Abstract
Innate immunity represents the first line of inducible defence against microbial infection in plants and animals1,2,3. In both kingdoms, recognition of pathogen- or microbe-associated molecular patterns (PAMPs or MAMPs, respectively), such as flagellin, initiates convergent signalling pathways involving mitogen-activated protein kinase (MAPK) cascades and global transcriptional changes to boost immunity1,2,3,4. Although Ca2+ has long been recognized as an essential and conserved primary mediator in plant defence responses, how Ca2+ signals are sensed and relayed into early MAMP signalling is unknown5,6. Using a functional genomic screen and genome-wide gene expression profiling, here we show that four calcium-dependent protein kinases (CDPKs) are Ca2+-sensor protein kinases critical for transcriptional reprogramming in plant innate immune signalling. Unexpectedly, CDPKs and MAPK cascades act differentially in four MAMP-mediated regulatory programs to control early genes involved in the synthesis of defence peptides and metabolites, cell wall modifications and redox signalling. Transcriptome profile comparison suggests that CDPKs are the convergence point of signalling triggered by most MAMPs. Double, triple and quadruple cpk mutant plants display progressively diminished oxidative burst and gene activation induced by the 22-amino-acid peptide flg22, as well as compromised pathogen defence. In contrast to negative roles of calmodulin and a calmodulin-activated transcription factor in plant defence7,8, the present study reveals Ca2+ signalling complexity and demonstrates key positive roles of specific CDPKs in initial MAMP signalling.
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Gene Expression Omnibus
Data deposits
All microarray data are available at the Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE16557.
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Acknowledgements
We thank K. H. Liu and E. Baena-González for contributing to the CPK clone collection, O. R. Patharkar for initial microarray data analysis, B. Mueller and G. Tena for offering qPCR primers, M. N. Soler and S. Bolte for assistance on confocal microscopy, C. Laurière for discussions, S. P. Dinesh-Kumar for VIGS vectors, K. Schreiber and D. Desveaux for the seedling pathogen assay, and A. Gust and T. Nürnberger for the original PGN microarray data. We thank the Salk Institute, Syngenta Biotechnology and A. Sessions for sharing the Arabidopsis T-DNA collections, and the Arabidopsis Biological Resource Center for providing Arabidopsis mutant seeds. This research has been supported by a Marie Curie International fellowship within the 6th European Community Framework Program to M.B., an NSF predoctoral fellowship to M.R.W., and grants from the National Science Foundation and the National Institute of Health, and the MGH CCIB fund to J.S.
Author Contributions J.S. and M.R.W. initiated the project; M.B., M.R.W. and J.S. designed the experiments; M.R.W., M.B. and S.-H.C. built the CPK clone collection; M.B., M.R.W., H.L., L.S., P.H. and J.S. conducted the experiments and analysed the data; M.M., J.S. and M.B. analysed microarray data; J.B. managed plants; M.B., J.S. and M.M. prepared the manuscript with inputs from all co-authors.
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Supplementary Information
This file contains Supplementary Figures 1-14 with Legends, Supplementary Tables 1and 9-14 (see separate files for Supplementary Tables 2-8), Supplementary Methods and Supplementary References. (PDF 12933 kb)
Supplementary Table 2
This table shows the total target gene list of CPK5 and CPK11, with the induction level (log2 ratio) by CPK and flg22 treatments in protoplasts, leaves and seedlings. For each gene, the Affymetrix ID, AGI number and TAIR annotations are provided. (XLS 205 kb)
Supplementary Table 3
This table shows shared CPK5 and CPK11 target genes overlapping with early flg22 responsive genes. For each gene, the induction level (log2 ratio) by CPK, the Affymetrix ID, AGI number, MapMan function category and annotations, Sheen lab annotations and TAIR annotations are provided. (XLS 126 kb)
Supplementary Table 4
This table shows CPK5-specific target genes overlapping with early flg22 responsive genes. For each gene, the induction level (log2 ratio) by CPK, the Affymetrix ID, AGI number, MapMan function category and annotations, Sheen lab annotations and TAIR annotations are provided. (XLS 106 kb)
Supplementary Table 5
This table shows CPK11-specific target genes overlapping with early flg22 responsive genes. For each gene, the induction level (log2 ratio) by CPK, the Affymetrix ID, AGI number, MapMan function category and annotations, Sheen lab annotations and TAIR annotations are provided. (XLS 76 kb)
Supplementary Table 6
This table shows CPK5 and CPK11 target genes (shared and specific) overlapping with early flg22 responsive genes. For each gene, the induction level (log2 ratio) by CPK and flg22 treatments in protoplasts, leaves and seedlings, the Affymetrix ID, AGI number and TAIR annotations are provided. (XLS 57 kb)
Supplementary Table 7
This table shows CPK5 and CPK11 target genes (shared and specific) overlapping with early MAMP responsive genes. For each gene, the induction level (log2 ratio) by CPK, flg22 treatments in leaves and seedlings and ABA treatment, the Affymetrix ID, AGI number and TAIR annotations are provided. (XLS 52 kb)
Supplementary Table 8
This table shows CPK5 and CPK11 target genes (shared and specific) overlapping with microbe responsive genes. For each gene, the induction level (log2 ratio) by CPK and microbes, the Affymetrix ID, AGI number and TAIR annotations are provided. (XLS 44 kb)
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Boudsocq, M., Willmann, M., McCormack, M. et al. Differential innate immune signalling via Ca2+ sensor protein kinases. Nature 464, 418–422 (2010). https://doi.org/10.1038/nature08794
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DOI: https://doi.org/10.1038/nature08794
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