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Gene regulatory networks underpin stress response pathways in plants. However, parsing these networks to prioritize key genes underlying a particular trait is challenging. Here, we have built the Gene Regulation and Association Network (GRAiN) of rice ( Oryza sativa ). GRAiN is an interactive query-based web-platform that allows users to study functional relationships between transcription factors (TFs) and genetic modules underlying abiotic-stress responses. We built GRAiN by applying a combination of different network inference algorithms to publicly available gene expression data. We propose a supervised machine learning framework that complements GRAiN in prioritizing genes that regulate stress signal transduction and modulate gene expression under drought conditions. Our framework converts intricate network connectivity patterns of 2160 TFs into a single drought score. We observed that TFs with the highest drought scores define the functional, structural, and evolutionary characteristics of drought resistance in rice. Our approach accurately predicted the function of OsbHLH148 TF, which we validated using in vitro protein-DNA binding assays and mRNA sequencing loss-of-function mutants grown under control and drought stress conditions. Our network and the complementary machine learning strategy lends itself to predicting key regulatory genes underlying other agricultural traits and will assist in the genetic engineering of desirable rice varieties. 

Transcription factors (TFs) play a central role in regulating molecular level responses of plants to external stresses such as water limiting conditions, but identification of such TFs in the genome remains a challenge. Here, we describe a network-based supervised machine learning framework that accurately predicts and ranks all TFs in the genome according to their potential association with drought tolerance. We show that top ranked regulators fall mainly into two ‘age’ groups; genes that appeared first in land plants and genes that emerged later in the Oryza clade. TFs predicted to be high in the ranking belong to specific gene families, have relatively simple intron/exon and protein structures, and functionally converge to regulate primary and secondary metabolism pathways. Repeated trials of nested cross-validation tests showed that models trained only on regulatory network patterns, inferred from large transcriptome datasets, outperform models trained on heterogenous genomic features in the prediction of known drought response regulators. A new R/Shiny based web application, called the DroughtApp, provides a primer for generation of new testable hypotheses related to regulation of drought stress response. Furthermore, to test the system we experimentally validated predictions on the functional role of the rice transcription factor OsbHLH148, using RNA sequencing of knockout mutants in response to drought stress and protein-DNA interaction assays. Our study exemplifies the integration of domain knowledge for prioritization of regulatory genes in biological pathways of well-studied agricultural traits.