The global response regulator ExpA controls virulence gene expression through RsmA-mediated and RsmA-independent pathways in Pectobacterium wasabiae SCC3193.

ExpA (GacA) is a global response regulator that controls the expression of major virulence genes, such as those encoding plant cell wall-degrading enzymes (PCWDEs) in the model soft rot phytopathogen Pectobacterium wasabiae SCC3193. Several studies with pectobacteria as well as related phytopathogenic gammaproteobacteria, such as Dickeya and Pseudomonas, suggest that the control of virulence by ExpA and its homologues is executed partly by modulating the activity of RsmA, an RNA-binding posttranscriptional regulator. To elucidate the extent of the overlap between the ExpA and RsmA regulons in P. wasabiae, we characterized both regulons by microarray analysis. To do this, we compared the transcriptomes of the wild-type strain, an expA mutant, an rsmA mutant, and an expA rsmA double mutant. The microarray data for selected virulence-related genes were confirmed through quantitative reverse transcription (qRT-PCR). Subsequently, assays were performed to link the observed transcriptome differences to changes in bacterial phenotypes such as growth, motility, PCWDE production, and virulence in planta. An extensive overlap between the ExpA and RsmA regulons was observed, suggesting that a substantial portion of ExpA regulation appears to be mediated through RsmA. However, a number of genes involved in the electron transport chain and oligogalacturonide metabolism, among other processes, were identified as being regulated by ExpA independently of RsmA. These results suggest that ExpA may only partially impact fitness and virulence via RsmA.

Pathogenicity of and plant immunity to soft rot pectobacteria.

Soft rot pectobacteria are broad host range enterobacterial pathogens that cause disease on a variety of plant species including the major crop potato. Pectobacteria are aggressive necrotrophs that harbor a large arsenal of plant cell wall-degrading enzymes as their primary virulence determinants. These enzymes together with additional virulence factors are employed to macerate the host tissue and promote host cell death to provide nutrients for the pathogens. In contrast to (hemi)biotrophs such as Pseudomonas, type III secretion systems (T3SS) and T3 effectors do not appear central to pathogenesis of pectobacteria. Indeed, recent genomic analysis of several Pectobacterium species including the emerging pathogen Pectobacterium wasabiae has shown that many strains lack the entire T3SS as well as the T3 effectors. Instead, this analysis has indicated the presence of novel virulence determinants. Resistance to broad host range pectobacteria is complex and does not appear to involve single resistance genes. Instead, activation of plant innate immunity systems including both SA (salicylic acid) and JA (jasmonic acid)/ET (ethylene)-mediated defenses appears to play a central role in attenuation of Pectobacterium virulence. These defenses are triggered by detection of pathogen-associated molecular patterns (PAMPs) or recognition of modified-self such as damage-associated molecular patterns (DAMPs) and result in enhancement of basal immunity (PAMP/DAMP-triggered immunity or pattern-triggered immunity, PTI). In particular plant cell wall fragments released by the action of the degradative enzymes secreted by pectobacteria are major players in enhanced immunity toward these pathogens. Most notably bacterial pectin-degrading enzymes release oligogalacturonide (OG) fragments recognized as DAMPs activating innate immune responses. Recent progress in understanding OG recognition and signaling allows novel genetic screens for OG-insensitive mutants and will provide new insights into plant defense strategies against necrotrophs such as pectobacteria.

Characterization of the hrp pathogenicity cluster of Erwinia carotovora subsp. carotovora: high basal level expression in a mutant is associated with reduced virulence.

