scholarly journals Expression of avrPphB, an Avirulence Gene from Pseudomonas syringae pv. phaseolicola, and the Delivery of Signals Causing the Hypersensitive Reaction in Bean

1997 ◽  
Vol 10 (2) ◽  
pp. 247-256 ◽  
Author(s):  
Nakul Puri ◽  
Carol Jenner ◽  
Mark Bennett ◽  
Ruth Stewart ◽  
John Mansfield ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Induction Time ◽  
Phenotypic Expression ◽  
Constitutive Expression ◽  
Hypersensitive Reaction ◽  
Full Length ◽  
Avirulence Gene ◽  
Over Expression ◽  
E Coli ◽  
Induction Kinetics

Protein production encoded by the avirulence gene avrPphB from Pseudomonas syringae pv. phaseolicola was examined. Incorporation of [35S]-labeled methionine into the AvrPphB protein indicated processing of the full-length peptide in Escherichia coli to give a major 28-kDa product. The 28-kDa native peptide was isolated from E. coli following over-expression of avrPphB and found not to elicit the hypersensitive response (HR) after infiltration into bean leaves. Antiserum raised to the 28-kDa peptide allowed expression of avrPphB and processing of AvrPphB protein to be examined in P. syringae pv. phaseolicola; immunoreactive peptides of both 35 and 28-kDa were detected in races 3 and 4 (which contain avrPphB) only after induction in minimal medium + 10 mM sucrose. Antiserum raised to a synthetic peptide, derived from the sequence of the 62 amino acids found to be cleaved from the full-length AvrPphB protein, revealed the accumulation of peptides corresponding to the smaller cleavage products, in both E. coli and P. syringae pv. phaseolicola. Biochemical localization experiments showed that all AvrPphB peptides were cytoplasmic in P. syringae pv. phaseolicola. No AvrPphB peptides were produced in a hrpL mutant unless expression of the gene was directed by a strong vector promoter; induction kinetics similar to wild type were observed in a hrpY - strain, although it also failed to cause a confluent HR. Growth of P. syringae pv. phaseolicola under inducing conditions removed the requirement for rifampicin-sensitive mRNA synthesis by bacteria to allow HR development (the induction time) in bean and lettuce leaves. Constitutive expression of hrpL reduced but did not remove the induction time. Expression of the hrp gene cluster of P. syringae pv. phaseolicola from plasmid pPPY430 in E. coli enabled phenotypic expression of avrPphE (also carried by pPPY430) and avrPphB (if over-expressed from pPPY3031). Despite constitutive expression of the hrp and avr genes in E. coli, a protein synthesis dependent induction time was still required for development of the HR in bean genotypes with matching resistance genes. The significance of processing for the function of AvrPphB peptides and the delivery of elicitors of the HR are discussed.

scholarly journals A Salicylic Acid–Induced Lectin-Like Protein Plays a Positive Role in the Effector-Triggered Immunity Response of Arabidopsis thaliana to Pseudomonas syringae Avr-Rpm1

2013 ◽  
Vol 26 (12) ◽  
pp. 1395-1406 ◽  
Author(s):  
Grace Armijo ◽  
Paula Salinas ◽  
Mariela Inés Monteoliva ◽  
Aldo Seguel ◽  
Consuelo García ◽  
...  
Keyword(s):  
Cell Death ◽  
Salicylic Acid ◽  
Pseudomonas Syringae ◽  
Functional Characterization ◽  
Constitutive Expression ◽  
Hypersensitive Reaction ◽  
Positive Role ◽  
Legume Lectin ◽  
Immunity Response ◽  
Effector Triggered Immunity

