Expression of an entire bacterial operon in plants.

Multigene expression is required for metabolic engineering, i.e. coregulated expression of all genes in a metabolic pathway for the production of a desired secondary metabolite. To that end, several transgenic approaches have been attempted with limited success. Better success has been achieved by transforming plastids with operons. IL-60 is a platform of constructs driven from the geminivirus Tomato yellow leaf curl virus. We demonstrate that IL-60 enables nontransgenic expression of an entire bacterial operon in tomato (Solanum lycopersicum) plants without the need for plastid (or any other) transformation. Delivery to the plant is simple, and the rate of expressing plants is close to 100%, eliminating the need for selectable markers. Using this platform, we show the expression of an entire metabolic pathway in plants and delivery of the end product secondary metabolite (pyrrolnitrin). Expression of this unique secondary metabolite resulted in the appearance of a unique plant phenotype disease resistance. Pyrrolnitrin production was already evident 2 d after application of the operon to plants and persisted throughout the plant's life span. Expression of entire metabolic pathways in plants is potentially beneficial for plant improvement, disease resistance, and biotechnological advances, such as commercial production of desired metabolites.

Differential effectiveness of Serratia plymuthica IC1270-induced systemic resistance against hemibiotrophic and necrotrophic leaf pathogens in rice.

BACKGROUND: Induced resistance is a state of enhanced defensive capacity developed by a plant reacting to specific biotic or chemical stimuli. Over the years, several forms of induced resistance have been characterized, including systemic acquired resistance, which is induced upon localized infection by an avirulent necrotizing pathogen, and induced systemic resistance (ISR), which is elicited by selected strains of nonpathogenic rhizobacteria. However, contrary to the relative wealth of information on inducible defense responses in dicotyledoneous plants, our understanding of the molecular mechanisms underlying induced resistance phenomena in cereal crops is still in its infancy. Using a combined cytomolecular and pharmacological approach, we analyzed the host defense mechanisms associated with the establishment of ISR in rice by the rhizobacterium Serratia plymuthica IC1270. RESULTS: In a standardized soil-based assay, root treatment with IC1270 rendered foliar tissues more resistant to the hemibiotrophic pathogen Magnaporthe oryzae, causal agent of the devastating rice blast disease. Analysis of the cytological and biochemical alterations associated with restriction of fungal growth in IC1270-induced plants revealed that IC1270 primes rice for enhanced attacker-induced accumulation of reactive oxygen species (ROS) and autofluorescent phenolic compounds in and near epidermal cells displaying dense cytoplasmic granulation. Similar, yet more abundant, phenotypes of hypersensitively dying cells in the vicinity of fungal hyphae were evident in a gene-for-gene interaction with an avirulent M. oryzae strain, suggesting that IC1270-inducible ISR and R protein conditioned effector-triggered immunity (ETI) target similar defense mechanisms. Yet, this IC1270-inducible ISR response seems to act as a double-edged sword within the rice defense network as induced plants displayed an increased vulnerability to the necrotrophic pathogens Rhizoctonia solani and Cochliobolus miyabeanus. Artificial enhancement of ROS levels in inoculated leaves faithfully mimicked the opposite effects of IC1270 bacteria on aforementioned pathogens, suggesting a central role for oxidative events in the IC1270-induced resistance mechanism. CONCLUSION: Besides identifying ROS as modulators of antagonistic defense mechanisms in rice, this work reveals the mechanistic similarities between S. plymuthica-mediated ISR and R protein-dictated ETI and underscores the importance of using appropriate innate defense mechanisms when breeding for broad-spectrum rice disease resistance.

SprI/SprR Quorum Sensing System of Serratia proteamaculans 94.

In this study, we investigated the quorum sensing (QS) regulatory system of the psychrotrophic strain Serratia proteamaculans 94 isolated from spoiled refrigerated meat. The strain produced several N-acyl-L-homoserine-lactone (AHL) QS signal molecules, with N-(3-oxo-hexanoyl)-L-homoserine lactone and N-(3-hydroxy-hexanoyl)-L-homoserine lactone as two main types. The sprI and sprR genes encoding an AHL synthase and a receptor regulatory protein, respectively, were cloned and sequenced. Analysis of their nucleotide sequence showed that these genes were transcribed convergently and that their reading frames partly overlapped by 23 bp in the terminal regions. The genes were highly similar to the luxI/luxR-type QS genes of other Gram-negative bacteria. An spr-box (analog of the lux-box) was identified upstream of the sprR gene and found to be overlapped with the sequence of -10 sequence site in the promoter region of this gene. Inactivation of the sprI gene led to the absence of AHL synthesis, chitinolytic activity, and swimming motility; decrease of extracellular proteolytic activity; affected the cellular fatty acid composition; and reduced suppression of the fungal plant pathogen mycelium growth by volatile compounds emitted by strain S. proteamaculans 94. The data obtained demonstrated the important role of the QS system in the regulation of cellular processes in S. proteamaculans 94.

