Alginate gene expression by Pseudomonas syringae pv. tomato DC3000 in host and non-host plants

Microbiology ◽  
2003 ◽  
Vol 149 (5) ◽  
pp. 1127-1138 ◽  
Author(s):  
Ronald C. Keith ◽  
Lisa M. W. Keith ◽  
Gustavo Hernández-Guzmán ◽  
Srinivasa R. Uppalapati ◽  
Carol L. Bender
Keyword(s):  
Gene Expression ◽  
Pseudomonas Syringae ◽  
Host Plants ◽  
Rio Grande ◽  
Histochemical Staining ◽  
Post Inoculation ◽  
Bacterial Cells ◽  
In Planta ◽  
Tomato Dc3000 ◽  
Susceptible Tomato

Pseudomonas syringae produces the exopolysaccharide alginate, a copolymer of mannuronic and guluronic acid. Although alginate has been isolated from plants infected by P. syringae, the signals and timing of alginate gene expression in planta have not been described. In this study, an algD : : uidA transcriptional fusion, designated pDCalgDP, was constructed and used to monitor alginate gene expression in host and non-host plants inoculated with P. syringae pv. tomato DC3000. When leaves of susceptible collard plants were spray-inoculated with DC3000(pDCalgDP), algD was activated within 72 h post-inoculation (p.i.) and was associated with the development of water-soaked lesions. In leaves of the susceptible tomato cv. Rio Grande-PtoS, algD activity was lower than in collard and was not associated with water-soaking. The expression of algD was also monitored in leaves of tomato cv. Rio Grande-PtoR, which is resistant to P. syringae pv. tomato DC3000. Within 12 h p.i., a microscopic hypersensitive response (micro-HR) was observed in Rio Grande-PtoR leaves spray-inoculated with P. syringae pv. tomato DC3000(pDCalgDP). As the HR progressed, histochemical staining indicated that individual bacterial cells on the surface of resistant tomato leaves were expressing algD. These results indicate that algD is expressed in both susceptible (e.g. collard, tomato) and resistant (Rio Grande-PtoR) host plants. The expression of algD in an incompatible host–pathogen interaction was further explored by monitoring transcriptional activity in leaves of tobacco, which is not a host for P. syringae pv. tomato. In tobacco inoculated with DC3000(pDCalgDP), an HR was evident within 12 h p.i., and algD expression was evident within 8-12 h p.i. However, when tobacco was inoculated with an hrcC mutant of DC3000, the HR did not occur and algD expression was substantially lower. These results suggest that signals that precede the HR may stimulate alginate gene expression in P. syringae. Histochemical staining with nitro blue tetrazolium indicated that the superoxide anion () is a signal for algD activation in planta. This study indicates that algD is expressed when P. syringae attempts to colonize both susceptible and resistant plant hosts.

scholarly journals Identification of Tomato Leaf Factors that Activate Toxin Gene Expression in Pseudomonas syringae pv. tomato DC3000

1998 ◽  
Vol 88 (10) ◽  
pp. 1094-1100 ◽  
Author(s):  
Xiu-Zhen Li ◽  
Alvin N. Starratt ◽  
Diane A. Cuppels
Keyword(s):  
Gene Expression ◽  
Pseudomonas Syringae ◽  
High Performance ◽  
Shikimic Acid ◽  
Ice Nucleation ◽  
Tomato Leaf ◽  
Toxin Gene ◽  
In Planta ◽  
Active Components ◽  
Tomato Dc3000

Coronatine is a non-host-specific chlorosis-inducing phytotoxin produced by the tomato and crucifer pathogen Pseudomonas syringae pv. tomato DC3000. How the chromosomal gene cluster controlling toxin synthesis in this strain is regulated in planta is unknown. Ice nucleation-active cor:inaZ marker-exchange derivatives of strain DC3000 were used to determine coronatine gene expression in various host and nonhost plants and in a minimal medium supplemented with selected tomato plant constituents. Ice nucleation activity, which was first detected 4 h after inoculation, was highest in cabbage, tomato, and soybean and lowest in melon and cucumber. No correlation existed between bacterial population size and expression level on the various plants. Crude tomato leaf extract and intercellular fluid were strong inducers of toxin synthesis. Based on high-performance liquid chromatography analyses and bioassays, we concluded that the active components of both preparations were malic and citric acids, with minor contributions coming from shikimic and quinic acid. Although several compounds including glucose and inositol activated the toxin genes when tested at high concentrations (3 to 5 mM), shikimic and quinic acids were the only ones with activity at concentrations below 0.1 mM. Neither acid could be used as a sole carbon source by strain DC3000. The signal activity of shikimic acid was enhanced 10-fold by the addition of glucose. None of the plant phenolics that we screened affected coronatine gene expression.