Salicylic acid (SA) is one of the key hormones that orchestrate the pathogen-induced immune response in plants. This response is often characterized by the activation of a local hypersensitive reaction involving programmed cell death, which constrains proliferation of biotrophic pathogens. Here, we report the identification and functional characterization of an SA-induced legume lectin-like protein 1 (SAI-LLP1), which is coded by a gene that belongs to the group of early SA-activated Arabidopsis genes. SAI-LLP1 expression is induced upon inoculation with avirulent strains of Pseudomonas syringae pv. tomato via an SA-dependent mechanism. Constitutive expression of SAI-LLP1 restrains proliferation of P. syringae pv. tomato Avr-Rpm1 and triggers more cell death in inoculated leaves. Cellular and biochemical evidence indicates that SAI-LLP1 is a glycoprotein located primarily at the apoplastic side of the plasma membrane. This work indicates that SAI-LLP1 is involved in resistance to P. syringae pv. tomato Avr-Rpm1 in Arabidopsis, as a component of the SA-mediated defense processes associated with the effector-triggered immunity response.

scholarly journals Virulence of the Phytopathogen Pseudomonas syringae pv. Maculicola Is rpoN Dependent

2000 ◽  
Vol 182 (12) ◽  
pp. 3498-3507 ◽  
Author(s):  
Erik L. Hendrickson ◽  
Pablo Guevera ◽  
Alejandro Peñaloza-Vàzquez ◽  
Jing Shao ◽  
Carol Bender ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Constitutive Expression ◽  
Nitrogen Sources ◽  
Dicarboxylic Acids ◽  
Avirulence Gene ◽  
Gene Encoding ◽  
Direct Role ◽  
Mrna Accumulation ◽  
Genes Encoding ◽  
Mutant Phenotypes

ABSTRACT We cloned the rpoN (ntrA andglnF) gene encoding ς54 from the phytopathogen Pseudomonas syringae pv. maculicola strain ES4326. The P. syringae ES4326 rpoN gene complemented Pseudomonas aeruginosa, Escherichia coli, and Klebsiella aerogenes rpoN mutants for a variety of rpoN mutant phenotypes, including the inability to utilize nitrate as sole nitrogen source. DNA sequence analysis of the P. syringae ES4326 rpoN gene revealed that the deduced amino acid sequence was most similar (86% identity; 95% similarity) to the ς54 protein encoded by thePseudomonas putida rpoN gene. A marker exchange protocol was used to construct an ES4326 rpoN insertional mutation,rpoN::Kmr. In contrast to wild-type ES4326, ES4326 rpoN::Kmr was nonmotile and could not utilize nitrate, urea, C4-dicarboxylic acids, several amino acids, or concentrations of ammonia below 2 mM as nitrogen sources.rpoN was essential for production of the phytotoxin coronatine and for expression of the structural genes encoding coronamic acid. In addition, ES4326rpoN::Kmr did not multiply or elicit disease symptoms when infiltrated into Arabidopsis thalianaleaves, did not elicit the accumulation of severalArabidopsis defense-related mRNAs, and did not elicit a hypersensitive response (HR) when infiltrated into tobacco (Nicotiana tabacum) leaves. Furthermore, whereas P. syringae ES4326 carrying the avirulence gene avrRpt2elicited an HR when infiltrated into Arabidopsis ecotype Columbia leaves, ES4326 rpoN::Kmrcarrying avrRpt2 elicited no response. Constitutive expression of ES4326 hrpL in ES4326rpoN::Kmr partially restored defense-related mRNA accumulation, showing a direct role for thehrp cluster in host defense gene induction in a compatible host-pathogen interaction. However, constitutive expression ofhrpL in ES4326 rpoN::Kmrdid not restore coronatine production, showing that coronatine biosynthesis requires factors other than hrpL.

scholarly journals Confocal Imaging of Pseudomonas syringae pv. phaseolicola Colony Development in Bean Reveals Reduced Multiplication of Strains Containing the Genomic Island PPHGI-1

2010 ◽  
Vol 23 (10) ◽  
pp. 1294-1302 ◽  
Author(s):  
S. A. C. Godfrey ◽  
J. W. Mansfield ◽  
D. S. Corry ◽  
H. C. Lovell ◽  
R. W. Jackson ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Genomic Island ◽  
Effective Means ◽  
Leaf Tissue ◽  
Hypersensitive Reaction ◽  
Confocal Imaging ◽  
Avirulence Gene ◽  
Colony Development ◽  
Bacterial Survival ◽  
In Planta

