A quick and robust method for quantification of the hypersensitive response in plants

One of the most studied defense reactions of plants against microbial pathogens is the hypersensitive response (HR). The HR is a complex multicellular process that involves programmed cell death at the site of infection. A standard method to quantify plant defense and the HR is to measure the release of cellular electrolytes into water after infiltration with pathogenic bacteria. In this type of experiment, the bacteria are typically delivered into the plant tissue through syringe infiltration. Here we report the development of a vacuum infiltration protocol that allows multiple plant lines to be infiltrated simultaneously and assayed for defense responses. Vacuum infiltration did not induce more wounding response in Arabidopsis leaf tissue than syringe inoculation, whereas throughput and reproducibility were improved. The method was used to study HR-induced electrolyte loss after treatment with the bacteriumPseudomonas syringaepv.tomatoDC3000 harboring the effector AvrRpm1, AvrRpt2 or AvrRps4. Specifically, the influence of bacterial titer on AvrRpm1-induced HR was investigated. Not only the amplitude, but also the timing of the maximum rate of the HR reaction was found to be dose-dependent. Finally, using vacuum infiltration, we were able quantify induction of phospholipase D activity after AvrRpm1 recognition in leaves labeled with33PO4.

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The HopZ Family of Pseudomonas syringae Type III Effectors Require Myristoylation for Virulence and Avirulence Functions in Arabidopsis thaliana

Journal of Bacteriology ◽

10.1128/jb.01702-07 ◽

2008 ◽

Vol 190 (8) ◽

pp. 2880-2891 ◽

Cited By ~ 68

Author(s):

Jennifer D. Lewis ◽

Wasan Abada ◽

Wenbo Ma ◽

David S. Guttman ◽

Darrell Desveaux

Keyword(s):

Arabidopsis Thaliana ◽

Pseudomonas Syringae ◽

Hypersensitive Response ◽

Pathogenic Bacteria ◽

Functional Characterization ◽

Defense Responses ◽

Effector Proteins ◽

Type Iii Effectors ◽

Type Iii ◽

Virulence Protein

ABSTRACT Pseudomonas syringae utilizes the type III secretion system to translocate effector proteins into plant cells, where they can contribute to the pathogen's ability to infect and cause disease. Recognition of these effectors by resistance proteins induces defense responses that typically include a programmed cell death reaction called the hypersensitive response. The YopJ/HopZ family of type III effector proteins is a common family of effector proteins found in animal- and plant-pathogenic bacteria. The HopZ family in P. syringae includes HopZ1aPsyA2, HopZ1bPgyUnB647, HopZ1cPmaE54326, HopZ2Ppi895A and HopZ3PsyB728a. HopZ1a is predicted to be most similar to the ancestral hopZ allele and causes a hypersensitive response in multiple plant species, including Arabidopsis thaliana. Therefore, it has been proposed that host defense responses have driven the diversification of this effector family. In this study, we further characterized the hypersensitive response induced by HopZ1a and demonstrated that it is not dependent on known resistance genes. Further, we identified a novel virulence function for HopZ2 that requires the catalytic cysteine demonstrated to be required for protease activity. Sequence analysis of the HopZ family revealed the presence of a predicted myristoylation sequence in all members except HopZ3. We demonstrated that the myristoylation site is required for membrane localization of this effector family and contributes to the virulence and avirulence activities of HopZ2 and HopZ1a, respectively. This paper provides insight into the selective pressures driving virulence protein evolution by describing a detailed functional characterization of the diverse HopZ family of type III effectors with the model plant Arabidopsis.

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Signal Signature and Transcriptome Changes of Arabidopsis During Pathogen and Insect Attack

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-18-0923 ◽

2005 ◽

Vol 18 (9) ◽

pp. 923-937 ◽

Cited By ~ 608

Author(s):

Martin De Vos ◽

Vivian R. Van Oosten ◽

Remco M. P. Van Poecke ◽

Johan A. Van Pelt ◽

Maria J. Pozo ◽

...

