Exogenous Application of Polycationic Nanobactericide on Tomato Plants Reduces the Candidatus Liberibacter Solanacearum Infection.

Candidatus Liberibacter solanacearum (CaLso) is associated with diseases in tomato crops and transmitted by the tomato psyllid Bactericera cockerelli. A polymeric water-dispersible nanobactericide (PNB) was evaluated against CaLso as a different alternative. PNB is a well-defined polycationic diblock copolymer designed to permeate into the vascular system of plants. Its assessment under greenhouse conditions was carried out with tomato plants previously infected with CaLso. Using a concentration as low as 1.0 mg L-1, a small but significant reduction in the bacterial load was observed by real-time qPCR. Thus, to achieve an ecologically friendly dosage and set an optimum treatment protocol, we performed experiments to determine the effective concentration of PNB to reduce ~65% of the initial bacterial load. In a first bioassay, a 40- or 70-fold increase was used to reach that objective. At this concentration level, other bioassays were explored to determine the effect as a function of time. Surprisingly, a real reduction in the symptoms was observed after three weeks, and there was a significant decrease in the bacterial load level (~98%) compared to the untreated control plants. During this period, flowering and formation of tomato fruits were observed in plants treated with PNB.

A 14-20 kDa protein binds to the upstream region of the phtM operon involved in the synthesis of phaseolotoxin in Pseudomonas syringae pv. phaseolicola NPS3121 / Una proteína de 14-20 kDa se une a la región río arriba del operón phtM involucrado en la síntesis de faseolotoxina en Pseudomonas syringae pv. phaseolicola NPS3121

Pseudomonas syringae pv. phaseolicola is a phytopathogenic bacterium in beans that produces a phytotoxin called phaseolotoxin, in whose synthesis a group of genes that belong to the "Pht cluster" are involved. This cluster comprises 23 genes arranged in 5 transcriptional units, two monocistronic (argK, phtL) and three polycistronic (phtA, phtD, phtM) operons, whose expression is increased at 18°C, correlating with the production of phaseolotoxin by the bacterium. So far, the regulatory mechanisms involved in phaseolotoxin synthesis are poorly understood and only the requirement of low temperatures for its synthesis has been demon strated. Therefore, in this study we searched for regulatory proteins that could be involved in the phaseolotoxin synthesis, focusing on the regulation of the phtM operon. Gel shift assays showed that the promoter region of the phtM operon contains binding sites for putative regulatory proteins, which are encoded outside the Pht cluster and are independent of the GacS-GacA two-component system. Deletion assays with the promoter region of the phtM operon show that the binding site for a putative transcription factor is located within a 58 bp region. The putative transcription factor of the phtM operon has an apparent molecular mass in the 14-20 kDa range. Furthermore, the results demonstrate that the transcription factor recognizes and binds the upstream phtM region as monomer o multimer of a single polypeptide. Our findings provide new insights into the regulatory mechanisms involved in phaseolotoxin production, and suggest that the Pht cluster was integrated into the global regulatory mechanism of P. syringae pv. phaseolicola.

A 14-20kDa protein binds to the upstream region of the phtM operon involved in the synthesis of phaseolotoxin in Pseudomonas syringae pv. phaseolicola NPS3121.

Pseudomonas syringae pv. phaseolicola is a phytopathogenic bacterium in beans that produces a phytotoxin called phaseolotoxin, in whose synthesis a group of genes that belong to the "Pht cluster" are involved. This cluster comprises 23 genes arranged in 5 transcriptional units, two monocistronic (argK, phtL) and three polycistronic (phtA, phtD, phtM) operons, whose expression is increased at 18°C, correlating with the production of phaseolotoxin by the bacterium. So far, the regulatory mechanisms involved in phaseolotoxin synthesis are poorly understood and only the requirement of low temperatures for its synthesis has been demonstrated. Therefore, in this study we searched for regulatory proteins that could be involved in the phaseolotoxin synthesis, focusing on the regulation of the phtM operon. Gel shift assays showed that the promoter region of the phtM operon contains binding sites for putative regulatory proteins, which are encoded outside the Pht cluster and are independent of the GacS-GacA two-component system. Deletion assays with the promoter region of the phtM operon show that the binding site for a putative transcription factor is located within a 58bp region. The putative transcription factor of the phtM operon has an apparent molecular mass in the 14-20kDa range. Furthermore, the results demonstrate that the transcription factor recognizes and binds the upstream phtM region as monomer o multimer of a single polypeptide. Our findings provide new insights into the regulatory mechanisms involved in phaseolotoxin production, and suggest that the Pht cluster was integrated into the global regulatory mechanism of P. syringae pv. phaseolicola.

