Cell-specific reprogramming of legume roots for endosymbiotic infection

绝大多数陆地植物可以建立丛枝菌根(AM)，这是一种重要的农学共生真菌，可以改善矿物质和水的供应。豆科植物使用一种古老的信号转导程序，从祖先的AM中挑选出来，在根瘤共生中与固氮根瘤菌建立细胞内(内共生)相互作用。成功的共生感染依赖于一种古老的细胞分化程序，以适应微生物在细胞内的适应。虽然已经通过遗传方法确定了一些对感染过程至关重要的重要共生基因，但细菌在根部定植的确切细胞和分子事件却知之甚少。该项目将专注于核信号事件和相关的转录重编程，这些事件发生在参与根瘤菌感染的宿主细胞中。通过在活细胞中开发尖端细胞成像策略，图卢兹团队最近证明，钙尖峰和共生ERF转录因子(TF)ERN1的局部积累是与感染早期相关的特异性核反应。通过进一步推动这些技术，该项目旨在破译钙尖峰与感染前宿主细胞转录重编程之前TF积累和复杂构象动态之间的时空联系。我们将集中在两个关键的感染相关的转录因子ERN1和独眼巨人，后者与CCaMK，一个重要的早期整合钙信号的复合体发挥作用。慕尼黑团队观察到独眼巨人的磷酸化对其转录活性是必不可少的，并与独眼巨人二聚体的结构变化有关。基于FRET的策略将被用来跟踪感染期间活细胞中独眼动物复合体的形成，以及这与钙信号的相关性。慕尼黑研究小组发现，独眼巨人启动子对于成功的共生信号至关重要。我们将确定决定独眼巨人复杂表达模式的顺式和反式作用因子。我们将使用完整(在特定细胞类型中标记的核的分离)技术来专门针对正在进行重新编程的一小部分细胞，并将其与RNA测序结合起来，以访问与根瘤菌感染早期阶段特定相关的基因转录网络。野生型和感染缺陷型突变体的细胞特异性转录本的比较将有助于鉴定与内生菌进入直接相关的基因。因此，该项目将为内共生植物根部定植过程中的细胞重新编程提供新的见解。

The vast majority of land plants can establish arbuscular mycorrhiza (AM), an agronomically important symbiosis with fungi to improve mineral and water supply. Legumes use an ancient signal transduction program, co-opted from the ancestral AM, to establish intracellular (endosymbiotic) interactions with nitrogen-fixing rhizobia, in the root nodule symbiosis. Successful symbiotic infection relies on an ancient cellular differentiation program for the intracellular accommodation of the microsymbionts. Although a number of important symbiosis genes essential for the infection process have been identified through genetic approaches, the precise cellular and molecular events underlying bacterial colonization of the root are poorly understood. The COME-IN project will focus on nuclear signalling events and associated transcriptional reprogramming that take place in host cells engaged in rhizobial infection. By developing cutting-edge cell imaging strategies in living cells, the Toulouse team has recently shown that calcium-spiking and the localized accumulation of the symbiotic ERF transcription factor (TF) ERN1 are specific nuclear responses which are associated with the early stages of infection. By pushing the technologies one step further, this project aims to decipher the spatio-temporal interconnection between calcium-spiking and the dynamics of TF accumulation and complex conformation preceding host cell transcriptional reprogramming for infection. We will focus on the two key infection-related TFs ERN1 and CYCLOPS, the latter acting in a complex with CCaMK, an important early integrator of calcium signalling. The Munich team has observed that CYCLOPS phosphorylation is essential for its transcriptional activity, and is associated with structural alterations of the CYCLOPS dimer. FRET-based strategies will be used to follow CYCLOPS complex formation in living cells during infection and how this correlates with calcium signalling. The Munich team has found the CYCLOPS promoter to be critical for successful symbiotic signalling. We will identify the cis- and trans-acting factors that determine the complex expression pattern of CYCLOPS. We will employ the INTACT (Isolation of Nuclei TAgged in specific Cell Types) technology to specifically target the small subpopulation of cells undergoing reprogramming and couple it with RNA sequencing to access gene transcription networks specifically associated with early stages of rhizobial infection. The comparison of cell-specific transcriptomes of wild type and infection-deficient mutants will aid in the identification of genes directly related to endosymbiotic entry. The COME-IN project will thus provide new insights into the cellular reprogramming during endosymbiotic root colonization.