Extracellularly targeted proteins are crucial for virulence of gram-negative phytopathogenic bacteria. Erwinia carotovora subsp. carotovora employs the so-called type II (GSP) pathway to secrete a number of pectinases and cellulases, which cause the typical tissue maceration symptoms of soft-rot disease. The type III (hrp) pathway is the major virulence determinant in the genera Pseudomonas, Ralstonia and Xanthomonas, and in non-macerating species of Erwinia. The hrp cluster was recently partially characterized from E. carotovora sp. carotovora, and shown to affect virulence during early stages of infection. Here we have isolated and characterized 15 hrp genes comprising the remaining part of the cluster. The genes hrpL, hrpXY and hrpS were deduced to be transcribed as separate units, whereas the 11 remaining genes from hrpJ to hrcU form a single large operon. The hrpX gene, which codes for the sensory kinase of the two-component regulatory locus hrpXY was insertionally inactivated by placing a transposon (entranceposon) in the gene. The resulting mutant bacterium expresses the hrp genes at high basal level even in a non-inducing medium. This relative overexpression was shown to be due to the hrpX::entranceposon insertion causing enhanced transcription of the downstream hrpY gene. The hrpX(-)-hrpYC mutant bacterium exhibited a slower growth rate and the appearance of disease symptoms in infected Arabidopsis plants was delayed, as compared to the wild-type strain. The need for hrp gene expression for virulence has been documented in both non-macerating plant pathogens and in soft-rotting Erwinia sp. but this is the first demonstration that high basal-level expression of hrp -regulated genes may actually have a negative impact on disease progress in a susceptible host plant.

Transgenic plants producing the bacterial pheromone N-acyl-homoserine lactone exhibit enhanced resistance to the bacterial phytopathogen Erwinia carotovora.

Bacterial pheromones, mainly different homoserine lactones, are central to a number of bacterial signaling processes, including those involved in plant pathogenicity. We previously demonstrated that N-oxoacyl-homoserine lactone (OHL) is essential for quorum sensing in the soft-rot phytopathogen Erwinia carotovora. In this pathogen, OHL controls the coordinate activation of genes encoding the main virulence determinants, extracellular plant cell wall degrading enzymes (PCWDEs), in a cell density-dependent manner. We suggest that E. carotovora employ quorum sensing to avoid the premature production of PCWDEs and subsequent activation of plant defense responses. To test whether modulating this sensory system would affect the outcome of a plant-pathogen interaction, we generated transgenic tobacco, producing OHL. This was accomplished by ectopic expression in tobacco of the E. carotovora gene expI, which is responsible for OHL biosynthesis. We show that expI-positive transgenic tobacco lines produced the active pheromone and partially complemented the avirulent phenotype of expI mutants. The OHL-producing tobacco lines exhibited enhanced resistance to infection by wild-type E. carotovora. The results were confirmed by exogenous addition of OHL to wild-type plants, which also resulted in increased resistance to E. carotovora.

Global regulators ExpA (GacA) and KdgR modulate extracellular enzyme gene expression through the RsmA-rsmB system in Erwinia carotovora subsp. carotovora.

The production of the main virulence determinants, the extracellular plant cell wall-degrading enzymes, and hence virulence of Erwinia carotovora subsp. carotovora is controlled by a complex regulatory network. One of the global regulators, the response regulator ExpA, a GacA homolog, is required for transcriptional activation of the extracellular enzyme genes of this soft-rot pathogen. To elucidate the mechanism of ExpA control as well as interactions with other regulatory systems, we isolated second-site transposon mutants that would suppress the enzyme-negative phenotype of an expA (gacA) mutant. Inactivation of kdgR resulted in partial restoration of extracellular enzyme production and virulence to the expA mutant, suggesting an interaction between the two regulatory pathways. This interaction was mediated by the RsmA-rsmB system. Northern analysis was used to show that the regulatory rsmB RNA was under positive control of ExpA. Conversely, the expression of rsmA encoding a global repressor was under negative control of ExpA and positive control of KdgR. This study indicates a central role for the RsmA-rsmB regulatory system during pathogenesis, integrating signals from the ExpA (GacA) and KdgR global regulators of extracellular enzyme production in E. carotovora subsp. carotovora.

Type III secretion contributes to the pathogenesis of the soft-rot pathogen Erwinia carotovora: partial characterization of the hrp gene cluster.

The virulence of soft-rot Erwinia species is dependent mainly upon secreted enzymes such as pectinases, pectin lyases, and proteases that cause maceration of plant tissue. Some soft-rot Erwinia spp. also harbor genes homologous to the hypersensitive reaction and pathogenesis (hrp) gene cluster, encoding components of the type III secretion system. The hrp genes are essential virulence determinants for numerous nonmacerating gram-negative plant pathogens but their role in the virulence of soft-rot Erwinia spp. is not clear. We isolated and characterized 11 hrp genes of Erwinia carotovora subsp. carotovora. Three putative sigmaL-dependent Hrp box promoter sequences were found. The genes were expressed when the bacteria were grown in Hrp-inducing medium. The operon structure of the hrp genes was determined by mRNA hybridization, and the results were in accordance with the location of the Hrp boxes. An E. carotovora strain with mutated hrcC, an essential hrp gene, was constructed. The hrcC- strain was able to multiply and cause disease in Arabidopsis, but the population kinetics were altered so that growth was delayed during the early stages of infection.

