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 Immunogold Labeling of Hrp Pili of Pseudomonas syringae pv. tomato Assembled in Minimal Medium and In Planta

2001 ◽  
Vol 14 (2) ◽  
pp. 234-241 ◽  
Author(s):  
Wenqi Hu ◽  
Jing Yuan ◽  
Qiao-Ling Jin ◽  
Patrick Hart ◽  
Sheng Yang He
Keyword(s):  
Arabidopsis Thaliana ◽  
Pseudomonas Syringae ◽  
Minimal Medium ◽  
Leaf Tissue ◽  
Hypersensitive Reaction ◽  
Immunogold Labeling ◽  
Structural Protein ◽  
In Planta ◽  
Hrp Genes

Hypersensitive reaction and pathogenicity (hrp) genes are required for Pseudomonas syringae pv. tomato (Pst) DC3000 to cause disease in susceptible tomato and Arabidopsis thaliana plants and to elicit the hypersensitive response in resistant plants. The hrp genes encode a type III protein secretion system known as the Hrp system, which in Pst DC3000 secretes HrpA, HrpZ, HrpW, and AvrPto and assembles a surface appendage, named the Hrp pilus, in hrp-gene-inducing minimal medium. HrpA has been suggested to be the Hrp pilus structural protein on the basis of copurification and mutational analyses. In this study, we show that an antibody against HrpA efficiently labeled Hrp pili, whereas antibodies against HrpW and HrpZ did not. Immunogold labeling of bacteria-infected Arabidopsis thaliana leaf tissue with an Hrp pilus antibody revealed a characteristic lineup of gold particles around bacteria and/or at the bacterium-plant contact site. These results confirm that HrpA is the major structural protein of the Hrp pilus and provide evidence that Hrp pili are assembled in vitro and in planta.

scholarly journals Components of the Pseudomonas syringae Type III Secretion System Can Suppress and May Elicit Plant Innate Immunity

2010 ◽  
Vol 23 (6) ◽  
pp. 727-739 ◽  
Author(s):  
Hye-Sook Oh ◽  
Duck Hwan Park ◽  
Alan Collmer
Keyword(s):  
Pseudomonas Syringae ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Leaf Tissue ◽  
Secretion System ◽  
Colony Development ◽  
Plant Responses ◽  
In Planta ◽  
Callose Deposition ◽  
Type Iii

The type III secretion system (T3SS) of Pseudomonas syringae translocates into plant cells multiple effectors that suppress pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). P. syringae pv. tomato DC3000 no longer delivers the T3SS translocation reporter AvrPto-Cya in Nicotiana benthamiana leaf tissue in which PTI was induced by prior inoculation with P. fluorescens(pLN18). Cosmid pLN18 expresses the T3SS system of P. syringae pv. syringae 61 but lacks the hopA1Psy61 effector gene. P. fluorescens(pLN18) expressing HrpHPtoDC3000 or HopP1PtoDC3000, two T3SS-associated putative lytic transglycosylases, suppresses PTI, based on multiple assays involving DC3000 challenge inoculum (AvrPto-Cya translocation, hypersensitive response elicitation, and colony development in planta) or on plant responses (vascular dye uptake or callose deposition). Analysis of additional mutations in pHIR11 derivatives revealed that the pLN18-encoded T3SS elicits a higher level of reactive oxygen species (ROS) than does P. fluorescens without a T3SS, that enhanced ROS production is dependent on the HrpK1 translocator, and that HopA1Psy61 suppresses ROS elicitation attributable to both the P. fluorescens PAMPs and the presence of a functional T3SS.

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.

