Ecological interactions sculpt genomes, but their complexity thwarts the use of forward genetics; only traits amendable to high-through-put (HTP) phenotyping meet the sample-size requirements of this most unbiased of genetic analyses. In EcoMAGIC unbiased forward genetics wedded with unbiased metabolomics and unbiased natural history-based field biology using sentinel insects and microbes, will uncover for the first time, the genetics behind the management of three complex ecological interactions: those with herbivores, root-recruited beneficial bacteria and fungi, and arbuscular mycorrhizal fungi (AMF). In a nine-year unprecedented effort, a Multi-parent Advanced Generation InterCross (MAGIC) population of 1950 recombinant inbred lines (RILs) was created from 26 parental wild tobacco accessions: it captures the species’ ecological phenotypic diversity, selected by phenotyping 230 accessions from 60 sites. By planting this MAGIC population into two native habitats with divergent ecologies in Utah and Arizona over three years, and using comprehensive HTP unbiased mass spectrometry-based metabolomics and RNA sequencing of leaf, flower and root tissues combined with ecological phenotyping, we will impute new regulatory loci for the plant’s management of these three complex co-occurring interactions. Ecological phenotyping will use innovative HTP screens: lepidopteran oral secretions to simulate species-specific herbivore attack; sentinel Empoasca leafhoppers that eavesdrop on jasmonate signaling, mirids that manipulate cytokinin signaling; predation assays revealing volatile-mediated indirect defenses; AMF-indicative foliar blumenol metabolites; and an established microbiome analysis pipeline. Imputed candidate protein-coding genes, lncRNAs and smRNAs will be directly silenced/edited or manipulated by silencing smRNA machinery components in RILs. These GM-RILs planted into hypothesis-structured populations will establish causality, which is best studied holistically in nature.