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.     

Identification and characterization of a well-defined series of coronatine biosynthetic mutants of Pseudomonas syringae pv. tomato DC3000

To identify Pseudomonas syringae pv. tomato genes involved in pathogenesis, we carried out a screen for Tn5 mutants of P. syringae pv. tomato DC3000 with reduced virulence on Arabidopsis thaliana. Several mutants defining both known and novel virulence loci were identified. Six mutants contained insertions in biosynthetic genes for the phytotoxin coronatine (COR). The P. syringae pv. tomato DC3000 COR genes are chromosomally encoded and are arranged in two separate clusters, which encode enzymes responsible for the synthesis of coronafacic acid (CFA) or coronamic acid (CMA), the two defined intermediates in COR biosynthesis. High-performance liquid chromatography fractionation and exogenous feeding studies confirmed that Tn5 insertions in the cfa and cma genes disrupt CFA and CMA biosynthesis, respectively. All six COR biosynthetic mutants were significantly impaired in their ability to multiply to high levels and to elicit disease symptoms on A. thaliana plants. To assess the relative contributions of CFA, CMA, and COR in virulence, we constructed and characterized cfa6 cmaA double mutant strains. These exhibited virulence phenotypes on A. thalliana identical to those observed for the cmaA or cfa6 single mutants, suggesting that reduced virulence of these mutants on A. thaliana is caused by the absence of the intact COR toxin. This is the first study to use biochemically and genetically defined COR mutants to address the role of COR in pathogenesis.     

Virulence systems of Pseudomonas syringae pv. tomato promote bacterial speck disease in tomato by targeting the jasmonate signaling pathway

Pseudomonas syringae pv. tomato strain DC3000 (Pst DC3000) causes bacterial speck disease on tomato. The pathogenicity of Pst DC3000 depends on both the type III secretion system that delivers virulence effector proteins into host cells and the phytotoxin coronatine (COR), which is thought to mimic the action of the plant hormone jasmonic acid (JA). We found that a JA-insensitive mutant (jai1) of tomato was unresponsive to COR and highly resistant to Pst DC3000, whereas host genotypes that are defective in JA biosynthesis were as susceptible to Pst DC3000 as wild-type (WT) plants. Treatment of WT plants with exogenous methyl-JA (MeJA) complemented the virulence defect of a bacterial mutant deficient in COR production, but not a mutant defective in the type III secretion system. Analysis of host gene expression using cDNA microarrays revealed that COR works through Jai1 to induce the massive expression of JA and wound response genes that have been implicated in defense against herbivores. Concomitant with the induction of JA and wound response genes, the type III secretion system and COR repressed the expression of pathogenesis-related (PR) genes in Pst DC3000-infected WT plants. Resistance of jai1 plants to Pst DC3000 was correlated with a high level of PR gene expression and reduced expression of JA/wound response genes. These results indicate that COR promotes bacterial virulence by activating the host's JA signaling pathway, and further suggest that the type III secretion system might also modify host defense by targeting the JA signaling pathway in susceptible tomato plants.     

Population Dynamics of Pseudomonas syringae pv. tomato Strains on Tomato Cultivars Rio Grande and Rio Grande-Pto under Field Conditions

We examined the effects of the Pto resistance locus on the population dynamics of Pseudomonas syringae pv. tomato (Pst) strains in field experiments with the nearly isogenic tomato lines Rio Grande (RG, susceptible to Pst races 0 and 1) and Rio Grande-Pto (RG-Pto, resistant to Pst race 0, susceptible to Pst race 1). Pst strain SM78-1Sm^r (race 0) grew well under field conditions and caused ample bacterial speck disease on susceptible RG plants. In contrast, strain DC3000 failed to establish large populations when inoculated onto field grown RG plants. Mean population sizes of SM78-1Sm^r were 4–5 orders of magnitude larger on RG than RG-Pto plants indicating that RG-Pto plants were highly effective in attenuating pathogen population development. Most of the sampled leaflets from RG-Pto field plots harboured small numbers of SM78-1Sm^r. However, population sizes SM78-1Sm^r as large as 10⁵–10⁶ CFU were found on a few leaflets. Bacteria isolated from these leaflets had phenotypes characteristic of Pst race 1 strains. In growth chamber plant assays, the bacterial strains grew well and caused typical speck lesions on RG-Pto plants. The strains appeared to be race-shift mutants of SM SM78-1Sm^r. Interestingly, results from DNA hybridization experiments demonstrated that the race-shift mutants were deleted for the avirulence gene, avrPto but not for avrPtoB.     

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.     

