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

在病原体攻击时，植物表现出多种诱导反应，这些反应在不同水平上受到控制。最近，利用全基因组方法强调了叶片组织中M/PAMP（微生物/病原体相关分子模式）触发免疫的翻译控制的重要性。在这里，uorf（上游开放阅读框）和r - motif（富含嘌呤）被鉴定为mrna定位序列，介导M/ pamp依赖的翻译控制。利用RNA测序和TRAPseq（翻译核糖体亲和纯化和RNA测序）技术，我们比较了维管致病性真菌黄萎病根的全基因组转录和翻译调控基因表达。与在叶片中获得的结果一致，在根中没有观察到转录和翻译之间的强相关性。由于目前没有公开的数据，本项目拟重点研究病原菌侵染根中转化控制的功能影响和调控机制。(1)在一项概念验证研究中，我们将评估从现有全基因组数据集中选择的候选基因的翻译控制。我们将重点关注翻译（而不是转录）诱导基因，这是实验室的研究重点，即SnRK1。α1，编码能量/资源管理中进化保守的中央调控因子（SnRK1， Snf1相关蛋白KINASE1）的催化亚基和下游转录调控因子bZIP1 （BASIC亮氨酸ZIPPER1）。由于植物防御需要大量的能量，因此可以想象与控制能量稳态电路的机制联系，但尚未确定。基于现有的分子工具，我们将研究翻译控制，并使用基因编辑技术来评估候选rna中存在的uORF和r -motif的体内功能。此外，我们将分析SnRK1-bZIP1信号在病原体防御中的功能影响。(2)在全基因组方法中，我们将通过引入Ribo-seq（核糖体足迹测序）进一步扩展转录组-翻译组数据集，并使用生物信息学工具定义翻译控制中的新的候选基序。为了确认它们的功能相关性，将使用功能损失和功能获得方法。综上所述，该项目将提供对病原体攻击时能量稳态控制的见解，并将拓宽我们对病原体控制基因表达调控的机制观点。所获得的知识将有助于制定新的策略来提高作物的抗病性。

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.