Molecular and functional analysis of novel regulators of defense hormone signaling

Abstract

Hormones are central regulators of plant immunity. They can trigger large-scale reprogramming of the plant’s transcriptome, and influence each other in an antagonistic or synergistic manner. To better understand the complexity of SA- and JA-mediated transcriptional reprogramming, we performed high-resolution RNA-seq time series in Arabidopsis leaves to define the transcriptional effects elicited by SA and JA. The overall aim of my PhD project was to advance our understanding of the SA- and JA- controlled immune signaling networks in Arabidopsis and discover novel master regulators of hormone-regulated plant immunity. To this end, we used the systems approach of high-density time series RNA-seq to unravel in detail the dynamics and architecture of the gene regulatory network that is activated in Arabidopsis in response to SA and JA. In Chapter 2, we used high-density time series RNA-seq to investigate the architecture and dynamics of the JA gene regulatory network. Using this approach, we uncovered several TFs, including MYB59 and bHLH27, as early network components with a role in pathogen and insect resistance. Analysis of subnetworks surrounding the TFs ORA47, MYB59 and bHLH27, using a combination of transcriptome profiling of mutants, plant bioassays with pathogens and insects, and protein-protein interaction studies highlighted their specific regulatory roles in defined modules of the JA network. In Chapter 3, we used high-density time series RNA-seq to unravel the architecture and dynamics of the SA gene regulatory network. Important roles for the sofar unidentified TFs ANAC061 and ANAC090 in SA-mediated immunity was unveiled using mutants in bioassays and transcriptome studies. In Chapter 4, we used a bioinformatics pipeline to identify groups of related proteins whose genes were responsive to SA, as determined by RNA-seq in Ch3, but as yet had no previously characterized function in plant immunity. One of these families consisted of eight genes encoding small proteins that contain a cysteine-rich transmembrane domain, which we named pathogen-induced cysteine-rich transmembrane proteins (PCMs). Stable PCM-overexpressing Arabidopsis lines displayed enhanced resistance against biotrophic pathogens confirming a role for members of this gene family in plant immunity. In Chapter 5, I provide a summarizing discussion on the outcomes of my PhD research and a reflection on the questions that remain for future research.