Pseudomonas syringae pv. phaseolicola is the seed borne causative agent of halo blight in the common bean Phaseolus vulgaris. Pseudomonas syringae pv. phaseolicola race 4 strain 1302A contains the avirulence gene hopAR1 (located on a 106-kb genomic island, PPHGI-1, and earlier named avrPphB), which matches resistance gene R3 in P. vulgaris cultivar Tendergreen (TG) and causes a rapid hypersensitive reaction (HR). Here, we have fluorescently labeled selected Pseudomonas syringae pv. phaseolicola 1302A and 1448A strains (with and without PPHGI-1) to enable confocal imaging of in-planta colony formation within the apoplast of resistant (TG) and susceptible (Canadian Wonder [CW]) P. vulgaris leaves. Temporal quantification of fluorescent Pseudomonas syringae pv. phaseolicola colony development correlated with in-planta bacterial multiplication (measured as CFU/ml) and is, therefore, an effective means of monitoring Pseudomonas syringae pv. phaseolicola endophytic colonization and survival in P. vulgaris. We present advances in the application of confocal microscopy for in-planta visualization of Pseudomonas syringae pv. phaseolicola colony development in the leaf mesophyll to show how the HR defense response greatly affects colony morphology and bacterial survival. Unexpectedly, the presence of PPHGI-1 was found to cause a reduction of colony development in susceptible P. vulgaris CW leaf tissue. We discuss the evolutionary consequences that the acquisition and retention of PPHGI-1 brings to Pseudomonas syringae pv. phaseolicola in planta.

scholarly journals Molecular Determinants Required for the Avirulence Function of AvrPphB in Bean and Other Plants

2002 ◽  
Vol 15 (3) ◽  
pp. 292-300 ◽  
Author(s):  
Anastasia P. Tampakaki ◽  
Marina Bastaki ◽  
John W. Mansfield ◽  
Nickolas J. Panopoulos
Keyword(s):  
Pseudomonas Syringae ◽  
Potato Virus X ◽  
Hypersensitive Reaction ◽  
Avirulence Gene ◽  
In Planta ◽  
Mutant Proteins ◽  
Type Iii ◽  
Amino Terminal ◽  
Plant Recognition ◽  
Effector Recognition

The avirulence gene avrPphB from Pseudomonas syringae pv. phaseolicola determines incompatibility, manifested as a hypersensitive reaction (HR), on bean cultivars carrying the R3 resistance gene and also confers avirulence on other plants. The AvrPphB protein carries an embedded consensus myristoylation motif and is cleaved in bacteria and certain plants to yield fragments of about 6 and 28 kDa. We investigated plant recognition and type III translocation determinants in AvrPphB by constructing three N-terminally truncated and two site-directed mutants carrying substitutions in the conserved G63 residue of the myristoy-lation motif, which lies adjacent to the proteolytic cleavage site. The peptides were either delivered to plant cells by pseudomonads or were expressed transiently in planta via the Agrobacterium tumefaciens or Potato virus X. The 63 amino terminal residues were required for type III-mediated translocation from Pseudomonas strains to the plant, but were partially dispensable for effector recognition following in planta expression. Substitution of the G63 residue resulted in differential HR phenotypes in two different R3 cultivars of bean and abolished effector processing in Pseudomonas strains. Agrobacterium-mediated expression of the mutant proteins elicited HR in resistant bean hosts and in tomato but elicited no reaction in Nicotiana species.

Isolation and Characterization of Broad-Spectrum Disease-Resistant Arabidopsis Mutants

Genetics ◽  
2002 ◽  
Vol 160 (4) ◽  
pp. 1661-1671
Author(s):  
Klaus Maleck ◽  
Urs Neuenschwander ◽  
Rebecca M Cade ◽  
Robert A Dietrich ◽  
Jeffery L Dangl ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Systemic Acquired Resistance ◽  
Acquired Resistance ◽  
Constitutive Expression ◽  
Firefly Luciferase ◽  
Marker Genes ◽  
Arabidopsis Mutants ◽  
Isolation And Characterization ◽  
Firefly Luciferase Gene

