Mqo,a Tricarboxylic Acid Cycle Enzyme,Is Required for Virulence of Pseudomonas syringae pv. tomato Strain DC3000 on Arabidopsis thaliana

Plant pathogenic bacteria, such as Pseudomonas syringae pv. tomato strain DC3000, the causative agent of tomato bacterial speck disease, grow to high levels in the apoplastic space between plant cells. Colonization of plant tissue requires expression of virulence factors that modify the apoplast to make it more suitable for pathogen growth or facilitate adaptation of the bacteria to the apoplastic environment. To identify new virulence factors involved in these processes, DC3000 Tn5 transposon insertion mutants with reduced virulence on Arabidopsis thaliana were identified. In one of these mutants, the Tn5 insertion disrupted the malate:quinone oxidoreductase gene (mqo), which encodes an enzyme of the tricarboxylic acid cycle. mqo mutants do not grow to wild-type levels in plant tissue at early time points during infection. Further, plants infected with mqo mutants develop significantly reduced disease symptoms, even when the growth of the mqo mutant reaches wild-type levels at late stages of infection. Mutants lacking mqo function grow more slowly in culture than wild-type bacteria when dicarboxylates are the only available carbon source. To explore whether dicarboxylates are important for growth of DC3000 in the apoplast, we disrupted the dctA1 dicarboxylate transporter gene. DC3000 mutants lacking dctA1 do not grow to wild-type levels in planta, indicating that transport and utilization of dicarboxylates are important for virulence of DC3000. Thus, mqo may be required by DC3000 to meet nutritional requirements in the apoplast and may provide insight into the mechanisms underlying the important, but poorly understood process of adaptation to the host environment.One important aspect of interactions between plant pathogens and their hosts is the ability of the pathogen to obtain nutrients within the plant tissue. Nutrient acquisition is essential for growth within the host, since both cell division and DNA replication can be influenced by nutrient availability. Bacterial plant pathogens differ in the strategies they use to get necessary nutrients during infection. Some pathogens, such as Agrobacterium tumefaciens, elicit production of specific carbon and nitrogen sources by the plant (1). Other pathogens may rely on metabolites that are readily available in the plant apoplast or may stimulate the release of water or nutrients from surrounding plant cells (27).Little is known about how pathogenic Pseudomonas syringae strains acquire nutrients when growing in their hosts. P. syringae strains are gram-negative gammaproteobacteria, which as a group cause disease on many agriculturally important plants. For example, P. syringae pv. tomato strain DC3000 causes disease on tomato, A. thaliana, and several agriculturally important Brassicas, such as turnip, mustard, collard, and cauliflower (8, 51). Initially, DC3000 colonizes plant surfaces and then enters the plant tissue through natural openings (such as stomata) or wounds (34, 38). DC3000 then establishes itself in the plant apoplast, the intercellular space between plant cells (38). Once in the apoplast of susceptible hosts, DC3000 multiplies to high levels, and the infected plants develop disease symptoms, including chlorosis (yellowing) of the leaf tissue and necrotic spots or patches called lesions (38, 49). Pseudomonads, such as P. aeruginosa and P. fluorescens, preferentially utilize tricarboxylic acid (TCA) cycle intermediates (20, 29, 33, 44), and DC3000 utilizes these carbon sources in culture (19). Some studies to investigate nutrient acquisition of DC3000 have been carried out (3, 7, 40); however, it is not clear what carbon sources DC3000 utilizes when growing in plant tissue.Several virulence factors are necessary for DC3000 to enter, grow inside the plant, and cause disease. Like many other bacterial pathogens, DC3000 uses a type III secretion system (TTSS) (15), which is encoded by the hrp/hrc genes, to inject effector proteins into plant cells (16, 27). Many of these effectors suppress host defenses, and it is likely that some may be involved in modulating the apoplastic environment or nutrient acquisition (16). DC3000 also produces the phytotoxin coronatine, which promotes entry of the bacteria into the plant apoplast by stimulating the opening of stomata (34) and is required for bacterial growth in the apoplast by suppressing salicylic acid (SA)-dependent host defenses (4, 45). Coronatine also promotes disease symptom development via an SA-independent mechanism (4). While much emphasis has been placed on exploring how type III-secreted effectors and coronatine promote DC3000 virulence, other factors are also likely to be important during pathogenesis.To identify additional factors involved in pathogenesis, we undertook a genetic screen to identify novel virulence factors (5, 24). DC3000 mutants with reduced virulence were identified by assaying for their ability to elicit disease symptoms on A. thaliana and tomato plants (24, 37). One of these mutants, AK4C9, had reduced virulence on both hosts. The gene disrupted in this mutant is the malate:quinone oxidoreductase gene (mqo), which encodes an enzyme of the TCA cycle. mqo mutants grow more slowly than wild-type DC3000 in planta and in culture when dicarboxylates are the only carbon source, suggesting that dicarboxylates are important for the growth of DC3000 in the apoplast. In the present study, we explore the role of Mqo and a dicarboxylate transporter, DctA1, in DC3000 pathogenesis.     

