Abstract
The plant circadian clock drives temporal differences in susceptibility to pathogens. We investigated the role of TIMING OF CAB EXPRESSION 1 (TOC1) in the regulation of defence against Botrytis cinerea in Arabidopsis. The temporal variation in susceptibility to B. cinerea observed in wild-type Arabidopsis was abolished in TOC1-ox and toc1-2 plants under both diurnal and constant light conditions. In addition, TOC1-ox plants were more susceptible than Col-0 following inoculation at dawn, while inoculation at night led to enhanced resistance in toc1-2 plants versus C24 plants, suggesting that TOC1 is a negative regulator of immunity. RNA-seq analysis showed that the genes mis-regulated in toc1-2 plants had significant enrichment for terms related to biotic stress, an overrepresentation of G-box elements in their promoters and included genes encoding key transcription factors (TFs) involved in defence against necrotrophic pathogens. Chromatin immunoprecipitation-qPCR showed that TOC1 occupies G-box containing regions of the defence TFs ERF4, ORA47, ORA59 and WRKY33 in a pathogen-responsive and MYC2-dependent manner. We suggest that the phased TOC1 occupancy of defence gene promoters contributes to the gating of plant immunity against necrotrophic pathogens, while the MYC2-dependent release of TOC1 in response to pathogen detection allows plants to mount an acute immune response.
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Data availability
The RNA-seq datasets generated in this work have been deposited in NCBI under accession number PRJNA1270936. Trimmed Mean of M values (TMM)-normalised expression values for each sample in the RNA-seq experiment are provided in Supplementary Data 5. The source data used to generate Figs. 1 to 5 and Supplementary Fig. 2, 3 and 5 are provided in Supplemental Data 6. This study did not develop any custom code. All other data supporting the findings of this study are available from Robert Ingle (Robert.ingle@uct.ac.za) upon reasonable request.
References
Nozue, K. et al. Rhythmic growth explained by coincidence between internal and external cues. Nature 448, 358–361 (2007).
Bhardwaj, V., Meier, S., Petersen, L. N., Ingle, R. A. & Roden, L. C. Defence responses of Arabidopsis thaliana to infection by Pseudomonas syringae are regulated by the circadian clock. PLOS ONE 6, e26968 (2011).
Ingle, R. A. et al. Jasmonate signalling drives time-of-day differences in susceptibility of Arabidopsis to the fungal pathogen Botrytis cinerea. Plant J. 84, 937–948 (2015).
Wang, W. et al. Timing of plant immune responses by a central circadian regulator. Nature 470, 110–114 (2011).
Zhang, C. et al. LUX ARRHYTHMO mediates crosstalk between the circadian clock and defense in Arabidopsis. Nat. Commun. 10, 2543 (2019).
Zhang, C. et al. Crosstalk between the circadian clock and innate Immunity in Arabidopsis. PLOS Pathog. 9, e1003370 (2013).
Eichmann, R. & Schäfer, P. Growth versus immunity—a redirection of the cell cycle? Curr. Opin. Plant Biol. 26, 106–112 (2015).
Hevia, M. A., Canessa, P., Müller-Esparza, H. & Larrondo, L. F. A circadian oscillator in the fungus Botrytis cinerea regulates virulence when infecting Arabidopsis thaliana. Proc. Natl. Acad. Sci. 112, 8744–8749 (2015).
Schaffer, R. et al. The late elongated hypocotyl mutation of Arabidopsis disrupts circadian rhythms and the photoperiodic control of flowering. Cell 93, 1219–1229 (1998).
Strayer, C. et al. Cloning of the Arabidopsis clock gene TOC1, an autoregulatory response regulator homolog. Science 289, 768–771 (2000).
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, 1207–1217 (1998).
Alabadı́, D. et al. Reciprocal regulation between TOC1 and LHY/CCA1 within the Arabidopsis circadian clock. Science 293, 880–883 (2001).
Gendron, J. M. et al. Arabidopsis circadian clock protein, TOC1, is a DNA-binding transcription factor. Proc. Natl. Acad. Sci. 109, 3167–3172 (2012).
Huang, W. et al. Mapping the core of the Arabidopsis circadian clock defines the network structure of the oscillator. Science 336, 75–79 (2012).
Pruneda-Paz, J. L., Breton, G., Para, A. & Kay, S. A. A functional genomics approach reveals CHE as a component of the Arabidopsis circadian clock. Science 323, 1481–1485 (2009).
Jones, J. D. G. & Dangl, J. L. The plant immune system. Nature 444, 323–329 (2006).
Ngou, B. P. M., Ding, P. & Jones, J. D. G. Thirty years of resistance: Zig-zag through the plant immune system. Plant Cell 34, 1447–1478 (2022).
