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Artykuły w czasopismach na temat "Pseudomonas – Genetics"

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Freitas, J. Renato de, i James J. Germida. "Pseudomonas cepacia and Pseudomonas putida as winter wheat inoculants for biocontrol of Rhizoctonia solani". Canadian Journal of Microbiology 37, nr 10 (1.10.1991): 780–84. http://dx.doi.org/10.1139/m91-134.

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Pseudomonas cepacia R55 and R85 and Pseudomonas putida R104, antagonistic towards plant pathogenic fungi in vitro, were assessed as seed inoculants for winter wheat (cv. Norstar) grown in a growth chamber in soil infested with Fusarium solani or Rhizoctonia solani isolate AG-1, AG 2-1, or AG-3. Infestation of soil with R. solani AG-1 or AG 2-1 reduced root dry weight of uninoculated plants by 62 and 78%, respectively, whereas R. solani AG-3 or F. solani had no effect on plant biomass. Pseudomonad inoculants increased (relative to plants subjected to disease) the winter wheat root dry weight by 92–128% and shoot dry weight by 28–48% in the soil infested with R. solani AG-1. The shoot material of all plants inoculated with pseudomonads also had significantly (P < 0.05) higher total Fe contents than the uninoculated treatment in the R. solani AG-1 infested soil. Pseudomonas cepacia R55 produced the highest (P < 0.01) total Fe contents in the shoots, but it had no effect on N and P content. Pseudomonas cepacia R85 significantly increased total N (P < 0.05) and total P (P < 0.01) of wheat shoots, and P. putida R104 increased the percentage (P < 0.05) and (or) total P content (P < 0.01) in the soil infested with R. solani AG-1. Pseudomonas cepacia R85 also significantly (P < 0.05) increased wheat shoot biomass in R. solani AG-3 infested soil. All three pseudomonads produced fluorescent siderophores when cultured in a low-iron medium. These results suggest an in situ antibiosis activity of three fluorescent pseudomonad strains towards phytopathogenic fungi and suggest that the plant growth response was probably due to protection against damage caused by R. solani. Key words: biocontrol, pseudomonads, Rhizoctonia solani, winter wheat, siderophores.
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Beaulieu, C., S. Gill, L. Miville i P. Dion. "Genetic regions of Pseudomonas aureofaciens strain 211 involved in nopaline catabolism". Canadian Journal of Microbiology 34, nr 7 (1.07.1988): 843–49. http://dx.doi.org/10.1139/m88-145.

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A DNA fragment from the nopaline catabolism region of the Ti plasmid of Agrobacterium tumefaciens showed no detectable homology to the total DNA of several nopaline-utilizing strains of Pseudomonas spp. From one of these pseudomonads, Pseudomonas aureofaciens strain 211, mutants defective in the catabolism of nopaline but not arginine, have been obtained by mutagenesis with transposon Tn5, and also with TnV using a new suicide plasmid vector. The DNA fragment bearing the TnV insertion has been cloned and found to hybridize with DNA of every pseudomonad tested, independently of the capacity to utilize nopaline, but not with Agrobacterium DNA. In a separate experiment, nonmutagenized DNA of strain 211 was cloned in a cosmid vector. Transfer of one of these clones to Pseudomonas putida strain KT2440 conferred on the recipient the capacity for nopaline utilization. However, this cosmid clone was only partially functional, since it did not complement a TnV mutant.
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Katsuwon, J., R. Zdor i A. J. Anderson. "Superoxide dismutase activity in root-colonizing pseudomonads". Canadian Journal of Microbiology 39, nr 4 (1.04.1993): 420–29. http://dx.doi.org/10.1139/m93-061.

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Several saprophytic fluorescent pseudomonads that are aggressive root colonizers express similar specific activities of superoxide dismutase during growth in liquid culture. The pseudomonads have the potential to produce hydrogen peroxide sensitive and hydrogen peroxide insensitive isoforms of superoxide dismutase with distinct mobilities in nondenaturing polyacrylamide gel electrophoresis. Synthesis of the hydrogen peroxide insensitive form is enhanced by limited iron availability, by exposure to Mn2+, and to a lesser extent by external sources of superoxide anion. Unlike Pseudomonas aeruginosa, a root-colonizing strain of Pseudomonas putida did not show regulation of isoform pattern by phosphate availability. A plasmid potentially encoding the pseudomonad hydrogen peroxide sensitive form complemented the superoxide dismutase deficiency in a mutant of Escherichia coli lacking expression of both Fe and Mn genes. Contact between the plant root and pseudomonad or E. coli cells that lack or express superoxide dismutase did not influence superoxide anion production from root surface enzymes. The pseudomonad and the superoxide dismutase deficient and producing E. coli strains survived exposure to the root equally well. Only the hydrogen peroxide sensitive isoform of superoxide dismutase was detected in P. putida cells associated with bean root surfaces.Key words: pseudomonads, activated oxygen, root surface colonization.
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Campbell, James N., Kenneth Conn, Linnea Sorlie i Fred D. Cook. "Inhibition of growth in canola seedlings caused by an opportunistic Pseudomonas sp. Under laboratory and field conditions". Canadian Journal of Microbiology 32, nr 3 (1.03.1986): 201–7. http://dx.doi.org/10.1139/m86-041.

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If Pseudomonas rp2, a field-isolated fluorescent pseudomonad, is present on canola (rape) seeds at the time of sprouting, it causes an inhibition of root growth leading to death or delayed maturation of the plant. Inhibitory strains of this type comprise less than 10% of the fluorescent pseudomonads isolated from local field samples, but they were found in widely dispersed sources. By present standards, these Pseudomonas strains would be considered soil saprophytes, since they survive in sterile soil at 4 and −20 °C in the absence of plant material, and since they do not match taxonomically with established plant pathogenic Pseudomonas spp. tested. Under laboratory conditions, inoculation of seeds with Pseudomonas rp2 caused death in 30%, and delayed development in 68% of infected plants. Minimum bacterial load, recoverable from the seed surface and capable of causing inhibition, was 10–20 colony-forming units per seed. The effect of inoculation of seeds with Pseudomonas rp2 on stand, rate of growth, and seed yield was quantitated under field conditions. The results reflected values obtained under laboratory conditions. The potential economic and ecological significance of this type of infection is discussed.
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Cabrera, Ma Ángeles, Sebastián L. Márquez i José M. Pérez-Donoso. "Comparative Genomic Analysis of Antarctic Pseudomonas Isolates with 2,4,6-Trinitrotoluene Transformation Capabilities Reveals Their Unique Features for Xenobiotics Degradation". Genes 13, nr 8 (28.07.2022): 1354. http://dx.doi.org/10.3390/genes13081354.

