scholarly journals Arabidopsis thaliana Expresses Multiple Lines of Defense to Counterattack Erwinia chrysanthemi

2007 ◽  
Vol 20 (7) ◽  
pp. 794-805 ◽  
Author(s):  
Mathilde Fagard ◽  
Alia Dellagi ◽  
Camille Roux ◽  
Claude Périno ◽  
Martine Rigault ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Signaling Pathways ◽  
Oxidative Burst ◽  
Pectate Lyase ◽  
Erwinia Chrysanthemi ◽  
Secreted Proteins ◽  
Type Ii Secretion ◽  
Susceptible Host ◽  
Pectate Lyases ◽  
Lyase Activity

Many taxonomically diverse plant species are attacked by Erwinia chrysanthemi, a member of the causal agents of soft-rotting diseases. Symptom development is due to the collective action of pectin-degrading enzymes secreted by the bacterium through a type II secretion system (T2SS). Using Arabidopsis thaliana as a susceptible host, we show that plants respond to E. chrysanthemi 3937 by expressing cell-wall reactions, production of an oxidative burst, and activation of salicylic acid (SA) and jasmonic acid (JA) or ethylene (ET) signaling pathways. We found that the oxidative burst is mainly generated via the expression of the AtrbohD gene, constitutes a barrier of resistance to bacterial attack, and acts independently of the SA-mediated response. To determine the importance of T2SS-secreted proteins in elicitation of these defenses, we used a T2SS deficient mutant and purified enzymatic preparations of representative members of strain 3937 pectate lyase activity. The T2SS-secreted proteins were responsible only partially for the activation of SA and JA or ET signaling pathways observed after infection with the wild-type bacterium and were not involved in the expression of other identified defense reactions. Our study shows the differential role played by pectate lyases isoenzymes in this process and highlights the complexity of the host immune network, which is finely controlled by the bacterium.

scholarly journals The Exopolygalacturonate Lyase PelW and the Oligogalacturonate Lyase Ogl, Two Cytoplasmic Enzymes of Pectin Catabolism in Erwinia chrysanthemi 3937

1999 ◽  
Vol 181 (13) ◽  
pp. 3912-3919 ◽  
Author(s):  
Vladimir E. Shevchik ◽  
Guy Condemine ◽  
Janine Robert-Baudouy ◽  
Nicole Hugouvieux-Cotte-Pattat
Keyword(s):  
Yersinia Pseudotuberculosis ◽  
Divalent Cations ◽  
Pectate Lyase ◽  
Erwinia Carotovora ◽  
Erwinia Chrysanthemi ◽  
Pectate Lyases ◽  
Acid Protein ◽  
Lyase Activity ◽  
Bacterial Cytoplasm ◽  
High Degree

ABSTRACT Erwinia chrysanthemi 3937 secretes into the external medium several pectinolytic enzymes, among which are eight isoenzymes of the endo-cleaving pectate lyases: PelA, PelB, PelC, PelD, and PelE (family 1); PelI (family 4); PelL (family 3); and PelZ (family 5). In addition, one exo-cleaving pectate lyase, PelX (family 3), has been found in the periplasm of E. chrysanthemi. The E. chrysanthemi 3937 gene kdgC has been shown to exhibit a high degree of similarity to the genes pelY ofYersinia pseudotuberculosis and pelB ofErwinia carotovora, which encode family 2 pectate lyases. However, no pectinolytic activity has been assigned to the KdgC protein. After verification of the corresponding nucleotide sequence, we cloned a longer DNA fragment and showed that this gene encodes a 553-amino-acid protein exhibiting an exo-cleaving pectate lyase activity. Thus, the kdgC gene was renamed pelW. PelW catalyzes the formation of unsaturated digalacturonates from polygalacturonate or short oligogalacturonates. PelW is located in the bacterial cytoplasm. In this compartment, PelW action could complete the degradation of pectic oligomers that was initiated by the extracellular or periplasmic pectinases and precede the action of the cytoplasmic oligogalacturonate lyase, Ogl. Both cytoplasmic pectinases, PelW and Ogl, seem to act in sequence during oligogalacturonate depolymerization, since oligomers longer than dimers are very poor substrates for Ogl but are good substrates for PelW. The estimated number of binding subsites for PelW is three, extending from subsite −2 to +1, while it is probably two for Ogl, extending from subsite −1 to +1. The activities of the two cytoplasmic lyases, PelW and Ogl, are dependent on the presence of divalent cations, since both enzymes are inhibited by EDTA. In contrast to the extracellular pectate lyases, Ca2+ is unable to restore the activity of PelW or Ogl, while several other cations, including Co2+, Mn2+, and Ni2+, can activate both cytoplasmic lyases.

