedlings may become severely wilted (Fig. 1B). The plants usually die when infected at the seedling stage. The leaf blight phase occurs when mature plants are infected (Fig. 1C). The bacteria enter the xylem and cause long linear yellow-grey lesions with a wavy margin that run parallel to the leaf veins. These lesions later turn necrotic and dark in colour. The leaf blight phase is most apparent after tasselling and does not generally cause death of the plant. In addition, the bacteria can sometimes break out of the xylem and cause pith rot in mature sweet corn plants. In resistant varieties, lesions are usually limited to only a few centimetres depending on the level of resistance of the particular hybrid (Claflin, 2000; Pataky, 2003). USEFUL WEBSITES: http://www.apsnet.org/publications/apsnetfeatures/Pages/StewartsWilt.aspx.     

Biological Role of Pigment Production for the Bacterial Phytopathogen Pantoea stewartii subsp. stewartii

Pantoea stewartii subsp. stewartii, the causal agent of Stewart's wilt of sweet corn, produces a yellow carotenoid pigment. A nonpigmented mutant was selected from a bank of mutants generated by random transposon mutagenesis. The transposon insertion site was mapped to the crtB gene, encoding a putative phytoene synthase, an enzyme involved in the early steps of carotenoid biosynthesis. We demonstrate here that the carotenoid pigment imparts protection against UV radiation and also contributes to the complete antioxidant pathway of P. stewartii. Moreover, production of this pigment is regulated by the EsaI/EsaR quorum-sensing system and significantly contributes to the virulence of the pathogen in planta.     

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. Sequencing of the hrp cluster revealed a putative harpin gene, hrpN. The product of this gene was overexpressed in Escherichia coli and shown to elicit the HR in tobacco and systemic resistance in radishes. The protein was designated HrpN(Pnss). Like other harpins, it was heat stable and protease sensitive, although it was three- to fourfold less active biologically than Erwinia amylovora harpin. We used antibodies to purified HrpN(Pnss) to verify that hrpN mutants could not produce harpin. This protein was secreted into the culture supernatant and was produced by strains of P. stewartii subsp. indologenes. In order to determine the importance of HrpN(Pnss) in pathogenesis on sweet corn, three hrpN::Tn5 mutants were compared with the wild-type strain with 50% effective dose, disease severity, response time, and growth rate in planta as parameters. In all tests, HrpN(Pnss) was not required for infection, growth, or virulence in corn or endophytic growth in related grasses.     

Pantoea stewartii subsp. stewartii exhibits surface motility, which is a critical aspect of Stewart's wilt disease development on maize

Pantoea stewartii subsp. stewartii is a plant-pathogenic bacterium that causes Stewart's vascular wilt in maize. The organism is taxonomically described as aflagellated and nonmotile. We recently showed that P. stewartii colonizes the xylem of maize as sessile, cell-wall-adherent biofilms. Biofilm formation is a developmental process that generally involves some form of surface motility. For that reason, we reexamined the motility properties of P. stewartii DC283 based on the assumption that the organism requires some form of surface motility for biofilm development. Here, we show that the organism is highly motile on agar surfaces. This motility is flagella dependent, shown by the fact that a fliC mutant, impaired in flagellin subunit synthesis, is nonmotile. Motility also requires the production of stewartan exopolysaccharide. Moreover, surface motility plays a significant role in the colonization of the plant host.     

玉米细菌性枯萎病及其病原菌的检测技术

玉米细菌性枯萎病是影响玉米生产的重要病害 ,该病害通过带菌的种子进行远距离传播 ,对该病菌的检测成为防止和控制该病害的重要手段。介绍现有的检测该病菌的各种方法 ,即黑色素选择培养法、double sandwichELISA以及RAPD PCR ,LCR PCR ,Nested PCR ,multiplePCR和荧光实时PCR等分子生物学检测方法 ,并对这些方法的特性进行比较和探讨。     

