Riboflavin-induced Priming for Pathogen Defense in Arabidopsis thaliana

Riboflavin (vitamin B2) participates in a variety of redox processes that affect plant defense responses. Previously we have shown that riboflavin induces pathogen resistance in the absence of hypersensitive cell death (HCD) in plants. Herein, we report that riboflavin induces priming of defense responses in Arabidopsis thaliana toward infection by virulent Pseudomonas syringae pv. Tomato DC3000 (Pst). Induced resistance was mechanistically connected with the expression of defense response genes and cellular defense events, including H2O2 burst, HCD, and callose deposition in the plant. Riboflavin treatment and inoculation of plants with Pst were neither active but both synergized to induce defense responses. The priming process needed NPR1 (essential regulator of systemic acquired resistance) and maintenance of H2O2 burst but was independent of salicylic acid, jasmonic acid, ethylene, and abscisic acid. Our results suggest that the role of riboflavin in priming defenses is subject to a signaling process distinct from the known pathways of hormone signal transduction.     

Priming for enhanced defence responses by specific inhibition of the Arabidopsis response to coronatine

The priming agent β-aminobutyric acid (BABA) is known to enhance Arabidopsis resistance to the bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000 by potentiating salicylic acid (SA) defence signalling, notably PR1 expression. The molecular mechanisms underlying this phenomenon remain unknown. A genome-wide microarray analysis of BABA priming during Pst DC3000 infection revealed direct and primed up-regulation of genes that are responsive to SA, the SA analogue benzothiadiazole and pathogens. In addition, BABA was found to inhibit the Arabidopsis response to the bacterial effector coronatine (COR). COR is known to promote bacterial virulence by inducing the jasmonic acid (JA) response to antagonize SA signalling activation. BABA specifically repressed the JA response induced by COR without affecting other plant JA responses. This repression was largely SA-independent, suggesting that it is not caused by negative cross-talk between SA and JA signalling cascades. Treatment with relatively high concentrations of purified COR counteracted BABA inhibition. Under these conditions, BABA failed to protect Arabidopsis against Pst DC3000. BABA did not induce priming and resistance in plants inoculated with a COR-deficient strain of Pst DC3000 or in the COR-insensitive mutant coi1-16. In addition, BABA blocked the COR-dependent re-opening of stomata during Pst DC3000 infection. Our data suggest that BABA primes for enhanced resistance to Pst DC3000 by interfering with the bacterial suppression of Arabidopsis SA-dependent defences. This study also suggests the existence of a signalling node that distinguishes COR from other JA responses.     

A genetic screen reveals Arabidopsis stomatal and/or apoplastic defenses against Pseudomonas syringae pv. tomato DC3000

Bacterial infection of plants often begins with colonization of the plant surface, followed by entry into the plant through wounds and natural openings (such as stomata), multiplication in the intercellular space (apoplast) of the infected tissues, and dissemination of bacteria to other plants. Historically, most studies assess bacterial infection based on final outcomes of disease and/or pathogen growth using whole infected tissues; few studies have genetically distinguished the contribution of different host cell types in response to an infection. The phytotoxin coronatine (COR) is produced by several pathovars of Pseudomonas syringae. COR-deficient mutants of P. s. tomato (Pst) DC3000 are severely compromised in virulence, especially when inoculated onto the plant surface. We report here a genetic screen to identify Arabidopsis mutants that could rescue the virulence of COR-deficient mutant bacteria. Among the susceptible to coronatine-deficient Pst DC3000 (scord) mutants were two that were defective in stomatal closure response, two that were defective in apoplast defense, and four that were defective in both stomatal and apoplast defense. Isolation of these three classes of mutants suggests that stomatal and apoplastic defenses are integrated in plants, but are genetically separable, and that COR is important for Pst DC3000 to overcome both stomatal guard cell- and apoplastic mesophyll cell-based defenses. Of the six mutants defective in bacterium-triggered stomatal closure, three are defective in salicylic acid (SA)-induced stomatal closure, but exhibit normal stomatal closure in response to abscisic acid (ABA), and scord7 is compromised in both SA- and ABA-induced stomatal closure. We have cloned SCORD3, which is required for salicylic acid (SA) biosynthesis, and SCORD5, which encodes an ATP-binding cassette (ABC) protein, AtGCN20/AtABCF3, predicted to be involved in stress-associated protein translation control. Identification of SCORD5 begins to implicate an important role of stress-associated protein translation in stomatal guard cell signaling in response to microbe-associated molecular patterns and bacterial infection.     

