The Use of Bioluminescence for Monitoring in planta Growth Dynamics of a Pseudomonas syringae Plant Pathogen

ABSTRACTThe plant pathogenPseudomonas syringaepv. syringae B728a grows and survives on leaf surfaces and in the leaf apoplast of its host, bean (Phaseolus vulgaris). To understand the contribution of distinct regulators to B728a fitness and pathogenicity, we performed a transcriptome analysis of strain B728a and nine regulatory mutants recovered from the surfaces and interior of leaves and exposed to environmental stresses in culture. The quorum-sensing regulators AhlR and AefR influenced few genesin plantaorin vitro. In contrast, GacS and a downstream regulator, SalA, formed a large regulatory network that included a branch that regulated diverse traits and was independent of plant-specific environmental signals and a plant signal-dependent branch that positively regulated secondary metabolite genes and negatively regulated the type III secretion system. SalA functioned as a central regulator of iron status based on its reciprocal regulation of pyoverdine and achromobactin genes and also sulfur uptake, suggesting a role in the iron-sulfur balance. RetS functioned almost exclusively to repress secondary metabolite genes when the cells were not on leaves. Among the sigma factors examined, AlgU influenced many more genes than RpoS, and most AlgU-regulated genes depended on RpoN. RpoN differentially impacted many AlgU- and GacS-activated genes in cells recovered from apoplastic versus epiphytic sites, suggesting differences in environmental signals or bacterial stress status in these two habitats. Collectively, our findings illustrate a central role for GacS, SalA, RpoN, and AlgU in global regulation in B728ain plantaand a high level of plasticity in these regulators’ responses to distinct environmental signals.IMPORTANCELeaves harbor abundant microorganisms, all of which must withstand challenges such as active plant defenses and a highly dynamic environment. Some of these microbes can influence plant health. Despite knowledge of individual regulators that affect the fitness or pathogenicity of foliar pathogens, our understanding of the relative importance of various global regulators to leaf colonization is limited.Pseudomonas syringaestrain B728a is a plant pathogen and a good colonist of both the surfaces and interior of leaves. This study used global transcript profiles of strain B728a to investigate the complex regulatory network of putative quorum-sensing regulators, two-component regulators, and sigma factors in cells colonizing the leaf surface and leaf interior under stressfulin vitroconditions. The results highlighted the value of evaluating these networksin plantadue to the impact of leaf-specific environmental signals and suggested signal differences that may enable cells to differentiate surface versus interior leaf habitats.

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Genome-wide identification ofPseudomonas syringaegenes required for competitive fitness during colonization of the leaf surface and apoplast

10.1101/637496 ◽

2019 ◽

Cited By ~ 1

Author(s):

Tyler C. Helmann ◽

Adam M. Deutschbauer ◽

Steven E. Lindow

Keyword(s):

