Abstract
The war between plants and their pathogens is endless, with plant resistance genes offering protection against pathogens until the pathogen evolves a way to overcome this resistance. Given how quickly new pathogen strains can arise and defeat plant defenses, it is critical to more rapidly identify and examine the specific genomic characteristics new virulent strains have gained which give them the upper hand. An indispensable tool is bioinformatics. Genome sequencing has advanced rapidly in the last decade, and labs are frequently uploading high-quality genomes of various organisms, including plant pathogenic bacteria such as Pseudomonas syringae. Pseudomonas syringae strains inject several effector proteins into host cells which often overcome host defenses. Probing online genomes provides a way to quickly and accurately predict effector repertoires of Pseudomonas, enabling the cloning of complete effector libraries of newly emerged strains. Here, we describe detailed protocols to rapidly clone bioinformatically predicted P. syringae effectors for various screening applications.
Jay Jayaraman and Morgan K. Halane are co-first authors.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Jones JD, Dangl JL (2006) The plant immune system. Nature 444(7117):323–329. https://doi.org/10.1038/nature05286
Templeton MD, Warren BA, Andersen MT et al (2015) Complete DNA sequence of pseudomonas syringae pv. actinidiae, the causal agent of Kiwifruit canker disease. Genome Announc 3(5):e01054. https://doi.org/10.1128/genomeA.01054-15
McCann HC, Rikkerink EH, Bertels F et al (2013) Genomic analysis of the Kiwifruit pathogen Pseudomonas syringae pv. actinidiae provides insight into the origins of an emergent plant disease. PLoS Pathog 9(7):e1003503. https://doi.org/10.1371/journal.ppat.1003503
Choi S, Jayaraman J, Segonzac C et al (2017) Pseudomonas syringae pv. actinidiae type III effectors localized at multiple cellular compartments activate or suppress innate immune responses in nicotiana benthamiana. Front Plant Sci 8:2157. https://doi.org/10.3389/fpls.2017.02157
Engler C, Youles M, Gruetzner R et al (2014) A golden gate modular cloning toolbox for plants. ACS Synth Biol 3(11):839–843. https://doi.org/10.1021/sb4001504
Altschul SF, Gish W, Miller W et al (1990) Basic local alignment search tool. J Mol Biol 215(3):403–410. https://doi.org/10.1016/S0022-2836(05)80360-2
Lindeberg M, Stavrinides J, Chang JH et al (2005) Proposed guidelines for a unified nomenclature and phylogenetic analysis of type III Hop effector proteins in the plant pathogen Pseudomonas syringae. Mol Plant-Microbe Interact 18(4):275–282. https://doi.org/10.1094/MPMI-18-0275
Stamatakis A (2014) RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30(9):1312–1313. https://doi.org/10.1093/bioinformatics/btu033
Engler C, Kandzia R, Marillonnet S (2008) A one pot, one step, precision cloning method with high throughput capability. PLoS One 3(11):e3647. https://doi.org/10.1371/journal.pone.0003647
Thomas WJ, Thireault CA, Kimbrel JA et al (2009) Recombineering and stable integration of the Pseudomonas syringae pv. syringae 61 hrp/hrc cluster into the genome of the soil bacterium Pseudomonas fluorescens Pf0-1. Plant J 60(5):919–928. https://doi.org/10.1111/j.1365-313X.2009.03998.x
Jayaraman J, Choi S., Prokchorchik M, Choi DS, Spiandore A, Rikkerink EH, Templeton MD, Segonzac C, Sohn KH (2017) A bacterial acetyltransferase triggers immunity in Arabidopsis thaliana independent of hypersensitive response. Sci Rep 7:3557. https://doi.org/10.1111/j.1365-313X.2009.03998.x
Weber E, Engler C, Gruetzner R, Werner S, Marillonnet S (2011) A modular cloning system for standardized assembly of multigene constructs. PLoS ONE 6(2):e16765. https://doi.org/10.1371/journal.pone.0016765
Segonzac C, Newman TE, Choi S, Jayaraman J, Choi DS, Jung, GY, Cho H, Lee YK, Sohn KH (2017) A conserved EAR motif is required for avirulence and stability of the Ralstonia solanacearum effector PopP2 in planta. Front Plant Sci 8. https://doi.org/10.3389/fpls.2017.01330
Baltrus DA, Nishimura MT, Romanchuk A et al (2011) Dynamic evolution of pathogenicity revealed by sequencing and comparative genomics of 19 Pseudomonas syringae isolates. PLoS Pathog 7(7):e1002132. https://doi.org/10.1371/journal.ppat.1002132
Hulin MT, Armitage AD, Vicente JG et al (2018) Comparative genomics of Pseudomonas syringae reveals convergent gene gain and loss associated with specialization onto cherry (Prunus avium). New Phytol 219(2):672–696. https://doi.org/10.1111/nph.15182
Vaghchhipawala Z, Rojas CM, Senthil-Kumar M et al (2011) Agroinoculation and agroinfiltration: simple tools for complex gene function analyses. Methods Mol Biol 678:65–76. https://doi.org/10.1007/978-1-60761-682-5_6
Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16(6):735–743
Fields S, Song O (1989) A novel genetic system to detect protein-protein interactions. Nature 340(6230):245–246. https://doi.org/10.1038/340245a0
Acknowledgments
This work was carried out with the support of the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2016R1D1A1B03934707), Republic of Korea.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Jayaraman, J., Halane, M.K., Choi, S., McCann, H.C., Sohn, K.H. (2019). Using Bioinformatics and Molecular Biology to Streamline Construction of Effector Libraries for Phytopathogenic Pseudomonas syringae Strains. In: Gassmann, W. (eds) Plant Innate Immunity. Methods in Molecular Biology, vol 1991. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9458-8_1
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9458-8_1
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-9457-1
Online ISBN: 978-1-4939-9458-8
eBook Packages: Springer Protocols