Nanogram amounts of salicylic acid produced by the rhizobacterium Pseudomonas aeruginosa 7NSK2 activate the systemic acquired resistance pathway in bean

Root colonization by specific nonpathogenic bacteria can induce a systemic resistance in plants to pathogen infections. In bean, this kind of systemic resistance can be induced by the rhizobacterium Pseudomonas aeruginosa 7NSK2 and depends on the production of salicylic acid by this strain. In a model with plants grown in perlite we demonstrated that Pseudomonas aeruginosa 7NSK2-induced resistance is equivalent to the inclusion of 1 nM salicylic acid in the nutrient solution and used the latter treatment to analyze the molecular basis of this phenomenon. Hydroponic feeding of 1 nM salicylic acid solutions induced phenylalanine ammonia-lyase activity in roots and increased free salicylic acid levels in leaves. Because pathogen-induced systemic acquired resistance involves similar changes it was concluded that 7NSK2-induced resistance is mediated by the systemic acquired resistance pathway. This conclusion was validated by analysis of phenylalanine ammonia-lyase activity in roots and of salicylic acid levels in leaves of soil-grown plants treated with Pseudomonas aeruginosa. The induction of systemic acquired resistance by nanogram amounts of salicylic acid is discussed with respect to long-distance signaling in systemic acquired resistance.     

Isolation of an N-alkylated benzylamine derivative from Pseudomonas putida BTP1 as elicitor of induced systemic resistance in bean

Root treatment of Phaseolus vulgaris with the nonpathogenic Pseudomonas putida BTP1 led to significant reduction of the disease caused by the pathogen Botrytis cinerea on leaves. The molecular determinant of P. putida BTP1 mainly responsible for the induced systemic resistance (ISR) was isolated from cell-free culture fluid after growth of the strain in the iron-poor casamino acid medium. Mass spectrometry analyses performed on both the bacterial product and synthetic analogues revealed a polyalkylated benzylamine structure, with the quaternary ammonium substituted by methyl, ethyl, and C13 aliphatic groups responsible for the relative hydrophobicity of the molecule. The specific involvement of the N-alkylated benzylamine derivative (NABD) in ISR elicitation was first evidenced by testing the purified compound that mimicked the protective effect afforded by crude supernatant samples. The evidence was supported by the loss of elicitor activity of mutants impaired in NABD biosynthesis. Our experiments also showed that other iron-regulated metabolites secreted by the strain are not involved in ISR stimulation. Thus, these results indicate a wider variety of Pseudomonas determinants for ISR than reported to date.     

Production of rhamnolipids by Pseudomonas chlororaphis, a nonpathogenic bacterium

Rhamnolipids, naturally occurring biosurfactants constructed of rhamnose sugar molecules and beta-hydroxyalkanoic acids, have a wide range of potential commercial applications. In the course of a survey of 33 different bacterial isolates, we have identified, using a phenotypic assay for rhamnolipid production, a strain of the nonpathogenic bacterial species Pseudomonas chlororaphis that is capable of producing rhamnolipids. Rhamnolipid production by P. chlororaphis was achieved by growth at room temperature in static cultures of a mineral salts medium containing 2% glucose. We obtained yields of roughly 1 g/liter of rhamnolipids, an amount comparable to the production levels reported in Pseudomonas aeruginosa grown with glucose as the carbon source. The rhamnolipids produced by P. chlororaphis appear to be exclusively the mono-rhamnolipid form. The most prevalent molecular species had one monounsaturated hydroxy fatty acid of 12 carbons and one saturated hydroxy fatty acid of 10 carbons. P. chlororaphis, a nonpathogenic saprophyte of the soil, is currently employed as a biocontrol agent against certain types of plant fungal diseases. The pathogenic nature of all bacteria previously known to produce rhamnolipids has been a major obstacle to commercial production of rhamnolipids. The use of P. chlororaphis therefore greatly simplifies this matter by removing the need for containment systems and stringent separation processes in the production of rhamnolipids.     

The involvement of Pseudomonas bacteria in induced systemic resistance in plants (review)

This article reviews the most recent results of studies on the mechanism of induced systemic resistance (ISR) elicited in plants by non-pathogenic bacteria of the genus Pseudomonas. Several examples of Pseudomonas strains eliciting resistance against fungal phytopathogens in different species of crop plants are presented. Literature data dealing with bacterial elicitors and the effect of their interaction with plant receptors are quoted. Special focus is focused on the controversial issue of the correlation between the synthesis of pathogenesis-related proteins (PRs) and ISR.     