Elevation of rat intestinal permeability by enterotoxigenic Escherichia coli in comparison to non-toxigenic E. coli and Salmonella typhimurium. Application of fluorescent dextran 3000 as permeability probe

Abstract The effect of bacterial enterotoxins on rat intestinal permeability properties was studied by comparing the effect of toxin-positive and toxin-negative Escherichia coli and Salmonella typhimurium inoculated into a segment of rat small intestine. Fluoresceinated dextran 3000 (FITC-D3; M _r 3000) was applied as permeability marker. The E. coli strain C922a-1 producing heat-labile (LT) and heat-stable (ST) enterotoxins and colonising factor CFA/II increased the transmural passage of the dextran probe into portal blood. In contrast, its plasmid-negative variant, a non-toxin producer lacking CFA, caused permeability changes indistinguishable from the bacteria-free nutrient broth control. Another pair of enterotoxigenic E. coli strains, 1628–14 (LT⁺, ST⁺, CFA/I⁺) and 1628–15 (LT⁺, ST⁻ and CFA/I⁻) both increased the intestinal permeability. The observations indicate that the LT⁺-only E. coli strain 1628–15 has the ability to promote permeability of rat intestine. The toxin-negative, rough S. typhimurium 395MR10 bacteria had a very small effect on the permeability, which was also achieved with culture filtrate only.
It is concluded that enterotoxigenic E. coli (ETEC) can alter the properties of the mucosal barrier towards intermediate-sized molecules that could be of antigenic significance, or which could play a crucial role in the nutritional status of the host organism.     

Pseudomonas syringae pv. tomato DC3000 polymutants deploying coronatine and two type III effectors produce quantifiable chlorotic spots from individual bacterial colonies in Nicotiana benthamiana leaves

Primary virulence factors of Pseudomonas syringae pv. tomato DC3000 include the phytotoxin coronatine (COR) and a repertoire of 29 effector proteins injected into plant cells by the type III secretion system (T3SS). DC3000 derivatives differentially producing COR, the T3SS machinery and subsets of key effectors were constructed and assayed in leaves of Nicotiana benthamiana. Bacteria were inoculated by the dipping of whole plants and assayed for population growth and the production of chlorotic spots on leaves. The strains fell into three classes. Class I strains are T3SS⁺ but functionally effectorless, grow poorly in planta and produce faint chlorotic spots only if COR⁺. Class II strains are T3SS^– or, if T3SS⁺, also produce effectors AvrPtoB and HopM1. Class II strains grow better than class I strains in planta and, if COR⁺, produce robust chlorotic spots. Class III strains are T3SS⁺ and minimally produce AvrPtoB, HopM1 and three other effectors encoded in the P. syringae conserved effector locus. These strains differ from class II strains in growing better in planta, and produce chlorotic spots without COR if the precursor coronafacic acid is produced. Assays for chlorotic spot formation, in conjunction with pressure infiltration of low‐level inoculum and confocal microscopy of fluorescent protein‐labelled bacteria, revealed that single bacteria in the apoplast are capable of producing colonies and associated leaf spots in a 1 : 1 : 1 manner. However, COR makes no significant contribution to the bacterial colonization of the apoplast, but, instead, enables a gratuitous, semi‐quantitative, surface indicator of bacterial growth, which is determined by the strain's effector composition.     

The phytotoxin coronatine induces light-dependent reactive oxygen species in tomato seedlings

The phytotoxin coronatine (COR), which is produced by Pseudomonas syringae pv. tomato DC3000 (DC3000), has multiple roles in virulence that lead to chlorosis and a reduction in chlorophyll content. However, the physiological significance of COR-induced chlorosis in disease development is still largely unknown. Global expression analysis demonstrated that DC3000 and COR, but not the COR-defective mutant DB29, caused reduced expression of photosynthesis-related genes and result in a 1.5- to 2-fold reduction in maximum quantum efficiency of photosystem II (F(V)/F(M)). Tomato (Solanum lycopersicum) seedlings inoculated with DC3000 and incubated in a long daily photoperiod showed more necrosis than inoculated seedlings incubated in either dark or a short daily photoperiod. The accumulation of reactive oxygen species (ROS) was detected in cotyledons inoculated with either purified COR or DC3000 but not in tissues inoculated with DB29. Interestingly, COR-induced ROS accumulated only in light and was inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea and diphenylene iodonium, which function to inhibit electron transport from PSII. Furthermore, COR and DC3000 suppressed expression of the gene encoding the thylakoid Cu/Zn superoxide dismutase but not the cytosolic form of the same enzyme. In conclusion, these results demonstrate a role for COR-induced effects on photosynthetic machinery and ROS in modulating necrotic cell death during bacterial speck disease of tomato.     