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The nitroaromatic explosive 2,4,6-trinitrotoluene (TNT) is a highly toxic and persistent environmental pollutant. Since physicochemical methods for remediation are poorly effective, the use of microorganisms has gained interest as an alternative to restore TNT-contaminated sites. We previously demonstrated the high TNT-transforming capability of three novel Pseudomonas spp. isolated from Deception Island, Antarctica, which exceeded that of the well-characterized TNT-degrading bacterium Pseudomonas putida KT2440. In this study, a comparative genomic analysis was performed to search for the metabolic functions encoded in the genomes of these isolates that might explain their TNT-transforming phenotype, and also to look for differences with 21 other selected pseudomonads, including xenobiotics-degrading species. Comparative analysis of xenobiotic degradation pathways revealed that our isolates have the highest abundance of key enzymes related to the degradation of fluorobenzoate, TNT, and bisphenol A. Further comparisons considering only TNT-transforming pseudomonads revealed the presence of unique genes in these isolates that would likely participate directly in TNT-transformation, and others involved in the β-ketoadipate pathway for aromatic compound degradation. Lastly, the phylogenomic analysis suggested that these Antarctic isolates likely represent novel species of the genus Pseudomonas, which emphasizes their relevance as potential agents for the bioremediation of TNT and other xenobiotics.
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Martusevich, Andrew K., Ivan V. Bocharin, Elena A. Kochkurova i Natalia A. Ronzhina. "INFLUENCE OF DISINFECTANT ON CRYSTALLOGENIC ACTIVITY OF PSEUDOMONAS AERUGINOSA IN VITRO". Siberian Journal of Life Sciences and Agriculture 13, nr 5 (29.10.2021): 191–204. http://dx.doi.org/10.12731/2658-6649-2021-13-5-191-204.

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The purpose of this work was to clarify the crystallogenic properties of pseudomonads under the action of an antiseptic. Material and methods. The material for the study was 8 strains of P. aeruginosa isolated from patients of the burn Department. In accordance with the purpose and objectives of the study, the work was performed in 3 stages: assessment of the biological properties of isolated pseudomonad strains; determination of sensitivity to disinfectants by the square method; assessment of the crystallogenic (initiating) activity of pseudomonads in individual and joint form with the disinfectant. The tested antiseptic was “Desam” in the form of a standard 1% solution used for disinfection of surfaces and medical instruments. Results. It was found that all the studied Pseudomonas strains have the ability to activate the crystallogenesis of the basic substance (0.9% sodium chloride solution), which manifests itself both in qualitative and quantitative changes in the thesigraphic picture. It is shown that the addition of a common disinfectant (“Desam”) to the system “Pseudomonas aeruginosa – 0.9% sodium chloride solution” significantly transforms its dehydration structuring. At the same time, strains of the microorganism resistant to disinfectants moderately reduce the crystal’s gene activity (according to the main teziographic coefficient and belt coefficient). On the contrary, sensitive strains demonstrate a pronounced inhibition of the crystallogenesis of the basic substance. It allows to develop a new express method for determining the sensitivity of microorganisms to disinfectants.
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Pyle, Barry H., i Gordon A. McFeters. "Population dynamics of pseudomonads after iodination". Canadian Journal of Microbiology 36, nr 11 (1.11.1990): 801–3. http://dx.doi.org/10.1139/m90-137.

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The population dynamics of pseudomonads grown under rich or low nutrient conditions were examined following iodination. Iodinated and untreated controls of Pseudomonas aeruginosa or Pseudomonas cepacia were resuspended in phosphate buffer and incubated at room temperature. Viable populations of iodine-treated cultures increased faster in phosphate-buffered water than uniodinated controls. Thus, bacteria in iodinated water systems may recover or multiply during storage and distribution after disinfection and may pose a significant health risk. Key words: iodine, disinfection, pseudomonads.
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Giles, Courtney D., Pei-Chun (Lisa) Hsu, Alan E. Richardson, Mark R. H. Hurst i Jane E. Hill. "The role of gluconate production by Pseudomonas spp. in the mineralization and bioavailability of calcium–phytate to Nicotiana tabacum". Canadian Journal of Microbiology 61, nr 12 (grudzień 2015): 885–97. http://dx.doi.org/10.1139/cjm-2015-0206.

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Organic phosphorus (P) is abundant in most soils but is largely unavailable to plants. Pseudomonas spp. can improve the availability of P to plants through the production of phytases and organic anions. Gluconate is a major component of Pseudomonas organic anion production and may therefore play an important role in the mineralization of insoluble organic P forms such as calcium–phytate (CaIHP). Organic anion and phytase production was characterized in 2 Pseudomonas spp. soil isolates (CCAR59, Ha200) and an isogenic mutant of strain Ha200, which lacked a functional glucose dehydrogenase (Gcd) gene (strain Ha200 gcd::Tn5B8). Wild-type and mutant strains of Pseudomonas spp. were evaluated for their ability to solubilize and hydrolyze CaIHP and to promote the growth and assimilation of P by tobacco plants. Gluconate, 2-keto-gluconate, pyruvate, ascorbate, acetate, and formate were detected in Pseudomonas spp. supernatants. Wild-type pseudomonads containing a functional gcd could produce gluconate and mineralize CaIHP, whereas the isogenic mutant could not. Inoculation with Pseudomonas improved the bioavailability of CaIHP to tobacco plants, but there was no difference in plant growth response due to Gcd function. Gcd function is required for the mineralization of CaIHP in vitro; however, further studies will be needed to quantify the relative contribution of specific organic anions such as gluconate to plant growth promotion by soil pseudomonads.
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Brown, Gerry, Zeayen Khan i Ran Lifshitz. "Plant growth promoting rhizobacteria: strain identification by restriction fragment length polymorphisms". Canadian Journal of Microbiology 36, nr 4 (1.04.1990): 242–48. http://dx.doi.org/10.1139/m90-042.

