scholarly journals A rapid and non-destructive method for spatial–temporal quantification of colonization by Pseudomonas syringae pv. tomato DC3000 in Arabidopsis and tomato

Plant Methods ◽  
2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Leonardo Furci ◽  
David Pascual-Pardo ◽  
Jurriaan Ton
Keyword(s):  
Pseudomonas Syringae ◽  
Time Course ◽  
Infected Leaf ◽  
Cmos Camera ◽  
Basal Resistance ◽  
Additional Information ◽  
Spray Inoculation ◽  
Colony Counting ◽  
Leaf Pathogen ◽  
Non Destructive

Abstract Background The bacterial leaf pathogen Pseudomonas syringae pv tomato (Pst) is the most popular model pathogen for plant pathology research. Previous methods to study the plant-Pst interactions rely on destructive quantification of Pst colonisation, which can be labour- and time-consuming and does not allow for spatial–temporal monitoring of the bacterial colonisation. Here, we describe a rapid and non-destructive method to quantify and visualise spatial–temporal colonisation by Pst in intact leaves of Arabidopsis and tomato. Results The method presented here uses a bioluminescent Pst DC3000 strain that constitutively expresses the luxCDABE operon from Photorhabdus luminescens (Pst::LUX) and requires a common gel documentation (Gel Doc) system with a sensitive CCD/CMOS camera and imaging software (Photoshop or Image J). By capturing bright field and bioluminescence images from Pst::LUX-infected leaves, we imaged the spatiotemporal dynamics of Pst infection. Analysis of bioluminescence from live Pst bacteria over a 5-day time course after spray inoculation of Arabidopsis revealed transition of the bacterial presence from the older leaves to the younger leaves and apical meristem. Colonisation by Pst:LUX bioluminescence was obtained from digital photos by calculating relative bioluminescence values, which is adjusted for bioluminescence intensity and normalised by leaf surface. This method detected statistically significant differences in Pst::LUX colonisation between Arabidopsis genotypes varying in basal resistance, as well as statistically significant reductions in Pst::LUX colonisation by resistance-inducing treatments in both Arabidopsis and tomato. Comparison of relative bioluminescence values to conventional colony counting on selective agar medium revealed a statistically significant correlation, which was reproducible between different Gel Doc systems. Conclusions We present a non-destructive method to quantify colonisation by bioluminescent Pst::LUX in plants. Using a common Gel Doc system and imaging software, our method requires less time and labour than conventional methods that are based on destructive sampling of infected leaf material. Furthermore, in contrast to conventional strategies, our method provides additional information about the spatial–temporal patterns of Pst colonisation.

scholarly journals A Review of the Studies and Interactions ofPseudomonas syringaePathovars on Wheat

2012 ◽  
Vol 2012 ◽  
pp. 1-5 ◽  
Author(s):  
Alberto J. Valencia-Botín ◽  
María E. Cisneros-López
Keyword(s):  
Pseudomonas Syringae ◽  
Seed Yield ◽  
Yield Components ◽  
Aleurone Layer ◽  
Genomic Evolution ◽  
Good Tool ◽  
Rapd Pcr ◽  
Spray Inoculation ◽  
Source Sink

Wheat is affected by some pathovars ofPseudomonas syringaeand by otherPseudomonasspecies. Of these,P. syringaepv.syringaeis the major one responsible for reduction. Recent studies have been made to characterize and identify the pathogen and to determine its aggressiveness and the pattern of colonization in seed and its effects on seed yield, yield components, and source-sink relationships during postanthesis. It was found that the reduction in the aerial biomass production is the best way to evaluate the aggressiveness of this bacterium, and the spray inoculation is good tool to make evaluations at seedling stage. The characterization of bacteria fingerprintings with molecular markers such as RAPD-PCR, ERIC, and REP-PCR is available. Genomic evolution has been elucidated with next-generation genome sequencing. Also, the colonization pattern shows that, early on, microcolonies are frequently detected in the aleurone layer, later in the endosperm and finally close to the crease and even in some cells of the embryo itself. In the wheat cultivars Seri M82 and Rebeca F2000 seed yield and its components are negatively affected. In general,P. syringaepv.syringaereduces the plant height, seed yield, and yield components, as well as the growth of most organs. When this bacterium attacks, the stems are the predominant sink organs and the leaf laminae and panicles are the predominant source organs.

