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.     

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.     

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.     

Pantoea stewartii subsp. stewartii produces an endoglucanase that is required for full virulence in sweet corn

Pantoea stewartii subsp. stewartii, a xylem-dwelling bacterium, is the causal agent of Stewart's wilt and blight of sweet corn. The goal of this study was to characterize the only gene in the P. stewartii subsp. stewartii genome predicted to encode an endoglucanase (EGase); this gene was designated engY. Culture supernatants from P. stewartii subsp. stewartii and Escherichia coli expressing recombinant EngY protein possessed both EGase and xylanase activities. Deletion of engY abolished EGase and xylanase activity, demonstrating that EngY appears to be the major EGase or xylanase produced by P. stewartii subsp. stewartii. Most importantly, our results show that EngY contributes to movement in the xylem and disease severity during the wilting phase of Stewart's wilt but is not required for water-soaked lesion formation.     

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.     

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.     

Constitutive expression of hrap gene in transgenic tobacco plant enhances resistance against virulent bacterial pathogens by induction of a hypersensitive response

Hypersensitive response-assisting protein (HRAP) has been previously reported as an amphipathic plant protein isolated from sweet pepper that intensifies the harpin(Pss)-mediated hypersensitive response (HR). The hrap gene has no appreciable similarity to any other known sequences, and its activity can be rapidly induced by incompatible pathogen infection. To assess the function of the hrap gene in plant disease resistance, the CaMV 35S promoter was used to express sweet pepper hrap in transgenic tobacco. Compared with wild-type tobacco, transgenic tobacco plants exhibit more sensitivity to harpin(Pss) and show resistance to virulent pathogens (Pseudomonas syringae pv. tabaci and Erwinia carotovora subsp. carotovora). This disease resistance of transgenic tobacco does not originate from a constitutive HR, because endogenous level of salicylic acid and hsr203J mRNA showed similarities in transgenic and wildtype tobacco under noninfected conditions. However, following a virulent pathogen infection in hrap transgenic tobacco, hsr203J was rapidly induced and a micro-HR necrosis was visualized by trypan blue staining in the infiltration area. Consequently, we suggest that the disease resistance of transgenic plants may result from the induction of a HR by a virulent pathogen infection.     

EcbCSL101菌株hrpN基因的克隆、表达及HarpinEcbCSL101蛋白的生物学活性

胡萝卜软腐欧文氏菌甜菜亚种(Erwinia carotovora subsp. betavasculorum) EcbCSL101菌株具有很强胞外酶分泌活性, 接种非寄主植物烟草引起过敏反应。Southern blotting结果表明EcbCSL101菌株中含有hrpN 基因。PCR扩增含EcbCSL101完整开放阅读框的DNA片段并克隆到表达载体pET28a(+)中。核苷酸序列分析表明, EcbCSL101菌株的hrpN 基因的ORF为1113 bp, 编码36.65 kD HarpinEcbCSL101蛋白(GenBank, DQ355519),与其它几种软腐欧文氏菌Harpin蛋白有较高的同源性。将含有hrpNEcbCSL101基因的重组质粒转化到大肠杆菌JM109(DE3)中进行表达,纯化后的HarpinEcbCSL101能诱导烟草发生过敏反应。     

EcbCSL101菌株hrpN基因的克隆、表达及HarpinEcbCSL101蛋白的生物学活性

胡萝卜软腐欧文氏茵甜菜亚种(Erwinia carotovora subsp.betavasculorum)EcbCSL101菌株具有很强胞外酶分泌活性,接种非寄主植物烟草引起过敏反应.Southern blotting结果表明EcbCSL101菌株中含有hrpN基因.PCR扩增含EcbCSL101完整开放阅读框的DNA片段并克隆到表达载体pET28a( )中.核苷酸序列分析表明,EcbCSL101菌株的hrpN基因的ORF为1113 bp,编码36.65 kD HarpinEcbCSL101蛋白(GenBank,DQ355519),与其它几种软腐欧文氏菌Harpin蛋白有较高的同源性.将含有hrpNEcbCSL101基因的重组质粒转化到大肠杆菌JM109(DE3)中进行表达,纯化后的HarpinEcbCSL101能诱导烟草发生过敏反应.     

