The MtDMI2-MtPUB2 Negative Feedback Loop Plays a Role in Nodulation Homeostasis

Abstract

DOES NOT MAKE INFECTION 2 (MtDMI2) is a Leu rich repeat-type receptor kinase required for signal transduction in the <i>Medicago truncatula/Sinorhizobium meliloti</i> symbiosis pathway. However, the mechanisms through which MtDMI2 participates in nodulation homeostasis are poorly understood. In this study, we identified MtPUB2-a novel plant U-box (PUB)-type E3 ligase-and showed that it interacts with MtDMI2. MtDMI2 and MtPUB2 accumulation were shown to be similar in various tissues. Roots of plants in which <i>MtPUB2</i> was silenced by RNAi (<i>MtPUB2-</i>RNAi plants) exhibited impaired infection threads, fewer nodules, and shorter primary root lengths compared to those of control plants transformed with empty vector. Using liquid chromatography-tandem mass spectrometry, we showed that MtDMI2 phosphorylates MtPUB2 at Ser-316, Ser-421, and Thr-488 residues. When <i>MtPUB2</i>-RNAi plants were transformed with <i>MtPUB2^S421D</i> , which mimics the phosphorylated state, MtDMI2 was persistently ubiquitinated and degraded by MtPUB2^S421D, resulting in fewer nodules than observed in <i>MtPUB2</i>/<i>MtPUB2</i>-RNAi-complemented plants. However, <i>MtPUB2^S421A</i> /<i>MtPUB2</i>-RNAi-complemented plants showed no MtPUB2 ubiquitination activity, and their nodulation phenotype was similar to that of <i>MtPUB2</i>-RNAi plants transformed with empty vector. Further studies demonstrated that these proteins form a negative feedback loop of the prey (MtDMI2)-predator (MtPUB2) type. Our results suggest that the MtDMI2-MtPUB2 negative feedback loop, which displays crosstalk with the long-distance autoregulation of nodulation via MtNIN, plays an important role in nodulation homeostasis.