LIPME, Université de Toulouse, INRAE, CNRS, Castanet-Tolosan, France.
Université Paris-Saclay, CNRS, INRAE, Université Evry, Institute of Plant Sciences Paris-Saclay (IPS2), Orsay 91405, France.
Plant Physiol. 2022 Jun 27;189(3):1587-1607. doi: 10.1093/plphys/kiac177.
Rhizobium-legume nitrogen-fixing symbiosis involves the formation of a specific organ, the root nodule, which provides bacteria with the proper cellular environment for atmospheric nitrogen fixation. Coordinated differentiation of plant and bacterial cells is an essential step of nodule development, for which few transcriptional regulators have been characterized. Medicago truncatula ETHYLENE RESPONSE FACTOR REQUIRED FOR NODULE DIFFERENTIATION (MtEFD) encodes an APETALA2/ETHYLENE RESPONSIVE FACTOR (ERF) transcription factor, the mutation of which leads to both hypernodulation and severe defects in nodule development. MtEFD positively controls a negative regulator of cytokinin signaling, the RESPONSE REGULATOR 4 (MtRR4) gene. Here we showed that that the Mtefd-1 mutation affects both plant and bacterial endoreduplication in nodules, as well as the expression of hundreds of genes in young and mature nodules, upstream of known regulators of symbiotic differentiation. MtRR4 expressed with the MtEFD promoter complemented Mtefd-1 hypernodulation but not the nodule differentiation phenotype. Unexpectedly, a nonlegume homolog of MtEFD, AtERF003 in Arabidopsis (Arabidopsis thaliana), could efficiently complement both phenotypes of Mtefd-1, in contrast to the MtEFD paralog MtEFD2 expressed in the root and nodule meristematic zone. A domain swap experiment showed that MtEFD2 differs from MtEFD by its C-terminal fraction outside the DNA binding domain. Furthermore, clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9 (CRISPR-Cas9) mutagenesis of MtEFD2 led to a reduction in the number of nodules formed in Mtefd-1, with downregulation of a set of genes, including notably NUCLEAR FACTOR-YA1 (MtNF-YA1) and MtNF-YB16, which are essential for nodule meristem establishment. We, therefore, conclude that nitrogen-fixing symbiosis recruited two proteins originally expressed in roots, MtEFD and MtEFD2, with distinct functions and neofunctionalization processes for each of them.
根瘤菌-豆科植物共生固氮涉及形成一种特殊的器官，根瘤，为细菌提供大气氮固定的适当细胞环境。植物和细菌细胞的协调分化是结瘤发育的重要步骤，其中少数转录调节剂已被表征。百脉根乙烯反应因子（MtEFD）编码一个 APETALA2/ETHYLENE RESPONSE FACTOR（ERF）转录因子，其突变导致过度结瘤和结瘤发育的严重缺陷。MtEFD 正向调控细胞分裂素信号的负调节剂 RESPONSE REGULATOR 4（MtRR4）基因。在这里，我们表明 Mtefd-1 突变影响结瘤中的植物和细菌内复制，以及数百个在年轻和成熟结瘤中表达的基因，这些基因位于共生分化的已知调节剂的上游。MtRR4 与 MtEFD 启动子一起表达，可补充 Mtefd-1 的过度结瘤，但不能补充结瘤分化表型。出乎意料的是，拟南芥中的 MtEFD 的非豆科同源物 AtERF003，与在根和根瘤分生组织区表达的 MtEFD 旁系同源物 MtEFD2 相反，可以有效地补充 Mtefd-1 的两种表型。结构域交换实验表明，MtEFD2 与 MtEFD 的不同之处在于其 DNA 结合域外的 C 末端部分。此外，CRISPR-Cas9 突变使 MtEFD2 发生聚类规则间隔短回文重复序列（CRISPR）相关蛋白 9（CRISPR-Cas9）突变，导致 Mtefd-1 中形成的结节数量减少，一组基因下调，包括核因子-YA1（MtNF-YA1）和 MtNF-YB16，它们对结节分生组织的建立至关重要。因此，我们得出结论，固氮共生作用招募了最初在根中表达的两种蛋白质，MtEFD 和 MtEFD2，它们具有不同的功能，并为它们各自的功能进行了新功能化过程。