Gene Expression ◽

Pseudomonas Syringae ◽

Plant Pathogen ◽

Pathogenic Bacteria ◽

Leaf Surface ◽

In Planta ◽

Competitive Fitness ◽

Genome Wide ◽

A Genome ◽

Leaf Surfaces

AbstractThe foliar plant pathogenPseudomonas syringaecan establish large epiphytic populations on leaf surfaces before infection. However, the bacterial genes that contribute to these lifestyles have not been completely defined. The fitness contributions of most genes inP. syringaepv.syringaeB728a were determined by genome-wide fitness profiling with a randomly barcoded transposon mutant library that was grown on the leaf surface and in the apoplast of the susceptible plantPhaseolus vulgaris. Genes within the functional categories of amino acid and polysaccharide (including alginate) biosynthesis contributed most to fitness both on the leaf surface (epiphytic) or in the leaf interior (apoplast), while genes in the type III secretion system and syringomycin synthesis were primarily important in the apoplast. Numerous other genes that had not been previously associated within plantagrowth were also required for maximum epiphytic or apoplastic fitness. Many hypothetical proteins and uncategorized glycosyltransferases were also required for maximum competitive fitness in and on leaves. For most genes, no relationship was seen between fitnessin plantaand either the magnitude of their expressionin plantaor degree of inductionin plantacompared toin vitroconditions measured in other studies. A lack of association of gene expression and fitness has important implications for the interpretation of transcriptional information and our broad understanding of plant-microbe interactions.Significance StatementMany plant pathogenic bacteria can extensively colonize leaf surfaces before entry and multiplication within the leaf to cause disease. While these habitats presumably require distinct adaptations, the genes required in these habitats and how they would differ was unknown. Using a genome-wide library of barcoded insertional mutants in the plant pathogenPseudomonas syringae, we ascertained the common and unique genes required to colonize these habitats. A lack of association between gene expression and contribution to fitness suggests that many genes that are highly expressed or inducedin plantaare dispensable or redundant. As a model bacterium for plant pathogenesis and colonization, our comprehensive genetic dataset allows us to better understand the traits needed for association with leaves.

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Hcp2, a Secreted Protein of the Phytopathogen Pseudomonas syringae pv. Tomato DC3000, Is Required for Fitness for Competition against Bacteria and Yeasts

Journal of Bacteriology ◽

10.1128/jb.00611-12 ◽

2012 ◽

Vol 194 (18) ◽

pp. 4810-4822 ◽

Cited By ~ 58

Author(s):

Minna Haapalainen ◽

Hanna Mosorin ◽

Federico Dorati ◽

Ru-Fen Wu ◽

Elina Roine ◽

...

Keyword(s):

Pseudomonas Syringae ◽

Plant Pathogen ◽

Expression Patterns ◽

Stationary Growth Phase ◽

Plant Colonization ◽

In Planta ◽

Type Vi Secretion System ◽

Tomato Dc3000 ◽

Content Type ◽

Covalently Linked

ABSTRACTWhen analyzing the secretome of the plant pathogenPseudomonas syringaepv. tomato DC3000, we identified hemolysin-coregulated protein (Hcp) as one of the secreted proteins. Hcp is assumed to be an extracellular component of the type VI secretion system (T6SS). Two copies ofhcpgenes are present in theP. syringaepv. tomato DC3000 genome,hcp1(PSPTO_2539) andhcp2(PSPTO_5435). We studied the expression patterns of thehcpgenes and tested the fitness ofhcpknockout mutants in host plant colonization and in intermicrobial competition. We found that thehcp2gene is expressed most actively at the stationary growth phase and that the Hcp2 protein is secreted via the T6SS and appears in the culture medium as covalently linked dimers. Expression ofhcp2is not inducedin plantaand does not contribute to virulence in or colonization of tomato orArabidopsisplants. Instead,hcp2is required for survival in competition with enterobacteria and yeasts, and its function is associated with the suppression of the growth of these competitors. This is the first report on bacterial T6SS-associated genes functioning in competition with yeast. Our results suggest that the T6SS ofP. syringaemay play an important role in bacterial fitness, allowing this plant pathogen to survive under conditions where it has to compete with other microorganisms for resources.

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Malate : Quinone Oxidoreductase and Malic Enzyme are required for the Plant Pathogen Pseudomonas syringae pv. tomato DC3000 to Utilize Malate

American Journal of Undergraduate Research ◽

10.33697/ajur.2016.016 ◽

2016 ◽

Vol 13 (2) ◽

Author(s):

Zabrina Ebert ◽

Preston Jacob ◽

Katrina Jose ◽

Lina Fouad ◽

Katherine Vercellino ◽

...

