Elucidation of translational regulatory mechanisms of plant immune responses

由于植物不具有动物中存在的特化免疫细胞，植物中免疫的激活涉及从产生生长促进蛋白质到防御蛋白质的转变。尽管产生防御蛋白模板（RNA转录物）的过程已被广泛研究，但模板如何选择性地翻译成蛋白质仍在很大程度上未知。这一重大的知识差距严重阻碍了在农业中以及一般生物工程中开发工程抗病作物的新策略的进展，因为受控的蛋白质生产对大多数生物功能至关重要。该项目的长期目标是阐明植物在免疫应答过程中产生防御蛋白的调节机制。为了实现这一目标，已经开发或改造了新技术来检查连接RNA转录物和蛋白质合成的多个步骤。利用翻译开关来加强对防御蛋白翻译的调控，将最大限度地减少通常与广谱抗病性相关的产量损失，并彻底改变主要集中在疾病特异性类型抗性上的作物病害防治实践。该项目也可能通过提高重组蛋白的产量而使其他行业和医药受益。该项目的成功还将增加从高中生到博士后研究人员的科学家管道，这些科学家在进行前沿研究方面接受了强大的STEM培训。在植物免疫应答过程中具有改变的翻译效率的基因的鉴定导致了新的调节剂的发现。令人惊讶的是，植物中与免疫相关的翻译重编程已被发现与酵母和哺乳动物中发现的整合应激反应途径不同，这表明植物使用替代调节机制来控制防御蛋白质组翻译。为了阐明这些调节机制，对模式触发免疫（PTI）和效应物触发免疫（ETI）进行了全基因组翻译组分析，并在PTI期间翻译诱导mRNA的5 '-前导序列中发现了主要由嘌呤组成的共有序列（“R基序”）。发现R基序对于PTI诱导的翻译不仅是必需的，而且是充分的。其调节是通过与聚（A）结合蛋白（PABP）的相互作用。此外，已经建立了m6 A-seq和SHAPE-MaP来分别检查mRNA修饰和体内mRNA结构动力学。随着这些尖端技术的建立，本项目将集中于两个具体目标：目标1：研究上游开放阅读框架（uORF）对PTI诱导的主要ORF（mORF）翻译的影响，以验证RNA修饰和二级结构是核糖体可及性的关键决定因素的假设，以控制PTI期间mORF的翻译。目的2：利用正向和反向遗传学方法阐明PTI过程中导致翻译体变化的信号通路。最终目标是将各种翻译顺式和反式元件与目前已知的防御信号网络连接起来，以填补植物如何重新编程蛋白质组以启动免疫反应的知识空白。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Since plants do not have the specialized immune cells present in animals, activation of immunity in plants involves switching from producing growth-promoting proteins to defense proteins. Even though the process of generating templates (RNA transcripts) for defense proteins has been studied extensively, how the templates are selectively translated into proteins remains largely unknown. This major knowledge gap significantly impedes progress in developing new strategies of engineering disease resistant crops in agriculture as well as in bioengineering in general because controlled protein production is central to most biological functions. The long-term goal of this project is to elucidate the regulatory mechanisms by which plants produce defense proteins during immune responses. To achieve this goal, new technologies have been developed or adapted to examine the multiple steps connecting RNA transcripts to protein synthesis. Using translational switches to tighten the regulation of defense protein translation will minimize the yield penalty often associated with broad-spectrum disease resistance and revolutionize the practice of controlling crop diseases, which has mainly been focused on the disease-specific type of resistance. This project may also benefit other industries and medicine by improving recombinant protein production. The success of the project will also add to the pipeline of scientists from high school students to postdoctoral researchers with strong STEM training in performing frontier research. Identification of genes with altered translation efficiency during plant immune responses led to the discovery of novel regulators. Surprisingly, immune-related translation reprogramming in plants has been found to be distinct from the integrated stress response pathway found in yeast and mammals, indicating that plants use alternative regulatory mechanisms to control defense proteome translation. To elucidate these regulatory mechanisms, whole genome translatome analyses were performed for both pattern-triggered immune (PTI) and effector-triggered immunity (ETI) and a consensus sequence consisting of mostly purines (“R-motif”) was discovered in the 5’-leader sequences of translationally induced mRNAs during PTI. The R-motif was found to be not only necessary, but also sufficient for the PTI-induced translation. Its regulation is through the interaction with poly(A)-binding proteins (PABPs). Moreover, m6A-seq and SHAPE-MaP have been established to examine mRNA modifications and in vivo mRNA structural dynamics, respectively. With the establishment of these cutting-edge technologies, this project will focus on two specific aims: Aim 1: Study the effects of upstream open reading frames (uORF) on translation of the main ORF (mORF) in response PTI induction to test the hypothesis that RNA modification and secondary structure are critical determinants of ribosome accessibility to uORFs in controlling mORFs translation during PTI. Aim 2: Elucidate the signaling pathway leading to translatomic changes during PTI using both forward and reverse genetic approaches. The ultimate goal is to connect the various translational cis- and trans-elements with the currently known defense signaling network to fill the knowledge gap on how plants reprogram the proteome to mount immune responses.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.