Abstract
In temperate legumes, endosymbiotic nitrogen-fixing rhizobia gain access to inner root tissues via a specialized transcellular apoplastic compartment known as the infection thread (IT). To study IT development in living root hairs, a protocol has been established for Medicago truncatula that allows confocal microscopic observations of the intracellular dynamics associated with IT growth. Fluorescent labeling of both the IT envelope (AtPIP2;1-green fluorescent protein) and the host endoplasmic reticulum (green fluorescent protein-HDEL) has revealed that IT growth is a fundamentally discontinuous process and that the variable rate of root hair invagination is reflected in changes in the host cell cytoarchitecture. The concomitant use of fluorescently labeled Sinorhizobium meliloti has further revealed that a bacteria-free zone is frequently present at the growing tip of the IT, thus indicating that bacterial contact is not essential for thread progression. Finally, these in vivo studies have shown that gaps within the bacterial file are a common feature during the early stages of IT development, and that segments of the file are able to slide collectively down the thread. Taken together, these observations lead us to propose that (1) IT growth involves a host-driven cellular mechanism analogous to that described for intracellular infection by arbuscular mycorrhizal fungi; (2) the non-regular growth of the thread is a consequence of the rate-limiting colonization by the infecting rhizobia; and (3) bacterial colonization involves a combination of bacterial cell division and sliding movement within the extracellular matrix of the apoplastic compartment.
Highlights
In temperate legumes, endosymbiotic nitrogen-fixing rhizobia gain access to inner root tissues via a specialized transcellular apoplastic compartment known as the infection thread (IT)
To perform confocal microscopy studies of rhizobial infection in M. truncatula root hairs, we have developed an experimental procedure similar to that previously used for arbuscular mycorrhizas (AMs) infection (Genre et al, 2005), which is described in detail in “Materials and Methods.”
We describe an experimental protocol for performing in vivo confocal studies of rhizobial infection within root hairs of the model legume M. truncatula and the identification of appropriate fluorescent markers, which allow concomitant visualization of the growing IT, the infecting microsymbiont, and various cellular components that play an active role in IT development
Summary
Endosymbiotic nitrogen-fixing rhizobia gain access to inner root tissues via a specialized transcellular apoplastic compartment known as the infection thread (IT). Glomeromycota fungi are able to associate with the majority of vascular land plants to form so-called arbuscular mycorrhizas (AMs), whereas root nodulation involving nitrogen-fixing soil bacteria of the Rhizobiaceae family is restricted to the legume family In these tightly regulated biotrophic associations, hostmicrobe recognition and initial root entry are crucial steps in the establishment of these two endosymbioses, and, in both cases, outer root penetration by the infecting microsymbiont is transcellular, involving the. Recent in vivo confocal imaging techniques using GFP tagging of cellular components have provided important information about host cell reorganization both prior to and during AM infection (Genre et al, 2005, 2008) These studies have revealed that initial AM infection of non-root hair epidermal cells is preceded by complex intracellular remodeling involving transcellular nuclear migration associated with the formation of a transient cytoplasmic assembly comprising cytoskeletal and endoplasmic reticulum (ER) components. Little is currently known about the dynamics of rhizobial infection and, in particular, the subcellular remodeling in the host cell that accompanies IT initiation and development, as well as the coordination with bacterial colonization of the thread
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