Genome-wide disruption of a dynamic transcription factor network is coupled to knockout of a barley NLR immune receptor

Cellular pathways that control plant immunity are guided by complex transcriptional networks. Activation of these networks is often mediated by nucleotide-binding leucine-rich repeat (NLR) immune receptors. We used the interaction between an archetype NLR receptor, barley MLA, and the biotrophic powdery mildew fungus, Blumeria hordei (Bh), to interrogate the temporal regulation of immune signaling via computation of a Gene Regulatory Network (GRN). The GRN comprises 72% of 34603 high-confidence genes in the barley genome, including 1214 transcription factors (TF), 20125 targets, and 54698 interactions. In silico gene knockouts were used to evaluate network nodes, and the most essential TF, i.e., those that induce high interference values in the remaining nodes of the network, were selected for antibody design. The GRN was then verified via time-course chromatin immunoprecipitation sequencing (ChIP-Seq), where knockout of a functional MLA6 NLR was associated with genome-wide disruption of TF-promoter interactions and TF-target gene expression. A newly identified NAM-ATAF1,2-CUC2 (NAC) TF influences the activation of Mla, along with feedback loops linking homeobox (HB), WRKY2 and MADS box proteins that cooperatively control hundreds of genes at discrete times during fungal infection, including penetration of host epidermal cells and development of haustorial feeding structures. These results suggest a novel auto-regulatory mechanism of the immune receptor that drives the response to powdery mildew over the course of disease progression.