Translational expression control in land plant arginine decarboxylase transcripts

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Polyamines (PAs) are metabolites with two or more amino groups that exist throughout all three domains of life: eukaryotes, bacteria, and archaea. The ubiquity of PAs in living cells inherently implies that their existence is imperative, and while their biosynthesis is well understood, a singular concept that unifies their molecular role is lacking. In plants, arginine decarboxylase (ADC) catalyzes the synthesis of putrescine, the precursor of all PAs. Previously, we have found that the bacterial plant pathogen Ralstonia solanacearum injects a transcription-activator-like effector (TALE) protein into host plant cells, which binds to and transcriptionally activates plant ADC genes to boost host PA levels. The TALE protein binds within a 50bp-motif upstream of ADC genes, which is conserved across all land plant species, and was designated as the ADC-box. In native ADC mRNAs, the transcribed ADC-box is part of the 5’UTR and is predicted to form a hairpin structure that inhibits movement of the ribosome towards the AUG start codon. In agreement with this model, the ADC-box has been shown to inhibit translation of ADC transcripts at high PA levels, and stands to reason that the ADC-box could be part of an autoregulatory expressional control system for PA homeostasis. Within the current research proposal we aim to elucidate the molecular basis of translational regulation in land plant ADC transcripts. The research proposal is topically divided into two sections:1.) Identify sequence elements within ADC transcript 5’UTRs that contribute to expressional regulation in the plant model species tomato and Arabidopsis. To do so, we will study ADC mutant alleles generated by either CRISPR or site-directed mutagenesis.2.) Uncover cellular components that mediate feedback control in translation of ADC transcripts. Here, reporter-based assays will identify PA-related proteins and/or metabolites that affect translational activity of ADC transcripts. This functional approach will be complemented with interaction-based strategies to uncover regulatory components that bind to ADC transcript 5’UTRs.The aforementioned studies will bring to light the intricacies of the translational regulation of ADC transcripts and PA homeostasis. Once we have decoded the mechanistic principles of translational regulation in ADC transcripts, we can use the identified PA-sensory modules of this regulatory circuit to engineer genetically-encoded PA biosensors. Such biosensors will enable us to monitor changes in PA levels in vivo with high spatial and temporal resolution and thus provide a key tool to uncover PA-regulated biological processes.