Vascular mobile signals prime plant immune responses through chromatin manipulation

Abstract

Globally, disease is responsible for reducing crop yield by over 20% every year. Mitigating these losses will drastically improve global food security, aiding efforts to feed the growing human population. Traditionally, pesticides and breeding are used to reduce disease in plants, but breeding proves too slow to combat rapidly emerging pathogens, pesticides are increasingly regulated due to toxic side effects, and both are losing efficacy as established pathogens evolve. Initial infection can immunise plants to establish systemic acquired resistance (SAR) against future pathogen attacks. SAR is associated with the priming of immune responses, leading to faster and stronger immune activation against a wide range of pathogens. Vascular mobile signals that travel through the phloem are vital for the establishment of SAR throughout the entire plant. The aim of this study is to investigate the roles of mobile signals in immune priming, as these pose a promising avenue for harnessing the plant’s own immune system to improve disease resistance.

Phloem mobile phytohormones have been identified as the signals that establish the primed state, but how they induce priming remains unknown. Here, I focus on the interactions between mobile signals and the immune hormone salicylic acid (SA), a key component of SAR. In Chapter 3, I assessed interplay between the mobile signal azelaic acid (AzA) and SA in terms of SA-responsive gene expression and SA-mediated immunity. Using sequential AzA and SA treatments I show that AzA pre-treatment modulates the expression of 74.1% of SA-dependent transcripts, primarily by reducing their SA-responsiveness. GO term analysis revealed that many of the genes targeted by AzA are involved in hypersensitive cell death and suppression of photosynthesis, suggesting AzA acts to balance investment in growth and hypersensitive defence during activation of a primed immune response. Accordingly, AzA was unable to enhance SA-responsiveness of immune responses against Pseudomonas syringae, suggesting other signals act to boost immunity, while AzA may balance energy investment.

N-hydroxy-pipecolic acid (NHP) is a phloem mobile signal that has been shown to directly prime plant immunity. In Chapter 4, I investigated the interaction between NHP and SA in establishing immunity. I discovered that NHP affects 75.2% of SA-responsive transcriptome, primarily by priming or cumulatively increasing their expression. GO term analysis showed that NHP primed immune-responsive genes but not those linked to programmed cell death, showing little overlap with AzA targets. I also show that NHP primed SA-mediated immunity against pathogen infection such that in presence of NHP, previously inert concentrations of SA induced resistance against P. syringae. Thus, NHP-mediated priming of the SA-responsive transcriptome enhances immune responsiveness. In search of an NHP regulator, I found that NHP stabilised the levels of SA-induced NPR1 protein, a master transcription coactivator of SAR, possibly through post-translational modification. Overall, these data show that NHP primes SA-responsiveness to increase the strength of immune responses, possibly through manipulation of transcriptional regulators such as NPR1.

To understand how NHP primes SA-responsive genes, I then searched for binding motifs in NHP-primed genes. Strikingly, NHP-primed genes were enriched with WRKY family transcription factor binding sites, and after testing several mutants I found that WRKY38 and WRKY62 are indispensable for priming. Through RNA sequencing (RNA-seq) I demonstrate that in wrky38/62 double mutants the majority of NHP responses are iv dysregulated and immune priming by NHP is lost. Because WRKY38/62 may be involved in chromatin remodelling, I investigated the impact of NHP on chromatin accessibility through Assay for Transposase-Accessible Chromatin sequencing (ATAC-seq). Here, I found that NHP dramatically reorganises chromatin accessibility, but this effect is largely abolished in absence of WRKY38/62. Comparison between NHP-primed genes and NHP-dependent chromatin reorganisation showed that NHP mediates priming primarily through WRKY38/62-dependent chromatin remodelling.

In summary, I demonstrate phloem mobile signals have previously unrecognised roles in SAR and suggest that several phytohormones work in tandem to optimise and regulate the process of immune priming. By identifying the key priming regulators WRKY38 and WRKY62, I propose targets for either chemically-induced or gene-edited induction of priming, which could be used to protect a variety of crop species without limiting yield or damaging the environment.