Visualization and characterization of Pseudomonas syringae pv. tomato DC3000 pellicles

Cellulose, whose production is controlled by c-di-GMP, is a commonly found exopolysaccharide in bacterial biofilms. Pseudomonas syringae pv. tomato (Pto) DC3000, a model organism for molecular studies of plant–pathogen interactions, carries the wssABCDEFGHI operon for the synthesis of acetylated cellulose. The high intracellular levels of the second messenger c-di-GMP induced by the overexpression of the heterologous diguanylate cyclase PleD stimulate cellulose production and enhance air–liquid biofilm (pellicle) formation. To characterize the mechanisms involved in Pto DC3000 pellicle formation, we studied this process using mutants lacking flagella, biosurfactant or different extracellular matrix components, and compared the pellicles produced in the absence and in the presence of PleD. We have discovered that neither alginate nor the biosurfactant syringafactin are needed for their formation, whereas cellulose and flagella are important but not essential. We have also observed that the high c-di-GMP levels conferred more cohesion to Pto cells within the pellicle and induced the formation of intracellular inclusion bodies and extracellular fibres and vesicles. Since the pellicles were very labile and this greatly hindered their handling and processing for microscopy, we have also developed new methods to collect and process them for scanning and transmission electron microscopy. These techniques open up new perspectives for the analysis of fragile biofilms in other bacterial strains.     

Tomato SlSAP3, a member of the stress-associated protein family,is a positive regulator of immunity against Pseudomonas syringae pv. tomato DC3000

Tomato stress-associated proteins (SAPs) belong to A20/AN1 zinc finger protein family, some of which have been shown to play important roles in plant stress responses. However, little is known about the functions and underlying molecular mechanisms of SAPs in plant immune responses. In the present study, we reported the function of tomato SlSAP3 in immunity to Pseudomonas syringae pv. tomato (Pst) DC3000. Silencing of SlSAP3 attenuated while overexpression of SlSAP3 in transgenic tomato increased immunity to Pst DC3000, accompanied with reduced and increased Pst DC3000-induced expression of SA signalling and defence genes, respectively. Flg22-induced reactive oxygen species (ROS) burst and expression of PAMP-triggered immunity (PTI) marker genes SlPTI5 and SlLRR22 were strengthened in SlSAP3-OE plants but were weakened in SlSAP3-silenced plants. SlSAP3 interacted with two SlBOBs and the A20 domain in SlSAP3 is critical for the SlSAP3-SlBOB1 interaction. Silencing of SlBOB1 and co-silencing of all three SlBOB genes conferred increased resistance to Pst DC3000, accompanied with increased Pst DC3000-induced expression of SA signalling and defence genes. These data demonstrate that SlSAP3 acts as a positive regulator of immunity against Pst DC3000 in tomato through the SA signalling and that SlSAP3 may exert its function in immunity by interacting with other proteins such as SlBOBs, which act as negative regulators of immunity against Pst DC3000 in tomato.     