Keyword(s):

Arabidopsis Thaliana ◽

Citric Acid ◽

Pseudomonas Syringae ◽

Plant Pathogen ◽

Malic Enzyme ◽

Citric Acid Cycle ◽

Wild Type ◽

In Planta ◽

Quinone Oxidoreductase ◽

Acid Cycle

Pseudomonas syringae pv. tomato strain DC3000 (DC3000) is a gram-negative bacterial plant pathogen that causes disease on tomato and the model plant Arabidopsis thaliana. Interestingly, previous studies showed that malate:quinone oxidoreductase (Mqo), an enzyme in the citric acid cycle, is required for DC3000 to cause disease on these plants. In addition, growth of DC3000 lacking the mqo gene in minimal medium with malate was significantly delayed, but eventually reached wild-type levels of growth, which is similar to growth in planta. This suggests that malate may be an important carbon source for DC3000. One reason the mqo::KO bacteria may be able to reach wild-type levels of growth in culture and plants is that the DC3000 malic enzyme may be used to complete the citric acid cycle. Our research shows that a mutant strain lacking a functional mqo gene and malic enzyme gene (mqo::KO;ME::pJP) fails to grow in minimal media cultures with malate and has reduced growth in media with citrate, indicating that both Mqo and ME are required for normal growth when utilizing these carbon sources. Future studies looking at growth of this double mutant in plants will identify how important the activities of both of these genes are for DC3000 to cause disease in plants. KEY WORDS: Malate:quinone Oxidoreductase; Malic Enzyme; MQO; Pseudomonas syringae; Arabidopsis thaliana; Malate; Citrate; DC3000

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Zinc phosphate protects tomato plants against Pseudomonas syringae pv. tomato

Journal of Plant Diseases and Protection ◽

10.1007/s41348-021-00444-z ◽

2021 ◽

Author(s):

Mara Quaglia ◽

Marika Bocchini ◽

Benedetta Orfei ◽

Roberto D’Amato ◽

Franco Famiani ◽

...

Keyword(s):

Disease Severity ◽

Pseudomonas Syringae ◽

Zinc Phosphate ◽

Plant Defence ◽

In Planta ◽

Tomato Plants ◽

Pathogenesis Related Protein ◽

Defence Responses ◽

Plant Defence Responses

AbstractThe purpose of this study was to determine whether zinc phosphate treatments of tomato plants (Solanum lycopersicum L.) can attenuate bacterial speck disease severity through reduction of Pseudomonas syringae pv. tomato (Pst) growth in planta and induce morphological and biochemical plant defence responses. Tomato plants were treated with 10 ppm (25.90 µM) zinc phosphate and then spray inoculated with strain DAPP-PG 215, race 0 of Pst. Disease symptoms were recorded as chlorosis and/or necrosis per leaf (%) and as numbers of necrotic spots. Soil treatments with zinc phosphate protected susceptible tomato plants against Pst, with reductions in both disease severity and pathogen growth in planta. The reduction of Pst growth in planta combined with significantly higher zinc levels in zinc-phosphate-treated plants indicated direct antimicrobial toxicity of this microelement, as also confirmed by in vitro assays. Morphological (i.e. callose apposition) and biochemical (i.e., expression of salicylic-acid-dependent pathogenesis-related protein PR1b1 gene) defence responses were induced by the zinc phosphate treatment, as demonstrated by histochemical and qPCR analyses, respectively. In conclusion, soil treatments with zinc phosphate can protect tomato plants against Pst attacks through direct antimicrobial activity and induction of morphological and biochemical plant defence responses.

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Prediction and Activity of a Cationic α-Helix Antimicrobial Peptide ZM-804 from Maize

International Journal of Molecular Sciences ◽

10.3390/ijms22052643 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2643

Author(s):

Mohamed F. Hassan ◽

Abdelrahman M. Qutb ◽

Wubei Dong

Keyword(s):