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A genomic library of the Pseudomonas putida strain GR12-2 was screened to identify both genus-universal and strain-specific 8-kilobase inserts. The genus-universal clone (pAM141), in combination with the restriction enzymes EcoRI, PstI, and PvuII, was used to generate unique restriction fragment length polymorphisms for 20 related plant growth promoting rhizobacteria and seven reference strains. Strain restriction fragment length polymorphism profiles based on the genus-universal clone pAM141 allow positive identification of individual pseudomonad strains. The strain-specific clone (pAM227) clearly distinguished the parent strain (GR12-2) from 16 other pseudomonad strains, including 8 P. putida, 7 P. fluorescens, and 1 P. cepacia. pAM227 may be useful for monitoring the fate of the P. putida strain GR12-2 in the environment. Key words: pseudomonads, rhizobacteria, restriction fragment length polymorphism, strain identification.
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Outryve, M. F. Van, F. Gosselé, K. Kersters i J. Swings. "The composition of the rhizosphere of chicory (Cichorium intybus L. var. foliosum Hegi)". Canadian Journal of Microbiology 34, nr 11 (1.11.1988): 1203–8. http://dx.doi.org/10.1139/m88-211.

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The bacterial composition of the chicory rhizosphere (Cichorium intybus L. var. foliosum Hegi) was examined at four different growth stages in the field and also after 1 month storage of the roots. Based on protein fingerprints (SDS – polyacrylamide gel electrophoresis of total cell proteins) 233 isolates were grouped into 117 different groups. Forty percent of the isolates belonged to one of three groups: CH001, CH002, or CH213. Fingerprint type CH001 and CH002 were comprised of fluorescent pseudomonads. Fingerprint type CH213 was identified as Alcaligenes paradoxus. Fingerprint type CH213 strains, normally isolated early in the growing season, were inhibited in vitro by fingerprint type CH001 strains which appeared later in the growing season. Gram-negative isolates were predominant among the remaining fingerprint types: Pseudomonas paucimobilis, Xanthomonas maltophilia, Agrobacterium radiobacter, and Flavobacterium spp. A few Gram-positive isolates were found at the beginning of the growing season, i.e., Bacillus spp. and Streptomyces spp. The production of antifungal compounds was restricted to the 11 isolates among which were fluorescent Pseudomonas and Bacillus spp. Twenty-four fluorescent pseudomonad isolates from the rhizosphere were pathogenic on chicory leaves.
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Rozprawy doktorskie na temat "Pseudomonas – Genetics"

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Haubold, Bernhard. "The population genetics of fluorescent pseudomonas". Thesis, University of Oxford, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.390493.

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Gifford, Danna R. "Population genetics of rifampicin-resistant Pseudomonas aeruginosa". Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:044b9258-4f10-4e77-9ff6-aa4035cec33b.

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Antibiotic resistance is generally associated with a cost in terms of reduced competitive fitness in the absence of antibiotics. Despite this 'cost of resistance', the cessation of antibiotic treatment does not result in significant reductions in the prevalence of resistance. The maintenance of resistance, in spite of the costs, has been attributed to the rarity of reversion mutations, relative to compensatory mutations at other loci in the genome. However, the large size of bacteria populations, and the potential for migration, suggest that reversion mutations should occasionally be introduced to resistant populations. In this thesis, I show that additional mechanisms can prevent fixation of reversion mutations even if they do occur. Using an experimental evolution approach, with rifampicin resistance in Pseudomonas aeruginosa as a model system, I measured the costs of resistance in several environments and followed the adaptive dynamics of resistant populations where a sensitive lineage had invaded by migration. The results suggest that several additional mechanisms contribute to the maintenance of antibiotic resistance. Most rifampicin resistance mutations are not unconditionally costly in all environments, suggesting that migration between environments could maintain a resistant reservoir population. In environments where resistance is initially costly, the fixation of a revertant is not guaranteed, even if introduced through migration. Revertant fixation was impeded or prevented by clonal interference from adaptation in the resistant strain. Revertants that did successfully replace the resistant strain were forced to adapt to do so. Contrary to assumptions in the existing literature, fitness in the resistant strains was not recovered by general compensatory mutations, but instead by adaptive mutations specific to the environment. The data challenge several assumptions about the maintenance of antibiotic resistance: that resistance mutations are always costly, that the rarity of back mutations prevents the reversion of resistance, and that resistant strains recover fitness by compensatory mutations.
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Hughes, M. A. "Transfer RNA genes in Pseudomonas aeruginosa". Thesis, University of Liverpool, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.370854.

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Allen, Rebecca Louise. "A study of gene expression in Pseudomonas". Thesis, University of Leicester, 1989. http://hdl.handle.net/2381/35185.

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An inherent problem in the study of the genetics of the interesting and potentially commercially useful properties of the pseudomonads is that of gene expression, since many of the genes encoding these properties are not well expressed in an E.coli background. The evidence available at the present time indicates that some Pseudomonas genes may possess different promoter sequences not recognised by E.coli RNA polymerase. An in vitro coupled transcription/translation system based on P.putida has been developed. A comparison of E.coli and broad host range plasmid DNA in this and the equivalent E. coli system showed that although cloned E. coli and vector polypeptides were synthesised in both systems, there was a difference in the polypeptide products directed by broad host range plasmid DNA in the two systems. In particular RSF1010 directed the synthesis of a 73kD polypeptide uniquely in the P.putida system. This was shown to be a polypeptide involved in mobilisation of the plasmid. A broad host rsmge proraoter-probe vector based on RSFlOlO was constructed and used for the shotgun cloning of P.putida promoters. A small subset of fragments which were active as promoters in P.putida but exhibited much lower activity in E.coli were isolated, sequenced and analysed with respect to concensus E. coli and nitrogen-regulated promoter sequences. These isolated DNA fragments may represent promoters which have sequences specifically recognised by Pseudomonas RNA polymerase. An analysis of published Pseudomonas chromosomally-encoded promoters revealed putative Pseudomonas-specific concensus regions.
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Moulton, Paul Jonathan. "The molecular genetics of Pseudomonas syringae pv. pisi". Thesis, University of the West of England, Bristol, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.278900.

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林嘉敏 i Ka-man Amy Lam. "Characterization and heterologous expression of a dehalogenase gene from pseudomonas putida STRAIN A". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 1999. http://hub.hku.hk/bib/B31221099.