Synthetic biology approaches to establish a heterologous production system for coronatines

Coronatine (COR) represents a phytotoxin produced by several pathovars of Pseudomonas syringae. It mediates multiple virulence activities by mimicking the plant stress hormone jasmonoyl-l-isoleucine. Structurally, COR consists of a bicyclic polyketide moiety, coronafacic acid (CFA), which is linked via an amide bond to an unusual ethylcyclopropyl amino acid moiety, coronamic acid (CMA). In our studies, we aimed at establishing and engineering of heterologous COR and CFA production platforms using P. putida KT2440 as host. Based on genetic information of the native producer P. syringae pv. tomato DC3000 a COR biosynthetic gene cluster was designed and reconstituted from synthetic DNA fragments. The applied constructional design facilitated versatile pathway modifications and the generation of various expression constructs, which were evaluated for the production of CFA, COR and its derivatives. By modifications of the gene cluster composition production profiles were directed towards target compounds and valuable information about the function and impact of selected pathway proteins on COR biosynthesis were obtained. Additional engineering of expression vector features, including the use of the constitutive PrpsH promoter and a p15Aori-based transposon backbone, led to the development of an expression strain with promising CFA production yields of > 90 mg/l.     

The Arabidopsis thaliana JASMONATE INSENSITIVE 1 gene is required for suppression of salicylic acid-dependent defenses during infection by Pseudomonas syringae

Many plant pathogens suppress antimicrobial defenses using virulence factors that modulate endogenous host defenses. The Pseudomonas syringae phytotoxin coronatine (COR) is believed to promote virulence by acting as a jasmonate analog, because COR-insensitive 1 (coil) Arabidopsis thaliana and tomato mutants are impaired in jasmonate signaling and exhibit reduced susceptibility to P. syringae. To further investigate the role of jasmonate signaling in disease development, we analyzed several jasmonate-insensitive A. thaliana mutants for susceptibility to P. syringae pv. tomato strain DC3000 and sensitivity to COR. Jasmonate-insensitive 1 (jin1) mutants exhibit both reduced susceptibility to P. syringae pv. tomato DC3000 and reduced sensitivity to COR, whereas jasmonate-resistant 1 (jar1) plants exhibit wild-type responses to both COR and P. syringae pv. tomato DC3000. A jin1 jar1 double mutant does not exhibit enhanced jasmonate insensitivity, suggesting that JIN1 functions downstream of jasmonic acid-amino acid conjugates synthesized by JAR1. Reduced disease susceptibility in jin1 mutants is correlated with elevated expression of pathogenesis-related 1 (PR-1) and is dependent on accumulation of salicylic acid (SA). We also show that JIN1 is required for normal P. syringae pv. tomato DC3000 symptom development through an SA-independent mechanism. Thus, P. syringae pv. tomato DC3000 appears to utilize COR to manipulate JIN1-dependent jasmonate signaling both to suppress SA-mediated defenses and to promote symptom development.     

Features of basal and race-specific defences in photosynthetic Arabidopsis thaliana suspension cultured cells

Plant suspension cell cultures display many features of the innate immune responses observed in planta and have been extensively applied to the study of basal and race-specific defences. However, no single model including photosynthetic cultured cells has been used for the exhaustive characterization of both basal and race-specific defences to date. In this article, we report the activation of basal and race-specific defences in green cultured cells from Arabidopsis thaliana . Inoculation of cultured cells with isogenic virulent or avirulent strains of Pseudomonas syringae pv. tomato DC3000 (Pst) was used to evaluate race-specific defences. The proliferation of avirulent Pst was found to be lower than that of virulent Pst in the inoculated cultures. Extracellular pH changes, sustained oxidative burst (5–13 h post-inoculation), enhancement of salicylic acid, and massive cell death were specifically stimulated by the avirulent bacterium. Neither avirulent nor virulent Pst induced markers of basal resistance, such as callose deposition or early oxidative burst (1–5 h post-inoculation). However, both basal defences were activated when cells were exposed to Pseudomonas syringae pv. phaseolicola or to the Pst mutant defective in the type III secretion system (TTSS), Pst-hrpL⁻. Thus, in these cells, basal defences may be inhibited by Pst in a TTSS-dependent manner. Recapitulation of classical defence features demonstrates the usefulness of this system for the fine characterization of plant innate immune components.     

Two mutants of Arabidopsis thaliana that become chlorotic in atmospheres enriched with CO2

Abstract. Two nonallelic, nuclear recessive mutants of Arabidopsis thaliana (L.) Heynh. which become chlorotic when grown in an atmosphere enriched to 20000 cm³ CO₂ m^-3 have been isolated. For one of the mutants, chlorosis begins at the veins and gradually spreads to the interveinal regions. A minimum photon flux density of ca 50 μmol m^-2 s^-1 is required for this response. For the other mutant, the yellowing is independent of the light intensity and begins at the basal regions of the leaves and spreads to the tips. The injurious effects of CO₂ seem to be restricted to photosynthetic tissues, since root elongation and callus growth were not inhibited by a high atmospheric CO₂ concentration for either mutant. Neither mutant became chlorotic in a low O₂ atmosphere that suppressed photorespiration as effectively as the elevated CO₂ does. Thus, the mutations do not impose a requirement for photorespiration. The possibilities that the high CO₂-sensitive phenotypes are caused by an effect of CO₂ in stomata, on ethylene synthesis, or on mineral uptake are discussed but are considered unlikely.     