Jasmonate-dependent induction of indole glucosinolates in Arabidopsis by culture filtrates of the nonspecific pathogen Erwinia carotovora.

Elicitors from the plant pathogen Erwinia carotovora trigger coordinate induction of the tryptophan (Trp) biosynthesis pathway and Trp oxidizing genes in Arabidopsis. To elucidate the biological role of such pathogen-induced activation we characterized the production of secondary defense metabolites such as camalexin and indole glucosinolates derived from precursors of this pathway. Elicitor induction was followed by a specific increase in 3-indolylmethylglucosinolate (IGS) content, but only a barely detectable accumulation of the indole-derived phytoalexin camalexin. The response is mediated by jasmonic acid as shown by lack of IGS induction in the jasmonate-insensitive mutant coi1-1. In accordance with this, methyl jasmonate was able to trigger IGS accumulation in Arabidopsis. In contrast, ethylene and salicylic acid seem to play a minor role in the response. They did not trigger alterations in IGS levels, and methyl jasmonate- or elicitor-induced IGS accumulation in NahG and ethylene-insensitive ein2-1 mutant plants was similar as in the wild type. The breakdown products of IGS and other glucosinolates were able to inhibit growth of E. carotovora. The results suggest that IGS is of importance in the defense against bacterial pathogens.

Stress-induced accumulation and tissue-specific localization of dehydrins in Arabidopsis thaliana.

Stress-induced accumulation of five (COR47, LTI29, ERD14, LTI30 and RAB18) and tissue localization of four (LTI29, ERD14, LTI30 and RAB18) dehydrins in Arabidopsis were characterized immunologically with protein-specific antibodies. The five dehydrins exhibited clear differences in their accumulation patterns in response to low temperature, ABA and salinity. ERD14 accumulated in unstressed plants, although the protein level was up-regulated by ABA, salinity and low temperature. LTI29 mainly accumulated in response to low temperature, but was also found in ABA- and salt-treated plants. LTI30 and COR47 accumulated primarily in response to low temperature, whereas RAB18 was only found in ABA-treated plants and was the only dehydrin in this study that accumulated in dry seeds. Immunohistochemical localization of LTI29, ERD14 and RAB18 demonstrated tissue and cell type specificity in unstressed plants. ERD14 was present in the vascular tissue and bordering parenchymal cells, LTI29 and ERD14 accumulated in the root tip, and RAB18 was localized to stomatal guard cells. LTI30 was not detected in unstressed plants. The localization of LTI29, ERD14 and RAB18 in stress-treated plants was not restricted to certain tissues or cell types. Instead these proteins accumulated in most cells, although cells within and surrounding the vascular tissue showed more intense staining. LTI30 accumulated primarily in vascular tissue and anthers of cold-treated plants. This study supports a physiological function for dehydrins in certain plant cells during optimal growth conditions and in most cell types during ABA or cold treatment. The differences in stress specificity and spatial distribution of dehydrins in Arabidopsis suggest a functional specialization for the members of this protein family.

The low-temperature- and salt-induced RCI2A gene of Arabidopsis complements the sodium sensitivity caused by a deletion of the homologous yeast gene SNA1.

Two closely related, tandemly arranged, low-temperature- and salt-induced Arabidopsis genes, corresponding to the previously isolated cDNAs RCI2A and RCI2B, were isolated and characterized. The RCI2A transcript accumulated primarily in response to low temperature or high salinity, and to a lesser extent in response to ABA treatment or water deficit stress. The RCI2B transcript was present at much lower levels than RCI2A, and could only be detected by reverse transcription-PCR amplification. The predicted 6 kDa RCI2 proteins are highly hydrophobic and contain two putative membrane-spanning regions. The polypeptides exhibit extensive similarity to deduced low-temperature- and/or salt-induced proteins from barley, wheat grass and strawberry, and to predicted proteins from bacteria, fungi, nematodes and yeast. Interestingly, we found that a deletion of the RCI2 homologous gene, SNA1 (YRD276c), in yeast causes a salt-sensitive phenotype. This effect is specific for sodium, since no growth defect was observed for the sna1 mutant on 1.7 M sorbitol, 1 M KCl or 0.6 M LiCl. Finally, we found that the Arabidopsis RCI2A cDNA can complement the sna1 mutant when expressed in yeast, indicating that the plant and yeast proteins have similar functions during high salt stress.

