Nitrogen-fixing root nodulation in legumes challenged with nitrogen-limiting conditions requires infection of the root hairs by soil symbiotic bacteria, collectively referred to as rhizobia, and the initiation of cell divisions in the root cortex. Cytokinin hormones are critical for early nodulation to coordinate root nodule organogenesis and the progression of bacterial infections. Cytokinin signaling involves regulation of the expression of cytokinin primary response genes by type-B response regulator (RRB) transcription factors. RNA interference or mutation of MtRRB3, the RRB-encoding gene most strongly expressed in Medicago truncatula roots and nodules, significantly decreased the number of nodules formed, indicating a function of this RRB in nodulation initiation. Fewer infection events were also observed in rrb3 mutant roots associated with a reduced Nod factor induction of the Early Nodulin 11 (MtENOD11) infection marker, and of the cytokinin-regulated Nodulation Signaling Pathway 2 (MtNSP2) gene. Rhizobial infections correlate with an expansion of the nuclear area, suggesting the activation of endoreduplication cycles linked to the cytokinin-regulated Cell Cycle Switch 52A (MtCCS52A) gene. Although no significant difference in nucleus size and endoreduplication were detected in rhizobia-infected rrb3 mutant roots, expression of the MtCCS52A endoreduplication marker was reduced. As the MtRRB3 expression pattern overlaps with those of MtNSP2 and MtCCS52A in roots and nodule primordia, chromatin immunoprecipitation-quantitative PCR and protoplast trans-activation assays were used to show that MtRRB3 can interact with and trans-activate MtNSP2 and MtCCS52A promoters. Overall, we highlight that the MtRRB3 cytokinin signaling transcription factor coordinates the expression of key early nodulation genes.

在受氮限制条件挑战的豆类中，固氮根瘤需要根系土壤共生细菌（统称为根瘤菌）感染根毛，并启动根皮层中的细胞分裂。细胞分裂素激素对于早期结节以协调根瘤的器官发生和细菌感染的进展至关重要。细胞分裂素信号传导涉及通过B型反应调节剂（RRB）转录因子调节细胞分裂素初级反应基因的表达。RNA干扰或MtRRB3突变，这是在苜蓿中最强烈表达的RRB编码基因根和结节显着减少了结节形成的数量，表明该RRB在结瘤起始中发挥了作用。在与早期Nodulin 11（MtENOD11）感染标记物和细胞分裂素调节的Nodulation Signaling Pathway 2（Mt NSP2）基因的Nod因子诱导降低相关的rrb3突变根中，也观察到了较少的感染事件。根瘤菌感染与核区域的扩大有关，表明核内复制周期的激活与细胞分裂素调节的细胞周期开关52A（Mt CCS52A）基因。尽管在根瘤菌感染的rrb3突变体根中未检测到核大小和核内复制的显着差异，但MtCCS52A核内复制标记的表达降低了。由于MtRRB3的表达模式与MtNSP2和MtCCS52A的根和结节原基重叠，因此染色质免疫沉淀定量PCR和原生质体反式激活法被用于显示MtRRB3可以与MtNSP2和MtCCS52A启动子相互作用并反式激活。总体而言，我们强调了MtRRB3细胞分裂素信号传导转录因子可协调关键早期结节基因的表达。