scholarly journals Induction and Suppression of PEN3 Focal Accumulation During Pseudomonas syringae pv. tomato DC3000 Infection of Arabidopsis

2013 ◽  
Vol 26 (8) ◽  
pp. 861-867 ◽  
Author(s):  
Xiu-Fang Xin ◽  
Kinya Nomura ◽  
William Underwood ◽  
Sheng Yang He
Keyword(s):  
Pseudomonas Syringae ◽  
Type Iii Secretion ◽  
Cellular Response ◽  
Fluorescent Protein ◽  
Bacterial Pathogen ◽  
Tomato Dc3000 ◽  
Positive Role ◽  
Powdery Mildew Fungi ◽  
Combined Action ◽  
Green Fluorescent

The pleiotropic drug resistance (PDR) proteins belong to the super-family of ATP-binding cassette (ABC) transporters. AtPDR8, also called PEN3, is required for penetration resistance of Arabidopsis to nonadapted powdery mildew fungi. During fungal infection, plasma-membrane-localized PEN3 is concentrated at fungal entry sites, as part of the plant's focal immune response. Here, we show that the pen3 mutant is compromised in resistance to the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. P. syringae pv. tomato DC3000 infection or treatment with a flagellin-derived peptide, flg22, induced strong focal accumulation of PEN3-green fluorescent protein. Interestingly, after an initial induction of PEN3 accumulation, P. syringae pv. tomato DC3000 but not the type-III-secretion-deficient mutant hrcC could suppress PEN3 accumulation. Moreover, transgenic overexpression of the P. syringae pv. tomato DC3000 effector AvrPto was sufficient to suppress PEN3 focal accumulation in response to flg22. Analyses of P. syringae pv. tomato DC3000 effector deletion mutants showed that individual effectors, including AvrPto, appear to be insufficient to suppress PEN3 accumulation when delivered by bacteria, suggesting a requirement for a combined action of multiple effectors. Collectively, our results indicate that PEN3 plays a positive role in plant resistance to a bacterial pathogen and show that focal accumulation of PEN3 protein may be a useful cellular response marker for the Arabidopsis–P. syringae interaction.

scholarly journals Pseudomonas syringae HrpP Is a Type III Secretion Substrate Specificity Switch Domain Protein That Is Translocated into Plant Cells but Functions Atypically for a Substrate-Switching Protein

2009 ◽  
Vol 191 (9) ◽  
pp. 3120-3131 ◽  
Author(s):  
Joanne E. Morello ◽  
Alan Collmer
Keyword(s):  
Substrate Specificity ◽  
Pseudomonas Syringae ◽  
Type Iii Secretion ◽  
Fluorescent Protein ◽  
Plant Cells ◽  
Effector Proteins ◽  
Type Iii ◽  
Native Promoter ◽  
C Terminus ◽  
Green Fluorescent

ABSTRACT Pseudomonas syringae delivers virulence effector proteins into plant cells via an Hrp1 type III secretion system (T3SS). P. syringae pv. tomato DC3000 HrpP has a C-terminal, putative T3SS substrate specificity switch domain, like Yersinia YscP. A ΔhrpP DC3000 mutant could not cause disease in tomato or elicit a hypersensitive response (HR) in tobacco, but the HR could be restored by expression of HrpP in trans. Though HrpP is a relatively divergent protein in the T3SS of different P. syringae pathovars, hrpP from P. syringae pv. syringae 61 and P. syringae pv. phaseolicola 1448A restored HR elicitation and pathogenicity to DC3000 ΔhrpP. HrpP was translocated into Nicotiana benthamiana cells via the DC3000 T3SS when expressed from its native promoter, but it was not secreted in culture. N- and C-terminal truncations of HrpP were tested for their ability to be translocated and to restore HR elicitation activity to the ΔhrpP mutant. No N-terminal truncation completely abolished translocation, implying that HrpP has an atypical T3SS translocation signal. Deleting more than 20 amino acids from the C terminus abolished the ability to restore HR elicitation. HrpP fused to green fluorescent protein was no longer translocated but could restore HR elicitation activity to the ΔhrpP mutant, suggesting that translocation is not essential for the function of HrpP. No T3SS substrates were detectably secreted by DC3000 ΔhrpP except the pilin subunit HrpA, which unexpectedly was secreted poorly. HrpP may function somewhat differently than YscP because the P. syringae T3SS pilus likely varies in length due to differing plant cell walls.

scholarly journals A Gene in the Pseudomonas syringae pv. tomato Hrp Pathogenicity Island Conserved Effector Locus, hopPtoA1, Contributes to Efficient Formation of Bacterial Colonies in Planta and Is Duplicated Elsewhere in the Genome