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. WtsE, a type III secreted effector protein, is essential for the virulence of Pnss on corn. It belongs to the AvrE family of effectors, which includes DspA/E from Erwinia amylovora and AvrE1 from Pseudomonas syringae . Previously, WtsE was shown to cause disease-associated cell death in its host plant, sweet corn. Here, we examine the biological activity of WtsE in several non-host plants. WtsE induced cell death in Nicotiana benthamiana , tobacco, beet and Arabidopsis thaliana when it was transiently produced in plant cells following agroinfiltration or translocated into plant cells from Pnss , Escherichia coli or Pseudomonas syringae pv. phaseolicola ( Pph ). WtsE-induced cell death in N. benthamiana , tobacco and beet resembled a hypersensitive response and in N. benthamiana it was delayed by cycloheximide. Interestingly, WtsE strongly promoted the growth of Pnss in N. benthamiana prior to the onset of cell death. Deletion derivatives of WtsE that failed to induce cell death in N. benthamiana and tobacco also did not complement wtsE mutants of Pnss for virulence in sweet corn, indicating a correlation between the two activities. WtsE also induced cell death in A. thaliana , where it suppressed basal defences induced by Pph . Thus, WtsE has growth-promoting, defence-suppressing and cell death-inducing activities in non-host plants. Expression of WtsE also prevented the growth of yeast, possibly due to an innate toxicity to eukaryotic cells.     

Population densities of corn flea beetle (Coleoptera: Chrysomelidae) and incidence of Stewart's wilt in sweet corn

To quantify populations of the corn flea beetle, Chaetocnema pulicaria Melsheimer (Coleoptera: Chrysomelidae), and refine estimates of a threshold for its control to prevent Stewart's wilt caused by Erwinia stewartii, sequential plantings of 'Jubilee' sweet corn were made at 2-wk intervals from April or May through August or September 2001 and 2002 at four locations from southern to northern Illinois: Simpson, Brownstown, Champaign, and Mendota. Densities of C. pulicaria and incidence of Stewart's wilt were monitored weekly. At Mendota, where C. pulicaria populations were decimated by cold temperatures during winter 2000-2001, densities reached 33.3 beetles per 15-cm yellow sticky trap per day by September 2002, after a mild 2001-2002 winter. Maximum incidence of Stewart's wilt in single plots at Simpson, Brownstown, Champaign, and Mendota was 22, 36, 39, and 2%, respectively, in 2001, and 33, 47, 99, and 87%, respectively, in 2002. In 24 plots where beetle densities were < or =2 per trap per day, Stewart's wilt incidence was <5% in 20 plots. We propose that two corn flea beetles per trap per day be used as a threshold for insecticide application to seedlings to control C. pulicaria and minimize subsequent incidence of Stewart's wilt in processing sweet corn. Enzyme-linked immunosorbent assays indicated that E. stewartii incidence in C. pulicaria peaked at 67, 62, and 54%, respectively, at Simpson, Brownstown, and Champaign, in 2001, and at 71, 76, and 60%, respectively, in 2002. Further studies might allow the use of areawide or field-specific estimates of E. stewartii incidence in corn flea beetles for adjusting management decisions.     

Miniprimer PCR assay targeting multiple genes: a new rapid and reliable tool for genotyping Pantoea stewartii subsp. stewartii