scholarly journals HrpW of Erwinia amylovora, a New Harpin That Contains a Domain Homologous to Pectate Lyases of a Distinct Class

1998 ◽  
Vol 180 (19) ◽  
pp. 5203-5210 ◽  
Author(s):  
Jihyun F. Kim ◽  
Steven V. Beer
Keyword(s):  
Plant Cell Wall ◽  
Erwinia Amylovora ◽  
Pectate Lyase ◽  
Hypersensitive Reaction ◽  
Host Tissue ◽  
Wild Type ◽  
Pectate Lyases ◽  
Heat Stable ◽  
Lyase Activity ◽  
Type Strains

ABSTRACT Harpins, such as HrpN of Erwinia amylovora, are extracellular glycine-rich proteins that elicit the hypersensitive reaction (HR). We identified hrpW of E. amylovora, which encodes a protein similar to known harpins in that it is acidic, rich in glycine and serine, and lacks cysteine. A putative HrpL-dependent promoter was identified upstream ofhrpW, and Western blot analysis of hrpL mutants indicated that the production of HrpW is regulated by hrpL. HrpW is secreted via the Hrp (type III) pathway based on analysis of wild-type strains and hrp secretion mutants. When infiltrated into plants, HrpW induced rapid tissue collapse, which required active plant metabolism. The HR-eliciting activity was heat stable and protease sensitive. Thus, we concluded that HrpW is a new harpin. HrpW of E. amylovora consists of two domains connected by a Pro and Ser-rich sequence. A fragment containing the N-terminal domain was sufficient to elicit the HR. Although no pectate lyase activity was detected, the C-terminal region of HrpW is homologous to pectate lyases of a unique class, suggesting that HrpW may be targeted to the plant cell wall. Southern analysis indicated that hrpW is conserved among several Erwiniaspecies, and hrpW, provided in trans, enhanced the HR-inducing ability of a hrpN mutant. However, HrpW did not increase the virulence of a hrpN mutant in host tissue, and hrpW mutants retained the wild-type ability to elicit the HR in nonhosts and to cause disease in hosts.

scholarly journals Molecular Cloning, Characterization, and Mutagenesis of a pel Gene from Pseudomonas syringae pv. lachrymans Encoding a Member of the Erwinia chrysanthemi PelADE Family of Pectate Lyases

1997 ◽  
Vol 10 (3) ◽  
pp. 369-379 ◽  
Author(s):  
David W. Bauer ◽  
Alan Collmer
Keyword(s):  
Host Range ◽  
Pseudomonas Syringae ◽  
Xanthomonas Campestris ◽  
Pectate Lyase ◽  
Erwinia Chrysanthemi ◽  
Broad Host Range ◽  
Protein Sequence Analysis ◽  
Pectate Lyases ◽  
Dna Libraries ◽  
Cucumber Cotyledons