Identification of a DNA region required for growth of Pseudomonas syringae pv. tomato on tomato plants

1992 ◽  
Vol 38 (9) ◽  
pp. 883-890 ◽  
Author(s):  
Dennis P. Jackson ◽  
Douglas A. Gray ◽  
Vincent L. Morris ◽  
Diane A. Cuppels
Keyword(s):  
Organic Acids ◽  
Pseudomonas Syringae ◽  
Acid Metabolism ◽  
Carbon Sources ◽  
Hypersensitive Reaction ◽  
Wild Type ◽  
In Planta ◽  
Tomato Plants ◽  
Tartaric Acids ◽  
Nonpathogenic Strain

The prototrophic Pseudomonas syringae pv. tomato mutant DC3481, which is the result of a single-site Tn5 insertion, cannot grow and cause disease on tomato plants and cannot use the major organic acids of tomato, i.e., citric, malic, succinic, and tartaric acids, as sole carbon sources. Although nonpathogenic, strain DC3481 can still induce a hypersensitive reaction in nonhost plants. We have identified a 30-kb fragment of P. syringae pv. tomato wild-type DNA that can complement this mutant. EcoRI fragments from this region were subcloned and individually subjected to functional complementation analysis. The 3.8-kb fragment, which was the site of the Tn5 insertion, restored pathogenicity and the ability to use all the major organic acids of tomato as carbon sources. It shares sequence homology with several P. syringae pathovars but not other bacterial tomato pathogens. Our results indicate that sequences on the 3.8-kb EcoRI fragment are required for both the ability to grow on tomato leaves (and thus cause disease) and the utilization of carboxylic acids common to tomato. The 3.8-kb fragment may contain a sequence (or sequences) that regulates both traits. Key words: Pseudomonas syringae pv. tomato, phytopathogenicity, Tn5, tricarboxylic acid metabolism, bacterial speck, growth in planta.

The 73-kb pIAA plasmid increases competitive fitness of Pseudomonas syringae subspecies savastanoi in oleander

1993 ◽  
Vol 39 (7) ◽  
pp. 659-664 ◽  
Author(s):  
Sara E. Silverstone ◽  
David G. Gilchrist ◽  
Richard M. Bostock ◽  
Tsune Kosuge
Keyword(s):  
Population Density ◽  
Pseudomonas Syringae ◽  
Indoleacetic Acid ◽  
Leaf Tissue ◽  
Insertion Mutant ◽  
Wild Type ◽  
In Planta ◽  
Bacterial Strains ◽  
Growth And Survival ◽  
Iaa Production

Pseudomonas syringae subsp. savastanoi causes tumors on olive and oleander by producing the plant growth regulators indoleacetic acid (IAA) and cytokinins following infection of the plant. The contribution of IAA production to the ability of P. syringae subsp. savastanoi to grow and survive in oleander leaf tissue was studied. Bacterial strains differing only with respect to IAA production were characterized. Growth and survival of wild-type and two mutant strains of P. syringae subsp. savastanoi in oleander leaf tissue were monitored by weekly colony counts and IAA plate assays. Growth rate of the three strains in culture and in planta did not differ significantly. However, the wild-type strain reached a higher population density and maintained its maximum density at least 9 weeks longer than either mutant population. An insertion mutant containing the IAA plasmid (pIAA), but incapable of IAA production, did not maintain a higher population density than a strain cured of the IAA plasmid. The pIAA-cured strain maintained a higher population density when coinoculated with an IAA-producing strain than when inoculated alone. These results suggest that IAA production may contribute to the fitness of P. syringae subsp. savastanoi in oleander tissue and that the iaa operon alone may be responsible for the competitive advantage of cells harboring pIAA.Key words: indoleacetic acid, bacterial ecology.

scholarly journals Biophysical and proteomic analyses suggest functions of Pseudomonas syringae pv tomato DC3000 extracellular vesicles in bacterial growth during plant infection

2021 ◽  
Author(s):  
Martin Janda ◽  
Christina Ludwig ◽  
Katarzyna Rybak ◽  
Chen Meng ◽  
Egidio Stigliano ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Extracellular Vesicles ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Bioinformatic Analysis ◽  
Potential Contribution ◽  
Bacterial Cells ◽  
Bacterial Survival ◽  
In Planta ◽  
Plant Infection