Temperature-mediated biosynthesis of the phytotoxin phaseolotoxin by Pseudomonas syringae pv. phaseolicola depends on the autoregulated expression of the phtABC genes.

Pseudomonas syringae pv. phaseolicola produces phaseolotoxin in a temperature dependent manner, being optimally synthesized between 18°C and 20°C, while no detectable amounts are present above 28°C. The Pht cluster, involved in the biosynthesis of phaseolotoxin, contains 23 genes that are organized in five transcriptional units. The function of most of the genes from the Pht cluster is still unknown and little information about the regulatory circuitry leading to expression of these genes has been reported. The purpose of the present study was to investigate the participation of pht genes in the regulation of the operons coded into the Pht cluster. We conducted Northern blot, uidA fusions and reverse transcription-PCR assays of pht genes in several mutants unable to produce phaseolotoxin. This allowed us to determine that, in P. syringae pv. phaseolicola NPS3121, genes phtABC are essential to prevent their own expression at 28°C, a temperature at which no detectable amounts of the toxin are present. We obtained evidence that the phtABC genes also participate in the regulation of the phtD, phtM and phtL operons. According to our results, we propose that PhtABC and other Pht product activities could be involved in the synthesis of the sulfodiaminophosphinyl moiety of phaseolotoxin, which indirectly could be involved in the transcriptional regulation of the phtA operon.

The PhtL protein of Pseudomonas syringae pv. phaseolicola NPS3121 affects the expression of both phaseolotoxin cluster (Pht) and Non-Pht encoded genes.

Pseudomonas syringae pv. phaseolicola, the causal agent of halo blight disease in bean, produces a toxin known as phaseolotoxin, whose synthesis involves the products of some of the genes found within the Pht region. This region, considered a pathogenicity island, comprises 23 genes arranged in five transcriptional units: two single-gene units (argK, phtL) and three arranged as operons (phtA, phtD, phtM), most with unknown function. In P. syringae pv. phaseolicola, maximal expression of most of the genes encoded in the Pht region and the synthesis of phaseolotoxin require the product of the phtL gene, of unknown function but that has been proposed to have a regulatory role. In order to evaluate the role of phtL gene in P. syringae pv. phaseolicola, we performed a comparative transcriptional analysis with the wild type and a phtL(-) mutant strains using microarrays. The microarray data analysis showed that PhtL regulates the expression not only of genes within the Pht region, but also alters the expression of genomic genes outside it, indicating that this gene has been integrated into the regulatory machinery of the bacterium. The expression changes of many of those genes were confirmed by RT-PCR. This study also demonstrated the importance of the PhtL protein in the process of iron response, and suggests that the effect of PhtL on the expression of pathogenicity related, respiration and oxidative stress genes, observed in this study, appears to be indirect through its influence on the Fur protein expression.

Transcriptional profile of P. syringae pv. phaseolicola NPS3121 at low temperature: physiology of phytopathogenic bacteria.

BACKGROUND: Low temperatures play key roles in the development of most plant diseases, mainly because of their influence on the expression of various virulence factors in phytopathogenic bacteria. Thus far, studies regarding this environmental parameter have focused on specific themes and little is known about phytopathogenic bacteria physiology under these conditions. To obtain a global view regarding phytopathogenic bacteria strategies in response to physiologically relevant temperature changes, we used DNA microarray technology to compare the gene expression profile of the model bacterial pathogen P. syringae pv. phaseolicola NPS3121 grown at 18°C and 28°C. RESULTS: A total of 236 differentially regulated genes were identified, of which 133 were up-regulated and 103 were down-regulated at 18°C compared to 28°C. The majority of these genes are involved in pathogenicity and virulence processes. In general, the results of this study suggest that the expression profile obtained may be related to the fact that low temperatures induce oxidative stress in bacterial cells, which in turn influences the expression of iron metabolism genes. The expression also appears to be correlated with the profile expression obtained in genes related to motility, biofilm production, and the type III secretion system. CONCLUSIONS: From the data obtained in this study, we can begin to understand the strategies used by this phytopathogen during low temperature growth, which can occur in host interactions and disease development.