Deslandes, L. & Rivas, S. Catch me if you can: bacterial effectors and plant targets. Trends Plant Sci. 17, 644–655 (2012).
Xiang, T. et al. Pseudomonas syringae effector AvrPto blocks innate immunity by targeting receptor kinases. Curr. Biol. 18, 74–80 (2008).
Chen, J. et al. NLR surveillance of pathogen interference with hormone receptors induces immunity. Nature 613, 145–152 (2023).
Fu, M. et al. A pathogen effector HaRxL10 hijacks the circadian clock component CHE to perturb both plant development and immunity. Nat. Commun. 16, 1538 (2025).
Yuan, M., Ngou, B. P. M., Ding, P. & Xin, X.-F. PTI-ETI crosstalk: an integrative view of plant immunity. Curr. Opin. Plant Biol. 62, 102030 (2021).
Mine, A. et al. The defense phytohormone signaling network enables rapid, high-amplitude transcriptional reprogramming during effector-triggered immunity. Plant Cell 30, 1199–1219 (2018).
Shigenaga, A. M., Berens, M. L., Tsuda, K. & Argueso, C. T. Towards engineering of hormonal crosstalk in plant immunity. Curr. Opini. Plant Biol. 38, 164–172 (2017).
Aerts, N., Pereira Mendes, M. & Van Wees, S. C. M. Multiple levels of crosstalk in hormone networks regulating plant defense. Plant J. 105, 489–504 (2021).
Melotto, M., Underwood, W., Koczan, J., Nomura, K. & He, S. Y. Plant stomata function in innate immunity against bacterial invasion. Cell 126, 969–980 (2006).
Melotto, M., Zhang, L., Oblessuc, P. R. & He, S. Y. Stomatal defense a decade later. Plant Physiol. 174, 561–571 (2017).
Covington, M. F., Maloof, J. N., Straume, M., Kay, S. A. & Harmer, S. L. Global transcriptome analysis reveals circadian regulation of key pathways in plant growth and development. Genome Biol. 9, R130 (2008).
Mizuno, T. & Yamashino, T. Comparative transcriptome of diurnally oscillating genes and hormone-responsive genes in Arabidopsis thaliana: Insight into circadian clock-controlled daily responses to common ambient stresses in plants. Plant Cell Physiol. 49, 481–487 (2008).
Goodspeed, D., Chehab, E. W., Min-Venditti, A., Braam, J. & Covington, M. F. Arabidopsis synchronizes jasmonate-mediated defense with insect circadian behavior. Proc. Natl. Acad. Sci. 109, 4674–4677 (2012).
Nagel, D. H. et al. Genome-wide identification of CCA1 targets uncovers an expanded clock network in Arabidopsis. Proc. Natl. Acad. Sci. 112, E4802–E4810 (2015).
Shin, J., Heidrich, K., Sanchez-Villarreal, A., Parker, J. E. & Davis, S. J. TIME FOR COFFEE represses accumulation of the MYC2 transcription factor to provide time-of-day regulation of jasmonate signaling in Arabidopsis. Plant Cell 24, 2470–2482 (2012).
Chini, A. et al. The JAZ family of repressors is the missing link in jasmonate signalling. Nature 448, 666–671 (2007).
Dombrecht, B. et al. MYC2 differentially modulates diverse jasmonate-dependent functions in Arabidopsis. Plant Cell 19, 2225–2245 (2007).
Joseph, R., Odendaal, J. L., Ingle, R. A. & Roden, L. C. The role of the jasmonate signalling transcription factors MYC2/3/4 in circadian clock-mediated regulation of immunity in Arabidopsis. Philos. Trans. R. Soc. B 380, 20230338 (2025).
Más, P., Alabadí, D., Yanovsky, M. J., Oyama, T. & Kay, S. A. Dual role of TOC1 in the control of circadian and photomorphogenic responses in Arabidopsis. Plant Cell 15, 223–236 (2003).
Makino, S., Matsushika, A., Kojima, M., Yamashino, T. & Mizuno, T. The APRR1/TOC1 quintet implicated in circadian rhythms of Arabidopsis thaliana: I. Characterization with APRR1-overexpressing Plants. Plant Cell Physiol. 43, 58–69 (2002).
Yamashino, T. et al. Involvement of Arabidopsis clock-associated pseudo-response regulators in diurnal oscillations of gene expression in the presence of environmental time cues. Plant Cell Physiol. 49, 1839–1850 (2008).
Más, P., Kim, W.-Y., Somers, D. E. & Kay, S. A. Targeted degradation of TOC1 by ZTL modulates circadian function in Arabidopsis thaliana. Nature 426, 567–570 (2003).