Pseudomonas syringae elicits emission of the terpenoid (E,E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene in Arabidopsis leaves via jasmonate signaling and expression of the terpene synthase TPS4

Volatile, low-molecular weight terpenoids have been implicated in plant defenses, but their direct role in resistance against microbial pathogens is not clearly defined. We have examined a possible role of terpenoid metabolism in the induced defense of Arabidopsis thaliana plants against leaf infection with the bacterial pathogen Pseudomonas syringae. Inoculation of plants with virulent or avirulent P. syringae strains induces the emission of the terpenoids (E,E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene (TMTT), beta-ionone and alpha-farnesene. While the most abundant volatile, the C16-homoterpene TMTT, is produced relatively early in compatible and incompatible interactions, emission of both beta-ionone and alpha-farnesene only increases in later stages of the compatible interaction. Pathogen-induced synthesis of TMTT is controlled through jasmonic acid (JA)-dependent signaling but is independent of a functional salicylic acid (SA) pathway. We have identified Arabidopsis T-DNA insertion lines with defects in the terpene synthase gene TPS4, which is expressed in response to P. syringae inoculation. The tps4 knockout mutant completely lacks induced emission of TMTT but is capable of beta-ionone and alpha-farnesene production, demonstrating that TPS4 is specifically involved in TMTT formation. The tps4 plants display at least wild type-like resistance against P. syringae, indicating that TMTT per se does not protect against the bacterial pathogen in Arabidopsis leaves. Similarly, the ability to mount SA-dependent defenses and systemic acquired resistance (SAR) is barely affected in tps4, which excludes a signaling function of TMTT during SAR. Besides P. syringae challenge, intoxication of Arabidopsis leaves with copper sulfate, a treatment that strongly activates JA biosynthesis, triggers production of TMTT, beta-ionone, and alpha-farnesene. Taken together, our data suggest that induced TMTT production in Arabidopsis is a by-product of activated JA signaling, rather than an effective defense response that contributes to resistance against P. syringae.     

Suppression of host defense in compatible plant-Pseudomonas syringae interactions

Despite impressive advances in the study of plant resistance to pathogens, little is known about the molecular basis of plant susceptibility to virulent pathogens. Recent progress in susceptible plant-Pseudomonas syringae interactions has provided a glimpse into the battles fought between plants and bacterial pathogens. A key step for pathogenesis appears to be the suppression of host defenses. Suppression of host defenses, including basal defense, gene-for-gene resistance and nonhost resistance, is a key step for pathogenesis. Defense suppression is mediated by bacterial effector proteins, which are secreted through the type III secretion system, and by coronatine, a bacterial toxin that structurally and functionally mimics methyl jasmonate, a plant defense signaling molecule.     

A deletion in the nitrate high affinity transporter NRT2.1 alters metabolomic and transcriptomic responses to Pseudomonas syringae

A deletion in the high affinity nitrate trasporter NRT2.1 in Arabidopsis results in a reduced susceptibility to Pseudomonas syringae by two different mechanisms, the SA priming and an interference in the effector triggered susceptibility. In the present research we further characterized the metabolic and genetic profiles of the mutant nrt2 in the interaction with P. syringae. Despite the priming found in the SA-dependent pathway, the metabolic changes in nrt2 compared with wild-type plants are more remarkable prior infection. This is associated mainly to a pre-existing over representation of signals attributed to aromatic amino acids and phenylpropanoids in the nrt2. Genomic analysis confirms the implication of aromatic aminoacids and phenylpropanoids, but additionally, suggests a new role in ribosomal proteins as the major changes observed in nrt2 upon infection by the bacterium.     