FleQ Coordinates Flagellum-Dependent and -Independent Motilities in Pseudomonas syringae pv. tomato DC3000

Motility plays an essential role in bacterial fitness and colonization in the plant environment, since it favors nutrient acquisition and avoidance of toxic substances, successful competition with other microorganisms, the ability to locate the preferred hosts, access to optimal sites within them, and dispersal in the environment during the course of transmission. In this work, we have observed that the mutation of the flagellar master regulatory gene, fleQ, alters bacterial surface motility and biosurfactant production, uncovering a new type of motility for Pseudomonas syringae pv. tomato DC3000 on semisolid surfaces. We present evidence that P. syringae pv. tomato DC3000 moves over semisolid surfaces by using at least two different types of motility, namely, swarming, which depends on the presence of flagella and syringafactin, a biosurfactant produced by this strain, and a flagellum-independent surface spreading or sliding, which also requires syringafactin. We also show that FleQ activates flagellum synthesis and negatively regulates syringafactin production in P. syringae pv. tomato DC3000. Finally, it was surprising to observe that mutants lacking flagella or syringafactin were as virulent as the wild type, and only the simultaneous loss of both flagella and syringafactin impairs the ability of P. syringae pv. tomato DC3000 to colonize tomato host plants and cause disease.     

Relatedness of Pseudomonas syringae pv. tomato, Pseudomonas syringae pv. maculicola and Pseudomonas syringae pv. antirrhini

The relationships among strains of Pseudomonas syringae pv. tomato, Ps. syr. antirrhini, Ps. syr. maculicola, Ps. syr. apii and a strain isolated from squash were examined by restriction fragment length polymorphism (RFLP) patterns, nutritional characteristics, host of origin and host ranges. All strains tested except for Ps. syr. maculicola 4326 isolated from radish ( Raphanus sativus L.) constitute a closely related group. No polymorphism was seen among strains probed with the 5.7 and 2.3 kb Eco RI fragments which lie adjacent to the hrp cluster of Ps. syr. tomato and the 8.6 kb Eco RI insert of pBG2, a plasmid carrying the β-glucosidase gene(s). All strains tested had overlapping host ranges. In contrast to this, comparison of strains by RFLP patterns of sequences homologous to the 4.5 kb Hind III fragment of pRut2 and nutritional properties distinguished four groups. Group 1, consisting of strains of pathovars maculicola, tomato and apii , had similar RFLP patterns and used homoserine but not sorbitol as carbon sources. Group 2, consisting of strains of pathovars maculicola and tomato , differed from Group 1 in RFLP patterns and did not use either homoserine or sorbitol. Group 3 was similar to Group 2 in RFLP patterns but utilized homoserine and sorbitol. This group included strains of the pathovars tomato and antirrhini , and a strain isolated from squash. Group 4, a single strain of Ps. syr. maculicola isolated from radish, had unique RFLP patterns and resembled Group 3 nutritionally. The evolutionary relationships of these strains are discussed.     

Regulons of Three Pseudomonas syringae pv. tomato DC3000 Iron Starvation Sigma Factors

Pseudomonas syringae pv. tomato DC3000 contains genes for 15 sigma factors. The majority are members of the extracytoplasmic function class of sigma factors, including five that belong to the iron starvation subgroup. In this study, we identified the genes controlled by three iron starvation sigma factors. Their regulons are composed of a small number of genes likely to be involved in iron uptake.     