Aim: Development of a ‘miniprimer’ PCR assay for genotyping Pantoea stewartii subsp. stewartii, the causal agent of the Stewart’s bacterial wilt on maize. Methods and Results: Four 10‐nucleotide (10‐nt) ‘miniprimer’ sets were designed and evaluated in the presence of Titanium Taq DNA polymerase. Under optimal reaction conditions, the miniprimer pair Uni‐BacF‐10/Uni‐BacR‐10 reproducibly generated identical banding patterns among 10 strains of P. stewartii subsp. stewartii, different patterns from strains of P. stewartii subsp. indologenes, other Panteoa species, Clavibacter michiganensis, Pectobacterium spp., Pseudomonas spp. and other bacterial species. The amplicons of Pantoea stewartii subsp. stewartii were cloned and sequenced to identify genes or DNA fragments that are targeted by the miniprimer PCR assay. Of the 14 ‘clone types’ identified, sequences of a 1·23‐kb fragment had a 99·8% similarity to part of the Pantoea stewartii zeaxanthin diglucoside biosynthetic operon ( AY166713 ). Other dominant cloned fragments included a 411‐bp amplicon that exhibited 99·8% similarity to the psaU gene (syn:ysaU; GQ249669 ), a type III protein‐secretion system complex of P. stewartii subsp. stewartii strain DC283, and a 548‐bp fragment showed 63% homology to the Asp/Glu racemase encoding gene in Erwinia tasmaniensis strain ET1/99. Conclusion: The miniprimer PCR assay reported here is highly discriminatory and reproducible in genotyping Pantoea stewartii subsp. stewartii. Significance and Impact of the study: This miniprimer PCR assay could be a new reliable and rapid tool for fingerprinting the Stewart’s wilt pathogen of maize.     

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 single genetic locus in the phytopathogen Pantoea stewartii enables gut colonization and pathogenicity in an insect host

Aphids are typically exposed to a variety of epiphytic and phytopathogenic bacteria, many of which have entomopathogenic potential. Here we describe the interaction between Pantoea stewartii ssp. stewartii DC283 (DC283), an enteric phytopathogen and causal agent of Stewart's wilt, and the pea aphid, Acyrthosiphon pisum . When ingested by aphids, DC283 establishes and aggregates in the crop and gut, preventing honeydew flow and excretion, resulting in aphid death in 72 h. A mutagenesis screen identified a single locus, termed ucp1 (yo u c annot p ass), whose disruption abolishes aphid pathogenicity. Moreover, the expression of ucp1 in Escherichia coli is sufficient to mediate the hindgut aggregation phenotype by this normally avirulent species. Ucp1 is related to six other proteins in the DC283 genome, each having a common N-terminal region and a divergent C-terminus, but only ucp1 has a role in pathogenicity. Based on predicted motifs and secondary structure, Ucp1 is a membrane-bound protein that functions in bacterial adhesion and promotes the formation of aggregates that are lethal to the insect host. These results illustrate that the enteric plant pathogenic bacteria have the capacity to exploit alternative non-plant hosts, and retain genetic determinants for colonizing the gut.     

Probing the impact of ligand binding on the acyl-homoserine lactone-hindered transcription factor EsaR of Pantoea stewartii subsp. stewartii

The quorum-sensing regulator EsaR from Pantoea stewartii subsp. stewartii is a LuxR homologue that is inactivated by acyl-homoserine lactone (AHL). In the corn pathogen P. stewartii, production of exopolysaccharide (EPS) is repressed by EsaR at low cell densities. However, at high cell densities when high concentrations of its cognate AHL signal are present, EsaR is inactivated and derepression of EPS production occurs. Thus, EsaR responds to AHL in a manner opposite to that of most LuxR family members. Depending on the position of its binding site within target promoters, EsaR serves as either a repressor or activator in the absence rather than in the presence of its AHL ligand. The effect of AHL on LuxR homologues has been difficult to study in vitro because AHL is required for purification and stability. EsaR, however, can be purified without AHL enabling an in vitro analysis of the response of the protein to ligand. Western immunoblots and pulse-chase experiments demonstrated that EsaR is stable in vivo in the absence or presence of AHL. Limited in vitro proteolytic digestions of a biologically active His-MBP tagged version of EsaR highlighted intradomain and interdomain conformational changes that occur in the protein in response to AHL. Gel filtration chromatography of the full-length fusion protein and cross-linking of the N-terminal domain both suggest that this conformational change does not impact the multimeric state of the protein. These findings provide greater insight into the diverse mechanisms for AHL responsiveness found within the LuxR family.     

