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），这是一种重要的农艺学上与真菌的共生关系，可以改善矿物质和水的供应。豆科植物利用一种古老的信号转导程序，从祖先的AM中选择，在根瘤共生中与固氮根瘤菌建立细胞内（共生内）相互作用。成功的共生感染依赖于一个古老的细胞分化程序，用于微共生体的细胞内调节。虽然通过遗传方法已经确定了一些重要的共生基因对感染过程至关重要，但细菌定植根部的精确细胞和分子事件知之甚少。COME-IN项目将重点研究根瘤菌感染宿主细胞中发生的核信号事件和相关的转录重编程。通过在活细胞中开发先进的细胞成像策略，图卢兹团队最近表明，钙峰值和共生ERF转录因子（TF） ERN1的局部积累是与感染早期阶段相关的特异性核反应。通过进一步推动这些技术，该项目旨在破译钙峰值与宿主细胞转录重编程感染前TF积累和复杂构象动力学之间的时空联系。我们将重点关注两个关键的感染相关tf - ERN1和CYCLOPS，后者与CCaMK复合物起作用，CCaMK是钙信号的重要早期整合子。慕尼黑研究小组观察到，CYCLOPS磷酸化对其转录活性至关重要，并且与CYCLOPS二聚体的结构改变有关。基于fret的策略将用于跟踪感染期间活细胞中CYCLOPS复合物的形成以及它如何与钙信号传导相关。慕尼黑研究小组发现，CYCLOPS启动子对成功的共生信号传导至关重要。我们将确定决定CYCLOPS复杂表达模式的顺式和反式因子。我们将采用完好无损（在特定细胞类型中标记细胞核的分离）技术，专门针对正在进行重编程的细胞的小亚群，并将其与RNA测序结合，以获取与根瘤菌感染早期阶段特异性相关的基因转录网络。比较野生型和感染缺陷突变体的细胞特异性转录组将有助于鉴定与内共生进入直接相关的基因。因此，COME-IN项目将为内共生根定植过程中的细胞重编程提供新的见解。

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.