Timing of plant immune responses by a central circadian regulator

Journal name:
Date published:
Published online

The principal immune mechanism against biotrophic pathogens in plants is the resistance (R)-gene-mediated defence1. It was proposed to share components with the broad-spectrum basal defence machinery2. However, the underlying molecular mechanism is largely unknown. Here we report the identification of novel genes involved in R-gene-mediated resistance against downy mildew in Arabidopsis and their regulatory control by the circadian regulator, CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1). Numerical clustering based on phenotypes of these gene mutants revealed that programmed cell death (PCD) is the major contributor to resistance. Mutants compromised in the R-gene-mediated PCD were also defective in basal resistance, establishing an interconnection between these two distinct defence mechanisms. Surprisingly, we found that these new defence genes are under circadian control by CCA1, allowing plants to ‘anticipate’ infection at dawn when the pathogen normally disperses the spores and time immune responses according to the perception of different pathogenic signals upon infection. Temporal control of the defence genes by CCA1 differentiates their involvement in basal and R-gene-mediated defence. Our study has revealed a key functional link between the circadian clock and plant immunity.

At a glance


  1. Phenotypic analyses discovered two distinct RPP4-mediated resistance responses against Hpa Emwa1.
    Figure 1: Phenotypic analyses discovered two distinct RPP4-mediated resistance responses against Hpa Emwa1.

    a, Phenotype scores (percentage in 40 leaves per genotype). SPP, sporangiophore; TRN, trailing necrosis; OOS, oospore; FRH, free hypha; FHI, free hyphal intermediate; EXH, expanding hypersensitive response; DIH, discrete hypersensitive response. *P<0.05. b, Mutants were clustered on the basis of their phenotype scores in Fig. 1a. Second allele, ‘A’. Group 1, red; Group 2, blue. au, Approximately unbiased P-values (0–100%, the higher the number the more significant). c, Eigenvectors derived from PCA. The percentage of phenotypic variations captured by each PC is shown. d, A diagram showing that the Group 1 mutants are defective in RPP4-mediated PCD, whereas the Group 2 mutants are compromised in formation of physical/chemical barriers with intact PCD.

  2. Some of the RPP4-mediated resistance mutants are also compromised in basal defence.
    Figure 2: Some of the RPP4-mediated resistance mutants are also compromised in basal defence.

    a, Enhanced disease susceptibility to Hpa Noco2 based on sporangiospore count 7dpi (n = 3). b, Summary of the infection tests on the 22 defence gene mutants (22 mts) using different Hpa isolates. S, susceptible; R, resistant; EDS, enhanced disease susceptibility. c, Root length measurements 9days after elf18 treatment (n = 3). efr, efl18 receptor mutant. d, Fresh weight measurements 6days after elf18 treatment (n = 3). *P<0.05, **P<0.01, ***P<0.001.

  3. The circadian regulator, CCA1, controls the defence gene expression and the timing of immune responses.
    Figure 3: The circadian regulator, CCA1, controls the defence gene expression and the timing of immune responses.

    a, Enrichment of evening element (EE) (P<10−5). NMF, non-negative matrix factorization; CCA1, CCA1-binding sites; Circadian correl., circadian correlations14. +, sense; −, antisense. b, SPP count 7dpi by Hpa Emwa1 (n = 3). c, Time-course expression of NMF Cluster 1 genes. CI, confidence interval; CK, control; EMWA1, Hpa Emwa1 inoculated. White bars, day; black bars, night. d, SPP count after Hpa Emwa1 infection at dawn or dusk (n = 3). e, Occurrence of DIH 7dpi by Hpa Emwa. f, A model showing circadian regulation of the defence genes in anticipation of infection under normal conditions, in basal and R-gene-mediated resistance. The blocked arrows represent defence against infection.

