The subunit 3 of the SUPERKILLER 3 (SKI3) complex mediates miR172-directed cleavage of Nodule Number Control 1 (NNC1) to modulate nodulation in Medicago truncatula

Legumes and rhizobia establish a nitrogen-fixing symbiosis that involves the formation of a lateral root organ, the nodule, and the infection process that allows intracellular accommodation of rhizobia within nodule cells. This process involves significant gene expression changes regulated at the transcriptional and post-transcriptional levels. We have previously shown that a transcript encoding the subunit 3 of the Superkiller Complex (SKI), which guides mRNAs to the exosome for 3´-to-5´ degradation, is required for nodule formation and bacterial persistence within the nodule, as well as the induction of early nodulation genes (e.g., MtENOD40) during the Medicago truncatula-Sinorhizobium meliloti symbiosis. Here, we reveal through transcript degradome and small RNA sequencing analysis that knockdown of MtSKI3 impairs the miR172-directed endonucleolytic cleavage of the mRNA encoding Nodule Number Control 1 (MtNNC1), an APETALA2 transcription factor that negatively modulates nodulation. Knockdown of MtNNC1 enhances nodule number, bacterial infection, and the induction of MtENOD40 upon inoculation with S. meliloti whereas overexpression of a miR172-resistant form of MtNNC1 significantly reduces nodule formation. This work identifies miR172 cleavage of MtNNC1 and its control by MtSKI3, a component of the 3´-to- 5´mRNA degradation pathway, as a new regulatory hub controlling indeterminate nodulation. Small RNA Seq in GUS RNAi mock and SKI3 RNAi mock Medicago truncatula roots

豆科植物与根瘤菌可构建固氮共生关系，该过程涉及侧生器官根瘤的形成，以及使根瘤菌能够在根瘤细胞内完成胞内定殖的侵染过程。该过程涉及大量在转录及转录后水平受到精准调控的基因表达变化。我们既往研究已证实，在蒺藜苜蓿（Medicago truncatula）与苜蓿中华根瘤菌（Sinorhizobium meliloti）的共生体系中，编码超级杀伤复合物（Superkiller Complex, SKI）亚基3的转录本——该亚基负责引导mRNA进入外切体以进行3'→5'降解——对根瘤形成、根瘤内细菌持续定殖，以及早期结瘤基因（如MtENOD40）的诱导表达均不可或缺。本研究通过降解组测序与小RNA测序分析发现，敲低MtSKI3会损害miR172介导的结瘤数量调控因子1（Nodule Number Control 1, MtNNC1）编码mRNA的核酸内切切割过程；MtNNC1属于APETALA2类转录因子，可负向调控结瘤进程。敲低MtNNC1可提升结瘤数量、增强细菌侵染能力，并促进接种苜蓿中华根瘤菌后MtENOD40的诱导表达；而过表达抗miR172的MtNNC1突变体，则会显著抑制根瘤的形成。本研究明确了miR172对MtNNC1的切割作用，以及3'→5' mRNA降解通路组分MtSKI3对该过程的调控，将其鉴定为调控不定型根瘤形成的全新调控枢纽。本数据集为经GUS RNAi（RNA干扰）空白对照与SKI3 RNAi空白对照处理的蒺藜苜蓿根系的小RNA测序数据。