Age-related resistance in Arabidopsis is a developmentally regulated defense response to Pseudomonas syringae

Age-related resistance (ARR) has been observed in a number of plant species; however, little is known about the biochemical or molecular mechanisms involved in this response. Arabidopsis becomes more resistant, or less susceptible, to virulent Pseudomonas syringae (pv tomato or maculicola) as plants mature (in planta bacterial growth reduction of 10- to 100-fold). An ARR-like response also was observed in response to certain environmental conditions that accelerate Arabidopsis development. ARR occurs in the Arabidopsis mutants pad3-1, eds7-1, npr1-1, and etr1-4, suggesting that ARR is a distinct defense response, unlike the induced systemic resistance or systemic acquired resistance responses. However, three salicylic acid (SA) accumulation-deficient plant lines, NahG, sid1, and sid2, did not exhibit ARR. A heat-stable antibacterial activity was detected in intercellular washing fluids in response to Pst inoculation in wild-type ARR-competent plants but not in NAHG: These data suggest that the ability to accumulate SA is necessary for the ARR response and that SA may act as a signal for the production of the ARR-associated antimicrobial compound(s) and/or it may possess direct antibacterial activity against P. syringae.     

Targeting novel chemical and constitutive primed metabolites against Plectosphaerella cucumerina

Priming is a physiological state for protection of plants against a broad range of pathogens, and is achieved through stimulation of the plant immune system. Various stimuli, such as beneficial microbes and chemical induction, activate defense priming. In the present study, we demonstrate that impairment of the high‐affinity nitrate transporter 2.1 (encoded by NRT2.1) enables Arabidopsis to respond more quickly and strongly to Plectosphaerella cucumerina attack, leading to enhanced resistance. The Arabidopsis thaliana mutant lin1 (affected in NRT2.1) is a priming mutant that displays constitutive resistance to this necrotroph, with no associated developmental or growth costs. Chemically induced priming by β–aminobutyric acid treatment, the constitutive priming mutant ocp3 and the constitutive priming present in the lin1 mutant result in a common metabolic profile within the same plant–pathogen interactions. The defense priming significantly affects sugar metabolism, cell‐wall remodeling and shikimic acid derivatives levels, and results in specific changes in the amino acid profile and three specific branches of Trp metabolism, particularly accumulation of indole acetic acid, indole‐3–carboxaldehyde and camalexin, but not the indolic glucosinolates. Metabolomic analysis facilitated identification of three metabolites in the priming fingerprint: galacturonic acid, indole‐3–carboxylic acid and hypoxanthine. Treatment of plants with the latter two metabolites by soil drenching induced resistance against P. cucumerina, demonstrating that these compounds are key components of defense priming against this necrotrophic fungus. Here we demonstrate that indole‐3–carboxylic acid induces resistance by promoting papillae deposition and H₂O₂ production, and that this is independent of PR1, VSP2 and PDF1.2 priming.     

