Translational control of pathogen-regulated gene expression in the Arabidopsis root: global and gene-specific approaches

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Upon pathogen challenge, plants display a multitude of induced responses which are controlled on various levels. Very recently, the importance of translational control in M/PAMP (Microbe/Pathogen Associated Molecular Pattern) triggered immunity has been highlighted in leaf tissue using a genome-wide approach. Here, uORFs (upstream open reading frames) and R–motifs (enriched in purines) have been identified as mRNA-localized sequences mediating M/PAMP-dependent translational control. Using RNA sequencing and TRAPseq (Translating Ribosome Affinity Purification and RNA Sequencing), we have compared genome-wide transcriptionally and translationally regulated gene expression in roots infected with the vascular pathogenic fungus Verticillium longisporum. In line with the results obtained in leaves, no strong correlation between transcription and translation has been observed in roots. As no published data are currently available, this project proposes to focus on the functional impact and the regulatory mechanisms of translational control in pathogen infected roots. (1) In a proof-of-concept study, we will evaluate translational control of candidate genes selected from the available genome-wide data-set. We will focus on translationally (but not transcriptionally) induced genes, which are in the research focus of the lab, namely SnRK1.α1 which encodes a catalytic subunit of an evolutionary conserved central regulator in energy/resource management (SnRK1, Snf1 RELATED PROTEIN KINASE1) and a downstream transcriptional regulator bZIP1 (BASIC LEUCINE ZIPPER1). As plant defenses are highly energy demanding, a mechanistic link to the circuit controlling energy homeostasis is conceivable, however not yet established. Based on available molecular tools, we will study translational control and use gene editing techniques to evaluate the in vivo function of uORF and R-motifs present in the candidate RNAs. Moreover, we will analyze the functional impact of SnRK1-bZIP1 signaling in pathogen defense. (2) In a genome-wide approach, we will further extend the transcriptome – translatome data-set by introducing Ribo-seq (ribosome footprint sequencing) and use bioinformatic tools to define novel candidate motifs in translational control. To confirm their functional relevance, loss- and gain-of-function approaches will be used. Taken together, this project will provide insight in the control of energy homeostasis upon pathogen attack and will broaden our mechanistic view on the regulation of pathogen-controlled gene expression. The knowledge gained will enable novel strategies to improve pathogen resistance in crops.