The involvement of Pseudomonas bacteria in induced systemic resistance in plants (Review)

This article reviews the most recent results of studies on the mechanism of induced systemic resistance (ISR) elicited in plants by non-pathogenic bacteria of the genus Pseudomonas. Several examples of Pseudomonas strains eliciting resistance against fungal phytopathogens in different species of crop plants are presented. Literature data dealing with bacterial elicitors and the effect of their interaction with plant receptors are quoted. Special focus is focused on the controversial issue of the correlation between the synthesis of pathogenesis-related proteins (PRs) and ISR.     

Multiple determinants influence root colonization and induction of induced systemic resistance by Pseudomonas chlororaphis O6

Colonization of the roots of tobacco by Pseudomonas chlororaphis O6 induces systemic resistance to the soft-rot pathogen, Erwinia carotovora ssp. carotovara SCC1. A screen of the transposon mutants of P. chlororaphis O6 showed mutants with about a fivefold reduction in ability to induce systemic resistance to the soft-rot disease. These mutations disrupted genes involved in diverse functions: a methyl-accepting chemotaxis protein, biosynthesis of purines, phospholipase C, transport of branched-chain amino acids and an ABC transporter. Additional mutations were detected in the intergenic spacer regions between genes encoding a GGDEF protein and fumarate dehydratase, and in genes of unknown function. The mutants in the ABC transporters did not display reduced root colonization. However, the other mutants had up to 100-fold reduced colonization levels. Generally the production of metabolites important for interactions in the rhizosphere, phenazines and siderophores, was not altered by the mutations. A reduced induction of systemic resistance by a purine biosynthesis mutant with a disrupted purM gene correlated with poor growth rate, lesser production of phenazines and siderophore and low levels of root colonization. These studies showed that multiple determinants are involved in the induction of systemic resistance, with there being a requirement for strong root colonization.     

Dependency of the Paf-acether induced bronchospasm on the lipoxygenase pathway in the guinea-pig

The Paf-acether (platelet-activating factor) induced bronchospasm (Paf-BCS) was studied in the anesthetized guinea-pig. The SRS antagonist, FPL-55712, as well as inhibitors of both lipoxygenase and cyclooxygenase, phenidone, nordihydroguaiaretic acid (NDGA), and benoxaprofen, caused a dose-related antagonism of Paf-BCS. By contrast, selective inhibitors of cyclooxygenase, indomethacin and aspirin, exerted moderate antagonism at intermediate doses, but had no effect at high doses. Furthermore, diethylmaleate (DEM), which impairs leukotriene synthesis by interfering with glutathione (GSH), suppressed Paf-BCS. Taken together, these results demonstrate that the lipoxygenase pathway plays a major part in the bronchospasmogenic effect of Paf-acether in the guinea-pig.     

High-level expression of ice nuclei in a Pseudomonas syringae strain is induced by nutrient limitation and low temperature.

Attempts were made to maximize the expression of ice nuclei in Pseudomonas syringae T1 isolated from a tomato leaf. Nutritional starvation for nitrogen, phosphorous, sulfur, or iron but not carbon at 32 degrees C, coupled to a shift to 14 to 18 degrees C, led to the rapid induction of type 1 ice nuclei (i.e., ice nuclei active at temperatures warmer than -5 degrees C). Induction was most pronounced in stationary-phase cells that were grown with sorbitol as the carbon source and cooled rapidly, and under optimal conditions, the expression of type 1 ice nuclei increased from < 1 per 10(7) cells (i.e., not detectable) to 1 in every cell in 2 to 3 h. The induction was blocked by protein and RNA synthesis inhibitors, indicative of new gene expression. Pulse-labeling of nongrowing cultures with [35S]methionine after a shift to a low temperature demonstrated that the synthesis of a new set of "low-temperature" proteins was induced. Induced ice nuclei were stable at a low temperature, with no loss in activity at 4 degrees C after 8 days, but after a shift back to 32 degrees C, type 1 ice nuclei completely disappeared, with a half-life of approximately 1 h. Repeated cycles of low-temperature induction and high-temperature turnover of these ice nuclei could be demonstrated with the same nongrowing cells. Not all P. syringae strains from tomato or other plants were fully induced under the same culture conditions as strain T1, but all showed increased expression of type 1 ice nuclei after the shift to the low temperature. In support of this view, analysis of the published DNA sequence preceding the translational start site of the inaZ gene (R. L. Green and G. Warren, Nature [London] 317:645-648, 1985) suggests the presence of a gearbox-type promoter (M. Vincente, S. R. Kushner, T. Garrido, and M. Aldea, Mol. Microbiol. 5:2085-2091, 1991).     

