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Effector Translocation: Cya Reporter Assay

  • Suma Chakravarthy
  • Bethany Huot
  • Brian H. Kvitko
Part of the Methods in Molecular Biology book series (MIMB, volume 1615)

Abstract

An accurate and complete roster of the Type III effector (T3E) proteins translocated by the P. syringae Type III secretion system (T3SS) into host cells is critical to understanding the pathogen’s interactions with plants. The adenylate cyclase (Cya) reporter offers a highly sensitive and robust assay for monitoring the translocation of T3Es. T3Es are fused to the calmodulin-dependent adenylate-cyclase domain of CyaA. The T3E targets Cya for translocation through the T3SS into the host cell at which point it is activated by calmodulin and converts adenosine triphosphate into cyclic adenosine monophosphate (cAMP). The T3SS translocation-dependent increase in cAMP concentration in plant cells is then measured with an enzyme-linked immunosorbent assay kit. The Cya reporter can be used to determine whether a candidate protein is translocated by T3SS or to measure relative levels of T3SS translocation in a semiquantitative manner.

Key words

Pseudomonas syringae Type III secretion system Type III translocation Translocation reporter Adenylate cyclase Calmodulin cAMP ELISA 

Notes

Acknowledgments

The authors would like to thank Dr. Lisa Schechter, Dr. Hai Li Wei, Dr. Sebastien Cunnac, Dr. Alan Collmer, and Dr. Sheng Yang He for significant contributions to the development and refinement of the procedure described in this chapter. This work was supported by National Science Foundation grant IOS-1025642 and the Gordon and Betty Moore Foundation (GBMF3037).

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Suma Chakravarthy
    • 1
  • Bethany Huot
    • 2
  • Brian H. Kvitko
    • 3
  1. 1.Plant Pathology and Plant-Microbe Biology SectionSchool of Integrative Plant Science, Cornell UniversityIthacaUSA
  2. 2.Department of Energy, Plant Research LaboratoryMichigan State UniversityEast LansingUSA
  3. 3.Department of Plant PathologyUniversity of GeorgiaAthensUSA

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