2002 ◽  
Vol 15 (10) ◽  
pp. 1014-1024 ◽  
Author(s):  
J. L. Badel ◽  
A. O. Charkowski ◽  
W.-L. Deng ◽  
A. Collmer
Keyword(s):  
Pseudomonas Syringae ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Pathogenicity Island ◽  
Effector Proteins ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
In Planta ◽  
Tomato Dc3000 ◽  
Green Fluorescent

The ability of Pseudomonas syringae to grow in planta is thought to be dependent upon the Hrp (type III secretion) system and multiple effector proteins that this system injects into plant cells. ORF5 in the conserved effector locus of the P. syringae pv. tomato DC3000 Hrp pathogenicity island was shown to encode a Hrp-secreted protein and to have a similarly secreted homolog encoded in an effector-rich pathogenicity island located elsewhere in the genome. These putative effector genes were designated hopPtoA1 and hopPtoA2, respectively. DNA gel blot analysis revealed that sequences hybridizing with hopPtoA1 were widespread among P. syringae pathovars, and some strains, like DC3000, appear to have two copies of the gene. uidA transcriptional fusions revealed that expression of hopPtoA1 and hopPtoA2 can be activated by the HrpL alternative sigma factor. hopPtoA1 and hopPtoA1/hopPtoA2 double mutants were not obviously different from wild-type P. syringae pv. tomato DC3000 in their ability to produce symptoms or to increase their total population size in host tomato and Arabidopsis leaves. However, confocal laser-scanning microscopy of GFP (green fluorescent protein)-labeled bacteria in Arabidopsis leaves 2 days after inoculation revealed that the frequency of undeveloped individual colonies was higher in the hopPtoA1 mutant and even higher in the hopPtoA1/hopPtoA2 double mutant. These results suggest that hopPtoA1 and hopPtoA2 contribute redundantly to the formation of P. syringae pv. tomato DC3000 colonies in Arabidopsis leaves.

scholarly journals Spatiotemporal Monitoring of Pseudomonas syringae Effectors via Type III Secretion Using Split Fluorescent Protein Fragments

2017 ◽  
Vol 29 (7) ◽  
pp. 1571-1584 ◽  
Author(s):  
Eunsook Park ◽  
Hye-Young Lee ◽  
Jongchan Woo ◽  
Doil Choi ◽  
Savithramma P. Dinesh-Kumar
Keyword(s):  
Pseudomonas Syringae ◽  
Type Iii Secretion ◽  
Fluorescent Protein ◽  
Protein Fragments ◽  
Type Iii

scholarly journals A revised model for the role of GacS/GacA in regulating type III secretion by Pseudomonas syringae pv. tomato DC3000

2019 ◽  
Vol 21 (1) ◽  
pp. 139-144 ◽  
Author(s):  
Megan R. O’Malley ◽  
Ching‐Fang Chien ◽  
Scott C. Peck ◽  
Nai‐Chun Lin ◽  
Jeffrey C. Anderson
Keyword(s):  
Pseudomonas Syringae ◽  
Type Iii Secretion ◽  
Tomato Dc3000 ◽  
Type Iii

Inhibition of the type III secretion system of Pseudomonas syringae pv. tomato DC3000 by resveratrol oligomers identified in Vitis vinifera L

2020 ◽  
Vol 76 (7) ◽  
pp. 2294-2303 ◽  
Author(s):  
Ji Eun Kang ◽  
Byeong Jun Jeon ◽  
Min Young Park ◽  
Hye Ji Yang ◽  
Jaeyoung Kwon ◽  
...  
Keyword(s):  
Vitis Vinifera ◽  
Pseudomonas Syringae ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Vitis Vinifera L ◽  
Tomato Dc3000 ◽  
Type Iii

scholarly journals A C-Terminal Domain Targets the Pseudomonas aeruginosa Cytotoxin ExoU to the Plasma Membrane of Host Cells

2006 ◽  
Vol 74 (5) ◽  
pp. 2552-2561 ◽  
Author(s):  
Shira D. P. Rabin ◽  
Jeffrey L. Veesenmeyer ◽  
Kathryn T. Bieging ◽  
Alan R. Hauser
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Cell Death ◽  
Plasma Membrane ◽  
Hela Cells ◽  
Type Iii Secretion ◽  
Fluorescent Protein ◽  
Host Cells ◽  
Type Iii ◽  
C Terminus ◽  
Green Fluorescent

ABSTRACT ExoU, a phospholipase injected into host cells by the type III secretion system of Pseudomonas aeruginosa, leads to rapid cytolytic cell death. Although the importance of ExoU in infection is well established, the mechanism by which this toxin kills host cells is less clear. To gain insight into how ExoU causes cell death, we examined its subcellular localization following transfection or type III secretion/translocation into HeLa cells. Although rapid cell lysis precluded visualization of wild-type ExoU by fluorescence microscopy, catalytically inactive toxin was readily detected at the periphery of HeLa cells. Biochemical analysis confirmed that ExoU was targeted to the membrane fraction of transfected cells. Visualization of ExoU peptides fused with green fluorescent protein indicated that the domain responsible for this targeting was in the C terminus of ExoU, between residues 550 and 687. Localization to the plasma membrane occurred within 1 h of expression, which is consistent with the kinetics of cytotoxicity. Together, these results indicate that a domain between residues 550 and 687 of ExoU targets this toxin to the plasma membrane, a process that may be important in cytotoxicity.