Catalase released from beneficial and plant-pathogenic pseudomonads by water and chloroform treatments

1994 ◽  
Vol 40 (8) ◽  
pp. 630-636
Author(s):  
J. I. Pounder ◽  
A. J. Anderson
Keyword(s):  
Hydrogen Peroxide ◽  
Superoxide Dismutase ◽  
Water Treatment ◽  
Pseudomonas Syringae ◽  
Small Proportion ◽  
Plant Colonization ◽  
Periplasmic Proteins ◽  
Specific Activities ◽  
Leaf Pathogen ◽  
Glucose 6 Phosphate Dehydrogenase

Survival of pseudomonads during plant colonization may involve bacterial catalases to degrade the hydrogen peroxide produced by the plant. The specific activities of catalases in lysates from two saprophytic isolates of Pseudomonas putida and Pseudomonas fluorescens and three races of Pseudomonas syringae pv. glycinea were similar. To explore the location of the bacterial catalases, cells of the pathogenic and saprophytic pseudomonads were treated with chloroform, which is reported to release periplasmic proteins. Although catalase was released by chloroform treatment, the cytoplasmic enzymes isocitrate dehydrogenase, superoxide dismutase, and glucose-6-phosphate dehydrogenase were also detected. These proteins may have come from lysis of a small proportion of the cells rather than the periplasm. Water treatment of cells also released amounts of protein similar to those derived from chloroform treatment. Similar responses were found from both pathogenic and saprophytic strains. The release of catalase and proteins from the leaf pathogen P. syringae pv. glycinea race 0 and the root-associated saprophyte P. putida decreased as the cultures aged. With P. putida and P. syringae pv. glycinea race 0, the single isozyme of catalase released by water and chloroform treatment also was detected in lysates. Additional catalase isozymes were present in lysates as the cultures aged.Key words: periplasmic proteins, survival.

Perception and first defense responses against Pseudomonas syringae pv. phaseolicola in Phaseolus vulgaris L.: identification of Wall-Associated Kinase receptors

2021 ◽  
Author(s):  
Alfonso Gonzalo De la Rubia ◽  
María Luz Centeno ◽  
Victor Moreno-González ◽  
María De Castro ◽  
Penélope García-Angulo
Keyword(s):  
Phaseolus Vulgaris ◽  
Pseudomonas Syringae ◽  
Time Course ◽  
Early Time ◽  
Experimental System ◽  
Antioxidant Response ◽  
Defense Responses ◽  
Cellular Damage ◽  
Initial Increase ◽  
Phaseolus Vulgaris L

Common bean (Phaseolus vulgaris L.) is attacked by several pathogens such as the biotrophic gamma-proteobacterium Pseudomonas syringae pv. phaseolicola (Pph). In order to study the Pph-bean interaction during the first stages of infection, leaf disks of a susceptible bean variety named Riñón were infected with a pathogenic Pph. Using this experimental system, six new putative Wall-Associated Kinase (WAKs) receptors, previously identified in silico, were tested. These six bean WAKs (PvWAKs) showed high protein sequence homology to the well-described Arabidopsis WAK1 (AtWAK1) receptor and, by phylogenetic analysis, clustered together with AtWAKs. The expression of PvWAK1 increased at very early stages after the Pph infection. Time course experiments were performed to evaluate the accumulation of apoplastic H2O2, Ca2+ influx, total H2O2, antioxidant enzymatic activities, lipid peroxidation, and the concentrations of abscisic acid (ABA) and salicylic acid (SA), as well as the expression of six defense-related genes – MEKK-1, MAPKK, WRKY33, RIN4, PR1 and NPR1. The results showed that overexpression of PR1 occurred 2 h after Pph infection without a concomitant increase in SA levels. Although apoplastic H2O2 increased after infection, the oxidative burst was neither intense nor rapid and an efficient antioxidant response did not occur, suggesting that the observed cellular damage was due to the initial increase in total H2O2 at early time points after infection. In conclusion, the Riñón variety can perceive the presence of Pph, but this recognition only results in a modest and slow activation of host defenses, leading to high susceptibility to Pph.

scholarly journals Location and Survival of Leaf-Associated Bacteria in Relation to Pathogenicity and Potential for Growth within the Leaf

1999 ◽  
Vol 65 (4) ◽  
pp. 1435-1443 ◽  
Author(s):  
M. Wilson ◽  
S. S. Hirano ◽  
S. E. Lindow
Keyword(s):  
Pseudomonas Syringae ◽  
Plant Pathogens ◽  
Vacuum Infiltration ◽  
Associated Bacteria ◽  
Growth And Survival ◽  
Total Leaf ◽  
Spray Inoculation ◽  
Dry Conditions ◽  
Population Sizes ◽  
Better Than