Lipopolysaccharide alteration is associated with induced resistance of Neisseria gonorrhoeae to killing by human serum

On SDS-PAGE, solubilized and proteinase K treated preparations of Neisseria gonorrhoeae strain BS4 (agar) showed differences in silver stained lipopolysaccharide (LPS) patterns, before and after induction to resistance to serum killing by incubation for 3 h at 37 degrees C with low Mr fractions from lysates of guinea pig red blood cells. Preparations from the original serum susceptible gonococci and LPS purified from such bacteria showed two components, but the preparations from the serum resistant gonococci were deficient in the higher Mr component. Furthermore, on immunoblotting with fresh human serum (FHS), the two LPS components of the susceptible gonococci reacted strongly with IgM. With preparations from the serum resistant gonococci there was no reaction in the area corresponding to the higher Mr component and a weaker reaction with the component of low Mr. Purified LPS from the susceptible gonococci neutralized the bactericidal activity of FHS against N. gonorrhoeae strain BS4 (agar) probably by reacting with the relevant antibody, since heated FHS was no longer bactericidal when mixed with a source of complement (human placental serum) after prior reaction with the LPS. These neutralization tests coupled with the results of immunoblotting strongly suggest that increased serum resistance is due to the lack of the high Mr LPS moiety.     

Promotion of plant growth, biological control and induced systemic resistance in maize by Pseudomonas aurantiaca JD37

Some Pseudomonas aurantiaca strains have been found to facilitate plant growth. A P. aurantiaca JD37 strain isolated from a suburb of Shanghai, China, was found to effectively colonize the rhizosphere soil and internal roots of maize (Zea mays L.) and promote maize growth. Agar diffusion assays and biocontrol effect experiments showed that strain JD37 had significant antagonistic activity against Bipolaris maydis, as well as a high biocontrol effect on southern maize leaf blight caused by B. maydis. PCR detection, associated with reverse-phase high-performance liquid chromatography assays, showed that strain JD37 might produce a number of important antibacterial substances, such as phenazine-1-carboxylic acid, pyrrolnitrin and 2,4-diacetylphloroglucinol. The crude bacterial extracts and the cell-free supernatant of strain JD37 were found to induce resistance in maize against B. maydis and reduce plant disease. Our results indicate the potential of some bacteria for producing bacterial compounds that serve as inducers of disease resistance, which is an attractive alternative to the application of chemical fertilizers, pesticides and supplement in agricultural practices.     

Repression of the pea lipoxygenase gene loxg is associated with carpel development

A cDNA clone (loxg) corresponding to a gene repressed during carpel development has been isolated from a cDNA library of unpollinated carpels induced to grow by treatment with gibberellic acid (GA₃). The sequences of loxg cDNA and the deduced polypeptide have a high similarity with legume type 2 lipoxygenases, especially with Phaseolus lox1 (78.5% similarity at the protein level) and pea and soybean lox3 (83.6% and 85.4%, respectively). loxg expression is constant in unstimulated carpels but it decreases in carpels induced to keep growing by fertilization or hormone treatment. A similar pattern of repression was observed in lipoxygenase activity of pea and tomato carpels. In situ hybridization studies showed that loxg mRNAs are present in the endocarp and the mesocarp of pea pods; no loxg expression was detectable either in the pod exocarp or in the ovules. Loxg is also expressed in other young growing tissues, especially in flower organs. Nevertheless, the natural pattern of flower and fruit development is associated with loxg repression.     

Platelet activating factor. Stimulation of the lipoxygenase pathway in polymorphonuclear leukocytes by 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine

1-O-Alkyl-2-O-acetyl-sn-glycero-3-phosphocholine (AAGPC) triggered the release of [3H]arachidonate but not [14C]stearate from cellular phospholipids in cytochalasin B-treated rabbit polymorphonuclear leukocytes. Concentrations of AAGPC up to 20 nM caused a dose-dependent release and subsequent metabolism of the released [3H]arachidonic acid. Most of the release of the [3H]arachidonate had taken place within the first 2 min of stimulation. Phosphatidylinositol and phosphatidylcholine served as the sources of [3H]arachidonate with about 50% of the label coming from each pool. Challenge of cytochalasin B-treated polymorphonuclear leukocytes with AAPGC led to the production of [3H]hydroxyeicosatetraenoic acids and [3H]dihydroxyeicosatetraenoic acids. No significant production of [3H]prostaglandins or [3H]thromboxanes was detected. AAGPC also caused a dose-dependent degranulation of cytochalasin B-treated rabbit polymorphonuclear leukocytes as shown by the release of beta-glucuronidase and lysozyme. Both the AAGPC-stimulated production of arachidonate metabolites and the degranulation response were blocked by eicosatetraynoic acid and non-dihydroguaiaretic acid at similar inhibitor concentrations. These findings suggest the bioactions of AAGPC on polymorphonuclear leukocytes may be mediated by the release of arachidonic acid and the production of mono- and dihydroxyeicosatetraenoic acids.     