Expression of the gene for resistance to phaseolotoxin (argK) depends on the activity of genes phtABC in Pseudomonas syringae pv. phaseolicola.

The bacterium Pseudomonas syringae pv. phaseolicola produces phaseolotoxin in a temperature dependent manner, being optimally produced between 18°C and 20°C, while no detectable amounts are present above 28°C. Phaseolotoxin is an effective inhibitor of ornithine carbamoyltransferase (OCTase) activity from plant, mammalian and bacterial sources and causes a phenotypic requirement for arginine. To protect the cell from its own toxin, P. syringae pv. phaseolicola synthesizes a phaseolotoxin-resistant OCTase (ROCT). The ROCT is the product of the argK gene and is synthesized only under conditions leading to phaseolotoxin synthesis. The argK gene is included in a chromosomal fragment named Pht cluster, which contains genes involved in the synthesis of phaseolotoxin. The aim of the present work was to investigate the possible involvement of other genes included in the Pht cluster in the regulation of gene argK. We conducted transcriptional analyses of argK in several mutants unable to produce phaseolotoxin, transcriptional fusions and electrophoretic mobility shift assays, which allowed us to determine that genes phtABC, located within the Pht cluster, participate in the transcriptional repression of gene argK at temperatures not permissive for phaseolotoxin biosynthesis. This repression is mediated by a protein present in both toxigenic and nontoxigenic strains of P. syringae and in E. coli, and requires the coordinated participation of phtA, phtB and phtC products in order to carry out an efficient argK repression.

Integration Host Factor (IHF) binds to the promoter region of the phtD operon involved in phaseolotoxin synthesis in P. syringae pv. phaseolicola NPS3121.

BACKGROUND: Pseudomonas syringae pv. phaseolicola, the causal agent of halo blight disease in beans, produces a toxin known as phaseolotoxin, in whose synthesis participate a group of genes organized within the genome in a region known as the "Pht cluster". This region, which is thought to have been acquired by horizontal gene transfer, includes 5 transcriptional units, two monocistronic (argK, phtL) and three polycistronic (phtA, phtD, phtM), whose expression is temperature dependent. So far, the regulatory mechanisms involved in phaseolotoxin synthesis have not been elucidated and the only well-established fact is the requirement of low temperatures for its synthesis. In this work, we searched for regulatory proteins that could be involved in phaseolotoxin synthesis, focusing on the regulation of the phtD operon. RESULTS: In this study we identified the global regulator IHF (Integration Host Factor), which binds to the promoter region of the phtD operon, exerting a negative effect on the expression of this operon. This is the first regulatory protein identified as part of the phaseolotoxin synthesis system. Our findings suggest that the Pht cluster was similarly regulated in the ancestral cluster by IHF or similar protein, and integrated into the global regulatory mechanism of P. syringae pv. phaseolicola, after the horizontal gene transfer event by using the host IHF protein. CONCLUSION: This study identifies the IHF protein as one element involved in the regulation of phaseolotoxin synthesis in P. syringae pv. phaseolicola NPS3121 and provides new insights into the regulatory mechanisms involved in phaseolotoxin production.

Gene expression of Pht cluster genes and a putative non-ribosomal peptide synthetase required for phaseolotoxin production is regulated by GacS/GacA in Pseudomonas syringae pv. phaseolicola.

Pseudomonas syringae pv. phaseolicola is the causal agent of halo blight disease of beans (Phaseolus vulgaris L.), which is characterized by watersoaked lesions surrounded by a chlorotic halo resulting from the action of a non-host specific toxin known as phaseolotoxin. This toxin inhibits the enzyme ornithine carbamoyltransferase involved in the arginine biosynthesis pathway. It was previously reported that genes within the Pht cluster were involved in the regulation and synthesis of phaseolotoxin. The GacS/GacA two-component signal transduction system controls important pathogenicity and virulence mechanisms in several Gram-negative bacteria. Tox(-) phenotype gacA(-) and gacS(-) mutants were obtained and gacA(-) transcriptome analysis revealed that this response activator controls expression of genes within the Pht cluster as well as another gene located in a different region in the bacterial chromosome and that has been unambiguously shown to be directly involved in phaseolotoxin biosynthesis. Results presented in this work suggest that phaseolotoxin biosynthesis involve elements within and outside the Pht Cluster, and that the GacS/GacA two-component system exerts control over them.

