Table_1_Auxin Response Factor 2 (ARF2), ARF3, and ARF4 Mediate Both Lateral Root and Nitrogen Fixing Nodule Development in Medicago truncatula.DOCX

Auxin Response Factors (ARFs) constitute a large family of transcription factors that mediate auxin-regulated developmental programs in plants. ARF2, ARF3, and ARF4 are post-transcriptionally regulated by the microRNA390 (miR390)/trans-acting small interference RNA 3 (TAS3) module through the action of TAS3-derived trans-acting small interfering RNAs (ta-siRNA). We have previously reported that constitutive activation of the miR390/TAS3 pathway promotes elongation of lateral roots but impairs nodule organogenesis and infection by rhizobia during the nitrogen-fixing symbiosis established between Medicago truncatula and its partner Sinorhizobium meliloti. However, the involvement of the targets of the miR390/TAS3 pathway, i.e., MtARF2, MtARF3, MtARF4a, and MtARF4b, in root development and establishment of the nitrogen-fixing symbiosis remained unexplored. Here, promoter:reporter fusions showed that expression of both MtARF3 and MtARF4a was associated with lateral root development; however, only the MtARF4a promoter was active in developing nodules. In addition, up-regulation of MtARF2, MtARF3, and MtARF4a/b in response to rhizobia depends on Nod Factor perception. We provide evidence that simultaneous knockdown of MtARF2, MtARF3, MtARF4a, and MtARF4b or mutation in MtARF4a impaired nodule formation, and reduced initiation and progression of infection events. Silencing of MtARF2, MtARF3, MtARF4a, and MtARF4b altered mRNA levels of the early nodulation gene nodulation signaling pathway 2 (MtNSP2). In addition, roots with reduced levels of MtARF2, MtARF3, MtARF4a, and MtARF4b, as well as arf4a mutant plants exhibited altered root architecture, causing a reduction in primary and lateral root length, but increasing lateral root density. Taken together, our results suggest that these ARF members are common key players of the morphogenetic programs that control root development and the formation of nitrogen-fixing nodules.生长素响应因子（Auxin Response Factors, ARFs）是一类庞大的转录因子家族，可介导植物体内生长素调控的各类发育程序。ARF2、ARF3与ARF4可通过TAS3衍生的反式作用小干扰RNA（ta-siRNA）的作用，受microRNA390（miR390）/反式作用小干扰RNA3（trans-acting small interference RNA 3, TAS3）模块进行转录后调控。我们此前的研究表明，miR390/TAS3通路的组成型激活可促进蒺藜苜蓿（Medicago truncatula）与其共生伙伴苜蓿根瘤菌（Sinorhizobium meliloti）建立固氮共生过程中的侧根伸长，但会损害根瘤器官发生与根瘤菌侵染过程。然而，miR390/TAS3通路的靶基因——即MtARF2、MtARF3、MtARF4a与MtARF4b——在根系发育与固氮共生建立过程中的功能仍未被解析。本研究中，启动子-报告基因融合实验显示，MtARF3与MtARF4a的表达均与侧根发育相关；但仅MtARF4a启动子在发育中的根瘤中具有活性。此外，MtARF2、MtARF3与MtARF4a/b在根瘤菌刺激下的上调表达依赖于结瘤因子（Nod Factor）的感知。我们的实验证据表明，同时敲低MtARF2、MtARF3、MtARF4a与MtARF4b，或MtARF4a发生突变，均会损害根瘤形成，并降低侵染事件的起始与进展速率。沉默MtARF2、MtARF3、MtARF4a与MtARF4b会改变早期结瘤基因结瘤信号通路2（MtNSP2）的mRNA水平。此外，MtARF2、MtARF3、MtARF4a与MtARF4b表达量降低的根系，以及arf4a突变体植株，均表现出根系构型改变：主根与侧根长度缩短，但侧根密度升高。综上，我们的研究结果表明，这些ARF家族成员是调控根系发育与固氮根瘤形成的形态发生程序的共同关键调控因子。