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.
References
Cunnac S, Chakravarthy S, Kvitko BH, Russell AB, Martin GB, Collmer A (2011) Genetic disassembly and combinatorial reassembly identify a minimal functional repertoire of type III effectors in Pseudomonas syringae. Proc Natl Acad Sci U S A 108:2975–2980
Fouts DE, Badel JL, Ramos AR, Rapp RA, Collmer A (2003) A pseudomonas syringae pv. tomato DC3000 Hrp (Type III secretion) deletion mutant expressing the Hrp system of bean pathogen P. syringae pv. syringae 61 retains normal host specificity for tomato. Mol Plant-Microbe Interact 16:43–52
Alfano JR, Collmer A (2004) Type III secretion system effector proteins: double agents in bacterial disease and plant defense. Annu Rev Phytopathol 42:385–414
Wei CF, Kvitko BH, Shimizu R, Crabill E, Alfano JR, Lin NC, Martin GB, Huang HC, Collmer A (2007) A Pseudomonas syringae pv. tomato DC3000 mutant lacking the type III effector HopQ1-1 is able to cause disease in the model plant Nicotiana benthamiana. Plant J 51:32–46
Schechter LM, Vencato M, Jordan KL, Schneider SE, Schneider DJ, Collmer A (2006) Multiple approaches to a complete inventory of Pseudomonas syringae pv. tomato DC3000 type III secretion system effector proteins. Mol Plant-Microbe Interact 19:1180–1192
Schechter LM, Roberts KA, Jamir Y, Alfano JR, Collmer A (2004) Pseudomonas syringae type III secretion system targeting signals and novel effectors studied with a Cya translocation reporter. J Bacteriol 186:543–555
Garcia JT, Ferracci F, Jackson MW, Joseph SS, Pattis I, Plano LR, Fischer W, Plano GV (2006) Measurement of effector protein injection by type III and type IV secretion systems by using a 13-residue phosphorylatable glycogen synthase kinase tag. Infect Immun 74:5645–5657
Sory MP, Cornelis GR (1994) Translocation of a hybrid YopE-adenylate cyclase from Yersinia enterocolitica into HeLa cells. Mol Microbiol 14:583–594
den Dulk-Ras A, Vergunst AC, Hooykaas PJ (2014) Cre reporter assay for translocation (CRAfT): a tool for the study of protein translocation into host cells. Methods Mol Biol 1197:103–121
Schechter LM, Valenta JC, Schneider DJ, Collmer A, Sakk E (2012) Functional and computational analysis of amino acid patterns predictive type III secretion system substrates in Pseudomonas syringae. PLoS One 7:e36038
Crabill E, Joe A, Block A, van Rooyen JM, Alfano JR (2010) Plant immunity directly or indirectly restricts the injection of type III effectors by the Pseudomonas syringae type III secretion system. Plant Physiol 154:233–244
Wei HL, Chakravarthy S, Worley JN, Collmer A (2013) Consequences of flagellin export through the type III secretion system of Pseudomonas syringae reveal a major difference in the innate immune systems of mammals and the model plant Nicotiana benthamiana. Cell Microbiol 15:601–618
Oh HS, Kvitko BH, Morello JE, Collmer A (2007) Pseudomonas syringae lytic transglycosylases coregulated with the type III secretion system contribute to the translocation of effector proteins into plant cells. J Bacteriol 189:8277–8289
Badel JL, Shimizu R, Oh HS, Collmer A (2006) A Pseudomonas syringae pv. Tomato avrE1/hopM1 mutant is severely reduced in growth and lesion formation in tomato. Mol Plant-Microbe Interact 19:99–111
Lam HN, Chakravarthy S, Wei HL, BuiNguyen H, Stodghill PV, Collmer A, Swingle BM, Cartinhour SW (2014) Global analysis of the HrpL regulon in the plant pathogen Pseudomonas syringae pv. tomato DC3000 reveals new regulon members with diverse functions. PLoS One 9:e106115
Kvitko BH, Ramos AR, Morello JE, Oh HS, Collmer A (2007) Identification of harpins in Pseudomonas syringae pv. tomato DC3000, which are functionally similar to HrpK1 in promoting translocation of type III secretion system effectors. J Bacteriol 189:8059–8072
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).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Chakravarthy, S., Huot, B., Kvitko, B.H. (2017). Effector Translocation: Cya Reporter Assay. In: Journet, L., Cascales, E. (eds) Bacterial Protein Secretion Systems. Methods in Molecular Biology, vol 1615. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7033-9_33
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7033-9_33
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7031-5
Online ISBN: 978-1-4939-7033-9
eBook Packages: Springer Protocols