Variation in conservation of the cluster for biosynthesis of the phytotoxin phaseolotoxin in Pseudomonas syringae suggests at least two events of horizontal acquisition.

Certain strains of Pseudomonas syringae pathovars phaseolicola and actinidiae and P. syringae pv. syringae strain CFBP3388 produce the chlorosis-inducing phytotoxin phaseolotoxin, which inhibits biosynthesis of arginine and polyamines. The 25 kb Pht cluster, responsible for phaseolotoxin biosynthesis, is included in a putative pathogenicity island that is nearly identical in selected strains of the pathovars phaseolicola and actinidiae, suggesting that it has been recently acquired by horizontal transfer. The history of pathogenicity islands is pivotal for our understanding of the evolution of virulence in plant pathogenic bacteria; nevertheless, our knowledge of the origins, biology and genetics of this island is currently rather limited. The aim of this work was to explore the conservation of phaseolotoxin biosynthesis genes in a broader collection of isolates and in strain CFBP3388, in order to better understand its evolution and gene dynamics. PCR, hybridization and sequence analysis showed that the island is highly conserved among a diversity of strains of pathovars phaseolicola and actinidiae, suggesting that it was acquired only once by each pathovar. Strain CFBP3388 contained DNA homologous to the Pht cluster, and an insertional mutant in the regulatory gene phtL did not synthesize the toxin. A 6.5 kb fragment from strain CFBP3388 was syntenic to the Pht cluster, but showed nucleotide identity of only 85.3%. This contrasts with an identity higher than 99.8% among clusters of pathovars phaseolicola and actinidiae, in spite of the fact that pv. syringae is phylogenetically closer to pv. phaseolicola. In addition, strain CFBP3388 lacked the four integrases that are putatively responsible for the mobility of the pathogenicity island. These results indicate that genes for the biosynthesis of phaseolotoxin have a complex evolutionary history and were acquired by pathovars of P. syringae at least twice during evolution.

Transcriptional profile of Pseudomonas syringae pv. phaseolicola NPS3121 in response to tissue extracts from a susceptible Phaseolus vulgaris L. cultivar.

BACKGROUND: Pseudomonas syringae pv. phaseolicola is a Gram-negative plant-pathogenic bacterium that causes "halo blight" disease of beans (Phaseolus vulgaris L.). This disease affects both foliage and pods, and is a major problem in temperate areas of the world. Although several bacterial genes have been determined as participants in pathogenesis, the overall process still remains poorly understood, mainly because the identity and function of many of the genes are largely unknown. In this work, a genomic library of P. syringae pv. phaseolicola NPS3121 was constructed and PCR amplification of individual fragments was carried out in order to print a DNA microarray. This microarray was used to identify genes that are differentially expressed when bean leaf extracts, pod extracts or apoplastic fluid were added to the growth medium. RESULTS: Transcription profiles show that 224 genes were differentially expressed, the majority under the effect of bean leaf extract and apoplastic fluid. Some of the induced genes were previously known to be involved in the first stages of the bacterial-plant interaction and virulence. These include genes encoding type III secretion system proteins and genes involved in cell-wall degradation, phaseolotoxin synthesis and aerobic metabolism. On the other hand, most repressed genes were found to be involved in the uptake and metabolism of iron. CONCLUSION: This study furthers the understanding of the mechanisms involved, responses and the metabolic adaptation that occurs during the interaction of P. syringae pv. phaseolicola with a susceptible host plant.

Functional characterization of the gene cluster from Pseudomonas syringae pv. phaseolicola NPS3121 involved in synthesis of phaseolotoxin.