scholarly journals GABA (γ-Aminobutyric Acid) Uptake Via the GABA Permease GabP Represses Virulence Gene Expression in Pseudomonas syringae pv. tomato DC3000

2016 ◽  
Vol 29 (12) ◽  
pp. 938-949 ◽  
Author(s):  
S. L. McCraw ◽  
D. H. Park ◽  
R. Jones ◽  
M. A. Bentley ◽  
A. Rico ◽  
...  
Keyword(s):  
Gene Expression ◽  
Amino Acid ◽  
Pseudomonas Syringae ◽  
Aminobutyric Acid ◽  
Gaba Uptake ◽  
Acid Uptake ◽  
Natural Setting ◽  
In Planta ◽  
Tomato Dc3000 ◽  
Virulence Gene Expression

The nonprotein amino acid γ-aminobutyric acid (GABA) is the most abundant amino acid in the tomato (Solanum lycopersicum) leaf apoplast and is synthesized by Arabidopsis thaliana in response to infection by the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (hereafter called DC3000). High levels of exogenous GABA have previously been shown to repress the expression of the type III secretion system (T3SS) in DC3000, resulting in reduced elicitation of the hypersensitive response (HR) in the nonhost plant tobacco (Nicotiana tabacum). This study demonstrates that the GABA permease GabP provides the primary mechanism for GABA uptake by DC3000 and that the gabP deletion mutant ΔgabP is insensitive to GABA-mediated repression of T3SS expression. ΔgabP displayed an enhanced ability to elicit the HR in young tobacco leaves and in tobacco plants engineered to produce increased levels of GABA, which supports the hypothesis that GABA uptake via GabP acts to regulate T3SS expression in planta. The observation that P. syringae can be rendered insensitive to GABA through loss of gabP but that gabP is retained by this bacterium suggests that GabP is important for DC3000 in a natural setting, either for nutrition or as a mechanism for regulating gene expression. [Formula: see text] Copyright © 2016 The Author(s). This is an open access article distributed under the CC BY Attribution 4.0 International license .

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 Reference genes for gene expression analysis in the fungal pathogen Neonectria ditissima and their use demonstrating expression up-regulation of candidate virulence genes

PLoS ONE ◽  
2020 ◽  
Vol 15 (11) ◽  
pp. e0238157
Author(s):  
Liz M. Florez ◽  
Reiny W. A. Scheper ◽  
Brent M. Fisher ◽  
Paul W. Sutherland ◽  
Matthew D. Templeton ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Analysis ◽  
Reference Genes ◽  
Time Course ◽  
Gene Expression Analysis ◽  
Virulence Genes ◽  
Functional Characterization ◽  
Post Inoculation ◽  
In Planta ◽  
Regulation Of Expression

European canker, caused by the necrotrophic fungal phytopathogen Neonectria ditissima, is one of the most damaging apple diseases worldwide. An understanding of the molecular basis of N. ditissima virulence is currently lacking. Identification of genes with an up-regulation of expression during infection, which are therefore probably involved in virulence, is a first step towards this understanding. Reverse transcription quantitative real-time PCR (RT-qPCR) can be used to identify these candidate virulence genes, but relies on the use of reference genes for relative gene expression data normalisation. However, no report that addresses selecting appropriate fungal reference genes for use in the N. ditissima-apple pathosystem has been published to date. In this study, eight N. ditissima genes were selected as candidate RT-qPCR reference genes for gene expression analysis. A subset of the primers (six) designed to amplify regions from these genes were specific for N. ditissima, failing to amplify PCR products with template from other fungal pathogens present in the apple orchard. The efficiency of amplification of these six primer sets was satisfactory, ranging from 81.8 to 107.53%. Analysis of expression stability when a highly pathogenic N. ditissima isolate was cultured under 10 regimes, using the statistical algorithms geNorm, NormFinder and BestKeeper, indicated that actin and myo-inositol-1-phosphate synthase (mips), or their combination, could be utilised as the most suitable reference genes for normalisation of N. ditissima gene expression. As a test case, these reference genes were used to study expression of three candidate virulence genes during a time course of infection. All three, which shared traits with fungal effector genes, had up-regulated expression in planta compared to in vitro with expression peaking between five and six weeks post inoculation (wpi). Thus, these three genes may well be involved in N. ditissima pathogenicity and are priority candidates for further functional characterization.

scholarly journals Reference genes for gene expression analysis in the fungal pathogen Neonectria ditissima and their use demonstrating expression up-regulation of candidate virulence genes