The pelS gene from Pseudomonas syringae pv. lachrymans 859 was cloned by heterologous expression in nonpectolytic P. syringae pv. syringae BUVS1, using genomic DNA libraries constructed with two novel broad-host-range cosmid vectors, pCPP34 and pCPP47. Screening of P. syringae pv. syringae transconjugants for the ability to pit pectate media at pH 6.0 and 8.5 yielded several overlapping clones of the same DNA region. Ultrathin-layer isoelectric focusing gels, activity-stained with diagnostically buffered substrate overlays, revealed that this region encoded a single pectate lyase (PelS) with a pI of 9.4. pelS was subcloned from cosmid pCPP5020 and sequenced, revealing it to encode a member of the Erwinia chrysanthemi PelADE family, with highest similarity to Pseudomonas viridiflava PelV. A pelS probe hybridized at high stringency in DNA gel blots with total DNA from P. syringae pv. lachrymans strains 859 and Pla5, P. syringae pv. tabaci, P. syringae pv. phaseolicola, P. syringae pv. glycinea, P. fluorescens (marginalis), P. viridiflava, and Xanthomonas campestris pv. campestris, but not with P. syringae pv. pisi, P. syringae pv. syringae, P. syringae pv. tomato, P. syringae pv. papulans, E. chrysanthemi, or Ralstonia (Pseudomonas or Burkholderia) solanacearum. The PelS sequence revealed an N-terminal signal peptide, whose processing in Escherichia coli was confirmed by protein sequence analysis. PelS was similar to E. chrysanthemi PelE in its substrate preference and ability to reduce the viscosity of pectate and to macerate potato tuber tissue. A pelS∷ΩKmr mutation was marker-exchanged into P. syringae pv. lachrymans Pla5. pelS was also subcloned into the broad-host-range expression vector pML122 under control of the vector nptII promoter, and then transformed into P. syringae pv. lachrymans Pla5 to produce a strain overproducing PelS. Necrotic lesions developed in cotyledons following inoculation with all of the P. syringae pv. lachrymans Pla5 derivatives, regardless of their Pel phenotype. However, only cotyledons infected with pelS+ strains showed evidence of maceration and yielded Pel activity upon extraction. In contrast, pelS+ P. syringae pv. syringae BUVS1(pCPP5020) produced no symptoms in cucumber cotyledons. Thus, PelS in P. syringae pv. lachrymans appears to alter the final symptoms in infected cucumber cotyledons without contributing to pathogenicity or altering host range.

scholarly journals Commensal Effect of Pectate Lyases Secreted fromDickeya dadantiion Proliferation ofEscherichia coliO157:H7 EDL933 on Lettuce Leaves

2010 ◽  
Vol 77 (1) ◽  
pp. 156-162 ◽  
Author(s):  
Akihiro Yamazaki ◽  
Jin Li ◽  
William C. Hutchins ◽  
Lixia Wang ◽  
Jincai Ma ◽  
...  
Keyword(s):  
Pectate Lyase ◽  
Type Ii Secretion ◽  
Broad Host Range ◽  
Deficient Mutant ◽  
In Planta ◽  
Enzyme Activity Assay ◽  
Dickeya Dadantii ◽  
Pectate Lyases ◽  
Σ Factor ◽  
Tissue Maceration

ABSTRACTThe outbreaks caused by enterohemorrhagicEscherichia coliO157:H7 on leafy greens have raised serious and immediate food safety concerns. It has been suggested that several phytopathogens aid in the persistence and proliferation of the human enteropathogens in the phyllosphere. In this work, we examined the influence of virulence mechanisms ofDickeya dadantii3937, a broad-host-range phytopathogen, on the proliferation of the human pathogenE. coliO157:H7 EDL933 (EDL933) on postharvest lettuce by coinoculation of EDL933 withD. dadantii3937 derivatives that have mutations in virulence-related genes. A type II secretion system (T2SS)-deficient mutant ofD. dadantii3937, A1919 (ΔoutC), lost the capability to promote the multiplication of EDL933, whereas Ech159 (ΔrpoS), a stress-responsive σ factor RpoS-deficient mutant, increased EDL933 proliferation on lettuce leaves. A spectrophotometric enzyme activity assay revealed that A1919 (ΔoutC) was completely deficient in the secretion of pectate lyases (Pels), which play a major role in plant tissue maceration. In contrast to A1919 (ΔoutC), Ech159 (ΔrpoS) showed more than 2-fold-greater Pel activity than the wild-typeD. dadantii3937. Increased expression ofpelD(encodes an endo-pectate lyase) was observed in Ech159 (ΔrpoS)in planta. These results suggest that the pectinolytic activity ofD. dadantii3937 is the dominant determinant of enhanced EDL933 proliferation on the lettuce leaves. In addition, RpoS, the general stress response σ factor involved in cell survival in suboptimal conditions, plays a role in EDL933 proliferation by controlling the production of pectate lyases inD. dadantii3937.