Quorum-sensing signaling is required for production of the antibiotic pyrrolnitrin in a rhizospheric biocontrol strain of Serratia plymuthica.

One mechanism that bacteria have adopted to regulate the production of antimicrobial compounds is population-density-dependent LuxRI-type quorum sensing (QS), exploiting the production of N-acyl homoserine lactone (AHL) autoinducer signals. In biocontrol bacteria, most known cases involve the AHL control of phenazine antibiotics production by rhizospheric pseudomonads. This work is the first to demonstrate that phenazines are not the only group of biocontrol-related antibiotics whose production is regulated by QS systems. Strain HRO-C48 of Serratia plymuthica isolated from the rhizosphere of oilseed rape and described as a chitinolytic bacterium, which protects crops against Verticillium wilt, was also shown to produce wide-range antibiotic pyrrolnitrin and several AHLs, including N-butanoyl-HSL, N-hexanoyl-HSL and N-3-oxo-hexanoyl-HSL (OHHL). The genes splI and splR, which are analogues of luxI and luxR genes from other Gram-negative bacteria, were cloned and sequenced. The mutant AHL-4 (splI::miniTn5) was simultaneously deficient in the production of AHLs and pyrrolnitrin, as well as in its ability to suppress the growth of several fungal plant pathogens in vitro. However, pyrrolnitrin production could be restored in this mutant by introduction of the splIR genes cloned into a plasmid or by addition of the conditioned medium from strain C48 or OHHL standard to the growth medium.

Influence of volatile organic compounds emitted by Pseudomonas and Serratia strains on Agrobacterium tumefaciens biofilms.

The ability to form biofilms plays an important role in bacteria-host interactions, including plant pathogenicity. In this work, we investigated the action of volatile organic compounds (VOCs) produced by rhizospheric strains of Pseudomonas chlororaphis 449, Pseudomonas fluorescens B-4117, Serratia plymuthica IC1270, as well as Serratia proteamaculans strain 94, isolated from spoiled meat, on biofilms formation by three strains of Agrobacterium tumefaciens which are causative agents of crown-gall disease in a wide range of plants. In dual culture assays, the pool of volatiles emitted by the tested Pseudomonas and Serratia strains suppressed the formation of biofilms of A. tumefaciens strains grown on polycarbonate membrane filters and killed Agrobacterium cells in mature biofilms. The individual VOCs produced by the tested Pseudomonas strains, that is, ketones (2-nonanone, 2-heptanone, 2-undecanone), and dimethyl disulfide (DMDS) produced by Serratia strains, were shown to kill A. tumefaciens cells in mature biofilms and suppress their formation. The data obtained in this study suggest an additional potential of some ketones and DMDS as protectors of plants against A. tumefaciens strains, whose virulence is associated with the formation of biofilms on the infected plants.

Silencing the mob: disrupting quorum sensing as a means to fight plant disease.

Bacteria are able to sense their population's density through a cell-cell communication system, termed 'quorum sensing' (QS). This system regulates gene expression in response to cell density through the constant production and detection of signalling molecules. These molecules commonly act as auto-inducers through the up-regulation of their own synthesis. Many pathogenic bacteria, including those of plants, rely on this communication system for infection of their hosts. The finding that the countering of QS-disrupting mechanisms exists in many prokaryotic and eukaryotic organisms offers a promising novel method to fight disease. During the last decade, several approaches have been proposed to disrupt QS pathways of phytopathogens, and hence to reduce their virulence. Such studies have had varied success in vivo, but most lend promising support to the idea that QS manipulation could be a potentially effective method to reduce bacterial-mediated plant disease. This review discusses the various QS-disrupting mechanisms found in both bacteria and plants, as well as the different approaches applied artificially to interfere with QS pathways and thus protect plant health.

Regulation of GacA in Pseudomonas chlororaphis Strains Shows a Niche Specificity.