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Wong, Chi-fat, i 黃志發. "Genome sequencing and comparative genomic analysis of Pseudomonas mendocina DLHK". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2012. http://hdl.handle.net/10722/197162.

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Nitrogen oxides are targeted as important gas pollutants to be eliminated. Biological removal of nitrogen oxides, like the use of bio-trickling reactor, is gaining popularity over conventional methods. A bacterium isolated from a bio-trickling reactor showing high performance in removing nitrogen oxides was identified to be Pseudomonas mendocina. The genome of this isolate was sequenced using 454 pyrosequencing technology to a high level of completeness with 33 contigs and N50 contig length of 273 kbp. The genome was annotated by Prokaryotic Genome Automatic Annotaion Pipeline (PGAAP)and the strain was named P. mendocina DLHK. Examination of P. mendocina DLHK genome annotation found nitrate reductase but not nitrite reductase, nitric oxide reductase and nitrous oxide reductase. Novel genes or pathways might be available in P. mendocina DLHK contributingits denitrification function in the bio-trickling reactor. To better understand the evolution of Pseudomonas, a comprehensive comparative study was performed. Genomes of the Pseudomonasgenus were reannotated. Core genes were extracted to construct a phylogenomic tree using supermatrix approach. Effectiveness of using single phylogenetic marker in constructing phylogeny of Pseudomonas was evaluated using rpoB gene marker. Both methods generated phylogenetic trees of very similar topology, suggesting that rpoB gene can be a very effective marker in the rapid construction of Pseudomonas phylogeny.It is surprising to note that Azotobacter vinelandii DJ was included in the large clade of Pseudomonas and have the closest relationship with P. stutzeri in both phylogenetic trees, providing evidence that this species should be reclassified into the genus Pseudomonas. Cluster of Orthologous Groups (COG)category distribution of all pseudomonads were analysed for physiological significance. The large amount of gene categorised into the COG for amino acid metabolism and transcription may imply the importance for these 2 functions on the survival of pseudomonads. It is again surprising to note the COG distribution pattern of A. vinelandii DJ is different from other pseudomonads, especially to its closest phylogenetic neighbour P. stutzeri. Horizontal gene transfer events inpseudomonads were investigated because of its importance in prokaryotic evolution. The high congruence of the phylogemonic tree to most core genes suggests that the phylogenomic tree is a good piece of evidence describing the evolution of pseudomonads. It is a bit surprising to observe that quite a number of core genes may have exhibited horizontal gene transfer which show incongruence of the gene tree to the core genes. The impact of horizontal gene transfer events on the functionality and evolution of pseudomonads remains to be investigated.
published_or_final_version
Biological Sciences
Master
Master of Philosophy
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Malik, A. N. "Genetic studies with Pseudomonas syringae pathovar pisi". Thesis, University of Greenwich, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.354390.

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Chung, Yiu-kay Wilson, i 鍾堯基. "Identification of the regulatory element of dehalogenase IVa of Burkholderia cepacia MBA4". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2003. http://hub.hku.hk/bib/B29517291.

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Roush, Wendy A. "Pyrimidine Genes in Pseudomonas Species". Thesis, University of North Texas, 2003. https://digital.library.unt.edu/ark:/67531/metadc4395/.

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This thesis is a comparative study of gene arrangements in Pseudomonas species, and is organized into three major sections. The first section compares gene arrangements for different pathways in Pseudomonas aeruginosa PAO1 to determine if the gene arrangements are similar to previous studies. It also serves as a reference for pyrimidine gene arrangements in P. aeruginosa. The second part compares the physical, and genetic maps of P. aeruginosa PAO1 with the genome sequence. The final section compares pyrimidine gene arrangements in three species of Pseudomonas. Pyrimidine biosynthesis and salvage genes will be aligned for P. aeruginosa PAO1, P. putida KT2440, and P. syringae DC3000. The whole study will gives insight into gene patterns in Pseudomonas, with a focus on pyrimidine genes.
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Książki na temat "Pseudomonas – Genetics"

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P, Gacesa, i Russell Nicholas J, red. Pseudomonas infection and alginates: Biochemistry, genetics, and pathology. London: Chapman and Hall, 1990.

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Media, Springer Science+Business, red. Pseudomonas: Methods and protocols. New York: Humana Press, 2014.

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Ramos, Juan L. Pseudomonas: Volume 6: Molecular Microbiology, Infection and Biodiversity. Dordrecht: Springer Science+Business Media B.V., 2010.

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International, Conference on Pseudomonas syringae Pathovers and Related Pathogens (6th 2002 Maratea Italy). Pseudomonas syringae and related pathogens: Biology and genetic. Dordrecht: Kluwer Academic Publishers, 2003.

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Clarke, Neil James. Molecular genetic characterisation of the mupirocin biosynthetic pathway of 'Pseudomonas fluorescens' NCIB 10586. Birmingham: University of Birmingham, 1989.

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Ramos, Juan-Luis. Pseudomonas. Springer, 2004.

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Xu, Guo-Wei. Molecular genetics of syringomycin production by Pseudomonas syringae pv. syringae. 1987.

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Pseudomonas: Volume 3 Biosynthesis of Macromolecules and Molecular Metabolism. Springer, 2012.

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(Editor), Nicola Sante Iacobellis, Alan Collmer (Editor), Steven W. Hutcheson (Editor), John W. Mansfield (Editor), Cindy E. Morris (Editor), Jesús Murillo (Editor), Norman W. Schaad (Editor), David E. Stead (Editor), Giuseppe Surico (Editor) i Matthias S. Ullrich (Editor), red. Pseudomonas syringae and Related Pathogens: Biology and Genetics. Springer, 2003.

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Ramos, Juan-Luis. Pseudomonas: Volume 3 Biosynthesis of Macromolecules and Molecular Metabolism. Springer London, Limited, 2012.

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Części książek na temat "Pseudomonas – Genetics"

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Alvarez-Morales, Ariel, Karina López-López i José Luis Hernández-Flores. "Current Ideas on the Genetics and Regulation of the Synthesis of Phaseolotoxin in Pseudomonas Syringae PV. Phaseolicola". W Pseudomonas, 159–80. Boston, MA: Springer US, 2004. http://dx.doi.org/10.1007/978-1-4419-9084-6_5.

