scholarly journals Imaging-based resistance assay using enhanced luminescence-tagged Pseudomonas syringae reveals a complex epigenetic network in plant defense signaling pathways

2021 ◽  
Author(s):  
Dinesh S. Pujara ◽  
Sung-Il Kim ◽  
Ji-Chul Nam ◽  
Jose Mayorga ◽  
Isabella Elmore ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Pseudomonas Syringae ◽  
High Throughput ◽  
Imaging System ◽  
Firefly Luciferase ◽  
In Planta ◽  
Basal Resistance ◽  
Defense Signaling ◽  
Enhanced Resistance ◽  
Selection Of

High-throughput resistance assays in plants have a limited selection of suitable pathogens. In this study, we developed a Pseudomonas syringae strain chromosomally tagged with the NanoLuc (NL) luciferase from the deep-sea shrimp Oplophorus gracilirostris, a bioluminescent marker significantly brighter than the conventional firefly luciferase. Our reporter strain tagged with NL was over 100 times brighter than P. syringae tagged with the LuxCDABE operon from Photorhabdus luminescens, one of the existing luciferase-based strains. In planta-imaging was improved by using the surfactant Silwet L-77, particularly at a lower reporter concentration. Using this imaging system, over 30 epigenetic mutants were analyzed for their resistance traits since the defense signaling pathway is known to be epigenetically regulated. SWC1, a defense-related chromatin remodeling complex, was found to be a positive defense regulator, which supported one of two earlier conflicting reports. Compromises in DNA methylation in the CG context led to enhanced resistance against virulent Pst. DCLs and AGOs, important in the biogenesis and exerting the effector function of small RNAs, respectively, showed modest but distinct requirements for ETI and basal resistance to Pst. In addition, the transcriptional expression of an epigenetic component was found to be a significant predictor of its immunity contribution. In summary, this study showcased how a high-throughput resistance assay enabled by a pathogen strain with an improved luminescent reporter could provide insightful knowledge on complex defense signaling pathways.

scholarly journals Arabidopsis ssi2-Conferred Susceptibility to Botrytis cinerea Is Dependent on EDS5 and PAD4

2005 ◽  
Vol 18 (4) ◽  
pp. 363-370 ◽  
Author(s):  
Ashis Nandi ◽  
Wolfgang Moeder ◽  
Pradeep Kachroo ◽  
Daniel F. Klessig ◽  
Jyoti Shah
Keyword(s):  
Cell Death ◽  
Botrytis Cinerea ◽  
Pseudomonas Syringae ◽  
Desaturase Activity ◽  
Peronospora Parasitica ◽  
Basal Resistance ◽  
Enhanced Resistance ◽  
Pathogenesis Related ◽  
Cell Death Phenotype ◽  
Dependent Mechanism

Loss of a stearoyl-ACP desaturase activity in the Arabidopsis thaliana ssi2 mutant confers susceptibility to the necrotroph, Botrytis cinerea. In contrast, the ssi2 mutant exhibits enhanced resistance to Pseudomonas syringae, Peronospora parasitica, and Cucumber mosaic virus. The altered basal resistance to these pathogens in the ssi2 mutant plant is accompanied by the constitutive accumulation of elevated salicylic acid (SA) level and expression of the pathogenesis-related 1 (PR1) gene, the inability of jasmonic acid (JA) to activate expression of the defensin gene, PDF1.2, and the spontaneous death of cells. Here, we show that presence of the eds5 and pad4 mutant alleles compromises the ssi2-conferred resistance to Pseudomonas syringae pv. maculicola. In contrast, resistance to B. cinerea was restored in the ssi2 eds5 and ssi2 pad4 double-mutant plants. However, resistance to B. cinerea was not accompanied by the restoration of JA responsiveness in the ssi2 eds5 and ssi2 pad4 plants. The ssi2 eds5 and ssi2 pad4 plants retain the ssi2-conferred spontaneous cell death phenotype, suggesting that cell death is not a major factor that predisposes the ssi2 mutant to infection by B. cinerea. Furthermore, the high SA content of the ssi2 pad4 plant, combined with our previous observation that the SA-deficient ssi2 nahG plant succumbs to infection by B. cinerea, suggests that elevated SA level does not have a causal role in the ssi2-conferred susceptibility to B. cinerea. Our results suggest that interaction between an SSI2-dependent factor or factors and an EDS5- and PAD4- dependent mechanism or mechanisms modulates defense to B. cinerea.

scholarly journals Basal Resistance Against Pseudomonas syringae in Arabidopsis Involves WRKY53 and a Protein with Homology to a Nematode Resistance Protein