Cdna Library ◽

Pseudomonas Syringae ◽

Inbred Line ◽

Mammalian Cells ◽

Laser Scanning ◽

Pathogenic Bacteria ◽

Antimicrobial Activities ◽

Confocal Laser ◽

In Planta ◽

Minimal Inhibitory Concentrations

Antimicrobial peptides (AMPs) are small molecules consisting of less than fifty residues of amino acids. Plant AMPs establish the first barrier of defense in the innate immune system in response to invading pathogens. The purpose of this study was to isolate new AMPs from the Zea mays L. inbred line B73 and investigate their antimicrobial activities and mechanisms against certain essential plant pathogenic bacteria. In silico, the Collection of Anti-Microbial Peptides (CAMPR3), a computational AMP prediction server, was used to screen a cDNA library for AMPs. A ZM-804 peptide, isolated from the Z. mays L. inbred line B73 cDNA library, was predicted as a new cationic AMP with high prediction values. ZM-804 was tested against eleven pathogens of Gram-negative and Gram-positive bacteria and exhibited high antimicrobial activities as determined by the minimal inhibitory concentrations (MICs) and the minimum bactericidal concentrations (MBCs). A confocal laser scanning microscope observation showed that the ZM-804 AMP targets bacterial cell membranes. SEM and TEM images revealed the disruption and damage of the cell membrane morphology of Clavibacter michiganensis subsp. michiganensis and Pseudomonas syringae pv. tomato (Pst) DC3000 caused by ZM-804. In planta, ZM-804 demonstrated antimicrobial activity and prevented the infection of tomato plants by Pst DC3000. Moreover, four virulent phytopathogenic bacteria were prevented from inducing hypersensitive response (HR) in tobacco leaves in response to low ZM-804 concentrations. ZM-804 exhibits low hemolytic activity against mouse red blood cells (RBCs) and is relatively safe for mammalian cells. In conclusion, the ZM-804 peptide has a strong antibacterial activity and provides an alternative tool for plant disease control. Additionally, the ZM-804 peptide is considered a promising candidate for human and animal drug development.

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HopA1 Effector from Pseudomonas syringae pv syringae Strain 61 Affects NMD Processes and Elicits Effector-Triggered Immunity

International Journal of Molecular Sciences ◽

10.3390/ijms22147440 ◽

2021 ◽

Vol 22 (14) ◽

pp. 7440

Author(s):

Shraddha K. Dahale ◽

Daipayan Ghosh ◽

Kishor D. Ingole ◽

Anup Chugani ◽

Sang Hee Kim ◽

...

Keyword(s):

Pseudomonas Syringae ◽

Disease Susceptibility ◽

Null Mutant ◽

In Planta ◽

Host Range Expansion ◽

Unique System ◽

Effector Triggered Immunity ◽

Transcriptomic Changes ◽

Immune Detection

Pseudomonas syringae-secreted HopA1 effectors are important determinants in host range expansion and increased pathogenicity. Their recent acquisitions via horizontal gene transfer in several non-pathogenic Pseudomonas strains worldwide have caused alarming increase in their virulence capabilities. In Arabidopsis thaliana, RESISTANCE TO PSEUDOMONAS SYRINGAE 6 (RPS6) gene confers effector-triggered immunity (ETI) against HopA1pss derived from P. syringae pv. syringae strain 61. Surprisingly, a closely related HopA1pst from the tomato pathovar evades immune detection. These responsive differences in planta between the two HopA1s represents a unique system to study pathogen adaptation skills and host-jumps. However, molecular understanding of HopA1′s contribution to overall virulence remain undeciphered. Here, we show that immune-suppressive functions of HopA1pst are more potent than HopA1pss. In the resistance-compromised ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) null-mutant, transcriptomic changes associated with HopA1pss-elicited ETI are still induced and carry resemblance to PAMP-triggered immunity (PTI) signatures. Enrichment of HopA1pss interactome identifies proteins with regulatory roles in post-transcriptional and translational processes. With our demonstration here that both HopA1 suppress reporter-gene translations in vitro imply that the above effector-associations with plant target carry inhibitory consequences. Overall, with our results here we unravel possible virulence role(s) of HopA1 in suppressing PTI and provide newer insights into its detection in resistant plants.

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The algT Gene of Pseudomonas syringae pv. glycinea andNew Insights into the Transcriptional Organization of thealgT-muc Gene Cluster

Journal of Bacteriology ◽

10.1128/jb.01160-06 ◽

2006 ◽

Vol 188 (23) ◽

pp. 8013-8021 ◽

Cited By ~ 14

Author(s):

Alexander Schenk ◽

Michael Berger ◽

Lisa M. Keith ◽

Carol L. Bender ◽

Georgi Muskhelishvili ◽

...