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Ohman, D. E., i J. B. Goldberg. "Genetics of alginate biosynthesis in Pseudomonas aeruginosa". W Pseudomonas Infection and Alginates, 206–20. Dordrecht: Springer Netherlands, 1990. http://dx.doi.org/10.1007/978-94-009-1836-8_11.

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Ingraham, John L. "Genetics of Denitrification in Pseudomonas Aeruginosa and Stutzeri". W Denitrification in the Nitrogen Cycle, 67–78. Boston, MA: Springer US, 1985. http://dx.doi.org/10.1007/978-1-4757-9972-9_5.

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Schell, Mark A., Daniel P. Roberts i Timothy P. Denny. "Analysis of the Spontaneous Mutation to Avirulence by Pseudomonas Solanacearum". W Molecular genetics of plant-microbe interactions, 61–66. Dordrecht: Springer Netherlands, 1987. http://dx.doi.org/10.1007/978-94-009-4482-4_13.

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Boucher, C., A. Martinel, P. Barberis, G. Alloing i C. Zischek. "Genetics of Virulence in the Wilt Pathogen, Pseudomonas Solanacearum". W Plant Pathogenic Bacteria, 412. Dordrecht: Springer Netherlands, 1987. http://dx.doi.org/10.1007/978-94-009-3555-6_81.

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Seaton, Sarah Craven, i Mark W. Silby. "Genetics and Functional Genomics of the Pseudomonas fluorescens Group". W Genomics of Plant-Associated Bacteria, 99–125. Berlin, Heidelberg: Springer Berlin Heidelberg, 2014. http://dx.doi.org/10.1007/978-3-642-55378-3_5.

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Leary, J. V., J. W. Willis i D. Trollinger. "Molecular Genetics of Coronatine Production by Pseudomonas Syringae Pv. Glycinea". W Plant Pathogenic Bacteria, 498–508. Dordrecht: Springer Netherlands, 1987. http://dx.doi.org/10.1007/978-94-009-3555-6_106.

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Ausubel, Frederick M., Jane Glazebrook, Jean Greenberg, Michael Mindrinos i Guo-Liang Yu. "Analysis of the Arabidopsis Defense Response to Pseudomonas Pathogens". W Advances in Molecular Genetics of Plant-Microbe Interactions, Vol. 2, 393–403. Dordrecht: Springer Netherlands, 1993. http://dx.doi.org/10.1007/978-94-017-0651-3_43.

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Keen, Noel, Sharon L. Midland, Carol Boyd, Irem Yucel, Tetsu Tsurushima, Jennifer Lorang i James J. Sims. "Syringolide Elicitors Specified by Avirulence Gene D Alleles in Pseudomonas Syringae". W Advances in Molecular Genetics of Plant-Microbe Interactions, 41–48. Dordrecht: Springer Netherlands, 1994. http://dx.doi.org/10.1007/978-94-011-0177-6_7.

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Leong, J., W. Bitter, M. Koster, J. D. Marugg i P. J. Weisbeek. "Genetics of iron transport in plant growth-promoting Pseudomonas putida WCS358". W The Rhizosphere and Plant Growth, 271–78. Dordrecht: Springer Netherlands, 1991. http://dx.doi.org/10.1007/978-94-011-3336-4_54.

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Streszczenia konferencji na temat "Pseudomonas – Genetics"

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Rotaru, Vladimir. "Ifluenţa fosforului si tulpinilor rizobacteriene asupra dezvoltării sistemului radicular la plante de soia (Glycine max L. MERR.) în condiţii deficitului de fosfor si umidiate". W VIIth International Scientific Conference “Genetics, Physiology and Plant Breeding”. Institute of Genetics, Physiology and Plant Protection, Republic of Moldova, 2021. http://dx.doi.org/10.53040/gppb7.2021.24.

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Drought and nutrient deficiency are major abiotic factors that limits crop production. This study determined the effect of phosphorus (P) and rhizobacteria application on root system development of soybean plants subjected to P deficiency and drought. The P application alone or in combination with bacteria strains (Pseudomonas fluorescence and Azotobacter chroococcum) increased total roots length irrespective of soil moisture. Root growth of cultivar Horboveanca responded more evidently to treatment with rhizobacteria than cultivar Zodiac under P deficiency. Thus, the experimental results demonstrated that the effectiveness of integrated use of P and rhizobacteria (Pseudomonas fluorescence and Azotobacter chroococcum) promotes roots development of soybean plants under normal soil moisture as well as under temporary drought.
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Самойлова, Анна. "Бактериофаги Pseudomonas syringae pv. syringae перспективные в подавлении развития бактериального рака плодовых". W VIIth International Scientific Conference “Genetics, Physiology and Plant Breeding”. Institute of Genetics, Physiology and Plant Protection, Republic of Moldova, 2021. http://dx.doi.org/10.53040/gppb7.2021.88.

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Five Pseudomonas syringae pv. syringae bacteriophages were isolated from the quince, apple and pear. After a detailed study, the isolated bacteriophages could be used for biocontrol of the bacterial canker patho-gen. One of the isolated phages was active against the causative agents of bacterial canker and fire blight.
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Eklöf, Josefin, Mari-Anna Misiakou, Pradeesh Sivapalan, Karin Armbruster, Andrea Browatzki, Thyge L. Nielsen, Therese S. Lapperre i in. "Persistence and genetic adaptation of Pseudomonas aeruginosa in patients with COPD". W ERS International Congress 2020 abstracts. European Respiratory Society, 2020. http://dx.doi.org/10.1183/13993003.congress-2020.4932.

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Sadeeva, Zulfirya, Irina Novikova, Natalya Alyabyeva, Anna Lazareva, Yliya Gorinova i Olga Simonova. "Molecular genetic characteristics of Pseudomonas aeruginosa strains isolated from children with cystic fibrosis in Moscow". W ERS International Congress 2021 abstracts. European Respiratory Society, 2021. http://dx.doi.org/10.1183/13993003.congress-2021.pa3378.

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Hoke, John L., John G. Georgiadis i Rafael Jimenez-Flores. "Freezing of Aqueous Solutions of Glycosylated Bovine Beta-Casein". W ASME 1998 International Mechanical Engineering Congress and Exposition. American Society of Mechanical Engineers, 1998. http://dx.doi.org/10.1115/imece1998-0813.