The GacS/GacA two-component system plays a central role in the regulation of a broad range of biological functions in many bacteria. In the biocontrol organism Pseudomonas chlororaphis, the Gac system has been shown to positively control quorum sensing, biofilm formation, and phenazine production, but has an overall negative impact on motility. These studies have been performed with strains originated from the rhizosphere predominantly. To investigate the level of conservation between the GacA regulation of biocontrol-related traits in P. chlororaphis isolates from different habitats, the studies presented here focused on the endophytic isolate G5 of P. chlororaphis subsp. aurantiaca. A gacA mutant deficient in the production of N-acylhomoserine lactones (AHLs) and phenazine was isolated through transposon mutagenesis. Further phenotypic characterization revealed that in strain G5, similar to other P. chlororaphis strains, a gacA mutation caused inability to produce biocontrol factors such as phenazine, HCN and proteases responsible for antifungal activity, but overproduced siderophores. LC-MS/MS analysis revealed that AHL production was also practically abolished in this mutant. However, the wild type exhibited an extremely diverse AHL pattern which has never been identified in P. chlororaphis. In contrast to other isolates of this organism, GacA in strain G5 was shown to negatively regulate biofilm formation and oxidative stress response whilst positively regulating cell motility and biosynthesis of indole-3-acetic acid (IAA). To gain a better understanding of the overall impact of GacA in G5, a comparative proteomic analysis was performed revealing that, in addition to some of the traits like phenazine mentioned above, GacA also negatively regulated lipopolysaccharide (LPS) and trehalose biosynthesis whilst having a positive impact on energy metabolism, an effect not previously described in P. chlororaphis. Consequently, GacA regulation shows a differential strain dependency which is likely to be in line with their niche of origin.

Inhibitory and toxic effects of volatiles emitted by strains of Pseudomonas and Serratia on growth and survival of selected microorganisms, Caenorhabditis elegans, and Drosophila melanogaster.

In previous research, volatile organic compounds (VOCs) emitted by various bacteria into the chemosphere were suggested to play a significant role in the antagonistic interactions between microorganisms occupying the same ecological niche and between bacteria and target eukaryotes. Moreover, a number of volatiles released by bacteria were reported to suppress quorum-sensing cell-to-cell communication in bacteria, and to stimulate plant growth. Here, volatiles produced by Pseudomonas and Serratia strains isolated mainly from the soil or rhizosphere exhibited bacteriostatic action on phytopathogenic Agrobacterium tumefaciens and fungi and demonstrated a killing effect on cyanobacteria, flies (Drosophila melanogaster), and nematodes (Caenorhabditis elegans). VOCs emitted by the rhizospheric Pseudomonas chlororaphis strain 449 and by Serratia proteamaculans strain 94 isolated from spoiled meat were identified using gas chromatography-mass spectrometry analysis, and the effects of the main headspace compounds--ketones (2-nonanone, 2-heptanone, 2-undecanone) and dimethyl disulfide--were inhibitory toward the tested microorganisms, nematodes, and flies. The data confirmed the role of bacterial volatiles as important compounds involved in interactions between organisms under natural ecological conditions.

Quorum-sensing quenching by rhizobacterial volatiles.

We show that volatile organic compounds (VOCs) produced by rhizospheric strains Pseudomonas fluorescens B-4117 and Serratia plymuthica IC1270 may act as inhibitors of the cell-cell communication quorum-sensing (QS) network mediated by N-acyl homoserine lactone (AHL) signal molecules produced by various bacteria, including strains of Agrobacterium, Chromobacterium, Pectobacterium and Pseudomonas. This quorum-quenching effect was observed when AHL-producing bacteria were treated with VOCs emitted by strains B-4117 and IC1270 or with dimethyl disulfide (DMDS), the major volatile produced by strain IC1270. LC-MS/MS analysis revealed that treatment of strains Pseudomonas chlororaphis 449, Pseudomonas aeruginosa PAO1 or Ps. fluorescens 2-79 with VOCs emitted by strain IC1270 or DMDS drastically decreases the amount of AHLs produced by these bacteria. Volatile organic compounds produced by Ps. chlororaphis 449 were able to suppress its own QS-induction activity, suggesting a negative interaction between VOCs and AHL molecules in the same strain. Quantitative RT-PCR analysis showed that treatment of Ps. chlororaphis 449 with VOCs emitted by cells of IC1270, B-4117 or 449 itself, or with DMDS, leads to significant suppression of transcription of AHL synthase genes phzI and csaI. Thus, along with AHLs, bacterial volatiles might be considered another type of signal molecule involved in microbial communication in the rhizosphere.

