Erwinia carotovora subsp. carotovora and Erwinia-derived elicitors HrpN and PehA trigger distinct but interacting defense responses and cell death in Arabidopsis

We have used an hrp-positive strain of the soft rot pathogen Erwinia carotovora subsp. carotovora to elucidate plant responses to this bacterial necrotroph. Purified virulence determinants, harpin (HrpN) and polygalacturonase (PehA), were used as tools to facilitate this analysis. We show that HrpN elicits lesion formation in Arabidopsis and tobacco and triggers systemic resistance in Arabidopsis. Establishment of resistance is accompanied by the expression of salicylic acid (SA)-dependent, but also jasmonate/ethylene (JA/ET)-dependent, marker genes PR1 and PDF1.2, respectively, suggesting that both SA-dependent and JA/ET-dependent defense pathways are activated. Use of pathway-specific mutants and transgenic NahG plants show that both pathways are required for the induction of resistance. Arabidopsis plants treated simultaneously with both elictors PehA, known to trigger only JA/ET-dependent defense signaling, and HrpN react with accelerated and enhanced induction of the marker genes PR1 and PDF1.2 both locally and systemically. This mutual amplification of defense gene expression involves both SA-dependent and JA/ET-dependent defense signaling. The two elicitors produced by E. carotovora subsp. carotovora also cooperate in triggering increased production of superoxide and lesion formation.     

Loss of NECROTIC SPOTTED LESIONS 1 associates with cell death and defense responses in Arabidopsis thaliana

We isolated a lesion mimic mutant, n ecrotic s potted l esions 1 (nsl1), from Ds-tagged Arabidopsis thaliana accession No-0. The nsl1 mutant exhibits a growth retardation phenotype and develops spotted necrotic lesions on its rosette and cauline leaves. These phenotypes occur in the absence of pathogens indicating that nsl1 mutants may constitutively express defense responses. Consistent with this idea, nsl1 accumulates high levels of callose and autofluorescent phenolic compounds localized to the necrotic lesions. Furthermore RNA gel blot analysis revealed that genes associated with disease resistance activation are upregulated in the nsl1 mutants and these plants contain elevated levels of salicylic acid (SA). Crossing nsl1 with an SA deficient mutant, eds16-1, revealed that the nsl1 lesions and growth retardation are dependent upon SA. The nsl1 phenotypes are not suppressed under either the rar1-10 or sgt1b-1 genetic background. NSL1 encodes a novel 612aa protein which contains a membrane-attack complex/perforin (MACPF) domain, which is conserved in bacteria, fungi, mammals and plants. The possible modes of action of NSL1 protein in negative regulation of cell death programs and defense responses are discussed.     

Secretion and regulation Hrp mutants of Erwinia amylovora trigger different responses in apple

The interaction between two Hrp mutants of Erwinia amylovora, the parental virulent strain, and a host plant (Malus sp.) was studied with apple seedlings and apple flowers, using inoculation procedures which reduce the possibility of injury. We showed that one hrp regulatory mutant protected apple tissue against the virulent strain to a greater degree than a secretion mutant. The dynamics of bacterial growth in this interaction indicated that the protection was associated with the inhibition of the multiplication of the virulent strain. The activity of two enzymes involved in plant defence responses, phenylalanine-ammonia lyase and guaiacol peroxidase, increased in leaves treated with the regulatory mutant, but not in the leaves treated with the secretion mutant. The role of regulatory genes of the hrp cluster in this interaction is discussed.     

Arabidopsis thaliana expresses multiple lines of defense to counterattack Erwinia chrysanthemi

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.     

Reduced cellulose synthesis invokes lignification and defense responses in Arabidopsis thaliana

The cell wall determines the shape of plant cells and is also the primary interface for pathogen interactions. The structure of the cell wall can be modified in response to developmental and environmental cues, for example to strengthen the wall and to create barriers to pathogen ingress. The ectopic lignin 1-1 and 1-2 (eli1-1 and eli1-2) mutations lead to an aberrant deposition of lignin, a complex phenylpropanoid polymer. We show that the eli1 mutants occur in the cellulose synthase gene CESA3 in Arabidopsis thaliana and cause reduced cellulose synthesis, providing further evidence for the function of multiple CESA subunits in cellulose synthesis. We show that reduced levels of cellulose synthesis, caused by mutations in cellulose synthase genes and in genes affecting cell expansion, activate lignin synthesis and defense responses through jasmonate and ethylene and other signaling pathways. These observations suggest that mechanisms monitoring cell wall integrity can activate lignification and defense responses.     