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Abstract Genetic engineering of milk proteins allows control of their physicochemical properties in foods and dairy products during processing, storage, and consumption. Robust methods for the estimation of the quality and function of the proteins during downstream processing are sought. The focus of this study is a systematic microscopic investigation of the freezing of sub-microliter pendent droplets of buffer solution of glycosylated bovine beta-casein. The freezing and crystallization is observed with a scanning confocal microscope fitted with a stage cooled with vapor boiled off a liquid nitrogen dewar. Four liquid samples (with glycosylated bovine beta-casein concentrations of 35, 125, 500, and 1000 μg/ml) are compared against the control (10 mg/ml of wild type non-glycosylated bovine beta-casein). The freezing of similar size samples consisting of de-ionized water, ice-nucleating mixture (Pseudomonas syringae), and pure buffer solution is also examined for comparison. Higher concentrations of the engineered-beta casein result in increasing antifreeze action, corresponding to depression of the freezing point, and thermal stabilization of the supercooled liquid. This conclusion is supported by a non-parametric statistical analysis based on the Jonckheere test. A freezing point assay can thus be made to assess the quality of glyco-bovine beta-casein.
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Spagnuolo, Lorenza, Maura De Simone, Nicola Lorè, Cristina Cigana, Ida De Fino, Veronica Basso, Anna Mondino i Alessandra Bragonzi. "LSC Abstract – The host genetic background impacts on pseudomonas aeruginosa-induced T cell responses and individual susceptibility/resistance to respiratory infection". W Annual Congress 2015. European Respiratory Society, 2015. http://dx.doi.org/10.1183/13993003.congress-2015.pa2661.

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Raporty organizacyjne na temat "Pseudomonas – Genetics"

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Lewis, Kim. Genetics of Persister Formation in Pseudomonas aeruginosa. Fort Belvoir, VA: Defense Technical Information Center, grudzień 2012. http://dx.doi.org/10.21236/ada580295.

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Lindow, Steven E., Shulamit Manulis, Dan Zutra i Dan Gaash. Evaluation of Strategies and Implementation of Biological Control of Fire Blight. United States Department of Agriculture, lipiec 1993. http://dx.doi.org/10.32747/1993.7568106.bard.

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The main objective of this study was to develop data that would facilitate a consistently effective method of biological control of fire blight disease to be developed and to enable its implementation for disease control by ensuring its compatibility with variations in the biological, environmental, and chemical conditions present in pear orchards. As considerable information on the pathogen and biological control of fire blight was already gathered from studies in California and elsewhere, an emphasis was placed on investigating the genetics and ecology of Erwinia amylovora, the causal agent of fire blight in Israel. Studies of plasmid profile, virulence on several host, serological characteristics, as well as DNA fingerprints with selected primers all revealed E. amylovora strains in Israel to be homogeneous. Strains did vary in their resistance to streptomycin, with those from more northern locations being resistant while those in the southern costal plain were all sensitive to streptomycin. Resistance appeared to be conferred by chromosomal mutations as in streptomycin-resistant strains in California. The biological control agent Pseudomonas fluorescens strain A506 colonized flowers of both the Costia and Spodona pear cultivars in Israel as well as Bartlett pear in California. Flowers that were open at the time of spray inoculation of trees subsequently harbored from 105 to 107 cells of strain A506 per flower, while those that opened subsequent to spraying developed population sizes of about 105 cells/flower within 5 days. The incidence of fire blight infections were reduced about 3-fold in several trials in which moderate amounts of disease occurred in the plot areas; this degree of biological control is similar to that observed in California and elsewhere. On two occasions warm and moist weather that favored disease led to epidemics in which nearly all flowers became infected and which was so severe that neither P. fluorescens strain A506 nor chemical bactericides reduced disease incidence. A novel method for identifying antagonistic microorganisms for biological control of fire blight and other diseases was developed. A bacterial ice nucleation gene was introduced into E. amylovora to confer an Ice+ phenotype and the population sizes of this modified pathogen on flowers that had been pre-treated with potential control agents was estimated by measuring the freezing temperature of colonized flowers. Antagonistic strains that prevented the growth of E. amylovora in flowers were readily detected as those in which flowers froze at a low temperature. The method is both rapid and unbiased and several bacterial strains with substantial biological control potential have been identified using this method.
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Sessa, Guido, i Gregory B. Martin. molecular link from PAMP perception to a MAPK cascade associated with tomato disease resistance. United States Department of Agriculture, styczeń 2012. http://dx.doi.org/10.32747/2012.7597918.bard.

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The research problem: The detection of pathogen-associated molecular patterns (PAMPs) by plant pattern recognition receptors (PRRs) is a key mechanism by which plants activate an effective immune response against pathogen attack. MAPK cascades are important signaling components downstream of PRRs that transduce the PAMP signal to activate various defense responses. Preliminary experiments suggested that the receptor-like cytoplasmickinase (RLCK) Mai5 plays a positive role in pattern-triggered immunity (PTI) and interacts with the MAPKKK M3Kε. We thus hypothesized that Mai5, as other RLCKs, functions as a component PRR complexes and acts as a molecular link between PAMP perception and activation of MAPK cascades. Original goals: The central goal of this research was to investigate the molecular mechanisms by which Mai5 and M3Kε regulate plant immunity. Specific objectives were to: 1. Determine the spectrum of PAMPs whose perception is transmitted by M3Kε; 2. Identify plant proteins that act downstream of M3Kε to mediate PTI; 3. Investigate how and where Mai5 interacts with M3Kε in the plant cell; 4. Examine the mechanism by which Mai5 contributes to PTI. Changes in research directions: We did not find convincing evidence for the involvement of M3Kε in PTI signaling and substituted objectives 1 and 3 with research activities aimed at the analysis of transcriptomic profiles of tomato plants during the onset of plant immunity, isolation of the novel tomato PRR FLS3, and investigation of the involvement of the RLCKBSKs in PTI. Main achievements during this research program are in the following major areas: 1. Functional characterization of Mai5. The function of Mai5 in PTI signaling was demonstrated by testing the effect of silencing the Mai5 gene by virus-induced gene silencing (VIGS) experiments and in cell death assays. Domains of Mai5 that interact with MAPKKKs and subcellular localization of Mai5 were analyzed in detail. 2. Analysis of transcriptional profiles during the tomato immune responses to Pseudomonas syringae (Pombo et al., 2014). We identified tomato genes whose expression is induced specifically in PTI or in effector-triggered immunity (ETI). Thirty ETI-specific genes were examined by VIGS for their involvement in immunity and the MAPKKK EPK1, was found to be required for ETI. 3. Dissection of MAP kinase cascades downstream of M3Kε (Oh et al., 2013; Teper et al., 2015). We identified genes that encode positive (SGT and EDS1) and negative (WRKY1 and WRKY2) regulators of the ETI-associated cell death mediated by M3Kε. In addition, the MKK2 MAPKK, which acts downstream of M3Kε, was found to interact with the MPK3 MAPK and specific MPK3 amino acids involved interaction were identified and found to be required for induction of cell death. We also identified 5 type III effectors of the bacterial pathogen Xanthomonaseuvesicatoria that inhibited cell death induced by components of ETI-associated MAP kinase cascades. 4. Isolation of the tomato PRR FLS3 (Hind et al., submitted). FLS3, a novel PRR of the LRR-RLK family that specifically recognizes the flagellinepitope flgII-28 was isolated. FLS3 was shown to bind flgII-28, to require kinase activity for function, to act in concert with BAK1, and to enhance disease resistance to Pseudomonas syringae. 5. Functional analysis of RLCKs of the brassinosteroid signaling kinase (BSK) family.Arabidopsis and tomato BSKs were found to interact with PRRs. In addition, certain ArabidospsisBSK mutants were found to be impaired in PAMP-induced resistance to Pseudomonas syringae. Scientific and agricultural significance: Our research activities discovered and characterized new molecular components of signaling pathways mediating recognition of invading pathogens and activation of immune responses against them. Increased understanding of molecular mechanisms of immunity will allow them to be manipulated by both molecular breeding and genetic engineering to produce plants with enhanced natural defense against disease.
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Sessa, Guido, i Gregory Martin. A functional genomics approach to dissect resistance of tomato to bacterial spot disease. United States Department of Agriculture, styczeń 2004. http://dx.doi.org/10.32747/2004.7695876.bard.