Accession codes

Primary accessions

Gene Expression Omnibus


  1. Jones, J. D. & Dangl, J. L. The plant immune system. Nature 444, 323329 (2006)
  2. Tao, Y. et al. Quantitative nature of Arabidopsis responses during compatible and incompatible interactions with the bacterial pathogen Pseudomonas syringae . Plant Cell 15, 317330 (2003)
  3. Roden, L. C. & Ingle, R. A. Lights, rhythms, infection: the role of light and the circadian clock in determining the outcome of plant–pathogen interactions. Plant Cell 21, 25462552 (2009)
  4. Lam, E., Kato, N. & Lawton, M. Programmed cell death, mitochondria and the plant hypersensitive response. Nature 411, 848853 (2001)
  5. Donofrio, N. M. & Delaney, T. P. Abnormal callose response phenotype and hypersusceptibility to Peronospoara parasitica in defence-compromised Arabidopsis nim1–1 and salicylate hydroxylase-expressing plants. Mol. Plant Microbe Interact. 14, 439450 (2001)
  6. Holub, E. B., Beynon, J. L. & Crute, I. R. Phenotypic and genotypic characterization of interactions between isolates of Peronospora parasitica and accessions of Arabidopsis thaliana . Mol. Plant Microbe Interact. 7, 223239 (1994)
  7. van der Biezen, E. A., Freddie, C. T., Kahn, K., Parker, J. E. & Jones, J. D. Arabidopsis RPP4 is a member of the RPP5 multigene family of TIR-NB-LRR genes and confers downy mildew resistance through multiple signalling components. Plant J. 29, 439451 (2002)
  8. McDowell, J. M. et al. Downy mildew (Peronospora parasitica) resistance genes in Arabidopsis vary in functional requirements for NDR1, EDS1, NPR1 and salicylic acid accumulation. Plant J. 22, 523529 (2000)
  9. Chinchilla, D. et al. A flagellin-induced complex of the receptor FLS2 and BAK1 initiates plant defence. Nature 448, 497500 (2007)
  10. Kemmerling, B. et al. The BRI1-associated kinase 1, BAK1, has a brassinolide-independent role in plant cell-death control. Curr. Biol. 17, 11161122 (2007)
  11. Heese, A. et al. The receptor-like kinase SERK3/BAK1 is a central regulator of innate immunity in plants. Proc. Natl Acad. Sci. USA 104, 1221712222 (2007)
  12. Harmer, S. L. et al. Orchestrated transcription of key pathways in Arabidopsis by the circadian clock. Science 290, 21102113 (2000)
  13. Harmer, S. L. & Kay, S. A. Positive and negative factors confer phase-specific circadian regulation of transcription in Arabidopsis . Plant Cell 17, 19261940 (2005)
  14. Michael, T. P. et al. Network discovery pipeline elucidates conserved time-of-day-specific cis-regulatory modules. PLoS Genet. 4, e14 (2008)
  15. Slusarenko, A. & Schlaich, N. L. Downy Mildew of Arabidopsis thaliana caused by Hyaloperonospora parasitica (formerly Peronospora parasitica). Mol. Plant Pathol. 4, 159170 (2003)
  16. Salome, P. A. & McClung, C. R. PSEUDO-RESPONSE REGULATOR 7 and 9 are partially redundant genes essential for the temperature responsiveness of the Arabidopsis circadian clock. Plant Cell 17, 791803 (2005)
  17. Wang, Z. Y. & Tobin, E. M. Constitutive expression of the CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) gene disrupts circadian rhythms and suppresses its own expression. Cell 93, 12071217 (1998)
  18. Schaffer, R. et al. The late elongated hypocotyl mutation of Arabidopsis disrupts circadian rhythms and the photoperiodic control of flowering. Cell 93, 12191229 (1998)
  19. Yeakley, J. M. et al. Profiling alternative splicing on fiber-optic arrays. Nature Biotechnol. 20, 353358 (2002)
  20. Tamayo, P. et al. Metagene projection for cross-platform, cross-species characterization of global transcriptional states. Proc. Natl Acad. Sci. USA 104, 59595964 (2007)
  21. Bryant, P. A., Trinder, J. & Curtis, N. Sick and tired: does sleep have a vital role in the immune system? Nature Rev. Immunol. 4, 457467 (2004)
  22. Tor, M. et al. Arabidopsis SGT1b is required for defense signaling conferred by several downy mildew resistance genes. Plant Cell 14, 9931003 (2002)
  23. Brady, S. M. et al. A high-resolution root spatiotemporal map reveals dominant expression patterns. Science 318, 801806 (2007)
  24. Bowling, S. A., Clarke, J. D., Liu, Y., Klessig, D. F. & Dong, X. The cpr5 mutant of Arabidopsis expresses both NPR1-dependent and NPR1-independent resistance. Plant Cell 9, 15731584 (1997)
  25. Suzuki, R. & Shimodaira, H. Pvclust: an R package for assessing the uncertainty in hierarchical clustering. Bioinformatics 22, 15401542 (2006)
  26. Adam, L. & Somerville, S. C. Genetic characterization of five powdery mildew resistance loci in Arabidopsis thaliana . Plant J. 9, 341356 (1996)
  27. Cao, H., Bowling, S. A., Gordon, S. & Dong, X. Characterization of an Arabidopsis mutant that is nonresponsive to inducers of systemic acquired resistance. Plant Cell 6, 15831592 (1994)

Download references

Author information

  1. These authors contributed equally to this work.

    • Wei Wang &
    • Jinyoung Yang Barnaby


  1. Department of Biology, P. O. Box 90338, Duke University, Durham, North Carolina 27708, USA

    • Wei Wang,
    • Jinyoung Yang Barnaby,
    • Yasuomi Tada,
    • Daniela Caldelari,
    • Dae-un Lee &
    • Xinnian Dong
  2. Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, California 92093, USA

    • Hairi Li &
    • Xiang-Dong Fu
  3. National Pollen and Aerobiology Research Unit (NPARU), University of Worcester, Henwick Grove, Worcester WR2 6AJ, UK

    • Mahmut Tör
  4. Present address: Crop Systems and Global Change Laboratory, United States Department of Agriculture, Agricultural Research Service, Plant Sciences Institute, Room 342, Building 001, BARC-West, 10300 Baltimore Avenue, Beltsville, Maryland 20705,USA

    • Jinyoung Yang Barnaby
  5. Present address: Life Science Research Centre, Institute of Research Promotion, Kagawa University, 2393 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0795, Japan

    • Yasuomi Tada
  6. Present address: Plant Molecular Biology, University of Lausanne, CH-1015 Lausanne, Switzerland

    • Daniela Caldelari


J.Y.B., W.W., Y.T., D.C. and D.-u.L. identified new components in R-gene-mediated resistance; J.Y.B., W.W. and Y.T. showed that RPP4 controls two major defence responses by phenotypic clustering analysis; J.Y.B., W.W. and M.T. demonstrated that R-gene-mediated resistance shares common components with basal defence machinery; W.W., J.Y.B., H.L., X.-D.F. and X.D. verified the circadian regulator CCA1 plays a key role in timing the different immune responses; W.W., J.Y.B. and X.D. wrote the manuscript with inputs from all co-authors. All authors discussed the results and commented on the manuscript.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to:

The microarray data presented in this publication have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE22274.

Author details

Supplementary information

PDF files

  1. Supplementary Information (2.2M)

    This file contains Supplementary Figures 1-12 with legends and Supplementary Tables 1-3.

Additional data