Thermodependence of growth and enzymatic activities implicated in pathogenicity of twoErwinia carotovorasubspecies (Pectobacteriumspp.)

2004 ◽  
Vol 50 (1) ◽  
pp. 19-27 ◽  
Author(s):  
Bruno Smadja ◽  
Xavier Latour ◽  
Sameh Trigui ◽  
Jean François Burini ◽  
Sylvie Chevalier ◽  
...  
Keyword(s):  
Growth Rate ◽  
Extracellular Enzymes ◽  
Pectate Lyase ◽  
Erwinia Carotovora ◽  
Optimal Growth ◽  
Ecological Data ◽  
Pectate Lyases ◽  
Lyase Activity ◽  
Active Multiplication

Erwinia carotovora subsp. atroseptica and Erwinia carotovora subsp. carotovora can cause substantial damage to economically important plant crops and stored products. The occurrence of the disease and the scale of the damage are temperature dependent. Disease development consists first of active multiplication of the bacteria in the infection area and then production of numerous extracellular enzymes. We investigated the effects of various temperatures on these two steps. We assayed the specific growth rate and the pectate lyase and protease activities for eight strains belonging to E. carotovora subsp. atroseptica and E. carotovora subsp. carotovora in vitro. The temperature effect on growth rate and on pectate lyase activity is different for the two subspecies, but protease activity appears to be similarly thermoregulated. Our results are in agreement with ecological data implicating E. carotovora subsp. atroseptica in disease when the temperature is below 20 °C. The optimal temperature for pathogenicity appears to be different from the optimal growth temperature but seems to be a compromise between this temperature and temperatures at which lytic activities are maximal.Key words: temperature, Pectobacterium atrosepticum, Pectobacterium carotovorum, growth, pectate lyases, proteases.

scholarly journals PehN, a Polygalacturonase Homologue with a Low Hydrolase Activity, Is Coregulated with the Other Erwinia chrysanthemi Polygalacturonases

2002 ◽  
Vol 184 (10) ◽  
pp. 2664-2673 ◽  
Author(s):  
Nicole Hugouvieux-Cotte-Pattat ◽  
Vladimir E. Shevchik ◽  
William Nasser
Keyword(s):  
Rna Polymerase ◽  
Direct Interaction ◽  
Pectate Lyase ◽  
Soft Rot ◽  
Hydrolase Activity ◽  
Erwinia Chrysanthemi ◽  
Receptor Protein ◽  
Glycosyl Hydrolases ◽  
Pectate Lyases ◽  
Almost All