Modulation of defense responses of Malus spp. during compatible and incompatible interactions with Erwinia amylovora

Erwinia amylovora is the causal agent of fire blight, a disease affecting members of subfamily Maloideae. In order to analyze mechanisms leading to compatible or incompatible interactions, early plant molecular events were investigated in two genotypes of Malus with contrasting susceptibility to fire blight, after confrontation with either E. amylovora or the incompatible tobacco pathogen Pseudomonas syringae pv. tabaci. Many defense mechanisms, including generation of an oxidative burst and accumulation of pathogenesis-related proteins, were elicited in both resistant and susceptible genotypes by the two pathogens at similar rates and according to an equivalent time course. This elicitation was linked with the functional hypersensitive reaction and pathogenicity (hrp) cluster of E. amylovora, because an hrp secretion mutant did not induce such responses. However, a delayed induction of several genes of various branch pathways of the phenylpropanoid metabolism was recorded in tissues of the susceptible genotype challenged with the wild-type strain of E. amylovora, whereas these genes were quickly induced in every other plant-bacteria interaction, including interactions with the hrp secretion mutant. This suggests the existence of hrp-independent elicitors of defense in the fire blight pathogen as well as hrp-dependant mechanisms of suppression of these nonspecific inductions.     

The HrpN(ea) harpin from Erwinia amylovora triggers differential responses on the nonhost Arabidopsis thaliana cells and on the host apple cells

Erwinia amylovora is a gram-negative necrogenic bacterium causing fire blight of the Maloideae subfamily of Rosaceae such as apple and pear. It provokes progressive necrosis in aerial parts of susceptible host plants (compatible interaction) and a hypersensitive reaction (HR) when infiltrated in nonhost plants (incompatible interaction). The HrpN(ea) harpin is a type three secretion system effector secreted by E. amylovora. This protein is involved in pathogenicity and HR-eliciting capacity of E. amylovora. In the present study, we showed that, in nonhost Arabidopsis thaliana cells, purified HrpN(ea) induces cell death and H2O2 production, two nonhost resistance responses, but failed to induce such responses in host MM106 apple cells. Moreover, HrpN(ea) induced an increase in anion current in host MM106 apple cells, at the opposite of the decrease of anion current previously shown to be necessary to induce cell death in nonhost A. thaliana cells. These results suggest that HrpN(ea) induced different signaling pathways, which could account for early induced compatible or incompatible interaction development.     

Harpin, a hypersensitive response elicitor from Erwinia amylovora, regulates ion channel activities in Arabidopsis thaliana suspension cells

HrpN, the hypersensitive response elicitor from Erwinia amylovora, stimulated K(+) outward rectifying currents in Arabidopsis thaliana suspension cells. It also decreased anion currents. These data demonstrate the ability of harpin to regulate different plasma membrane ion channels, putative components of signal transduction chains leading to defense responses and programmed cell death.     

Plant nitrate supply regulates Erwinia amylovora virulence gene expression in Arabidopsis

We showed previously that nitrogen (N) limitation decreases Arabidopsis resistance to Erwinia amylovora (Ea). We show that decreased resistance to bacteria in low N is correlated with lower apoplastic reactive oxygen species (ROS) accumulation and lower jasmonic acid (JA) pathway expression. Consistently, pretreatment with methyl jasmonate (Me-JA) increased the resistance of plants grown under low N. In parallel, we show that in planta titres of a nonvirulent type III secretion system (T3SS)-deficient Ea mutant were lower than those of wildtype Ea in low N, as expected, but surprisingly not in high N. This lack of difference in high N was consistent with the low expression of the T3SS-encoding hrp virulence genes by wildtype Ea in plants grown in high N compared to plants grown in low N. This suggests that expressing its virulence factors in planta could be a major limiting factor for Ea in the nonhost Arabidopsis. To test this hypothesis, we preincubated Ea in an inducing medium that triggers expression of hrp genes in vitro, prior to inoculation. This preincubation strongly enhanced Ea titres in planta, independently of the plant N status, and was correlated to a significant repression of JA-dependent genes. Finally, we identify two clusters of metabolites associated with resistance or with susceptibility to Ea. Altogether, our data showed that high susceptibility of Arabidopsis to Ea, under low N or following preincubation in hrp-inducing medium, is correlated with high expression of the Ea hrp genes in planta and low expression of the JA signalling pathway, and is correlated with the accumulation of specific metabolites.     

Fight to the death: Arabidopsis thaliana defense response to fungal necrotrophic pathogens

Necrotrophic fungi, being the largest class of fungal plant pathogens, pose a serious economic problem to crop production. They are the cause of heavy losses in agriculture worldwide. Understanding the process of plant infection by necrotrophic fungi, including subtle interaction networks connecting such evolutionarily distinct organisms has recently been given high research priority. Such studies are now possible mainly because of the utility of the model plant Arabidopsis thaliana. A. thaliana has a sequenced genome and thousands of mutants available, allowing investigation of virtually all aspects of plant pathogenesis. This review focuses on morphological and molecular changes in A. thaliana, which occur during response to infection by necrotrophic fungi. These responses in relation to resistance and susceptibility of the plant will be discussed.     