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The research problem. Bacterial spot disease in tomato is of great economic importance worldwide and it is particularly severe in warm and moist areas affecting yield and quality of tomato fruits. Causal agent of spot disease is the Gram-negative bacterium Xanthomonas campestris pv. vesicatoria (Xcv), which can be a contaminant on tomato seeds, or survive in plant debris and in association with certain weeds. Despite the economic significance of spot disease, plant protection against Xcvby cultural practices and chemical control have so far proven unsuccessful. In addition, breeding for resistance to bacterial spot in tomato has been undermined by the genetic complexity of the available sources of resistance and by the multiple races of the pathogen. Genetic resistance to specific Xcvraces have been identified in tomato lines that develop a hypersensitive response and additional defense responses upon bacterial challenge. Central goals of this research were: 1. To identify plant genes involved in signaling and defense responses that result in the onset of resistance. 2. To characterize molecular properties and mode of action of bacterial proteins, which function as avirulence or virulence factors during the interaction between Xcvand resistant or susceptible tomato plants, respectively. Our main achievements during this research program are in three major areas: 1. Identification of differentially expressed genes during the resistance response of tomato to Xcvrace T3. A combination of suppression subtractive hybridization and microarray analysis identified a large set of tomato genes that are induced or repressed during the response of resistant plants to avirulent XcvT3 bacteria. These genes were grouped in clusters based on coordinate expression kinetics, and classified into over 20 functional classes. Among them we identified genes that are directly modulated by expression of the type III effector protein AvrXv3 and genes that are induced also during the tomato resistance response to Pseudomonas syringae pv. tomato. 2. Characterization of molecular and biochemical properties of the tomato LeMPK3MAP kinase. A detailed molecular and biochemical analysis was performed for LeMPK3 MAP kinase, which was among the genes induced by XcvT3 in resistant tomato plants. LeMPK3 was induced at the mRNA level by different pathogens, elicitors, and wounding, but not by defense-related plant hormones. Moreover, an induction of LeMPK3 kinase activity was observed in resistant tomato plants upon Xcvinfection. LeMPK3 was biochemically defined as a dual-specificity MAP kinase, and extensively characterized in vitro in terms of kinase activity, sites and mechanism of autophosphorylation, divalent cation preference, Kₘand Vₘₐₓ values for ATP. 3. Characteriztion of molecular properties of the Xcveffector protein AvrRxv. The avirulence gene avrRxvis involved in the genetic interaction that determines tomato resistance to Xcvrace T1. We found that AvrRxv functions inside the plant cell, localizes to the cytoplasm, and is sufficient to confer avirulence to virulent Xcvstrains. In addition, we showed that the AvrRxv cysteine protease catalytic core is essential for host recognition. Finally, insights into cellular processes activated by AvrRxv expression in resistant plants were obtained by microarray analysis of 8,600 tomato genes. Scientific and agricultural significance: The findings of these activities depict a comprehensive and detailed picture of cellular processes taking place during the onset of tomato resistance to Xcv. In this research, a large pool of genes, which may be involved in the control and execution of plant defense responses, was identified and the stage is set for the dissection of signaling pathways specifically triggered by Xcv.
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Sessa, Guido, i Gregory Martin. MAP kinase cascades activated by SlMAPKKKε and their involvement in tomato resistance to bacterial pathogens. United States Department of Agriculture, styczeń 2012. http://dx.doi.org/10.32747/2012.7699834.bard.