The Pseudomonas syringae effector protein, AvrRPS4, requires in planta processing and the KRVY domain to function

A Pseudomonas syringae pv. pisi effector protein, AvrRPS4, triggers RPS4 -dependent immunity in Arabidopsis. We characterized biochemical and genetic aspects of AvrRPS4 function. Secretion of AvrRPS4 from Pst DC3000 is type III secretion-dependent, and AvrRPS4 is processed into a smaller form in plant cells but not in bacteria or yeast. Agrobacterium -mediated transient expression analysis of N-terminally truncated AvrRPS4 mutants revealed that the C-terminal 88 amino acids are sufficient to trigger the hypersensitive response in turnip. N-terminal sequencing of the processed AvrRPS4 showed that processing occurs between G133 and G134. The processing-deficient mutant, R112L, still triggers RPS4 -dependent immunity, suggesting that the processing is not required for the AvrRPS4 avirulence function. AvrRPS4 enhances bacterial growth when delivered by Pta 6606 into Nicotiana benthamiana in which AvrRPS4 is not recognized. Transgenic expression of AvrRPS4 in the Arabidopsis rps4 mutant enhances the growth of Pst DC3000 and suppresses PTI (PAMP-triggered immunity), showing that AvrRPS4 promotes virulence in two distinct host plants. Furthermore, full virulence activity of AvrRPS4 requires both proteolytic processing and the KRVY motif at the N-terminus of processed AvrRPS4. XopO, an Xcv effector, shares the amino acids required for AvrRPS4 processing and the KRVY motif. XopO is also processed into a smaller form in N. benthamiana , similar to AvrRPS4, suggesting that a common mechanism is involved in activation of the virulence activities of both AvrRPS4 and XopO.     

Transformation of Methanococcus maripaludis and identification of a Pst I-like restriction system

Abstract An optimized polyethylene glycol (PEG) method of transformation was developed for Methanococcus maripaludis using the pKAS102 integration vector. The frequency of transformation with 0.8 μg of plasmid and 3×10⁹ cells was 4.8×10⁻⁵ transformants cfu⁻¹, or 1.8×10⁵ transformants μg⁻¹, which was four orders of magnitude greater than with the natural transformation method. A Pst I restriction activity in M. maripaludis was also identified. Methylation of the plasmid with Pst I methylase increased the methanococcal transformation frequency at least four-fold. Also, chromosomal DNA from M. maripaludis was resistant to digestion by the Pst I endonuclease.     

The downy mildew effector proteins ATR1 and ATR13 promote disease susceptibility in Arabidopsis thaliana

The downy mildew (Hyaloperonospora parasitica) effector proteins ATR1 and ATR13 trigger RPP1-Nd/WsB- and RPP13-Nd-dependent resistance, respectively, in Arabidopsis thaliana. To better understand the functions of these effectors during compatible and incompatible interactions of H. parasitica isolates on Arabidopsis accessions, we developed a novel delivery system using Pseudomonas syringae type III secretion via fusions of ATRs to the N terminus of the P. syringae effector protein, AvrRPS4. ATR1 and ATR13 both triggered the hypersensitive response (HR) and resistance to bacterial pathogens in Arabidopsis carrying RPP1-Nd/WsB or RPP13-Nd, respectively, when delivered from P. syringae pv tomato (Pst) DC3000. In addition, multiple alleles of ATR1 and ATR13 confer enhanced virulence to Pst DC3000 on susceptible Arabidopsis accessions. We conclude that ATR1 and ATR13 positively contribute to pathogen virulence inside host cells. Two ATR13 alleles suppressed bacterial PAMP (for Pathogen-Associated Molecular Patterns)-triggered callose deposition in susceptible Arabidopsis when delivered by DC3000 DeltaCEL mutants. Furthermore, expression of another allele of ATR13 in plant cells suppressed PAMP-triggered reactive oxygen species production in addition to callose deposition. Intriguingly, although Wassilewskija (Ws-0) is highly susceptible to H. parasitica isolate Emco5, ATR13Emco5 when delivered by Pst DC3000 triggered localized immunity, including HR, on Ws-0. We suggest that an additional H. parasitica Emco5 effector might suppress ATR13-triggered immunity.