Capsulation and virulence in Erwinia amylovora

Evidence is presented that capsulation may be one virulence determinant for Erwinia amylovora, the fireblight pathogen. When 15 virulent and seven avirulent strains were grown on a medium containing asparagine as the only source of carbon and nitrogen, or yeast peptone agar, or on a sugar medium containing an inorganic source of nitrogen, capsule production and virulence were not correlated. However, if a sugar or sugar alcohol was added to the asparagine medium or to yeast peptone agar all the virulent strains produced some or many capsulated cells whereas six of the avirulent ones did not. Capsules were also produced by all the virulent strains during infection. The existence of a seventh avirulent strain which was capsulated on all media except unsupplemented asparagine agar, suggested that capsule production was not the only virulence determinant.     

Endophytic actinobacteria induce defense pathways in Arabidopsis thaliana

Endophytic actinobacteria, isolated from healthy wheat tissue, which are capable of suppressing a number wheat fungal pathogens both in vitro and in planta, were investigated for the ability to activate key genes in the systemic acquired resistance (SAR) or the jasmonate/ethylene (JA/ET) pathways in Arabidopsis thaliana. Inoculation of A. thaliana (Col-0) with selected endophytic strains induced a low level of SAR and JA/ET gene expression, measured using quantitative polymerase chain reaction. Upon pathogen challenge, endophyte-treated plants demonstrated a higher abundance of defense gene expression compared with the non-endophyte-treated controls. Resistance to the bacterial pathogen Erwinia carotovora subsp. carotovora required the JA/ET pathway. On the other hand, resistance to the fungal pathogen Fusarium oxysporum involved primarily the SAR pathway. The endophytic actinobacteria appear to be able to "prime" both the SAR and JA/ET pathways, upregulating genes in either pathway depending on the infecting pathogen. Culture filtrates of the endophytic actinobacteria were investigated for the ability to also activate defense pathways. The culture filtrate of Micromonospora sp. strain EN43 grown in a minimal medium resulted in the induction of the SAR pathway; however, when grown in a complex medium, the JA/ET pathway was activated. Further analysis using Streptomyces sp. strain EN27 and defense-compromised mutants of A. thaliana indicated that resistance to E. carotovora subsp. carotovora occurred via an NPR1-independent pathway and required salicylic acid whereas the JA/ET signaling molecules were not essential. In contrast, resistance to F. oxysporum mediated by Streptomyces sp. strain EN27 occurred via an NPR1-dependent pathway but also required salicylic acid and was JA/ET independent.     

The mitochondrion--an organelle commonly involved in programmed cell death in Arabidopsis thaliana

Plant cells undergoing programmed cell death (PCD) at late stages typically show chromatin condensation and endonucleolytic cleavage prior to obvious membrane or organelle ultrastructural changes. To investigate possible early PCD-associated events, we used microscopic observations and flow cytometry to quantitate mitochondrial membrane potential (DeltaPsim) changes during PCD at the single cell and population levels using Arabidopsis protoplasts. A DeltaPsim loss was commonly induced early during plant PCD and was important for PCD execution, as evidenced by the concomitant reduction of the change in DeltaPsim and PCD by cyclosporin A, which inhibits mitochondrial permeability transition pores in animal cells. DeltaPsim loss occurred prior to nuclear morphological changes and was only associated with mitochondrial cytochrome c release (an apoptotic trigger in animals) in response to one of three PCD elicitors. Three different stimuli in wild type implicated DeltaPsim changes in PCD: ceramide, protoporphyrin IX, and the hypersensitive response elicitor AvrRpt2. Additionally, the behavior of the conditional ectopic cell death mutant accelerated cell death2 and ACD2-overproducing plants also implicated DeltaPsim alteration as key for PCD execution. Because ACD2 is largely a chloroplast component in mature plants, the observation that the cell death in acd2 mutants requires changes in mitochondrial functions implicates communication between chloroplasts and mitochondria in mediating PCD activation. We suggest that DeltaPsim loss is a common early marker in plant PCD, similar to what has been documented in animals. However, unlike in animal cells, in plant cells, mitochondrial cytochrome c release is not an obligatory step in PCD control.     

