Department of Plant Biology, University of Minnesota, Saint Paul, MN, 55108, USA.
Supercomputing Institute for Advanced Computational Research, University of Minnesota, Minneapolis, MN, 55455, USA.
New Phytol. 2019 May;222(3):1538-1550. doi: 10.1111/nph.15697. Epub 2019 Feb 27.
Symbiotic nitrogen fixation in legumes is mediated by an interplay of signaling processes between plant hosts and rhizobial symbionts. In legumes, several secreted protein families have undergone expansions and play key roles in nodulation. Thus, identifying lineage-specific expansions (LSEs) of nodulation-associated genes can be a strategy to discover candidate gene families. Using bioinformatic tools, we identified 13 LSEs of nodulation-related secreted protein families, each unique to either Glycine, Arachis or Medicago lineages. In the Medicago lineage, nodule-specific Polycystin-1, Lipoxygenase, Alpha Toxin (PLAT) domain proteins (NPDs) expanded to five members. We examined NPD function using CRISPR/Cas9 multiplex genome editing to create Medicago truncatula NPD knockout lines, targeting one to five NPD genes. Mutant lines with differing combinations of NPD gene inactivations had progressively smaller nodules, earlier onset of nodule senescence, or ineffective nodules compared to the wild-type control. Double- and triple-knockout lines showed dissimilar nodulation phenotypes but coincided in upregulation of a DHHC-type zinc finger and an aspartyl protease gene, possible candidates for the observed disturbance of proper nodule function. By postulating that gene family expansions can be used to detect candidate genes, we identified a family of nodule-specific PLAT domain proteins and confirmed that they play a role in successful nodule formation.
豆科植物中的共生固氮是由植物宿主和根瘤共生体之间的信号转导过程相互作用介导的。在豆科植物中，几个分泌蛋白家族经历了扩张，在结瘤中发挥关键作用。因此，鉴定与结瘤相关的基因的谱系特异性扩张（LSE）可以是发现候选基因家族的一种策略。使用生物信息学工具，我们鉴定了 13 个与结瘤相关的分泌蛋白家族的 LSE，每个家族都特属于大豆、花生或紫花苜蓿谱系。在紫花苜蓿谱系中，结节特异性多囊蛋白 1、脂氧合酶、α毒素（NPD）蛋白扩展到五个成员。我们使用 CRISPR/Cas9 多重基因组编辑来研究 NPD 功能，以创建紫花苜蓿 truncatula NPD 敲除系，针对一个到五个 NPD 基因。与野生型对照相比，具有不同 NPD 基因失活组合的突变系的结节更小，结节衰老的起始更早，或无效的结节。双敲除和三敲除系表现出不同的结瘤表型，但在 DHHC 型锌指和天冬氨酸蛋白酶基因的上调上是一致的，这些基因可能是观察到的正确结瘤功能紊乱的候选基因。通过假设基因家族的扩张可以用来检测候选基因，我们鉴定了一个与结瘤相关的 PLAT 结构域蛋白家族，并证实它们在成功形成结瘤中发挥作用。