The AKT3 potassium channel protein interacts with the AtPP2CA protein phosphatase 2C.

The AKT3 potassium channel protein was identified as a strongly interacting partner of the Arabidopsis thaliana protein phosphatase 2C (AtPP2CA) in a yeast two-hybrid screen. A deletion analysis indicated that the catalytic domain of AtPP2CA was essential for the interaction with AKT3. Furthermore, the related PP2C phosphatase ABI1 did not interact with AKT3 in yeast.

Ectopic expression of ABI3 gene enhances freezing tolerance in response to abscisic acid and low temperature in Arabidopsis thaliana.

The plant hormone abscisic acid (ABA) regulates several physiological and developmental processes in plants, including stress adaptation and seed maturation. ABA-mediated processes appear to be central in plant cold acclimation and expression of cold acclimation-related genes. Ectopic expression of ABI3 encoding a seed-specific transcriptional activator confers on Arabidopsis vegetative tissues the ability to accumulate seed-specific transcripts in response to ABA, and also influences some ABA-mediated vegetative responses. In the present study we characterized the effect of ectopic expression of ABI3 on cold acclimation and development of freezing tolerance in Arabidopsis. We first determined the effect of ABI3 on ABA-induced expression of cold acclimation-related genes. Expression of ABI3 increased the ABA-induced accumulation of transcripts for several ABA/cold/drought-responsive genes such as RAB18 and LTI78. Enhanced expression of these genes was evident even after transient application of ABA, and the enhanced expression was correlated with increased freezing tolerance in ABI3 transgenic plants. Ectopic expression of ABI3 also appeared to modulate low temperature-induced freezing tolerance. The ABI3 transgenic plants acclimated faster than the wild-type plants, and the maximum tolerance obtained was significantly higher. These data showed that lower levels of ABA were needed to trigger the expression of the genes and to maintain the freezing-tolerant state in the ABI3 transgenic plants, and indicate that ectopic expression of ABI3 leads to enhanced responsiveness to ABA. The ectopic expression of ABI3 could provide a new strategy for engineering plant stress tolerance.

Novel receptor-like protein kinases induced by Erwinia carotovora and short oligogalacturonides in potato.

summary Identification of potato genes responsive to cell wall-degrading enzymes of Erwinia carotovora resulted in the isolation of cDNA clones for four related receptor-like protein kinases. One of the putative serine-threonine protein kinases might have arisen through alternative splicing. These potato receptor-like kinases (PRK1-4) were highly equivalent (91-99%), most likely constituting a family of related receptors. All PRKs and four other plant RLKs share in their extracellular domain a conserved bi-modular pattern of cysteine repeats distinct from that in previously characterized plant RLKs, suggesting that they represent a new class of receptors. The corresponding genes were rapidly induced by E. carotovora culture filtrate (CF), both in the leaves and tubers of potato. Furthermore, the genes were transiently induced by short oligogalacturonides. The structural identity of PRKs and their induction pattern suggested that they constitute part of the early response of potato to E. carotovora infection.

Purification of recombinant Arabidopsis thaliana dehydrins by metal ion affinity chromatography.

In this study we describe a novel method for purification of Arabidopsis thaliana dehydrins overproduced in Escherichia coli. The cDNAs corresponding to the four dehydrin genes RAB18, LTI29, LTI30, and COR47 were inserted into a bacterial expression vector under an isopropyl beta-d-thiogalactopyranoside (IPTG) inducible bacterial promoter. After IPTG induction all four proteins accumulated in high amounts. The recombinant proteins were efficiently purified to over 95% purity with a three-step purification scheme: heat fractionation, immobilized metal ion affinity chromatography (IMAC), and ion exchange chromatography. In this study we introduce the novel use of IMAC as an efficient purification method for native dehydrins. Characterization of the purified proteins was done by Edman degradation, mass spectrometry, reverse-phase chromatography, and analytical gel filtration under native and denaturing conditions. Yields of purified proteins were between 2.8 and 12.5 mg per liter of bacterial culture, sufficient for further biochemical studies.