ABSTRACT Erwinia chrysanthemi 3937 secretes an arsenal of pectinolytic enzymes, including at least eight endo-pectate lyases encoded by pel genes, which play a major role in the soft-rot disease caused by this bacterium on various plants. E. chrysanthemi also produces some hydrolases that cleave pectin. Three adjacent hydrolase genes, pehV, pehW, and pehX, encoding exo-poly-α-d-galacturonosidases, have been characterized. These enzymes liberate digalacturonides from the nonreducing end of pectin. We report the identification of a novel gene, named pehN, encoding a protein homologous to the glycosyl hydrolases of family 28, which includes mainly polygalacturonases. PehN has a low hydrolase activity on polygalacturonate and on various pectins. PehN action favors the activity of the secreted endo-pectate lyases, mainly PelB and PelC, and that of the periplasmic exo-pectate lyase PelX. However, removal of the pehN gene does not significantly alter the virulence of E. chrysanthemi. Regulation of pehN transcription was analyzed by using gene fusions. Like other pectinase genes, pehN transcription is dependent on several environmental conditions. It is induced by pectic catabolic products and is affected by growth phase, catabolite repression, osmolarity, anaerobiosis, nitrogen starvation, and the presence of calcium ions. The transcription of pehN is modulated by the repressor KdgR, which controls almost all the steps of pectin catabolism, and by cyclic AMP receptor protein (CRP), the global activator of sugar catabolism. The regulator PecS, which represses the transcription of the pel genes but activates that of pehV, pehW, and pehX, also activates transcription of pehN. The three regulators KdgR, PecS, and CRP act by direct interaction with the pehN promoter region. The sequences involved in the binding of these three regulators and of RNA polymerase have been precisely defined. Analysis of the simultaneous binding of these proteins indicates that CRP and RNA polymerase bind cooperatively and that the binding of KdgR could prevent pehN transcription. In contrast, the activator effect of PecS is not linked to competition with KdgR or to cooperation with CRP or RNA polymerase. This effect probably results from competition between PecS and an unidentified repressor involved in peh regulation.

scholarly journals The Flagellar Sigma Factor FliA Is Required for Dickeya dadantii Virulence

2008 ◽  
Vol 21 (11) ◽  
pp. 1431-1442 ◽  
Author(s):  
Courtney E. Jahn ◽  
David K. Willis ◽  
Amy O. Charkowski
Keyword(s):  
Sigma Factor ◽  
Biofilm Formation ◽  
Pectate Lyase ◽  
Secretion System ◽  
Erwinia Chrysanthemi ◽  
Growth Media ◽  
Flagellin Gene ◽  
Dickeya Dadantii ◽  
Disease Symptoms ◽  
Lyase Activity

The genome sequence of the Enterobacteriaceae phytopathogen Dickeya dadantii (formerly Erwinia chrysanthemi) revealed homologs of genes required for a complete flagellar secretion system and one flagellin gene. We found that D. dadantii was able to swim and swarm but that ability to swarm was dependent upon both growth media and temperature. Mutation of the D. dadantii fliA gene was pleiotropic, with the alternate sigma factor required for flagella production and development of disease symptoms but not bacterial growth in Nicotiana benthamiana leaves. The flagellar sigma factor was also required for multiple bacterial phenotypes, including biofilm formation in culture, bacterial adherence to plant tissue, and full expression of pectate lyase activity (but not cellulase or protease activity). Surprisingly, mutation of fliA resulted in the increased expression of avrL (a gene of unknown function in D. dadantii) and two pectate lyase gene homologs, pelX and ABF-0019391. Because FliA is a key contributor to virulence in D. dadantii, it is a new target for disease control.

scholarly journals Characterization of the Exopolygalacturonate Lyase PelX of Erwinia chrysanthemi 3937

1999 ◽  
Vol 181 (5) ◽  
pp. 1652-1663 ◽  
Author(s):  
Vladimir E. Shevchik ◽  
Harry C. M. Kester ◽  
Jacques A. E. Benen ◽  
Jaap Visser ◽  
Janine Robert-Baudouy ◽  
...  
Keyword(s):  
Pectate Lyase ◽  
Erwinia Chrysanthemi ◽  
Receptor Protein ◽  
Polymeric Chain ◽  
Pectate Lyases ◽  
Pectinolytic Enzymes ◽  
Terminal Amino ◽  
Almost All ◽  
Binding Subsites