Characterization of ACC deaminase from the biocontrol and plant growth-promoting agent Trichoderma asperellum T203.

1-aminocyclopropane-1-carboxylate (ACC) deaminase activity was evaluated in the biocontrol and plant growth-promoting fungus Trichoderma asperellum T203. Fungal cultures grown with ACC as the sole nitrogen source showed high enzymatic activity. The enzyme encoding gene (Tas-acdS) was isolated, and an average 3.5-fold induction of the gene by 3 mM ACC was detected by real-time PCR. Escherichia coli bacteria carrying the intron-free cDNA of Tas-acdS cloned into the vector pAlter-EX1 under the control of the tac promoter revealed specific ACC deaminase (ACCD) activity and the ability to promote canola (Brassica napus) root elongation in pouch assays. RNAi silencing of the ACCD gene in T. asperellum showed decreased ability of the mutants to promote root elongation of canola seedlings. These data suggest a role for ACCD in the plant root growth-promotion effect by T. asperellum.

Quorum-sensing effects in the antagonistic rhizosphere bacterium Serratia plymuthica HRO-C48.

The rhizosphere-associated bacterium Serratia plymuthica HRO-C48 is not only able to suppress symptoms caused by soil-borne pathogens but is also able to stimulate growth of plants. Detailed knowledge about the underlying mechanisms and regulation are crucial for the application in biocontrol strategies. To analyse the influence of N-acyl homoserine lactone (AHL)-mediated communication on the biocontrol activity, the AHL-degrading lactonase AiiA was heterologously expressed in the strain, resulting in abolished AHL production. The comparative analysis of the wild type and AHL negative mutants led to the identification of new AHL-regulated phenotypes. In the pathosystem Verticillium dahliae-oilseed rape, the essential role of AHL-mediated signaling for disease suppression was demonstrated. In vitro, the regulatory function of AHLs in the synthesis of the plant growth hormone indole-3-acetic acid is shown for the first time. Additionally, swimming motility was found to be negatively AHL regulated. In contrast, production of extracellular hydrolytic enzymes is shown to be positively AHL-regulated. HRO-C48 emits a broad spectrum of volatile organic compounds that are involved in antifungal activity and, interestingly, whose relative abundances are influenced by quorum sensing (QS). This study shows that QS is crucial for biocontrol activity of S. plymuthica and discusses the impact for the application of the strain as a biocontrol agent.

The global regulator genes from biocontrol strain Serratia plymuthica IC1270: cloning, sequencing, and functional studies.

The biocontrol activity of various fluorescent pseudomonads towards plant-pathogenic fungi is dependent upon the GacA/GacS-type two-component system of global regulators and the RpoS transcription sigma factor. In particular, these components are required for the production of antifungal antibiotics and exoenzymes. To investigate the effects of these global regulators on the expression of biocontrol factors by plant-associated bacteria other than Pseudomonas spp., gacA/gacS and rpoS homologues were cloned from biocontrol strain IC1270 of Serratia plymuthica, which produces a set of antifungal compounds, including chitinolytic enzymes and the antibiotic pyrrolnitrin. The nucleotide and deduced protein sequence alignments of the cloned gacA/gacS-like genes-tentatively designated grrA (global response regulation activator) and grrS (global response regulation sensor) and of the cloned rpoS gene revealed 64 to 93% identity with matching genes and proteins of the enteric bacteria Escherichia coli, Pectobacterium carotovora subsp. carotovora, and Serratia marcescens. grrA, grrS, and rpoS gene replacement mutants of strain IC1270 were deficient in the production of pyrrolnitrin, an exoprotease, and N-acylhomoserine lactone quorum-sensing signal molecules. However, neither mutant appeared to differ from the parental strain in the production of siderophores, and only grrA and grrS mutants were deficient in the production of a 58-kDa endochitinase, representing the involvement of other sigma factors in the regulation of strain IC1270's chitinolytic activity. Compared to the parental strain, the grrA, grrS, and rpoS mutants were markedly less capable of suppressing Rhizoctonia solani and Pythium aphanidermatum under greenhouse conditions, indicating the dependence of strain IC1270's biocontrol property on the GrrA/GrrS and RpoS global regulators.