Powdery mildew is among the most widespread foliar diseases of broadleaf trees. In the two European oak species, pedunculate oak (Quercus robur) and sessile oak (Q. petraea), Erysiphe alphitoides is the main cause of oak powdery mildew (OPM) [1–3]. Recurrent epidemics of OPM reduce photosynthetic capacity and lead to stunted growth of seedlings, thereby increasing management costs in nurseries and restoration plantings [4–6]. Oaks are of great ecological and economic importance across Europe [7], yet the molecular events that lead to OPM resistance or susceptibility, particularly within the early phase of contact, are poorly understood [8–10]. Plant immunity relies on rapid pathogen perception and signaling [11]. At the cell surface, pattern-recognition receptors and receptor-like kinases (RLKs) detect microbe-associated signals and trigger downstream cascades [12]. Canonical early outputs include mitogen-activated protein kinase activation and cytosolic Ca²⁺ fluxes decoded by calmodulin-like proteins (CMLs), transcriptional reprogramming via WRKY/ ethylene-responsive factors (ERFs) and cell-wall reinforcement with reactive oxygen species metabolism [13]. Hormonal crosstalk among salicylic acid, jasmonic acid, and ethylene modulates these responses in a context-dependent manner [14]. Phenylpropanoid biosynthesis contributes to both structural (lignin-related) and defensive (antimicrobial) metabolites [15]. In long-lived woody hosts, these processes may be temporally shifted or prolonged relative to annuals, underscoring the value of time-resolved studies in trees [16]. Despite these advances, molecular studies capturing the early responses of Q. robur to E. alphitoides remain scarce. Prior work has largely emphasized visible symptoms, priming effects, or later transcriptional adjustments in oak [8,9], and notably the transcriptomic study of Streptomyces-induced resistance by Kurth et al. [10] focused on systemic responses rather than the immediate signaling events that follow pathogen contact. However, identifying these early signaling pathways is essential for designing durable and sustainable management strategies against powdery mildew.

In this study, we present the results of gene expression changes in Q. robur detached leaves 30 hours after inoculation with E. alphitoides, to capture early host reprogramming events. Our results provide an early snapshot of the gene expression map of the oak-PM interaction that will guide future functional assays and inform selection of markers for screening pedigrees or provenances.

Pedunculate oak (Q. robur) seedlings were raised from acorns under controlled conditions. Healthy, fully expanded leaves were collected from independent seedlings of similar age and size prior to inoculation. Leaves were placed in sterile 90-100 mm Petri dishes lined with moistened tissue. For each condition (OPM-inoculated, mock), three biological replicates were used, each consisting of two leaves. Detached Q. robur leaves were inoculated on the adaxial surface with 2.5 µL droplets containing ~150 conidia of E. alphitoides or a water mock control, ensuring broad coverage of the leaf area [17]. Leaves were harvested at 30 hours post-inoculation (hpi) to capture early host transcriptional signaling following pathogen perception, after initial penetration but prior to extensive colonization or symptom development. For RNA-seq, we used three biological replicates per condition, each consisting of two pooled leaves. Total RNA was extracted, DNase-treated, poly(A)-selected, and sequenced (2 × 150 bp; NextSeq 2000; Illumina, Inc., San Diego, CA, USA). Reads were quality-checked, adapter- and quality-trimmed, and aligned to the Q. robur v2 reference genome [18] using CLC Genomics Workbench v24.0.1 (Qiagen, Hilden, Germany) with default RNA-seq analysis parameters. Gene expression was quantified at the gene level and normalized as transcripts per million values. Differential expression analysis was performed using a negative binomial model with Benjamini–Hochberg false discovery rate (FDR) correction; genes with |log2 fold change (FC)| ≥ 1 and FDR ≤ 0.05 were considered significant. Isoforms were collapsed to the longest transcript per gene prior to downstream analyses. The collapsed gene set comprised 12,946 genes. Evolutionary genealogy of genes by Non-supervised Orthologous Groups (eggNOG)-mapper v2 [19] returned annotations for 12,641 genes (Table S1). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) over-representation analyses (one-sided Fisher’s exact test; BH-FDR) used the annotated subsets: GO universe N = 5,717 genes with ≥1 GO term, and KEGG universe N = 5,819 genes with ≥1 K number: Gene-level eggNOG annotations for the differentially expressed gene (DEG) sets are provided in Table S2 (up-regulated) and Table S3 (down-regulated).

Six up-regulated RNA-seq targets (five DEGs with |log2 FC| ≥ 1 and FDR ≤ 0.05, plus one up-regulated candidate with FDR > 0.05) were selected based on (i) statistical significance and pathway coverage across RLK/Ca²+/ethylene modules and (ii) practical measurability; ensuring significant expression in mock controls to avoid zero-inflated fold change artifacts. Two CML19 loci were assayed with distinct primer pairs: CML19-81 (LOC126712681) and CML19-84 (LOC126712684). Relative expression was calculated by the Pfaffl method [20] using GAPDH and Actin as references; significance was assessed with two-sided Student’s t-tests. Primer sequences are provided in Table S4.

We profiled the leaf transcriptome of Q. robur at 30 hpi (n = 3 mock: S13–S15, n = 3 OPM: S16–S18). A principal component analysis of log2 (counts per million (CPM)+1) values showed separation between samples (PC1 = 34.9%, PC2 = 31.4%; Fig. 1A), indicating that condition effects dominated. The volcano plot (Fig. 1B) was shifted toward positive log2FC values, indicating an induction-biased response. With thresholds |log2 FC| ≥ 1 and FDR ≤ 0.05, we identified 195 up-regulated and 40 down-regulated genes. Effect sizes among up-regulated DEGs were moderate to strong (median log2 FC = 2.18; IQR 1.52–3.09; maximum = 6.62). A heat map highlighted a dominant induction module across inoculated samples with a comparatively smaller set of repressed transcripts (Fig. 1C). Gene-level eggNOG annotations for these DEGs are listed in Tables S2–S3.

We performed over-representation analysis separately on up- and down-regulated DEGs using a onesided Fisher’s exact test with Benjamini–Hochberg correction, against the GO-annotated background for GO terms and the KEGG-annotated background for KEGG pathways (ranked visualizations), shown in Fig. S1. Among up-regulated genes, enrichment clustered in defense-related Biological Process terms—most prominently defense response, response to chitin, defense response to other organism, and aligned response to biotic stimulus/wounding—with little signal in Molecular Function and sparse Cellular Component terms pointing to cell wall/external encapsulating structure, consistent with apoplastic/wall-associated defenses at this timepoint. For the downregulated DEGs, nominal enrichments in specialized metabolism and auxin transport were detected; however, none remained significant after FDR correction (q > 0.05), and the large apparent fold-enrichments reflect small background annotation counts (K). A summary of the major GO enrichment patterns is shown in Table 1; ranked visualizations are in Fig. S1A (GO) and Fig. S1B (KEGG); full outputs in Tables S5–S8; per-gene (one-per-LOC protein) eggNOG annotations for the DEG sets in Tables S2–S3.