A potato gene encoding a WRKY-like transcription factor is induced in interactions with Erwinia carotovora subsp. atroseptica and Phytophthora infestans and is coregulated with class I endochitinase expression.

A potato gene encoding a putative WRKY protein was isolated from a cDNA library enriched by suppression subtractive hybridization for sequences upregulated 1 h postinoculation with Erwinia carotovora subsp. atroseptica. The cDNA encodes a putative polypeptide of 172 amino acids, containing a single WRKY domain with a zinc finger motif and preceded by a potential nuclear localization site. St-WRKY1 was strongly upregulated in compatible, but only weakly in incompatible, interactions with Phytophthora infestans where, in all cases, it was coregulated with class I endochitinase, associating its expression with a known defense response. Whereas St-WRKY1 was strongly induced by E. carotovora culture filtrate (CF), confirming it to be an elicitor-induced gene, no such induction was detected after treatment with salicylic acid, methyl jasmonate, ethylene, or wounding. St-WRKY1 was upregulated by treatment of potato leaves with CFs from recombinant Escherichia coli containing plasmids expressing E. carotovora pectate lyase genes pelB and pelD, suggesting that either proteins encoded by these genes, or oligogalacturonides generated by their activity, elicit a potato defense pathway associated with St-WRKY1.

Quorum sensing in the plant pathogen Erwinia carotovora subsp. carotovora: the role of expR(Ecc).

The production of the main virulence determinants of the plant pathogen Erwinia carotovora subsp. carotovora, the extracellular cell wall-degrading enzymes, is partly controlled by the diffusible signal molecule N-(3-oxohexanoyl)-L-homoserine lactone (OHHL). OHHL is synthesized by the product of the expI/carI gene. Linked to expI we found a gene encoding a putative transcriptional regulator of the LuxR-family. This gene, expR(Ecc), is transcribed convergently to the expI gene and the two open reading frames are partially overlapping. The ExpR(Ecc) protein showed extensive amino acid sequence similarity to the repressor EsaR from Pantoea stewartii subsp. stewartii (formerly Erwinia stewartii subsp. stewartii) and to the ExpR(Ech) protein of Erwinia chrysanthemi. Inactivation of the E. carotovora subsp. carotovora expR(Ecc) gene caused no decrease in virulence or production of virulence determinants in vitro. In contrast, there was a slight increase in the maceration capacity of the mutant strain. The effects of ExpR(Ecc) were probably mediated by changes in OHHL levels. Inactivation of expR(Ecc) resulted in increased OHHL levels during early logarithmic growth. In addition, overexpression of expR(Ecc) caused a clear decrease in the production of virulence determinants and part of this effect was likely to be caused by OHHL binding to ExpR(Ecc). ExpR(Ecc) did not appear to exhibit transcriptional regulation of expI, but the effect on OHHL was apparently due to other mechanisms.

Interacting signal pathways control defense gene expression in Arabidopsis in response to cell wall-degrading enzymes from Erwinia carotovora.

We have characterized the role of salicylic acid (SA)-independent defense signaling in Arabidopsis thaliana in response to the plant pathogen Erwinia carotovora subsp. carotovora. Use of pathway-specific target genes as well as signal mutants allowed us to elucidate the role and interactions of ethylene, jasmonic acid (JA), and SA signal pathways in this response. Gene expression studies suggest a central role for both ethylene and JA pathways in the regulation of defense gene expression triggered by the pathogen or by plant cell wall-degrading enzymes (CF) secreted by the pathogen. Our results suggest that ethylene and JA act in concert in this regulation. In addition, CF triggers another, strictly JA-mediated response inhibited by ethylene and SA. SA does not appear to have a major role in activating defense gene expression in response to CF. However, SA may have a dual role in controlling CF-induced gene expression, by enhancing the expression of genes synergistically induced by ethylene and JA and repressing genes induced by JA alone.

A two-component regulatory system, pehR-pehS, controls endopolygalacturonase production and virulence in the plant pathogen Erwinia carotovora subsp. carotovora.