Herbivore-induced resistance against microbial pathogens in Arabidopsis

Caterpillars of the herbivore Pieris rapae stimulate the production of jasmonic acid (JA) and ethylene (ET) in Arabidopsis (Arabidopsis thaliana) and trigger a defense response that affects insect performance on systemic tissues. To investigate the spectrum of effectiveness of P. rapae-induced resistance, we examined the level of resistance against different pathogens. Although the necrotrophic fungus Alternaria brassicicola is sensitive to JA-dependent defenses, herbivore-induced resistance was not effective against this pathogen. By contrast, caterpillar feeding significantly reduced disease caused by the bacterial pathogens Pseudomonas syringae pv tomato and Xanthomonas campestris pv armoraciae. However, this effect was apparent only locally in caterpillar-damaged tissue. Arabidopsis mutants jar1, coi1, ein2, sid2, eds5, and npr1 showed wild-type levels of P. rapae-induced protection against P. syringae pv tomato, suggesting that this local, herbivore-induced defense response does not depend exclusively on either JA, ET, or salicylic acid (SA). Resistance against the biotroph Turnip crinkle virus (TCV) requires SA, but not JA and ET. Nevertheless, herbivore feeding strongly affected TCV multiplication and TCV lesion formation, also in systemic tissues. Wounding alone was not effective, but application of P. rapae regurgitate onto the wounds induced a similar level of protection. Analysis of SA-induced PATHOGENESIS RELATED-1 (PR-1) expression revealed that P. rapae grazing primed Arabidopsis leaves for augmented expression of SA-dependent defenses. Pharmacological experiments showed that ET acts synergistically on SA-induced PR-1, suggesting that the increased production of ET upon herbivore feeding sensitizes the tissue to respond faster to SA, thereby contributing to an enhanced defensive capacity toward pathogens, such as TCV, that trigger SA-dependent defenses upon infection.     

Resistance to Pseudomonas syringae conferred by an Arabidopsis thaliana coronatine-insensitive (coi1) mutation occurs through two distinct mechanisms

A new allele of the coronatine-insensitive locus (COI1) was isolated in a screen for Arabidopsis thaliana mutants with enhanced resistance to the bacterial pathogen Pseudomonas syringae. This mutant, designated coi1-20, exhibits robust resistance to several P. syringae isolates but remains susceptible to the virulent pathogens Erisyphe and cauliflower mosaic virus. Resistance to P. syringae strain PstDC3000 in coi1-20 plants is correlated with hyperactivation of PR-1 expression and accumulation of elevated levels of salicylic acid (SA) following infection, suggesting that the SA-mediated defense response pathway is sensitized in this mutant. Restriction of growth of PstDC3000 in coi1-20 leaves is partially dependent on NPR1 and fully dependent on SA, indicating that SA-mediated defenses are required for restriction of PstDC3000 growth in coi1-20 plants. Surprisingly, despite high levels of PstDC3000 growth in coi1-20 plants carrying the salicylate hydroxylase (nahG) transgene, these plants do not exhibit disease symptoms. Thus resistance to P. syringae in coi1-20 plants is conferred by two different mechanisms: (i) restriction of pathogen growth via activation of the SA-dependent defense pathway; and (ii) an SA-independent inability to develop disease symptoms. These findings are consistent with the hypotheses that the P. syringae phytotoxin coronatine acts to promote virulence by inhibiting host defense responses and by promoting lesion formation.     

A two-strain mixture of rhizobacteria elicits induction of systemic resistance against Pseudomonas syringae and Cucumber mosaic virus coupled to promotion of plant growth on Arabidopsis thaliana

We evaluated a commercial biopreparation of plant growth-promoting rhizobacteria (PGPR) strains Bacillus subtilis GB03 and B. amyloliquefaciens IN937a formulated with the carrier chitosan (BioYield) for its capacity to elicit growth promotion and induced systemic resistance against infection by Cucumber Mosaic Virus (CMV) and Pseudomonas syringae pv. tomato DC3000 in Arabidopsis thaliana. The biopreparation promoted plant growth of Arabidopsis hormonal mutants, which included auxin, gibberellic acid, ethylene, jasmonate, salicylic acid, and brassinosteroid insensitive lines as well as each wild-type. The biopreparation protected plants against CMV based on disease severity in wild-type plants. However, virus titre was not lower in control plants and those treated with biopreparation, suggesting that the biopreparation induced tolerance rather than resistance against CMV. Interestingly, the biopreparation induced resistance against CMV in NahG plants, as evidenced by both reduced disease severity and virus titer. The biopreparation also elicited induced resistance against P. syringae pv. tomato in the wild-type but not in NahG transgenic plants, which degrade endogenous salicylic acid, indicating the involvement of salicylic acid signaling. Our results indicate that some PGPR strains can elicit plant growth promotion by mechanisms that are different from known hormonal signaling pathways. In addition, the mechanism for elicitation of induced resistance by PGPR may be pathogen-dependent. Collectively, the two-Bacilli strain mixture can be utilized as a biological inoculant for both protection of plant against bacterial and viral pathogens and enhancement of plant growth.     