Pseudomonas syringae pv. phaseolicola is the causal agent of halo blight disease of beans (Phaseolus vulgaris L.), which is characterized by water-soaked lesions surrounded by a chlorotic halo resulting from the action of a non-host-specific toxin known as phaseolotoxin. This phytotoxin inhibits the enzyme ornithine carbamoyltransferase involved in arginine biosynthesis. Different evidence suggested that genes involved in phaseolotoxin production were clustered. Two genes had been previously identified in our laboratory within this cluster: argK, which is involved in the immunity of the bacterium to its own toxin, and amtA, which is involved in the synthesis of homoarginine. We sequenced the region around argK and amtA in P. syringae pv. phaseolicola NPS3121 to determine the limits of the putative phaseolotoxin gene cluster and to determine the transcriptional pattern of the genes comprising it. We report that the phaseolotoxin cluster (Pht cluster) is composed of 23 genes and is flanked by insertion sequences and transposases. The mutation of 14 of the genes within the cluster lead to a Tox(-) phenotype for 11 of them, while three mutants exhibited low levels of toxin production. The analysis of fusions of selected DNA fragments to uidA, Northern probing, and reverse transcription-PCR indicate the presence of five transcriptional units, two monocistronic and three polycistronic; one is internal to a larger operon. The site for transcription initiation has been determined for each promoter, and the putative promoter regions were identified. Preliminary results also indicate that the gene product of phtL is involved in the regulation of the synthesis of phaseolotoxin.

The global arginine regulator ArgR controls expression of argF in Pseudomonas syringae pv. phaseolicola but is not required for the synthesis of phaseolotoxin or for the regulated expression of argK.

In Pseudomonas syringae pv. phaseolicola the enzyme ornithine carbamoyltransferase (OCTase), encoded by argF, is negatively regulated by argR, similar to what has been reported for Pseudomonas aeruginosa. However, production of the phaseolotoxin-resistant OCTase encoded by argK, synthesis of phaseolotoxin, and infectivity for bean pods occur independently of the ArgR protein.

In Pseudomonas syringae pv. phaseolicola, expression of the argK gene, encoding the phaseolotoxin-resistant ornithine carbamoyltransferase, is regulated indirectly by temperature and directly by a precursor resembling carbamoylphosphate.

Pseudomonas syringae pv. phaseolicola synthesizes a non-host-specific toxin, phaseolotoxin, and also synthesizes a phaseolotoxin-resistant ornithine carbamoyltransferase (ROCT) to protect itself from its own toxin. ROCT is encoded by argK, which is expressed coordinately with phaseolotoxin synthesis at 18 degrees C. To investigate the regulatory mechanisms of this system, null mutants were constructed for argK, argF (encoding the phaseolotoxin-sensitive OCTase [SOCT]), and amtA (encoding an amidinotransferase involved in phaseolotoxin synthesis). The argF mutant did not exhibit arginine auxotrophy when grown in M9 medium at 28 degrees C, because under this condition SOCT was replaced by ROCT. This loss of thermoregulation of argK was apparently caused by accumulation of carbamoylphosphate, one of the substrates of SOCT. Carbamoylphosphate, which has a structure similar to that of the inorganic moiety of phaseolotoxin, was used in induction assays with wild-type P. syringae pv. phaseolicola and was shown to be able to induce argK expression in M9 medium at 28 degrees C. These results indicate that argK expression is independent of temperature and is regulated directly by a compound resembling the inorganic moiety of phaseolotoxin.

Physical map of the chromosome of the phytopathogenic bacterium Pseudomonas syringae pv. phaseolicola.

Pseudomonas syringae pv. phaseolicola (P.s. phaseolicola) is one of about 45 recognized pathovars within the P. syringae group and is the causal agent of halo-blight disease of beans. DNA from this bacterium digested to completion with two different restriction enzymes, PacI and PmeI, yielded 15 and 16 fragments, respectively. These were separated using PFGE and sized by comparison to known molecular mass markers. The P.s. phaseolicola chromosome was determined to be approximately 5.64 Mb in size. To link the different fragments obtained into a circular chromosome map for both enzymes, 150 random Tn5 mutants of P.s. phaseolicola were used as a source of DNA and the identification of the band carrying the transposon 'tag' in each mutant was done after PFGE and Southern hybridization of a complete chromosomal digestion using a Tn5 probe. Partial digestions of DNA from different Tn5 mutants 'tagging' specific bands were then generated and the complete and partial products of the digestion separated by PFGE and identified with a Tn5 probe. By calculating the size of the partial products, it was then possible to link different bands into a physical map. This is the first report on the construction of a physical map of a member of the P. syringae group and should be invaluable for molecular genetic analysis in this species and in evolutionary or taxonomic studies when compared to similar data obtained for any of the other recognized pathovars.