Architecture and dynamics of hormone-driven gene regulatory networks in plant immunity
Authors:   , Aerts Niels 
Published: December 2023

Plants defend themselves against pests and pathogens by activating an elaborate gene regulatory network (GRN). Large parts of this GRN are regulated by the defense-related plant hormones jasmonic acid (JA), abscisic acid (ABA) and salicylic acid (SA). The (sub-)GRNs activated by these hormones interact by reinforcing or attenuating each other, thus ensuring an optimized defense response. These interactions are known as hormone crosstalk. In this thesis, I describe our various lines of research aimed at increasing knowledge of immunity-related hormone crosstalk, with a focus on how the JA GRN is modulated by the ABA GRN and by specific components of the SA GRN. Because we already analyzed the JA and SA GRNs in earlier work, we also analyzed the ABA GRN by itself. In Chapter 1 we reviewed how crosstalk takes place at many levels of regulation: the network level, the protein level, the gene expression level and the hormone homeostasis level. In Chapter 2 and Chapter 3 we dove deeper into the ABA GRN (Chapter 2) and ABA/JA crosstalk (Chapter 3) by analyzing high-density time series RNA-seq data of plants treated with ABA, methyl JA (MeJA; a JA variant that is converted to free JA in the plant) and the combination, and integrating it with publicly available transcription factor (TF) binding site data and microarray data of ABA-treated plants that were inhibited in translation. We found that the ABA GRN was highly connected through a variety of TFs, of which the bZIP family was the most prominent (Chapter 2), and during some stages of its activation had significant similarities to the JA GRN (Chapter 3). We also found that ABA modulates the JA GRN at many of the levels described in Chapter 1: it affects the transcription of 2/3rd of all MeJA-responsive genes, including JA biosynthesis and catabolism genes, and affects protein accumulation of the JA master regulator ORA59 in protoplasts (Chapter 3). In Chapter 4 we investigated mechanisms of NPR1-mediated SA/JA crosstalk and found a function for nuclear localized NPR1. We found that two npr1 mutant lines that expressed NPR1 with altered cysteine residues (NPR1C82A and NPR1C82A) were disrupted in SA/JA crosstalk, but not in core SA responses. Further analysis of these lines led to the identification of SA- or SA/NPR1-responsive WRKY-family TFs that repress expression of the JA marker gene PDF1.2. We found that some of these WRKY TFs achieve this by reducing protein accumulation of ORA59, independent of its transcription. In this thesis, we investigated hormone crosstalk mostly at the level of transcriptional regulation, often measured by mRNA levels, as this is the current gold standard for approximation of (changes in) protein levels. However, many steps of regulation determine which gene is described and which mRNAs are translated to protein. These steps are often overlooked in research. Therefore, in Chapter 5 we summarized current knowledge on transcriptional regulation of plant innate immunity and gave an outlook to research in the future, where novel technologies can be used to investigate these overlooked levels in more detail.