Transgenic tobacco expressing the hrpN(EP) gene from Erwinia pyrifoliae triggers defense responses against botrytis cinerea

HrpN(EP), from the gram-negative pathogen, Erwinia pyrifoliae, is a member of the harpin group of proteins, inducing pathogen resistance and hypersensitive cell death in plants. When the hrpN(EP) gene driven by the OsCc1 promoter was introduced into tobacco plants via Agrobacterium-mediated transformation, their resistance to the necrotrophic fungal pathogen, Botrytis cinerea, increased. Resistance to B. cinerea was correlated with enhanced induction of SA-dependent genes such as PR-1a, PR2, PR3 and Chia5, of JA-dependent genes such as PR-1b, and of genes related to ethylene production, such as NT-EFE26, NT-1A1C, DS321, NT-ACS1 and NT-ACS2. However the expression of NPR1, which is thought to be essential for multiple-resistance, did not increase. Since the pattern of expression of defense-related genes in hrpN(EP)-expressing tobacco differed from that in plants expressing hpaG(Xoo) from Xanthomonas oryzae pv. Oryzae, these results suggest that different harpins can affect the expression of different defense-related genes, as well as resistance to different plant pathogens.     

hrp genes of Erwinia chrysanthemi 3937 are important virulence factors

We developed improved virulence assays for Erwinia chrysanthemi 3937 on African violet varieties and devised a new method for the construction of precise bacterial gene knockouts. These methods were tested by constructing mutations in genes suspected to be involved with plant interactions. The virulence of the hrpG and hrcC mutant strains (both gene products presumed to be involved in protein secretion) was greatly reduced on leaves of semitolerant African violet varieties. An hrpN mutant strain produced delayed symptoms on African violet leaves and an hrpN delta pel (delta pel = five major pectate lyase genes deleted) double mutant was nonpathogenic. The hrcC and hrpG mutants did not produce a rapid hypersensitive response (HR) in tobacco, unlike the wild-type bacterium, and the hrpN mutant gave a reduced HR. The results, therefore, establish the importance of hrp genes in the virulence of E. chrysanthemi and their ability to elicit HR on nonhosts. The data also suggest that other effector proteins secreted by the Hrp system are required for full virulence and HR elicitation.     

The C-terminal half of the HrpN virulence protein of the fire blight pathogen Erwinia amylovora is essential for its secretion and for its virulence and avirulence activities

The HrpN (harpin) protein of the fire blight pathogen Erwinia amylovora is an essential virulence factor secreted via the bacterial type III secretion system. HrpN also has avirulence activity when delivered to tobacco by E. amylovora and has defense elicitor activity when applied to plants as a cell-free protein extract. Here, we characterize a series of random mutations in hrpN that altered the predicted amino acid sequence of the protein. Amino acid substitutions and deletions in the highly conserved, C-terminal portion of HrpN disrupted the virulence and avirulence activities of the protein. Several of these mutations produced a dominant-negative effect on E. amylovora avirulence on tobacco. None of the mutations clearly separated the virulence and avirulence activities of HrpN. Some C-terminal mutations abolished secretion of HrpN by E. amylovora. The results indicate that the C-terminal half of HrpN is essential for its secretion by E. amylovora, for its virulence activity on apple and pear, and for its avirulence activity on tobacco. In contrast, the C-terminal half of HrpN was not required for cell-free elicitor activity. This suggests that the N-terminal and C-terminal halves of HrpN mediate cell-free elicitor activity and avirulence activity, respectively.     

