Exploring the Roles of GCN2 and the m6A RNA Modification in Plant Immunity

Plants have evolved a robust immune system to fend off pathogens. This response must be tightly regulated, as aberrant activation can have detrimental effects. Much work has been done to understand the transcriptional responses in plant immunity, but less is known about post-transcriptional mechanisms. Here I examine the roles of the general control nonderepressible 2 (GCN2) kinase and the N6-methyladenosine (m6A) RNA modification in regulating plant immunity at the post-transcriptional level.

A previous study revealed that the master immune regulator TBF1 (a transcription factor) is essential for mediating a defense response. TBF1 is under the control of 2 upstream open reading frames (uORFs) which inhibit translation of TBF1’s major open reading frame (mORF). These uORFs’ sequences are enriched with codons for aromatic amino acids, especially phenylalanine. Pathogen treatment resulted in the induction of uncharged tRNAphe and eIF2α phosphorylation. It was proposed that GCN2 would mediate eIF2α phosphorylation and allow readthrough of TBF1’s mORF, analogous to GCN2’s role in promoting translation of GCN4 upon amino acid starvation. To test this, eIF2α phosphorylation was examined in gcn2 mutant plants upon pathogen infection using a phoshpo-specific eIF2α antibody. eIF2α phosphorylation was not observed in gcn2 plants upon pathogen treatment and therefore GCN2 is likely to be the kinase responsible for this induced eIF2α phosphorylation. To test GCN2’s role in positively regulating TBF1, a bacterial infection assay was undertaken with gcn2 plants. Compared to wild-type plants, gcn2 plants did not have any significant difference in bacterial growth and therefore GCN2 is unlikely to play a role in regulating TBF1 or plant immunity.

The m6A modification is the most abundant internal modification present in mRNAs and has been found to regulate several aspects of mRNA metabolism and biological processes. There is less known about m6A in plants, though it has been found to regulate stability of transcripts and is important for development and the salt stress response. It is unknown whether m6A plays a role in plant immunity. To address this, m6A deficient plant lines were used and bacterial infection assays undertaken. These lines displayed significantly higher levels of bacterial growth (susceptibility) and thus m6A plays a positive role in plant immunity. Further assays found that m6A was essential for fully mediating pattern-triggered immunity (PTI) and salicylic acid (SA)-mediated immune responses. m6A-seq was used to map the dynamics of m6A across the transcriptome in response to the immune inducers SA and elf18 (a microbe-associated molecular pattern). Hundreds of pathogen-induced methylation sites were uncovered and gene ontology (GO) analysis revealed a predominance of defense/immune-related transcripts.

In summary, this dissertation work found that although GCN2 is required for pathogen induced eIF2α phosphorylation, it is dispensable for defense against a bacterial pathogen, and thus is unlikely to be a regulator of TBF1 or the immune response. On the other hand, m6A was established to be broadly essential and dynamic upon immune induction. These findings open the door for future studies to elucidate how m6A machinery is interacting with the defense response and establish a new area for post-transcriptional control of plant immunity.