2020 ◽  
Author(s):  
Liz M. Florez ◽  
Reiny W. A. Scheper ◽  
Brent M. Fisher ◽  
Paul W. Sutherland ◽  
Matthew D. Templeton ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Analysis ◽  
Reference Genes ◽  
Time Course ◽  
Gene Expression Analysis ◽  
Virulence Genes ◽  
Functional Characterization ◽  
Post Inoculation ◽  
In Planta ◽  
Qrt Pcr

AbstractEuropean canker, caused by the necrotrophic fungal phytopathogen Neonectria ditissima, is one of the most damaging apple diseases worldwide. An understanding of the molecular basis of N. ditissima virulence is currently lacking. Identification of genes with an up-regulation of expression during infection, which are therefore probably involved in virulence, is a first step towards this understanding. Real-time quantitative reverse transcription PCR (qRT-PCR) can be used to identify these candidate virulence genes, but relies on the use of reference genes for relative gene expression data normalisation. However, no report that addresses selecting appropriate fungal reference genes for use in the N. ditissima-apple pathosystem has been published to date. In this study, eight N. ditissima genes were selected as candidate qRT-PCR reference genes for gene expression analysis. A subset of the primers (six) designed to amplify regions from these genes were specific for N. ditissima, failing to amplify PCR products with template from other fungal pathogens present in the apple orchard. The efficiency of amplification of these six primer sets was satisfactory, ranging from 81.8 to 107.53%. Analysis of expression stability when a highly pathogenic N. ditissima isolate was cultured under 10 regimes, using the statistical algorithms geNorm, NormFinder and BestKeeper, indicated that actin and myo-inositol-1-phosphate synthase (mips), or their combination, could be utilised as the most suitable reference genes for normalisation of N. ditissima gene expression. As a test case, these reference genes were used to study expression of three candidate virulence genes during a time course of infection. All three, which shared traits with fungal effector genes, had up-regulated expression in planta compared to in vitro with expression peaking between five and six weeks post inoculation (wpi). Thus, these three genes may well be involved in N. ditissima pathogenicity and are priority candidates for further functional characterization.

scholarly journals The Majority of the Type III Effector Inventory of Pseudomonas syringae pv. tomato DC3000 Can Suppress Plant Immunity

2009 ◽  
Vol 22 (9) ◽  
pp. 1069-1080 ◽  
Author(s):  
Ming Guo ◽  
Fang Tian ◽  
Yashitola Wamboldt ◽  
James R. Alfano
Keyword(s):  
Pseudomonas Syringae ◽  
Plant Immunity ◽  
In Planta ◽  
Tomato Dc3000 ◽  
Type Iii Effector ◽  
Type Iii Effectors ◽  
Type Iii ◽  
Molecular Pattern ◽  
Effector Triggered Immunity ◽  
Type Iii Protein Secretion

The Pseudomonas syringae type III protein secretion system (T3SS) and the type III effectors it injects into plant cells are required for plant pathogenicity and the ability to elicit a hypersensitive response (HR). The HR is a programmed cell death that is associated with effector-triggered immunity (ETI). A primary function of P. syringae type III effectors appears to be the suppression of ETI and pathogen-associated molecular pattern–triggered immunity (PTI), which is induced by conserved molecules on microorganisms. We reported that seven type III effectors from P. syringae pv. tomato DC3000 were capable of suppressing an HR induced by P. fluorescens(pHIR11) and have now tested 35 DC3000 type III effectors in this assay, finding that the majority of them can suppress the HR induced by HopA1. One newly identified type III effector with particularly strong HR suppression activity was HopS2. We used the pHIR11 derivative pLN1965, which lacks hopA1, in related assays and found that a subset of the type III effectors that suppressed HopA1-induced ETI also suppressed an ETI response induced by AvrRpm1 in Arabidopsis thaliana. A. thaliana plants expressing either HopAO1 or HopF2, two type III effectors that suppressed the HopA1-induced HR, were reduced in the flagellin-induced PTI response as well as PTI induced by other PAMPs and allowed enhanced in planta growth of P. syringae. Collectively, our results suggest that the majority of DC3000 type III effectors can suppress plant immunity. Additionally, the construct pLN1965 will likely be a useful tool in determining whether other type III effectors or effectors from other types of pathogens can suppress either ETI, PTI, or both.

scholarly journals Negative Regulation of the Hrp Type III Secretion System in Pseudomonas syringae pv. phaseolicola

2010 ◽  
Vol 23 (5) ◽  
pp. 682-701 ◽  
Author(s):  
Inmaculada Ortiz-Martín ◽  
Richard Thwaites ◽  
John W. Mansfield ◽  
Carmen R. Beuzón
Keyword(s):  
Gene Expression ◽  
Pseudomonas Syringae ◽  
Type Iii Secretion ◽  
Pathogenic Bacteria ◽  
In Planta ◽  
Secretion Systems ◽  
Type Iii ◽  
Type Iii Secretion Systems ◽  
Bacterial Fitness