Exoenzyme S of Pseudomonas aeruginosa is secreted by a type III pathway

Exoenzyme S is an extracellular ADP-ribosyltransferase of Pseudomonas aeruginosa . Transposon mutagenesis of P. aeruginosa 388 was used to identify genes required for exoenzyme S production. Five Tn 5  Tc insertion mutants were isolated which exhibited an exoenzyme S-deficient phenotype (388::Tn 5  Tc 469, 550, 3453, 4885, and 5590). Mapping experiments demonstrated that 388::Tn 5  Tc 3453, 4885, and 5590 possessed insertions within a 5.0 kb Eco RI fragment that is not contiguous with the exoenzyme S trans -regulatory operon. 388::Tn 5  Tc 469 and 550 mapped to a region downstream of the trans -regulatory operon which has been previously shown to contain a promoter region that is co-ordinately regulated with exoenzyme S synthesis. Nucleotide sequence analysis of a 7.2 kb region flanking the 388::Tn 5  Tc 469 and 550 insertions, identified 12 contiguous open reading frames (ORFs). Database searches indicated that the first ORF, ExsD, is unique. The other 11 ORFs demonstrated high homology to the YscB–L proteins of the yersiniae Yop type III export apparatus. RNase-protection analysis of wild-type and mutant strains indicated that exsD and pscB–L form an operon. To determine whether ExoS was exported by a type III mechanism, derivatives consisting of internal deletions or lacking amino- or carboxy-terminal residues were expressed in P. aeruginosa . Deletion analyses indicated that the amino-terminal nine residues are required for ExoS export. Combined data from mutagenesis, regulatory, expression, and sequence analyses provide strong evidence that P. aeruginosa possesses a type III secretion apparatus which is required for the export of exoenzyme S and potentially other co-ordinately regulated proteins.     

Gramine increase associated with rapid and transient systemic resistance in barley seedlings induced by mechanical and biological stresses.

Systemic acquired resistance (SAR) is one of the intriguing issues for studying the mechanism in signal transduction system in a whole plant. We found that SAR and increase of an antifungal compound were induced rapidly and transiently in barley (Hordeum vulgare L. cv. Goseshikoku) by mechanical and biological stresses. One of the major antifungal compounds was identified as an indole alkaloid, gramine (N,N-dimethyl-3-aminomethylindole), by mass spectrum and NMR analyses. Gramine is well known as a constitutive compound of barley, but it increased significantly in the primary and secondary leaves of barley seedlings within 12 h after pruning or inoculating with the powdery mildew fungi of barley (Blumeria graminis f.sp. hordei) and wheat (B. graminis f.sp. tritici). However, in the leaf detached from unwounded seedlings or in the leaf inoculated with the barley powdery mildew fungus, gramine did not increase at all. In the water droplets contacted with barley leaves, the amount of leaked gramine increased dependently upon the time after the seedling was injured mechanically. We also found a tight correlation between gramine increase and enhancement of resistance to the barley powdery mildew fungus in barley leaves treated with an endogenous elicitor. Furthermore, such a systemic resistance was not observed in a barley cultivar Morex that lacks the biosynthetic pathway of gramine. From these results, we conclude that gramine is the excellent marker in rapid and transient systemic acquired resistance in barley.     

Thymine metabolism in Pseudomonas aeruginosa strain 1: the presence of a salvage pathway

Exogenous thymine was found to be taken up very slowly by Pseudomonas aeruginosa in comparison to other pyrimidines, and most of it was catabolized by the cell. The existence of a functional, although inefficient, thymine salvage pathway was demonstrated and this pathway operated more effectively when de novo thymidine nucleotide biosynthesis was inhibited by trimethoprim or methotrexate. The mechanism of thymine salvage by P. aeruginosa appears to be different from that of Escherichia coli and Pseudomonas acidovorans as thymidine was not incorporated into the DNA. Like P. acidovorans, P. aeruginosa lacked thymidine phosphorylase activity. Unsuccessful attempts were made to isolate thymine auxotrophs.