Keyword(s):

Gene Cluster ◽

Pseudomonas Syringae ◽

Azotobacter Vinelandii ◽

Regulatory Gene ◽

In Planta ◽

Phytopathogenic Bacterium ◽

Reverse Transcription Pcr ◽

Alginate Production ◽

Alginate Biosynthesis

ABSTRACT The phytopathogenic bacterium Pseudomonas syringae pv. glycinea infects soybean plants and causes bacterial blight. In addition to P. syringae, the human pathogen Pseudomonas aeruginosa and the soil bacterium Azotobacter vinelandii produce the exopolysaccharide alginate, a copolymer of d-mannuronic and l-guluronic acids. Alginate production in P. syringae has been associated with increased fitness and virulence in planta. Alginate biosynthesis is tightly controlled by proteins encoded by the algT-muc regulatory gene cluster in P. aeruginosa and A. vinelandii. These genes encode the alternative sigma factor AlgT (σ22), its anti-sigma factors MucA and MucB, MucC, a protein with a controversial function that is absent in P. syringae, and MucD, a periplasmic serine protease and homolog of HtrA in Escherichia coli. We compared an alginate-deficient algT mutant of P. syringae pv. glycinea with an alginate-producing derivative in which algT is intact. The alginate-producing derivative grew significantly slower in vitro growth but showed increased epiphytic fitness and better symptom development in planta. Evaluation of expression levels for algT, mucA, mucB, mucD, and algD, which encodes an alginate biosynthesis gene, showed that mucD transcription is not dependent on AlgT in P. syringae in vitro. Promoter mapping using primer extension experiments confirmed this finding. Results of reverse transcription-PCR demonstrated that algT, mucA, and mucB are cotranscribed as an operon in P. syringae. Northern blot analysis revealed that mucD was expressed as a 1.75-kb monocistronic mRNA in P. syringae.

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

Canadian Journal of Microbiology ◽

10.1139/m92-144 ◽

1992 ◽

Vol 38 (9) ◽

pp. 883-890 ◽

Cited By ~ 2

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.

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In planta bacterial multi-omics analysis illuminates regulatory principles underlying plant-pathogen interactions

10.1101/822932 ◽

2019 ◽

Cited By ~ 1

Author(s):

Tatsuya Nobori ◽

Yiming Wang ◽

Jingni Wu ◽

Sara Christina Stolze ◽

Yayoi Tsuda ◽

...

Keyword(s):

Gene Regulation ◽

Pseudomonas Syringae ◽

Protein Level ◽

Plant Pathogens ◽

Plant Immunity ◽

Bacterial Pathogen ◽

Bacterial Virulence ◽

In Planta ◽

Bacterial Gene ◽

Global Food Security

AbstractUnderstanding how gene expression is regulated in plant pathogens is crucial for pest control and thus global food security. An integrated understanding of bacterial gene regulation in the host is dependent on multi-omic datasets, but these are largely lacking. Here, we simultaneously characterized the transcriptome and proteome of a foliar bacterial pathogen, Pseudomonas syringae, in Arabidopsis thaliana and identified a number of bacterial processes influenced by plant immunity at the mRNA and the protein level. We found instances of both concordant and discordant regulation of bacterial mRNAs and proteins. Notably, the tip component of bacterial type III secretion system was selectively suppressed by the plant salicylic acid pathway at the protein level, suggesting protein-level targeting of the bacterial virulence system by plant immunity. Furthermore, gene co-expression analysis illuminated previously unknown gene regulatory modules underlying bacterial virulence and their regulatory hierarchy. Collectively, the integrated in planta bacterial omics approach provides molecular insights into multiple layers of bacterial gene regulation that contribute to bacterial growth in planta and elucidate the role of plant immunity in controlling pathogens.

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