Characterization of the Nrt2.6 Gene in Arabidopsis thaliana: A Link with Plant Response to Biotic and Abiotic Stress

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.     

Activation of defense responses in Chinese cabbage by a nonhost pathogen, Pseudomonas syringae pv. tomato

Pseudomonas syringae pv. tomato (Pst) causes a bacterial speck disease in tomato and Arabidopsis. In Chinese cabbage, in which host-pathogen interactions are not well understood, Pst does not cause disease but rather elicits a hypersensitive response. Pst induces localized cell death and H2O2 accumulation, a typical hypersensitive response, in infiltrated cabbage leaves. Pre-inoculation with Pst was found to induce resistance to Erwinia carotovora subsp. carotovora, a pathogen that causes soft rot disease in Chinese cabbage. An examination of the expression profiles of 12 previously identified Pst-inducible genes revealed that the majority of these genes were activated by salicylic acid or BTH; however, expressions of the genes encoding PR4 and a class IV chitinase were induced by ethephon, an ethylene-releasing compound, but not by salicylic acid, BTH, or methyl jasmonate. This implies that Pst activates both salicylate-dependent and salicylate-independent defense responses in Chinese cabbage.     

Constitutive activation of jasmonate signaling in an Arabidopsis mutant correlates with enhanced resistance to Erysiphe cichoracearum,Pseudomonas syringae,and Myzus persicae

In Arabidopsis spp., the jasmonate (JA) response pathway generally is required for defenses against necrotrophic pathogens and chewing insects, while the salicylic acid (SA) response pathway is generally required for specific, resistance (R) gene-mediated defenses against both biotrophic and necrotrophic pathogens. For example, SA-dependent defenses are required for resistance to the biotrophic fungal pathogen Erysiphe cichoracearum UCSC1 and the bacterial pathogen Pseudomonas syringae pv. maculicola, and also are expressed during response to the green peach aphid Myzus persicae. However, recent evidence indicates that the expression of JA-dependent defenses also may confer resistance to E. cichoracearum. To confirm and to extend this observation, we have compared the disease and pest resistance of wild-type Arabidopsis plants with that of the mutants coil, which is insensitive to JA, and cev1, which has constitutive JA signaling. Measurements of the colonization of these plants by E. cichoracearum, P. syringae pv. maculicola, and M. persicae indicated that activation of the JA signal pathway enhanced resistance, and was associated with the activation of JA-dependent defense genes and the suppression of SA-dependent defense genes. We conclude that JA and SA induce alternative defense pathways that can confer resistance to the same pathogens and pests.     

Herbivory in the previous generation primes plants for enhanced insect resistance

Inducible defenses, which provide enhanced resistance after initial attack, are nearly universal in plants. This defense signaling cascade is mediated by the synthesis, movement, and perception of jasmonic acid and related plant metabolites. To characterize the long-term persistence of plant immunity, we challenged Arabidopsis (Arabidopsis thaliana) and tomato (Solanum lycopersicum) with caterpillar herbivory, application of methyl jasmonate, or mechanical damage during vegetative growth and assessed plant resistance in subsequent generations. Here, we show that induced resistance was associated with transgenerational priming of jasmonic acid-dependent defense responses in both species, caused caterpillars to grow up to 50% smaller than on control plants, and persisted for two generations in Arabidopsis. Arabidopsis mutants that are deficient in jasmonate perception (coronatine insensitive1) or in the biogenesis of small interfering RNA (dicer-like2 dicer-like3 dicer-like4 and nuclear RNA polymerase d2a nuclear RNA polymerase d2b) do not exhibit inherited resistance. The observation of inherited resistance in both the Brassicaceae and Solanaceae suggests that this trait may be more widely distributed in plants. Epigenetic resistance to herbivory thus represents a phenotypically plastic mechanism for enhanced defense across generations.     

