Visualization of Nod Factor Receptor Dynamics

Nodulation in legume roots is triggered by nodulation (Nod) factors secreted by the rhizobial bacteria that ultimately fix nitrogen within the nodule. The perception of Nod factors initiates events at the root epidermis and cortex in preparation for root infection and nodule formation (reviewed in Oldroyd and Downie, 2008; Murray, 2011). In Medicago truncatula, two different putative receptors have been implicated in the recognition of Nod factors secreted by its symbiotic partner, Sinorhizobium meliloti. Now, Haney et al. (pages 2774–2787) have elegantly used confocal microscopy to monitor the effects of rhizobia on the localization and dynamics of one of those receptors, the lysin motif receptor-like kinase LYK3 (believed to be homologous to Lotus japonicus NOD FACTOR RECEPTOR1). Haney et al. transformed M. truncatula LYK3 mutants with a construct designed to use the native LYK3 promoter to drive expression of green fluorescent protein (GFP)–tagged LYK3. These lines were rescued for nodule formation, showing that the fusion protein is functional. Consistent with LYK3 functioning as a Nod factor receptor at the plasma membrane, the GFP signal in the transformed lines was found at the cell periphery and was apparently associated with the plasma membrane. The dynamics of LYK3-GFP were especially interesting because inoculation with rhizobia caused immobilization of LYK3-GFP. In untreated root hair cells, LYK3-GFP was found in a punctate pattern that persisted for between 3 and 180 s. However, 1 d after inoculation with S. meliloti, the puncta were stable for several more minutes. The authors found that this stabilization was dependent on Nod factor structure but that Nod factor alone was not sufficient to induce it, suggesting that another signal in addition to Nod factor is required for the stabilization. In addition, inoculation with S. meliloti induced the colocalization of LYK3 with a membrane-associated protein, FLOTILLIN4 (FLOT4), that is required for infection (see Figure). Furthermore, they found that the distribution of FLOT4 in the absence of bacteria was influenced by LYK3. Inoculation of transgenic M. truncatula root hairs with S. meliloti (right) induces colocalization of tagged LYK3 and FLOT4 proteins. In uninoculated root hairs (left), both proteins are found in puncta, but they do not overlap. Bar = 2 μm. (From Figure 3 of Haney et al. [2011].) Together, these data provide a view of LYK3 behavior in its native cellular environment and, importantly, driven by its own promoter. In that regard, this work represents a breakthrough in imaging dynamic changes in proteins expressed at low levels. In addition, it presents information that will be useful not only for understanding the early events in legume-rhizobium symbioses but also in plasma membrane signaling.