A central circadian oscillator confers defense heterosis in hybrids without growth vigor costs

Plant immunity frequently incurs growth penalties, which known as the trade-off between immunity and growth. Heterosis, the phenotypic superiority of a hybrid over its parents, has been demonstrated for many traits but rarely for disease resistance. Here, we report that the central circadian oscillator, CCA1, confers heterosis for bacterial defense in hybrids without growth vigor costs, and it even significantly enhances the growth heterosis of hybrids under pathogen infection. The genetic perturbation of CCA1 abrogated heterosis for both defense and growth in hybrids. Upon pathogen attack, the expression of CCA1 in F1 hybrids is precisely modulated at different time points during the day by its rhythmic histone modifications. Before dawn of the first infection day, epigenetic activation of CCA1 promotes an elevation of salicylic acid accumulation in hybrids, enabling heterosis for defense. During the middle of every infection day, diurnal epigenetic repression of CCA1 leads to rhythmically increased chlorophyll synthesis and starch metabolism in hybrids, effectively eliminating the immunity-growth heterosis trade-offs in hybrids.

qPCR analyses of PR1's expression level of the wild-type and CCA1-mutated F 1 hybrids and parents at 24 hours post infiltration with Pst DC3000. The expression level of PR1 was shown as the mean ± SD (n = 3, n indicates biological replicates). Data are standardized for the abundance of the ACTIN2 transcript. '***' p value < 0.001 (twotailed Student's t test).
growth is expressed as the mean value of viable bacteria per gram of leaf tissue ± SD (n = 6, n indicates biological replicates). e Mid-parent heterosis (MPH) value calculated by bacterial number at 5 days post infiltration with Pst DC3000 for wild-type and CCA1mutated F 1 hybrids. Data are shown as the mean ± SD. The results in d and e are representative of three independent experiments, with measurements that were taken from independent samples grown and processed at different times. 8 of PR1's expression level of wild-type and LHY-mutated F 1 hybrids and parents at 24 hours post infiltration with Pst DC3000. The expression level of PR1 is shown as the mean ± SD (n = 3, n indicates biological replicates). Data are standardized for the abundance of the ACTIN2 transcript. The results in c to e are representative of three independent experiments, with measurements that were taken from independent samples grown and processed at different times. Supplementary Fig. 7. Mutation of TOC1 did not influence defence heterosis of FCS hybrids.
a Gene model of TOC1-CRISPR mutants in the Sei-0 background. White box, promoter; black boxes, exons; gray boxes, 3'UTR. Lines with arrowheads indicate the fragment deleted in CRISPR mutants. Primers used to test the transcript level of TOC1 are indicated by arrows. b Transcript level of TOC1 mutants and the wild-type at ZT12. The transcript level of TOC1 (ZT12, 12-day-old seedlings) is shown as the mean ± SD (n = 3, n indicates biological replicates) and standardized for the abundance of the ACTIN2 transcript. c Bacterial titer (log10) of the wild-type and TOC1-mutated F 1 hybrids and parents at 5 days post infiltration with Pst DC3000. Bacterial growth is expressed as the mean values of viable bacteria per gram of leaf tissue ± SD (n = 6, n indicates biological replicates). d Mid-parent heterosis (MPH) and Best-parent heterosis (BPH) values calculated by bacterial number at 5 days post infiltration with Pst DC3000 for the wildtype and TOC1-mutated F 1 hybrids. Data are shown as the mean ± SD. e qPCR analyses of PR1's expression level of the wild-type and TOC1-mutated F 1 hybrids and parents at 12, 24 and 36 hours post infiltration with Pst DC3000. The expression level of PR1 was shown as the mean ± SD (n = 3, n indicates biological replicates). Data are standardized for the abundance of the ACTIN2 transcript. The results in c to e are representative of three independent experiments, with measurements that were taken from independent samples grown and processed at different times.

FCS hybrids and parents at both 6 hpi and 21 hpi.
Numbers above the bands indicated the intensities of CCA1 protein normalized to ACTIN, which calculated by image J software. CCA1-OE was used as a positive control.
ACTIN was used as a loading control.

biosynthetic and starch degradation genes showed no difference between the FCS hybrids and parents at 3 hpi.
ChIP-qPCR analyses of promoter fragments that contained 'evening element' motif and ELF3-mutated F 1 hybrids. Data are shown as the mean ± SD. The results in c and d are representative of three independent experiments, with measurements that were taken from independent samples grown and processed at different times. e Fresh weight of the whole rosette of wild-type and ELF3-mutated F 1 hybrids and parents at 5 days post infiltration with Pst DC3000. Data are shown as the mean ± SD (n = 30 plants for each genotype). '**' p < 0.01 (two-tailed Student's t test). f MPH and BPH values calculated by the whole rosette fresh weight at 5 days post infiltration with Pst DC3000 for the wild-type and ELF3-mutated F 1 hybrids. Data are shown as the mean ± SD. The results in f are representative of three independent experiments, with measurements that were taken from independent samples grown and processed at different times.
for wild-type and ELF4-mutated F 1 hybrids. Data are shown as the mean ± SD. The results in c and d are representative of three replicates, with measurements that were taken from independent samples grown and processed at different times. e Fresh weight of the whole rosette of wild-type and ELF4-mutated F 1 hybrids and parents at 5 days post infiltration with Pst DC3000. Data are shown as mean ± SD (n = 30 plants for each genotype). '***' p value < 0.001 (two-tailed Student's t test). f MPH and BPH values calculated by the whole rosette fresh weight at 5 days post infiltration with Pst DC3000 for the wild-type and ELF4-mutated F 1 hybrids. Data are shown as the mean ± SD. The results in f are representative of three independent experiments, with measurements that were taken from independent samples grown and processed at different times. Supplementary Table 1. Genes for GO analysis in Supplementary Figure 1.