ABSTRACT The growth and survival of pathogenic and nonpathogenicPseudomonas syringae strains and of the nonpathogenic species Pantoea agglomerans, Stenotrophomonas maltophilia, and Methylobacterium organophilum were compared in the phyllosphere of bean. In general, the plant pathogens survived better than the nonpathogens on leaves under environmental stress. The sizes of the total leaf-associated populations of the pathogenic P. syringae strains were greater than the sizes of the total leaf-associated populations of the nonpathogens under dry conditions but not under moist conditions. In these studies the surface sterilants hydrogen peroxide and UV irradiation were used to differentiate cells that were fully exposed on the surface from nonexposed cells that were in “protected sites” that were inaccessible to these agents. In general, the population sizes in protected sites increased with time after inoculation of plants. The proportion of bacteria on leaves that were in protected sites was generally greater for pathogens than for nonpathogens and was greater under dry conditions than under moist conditions. When organisms were vacuum infiltrated into leaves, the sizes of the nonexposed “internal” populations were greater for pathogenic P. syringae strains than for nonpathogenic P. syringaestrains. The sizes of the populations of the nonpathogenic species failed to increase or even decreased. The sizes of nonexposed populations following spray inoculation were correlated with the sizes of nonexposed, internal populations which developed after vacuum infiltration and incubation. While the sizes of the populations of the pathogenic P. syringae strains increased on leaves under dry conditions, the sizes of the populations of the nonpathogenic strains of P. syringae, P. agglomerans, andS. maltophilia decreased when the organisms were applied to plants. The sizes of the populations on dry leaves were also correlated with the sizes of the nonexposed populations that developed following vacuum infiltration. Although pathogenicity was not required for growth in the phyllosphere under high-relative-humidity conditions, pathogenicity apparently was involved in the ability to access and/or multiply in certain protected sites in the phyllosphere and in growth on dry leaves.

scholarly journals Harpin Inactivates Mitochondria in Arabidopsis Suspension Cells

2004 ◽  
Vol 17 (2) ◽  
pp. 131-139 ◽  
Author(s):  
Maren Krause ◽  
Jörg Durner
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Pseudomonas Syringae ◽  
Stress Responses ◽  
Time Course ◽  
Defense Responses ◽  
Ros Generation ◽  
Suspension Cells ◽  
Mitochondrial Functions ◽  
Arabidopsis Cells

Harpin is a well-known proteinaceous bacterial elicitor that can induce an oxidative burst and programmed cell death in various host plants. Given the demonstrated roles of mitochondria in animal apoptosis, we investigated the effect of harpin from Pseudomonas syringae on mitochondrial functions in Arabidopsis suspension cells in detail. Fluorescence microscopy in conjunction with double-staining for reactive oxygen species (ROS) and mitochondria suggested co-localization of mitochondria and ROS generation. Plant defense responses or cell death after pathogen attack have been suggested to be regulated by the concerted action of ROS and nitric oxide (NO). However, although Arabidopsis cells respond to harpin treatment with NO generation, time course analyses suggest that NO generation is not involved in initial responses but, rather, is a consequence of cellular decay. Among the fast responses we observed was a decrease of the mitochondrial membrane potential Δψm and, possibly as a direct consequence, of ATP production. Furthermore, treatment of Arabidopsis cells with harpin protein induced a rapid cytochrome C release from mitochondria into the cytosol, which is regarded as a hallmark of programmed cell death or apoptosis. Northern and DNA array analyses showed strong induction of protecting or scavenging systems such as alternative oxidase and small heat shock proteins, components that are known to be associated with cellular stress responses. In sum, the presented data suggest that harpin inactivates mitochondria in Arabidopsis cells.

scholarly journals Functional requirement of the Arabidopsis importin-α nuclear transport receptor family in autoimmunity mediated by the NLR protein SNC1

2020 ◽  
Author(s):  
Daniel Lüdke ◽  
Charlotte Roth ◽  
Sieglinde A. Kamrad ◽  
Jana Messerschmidt ◽  
Denise Hartken ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Nuclear Import ◽  
Nuclear Transport ◽  
Basal Resistance ◽  
Protein Protein Interaction ◽  
Cargo Proteins ◽  
Importin Α ◽  
Localization Signal ◽  
Receptor Mutant ◽  
Transport Receptor

SUMMARYIMPORTIN-α3/MOS6 (MODIFIER OF SNC1, 6) is one of nine importin-α isoforms in Arabidopsis that recruit nuclear localization signal (NLS)-containing cargo proteins to the nuclear import machinery. IMP-α3/MOS6 is required genetically for full autoimmunity of the nucleotide-binding leucine-rich repeat (NLR) immune receptor mutant snc1 (suppressor of npr1-1, constitutive 1) and MOS6 also contributes to basal disease resistance. Here, we investigated the contribution of the other importin-α genes to both types of immune responses, and we analyzed potential interactions of all importin-α isoforms with SNC1. By using reverse-genetic analyses in Arabidopsis and protein-protein interaction assays in N. benthamiana we provide evidence that among the nine α-importins in Arabidopsis, IMP-α3/MOS6 is the main nuclear transport receptor of SNC1, and that IMP-α3/MOS6 is required selectively for autoimmunity of snc1 and basal resistance to mildly virulent Pseudomonas syringae in Arabidopsis.SIGNIFICANCE STATEMENTSpecific requirement for the Arabidopsis α-importin MOS6 in snc1-mediated autoimmunity is explained by selective formation of MOS6-SNC1 nuclear import complexes.