scholarly journals Ecological Basis of the Interaction betweenPseudozyma flocculosaand Powdery Mildew Fungi

2010 ◽  
Vol 77 (3) ◽  
pp. 926-933 ◽  
Author(s):  
Walid Hammami ◽  
Candy Quiroga Castro ◽  
Wilfried Rémus-Borel ◽  
Caroline Labbé ◽  
Richard R. Bélanger
Keyword(s):  
Powdery Mildew ◽  
Fluorescent Protein ◽  
Close Relative ◽  
Tomato Plants ◽  
Powdery Mildews ◽  
Biocontrol Activity ◽  
Cellobiose Lipids ◽  
Powdery Mildew Fungi ◽  
Green Fluorescent ◽  
Ecological Basis

ABSTRACTIn this work, we sought to understand how glycolipid production and the availability of nutrients could explain the ecology ofPseudozyma flocculosaand its biocontrol activity. For this purpose, we compared the development ofP. flocculosato that of a close relative, the plant pathogenUstilago maydis, under different environmental conditions. This approach was further supported by measuring the expression ofcyp1, a pivotal gene in the synthesis of unique antifungal cellobiose lipids of both fungi. On healthy cucumber and tomato plants, the expression ofcyp1remained unchanged over time inP. flocculosaand was undetected inU. maydis. At the same time, green fluorescent protein (GFP) strains of both fungi showed only limited green fluorescence on control leaves. On powdery mildew-infected cucumber leaves,P. flocculosainduced a complete collapse of the pathogen colonies, but glycolipid production, as studied bycyp1expression, was still comparable to that of controls. In complete contrast,cyp1was upregulated nine times whenP. flocculosawas applied toBotrytis cinerea-infected leaves, but the biocontrol fungus did not develop very well on the pathogen. Analysis of the possible nutrients that could stimulate the growth ofP. flocculosaon powdery mildew structures revealed that the complex Zn/Mn played a key role in the interaction. Other related fungi such asU. maydisdo not appear to have the same nutritional requirements and hence lack the ability to colonize powdery mildews. Whether production of antifungal glycolipids contributes to the release of nutrients from powdery mildew colonies is unclear, but the specificity of the biocontrol activity ofP. flocculosatoward Erysiphales does appear to be more complex than simple antibiosis.

scholarly journals Cytoplasmic Protein Mobility in Osmotically Stressed Escherichia coli

2008 ◽  
Vol 191 (1) ◽  
pp. 231-237 ◽  
Author(s):  
Michael C. Konopka ◽  
Kem A. Sochacki ◽  
Benjamin P. Bratton ◽  
Irina A. Shkel ◽  
M. Thomas Record ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cellular Response ◽  
Fluorescent Protein ◽  
Volume Fraction ◽  
E Coli ◽  
Surprising Effect ◽  
Wide Range ◽  
Underlying Causes ◽  
The Mean ◽  
Green Fluorescent

ABSTRACT Facile diffusion of globular proteins within a cytoplasm that is dense with biopolymers is essential to normal cellular biochemical activity and growth. Remarkably, Escherichia coli grows in minimal medium over a wide range of external osmolalities (0.03 to 1.8 osmol). The mean cytoplasmic biopolymer volume fraction (〈φ〉) for such adapted cells ranges from 0.16 at 0.10 osmol to 0.36 at 1.45 osmol. For cells grown at 0.28 osmol, a similar 〈φ〉 range is obtained by plasmolysis (sudden osmotic upshift) using NaCl or sucrose as the external osmolyte, after which the only available cellular response is passive loss of cytoplasmic water. Here we measure the effective axial diffusion coefficient of green fluorescent protein (D GFP) in the cytoplasm of E. coli cells as a function of 〈φ〉 for both plasmolyzed and adapted cells. For plasmolyzed cells, the median D GFP ( \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(D_{GFP}^{m}\) \end{document} ) decreases by a factor of 70 as 〈φ〉 increases from 0.16 to 0.33. In sharp contrast, for adapted cells, \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(D_{GFP}^{m}\) \end{document} decreases only by a factor of 2.1 as 〈φ〉 increases from 0.16 to 0.36. Clearly, GFP diffusion is not determined by 〈φ〉 alone. By comparison with quantitative models, we show that the data cannot be explained by crowding theory. We suggest possible underlying causes of this surprising effect and further experiments that will help choose among competing hypotheses. Recovery of the ability of proteins to diffuse in the cytoplasm after plasmolysis may well be a key determinant of the time scale of the recovery of growth.

