Hyaloperonospora arabidopsidis (Hpa) propagation and inoculation were performed as described6 ,22 (link). Ten-day-old plants were inoculated with the asexual spores suspension (5 × 105 spores per ml) of Hpa. Unless specified, the Hpa infection was always performed at dawn of the growth chamber’s photoperiod. Hpa Emwa1-inoculated samples were collected at 0, 0.5, 2 and 4 days post inoculation (dpi). ATH1 GeneChip (Affymetrix) was used for microarray. The arrays were normalized and analysed as described previously23 (link). Disease phenotypes were scored after trypan blue staining at 7 dpi24 (link). Significance of the phenotypic scores was determined based on binomial distribution. Disease phenotypic analysis was performed using hierarchical clustering with distance measured by the standard correlation (average linkage; scale 0–1). The significance of the clustering (bootstrap 100,000 times) was measured by the approximately unbiased P-values (0–100%, the higher the number the more significant25 (link)). Callose deposition was detected after aniline blue staining26 (link). Accumulation of phenolic compounds was examined under ultraviolet illumination (Leica). Root length and fresh weight assays for elf18 sensitivity were performed as described previously9 (link). The evening element enrichment was determined based on hypergeometric distribution. Samples for RASL-seq were prepared according to ref. 19 (link). Non-negative matrix factorization algorithm was used to cluster the genes20 (link). RNA extraction was performed as described previously27 (link). cDNA synthesis (Superscript III, Invitrogen) and quantitative PCR (SYBR Green, Qiagen) were performed according to the manufacturer’s protocols. For Pseudomonas infection, 4-week-old plants were inoculated with 10 mM MgCl2 or Pseudomonas syringae maculicola ES4326 with or without the effector AvrRpt2 (OD600=0.001). The in planta bacterial growth was measured at 3 dpi. For diurnal luciferase measurement, protein was extracted and bioluminescence intensity was measured using the Luciferase Assay System (Promega) according to manufacturer’s protocol. Ten-day-old plate-grown plants were used for free-running test (details in Methods).
Full Methods and any associated references are available in the online version of the paper at www.nature.com/nature.