Ultraviolet-B-induced oxidative stress and antioxidant defense system responses in ascorbate-deficient vtc1 mutants of Arabidopsis thaliana

Ultraviolet-B (UV-B) radiation has a negative impact on plant cells, and results in the generation of reactive oxygen species (ROS). In order to increase our understanding of the effects of UV-B on antioxidant processes, we investigated the response of an ascorbate-deficient Arabidopsis thaliana mutant vtc1 to short-term increased UV-B exposure. After UV-B supplementation, vtc1 mutants exhibited oxidative damage. Evidence for damage included an increase in H(2)O(2) content and the production of thiobarbituric acid reactive substances (TBARS); a decrease in chlorophyll content and chlorophyll fluorescence parameters were also reported. The vtc1 mutants had higher total glutathione than the wild type (WT) during the first day of UV-B treatment. We found reduced ratio of glutathione/total glutathione and increased ratio of dehydroascorbate/total ascorbate in the vtc1 mutants, compared to the WT plants. In addition, the enzymes responsible for ROS scavenging, including superoxide dismutase, catalase, and ascorbate peroxidase, had insufficient activity in the vtc1 mutants, compared to the WT plants. The same reduced activity in the vtc1 mutants was reported for the enzymes responsible for the regeneration of ascorbate and glutathione (including monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase). These results suggest that the ascorbate-deficient mutant vtc1 is more sensitive to supplementary UV-B treatment than WT plants and ascorbate can be considered an important antioxidant for UV-B radiation.     

A diffusible signal from germinating Orobanche ramosa elicits early defense responses in suspension-cultured Arabidopsis thaliana

In plant/parasitic plant interaction, little is known about the host plant response before the establishment of the parasite within the host. In the present work, we focused on host responses to parasitic plant, O. ramosa in the early stage of infection. We used a co-culture system of A. thaliana suspension cells and O. ramosa germinated-seeds to avoid parasite attachment. We showed that O. ramosa induced H₂O₂ generation and camalexin synthesis by A. thaliana followed by a drastic increase in cell death. We further demonstrated that a heat sensitive diffusible signal is responsible for this cell death. These data indicate that recognition of O. ramosa occurs before the attachment of the parasite and initiates plant defence responses.Key words: Orobanche ramosa, Arabidopsis thaliana, cell death, hydrogen peroxide, secondary metabolism     

Isolation and Characterization of Five Erwinia amylovora Bacteriophages and Assessment of Phage Resistance in Strains of Erwinia amylovora

Phages able to infect the fire blight pathogen Erwinia amylovora were isolated from apple, pear, and raspberry tissues and from soil samples collected at sites displaying fire blight symptoms. Among a collection of 50 phage isolates, 5 distinct phages, including relatives of the previously described phages Ea1 and Ea7 and 3 novel phages named Ea100, Ea125, and Ea116C, were identified based on differences in genome size and restriction fragment pattern. Ea1, the phage distributed most widely, had an approximately 46-kb genome which exhibited some restriction site variability between isolates. Phages Ea100, Ea7, and Ea125 each had genomes of approximately 35 kb and could be distinguished by their EcoRI restriction fragment patterns. Ea116C contained an approximately 75-kb genome. Ea1, Ea7, Ea100, Ea125, and Ea116C were able to infect 39, 36, 16, 20, and 40, respectively, of 40 E. amylovora strains isolated from apple orchards in Michigan and 8, 12, 10, 10, and 12, respectively, of 12 E. amylovora strains isolated from raspberry fields (Rubus spp.) in Michigan. Only 22 of 52 strains were sensitive to all five phages, and 23 strains exhibited resistance to more than one phage. Ea116C was more effective than the other phages at lysing E. amylovora strain Ea110 in liquid culture, reducing the final titer of Ea110 by >95% when added at a ratio of 1 PFU per 10 CFU and by 58 to 90% at 1 PFU per 10⁵ CFU.     

Mycotoxin Ochratoxin A-induced cell death and changes in oxidative metabolism of Arabidopsis thaliana

We evaluated the phytotoxicity of mycotoxin ochratoxin A (OTA) from Aspergillus and Penicillium strains on Arabidopsis thaliana. The results demonstrate that the growth of Arabidopsis thaliana on media containing OTA was inhibited significantly. Moreover, OTA induced necrotic lesions in detached leaves, which are reminiscent of hypersensitive response lesions that are activated during plant–pathogen interactions and other abiotic stress factors. From our study, we can see that OTA exposure stimulated a biphasic oxidative burst in the leaves, resulting in the generation of hydrogen peroxide (H₂O₂) and superoxide anion radicals (O₂^·−) and in the concomitant down-regulation of antioxidant enzyme defense responses and up-regulation of lipid peroxidation. These results suggested that OTA damage might result from reactive oxygen species pathways. Our experiments provide a useful model plant system for research on OTA-induced plant cell death.