Keyword(s):

Gene Expression ◽

Pseudomonas Syringae ◽

Defense Response ◽

Frankliniella Occidentalis ◽

Pieris Rapae ◽

Expression Profiles ◽

Defense Responses ◽

Alternaria Brassicicola ◽

Microbial Pathogens ◽

Primary Role

Plant defenses against pathogens and insects are regulated differentially by cross-communicating signaling pathways in which salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) play key roles. To understand how plants integrate pathogen- and insect-induced signals into specific defense responses, we monitored the dynamics of SA, JA, and ET signaling in Arabidopsis after attack by a set of microbial pathogens and herbivorous insects with different modes of attack. Arabidopsis plants were exposed to a pathogenic leaf bacterium (Pseudomonas syringae pv. tomato), a pathogenic leaf fungus (Alternaria brassicicola), tissue-chewing caterpillars (Pieris rapae), cell-content-feeding thrips (Frankliniella occidentalis), or phloem-feeding aphids (Myzus persicae). Monitoring the signal signature in each plant-attacker combination showed that the kinetics of SA, JA, and ET production varies greatly in both quantity and timing. Analysis of global gene expression profiles demonstrated that the signal signature characteristic of each Arabidopsis-attacker combination is orchestrated into a surprisingly complex set of transcriptional alterations in which, in all cases, stress-related genes are overrepresented. Comparison of the transcript profiles revealed that consistent changes induced by pathogens and insects with very different modes of attack can show considerable overlap. Of all consistent changes induced by A. brassicicola, Pieris rapae, and F. occidentalis, more than 50% also were induced consistently by P. syringae. Notably, although these four attackers all stimulated JA biosynthesis, the majority of the changes in JA-responsive gene expression were attacker specific. All together, our study shows that SA, JA, and ET play a primary role in the orchestration of the plant's defense response, but other regulatory mechanisms, such as pathway cross-talk or additional attacker-induced signals, eventually shape the highly complex attacker-specific defense response.

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Positive Regulation of the Hrp Type III Secretion System in Pseudomonas syringae pv. phaseolicola

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-23-5-0665 ◽

2010 ◽

Vol 23 (5) ◽

pp. 665-681 ◽

Cited By ~ 40

Author(s):

Inmaculada Ortiz-Martín ◽

Richard Thwaites ◽

Alberto P. Macho ◽

John W. Mansfield ◽

Carmen R. Beuzón

Keyword(s):

Gene Expression ◽

Pseudomonas Syringae ◽

Hypersensitive Response ◽

Type Iii Secretion ◽

Pathogenic Bacteria ◽

Type Iii Secretion System ◽

Secretion System ◽

Virulence Determinants ◽

Type Iii

Disease in compatible hosts and induction of the hypersensitive response in resistant plants by most plant-pathogenic bacteria require a functional type III secretion system (T3SS). Expression of T3SS genes responds to host and environmental factors and is induced within the plant. In Pseudomonas syringae, expression of the T3SS requires HrpL, which is transcriptionally upregulated by HrpR and HrpS. In some pathovars, expression of the hrpRS genes is upregulated by the GacA/S two-component system. Additionally, HrpA, the major component of the T3SS pilus, has also been linked to the regulation of the hrpRS gene expression. Previous studies concerning regulation of hypersensitive response and pathogenesis/hypersensitive response conserved (hrp/hrc) gene expression have used mostly in vitro inducing conditions, different pathovars, and methodology. Here, we analyze the roles of HrpL, GacA, and HrpA in the bean pathogen, using single, double, and triple mutants as well as strains ectopically expressing the regulators. We use real-time polymerase chain reaction analysis in vitro and in planta to quantify gene expression and competitive indices and other assays to assess bacterial fitness. Our results indicate that i) HrpL acts as a general virulence regulator that upregulates non-T3SS virulence determinants and downregulates flagellar function; ii) GacA modulates the expression of hrpL, and its contribution to virulence is entirely HrpL dependent; iii) there is a basal HrpL-independent expression of the T3SS genes in rich medium that is important for full activation of the system, maybe by keeping the system primed for rapid activation upon contact with the plant; and iv) HrpA upregulates expression of the T3SS genes and is essential to activate expression of the hrpZ operon upon contact with the plant.