Abstract To identify Arabidopsis mutants that constitutively express systemic acquired resistance (SAR), we constructed reporter lines expressing the firefly luciferase gene under the control of the SAR-inducible PR-1 promoter (PR-1/luc). After EMS mutagenesis of a well-characterized transgenic line, we screened 250,000 M2 plants for constitutive expression of the reporter gene in vivo. From a mutant collection containing several hundred putative mutants, we concentrated on 16 mutants lacking spontaneous hypersensitive response (HR) cell death. We mapped 4 of these constitutive immunity (cim) mutants to chromosome arms. Constitutive expression of disease resistance was established by analyzing responses to virulent Peronospora parasitica and Pseudomonas syringae strains, by RNA blot analysis for endogenous marker genes, and by determination of salicylic acid levels in the mutants. The variety of the cim phenotypes allowed us to define distinct steps in both the canonical SAR signaling pathway and a separate pathway for resistance to Erysiphe cichoracearum, active in only a subset of the mutants.

scholarly journals Identification of Three Putative Signal Transduction Genes Involved in R Gene-Specified Disease Resistance in Arabidopsis

Genetics ◽  
1999 ◽  
Vol 152 (1) ◽  
pp. 401-412 ◽  
Author(s):  
Randall F Warren ◽  
Peter M Merritt ◽  
Eric Holub ◽  
Roger W Innes
Keyword(s):  
Disease Resistance ◽  
Pseudomonas Syringae ◽  
Resistance Genes ◽  
Virulent Strain ◽  
Disease Resistance Genes ◽  
Avirulence Gene ◽  
Detectable Effect ◽  
Disease Resistance Gene ◽  
Protein Functions ◽  
Signal Transduction Genes

Abstract The RPS5 disease resistance gene of Arabidopsis mediates recognition of Pseudomonas syringae strains that possess the avirulence gene avrPphB. By screening for loss of RPS5-specified resistance, we identified five pbs (avrPphB susceptible) mutants that represent three different genes. Mutations in PBS1 completely blocked RPS5-mediated resistance, but had little to no effect on resistance specified by other disease resistance genes, suggesting that PBS1 facilitates recognition of the avrPphB protein. The pbs2 mutation dramatically reduced resistance mediated by the RPS5 and RPM1 resistance genes, but had no detectable effect on resistance mediated by RPS4 and had an intermediate effect on RPS2-mediated resistance. The pbs2 mutation also had varying effects on resistance mediated by seven different RPP (recognition of Peronospora parasitica) genes. These data indicate that the PBS2 protein functions in a pathway that is important only to a subset of disease-resistance genes. The pbs3 mutation partially suppressed all four P. syringae-resistance genes (RPS5, RPM1, RPS2, and RPS4), and it had weak-to-intermediate effects on the RPP genes. In addition, the pbs3 mutant allowed higher bacterial growth in response to a virulent strain of P. syringae, indicating that the PBS3 gene product functions in a pathway involved in restricting the spread of both virulent and avirulent pathogens. The pbs mutations are recessive and have been mapped to chromosomes I (pbs2) and V (pbs1 and pbs3).

scholarly journals Expression of a Truncated Yeast Ccc1 Vacuolar Transporter Increases the Accumulation of Endogenous Iron

Genes ◽  
2021 ◽  
Vol 12 (8) ◽  
pp. 1120
Author(s):  
Raquel Sorribes-Dauden ◽  
María Teresa Martínez-Pastor ◽  
Sergi Puig
Keyword(s):  
Iron Homeostasis ◽  
Sufficient Conditions ◽  
Constitutive Expression ◽  
Iron Bioavailability ◽  
Full Length ◽  
Yeast Cells ◽  
Deficiency Anemia ◽  
Functional Protein ◽  
Amino Terminal ◽  
In Cells