The genetic and structural basis of two distinct terminal side branch residues in stewartan and amylovoran exopolysaccharides and their potential role in host adaptation

Stewartan and amylovoran exopolysaccharide (EPS) produced by the plant pathogenic bacteria Pantoea stewartii and Erwinia amylovora are virulence factors in the cause of Stewart's vascular wilt and fire blight. The biosynthesis of amylovoran and stewartan is encoded by a set of homologous operons that have been partially characterized, although some annotations are solely on the basis of sequence homology. The major distinguishing features of these two EPS forms are the presence of a terminal pyruvate in amylovoran and glucose in stewartan, even though the gene systems to account for both are conserved and present in each bacterium. This study explores the genetic, structural and functional differences of amylovoran and stewartan, and their potential role in host adaptation. We report that the pyruvyl transferase gene in P. stewartii is non-functional, while the terminal glucosyl transferase is catalytically active. Conversely, in E. amylovora, the homologous glucosyl transferase activity appears to be relatively ineffective, while the pyruvyl transferase function predominates. We also show that the terminally pyruvylated versus glucosylated EPS require specific repeating unit translocases (Wzx). We discuss the evolutionary, functional and biological implications of the terminally pyruvylated and glucosylated polymers and their potential contribution to plant and insect host adaptation.     

Identification of Erwinia stewartii by a ligase chain reaction assay.

A PCR-coupled ligase chain reaction (LCR) assay was developed to distinguish the plant pathogenic bacterium Erwinia stewartii from other erwiniae. This new technique allows discrimination to the species level on the basis of a single-base-pair difference in the 16S rRNA gene which is unique to E. stewartii. Portions of the 16S rRNA genes of E. stewartii and the closely related Erwinia herbicola were sequenced. From comparison of the two 16S rRNA gene regions, two primer pairs were constructed such that only E. stewartii DNA gave a product in the LCR assay. The ligated product was separated from the radioactively labelled primers by denaturing polyacrylamide gel electrophoresis and visualized by autoradiography. Twenty-four different Erwinia species and strains were tested by PCR-coupled LCR to verify the specificity of the assay, and only E. stewartii strains gave a positive reaction. In addition, infected and healthy plant material was also assayed. E. stewartii was detected in infected plant material, even when large populations of epiphytic bacteria were present. No enrichment was necessary for detection of the pathogen in corn leaves. This assay has potential as a diagnostic technique for the detection of E. stewartii in infected plant and vector material.     

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. Seventeen open reading frames (ORFs) comprise seven genetic complementation groups. These ORFs share homology with hrp and dsp genes from Erwinia amylovora, Erwinia chrysanthemi, and Pseudomonas syringae pathovars and have been designated, in map order, wtsF, wtsE, hrpN, hrpV, hrpT, hrcC, hrpG, hrpF, hrpE, hrpD, hrcJ, hrpB, hrpA, hrpS, hrpY, hrpX, and hrpL. Putative hrp consensus promoter sequences were identified upstream of hrpA, hrpF, hrpN, and wtsE. Expression of the hrpA, hrpC, and wtsE operons was regulated by HrpS. Transposon mutations in all of the hrp operons abolished pathogenicity and HR elicitation, except for the hrpN and hrpV mutants, which were still pathogenic. hrpS, hrpXY, and hrpL regulatory mutations abolished HrpN synthesis, whereas secretory mutations in the hrpC, hrpA, and hrpJ operons permitted intracellular HrpN synthesis. wtsEF mutants were not pathogenic but still produced HrpN and elicited the HR. wtsE encodes a 201-kDa protein that is similar to DspE in E. amylovora and AvrE in P. syringae pv. tomato, suggesting that this protein is a major virulence factor involved in the elicitation of water-soaked lesions.     

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. Their molecular mass of ca. 5 MDa, when isolated from agar plates, decreases to ca. 1 MDa for E. amylovora amylovoran from freeze-dried supernatants from liquid cultures and to 2 MDa for freeze-dried preparations of P. stewartii stewartan. Size changes were also found following growth in various other media and for different strains. Stewartan, amylovoran and E. pyrifoliae pyrifolan were also shown to be completely degraded by a bacteriophage EPS depolymerase.