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Effectors from Wheat Rust Fungi Suppress Multiple Plant Defense Responses

Phytopathology ◽

10.1094/phyto-02-16-0083-r ◽

2017 ◽

Vol 107 (1) ◽

pp. 75-83 ◽

Cited By ~ 29

Author(s):

Sowmya R. Ramachandran ◽

Chuntao Yin ◽

Joanna Kud ◽

Kiwamu Tanaka ◽

Aaron K. Mahoney ◽

...

Keyword(s):

Plant Defense ◽

Pseudomonas Syringae ◽

Hypersensitive Response ◽

Defense Responses ◽

Effector Proteins ◽

R Gene ◽

Plant Defense Responses ◽

Functional Studies ◽

Rust Diseases ◽

Suppression Assay

Fungi that cause cereal rust diseases (genus Puccinia) are important pathogens of wheat globally. Upon infection, the fungus secretes a number of effector proteins. Although a large repository of putative effectors has been predicted using bioinformatic pipelines, the lack of available high-throughput effector screening systems has limited functional studies on these proteins. In this study, we mined the available transcriptomes of Puccinia graminis and P. striiformis to look for potential effectors that suppress host hypersensitive response (HR). Twenty small (<300 amino acids), secreted proteins, with no predicted functions were selected for the HR suppression assay using Nicotiana benthamiana, in which each of the proteins were transiently expressed and evaluated for their ability to suppress HR caused by four cytotoxic effector‐R gene combinations (Cp/Rx, ATR13/RPP13, Rpt2/RPS‐2, and GPA/RBP‐1) and one mutated R gene—Pto(Y207D). Nine out of twenty proteins, designated Shr1 to Shr9 (suppressors of hypersensitive response), were found to suppress HR in N. benthamiana. These effectors varied in the effector-R gene defenses they suppressed, indicating these pathogens can interfere with a variety of host defense pathways. In addition to HR suppression, effector Shr7 also suppressed PAMP-triggered immune response triggered by flg22. Finally, delivery of Shr7 through Pseudomonas fluorescens EtHAn suppressed nonspecific HR induced by Pseudomonas syringae DC3000 in wheat, confirming its activity in a homologous system. Overall, this study provides the first evidence for the presence of effectors in Puccinia species suppressing multiple plant defense responses.

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N-acyl homoserine lactone-mediated modulation of plant growth and defense against Pseudoperonospora cubensis in cucumber

Journal of Experimental Botany ◽

10.1093/jxb/eraa384 ◽

2020 ◽

Vol 71 (20) ◽

pp. 6638-6654

Author(s):

Sercan Pazarlar ◽

Nedim Cetinkaya ◽

Melike Bor ◽

Recep Serdar Kara

Keyword(s):

Salicylic Acid ◽

Plant Growth ◽

Pseudomonas Syringae ◽

Primary Root ◽

Homoserine Lactone ◽

Defense Responses ◽

Microbial Pathogens ◽

Transcriptional Analysis ◽

Pseudoperonospora Cubensis ◽

Callose Deposition

Abstract N-acyl-homoserine lactones (AHLs), a well-described group of quorum sensing molecules, may modulate plant defense responses and plant growth. However, there is limited knowledge regarding the defense responses of non-model crops to AHLs and the mechanism of action responsible for the modulation of defense responses against microbial pathogens. In the present study, long-chain N-3-oxo-tetradecanoyl-l-homoserine lactone (oxo-C14-HSL) was shown to have a distinct potential to prime cucumber for enhanced defense responses against the biotrophic oomycete pathogen Pseudoperonospora cubensis and the hemibiotrophic bacterium Pseudomonas syringae pv. lachrymans. We provide evidence that AHL-mediated enhanced defense against downy mildew disease is based on cell wall reinforcement by lignin and callose deposition, the activation of defense-related enzymes (peroxidase, β-1,3-glucanase, phenylalanine ammonia-lyase), and the accumulation of reactive oxygen species (hydrogen peroxide, superoxide) and phenolic compounds. Quantitative analysis of salicylic acid and jasmonic acid, and transcriptional analysis of several of genes associated with these phytohormones, revealed that defense priming with oxo-C14-HSL is commonly regulated by the salicylic acid signaling pathway. We also show that treatment with short- (N-hexanoyl-l-homoserine lactone) and medium-chain (N-3-oxo-decanoyl-l-homoserine lactone) AHLs promoted primary root elongation and modified root architecture, respectively, resulting in enhanced plant growth.

