(J.T.).Pyridine nucleotides, such as NAD, are crucial redox carriers and have emerged as important signaling molecules in stress responses. Previously, we have demonstrated in Arabidopsis (Arabidopsis thaliana) that the inducible NAD-overproducing nadC lines are more resistant to an avirulent strain of Pseudomonas syringae pv tomato (Pst-AvrRpm1), which was associated with salicylic acid-dependent defense. Here, we have further characterized the NAD-dependent immune response in Arabidopsis. Quinolinate-induced stimulation of intracellular NAD in transgenic nadC plants enhanced resistance against a diverse range of (a)virulent pathogens, including Pst-AvrRpt2, Dickeya dadantii, and Botrytis cinerea. Characterization of the redox status demonstrated that elevated NAD levels induce reactive oxygen species (ROS) production and the expression of redox marker genes of the cytosol and mitochondrion. Using pharmacological and reverse genetics approaches, we show that NAD-induced ROS production functions independently of NADPH oxidase activity and light metabolism but depends on mitochondrial respiration, which was increased at higher NAD. We further demonstrate that NAD primes pathogen-induced callose deposition and cell death. Mass spectrometry analysis reveals that NAD simultaneously induces different defense hormones and that the NAD-induced metabolic profiles are similar to those of defense-expressing plants after treatment with pathogen-associated molecular patterns. We thus conclude that NAD triggers metabolic profiles rather similar to that of pathogen-associated molecular patterns and discuss how signaling cross talk between defense hormones, ROS, and NAD explains the observed resistance to pathogens.
Erwinia amylovora causes fire blight in rosaceous plants. In nonhost Arabidopsis thaliana, E. amylovora triggers necrotic symptoms associated with transient bacterial multiplication, suggesting either that A. thaliana lacks a susceptibility factor or that it actively restricts E. amylovora growth. Inhibiting plant protein synthesis at the time of infection led to an increase in necrosis and bacterial multiplication and reduced callose deposition, indicating that A. thaliana requires active protein synthesis to restrict E. amylovora growth. Analysis of the callose synthase-deficient pmr4-1 mutant indicated that lack of callose deposition alone did not lead to increased sensitivity to E. amylovora. Transcriptome analysis revealed that approximately 20% of the genes induced following E. amylovora infection are related to defense and signaling. Analysis of mutants affected in NDR1 and EDS1, two main components of the defense-gene activation observed, revealed that E. amylovora multiplied ten times more in the eds1-2 mutant than in the wild type but not in the ndr1-1 mutant. Analysis of mutants affected in three WRKY transcription factors showing EDS1-dependent activation identified WRKY46 and WRKY54 as positive regulators and WRKY70 as a negative regulator of defense against E. amylovora. Altogether, we show that EDS1 is a positive regulator of nonhost resistance against E. amylovora in A. thaliana and hypothesize that it controls the production of several effective defenses against E. amylovora through the action of WRKY46 and WRKY54, while WRKY70 acts as a negative regulator.
Erwinia amylovora is the bacterium responsible for fire blight, a necrotic disease affecting plants of the rosaceous family. E. amylovora pathogenicity requires a functional type three secretion system (T3SS). We show here that E. amylovora triggers a T3SS-dependent cell death on Arabidopsis thaliana. The plants respond by inducing T3SS-dependent defense responses, including salicylic acid (SA)-independent callose deposition, activation of the SA defense pathway, reactive oxygen species (ROS) accumulation, and part of the jasmonic acid/ethylene defense pathway. Several of these reactions are similar to what is observed in host plants. We show that the cell death triggered by E. amylovora on A. thaliana could not be simply explained by the recognition of AvrRpt2 ea by the resistance gene product RPS2. We then analyzed the role of type three-secreted proteins (T3SPs) DspA/E, HrpN, and HrpW in the induction of cell death and defense reactions in A. thaliana following infection with the corresponding E. amylovora mutant strains. HrpN and DspA/E were found to play an important role in the induction of cell death, activation of defense pathways, and ROS accumulation. None of the T3SPs tested played a major role in the induction of SA-independent callose deposition. The relative importance of T3SPs in A. thaliana is correlated with their relative importance in the disease process on host plants, indicating that A. thaliana can be used as a model to study their role.
SummaryAtNRAMP3 and AtNRAMP4 are two Arabidopsis metal transporters sharing about 50% sequence identity with mouse NRAMP1. The NRAMP1/Slc11A1 metal ion transporter plays a crucial role in the innate immunity of animal macrophages targeted by intracellular bacterial pathogens. AtNRAMP3 and AtNRAMP4 localize to the vacuolar membrane. We found that AtNRAMP3 is upregulated in leaves challenged with the bacterial pathogens Pseudomonas syringae and Erwinia chrysanthemi, whereas AtNRAMP4 expression is not modified. Using single and double nramp3 and nramp4 mutants, as well as lines ectopically expressing either of these genes, we show that AtNRAMP3 and, to a lesser extent, AtNRAMP4 are involved in Arabidopsis thaliana resistance against the bacterial pathogen E. chrysanthemi. The susceptibility of the double nramp3 nramp4 mutant is associated with the reduced accumulation of reactive oxygen species and ferritin (AtFER1), an iron storage protein known to participate in A. thaliana defense. Interestingly, roots from infected plants accumulated transcripts of AtNRAMP3 as well as the iron-deficiency markers IRT1 and FRO2. This finding suggests the existence of a shoot-to-root signal reminiscent of an iron-deficiency signal activated by pathogen infection. Our data indicate that the functions of NRAMP proteins in innate immunity have been conserved between animals and plants.