SummaryVesiculation is a process employed by Gram-negative bacteria to release extracellular vesicles (EVs) into the environment. Bacterial EVs contain molecular cargo from the donor bacterium and play important roles in bacterial survival and growth. Here, we describe EV production in plant-pathogenic Pseudomonas syringae pv. tomato DC3000 (Pto DC3000), the causal agent of bacterial speck disease. Cultured Pto DC3000 exhibited EV structures both on the cell surface and in the vicinity of bacterial cells, observed as outer membrane vesicle (OMV) release. We used in-solution trypsin digestion coupled to mass spectrometry to identify 369 proteins enriched in EVs recovered from cultured Pto DC3000. The predicted localization profile of EV proteins supports the production of EVs also in the form of outer-inner-membrane vesicles (OIMVs). EV production varied slightly between bacterial lifestyles and also occurred in planta. The potential contribution of EVs to Pto DC3000 plant infection was assessed using plant treatments and bioinformatic analysis of the EV-enriched proteins. While these results identify immunogenic activities of the EVs, they also point at roles for EVs in bacterial defences and nutrient acquisition by Pto DC3000.

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 Mutational Analysis of the Arabidopsis RPS2 Disease Resistance Gene and the Corresponding Pseudomonas syringae avrRpt2 Avirulence Gene

2001 ◽  
Vol 14 (2) ◽  
pp. 181-188 ◽  
Author(s):  
Michael J. Axtell ◽  
Timothy W. McNellis ◽  
Mary Beth Mudgett ◽  
Caroline S. Hsu ◽  
Brian J. Staskawicz
Keyword(s):  
Disease Resistance ◽  
Resistance Gene ◽  
Pseudomonas Syringae ◽  
Resistance Genes ◽  
Mutational Analysis ◽  
Defense Responses ◽  
Avirulence Gene ◽  
Disease Resistance Gene ◽  
In Planta ◽  
Hypersensitive Cell Death

Plants have evolved a large number of disease resistance genes that encode proteins containing conserved structural motifs that function to recognize pathogen signals and to initiate defense responses. The Arabidopsis RPS2 gene encodes a protein representative of the nucleotide-binding site-leucine-rich repeat (NBS-LRR) class of plant resistance proteins. RPS2 specifically recognizes Pseudomonas syringae pv. tomato strains expressing the avrRpt2 gene and initiates defense responses to bacteria carrying avrRpt2, including a hypersensitive cell death response (HR). We present an in planta mutagenesis experiment that resulted in the isolation of a series of rps2 and avrRpt2 alleles that disrupt the RPS2-avrRpt2 gene-for-gene interaction. Seven novel avrRpt2 alleles incapable of eliciting an RPS2-dependent HR all encode proteins with lesions in the C-terminal portion of AvrRpt2 previously shown to be sufficient for RPS2 recognition. Ten novel rps2 alleles were characterized with mutations in the NBS and the LRR. Several of these alleles code for point mutations in motifs that are conserved among NBS-LRR resistance genes, including the third LRR, which suggests the importance of these motifs for resistance gene function.

scholarly journals First Report of the Crucifer Pathogen Pseudomonas cannabina pv. alisalensis Causing Bacterial Blight on Radish (Raphanus sativus) in Germany

Plant Disease ◽  
2012 ◽  
Vol 96 (6) ◽  
pp. 904-904 ◽  
Author(s):  
I. Rubio ◽  
G. Hiddink ◽  
M. Asma ◽  
C. T. Bull
Keyword(s):  
Pseudomonas Syringae ◽  
Raphanus Sativus ◽  
Bacterial Blight ◽  
Management Strategies ◽  
Leaf Tissue ◽  
Hypersensitive Reaction ◽  
Negative Control ◽  
Banding Patterns ◽  
First Report ◽  
Dna Fragment

