Rhizobia are rod-shaped bacteria that form nitrogen-fixing root nodules on leguminous plants; however, they don’t carry MreB, a key determinant of rod-like cell shape. Here, we introduced an actin-like mreB homolog from a pseudomonad into Mesorhizobium huakuii 7653R (a microsymbiont of Astragalus sinicus L.) and examined the molecular, cellular, and symbiotic phenotypes of the resultant mutant. Exogenous mreB caused an enlarged cell size and slower growth in laboratory medium. However, the mutant formed small, ineffective nodules on A. sinicus (Nod⁺ Fix⁻), and rhizobial cells in the infection zone were unable to differentiate into bacteroids. RNA sequencing analysis also revealed minor effects of mreB on global gene expression in free-living cells but larger effects for cells grown in planta. Differentially expressed nodule-specific genes include cell cycle regulators such as the tubulin-like ftsZ₁ and ftsZ₂. Unlike the ubiquitous FtsZ₁, an FtsZ₂ homolog was commonly found in Rhizobium, Sinorhizobium, and Mesorhizobium spp. but not in closely related nonsymbiotic species. Bacterial two-hybrid analysis revealed that MreB interacts with FtsZ₁ and FtsZ₂, which are targeted by the host-derived nodule-specific cysteine-rich peptides. Significantly, MreB mutation D283A disrupted the protein–protein interactions and restored the aforementioned phenotypic defects caused by MreB in M. huakuii. Together, our data indicate that MreB is detrimental for modern rhizobia and its interaction with FtsZ₁ and FtsZ₂ causes the symbiotic process to cease at the late stage of bacteroid differentiation. These findings led to a hypothesis that loss of mreB in the common ancestor of members of Rhizobiales and subsequent acquisition of ftsZ₂ are critical evolutionary steps leading to legume-rhizobial symbiosis.

Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

根瘤菌是杆状细菌，在豆科植物上形成固氮根瘤；然而，它们不携带 MreB，这是杆状细胞形状的关键决定因素。在这里，我们将来自假单胞菌的肌动蛋白样mreB同源物引入Mesorhizobium huakuii 7653R（黄芪的微共生体），并检查了所得突变体的分子、细胞和共生表型。外源性mreB在实验室培养基中导致细胞变大和生长缓慢。然而，突变体在A. sinicus上形成了小的、无效的结节（Nod ⁺ Fix ^-)，感染区的根瘤菌细胞无法分化为类杆菌。RNA 测序分析还揭示了mreB对自由生活细胞中整体基因表达的影响较小，但对植物中生长的细胞影响较大。差异表达的结节特异性基因包括细胞周期调节因子，如微管蛋白样ftsZ ₁和ftsZ ₂。与无处不在的 FtsZ ₁不同，FtsZ ₂同源物常见于根瘤菌、中华根瘤菌和中生根瘤菌属。但不是在密切相关的非共生物种中。细菌双杂交分析显示 MreB 与 FtsZ ₁相互作用和 FtsZ ₂，它们被宿主衍生的结节特异性富含半胱氨酸的肽靶向。值得注意的是，MreB 突变 D283A 破坏了蛋白质 - 蛋白质相互作用并恢复了上述由 MreB 在M. huakuii 中引起的表型缺陷。总之，我们的数据表明 MreB 对现代根瘤菌有害，它与 FtsZ ₁和 FtsZ _{2 的}相互作用导致共生过程在类杆菌分化的后期停止。这些发现导致了一个假设，即根瘤菌目成员的共同祖先中mreB 的缺失和随后获得ftsZ ₂ 是导致豆科植物-根瘤菌共生的关键进化步骤。

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