Windram, O. et al. Arabidopsis defense against Botrytis cinerea: Chronology and regulation deciphered by high-resolution temporal transcriptomic analysis. Plant Cell 24, 3530–3557 (2012).
Graf, A. et al. Parallel analysis of Arabidopsis circadian clock mutants reveals different scales of transcriptome and proteome regulation. Open Biol. 7, 160333 (2017).
Liu, T. L., Newton, L., Liu, M. J., Shiu, S. H. & Farré, E. M. A G-Box-like motif Is necessary for transcriptional regulation by circadian Pseudo-Response Regulators in Arabidopsis. Plant Physiol. 170, 528–539 (2016).
Yoo, S.-D., Cho, Y.-H., Tena, G., Xiong, Y. & Sheen, J. Dual control of nuclear EIN3 by bifurcate MAPK cascades in C2H4 signalling. Nature 451, 789–795 (2008).
Wang, H. et al. MED25 connects enhancer–promoter looping and MYC2-dependent activation of jasmonate signalling. Nat. Plants 5, 616–625 (2019).
Bonnot, T. & Nagel, D. H. Time of the day prioritizes the pool of translating mRNAs in response to heat stress. Plant Cell 33, 2164–2182 (2021).
Gao, M. et al. Circadian regulation of the GLYCINE-RICH RNA-BINDING PROTEIN gene by the master clock protein CIRCADIAN CLOCK-ASSOCIATED 1 is important for plant innate immunity. J. Exp. Bot. 74, 991–1003 (2022).
Chao, Q. et al. Activation of the ethylene gas response pathway in Arabidopsis by the nuclear protein ETHYLENE-INSENSITIVE3 and related proteins. Cell 89, 1133–1144 (1997).
Pré, M. et al. The AP2/ERF domain transcription factor ORA59 integrates jasmonic acid and ethylene signals in plant defense. Plant Physiol. 147, 1347–1357 (2008).
Solano, R., Stepanova, A., Chao, Q. & Ecker, J. R. Nuclear events in ethylene signaling: a transcriptional cascade mediated by ETHYLENE-INSENSITIVE3 and ETHYLENE-RESPONSE-FACTOR1. Genes Dev. 12, 3703–3714 (1998).
Frerigmann, H., Glawischnig, E. & Gigolashvili, T. The role of MYB34, MYB51 and MYB122 in the regulation of camalexin biosynthesis in Arabidopsis thaliana. Front Plant Sci. 6, 654 (2015).
Moffat, C. S. et al. ERF5 and ERF6 play redundant roles as positive regulators of JA/Et-mediated defense against Botrytis cinerea in Arabidopsis. PLOS One 7, e35995 (2012).
Yang, Z., Tian, L., Latoszek-Green, M., Brown, D. & Wu, K. Arabidopsis ERF4 is a transcriptional repressor capable of modulating ethylene and abscisic acid responses. Plant Mol. Biol. 58, 585–596 (2005).
McGrath, K. C. et al. Repressor- and activator-type ethylene response factors functioning in jasmonate signaling and disease resistance identified via a genome-wide screen of Arabidopsis transcription factor gene expression. Plant Physiol. 139, 949–959 (2005).
Birkenbihl, R. P., Diezel, C. & Somssich, I. E. Arabidopsis WRKY33 Is a key transcriptional regulator of hormonal and metabolic responses toward Botrytis cinerea Infection. Plant Physiol. 159, 266–285 (2012).
Chen, H.-Y. et al. ORA47 (octadecanoid-responsive AP2/ERF-domain transcription factor 47) regulates jasmonic acid and abscisic acid biosynthesis and signaling through binding to a novel cis-element. N. Phytol. 211, 599–613 (2016).
Soy, J. et al. Molecular convergence of clock and photosensory pathways through PIF3-TOC1 interaction and co-occupancy of target promoters. Proc. Natl. Acad. Sci. 113, 4870–4875 (2016).
Wasternack, C. & Hause, B. Jasmonates: biosynthesis, perception, signal transduction and action in plant stress response, growth and development. An update to the 2007 review in Annals of Botany. Ann. Bot. 111, 1021–1058 (2013).
Yan, J. et al. TOC1 clock protein phosphorylation controls complex formation with NF-YB/C to repress hypocotyl growth. EMBO J. 40, e108684 (2021).
Yan, J. et al. TOC1 phosphorylation disproportionally enhances chromatin binding at rhythmic gene promoters. Sci. Adv. 11, eadx7804 (2025).
Chico, J. M. et al. Repression of jasmonate-dependent defenses by shade involves differential regulation of protein stability of MYC transcription factors and their JAZ repressors in Arabidopsis. Plant Cell 26, 1967–1980 (2014).
