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Methods Mol Biol. 2017;1615:473-487. doi: 10.1007/978-1-4939-7033-9_33.

Effector Translocation: Cya Reporter Assay.

Author information

1
Plant Pathology and Plant-Microbe Biology Section, School of Integrative Plant Science, Cornell University, Ithaca, NY, 14853, USA.
2
Department of Energy, Plant Research Laboratory, Michigan State University, East Lansing, MI, 48824, USA.
3
Department of Plant Pathology, University of Georgia, Athens, GA, 30602, USA. bkvitko@uga.edu.

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.

KEYWORDS:

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

PMID:
28667631
DOI:
10.1007/978-1-4939-7033-9_33
[Indexed for MEDLINE]

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