Next-Generation Mapping of the Salicylic Acid Signaling Hub and Transcriptional Cascade

Abstract

For over 60 years, salicylic acid (SA) has been known as a key signaling molecule for local and systemic immunity. SA causes drastic cellular reprogramming in a phenomenon known as Systemic Acquired Resistance (SAR). During SAR, thousands of genes transcriptional respond. This transcriptional response is regulated by the cofactor NONEXPRESSER OF PR1 (NPR1), despite NPR1 not having a DNA binding domain. With no DNA binding domain, how this cofactor functions in this transcriptional reprogramming became a question at the forefront of the field. Despite decades of investigation, we have only superficially understood its function, largely due to the reliance on marker genes and low-throughput, molecular biology approaches. While higher throughput methodologies have been employed in previous attempts to identify NPR1 partners and target genes, all efforts yielded futile results. However, with the development of new targeted proteomic techniques and higher signal chromatin profiling techniques, we are poised to adapt these technologies to identify NPR1 proximal proteins and direct targets. Utilizing TurboID, we identified 234 NPR1 proximal partner proteins, including most known NPR1 interactors, validating our methodology. These proximal proteins are enriched with chromatin remodelers, splicing machinery, the Mediator complex, and transcriptional regulators. Strikingly these proximal partners can play either a positive or negative role in establishing SAR. Beyond NPR1 partners, we identified NPR1 target genes upon SA treatment using greenCUT&RUN. Despite a proposed transcription factor switching model, our time course greenCUT&RUN showed NPR1 binds consistently to the same loci after SA treatment through TGA transcription factors and induces a transcriptional cascade comprised of WRKY, NAC, ERF, and MYB transcription factors. This induction requires the condensate formation of NPR1 to stabilize the NPR1 TGA interaction. Through this induced cascade, WRKYs continue propagating this SA transcriptional reprogramming and activating many signaling kinases and defense genes. Beyond NPR1, many conical ideas about transcription induction seem to be amiss in SA-induced genes, including increased chromatin accessibility and histone acetylation, leading rise to the new yet old question; how does NPR1, and the subsequent SA-induce transcription factors, cause this transcriptional reprogramming?