Song, S. et al. MYC5 is Involved in jasmonate-regulated plant growth, leaf senescence and defense responses. Plant Cell Physiol. 58, 1752–1763 (2017).
Kazan, K. & Manners, J. M. MYC2: the master in action. Mol. Plant 6, 686–703 (2013).
Lorenzo, O., Chico, J. M., Sánchez-Serrano, J. J. & Solano, R. JASMONATE-INSENSITIVE1 encodes a MYC transcription factor essential to discriminate between different jasmonate-regulated defense responses in Arabidopsis. Plant Cell 16, 1938–1950 (2004).
Nickstadt, A. et al. The jasmonate-insensitive mutant jin1 shows increased resistance to biotrophic as well as necrotrophic pathogens. Mol. Plant Pathol. 5, 425–434 (2004).
Gautam, J. K., Giri, M. K., Singh, D., Chattopadhyay, S. & Nandi, A. K. MYC2 influences salicylic acid biosynthesis and defense against bacterial pathogens in Arabidopsis thaliana. Physiol. Plant 173, 2248–2261 (2021).
Liu, T., Carlsson, J., Takeuchi, T., Newton, L. & Farré, E. M. Direct regulation of abiotic responses by the Arabidopsis circadian clock component PRR7. Plant J. 76, 101–114 (2013).
Nakamichi, N. et al. Transcriptional repressor PRR5 directly regulates clock-output pathways. Proc. Natl. Acad. Sci. 109, 17123–17128 (2012).
Shen, C. et al. Structural Insight into DNA Recognition by CCT/NF-YB/YC Complexes in Plant Photoperiodic Flowering. Plant Cell 32, 3469–3484 (2020).
Para, A. et al. PRR3 Is a vascular regulator of TOC1 stability in the Arabidopsis circadian clock. Plant Cell 19, 3462–3473 (2007).
Wang, L., Fujiwara, S. & Somers, D. E. PRR5 regulates phosphorylation, nuclear import and subnuclear localization of TOC1 in the Arabidopsis circadian clock. EMBO J. 29, 1903–1915 (2010).
Nakamichi, N. et al. PSEUDO-RESPONSE REGULATORS 9, 7, and 5 are transcriptional repressors in the Arabidopsis circadian clock. Plant Cell 22, 594–605 (2010).
Fraser, O. J. P., Spoel, S. H. & van Ooijen, G. TOC1 supresses PAMP-triggered immunity in Arabidopsis. bioRxiv, 2025.2007.2016.665052 (2025).
Gimenez-Ibanez, S. et al. JAZ2 controls stomata dynamics during bacterial invasion. N. Phytol. 213, 1378–1392 (2017).
Carstens, M. et al. Increased resistance to biotrophic pathogens in the Arabidopsis constitutive induced resistance 1 mutant is EDS1 and PAD4-dependent and modulated by environmental temperature. PLOS ONE 9, e109853 (2014).
Ingle, R. A. & Roden, L. C. in Plant Circadian Networks: Methods and Protocols (ed Dorothee Staiger) 273-283 (Springer New York, 2014).
Rasmussen, R. in Rapid Cycle Real-Time PCR: Methods and Applications (eds Stefan Meuer, Carl Wittwer, & Kan-Ichi Nakagawara) 21-34 (Springer Berlin Heidelberg, 2001).
Haring, M. et al. Chromatin immunoprecipitation: optimization, quantitative analysis and data normalization. Plant Methods 3, 11 (2007).
Mruk, D. D. & Cheng, C. Y. Enhanced chemiluminescence (ECL) for routine immunoblotting: An inexpensive alternative to commercially available kits. Spermatogenesis 1, 121–122 (2011).
Acknowledgements
We acknowledge Riccardo Aiese Cigliano (Sequentia Biotech) for his analysis of the RNA-Seq dataset. We also thank Lara Donaldson (ICGEB Cape Town) for C24 seed, Paloma Más (Universitat Autònoma de Barcelona) for toc1-2, TOC1-ox and TMG seeds and Matthew Lewsey (La Trobe University) for MYC2-YPet seed. This work was funded by the National Research Foundation of South Africa (Competitive support for rated researcher grant numbers 105819 and 118504).
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L.C.R. and R.A.I. conceptualised the study and obtained funding. SS performed the experimental work. S.S. and R.A.I. analysed the data. All authors contributed to the design of experiments and to writing of the manuscript.
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Sparks, SL., Roden, L.C. & Ingle, R.A. The core clock transcription factor TOC1 binds directly to defence gene promoters regulating immunity in Arabidopsis. Commun Biol (2026). https://doi.org/10.1038/s42003-026-09667-y
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DOI: https://doi.org/10.1038/s42003-026-09667-y
