Transcriptomic profile (by Real-time quantitative PCR analysis) of oak leaves from the second growth cycle after leaf herbivory over two successive growth cycles

Priming of plant defenses provides increased plant protection against herbivores and reduces the allocation costs of defense. Defense priming in woody plants remains obscure, in particular due to plant development traits such as the endogenous rhythmic growth displayed by oaks (Quercus robur). By using bioassays with oak microcuttings, and by combining transcriptomic and metabolomic analyses, we investigated how leaf herbivory by Lymantria dispar and root inoculation with the ectomycorrhizal fungus Piloderma croceum prime oak defenses. We further investigated how defense priming is modulated by rhythmic growth of the oaks. A first herbivory challenge in oak leaves primed newly grown leaves for an enhanced induction of jamonic acid (JA)-related direct defenses, or enhanced emission of volatiles, depending on the specific growth stage at which the plants where challenged. Root inoculation with Piloderma abolished the enhanced induction of JA-related defenses and volatile emission. Our results indicate that a first herbivore attack primes direct and indirect defenses of newly formed oak leaves, and that the specific display of defense priming is modulated by rhythmic growth. Our results further show that the priming memory in oaks can be transmitted to the next growth cycle even to the leaves of the new shoot unit. Transcriptomic profile of oak leaves from the second growth cycle after leaf herbivory over two successive growth cycles, analyzed by the 96.96 Dynamic Array™ IFC chip for Gene Expression (Fluidigm, San Francisco, CA, USA) and the BioMark™ Systems for Genetic Analysis (Fluidigm). qPCR gene expression profiling. Oak microcuttings, during shoot flush (SF) or root flush (RT), were inoculated with the ECM fungus (Pc) and non-inoculated with the ECM fungus (Co). The oak microcuttings were exposed to different L. dispar treatments: 1) no_Ld (not exposed to L. dispar); 2) Ld_prim_Ld_trig (exposed to L. dispar in two successive growth cycles, in the first growth cycle as priming stimulus and in the following growth cycle as triggering stimulus); 3) Ld_prim (exposed to L. dispar only as priming stimulus) and 4) Ld_trig (exposed to L. dispar only as triggering stimulus). Every treatment includes three biological replicates with two technical replicates.