In 2008, a bacterial blight was observed on Raphanus sativus in the Pfalz region in Germany. Disease was sporadic but severe when present within R. sativus fields, which resulted in unmarketable crops. Symptoms consisted of small, angular, water-soaked flecks that often were surrounded by chlorotic haloes. Lesions were visible from adaxial and abaxial leaf surfaces and generally retained chlorotic borders. A gram-negative, bluefluorescing bacterium was isolated from surface-disinfested leaf tissue on King's medium B agar. The radish isolate was levan positive, oxidase negative, and arginine dihydrolase negative. The isolate did not rot potato slices but induced a hypersensitive reaction in tobacco. These reactions corresponded to Lelliot's LOPAT group 1 (2). Repetitive extragenic palindromic sequence (rep)-PCR assays using the BOXA1R primer resulted in different DNA fragment banding patterns between the radish isolate and the pathotype strain of Pseudomonas syringae pv. maculicola (CFBP 1657), but identical DNA fragment banding patterns between the radish isolate and the pathotype strain of P. cannabina pv. alisalensis (CFBP 6866). Unlike P. syringae pv. maculicola, P. cannabina pv. alisalensis and the radish isolate were lysed by bacteriophage PBS1 (1). Pathogenicity was evaluated on two hosts, radish (R. sativus cv. Comet) and broccoli raab (Brassica rapa cv. Sorrento). In each of two independent experiments, 3-week-old radish and broccoli raab plants were inoculated with either the radish isolate, P. cannabina pv. alisalensis, or P. syringae pv. maculicola. Inoculum was prepared by growing the bacteria on nutrient agar for 48 h at 27°C, suspending the bacteria in 0.01 M phosphate buffer (pH 7.0), and adjusting each suspension to 0.6 OD at 600 nm (approximately 1 × 108 CFU/ml). All plants were inoculated by spraying until runoff, incubated in a humidity chamber for 48 h, then placed in a greenhouse at 20 to 25°C for symptom development. Plants inoculated with P. cannabina pv. alisalensis or sprayed with buffer served as positive and negative control treatments, respectively. Seven to ten days postinoculation, the development of symptoms similar to those originally observed in the field were observed on plants inoculated with the radish isolate. In addition, symptoms on radish and broccoli raab plants caused by the radish isolate were similar to symptoms caused by P. cannabina pv. alisalensis in contrast to the lack of symptoms on plants inoculated with P. syringae pv. maculicola. Bacteria isolated from symptomatic tissue and surface-disinfested with sodium hypochlorite (0.525%) had identical characteristics to the radish isolate used to inoculate plants and to the P. cannabina pv. alisalensis pathotype for LOPAT reactions, rep-PCR DNA fragment banding pattern analysis, and sensitivity to phage PBS1, thus fulfilling Koch's postulates. To our knowledge, this is the first report of P. cannabina pv. alisalensis isolated from diseased crucifers in Germany. Verification of P. cannabina pv. alisalensis in Germany indicates that German crucifer growers should differentiate between outbreaks caused by P. cannabina pv. alisalensis and P. syringae pv. maculicola and apply appropriate, specific management strategies. References: (1) C. T. Bull et al. Syst. Appl. Microbiol. 33:105, 2010. (2) R. A. Lelliott. J. Appl. Bacteriol. 29:470, 1966.

scholarly journals Mutation in the avrBs1 Avirulence Gene of Xanthomonas campestris pv. vesicatoria Influences Survival of the Bacterium in Soil and Detached Leaf Tissue

1997 ◽  
Vol 87 (9) ◽  
pp. 960-966 ◽  
Author(s):  
Leonard W. O'Garro ◽  
Harold Gibbs ◽  
Anthony Newton
Keyword(s):  
Mutant Strain ◽  
Type Strain ◽  
Xanthomonas Campestris ◽  
Wild Type Strain ◽  
Leaf Tissue ◽  
Avirulence Gene ◽  
Soil Types ◽  
Bacterial Survival ◽  
Wild Type ◽  
Detached Leaf

The role of the avrBs1 avirulence gene of Xanthomonas campestris pv. vesicatoria in survival of the bacterium was investigated by testing two strains that differ in the structure and function of the gene in 37 different soil types of Barbados and detached leaf tissue of four pepper genotypes. One strain carried a mutation in the avrBs1 gene and lacked avirulence activity, while the other expressed wild-type avrBs1 activity. In 30 to 32 soil types and all leaf tissue tested, the mutant strain persisted longer and more abundantly than the wild-type strain over a 2- to 6-week period. During this time, the mutant strain generally replaced the wild-type strain completely in soil initially infested with a mixture of equal amounts of each strain and by a factor of 6.7 in similarly infested pepper leaves. Nine selected soil factors, namely pH, clay and cation content, and percent nitrogen, carbonates, carbon, K+, Na+, and Ca2+ did not affect bacterial survival significantly.

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