Type III secretion chaperones ShcS1 and ShcO1 from Pseudomonas syringae pv. tomato DC3000 bind more than one effector

Microbiology ◽  
2005 ◽  
Vol 151 (1) ◽  
pp. 269-280 ◽  
Author(s):  
Ute Kabisch ◽  
Angelika Landgraf ◽  
Jana Krause ◽  
Ulla Bonas ◽  
Jens Boch
Keyword(s):  
Pseudomonas Syringae ◽  
Type Iii Secretion ◽  
Gel Filtration ◽  
Effector Proteins ◽  
Yeast Two Hybrid ◽  
Hybrid Analysis ◽  
Tomato Dc3000 ◽  
Type Iii ◽  
Effector Genes ◽  
Two Hybrid

The hrp-type III secretion (TTS) system is a key pathogenicity factor of the plant pathogen Pseudomonas syringae pv. tomato DC3000 that translocates effector proteins into the cytosol of the eukaryotic host cell. The translocation of a subset of effectors is dependent on specific chaperones. In this study an operon encoding a TTS chaperone (ShcS1) and the truncated effector HopS1′ was characterized. Yeast two-hybrid analysis and pull-down assays demonstrated that these proteins interact. Using protein fusions to AvrRpt2 it was shown that ShcS1 facilitates the translocation of HopS1′, suggesting that ShcS1 is a TTS chaperone for HopS1′ and that amino acids 1 to 118 of HopS1′ are required for translocation. P. syringae pv. tomato DC3000 carries two shcS1 homologues, shcO1 and shcS2, which are located in different operons, and both operons include additional putative effector genes. Transcomplementation experiments showed that ShcS1 and ShcO1, but not ShcS2, can facilitate the translocation of HopS1′ : : AvrRpt2. To characterize the specificities of the putative chaperones, yeast two-hybrid interaction studies were performed between the three chaperones and putative target effectors. These experiments showed that both ShcS1 and ShcO1 bind to two different effectors, HopS1′ and HopO1-1, that share only 16 % amino acid sequence identity. Using gel filtration it was shown that ShcS1 forms homodimers, and this was confirmed by yeast two-hybrid experiments. In addition, ShcS1 is also able to form heterodimers with ShcO1. These data demonstrate that ShcS1 and ShcO1 are exceptional class IA TTS chaperones because they can bind more than one target effector.

scholarly journals Characterization of a Novel, Defense-Related Arabidopsis Mutant, cir1, Isolated By Luciferase Imaging

2002 ◽  
Vol 15 (6) ◽  
pp. 557-566 ◽  
Author(s):  
Shane L. Murray ◽  
Catherine Thomson ◽  
Andrea Chini ◽  
Nick D. Read ◽  
Gary J. Loake
Keyword(s):  
Signaling Pathways ◽  
Pseudomonas Syringae ◽  
Constitutive Expression ◽  
Bacterial Pathogen ◽  
Negative Regulator ◽  
Reactive Oxygen Intermediate ◽  
Peronospora Parasitica ◽  
Tomato Dc3000 ◽  
Pathogen Invasion ◽  
Arabidopsis Mutant

In order to identify components of the defense signaling network engaged following attempted pathogen invasion, we generated a novel PR-1∷luciferase (LUC) transgenic line that was deployed in an imaging-based screen to uncover defense-related mutants. The recessive mutant designated cir1 exhibited constitutive expression of salicylic acid (SA), jasmonic acid (JA)/ethylene, and reactive oxygen intermediate-dependent genes. Moreover, this mutation conferred resistance against the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 and a virulent oomycete pathogen Peronospora parasitica Noco2. Epistasis analyses were undertaken between cir1 and mutants that disrupt the SA (npr1, nahG), JA (jar1), and ethylene (ET) (ein2) signaling pathways. While resistance against both P. syringae pv. tomato DC3000 and Peronospora parasitica Noco2 was partially reduced by npr1, resistance against both of these pathogens was lost in an nahG genetic background. Hence, cir1-mediated resistance is established via NPR1-dependent and -independent signaling pathways and SA accumulation is essential for the function of both pathways. While jar1 and ein2 reduced resistance against P. syringae pv. tomato DC3000, these mutations appeared not to impact cir1-mediated resistance against Peronospora parasitica Noco2. Thus, JA and ET sensitivity are required for cir1-mediated resistance against P. syringae pv. tomato DC3000 but not Peronospora parasitica Noco2. Therefore, the cir1 mutation may define a negative regulator of disease resistance that operates upstream of SA, JA, and ET accumulation.

Sign in / Sign up

Export Citation Format

Share Document