Polygenic resistance is associated with altered early immune timing and changes in transcriptome network structure

Downy mildew, caused by Plasmopara viticola, is a major threat to grapevine. To investigate how pyramiding resistance loci influences early immune processes, we generated 36 time-resolved transcriptomes (0, 6, 24 hpi) from genotypes carrying single (Rpv12), double (Rpv12+1), or triple (Rpv12+1+3) resistance loci, together with a susceptible control. Aggregated expression divergence revealed that multilocus genotypes showed detectable baseline transcriptional differences prior to infection and distinct temporal trajectories following inoculation. Co-expression analysis identified five higher-order metamodules and uncovered non-additive network restructuring, with the triple-locus line showing markedly reduced co-expression density rather than incremental strengthening. Layer-associated genes related to recognition, signal integration, and defense action displayed genotype- and time-specific expression patterns, suggesting that each locus combination is associated with characteristic shifts in immune-layer dynamics. Transcriptome profiles suggested that Rpv12 involves elevated transcript abundance of several EDS1-associated components despite its CNL-type origin; adding Rpv1 introduced pronounced early changes in recognition- and signaling-related genes; and stacking Rpv3 coincided with rapid, transient induction of transcription factors (e.g., WRKY47, NAC29) and metabolic defense pathways. Together, these results show that resistance loci are associated with non-additive differences in timing, network connectivity, and layer allocation of immune-associated transcriptional programs, revealing systems-level mechanisms underlying multilocus resistance.