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Multiple Activities of the Plant Pathogen Type III Effector Proteins WtsE and AvrE Require WxxxE Motifs

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-22-6-0703 ◽

2009 ◽

Vol 22 (6) ◽

pp. 703-712 ◽

Cited By ~ 32

Author(s):

Jong Hyun Ham ◽

Doris R. Majerczak ◽

Kinya Nomura ◽

Christy Mecey ◽

Francisco Uribe ◽

...

Keyword(s):

G Proteins ◽

Pseudomonas Syringae ◽

Virulence Factors ◽

Pathogenic Bacteria ◽

Plant Pathogens ◽

Biological Activities ◽

Defense Responses ◽

Endoplasmic Reticulum Membrane ◽

Type Iii Effectors ◽

Type Iii

The broadly conserved AvrE-family of type III effectors from gram-negative plant-pathogenic bacteria includes important virulence factors, yet little is known about the mechanisms by which these effectors function inside plant cells to promote disease. We have identified two conserved motifs in AvrE-family effectors: a WxxxE motif and a putative C-terminal endoplasmic reticulum membrane retention/retrieval signal (ERMRS). The WxxxE and ERMRS motifs are both required for the virulence activities of WtsE and AvrE, which are major virulence factors of the corn pathogen Pantoea stewartii subsp. stewartii and the tomato or Arabidopsis pathogen Pseudomonas syringae pv. tomato, respectively. The WxxxE and the predicted ERMRS motifs are also required for other biological activities of WtsE, including elicitation of the hypersensitive response in nonhost plants and suppression of defense responses in Arabidopsis. A family of type III effectors from mammalian bacterial pathogens requires WxxxE and subcellular targeting motifs for virulence functions that involve their ability to mimic activated G-proteins. The conservation of related motifs and their necessity for the function of type III effectors from plant pathogens indicates that disturbing host pathways by mimicking activated host G-proteins may be a virulence mechanism employed by plant pathogens as well.

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Negative Autogenous Control of the Master Type III Secretion System Regulator HrpL in Pseudomonas syringae

mBio ◽

10.1128/mbio.02273-16 ◽

2017 ◽

Vol 8 (1) ◽

Cited By ~ 11

Author(s):

Christopher Waite ◽

Jörg Schumacher ◽

Milija Jovanovic ◽

Mark Bennett ◽

Martin Buck

Keyword(s):

