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Related Concept Videos

Reporter Genes02:11

Reporter Genes

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Reporter genes are a type of protein-coding gene that are often tagged to a gene of interest. Once inside a target cell, reporter genes usually produce visually identifiable characteristics like fluorescence and luminescence when expressed along with the gene of interest. Thus, reporter genes “report” the presence or absence of genes of interest in an organism, determine the gene expression pattern, or track the physical location of a DNA segment or protein in the cell.
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Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions
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Effector Translocation: Cya Reporter Assay.

Suma Chakravarthy1, Bethany Huot2, Brian H Kvitko3

  • 1Plant Pathology and Plant-Microbe Biology Section, School of Integrative Plant Science, Cornell University, Ithaca, NY, 14853, USA.

Methods in Molecular Biology (Clifton, N.J.)
|July 2, 2017
PubMed
Summary
This summary is machine-generated.

Identifying P. syringae Type III effector (T3E) proteins is crucial for understanding plant-pathogen interactions. The adenylate cyclase (Cya) reporter assay provides a sensitive method for monitoring T3E translocation via the Type III secretion system (T3SS).

Keywords:
Adenylate cyclaseCalmodulinELISAPseudomonas syringaeTranslocation reporterType III secretion systemType III translocationcAMP

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Area of Science:

  • Microbiology
  • Plant Pathology
  • Molecular Biology

Background:

  • Understanding pathogen-host interactions is vital in plant pathology.
  • The P. syringae Type III secretion system (T3SS) translocates effector proteins into host plant cells.
  • Accurate identification of translocated Type III effector (T3E) proteins is essential.

Purpose of the Study:

  • To establish a sensitive and robust assay for monitoring T3E translocation.
  • To enable the identification and quantification of T3Es translocated by the P. syringae T3SS.
  • To facilitate research into P. syringae-plant interactions.

Main Methods:

  • Utilizing the adenylate cyclase (Cya) reporter system.
  • Fusing T3Es to the calmodulin-dependent adenylate-cyclase domain of CyaA.
  • Measuring T3SS translocation-dependent increases in cyclic adenosine monophosphate (cAMP) using an enzyme-linked immunosorbent assay (ELISA).

Main Results:

  • The Cya reporter assay demonstrated high sensitivity and robustness for detecting T3E translocation.
  • The assay allows for the determination of whether a candidate protein is translocated by the T3SS.
  • Relative levels of T3SS translocation can be measured in a semiquantitative manner.

Conclusions:

  • The Cya reporter assay is a valuable tool for studying T3E translocation by the P. syringae T3SS.
  • This method aids in the comprehensive characterization of T3E repertoires.
  • The assay contributes to a deeper understanding of plant-pathogen molecular mechanisms.