Rhizobia induce SYMRK endocytosis in Phaseolus vulgaris root hair cells.

MAIN CONCLUSION: PvSYMRK-EGFP undergoes constitutive and rhizobia-induced endocytosis, which rely on the phosphorylation status of T589, the endocytic YXXØ motif and the kinase activity of the receptor. Legume-rhizobia nodulation is a complex developmental process. It initiates when the rhizobia-produced Nod factors are perceived by specific LysM receptors present in the root hair apical membrane. Consequently, SYMRK (Symbiosis Receptor-like Kinase) becomes active in the root hair and triggers an extensive signaling network essential for the infection process and nodule organogenesis. Despite its relevant functions, the underlying cellular mechanisms involved in SYMRK signaling activity remain poorly characterized. In this study, we demonstrated that PvSYMRK-EGFP undergoes constitutive and rhizobia-induced endocytosis. We found that in uninoculated roots, PvSYMRK-EGFP is mainly associated with the plasma membrane, although intracellular puncta labelled with PvSymRK-EGFP were also observed in root hair and nonhair-epidermal cells. Inoculation with Rhizobium etli producing Nod factors induces in the root hair a redistribution of PvSYMRK-EGFP from the plasma membrane to intracellular puncta. In accordance, deletion of the endocytic motif YXXØ (YKTL) and treatment with the endocytosis inhibitors ikarugamycin (IKA) and tyrphostin A23 (TyrA23), as well as brefeldin A (BFA), drastically reduced the density of intracellular PvSYMRK-EGFP puncta. A similar effect was observed in the phosphorylation-deficient (T589A) and kinase-dead (K618E) mutants of PvSYMRK-EGFP, implying these structural features are positive regulators of PvSYMRK-EGFP endocytosis. Our findings lead us to postulate that rhizobia-induced endocytosis of SYMRK modulates the duration and amplitude of the SYMRK-dependent signaling pathway.

Visualization of the Crossroads between a Nascent Infection Thread and the First Cell Division Event in Phaseolus vulgaris Nodulation.

The development of a symbiotic nitrogen-fixing nodule in legumes involves infection and organogenesis. Infection begins when rhizobia enter a root hair through an inward structure, the infection thread (IT), which guides the bacteria towards the cortical tissue. Concurrently, organogenesis takes place by inducing cortical cell division (CCD) at the infection site. Genetic analysis showed that both events are well-coordinated; however, the dynamics connecting them remain to be elucidated. To visualize the crossroads between IT and CCD, we benefited from the fact that, in Phaseolus vulgaris nodulation, where the first division occurs in subepidermal cortical cells located underneath the infection site, we traced a Rhizobium etli strain expressing DsRed, the plant cytokinesis marker YFP-PvKNOLLE, a nuclear stain and cell wall auto-fluorescence. We found that the IT exits the root hair to penetrate an underlying subepidermal cortical (S-E) cell when it is concluding cytokinesis.

Rhizobium etli CE3-DsRed pMP604: a useful biological tool to study initial infection steps in Phaseolus vulgaris nodulation.

MAIN CONCLUSION: Rhizobium etli CE3-DsRed pMP604 drives infection 12-24 h earlier than R. etli CE3-DsRed and it is an excellent tool in live-cell imaging studies of IT developement in P. vulgaris roots. The study of the cellular dynamics of nodulation has frequently been limited by the difficulty of performing live-cell imaging in nodule primordia and legume roots, which are constituted by multiple cell layers, such is the case of Phaseolus vulgaris. Seeking conditions to reduce the time it takes for rhizobia to infect P. vulgaris root, we decided to explore the nodulation properties of Rhizobium etli CE3 pMP604, a strain that constitutively produces Nod factors through a flavonoids-independent transcriptional activation which is often used to purify Nod factors. Even though the strain infects 12-24 h earlier than the parental R. etli CE3 strain, infection thread (IT) formation, nodule organogenesis processes and N2-fixation activity are similar for both strains. Additionally, we have confirmed that R. etli CE3-DsRed pMP604 is an excellent tool to trace IT development in P. vulgaris roots.