Remodeling of the Infection Chamber before Infection Thread Formation Reveals a Two-Step Mechanism for Rhizobial Entry into the Host Legume Root Hair

In many legumes, root entry of symbiotic nitrogen-fixing rhizobia occurs via host-constructed tubular tip-growing structures known as infection threads (ITs). Here, we have used a confocal microscopy live-tissue imaging approach to investigate early stages of IT formation in Medicago truncatula root hairs (RHs) expressing fluorescent protein fusion reporters. This has revealed that ITs only initiate 10 to 20 h after the completion of RH curling, by which time major modifications have occurred within the so-called infection chamber, the site of bacterial entrapment. These include the accumulation of exocytosis (M. truncatula Vesicle-Associated Membrane Protein721e)- and cell wall (M. truncatula EARLY NODULIN11)-associated markers, concomitant with radial expansion of the chamber. Significantly, the infection-defective M. truncatula nodule inception-1 mutant is unable to create a functional infection chamber. This underlines the importance of the NIN-dependent phase of host cell wall remodeling that accompanies bacterial proliferation and precedes IT formation, and leads us to propose a two-step model for rhizobial infection initiation in legume RHs.Legumes possess the remarkable capacity to improve their nutrition by establishing a nitrogen-fixing root nodule symbiosis (RNS) with soil bacteria collectively called rhizobia. In many legumes such as Medicago truncatula, rhizobia penetrate across the root epidermis and outer cortex to reach the differentiating nodule tissues via sequentially constructed transcellular compartments known as infection threads (ITs; Gage, 2004). It is now well established that this mode of entry through specialized infection compartments, often referred to as accommodation, is shared with the more ancient arbuscular mycorrhizal (AM) symbiosis, from which the legume-Rhizobium RNS is thought to have evolved (Parniske, 2008; Markmann and Parniske, 2009). Furthermore, strong evidence indicates that the signaling and cellular mechanisms underlying IT formation in legumes are closely related to those used for infection compartment formation during AM infection of epidermal and outer cortical tissues (Bapaume and Reinhardt, 2012; Oldroyd, 2013).Rhizobial infection is set in motion after an initial molecular dialogue between symbiotic partners, in which rhizobial lipo-chitooligosaccharide (LCO) Nod factors (NFs) are key signaling molecules (for review, see Oldroyd, 2013). Host responses to NF signaling include rapid and sustained nuclear-associated Ca²⁺ oscillations (Ca²⁺ spiking; Ehrhardt et al., 1996; Oldroyd and Downie, 2006; Sieberer et al., 2009; Capoen et al., 2011) and the rapid expression of early epidermal marker genes such as M. truncatula EARLY NODULIN11 (Charron et al., 2004). The activation of nuclear Ca²⁺ spiking is one of the most characteristic features of the so-called common symbiotic signaling pathway, common to both RNS and AM (Kistner and Parniske, 2002; Singh and Parniske, 2012). Whereas these preinfection responses to NFs are observed in the majority of elongating root hairs (RHs) early after rhizobial inoculation (Journet et al., 2001; Wais et al., 2002), ITs are only formed in a small subset of RHs, and MtENOD11 expression is strongly activated at these rhizobial infection sites (Journet et al., 2001; Boisson-Dernier et al., 2005).ITs are tubular plant-derived structures delimited by a membrane that is contiguous with the RH plasmalemma and a layer of cell wall-like material, thus isolating the rhizobia from the host cell cytoplasm (Gage, 2004). These apoplastic infection compartments are progressively constructed along the length of the RH with their growing tip connected via a cytoplasmic bridge to the migrating RH nucleus. This broad cytoplasmic column provides the cell machinery for tip growth, which involves targeted exocytosis of membrane and extracellular materials to the growing apex of the IT (Oldroyd et al., 2011; Bapaume and Reinhardt, 2012). It is presumed that this cytoplasmic bridge shares an equivalent role to the prepenetration apparatus (PPA) formed at the onset of AM fungal infection (Genre et al., 2005, 2008). We now know that the IT tip region is formed in advance of rhizobial colonization and is progressively populated by dividing rhizobia that also physically move down the thread (Gage, 2004; Fournier et al., 2008). It has been proposed that the matrix of the growing IT tip is