Pseudomonas Syringae ◽

Plant Pathogen ◽

Pathogenic Bacteria ◽

Plant Pathogens ◽

Plant Defenses ◽

Management Strategies ◽

Microbial Pathogens ◽

Defense Signaling ◽

Autogenous Control ◽

United Nations Food

ABSTRACT The type III secretion system (T3SS) is a principal virulence determinant of the model bacterial plant pathogen Pseudomonas syringae. T3SS effector proteins inhibit plant defense signaling pathways in susceptible hosts and elicit evolved immunity in resistant plants. The extracytoplasmic function sigma factor HrpL coordinates the expression of most T3SS genes. Transcription of hrpL is dependent on sigma-54 and the codependent enhancer binding proteins HrpR and HrpS for hrpL promoter activation. hrpL is oriented adjacently to and divergently from the HrpL-dependent gene hrpJ, sharing an intergenic upstream regulatory region. We show that association of the RNA polymerase (RNAP)-HrpL complex with the hrpJ promoter element imposes negative autogenous control on hrpL transcription in P. syringae pv. tomato DC3000. The hrpL promoter was upregulated in a ΔhrpL mutant and was repressed by plasmid-borne hrpL. In a minimal Escherichia coli background, the activity of HrpL was sufficient to achieve repression of reconstituted hrpL transcription. This repression was relieved if both the HrpL DNA-binding function and the hrp-box sequence of the hrpJ promoter were compromised, implying dependence upon the hrpJ promoter. DNA-bound RNAP-HrpL entirely occluded the HrpRS and partially occluded the integration host factor (IHF) recognition elements of the hrpL promoter in vitro, implicating inhibition of DNA binding by these factors as a cause of negative autogenous control. A modest increase in the HrpL concentration caused hypersecretion of the HrpA1 pilus protein but intracellular accumulation of later T3SS substrates. We argue that negative feedback on HrpL activity fine-tunes expression of the T3SS regulon to minimize the elicitation of plant defenses. IMPORTANCE The United Nations Food and Agriculture Organization has warned that agriculture will need to satisfy a 50% to 70% increase in global food demand if the human population reaches 9 billion by 2050 as predicted. However, diseases caused by microbial pathogens represent a major threat to food security, accounting for over 10% of estimated yield losses in staple wheat, rice, and maize crops. Understanding the decision-making strategies employed by pathogens to coordinate virulence and to evade plant defenses is vital for informing crop resistance traits and management strategies. Many plant-pathogenic bacteria utilize the needle-like T3SS to inject virulence factors into host plant cells to suppress defense signaling. Pseudomonas syringae is an economically and environmentally devastating plant pathogen. We propose that the master regulator of its entire T3SS gene set, HrpL, downregulates its own expression to minimize elicitation of plant defenses. Revealing such conserved regulatory strategies will inform future antivirulence strategies targeting plant pathogens. The United Nations Food and Agriculture Organization has warned that agriculture will need to satisfy a 50% to 70% increase in global food demand if the human population reaches 9 billion by 2050 as predicted. However, diseases caused by microbial pathogens represent a major threat to food security, accounting for over 10% of estimated yield losses in staple wheat, rice, and maize crops. Understanding the decision-making strategies employed by pathogens to coordinate virulence and to evade plant defenses is vital for informing crop resistance traits and management strategies. Many plant-pathogenic bacteria utilize the needle-like T3SS to inject virulence factors into host plant cells to suppress defense signaling. Pseudomonas syringae is an economically and environmentally devastating plant pathogen. We propose that the master regulator of its entire T3SS gene set, HrpL, downregulates its own expression to minimize elicitation of plant defenses. Revealing such conserved regulatory strategies will inform future antivirulence strategies targeting plant pathogens.

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Re-evaluation of a Tn5::gacAmutant ofPseudomonas syringaepv.tomatoDC3000 uncovers roles foruvrCandanmKin promoting virulence

10.1101/774711 ◽

2019 ◽

Author(s):

Megan R. O’Malley ◽

Alexandra J. Weisberg ◽

Jeff H. Chang ◽

Jeffrey C. Anderson

Keyword(s):

Pseudomonas Syringae ◽

Type Iii Secretion ◽

Pathogenic Bacteria ◽

Insertional Mutagenesis ◽

Leaf Tissue ◽

Wild Type ◽

Two Component System ◽

Gene Encoding ◽

Interior Space ◽

Damage Repair

ABSTRACTPseudomonas syringaeis a taxon of plant pathogenic bacteria that can colonize and proliferate within the interior space of leaf tissue. This process requiresP. syringaeto rapidly upregulate the production of virulence factors including a type III secretion system (T3SS) that suppress host defenses. GacS/A is a two-component system that regulates virulence of many plant and animal pathogenic bacteria includingP. syringae. We recently investigated the virulence defect of strain AC811, a Tn5::gacAmutant ofP. syringae pv.tomatoDC3000 that is less virulent on Arabidopsis. We discovered that decreased virulence of AC811 is not caused by loss of GacA function. Here, we report the molecular basis of the virulence defect of AC811. We show that AC811 possesses a nonsense mutation inanmK, a gene predicted to encode a 1,6-anhydromuramic acid kinase involved in cell wall recycling. Expression of a wild-type allele ofanmKpartially increased growth of AC811 in Arabidopsis leaves. In addition to the defectiveanmKallele, we also show that the Tn5insertion ingacAexerts a polar effect onuvrC, a downstream gene encoding a regulator of DNA damage repair. Expression of the wild-typeanmKallele together with increased expression ofuvrCfully restored the virulence of AC811 during infection of Arabidopsis. These results demonstrate that defects inanmKanduvrCare together sufficient to account for the decreased virulence of AC811, and suggest caution is warranted in assigning phenotypes to GacA function based on insertional mutagenesis of thegacA-uvrClocus.

