The Medicago truncatula DREPP Protein Triggers Microtubule Fragmentation in Membrane Nanodomains during Symbiotic Infections.

Chao Su,Pierre-Marc Delaux,Casandra Hernández-Reyes,Jean Keller,Beatrice Lace,Marie-Luise Klein,Thomas Ott,Morgane Batzenschlager,Franck Anicet Ditengou

doi:10.1105/tpc.19.00777

Abstract

The initiation of intracellular host cell colonization by symbiotic rhizobia in Medicago truncatula requires repolarization of root hairs, including the rearrangement of cytoskeletal filaments. The molecular players governing microtubule (MT) reorganization during rhizobial infections remain to be discovered. Here, we identified M. truncatula DEVELOPMENTALLY REGULATED PLASMA MEMBRANE POLYPEPTIDE (DREPP), a member of the MT binding DREPP/PCaP protein family, and investigated its functions during rhizobial infections. We show that rhizobial colonization of drepp mutant roots as well as transgenic roots overexpressing DREPP is impaired. DREPP relocalizes into symbiosis-specific membrane nanodomains in a stimulus-dependent manner. This subcellular segregation coincides with DREPP-dependent MT fragmentation and a partial loss of the ability to reorganize the MT cytoskeleton in response to rhizobia, which might rely on an interaction between DREPP and the MT-organizing protein SPIRAL2. Taken together, our results reveal that establishment of symbiotic associations in M. truncatula requires DREPP in order to regulate MT reorganization during initial root hair responses to rhizobia.

Highlights

The legume-Rhizobium root nodule symbiosis (RNS) represents an almost exceptional example for an intracellular accommodation of a bacterial mutualist
Earlier work demonstrated that prolonged exposition of root hairs to isolated Nod Factors (NFs) or application of rhizobia results in a re-organization of membrane-resident signaling proteins such as the entry receptor LYSIN MOTIF RECEPTOR KINASE 3 (LYK3) and the molecular scaffolds FLOT4 and SYMREM1 into membrane nanodomains (Haney et al, 2011; Liang et al, 2018)
While the precise molecular composition of this symbiosis-induced nanodomain remains elusive, FLOT4 and actin are required for the subsequent recruitment of SYMREM1 and LYK3, which has been hypothesized to mark the transition between initial signaling events and morphological changes to prepare the cell for infection (Liang et al, 2018)

Summary

Introduction

The legume-Rhizobium root nodule symbiosis (RNS) represents an almost exceptional example for an intracellular accommodation of a bacterial mutualist. Ectopic expression of DREPP-GFP resulted in a peripheral fluorescent signal in young root hairs, indicating an association of the protein with the plasma membrane (PM) (Fig. 2A). As shown before in the root epidermis, ectopic expression of DREPP resulted in a reorientation of MTs but the filaments appeared largely intact (Supplemental Figure 4) This pattern was observed in root hairs (Fig. 5). When performing co-immunoprecipitation experiments using anti-GFP nanobody traps on protein extracts from N. benthamiana leaves, we only obtained signals for SPR2 and EB1b (Supplemental Figure 12, line 1 and line 6), indicating that DREPP may predominantly associate with the plus end of the MTs and interferes with their elongation Taken together these results show that DREPP controls MT reorganization by interfering with their plus end elongation, predominantly upon localizing to membrane nanodomains after inoculation with rhizobia

Discussion

Findings

Materials and Methods