Many plant-pathogenic bacteria require type III secretion systems (T3SS) to cause disease in compatible hosts and to induce the hypersensitive response in resistant plants. T3SS gene expression is induced within the plant and responds to host and environmental factors. In Pseudomonas syringae, expression is downregulated by the Lon protease in rich medium and by HrpV under inducing conditions. HrpV acts as an anti-activator by binding HrpS. HrpG, which can also bind HrpV, has been reported to act as an anti-anti-activator. Previous studies have used mostly in vitro inducing conditions, different pathovars, and methodology. We have used single and double lon and hrpV mutants of P. syringae pv. phaseolicola 1448a, as well as strains ectopically expressing the regulators, to examine their role in coordinating expression of the T3SS. We applied real-time polymerase chain reaction to analyze gene expression both in vitro and in planta, and assessed bacterial fitness using competitive indices. Our results indicate that i) Lon downregulates expression of the hrp/hrc genes in all conditions, probably by constitutively degrading naturally unstable HrpR; ii) HrpV and HrpT downregulate expression of the hrp/hrc genes in all conditions; and iii) HrpG has an additional, HrpV-independent role, regulating expression of the hrpC operon.

scholarly journals The HopPtoF Locus of Pseudomonas syringae pv. tomato DC3000 Encodes a Type III Chaperone and a Cognate Effector

2004 ◽  
Vol 17 (5) ◽  
pp. 447-455 ◽  
Author(s):  
Libo Shan ◽  
Hye-sook Oh ◽  
Jianfu Chen ◽  
Ming Guo ◽  
Jianmin Zhou ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Type Iii Secretion ◽  
Pathogenic Bacteria ◽  
Host Cells ◽  
Whole Genome Sequence ◽  
Avirulence Gene ◽  
Bacterial Cells ◽  
Tomato Dc3000 ◽  
Virulence Proteins ◽  
Type Iii

Type III secretion systes are highly conserved among gram-negative plant and animal pathogenic bacteria. Through the type III secretion system, bacteria inject a number of virulence proteins into the host cells. Analysis of the whole genome sequence of Pseudomonas syringae pv. tomato DC3000 strain identified a locus, named HopPtoF, that is homologous to the avirulence gene locus avrPphF in P. syringae pv. phaseolicola. The HopPtoF locus harbors two genes, ShcFPto and HopFPto, that are preceded by a single hrp box promoter. We present evidence here to show that ShcFPto and HopFPto encode a type III chaperone and a cognate effector, respectively. ShcFPto interacts with and stabilizes the HopFPto protein in the bacterial cell. Translation of HopFPto starts at a rare initiation codon ATA that limits the synthesis of the HopFPto protein to a low level in bacterial cells.

scholarly journals Multiple CsrA Proteins Control Key Virulence Traits in Pseudomonas syringae pv. tomato DC3000

2018 ◽  
Vol 31 (5) ◽  
pp. 525-536 ◽  
Author(s):  
María-Dolores Ferreiro ◽  
Joaquina Nogales ◽  
Gabriela A. Farias ◽  
Adela Olmedilla ◽  
Juan Sanjuán ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Environmental Cues ◽  
Symptom Development ◽  
Regulatory Pathway ◽  
In Planta ◽  
Tomato Dc3000 ◽  
Phytopathogenic Bacterium ◽  
Virulence Traits ◽  
Expression Of Genes ◽  
Physiological Outcomes

The phytopathogenic bacterium Pseudomonas syringae pv. tomato DC3000 has a complex Gac-rsm global regulatory pathway that controls virulence, motility, production of secondary metabolites, carbon metabolism, and quorum sensing. However, despite the fact that components of this pathway are known, their physiological roles have not yet been established. Regarding the CsrA/RsmA type proteins, five paralogs, three of which are well conserved within the Pseudomonas genus (csrA1, csrA2, and csrA3), have been found in the DC3000 genome. To decipher their function, mutants lacking the three most conserved CsrA proteins have been constructed and their physiological outcomes examined. We show that they exert nonredundant functions and demonstrate that CsrA3 and, to a lesser extent, CsrA2 but not CsrA1 alter the expression of genes involved in a variety of pathways and systems important for motility, exopolysaccharide synthesis, growth, and virulence. Particularly, alginate synthesis, syringafactin production, and virulence are considerably de-repressed in a csrA3 mutant, whereas growth in planta is impaired. We propose that the linkage of growth and symptom development is under the control of CsrA3, which functions as a pivotal regulator of the DC3000 life cycle, repressing virulence traits and promoting cell division in response to environmental cues.

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