Dissecting the beta-aminobutyric acid-induced priming phenomenon in Arabidopsis

Plants treated with the nonprotein amino acid beta-aminobutyric acid (BABA) develop an enhanced capacity to resist biotic and abiotic stresses. This BABA-induced resistance (BABA-IR) is associated with an augmented capacity to express basal defense responses, a phenomenon known as priming. Based on the observation that high amounts of BABA induce sterility in Arabidopsis thaliana, a mutagenesis screen was performed to select mutants impaired in BABA-induced sterility (ibs). Here, we report the isolation and subsequent characterization of three T-DNA-tagged ibs mutants. Mutant ibs1 is affected in a cyclin-dependent kinase-like protein, and ibs2 is defective in AtSAC1b encoding a polyphosphoinositide phosphatase. Mutant ibs3 is affected in the regulation of the ABA1 gene encoding the abscisic acid (ABA) biosynthetic enzyme zeaxanthin epoxidase. To elucidate the function of the three IBS genes in plant resistance, the mutants were tested for BABA-IR against the bacterium Pseudomonas syringae pv tomato, the oomycete Hyaloperonospora parasitica, and BABA-induced tolerance to salt. All three ibs mutants were compromised in BABA-IR against H. parasitica, although to a different extent. Whereas ibs1 was reduced in priming for salicylate (SA)-dependent trailing necrosis, mutants ibs2 and ibs3 were affected in the priming for callose deposition. Only ibs1 failed to express BABA-IR against P. syringae, which coincided with a defect in priming for SA-inducible PR-1 gene expression. By contrast, ibs2 and ibs3 showed reduced BABA-induced tolerance to salt, which correlated with an affected priming for ABA-inducible gene expression. For all three ibs alleles, the defects in BABA-induced sterility and BABA-induced protection against P. syringae, H. parasitica, and salt could be confirmed in independent mutants. The data presented here introduce three novel regulatory genes involved in priming for different defense responses.     

Antagonism between salicylic and abscisic acid reflects early host–pathogen conflict and moulds plant defence responses

The importance of phytohormone balance is increasingly recognized as central to the outcome of plant–pathogen interactions. Recently it has been demonstrated that abscisic acid signalling pathways are utilized by the bacterial phytopathogen Pseudomonas syringae to promote pathogenesis. In this study, we examined the dynamics, inter-relationship and impact of three key acidic phytohormones, salicylic acid, abscisic acid and jasmonic acid, and the bacterial virulence factor, coronatine, during progression of P. syringae infection of Arabidopsis thaliana . We show that levels of SA and ABA, but not JA, appear to play important early roles in determining the outcome of the infection process. SA is required in order to mount a full innate immune responses, while bacterial effectors act rapidly to activate ABA biosynthesis. ABA suppresses inducible innate immune responses by down-regulating SA biosynthesis and SA-mediated defences. Mutant analyses indicated that endogenous ABA levels represent an important reservoir that is necessary for effector suppression of plant-inducible innate defence responses and SA synthesis prior to subsequent pathogen-induced increases in ABA. Enhanced susceptibility due to loss of SA-mediated basal resistance is epistatically dominant over acquired resistance due to ABA deficiency, although ABA also contributes to symptom development. We conclude that pathogen-modulated ABA signalling rapidly antagonizes SA-mediated defences. We predict that hormonal perturbations, either induced or as a result of environmental stress, have a marked impact on pathological outcomes, and we provide a mechanistic basis for understanding priming events in plant defence.