Expression of the Hypersensitive Response-assisting Protein in <Emphasis Type="Italic">Arabidopsis</Emphasis> Results in Harpin-dependent Hypersensitive Cell Death in Response to <Emphasis Type="Italic">Erwinia carotovora</Emphasis>

Active defense mechanisms of plants against pathogens often include a rapid plant cell death known as the hypersensitive cell death (HCD). Hypersensitive response-assisting protein (HRAP) isolated from sweet pepper intensifies the harpin_Pss-mediated HCD. Here we demonstrate that constitutive expression of the hrap gene in Arabidopsis results in an enhanced disease resistance towards soft rot pathogen, E. carotovora subsp. carotovora. This resistance was due to the induction of HCD since different HCD markers viz. Athsr3, Athsr4, ion leakage, H₂O₂ and protein kinase were induced. One of the elicitor harpin proteins, HrpN, from Erwinia carotovora subsp. carotovora was able to induce a stronger HCD in hrap-Arabidopsis than non-transgenic controls. To elucidate the role of HrpN, we used E. carotovora subsp. carotovora defective in HrpN production. The hrpN⁻ mutant did not induce disease resistance or HCD markers in hrap-Arabidopsis. These results imply that the disease resistance of hrap-Arabidopsis against a virulent pathogen is harpin dependent.     

Harpin induces disease resistance in Arabidopsis through the systemic acquired resistance pathway mediated by salicylic acid and the NIM1 gene

Harpin, the product of the hrpN gene of Erwinia amylovora, elicits the hypersensitive response and disease resistance in many plants. Harpin and known inducers of systemic acquired resistance (SAR) were tested on five genotypes of Arabidopsis thaliana to assess the role of SAR in harpin-induced resistance. In wild-type plants, harpin elicited systemic resistance to Peronospora parasitica and Pseudomonas syringae pv. tomato, accompanied by induction of the SAR genes PR-1 and PR-2. However, in experiments with transgenic Arabidopsis plants containing the nahG gene which prevents accumulation of salicylic acid (SA), harpin neither elicited resistance nor activated SAR gene expression. Harpin also failed to activate SAR when applied to nim1 (non-inducible immunity) mutants, which are defective in responding to SA and regulation of SAR. In contrast, mutants compromised in responsiveness to methyl jasmonate and ethylene developed the same resistance as did wild-type plants. Thus, harpin elicits disease resistance through the NIM1-mediated SAR signal transduction pathway in an SA-dependent fashion. The site of action of harpin in the SAR regulatory pathway is upstream of SA.     

Degeneration of hrpZ gene in Pseudomonas syringae pv. tabaci to evade tobacco defence: an arms race between tobacco and its bacterial pathogen

The HrpZ harpin of Pseudomonas syringae is known to induce a hypersensitive response (HR) in some plants. In P. syringae pv. tabaci (Pta), the harpin gene hrpZ has been spontaneously disrupted by an internal deletion in its open reading frame and a frame shift. The loss of the ability of the recombinant harpin polypeptide of Pta to induce HR despite the high sensitivity of tobacco plants to harpin led us to investigate the meaning of the disrupted hrpZ gene in the virulence of Pta 6605. The hrpZ gene from P. syringae pv. pisi was introduced into wild-type (WT) Pta. The hrpZ-complemented Pta secreted harpin into the culture medium, but failed to cause disease symptoms by both infiltration and spray inoculation. Inoculation with the hrpZ-complemented Pta induced defence responses in tobacco plants, whereas the defence responses of tobacco plants were not prominent on inoculation with WT Pta. These results indicate that an ancestor of Pta might have disrupted hrpZ by an internal deletion to evade plant defences and confer the ability to cause disease in tobacco plants.     

Harpin,An Elicitor of the Hypersensitive Response in Tobacco Caused by Erwinia amylovora,Elicits Active Oxygen Production in Suspension Cells

Active oxygen (AO) production and a K+/H+ exchange response (XR) are two concurrent early events associated with incompatible plant-bacteria interactions that result in a hypersensitive response (HR). Recently, a protein, termed harpin, produced by Erwinia amylovora has been reported to be the elicitor responsible for the HR caused by this pathogen. Although both the bacterium and harpin are reported to induce XR in tobacco (Nicotiana tabacum) cell suspensions, there have been no reports regarding the concurrent production of AO in this system. Here we report that E. amylovora stimulates the AO response, whereas an E. amylovora mutant that does not produce harpin does not elicit the AO response. In addition, a cell-free preparation of harpin induces AO production. This study indicates that harpin may be the bacterial elicitor of the XR and AO responses during the development of E. amylovora-induced HR.