ABSTRACT Erwinia chrysanthemi 3937 secretes several pectinolytic enzymes, among which eight isoenzymes of pectate lyases with an endo-cleaving mode (PelA, PelB, PelC, PelD, PelE, PelI, PelL, and PelZ) have been identified. Two exo-cleaving enzymes, the exopolygalacturonate lyase, PelX, and an exo-poly-α-d-galacturonosidase, PehX, have been previously identified in other E. chrysanthemi strains. Using a genomic bank of a 3937 mutant with the major pelgenes deleted, we cloned a pectinase gene identified aspelX, encoding the exopolygalacturonate lyase. The deduced amino acid sequence of the 3937 PelX is very similar to the PelX of another E. chrysanthemi strain, EC16, except in the 43 C-terminal amino acids. PelX also has homology to the endo-pectate lyase PelL of E. chrysanthemi but has a N-terminal extension of 324 residues. The transcription of pelX, analyzed by gene fusions, is dependent on several environmental conditions. It is induced by pectic catabolic products and affected by growth phase, oxygen limitation, nitrogen starvation, and catabolite repression. Regulation of pelX expression is dependent on the KdgR repressor, which controls almost all the steps of pectin catabolism, and on the global activator of sugar catabolism, cyclic AMP receptor protein. In contrast, PecS and PecT, two repressors of the transcription of most pectate lyase genes, are not involved inpelX expression. The pelX mutant displayed reduced pathogenicity on chicory leaves, but its virulence on potato tubers or Saintpaulia ionantha plants did not appear to be affected. The purified PelX protein has no maceration activity on plant tissues. Tetragalacturonate is the best substrate of PelX, but PelX also has good activity on longer oligomers. Therefore, the estimated number of binding subsites for PelX is 4, extending from subsites −2 to +2. PelX and PehX were shown to be localized in the periplasm ofE. chrysanthemi 3937. PelX catalyzed the formation of unsaturated digalacturonates by attack from the reducing end of the substrate, while PehX released digalacturonates by attack from the nonreducing end of the substrate. Thus, the two types of exo-degrading enzymes appeared complementary in the degradation of pectic polymers, since they act on both extremities of the polymeric chain.

scholarly journals Analysis of the Oxidative Burst and Its Relevant Signaling Pathways in Leptosphaeria maculans—Brassica napus Pathosystem

2021 ◽  
Vol 22 (9) ◽  
pp. 4812
Author(s):  
Cunchun Yang ◽  
W. G. Dilantha Fernando
Keyword(s):  
Cell Death ◽  
Signaling Pathways ◽  
Oxidative Burst ◽  
Defense Mechanisms ◽  
Leptosphaeria Maculans ◽  
Gene Interaction ◽  
Early Response ◽  
Marker Genes ◽  
Ros Accumulation ◽  
Microbe Interactions

An oxidative burst is an early response of plants to various biotic/abiotic stresses. In plant-microbe interactions, the plant body can induce oxidative burst to activate various defense mechanisms to combat phytopathogens. A localized oxidative burst is also one of the typical behaviors during hypersensitive response (HR) caused by gene-for-gene interaction. In this study, the occurrence of oxidative burst and its signaling pathways was studied from different levels of disease severity (i.e., susceptible, intermediate, and resistant) in the B. napus–L. maculans pathosystem. Canola cotyledons with distinct levels of resistance exhibited differential regulation of the genes involved in reactive oxygen species (ROS) accumulation and responses. Histochemical assays were carried out to understand the patterns of H2O2 accumulation and cell death. Intermediate and resistant genotypes exhibited earlier accumulation of H2O2 and emergence of cell death around the inoculation origins. The observations also suggested that the cotyledons with stronger resistance were able to form a protective region of intensive oxidative bursts between the areas with and without hyphal intrusions to block further fungal advancement to the uninfected regions. The qPCR analysis suggested that different onset patterns of some marker genes in ROS accumulation/programmed cell death (PCD) such as RBOHD, MPK3 were associated with distinct levels of resistance from B. napus cultivars against L. maculans. The observations and datasets from this article indicated the distinct differences in ROS-related cellular behaviors and signaling between compatible and incompatible interactions.

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