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The research problem: Pseudomonas syringae pv. tomato (Pst) and Xanthomonas campestrispv. vesicatoria (Xcv) are the causal agents of tomato bacterial speck and spot diseases, respectively. These pathogens colonize the aerial parts of the plant and cause economically important losses to tomato yield worldwide. Control of speck and spot diseases by cultural practices or chemicals is not effective and genetic sources of resistance are very limited. In previous research supported by BARD, by gene expression profiling we identified signaling components involved in resistance to Xcvstrains. Follow up experiments revealed that a tomato gene encoding a MAP kinase kinase kinase (MAPKKKe) is required for resistance to Xcvand Pststrains. Goals: Central goal of this research was to investigate the molecular mechanisms by which MAPKKKεand associated MAP kinase cascades regulate host resistance. Specific objectives were to: 1. Determine whether MAPKKKεplays a broad role in defense signaling in plants; 2. Identify components of MAP kinase cascades acting downstream of MAPKKKε; 3. Determine the role of phosphorylation-related events in the function of MAPKKKε; 4. Isolate proteins directly activated by MAPKKKε-associatedMAPK modules. Our main achievements during this research program are in the following major areas: 1. Characterization of MAPKKKεas a positive regulator of cell death and dissection of downstream MAP kinase cascades (Melech-Bonfil et al., 2010; Melech-Bonfil and Sessa, 2011). The MAPKKKεgene was found to be required for tomato resistance to Xcvand Pstbacterial strains and for hypersensitive response cell death triggered by different R gene/effector gene pairs. In addition, overexpression analysis demonstrated that MAPKKKεis a positive regulator of cell death, whose activity depends on an intact kinase catalytic domain. Epistatic experiments delineated a signaling cascade downstream of MAPKKKεand identified SIPKK as a negative regulator of MAPKKKε-mediated cell death. Finally, genes encoding MAP kinase components downstream of MAPKKKεwere shown to contribute to tomato resistance to Xcv. 2. Identification of tomato proteins that interact with MAPKKKεand play a role in plant immunity (Oh et al., 2011). We identified proteins that interact with MAPKKKε. Among them, the 14-3-3 protein TFT7 was required for cell death mediated by several R proteins. In addition, TFT7 interacted with the MAPKK SlMKK2 and formed homodimersin vivo. Thus, TFT7 is proposed to recruit SlMKK2 and MAPKKK client proteins for efficient signal transfer. 3. Development of a chemical genetic approach to identify substrates of MAPKKKε-activated MAP kinase cascades (Salomon et al., 2009, 2011). This approach is based on engineering the kinase of interest to accept unnatural ATP analogs. For its implementation to identify substrates of MAPKKKε-activated MAP kinase modules, we sensitized the tomato MAP kinase SlMPK3 to ATP analogs and verified its ability to use them as phosphodonors. By using the sensitized SlMPK3 and radiolabeled N6(benzyl)ATP it should be possible to tag direct substrates of this kinase. 4. Development of methods to study immunity triggered by pathogen-associated molecular patterns (PAMPs) in tomato and N. benthamiana plants (Kim et al., 2009; Nguyen et al. 2010). We developed protocols for measuring various PTI-associatedphenotypes, including bacterial populations after pretreatment of leaves with PAMPs, induction of reporter genes, callose deposition at the cell wall, activation of MAP kinases, and a luciferase-based reporter system for use in protoplasts. Scientific and agricultural significance: Our research activities discovered and characterized a signal transduction pathway mediating plant immunity to bacterial pathogens. Increased understanding of molecular mechanisms of immunity will allow them to be manipulated by both molecular breeding and genetic engineering to produce plants with enhanced natural defense against disease. In addition, we successfully developed new biochemical and molecular methods that can be implemented in the study of plant immunity and other aspects of plant biology.
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Sessa, Guido, i Gregory Martin. Role of GRAS Transcription Factors in Tomato Disease Resistance and Basal Defense. United States Department of Agriculture, 2005. http://dx.doi.org/10.32747/2005.7696520.bard.

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The research problem: Bacterial spot and bacterial speck diseases of tomato are causedby strains of Xanthomonas campestris pv. vesicatoria (Xcv) and Pseudomonas syringae pv.tomato (Pst), respectively. These bacteria colonize aerial parts of the plant and causesignificant losses in tomato production worldwide. Protection against Xcv and Pst bycultural practices or chemical control has been unsuccessful and there are only limitedsources of genetic resistance to these pathogens. In previous research supported in part byBARD IS-3237-01, we extensively characterized changes in tomato gene expression uponthe onset of spot and speck disease resistance. A remarkable finding of these studies wasthe inducibility in tomato leaves by both Xcv and Pst strains of genes encodingtranscriptional activator of the GRAS family, which has not been previously linked todisease resistance. Goals: Central goals of this research were to investigate the role of GRAS genes in tomatoinnate immunity and to assess their potential use for disease control.Specific objectives were to: 1. Identify GRAS genes that are induced in tomato during thedefense response and analyze their role in disease resistance by loss-of-function experiments.2. Overexpress GRAS genes in tomato and characterize plants for possible broad-spectrumresistance. 3. Identify genes whose transcription is regulated by GRAS family. Our main achievements during this research program are in three major areas:1. Identification of tomato GRAS family members induced in defense responses andanalysis of their role in disease resistance. Genes encoding tomato GRAS family memberswere retrieved from databases and analyzed for their inducibility by Pst avirulent bacteria.Real-time RT-PCR analysis revealed that six SlGRAS transcripts are induced during theonset of disease resistance to Pst. Further expression analysis of two selected GRAS genesshowed that they accumulate in tomato plants in response to different avirulent bacteria orto the fungal elicitor EIX. In addition, eight SlGRAS genes, including the Pst-induciblefamily members, were induced by mechanical stress in part in a jasmonic acid-dependentmanner. Remarkably, SlGRAS6 gene was found to be required for tomato resistance to Pstin virus-induced gene silencing (VIGS) experiments.2. Molecular analysis of pathogen-induced GRAS transcriptional activators. In aheterologous yeast system, Pst-inducible GRAS genes were shown to have the ability toactivate transcription in agreement with their putative function of transcription factors. Inaddition, deletion analysis demonstrated that short sequences at the amino-terminus ofSlGRAS2, SlGRAS4 and SlGRAS6 are sufficient for transcriptional activation. Finally,defense-related SlGRAS proteins were found to localize to the cell nucleus. 3. Disease resistance and expression profiles of transgenic plants overexpressing SlGRASgenes. Transgenic plants overexpressing SlGRAS3 or SlGRAS6 were generated. Diseasesusceptibility tests revealed that these plants are not more resistant to Pst than wild-typeplants. Gene expression profiles of the overexpressing plants identified putative direct orindirect target genes regulated by SlGRAS3 and SlGRAS6. Scientific and agricultural significance: Our research activities established a novel linkbetween the GRAS family of transcription factors, plant disease resistance and mechanicalstress response. SlGRAS6 was found to be required for disease resistance to Pstsuggesting that this and possibly other GRAS family members are involved in thetranscriptional reprogramming that takes place during the onset of disease resistance.Their nuclear localization and transcriptional activation ability support their proposed roleas transcription factors or co-activators. However, the potential of utilizing GRAS familymembers for the improvement of plant disease resistance in agriculture has yet to bedemonstrated.
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