Genes coding for the main virulence determinants of the plant pathogen Erwinia carotovora subsp. carotovora, the plant cell wall-degrading enzymes, are under the coordinate control of global regulator systems including both positive and negative factors. In addition to this global control, some virulence determinants are subject to specific regulation. We have previously shown that mutations in the pehR locus result in reduced virulence and impaired production of one of these enzymes, an endopolygalacturonase (PehA). In contrast, these pehR strains produce essentially wild-type levels of other extracellular enzymes including pectate lyases and cellulases. In this work, we characterized the pehR locus and showed that the DNA sequence is composed of two genes, designated pehR and pehS, present in an operon. Mutations in either pehR or pehS caused a Peh-negative phenotype and resulted in reduced virulence on tobacco seedlings. Complementation experiments indicated that both genes are required for transcriptional activation of the endopolygalacturonase gene, pehA, as well as restoration of virulence. Structural characterization of the pehR-pehS operon demonstrated that the corresponding polypeptides are highly similar to the two-component transcriptional regulators PhoP-PhoQ of both Escherichia coli and Salmonella typhimurium. Functional similarity of PehR-PehS with PhoP-PhoQ of E. coli and S. typhimurium was demonstrated by genetic complementation.

The response regulator expM is essential for the virulence of Erwinia carotovora subsp. carotovora and acts negatively on the sigma factor RpoS (sigma s).

The main virulence factors of Erwinia carotovora subsp. carotovora, the secreted, extracellular cell-wall-degrading enzymes, are controlled by several regulatory mechanisms. We have isolated transposon mutants with reduced virulence on tobacco. One of these mutants, with a mutation in a gene designated expM, was characterized in this study. This mutant produces slightly reduced amounts of extracellular enzymes in vitro and the secretion of the enzymes is also affected. The expM wild-type allele was cloned together with an upstream gene, designated expL, that has an unknown function. The expM gene was sequenced and found to encode a protein with similarity to the RssB/SprE protein of Escherichia coli and the MviA protein of Salmonella typhimurium. These proteins belong to a new type of two-component response regulators that negatively regulate the stability of the Sigma factor RpoS (sigma s) at the protein level. The results of this study suggest that ExpM has a similar function in E. carotovora subsp. carotovora. We also provide evidence that the overproduction of RpoS in the expM mutant is an important factor for the reduced virulence phenotype and that it partly causes the observed phenotype seen in vitro. However, an expM/rpoS double mutant is still affected in secretion of extracellular enzymes, suggesting that ExpM in addition to RpoS also acts on other targets.

Oligogalacturonide-mediated induction of a gene involved in jasmonic acid synthesis in response to the cell-wall-degrading enzymes of the plant pathogen Erwinia carotovora.

Identification of Arabidopsis thaliana genes responsive to plant cell-wall-degrading enzymes of Erwinia carotovora subsp. carotovora led to the isolation of a cDNA clone with high sequence homology to the gene for allene oxide synthase, an enzyme involved in the biosynthesis of jasmonates. Expression of the corresponding gene was induced by the extracellular enzymes from this pathogen as well as by treatment with methyl jasmonate and short oligogalacturonides (OGAs). This suggests that OGAs are involved in the induction of the jasmonate pathway during plant defense response to E. carotovora subsp. carotovora attack.

A novel flower-specific Arabidopsis gene related to both pathogen-induced and developmentally regulated plant beta-1,3-glucanase genes.

Beta-1,3-glucanases are usually associated with plant defense responses, although some are also developmentally or hormonally regulated. We characterized two Arabidopsis genes linked in a tandem array, BG4 and BG5, encoding putative novel isoforms of beta-1,3-glucanase. The deduced polypeptides, BG4 and BG5, were highly similar to each other (89% amino acid identity) but only moderately related (32 to 41% amino acid identity) to the different categories of previously characterized beta-1,3-glucanases, suggesting that BG4 and BG5 may represent a novel class of beta-1,3-glucanases in plants. Neither of the genes was responsive to pathogen or SA induction in contrast to the previously identified Arabidopsis beta-1,3-glucanases, nor could we detect any developmental or hormonally induced expression in the vegetative parts of the plants. Both RNA blot and in situ hybridization data demonstrated that the BG4 gene was specifically expressed in the style and septum of the ovary, suggesting that the corresponding protein is involved in the reproductive process of the plant.