Crystal structure of uroporphyrinogen III synthase from Pseudomonas syringae pv. tomato DC3000

Uroporphyrinogen III synthase (U3S) is one of the key enzymes in the biosynthesis of tetrapyrrole compounds. It catalyzes the cyclization of the linear hydroxymethylbilane (HMB) to uroporphyrinogen III (uro’gen III). We have determined the crystal structure of U3S from Pseudomonas syringae pv. tomato DC3000 (psU3S) at 2.5 Å resolution by the single wavelength anomalous dispersion (SAD) method. Each psU3S molecule consists of two domains interlinked by a two-stranded antiparallel β-sheet. The conformation of psU3S is different from its homologous proteins because of the flexibility of the linker between the two domains, which might be related to this enzyme’s catalytic properties. Based on mutation and activity analysis, a key residue, Arg219, was found to be important for the catalytic activity of psU3S. Mutation of Arg219 to Ala caused a decrease in enzymatic activity to about 25% that of the wild type enzyme. Our results provide the structural basis and biochemical evidence to further elucidate the catalytic mechanism of U3S.     

PslD Is a Secreted Protein Required for Biofilm Formation by Pseudomonas aeruginosa

The function of pslD, which is part of the psl operon from Pseudomonas aeruginosa, was investigated in this study. The psl operon is involved in exopolysaccharide biosynthesis and biofilm formation. An isogenic marker-free pslD deletion mutant of P. aeruginosa PAO1 which was deficient in the formation of differentiated biofilms was generated. Expression of only the pslD gene coding region restored the wild-type phenotype. A C-terminal, hexahistidine tag fusion enabled the identification of PslD. LacZ and PhoA translational fusions with PslD indicated that PslD is a secreted protein required for biofilm formation, presumably via its role in exopolysaccharide export.     

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.     

Identification of Bacteriophages for Biocontrol of the Kiwifruit Canker Phytopathogen Pseudomonas syringae pv. actinidiae

Pseudomonas syringae pv. actinidiae is a reemerging pathogen which causes bacterial canker of kiwifruit (Actinidia sp.). Since 2008, a global outbreak of P. syringae pv. actinidiae has occurred, and in 2010 this pathogen was detected in New Zealand. The economic impact and the development of resistance in P. syringae pv. actinidiae and other pathovars against antibiotics and copper sprays have led to a search for alternative management strategies. We isolated 275 phages, 258 of which were active against P. syringae pv. actinidiae. Extensive host range testing on P. syringae pv. actinidiae, other pseudomonads, and bacteria isolated from kiwifruit orchards showed that most phages have a narrow host range. Twenty-four were analyzed by electron microscopy, pulse-field gel electrophoresis, and restriction digestion. Their suitability for biocontrol was tested by assessing stability and the absence of lysogeny and transduction. A detailed host range was performed, phage-resistant bacteria were isolated, and resistance to other phages was examined. The phages belonged to the Caudovirales and were analyzed based on morphology and genome size, which showed them to be representatives of Myoviridae, Podoviridae, and Siphoviridae. Twenty-one Myoviridae members have similar morphologies and genome sizes yet differ in restriction patterns, host range, and resistance, indicating a closely related group. Nine of these Myoviridae members were sequenced, and each was unique. The most closely related sequenced phages were a group infecting Pseudomonas aeruginosa and characterized by phages JG004 and PAK_P1. In summary, this study reports the isolation and characterization of P. syringae pv. actinidiae phages and provides a framework for the intelligent formulation of phage biocontrol agents against kiwifruit bacterial canker.     

Functions of pipecolic acid on induced resistance against Botrytis cinerea and Pseudomonas syringae pv. tomato DC3000 in tomato plants

Amino acid metabolic pathways are involved in the plant immune system. Pipecolic acid (Pip), a lysine-derived non-protein amino acid, acts as an important regulator of disease resistance. Here, we report the functions of Pip on tomato disease resistance. Tomato seedlings treated with 0.5 mM Pip showed increased resistance to Pst DC3000 and B. cinerea compared with the control. After pathogen infection, the expression of defence-related genes increased in plants pretreated with Pip, while reactive oxygen species (ROS) accumulation decreased. These data demonstrated that exogenous application of Pip induced resistance against Pst DC3000 and B. cinerea in tomatoes, possibly through the regulation of ROS accumulation and defence-related gene expression.     

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.