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Domain Structure of HrpE, the Hrp Pilus Subunit of Xanthomonas campestris pv. vesicatoria

Journal of Bacteriology ◽

10.1128/jb.187.17.6175-6186.2005 ◽

2005 ◽

Vol 187 (17) ◽

pp. 6175-6186 ◽

Cited By ~ 25

Author(s):

Ernst Weber ◽

Ralf Koebnik

Keyword(s):

Amino Acid ◽

Cell Surface ◽

Gene Cluster ◽

Domain Structure ◽

Pseudomonas Syringae ◽

Hypersensitive Response ◽

Xanthomonas Campestris ◽

Pathogenic Bacteria ◽

Dominant Negative ◽

Reporter Protein

ABSTRACT The plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria possesses a type III secretion (TTS) system necessary for pathogenicity in susceptible hosts and induction of the hypersensitive response in resistant plants. This specialized protein transport system is encoded by a 23-kb hrp (hypersensitive response and pathogenicity) gene cluster. X. campestris pv. vesicatoria produces filamentous structures, Hrp pili, at the cell surface under hrp-inducing conditions. The Hrp pilus acts as a cell surface appendage of the TTS system and serves as a conduit for the transfer of bacterial effector proteins into the plant cell cytosol. The major pilus component, the HrpE pilin, is unique to xanthomonads and is encoded within the hrp gene cluster. In this study, functional domains of HrpE were mapped by linker-scanning mutagenesis and by reporter protein fusions to an N-terminally truncated avirulence protein (AvrBs3Δ2). Thirteen five-amino-acid peptide insertion mutants were obtained and could be grouped into six phenotypic classes. Three permissive mutations were mapped in the N-terminal half of HrpE, which is weakly conserved within the HrpE protein family. Four dominant-negative peptide insertions in the strongly conserved C-terminal region suggest that this domain is critical for oligomerization of the pilus subunits. Reporter protein fusions revealed that the N-terminal 17 amino acid residues act as an efficient TTS signal. From these results, we postulate a three-domain structure of HrpE with an N-terminal secretion signal, a surface-exposed variable region of the N-terminal half, and a C-terminal polymerization domain. Comparisons with a mutant study of HrpA, the Hrp pilin from Pseudomonas syringae pv. tomato DC3000, and hydrophobicity plot analyses of several nonhomologous Hrp pilins suggest a common architecture of Hrp pilins of different plant-pathogenic bacteria.

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Effects of the pseudomonad phytotoxin coronatine on tomato leaf structure and ultrastructure

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100140683 ◽

1995 ◽

Vol 53 ◽

pp. 862-863

Author(s):

D.A. Palmer ◽

C.L. Bender

Keyword(s):

Pseudomonas Syringae ◽

Membrane Integrity ◽

Leaf Tissue ◽

Cell Number ◽

Cell Ultrastructure ◽

Response To Treatment ◽

Prominent Symptom ◽

Chlorophylls A And B ◽

Cell Dimensions ◽

Structure And Ultrastructure

Coronatine is a non-host-specific phytotoxin produced by several members of the Pseudomonas syringae group of pathovars. The toxin acts as a virulence factor in P. syringae pv. tomato, allowing the organism to multiply to a higher population density and develop larger lesions than mutant strains unable to produce the toxin. The most prominent symptom observed in leaf tissue treated with coronatine is an intense spreading chlorosis; this has been attributed to a loss of chlorophylls a and b in tobacco. Coronatine's effects on membrane integrity and cell ultrastructure have not been previously investigated. The present study describes changes in tomato leaves in response to treatment with purified coronatine, infection by a coronatine-producing strain of P. syringae pv. tomato, and infection by a cor" mutant.In contrast to H2O-treated tissue, coronatine-treated tissue showed a diffuse chlorosis extending approximately 5 mm from the inoculation site. Leaf thickness, cell number, and cell dimensions were similar for both healthy and coronatine-treated, chlorotic tissue; however, the epidermal cell walls were consistently thicker in coronatine-treated leaves (Figs, la and lb).

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