The high affinity nitrate transport system in Arabidopsis thaliana involves one gene and potentially seven genes from the NRT1 and NRT2 family, respectively. Among them, NRT2.1, NRT2.2, NRT2.4 and NRT2.7 proteins have been shown to transport nitrate and are localized on the plasmalemma or the tonoplast membranes. NRT2.1, NRT2.2 and NRT2.4 play a role in nitrate uptake from soil solution by root cells while NRT2.7 is responsible for nitrate loading in the seed vacuole. We have undertaken the functional characterization of a third member of the family, the NRT2.6 gene. NRT2.6 was weakly expressed in most plant organs and its expression was higher in vegetative organs than in reproductive organs. Contrary to other NRT2 members, NRT2.6 expression was not induced by limiting but rather by high nitrogen levels, and no nitrate-related phenotype was found in the nrt2.6-1 mutant. Consistently, the over-expression of the gene failed to complement the nitrate uptake defect of an nrt2.1-nrt2.2 double mutant. The NRT2.6 expression is induced after inoculation of Arabidopsis thaliana by the phytopathogenic bacterium Erwinia amylovora. Interestingly, plants with a decreased NRT2.6 expression showed a lower tolerance to pathogen attack. A correlation was found between NRT2.6 expression and ROS species accumulation in response to infection by E. amylovora and treatment with the redox-active herbicide methyl viologen, suggesting a probable link between NRT2.6 activity and the production of ROS in response to biotic and abiotic stress.
The type III effector DspA/E is an essential pathogenicity factor of the phytopathogenic bacterium Erwinia amylovora. We showed that DspA/E was required for transient bacterial growth in nonhost Arabidopsis thaliana leaves, as an E. amylovora dspA/E mutant was unable to grow. We expressed DspA/E in A. thaliana transgenic plants under the control of an oestradiol-inducible promoter, and found that DspA/E expressed in planta restored the growth of a dspA/E mutant. DspA/E expression in these transgenic plants led to the modulation by at least two-fold of the expression of 384 genes, mostly induced (324 genes). Both induced and repressed genes contained high proportions of defence genes. DspA/E expression ultimately resulted in plant cell death without requiring a functional salicylic acid signalling pathway. Analysis of A. thaliana transgenic seedlings expressing a green fluorescent protein (GFP):DspA/E fusion indicated that the fusion protein could only be detected in a few cells per seedling, suggesting the degradation or absence of accumulation of DspA/E in plant cells. Consistently, we found that DspA/E repressed plant protein synthesis when injected by E. amylovora or when expressed in transgenic plants. Thus, we conclude that DspA/E is toxic to A. thaliana: it promotes modifications, among which the repression of protein synthesis could be determinant in the facilitation of necrosis and bacterial growth.
Dickeya dadantii is a broad host range phytopathogenic bacterium provoking soft rot disease on many plants including Arabidopsis. We showed that, after D. dadantii infection, the expression of the Arabidopsis BOS1 gene was specifically induced by the production of the bacterial PelB/C pectinases able to degrade pectin. This prompted us to analyze the interaction between the bos1 mutant and D. dadantii. The phenotype of the infected bos1 mutant is complex. Indeed, maceration symptoms occurred more rapidly in the bos1 mutant than in the wild type parent but at a later stage of infection, a necrosis developed around the inoculation site that provoked a halt in the progression of the maceration. This necrosis became systemic and spread throughout the whole plant, a phenotype reminiscent of that observed in some lesion mimic mutants. In accordance with the progression of maceration symptoms, bacterial population began to grow more rapidly in the bos1 mutant than in the wild type plant but, when necrosis appeared in the bos1 mutant, a reduction in bacterial population was observed. From the plant side, this complex interaction between D. dadantii and its host includes an early plant defence response that comprises reactive oxygen species (ROS) production accompanied by the reinforcement of the plant cell wall by protein cross-linking. At later timepoints, another plant defence is raised by the death of the plant cells surrounding the inoculation site. This plant cell death appears to constitute an efficient defence mechanism induced by D. dadantii during Arabidopsis infection.
Erwinia amylovora is responsible for fire blight of apple and pear trees. Its pathogenicity depends on a type III secretion system (T3SS) mediating the translocation of effectors into the plant cell. The DspA/E effector suppresses callose deposition on apple leaves. We found that E. amylovora and Pseudomonas syringae DC3000 tts mutants or peptide flg22 do not trigger callose deposition as strongly as the dspA/E mutant on apple leaves. This suggests that, on apple leaves, callose deposition is poorly elicited by pathogen-associated molecular patterns (PAMPs) such as flg22 or other PAMPs harbored by tts mutants and is mainly elicited by injected effectors or by the T3SS itself. Callose elicitation partly depends on HrpW because an hrpW-dspA/E mutant elicits lower callose deposition than a dspA/E mutant. Furthermore, an hrpN-dspA/E mutant does not trigger callose deposition, indicating that HrpN is required to trigger this plant defense reaction. We showed that HrpN plays a general role in the translocation process. Thus, the HrpN requirement for callose deposition may be explained by its role in translocation: HrpN could be involved in the translocation of other effectors inducing callose deposition. Furthermore, HrpN may also directly contribute to the elicitation process because we showed that purified HrpN induces callose deposition.
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