Iron is an essential micronutrient for all eukaryotic organisms because it participates as a redox cofactor in multiple metabolic processes. Iron bioavailability is highly restricted due to the low solubility of its oxidized form, frequently leading to iron deficiency anemia. The baker’s yeast Saccharomyces cerevisiae is used as a model organism for iron homeostasis studies, but also as a food supplement and fermentative microorganism in the food industry. Yeast cells use the vacuolar Ccc1 transporter to detoxify and store excess iron in the vacuoles. Here, we modulate CCC1 expression and properties to increase iron extraction from the environment. We show that constitutive expression of full-length CCC1 is toxic, whereas deletion of its cytosolic amino-terminal (Nt) domain (NtDCCC1) rescues this phenotype. Toxicity is exacerbated in cells lacking AFT1 transcription factor. Further characterization of NtDCcc1 protein suggests that it is a partially functional protein. Western blot analyses indicate that deletion of Ccc1 Nt domain does not significantly alter GFP-Ccc1 protein stability. A functional full-length GFP-Ccc1 protein localized to particular regions of the vacuolar membrane, whereas GFP-NtDCcc1 protein was evenly distributed throughout this endogenous membrane. Interestingly, expression of NtDCCC1 increased the accumulation of endogenous iron in cells cultivated under iron-sufficient conditions, a strategy that could be used to extract iron from media that are not rich in iron.

scholarly journals High-density transposon libraries utilising outward-oriented promoters identify mechanisms of action and resistance to antimicrobials

2020 ◽  
Vol 367 (22) ◽  
Author(s):  
Chris Coward ◽  
Gopujara Dharmalingham ◽  
Omar Abdulle ◽  
Tim Avis ◽  
Stephan Beisken ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Pathogenic Bacteria ◽  
Selective Advantage ◽  
Mechanisms Of Action ◽  
Over Expression ◽  
E Coli ◽  
Synthase Inhibitor ◽  
Established Technique ◽  
Bacterial Transposon

ABSTRACT The use of bacterial transposon mutant libraries in phenotypic screens is a well-established technique for determining which genes are essential or advantageous for growth in conditions of interest. Standard, inactivating, transposon libraries cannot give direct information about genes whose over-expression gives a selective advantage. We report the development of a system wherein outward-oriented promoters are included in mini-transposons, generation of transposon mutant libraries in Escherichia coli and Pseudomonas aeruginosa and their use to probe genes important for growth under selection with the antimicrobial fosfomycin, and a recently-developed leucyl-tRNA synthase inhibitor. In addition to the identification of known mechanisms of action and resistance, we identify the carbon–phosphorous lyase complex as a potential resistance liability for fosfomycin in E. coli and P. aeruginosa. The use of this technology can facilitate the development of novel mechanism-of-action antimicrobials that are urgently required to combat the increasing threat worldwide from antimicrobial-resistant pathogenic bacteria.

scholarly journals Construction and Characterization of a proU-gfp Transcriptional Fusion That Measures Water Availability in a Microbial Habitat

2002 ◽  
Vol 68 (9) ◽  
pp. 4604-4612 ◽  
Author(s):  
Catherine A. Axtell ◽  
Gwyn A. Beattie
Keyword(s):  
Pseudomonas Syringae ◽  
Water Availability ◽  
Water Deprivation ◽  
Bacterial Species ◽  
Transcriptional Fusion ◽  
Bacterial Biosensor ◽  
E Coli ◽  
Quantitative Manner ◽  
Microbial Habitat

ABSTRACT We constructed and characterized a transcriptional fusion that measures the availability of water to a bacterial cell. This fusion between the proU promoter from Escherichia coli and the reporter gene gfp was introduced into strains of E. coli, Pantoea agglomerans, and Pseudomonas syringae. The proU-gfp fusion in these bacterial biosensor strains responded in a quantitative manner to water deprivation caused by the presence of NaCl, Na2SO4, KCl, or polyethylene glycol (molecular weight, 8000). The fusion was induced to a detectable level by NaCl concentrations of as low as 10 mM in all three bacterial species. Water deprivation induced proU-gfp expression in both planktonic and surface-associated cells; however, it induced a higher level of expression in the surface-associated cells. Following the introduction of P. agglomerans biosensor cells onto bean leaves, the cells detected a significant decrease in water availability within only 5 min. After 30 min, the populations were exposed, on average, to a water potential equivalent to that imposed by approximately 55 mM NaCl. These results demonstrate the effectiveness of a proU-gfp-based biosensor for evaluating water availability on leaves. Furthermore, the inducibility of proU-gfp in multiple bacterial species illustrates the potential for tailoring proU-gfp-based biosensors to specific habitats.

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