2007 ◽  
Vol 20 (11) ◽  
pp. 1431-1438 ◽  
Author(s):  
Shane L. Murray ◽  
Robert A. Ingle ◽  
Lindsay N. Petersen ◽  
Katherine J. Denby
Keyword(s):  
Pseudomonas Syringae ◽  
Bacterial Pathogen ◽  
Nematode Resistance ◽  
Host Recognition ◽  
In Planta ◽  
Basal Resistance ◽  
Pathogen Effectors ◽  
Positive Regulators ◽  
Resistance Protein ◽  
Molecular Patterns

Basal resistance is the ultimately unsuccessful plant defense response to infection with a virulent pathogen. It is thought to be triggered by host recognition of pathogen-associated molecular patterns, with subsequent suppression of particular components by pathogen effectors. To identify novel components of Arabidopsis basal resistance against the bacterial pathogen Pseudomonas syringae pv. tomato, microarray expression profiling was carried out on the cir1 mutant, which displays enhanced resistance against P. syringae pv. tomato. This identified two genes, At4g23810 and At2g40000, encoding the transcription factor WRKY53 and the nematode resistance protein-like HSPRO2, whose expression was upregulated in cir1 prior to pathogen infection and in wild-type plants after P. syringae pv. tomato infection. WRKY53 and HSPRO2 are positive regulators of basal resistance. Knockout mutants of both genes were more susceptible to P. syringae pv. tomato infection than complemented lines, with increased growth of the pathogen in planta. WRKY53 and HSPRO2 appear to function downstream of salicylic acid and to be negatively regulated by signaling through jasmonic acid and ethylene.

scholarly journals A high-throughput screening assay based on automated microscopy for monitoring antibiotic susceptibility of Mycobacterium tuberculosis phenotypes

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Sadaf Kalsum ◽  
Blanka Andersson ◽  
Jyotirmoy Das ◽  
Thomas Schön ◽  
Maria Lerm
Keyword(s):  
Mycobacterium Tuberculosis ◽  
High Throughput ◽  
High Throughput Screening ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Imaging System ◽  
Aggregate Size ◽  
Live Cell ◽  
Screening Assay ◽  
High Throughput Screening Assay

Abstract Background Efficient high-throughput drug screening assays are necessary to enable the discovery of new anti-mycobacterial drugs. The purpose of our work was to develop and validate an assay based on live-cell imaging which can monitor the growth of two distinct phenotypes of Mycobacterium tuberculosis and to test their susceptibility to commonly used TB drugs. Results Both planktonic and cording phenotypes were successfully monitored as fluorescent objects using the live-cell imaging system IncuCyte S3, allowing collection of data describing distinct characteristics of aggregate size and growth. The quantification of changes in total area of aggregates was used to define IC50 and MIC values of selected TB drugs which revealed that the cording phenotype grew more rapidly and displayed a higher susceptibility to rifampicin. In checkerboard approach, testing pair-wise combinations of sub-inhibitory concentrations of drugs, rifampicin, linezolid and pretomanid demonstrated superior growth inhibition of cording phenotype. Conclusions Our results emphasize the efficiency of using automated live-cell imaging and its potential in high-throughput whole-cell screening to evaluate existing and search for novel antimycobacterial drugs.

Isolation and Characterization of Broad-Spectrum Disease-Resistant Arabidopsis Mutants

Genetics ◽  
2002 ◽  
Vol 160 (4) ◽  
pp. 1661-1671
Author(s):  
Klaus Maleck ◽  
Urs Neuenschwander ◽  
Rebecca M Cade ◽  
Robert A Dietrich ◽  
Jeffery L Dangl ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Systemic Acquired Resistance ◽  
Acquired Resistance ◽  
Constitutive Expression ◽  
Firefly Luciferase ◽  
Marker Genes ◽  
Arabidopsis Mutants ◽  
Isolation And Characterization ◽  
Firefly Luciferase Gene

Abstract To identify Arabidopsis mutants that constitutively express systemic acquired resistance (SAR), we constructed reporter lines expressing the firefly luciferase gene under the control of the SAR-inducible PR-1 promoter (PR-1/luc). After EMS mutagenesis of a well-characterized transgenic line, we screened 250,000 M2 plants for constitutive expression of the reporter gene in vivo. From a mutant collection containing several hundred putative mutants, we concentrated on 16 mutants lacking spontaneous hypersensitive response (HR) cell death. We mapped 4 of these constitutive immunity (cim) mutants to chromosome arms. Constitutive expression of disease resistance was established by analyzing responses to virulent Peronospora parasitica and Pseudomonas syringae strains, by RNA blot analysis for endogenous marker genes, and by determination of salicylic acid levels in the mutants. The variety of the cim phenotypes allowed us to define distinct steps in both the canonical SAR signaling pathway and a separate pathway for resistance to Erysiphe cichoracearum, active in only a subset of the mutants.

scholarly journals Zinc phosphate protects tomato plants against Pseudomonas syringae pv. tomato

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.

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

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.

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

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.

Sign in / Sign up

Export Citation Format

Share Document