Timing of plant immune responses by a central circadian regulator

Abstract

The principal immune mechanism against biotrophic pathogens in plants is the resistance (R)-gene-mediated defence. It was proposed to share components with the broad-spectrum basal defence machinery. 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.

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.

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 7 dpi (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 9 days after elf18 treatment (n=3). efr, efl18 receptor mutant. d, Fresh weight measurements 6 days after elf18 treatment (n=3). *P<0.05, **P<0.01, ***P<0.001.

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 7 dpiby 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 7 dpi 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.