scholarly journals Evaluation of lockdown effect on SARS-CoV-2 dynamics through viral genome quantification in waste water, Greater Paris, France, 5 March to 23 April 2020

2020 ◽  
Vol 25 (50) ◽  
Author(s):  
S Wurtzer ◽  
V Marechal ◽  
JM Mouchel ◽  
Y Maday ◽  
R Teyssou ◽  
...  
Keyword(s):  
Waste Water ◽  
Viral Genome ◽  
Time Course ◽  
Human Populations ◽  
Additional Information ◽  
Quantitative Monitoring ◽  
Stool Samples ◽  
Important Additional Information ◽  
Specific Symptoms

Introduction Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of coronavirus disease (COVID-19). People infected with SARS-CoV-2 may exhibit no or mild non-specific symptoms; thus, they may contribute to silent circulation of the virus among humans. Since SARS-CoV-2 RNA can be detected in stool samples, monitoring SARS-CoV-2 RNA in waste water (WW) has been proposed as a complementary tool to investigate virus circulation in human populations. Aim To test if the quantification of SARS-CoV-2 genomes in WW correlates with the number of symptomatic or non-symptomatic carriers. Method We performed a time-course quantitative analysis of SARS-CoV-2 by RT-qPCR in raw WW samples collected from several major WW treatment plants in Greater Paris. The study period was 5 March to 23 April 2020, including the lockdown period in France (from 17 March). Results We showed that the increase of genome units in raw WW accurately followed the increase of human COVID-19 cases observed at the regional level. Of note, the viral genome could be detected before the epidemic grew massively (around 8 March). Equally importantly, a marked decrease in the quantities of genome units was observed concomitantly with the reduction in the number of new COVID-19 cases, 29 days following the lockdown. Conclusion This work suggests that a quantitative monitoring of SARS-CoV-2 genomes in WW could generate important additional information for improved monitoring of SARS-CoV-2 circulation at local or regional levels and emphasises the role of WW-based epidemiology.

scholarly journals Characterization of a Tryptophan 2-Monooxygenase Gene from Puccinia graminis f. sp. tritici Involved in Auxin Biosynthesis and Rust Pathogenicity

2014 ◽  
Vol 27 (3) ◽  
pp. 227-235 ◽  
Author(s):  
Chuntao Yin ◽  
Jeong-Jin Park ◽  
David R. Gang ◽  
Scot H. Hulbert
Keyword(s):  
Pseudomonas Syringae ◽  
Rust Fungus ◽  
Infected Plant ◽  
Bacterial Pathogen ◽  
Leaf Tissue ◽  
Infected Leaf ◽  
Puccinia Graminis ◽  
Monooxygenase Gene ◽  
Microbe Interactions

The plant hormone indole-3-acetic acid (IAA) is best known as a regulator of plant growth and development but its production can also affect plant–microbe interactions. Microorganisms, including numerous plant-associated bacteria and several fungi, are also capable of producing IAA. The stem rust fungus Puccinia graminis f. sp. tritici induced wheat plants to accumulate auxin in infected leaf tissue. A gene (Pgt-IaaM) encoding a putative tryptophan 2-monooxygenase, which makes the auxin precursor indole-3-acetamide (IAM), was identified in the P. graminis f. sp. tritici genome and found to be expressed in haustoria cells in infected plant tissue. Transient silencing of the gene in infected wheat plants indicated that it was required for full pathogenicity. Expression of Pgt-IaaM in Arabidopsis caused a typical auxin expression phenotype and promoted susceptibility to the bacterial pathogen Pseudomonas syringae pv. tomato DC3000.

Applying of dynamic acoustic signal attributes for evaluation of the contact between pile and soils

2020 ◽  
pp. 126-137
Author(s):  
V. V. Kapustin ◽  
A. A. Churkin
Keyword(s):  
Data Analysis ◽  
Static Load ◽  
Geophysical Methods ◽  
Additional Information ◽  
Integrity Testing ◽  
Testing Data ◽  
Load Tests ◽  
Dynamic Attributes ◽  
Non Destructive ◽  
Pile Integrity

The low-strain impact method is one of the most commonly used non-destructive geophysical methods in pile integrity testing. Data analysis of the low-strain method in the frequency domain allows the researcher to get additional information about the studied foundation. The authors of the article propose a methodology for assessing the contact of piles with soils based on an analysis of the dynamic attributes of the frequency response. Conclusions drawn from using the above-described method can be used by engineers to study piles quality and to plan direct static load tests to determine the bearing capacity of piles.

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