initially in a fluid or gel-like state compatible with bacterial growth and movement (Brewin, 2004; Fournier et al., 2008). This relative plasticity could result in part from the presence of atypical extracellular (glyco) proteins such as the repetitive Pro-rich proteins MtENOD11/MtENOD12 because their low Tyr content is presumed to limit cross linking to other wall components (Scheres et al., 1990; Pichon et al., 1992; Journet et al., 2001).Nevertheless, the mechanism by which rhizobial IT formation is initiated in RHs is not clear. Whereas AM fungal hyphae form contact structures called hyphopodia on the exposed surface of nonhair epidermal cells prior to PPA formation and perifungal infection compartment formation (Genre et al., 2005), rhizobial entry requires that the bacteria first become entrapped between RH walls. Attachment of rhizobia close to a growing RH tip induces a continuous reorientation of tip growth, most likely the result of localized NF production (Esseling et al., 2003), eventually leading to RH curling and subsequent bacterial entrapment within a closed chamber in the center of the curl (Catoira et al., 2001; Geurts et al., 2005). Rhizobial entrapment can also occur between the cell walls of two touching RHs (Dart, 1974; Gage, 2004).The closed chamber in curled RHs has often been termed the infection pocket (e.g. Murray, 2011; Guan et al., 2013). However, because this term is also used to designate a quite different and larger structure formed in root subepidermal tissues of legumes during intercellular infection after crack entry and involving localized cell death (Goormachtig et al., 2004), we propose to use the term infection chamber to describe the unique enclosure formed during rhizobial RH infection.After entrapment, it has been proposed that rhizobia multiply to form a so-called microcolony (Gage et al., 1996; Limpens et al., 2003), and that IT polar growth initiates in front of this microcolony by local invagination of the RH plasmalemma combined with exocytosis of extracellular materials (Gage, 2004). Furthermore, it has been suggested that localized degradation of the chamber wall would allow the rhizobia to access the newly formed IT (Callaham and Torrey, 1981; Turgeon and Bauer, 1985). However, a detailed investigation of this particular stage of rhizobial infection is lacking, particularly concerning when and where the rhizobia/cell wall interface becomes modified. Such studies have been limited until now, notably because ITs develop only in a low proportion of curled RHs (Dart, 1974).To attempt to answer this question, we have used a live-tissue imaging approach developed for in vivo confocal microscopy in M. truncatula (Fournier et al., 2008; Cerri et al., 2012; Sieberer et al., 2012) and particularly well adapted to time-lapse studies of the initial stages of rhizobial infection, including RH curling and IT formation. To investigate modifications occurring at the RH interface with the enclosed rhizobia during these early stages, we prepared M. truncatula plants expressing fluorescent protein fusions aimed at detecting both exocytosis activity and cell wall remodeling during the initial construction of the IT apoplastic compartment. To this end, we made use of the M. truncatula Vesicle-Associated Membrane Protein721e (MtVAMP721e; Ivanov et al., 2012), recently shown to label exocytosis sites both in growing RHs and during AM colonization (Genre et al., 2012), as well as the infection- and cell wall-associated MtENOD11 Pro-rich glycoprotein (Journet et al., 2001). Our experiments have revealed that IT development in curled RHs only initiates after a lengthy interval of 10 to 20 h, during which sustained exocytosis and MtENOD11 secretion to the infection chamber are associated with radial expansion as well as remodeling of the surrounding walls. Importantly, it was found that the infection-defective M. truncatula nodule inception-1 (Mtnin-1) mutant (Marsh et al., 2007) is impaired in chamber remodeling. Our findings led us to propose a new model for IT formation in which the infection chamber first differentiates into a globular apoplastic compartment displaying similarities to the future IT, and in which the enclosed rhizobia multiply. This is then followed by a switch from radial to tubular growth corresponding to tip-driven IT growth and associated movement of rhizobia into the extending thread. Importantly, this two-step model no longer requires that the host cell wall is degraded to allow access of the colonizing rhizobia to the newly initiated IT.