Probing nod factor perception in legumes by fluorescence microspectroscopy

Plants of the family of legumes are capable of forming a symbiosis with Rhizobium bacteria. These Gram-negative bacteria invade the root system of a host legume and fix nitrogen in a specialized organ, the so-called root nodule. In exchange for sugars, the bacteria convert atmospheric nitrogen to ammonia which can be used by the plant. This remarkable alliance allows the plant to grow independently from nitrogen sources provided by the soil. Examples of leguminous plants are clover, pea, and soybean. The symbiosis is initiated by a molecular dialogue. The plant produces flavonoid compounds which are recognized by the bacterial NodD protein. The signaling pathway which is activated leads to the synthesis and secretion of lipo-chitooligosaccharides which are also called Nod factors. The production of Nod factors by the Rhizobium bacteria is an essential step for accomplishing symbiosis and also determines host specificity. The general structure of Nod factors comprises a chitin backbone of three to five b-1,4-linked N-acetylglucosamine units. A fatty acid of 16-20 carbon atoms is N-linked to the terminal non-reducing sugar residue. The exact molecular structure can comprise different acyl chains and a variety of decorations on the chitin backbone depending on the Rhizobium species. After successful recognition of the bacteria by the legume, a remarkable morphogenic process takes place, which is known as root hair curling. The root hair curls around the Rhizobium colony by which the bacteria are entrapped within the so-called shepherd's crook. Subsequently, the rhizobia enter the root hair through an infection thread, starting from the center of the curl. Via the infection thread several cell layers are crossed after which the bacteria are released in nodule primordium cells, where they differentiate into bacteroids that fix nitrogen. Nod factors in the absence of bacteria, either purified from Rhizobium cultures or chemically synthesized can elicit a wide variety of responses on a compatible legume host. When Nod factors are applied to roots, the earliest visible response takes place in root hairs. Root hairs are single tip-growing cells that develop from the epidermis of a root and grow perpendicular from the longitudinal axis of the root. Generally, root hairs that are terminating growth are susceptible to Nod factors and respond by swelling of the tip of the root hairs, followed by the re-initiation of tip growth in a random direction. This typical Nod factor response is referred to as root hair deformation and can be observed with a microscope 2-3 hours after addition of Nod factors. The perception of Nod factors by the plant, and the downstream signaling cascades that are activated are major research topics in the Rhizobium-legume interaction. The low concentration (down to 10-12 M) at which Nod factors can still induce root hair deformation and the dependence of the bioactivity on specific decorations of the Nod factor suggest that these molecules are perceived by receptors at the root hair. However, to date no such receptors are characterized. Moreover, it is far from clear where Nod factor recognition by root hairs takes place. Therefore an approach was taken in which fluorescent Nod factor derivatives are synthesized, allowing to probe the ligand binding sites on legume root hairs. The research described in this thesis focuses on the quantification, characterization and perception by legumes of Nod factors. In order to detect Nod factors at physiologically relevant concentrations sensitive techniques are required. A number of fluorescence spectroscopy and microscopy based techniques can be used to study fluorescent derivatives of signaling molecules. In chapter 1, the use of fluorescence microspectroscopic techniques available in the laboratory are discussed. Examples how these techniques can be used for the study of root hairs and other living cells are described. In chapter 2, two methods to quantify purified Nod factors are described. An enzymatic step which is crucial for the first method was analyzed in detail. The second method was optimized and validated using fluorescent and radiolabeled Nod factor derivatives. The chapter describes in detail how the two optimized methods can be used for quantifying Nod factors as well as potential pitfalls. In chapter 3, the spectral properties of three novel fluorescent Nod factor derivatives are described. It is checked whether these fluorescent Nod factors can still elicit root hair deformation on Vicia sativa roots. The properties of the amphiphilic signaling molecules were characterized in vitro in the absence and presence of micelles and model membrane systems using fluorescence spectroscopy. Time-correlated single photon counting fluorescence spectroscopy was used to measure rotational mobility of the fluorophore. These experiments are complemented with fluorescence correlation spectroscopy to examine diffusional mobility of the Nod factors. A lipid transfer assay was used to measure the rate of intermembrane transfer and intramembrane flip-flop of Nod factors. In chapter 4, a detailed study is reported describing the sites at which the fluorescent Nod factors accumulate. Fluorescence microscopy is used to examine the location of fluorescent Nod factors on root hairs during the initial perception and during root hair deformation. Subsequently, the diffusional mobility of the fluorescent Nod factors is measured in vivo using fluorescence correlation microscopy (FCM), allowing quantification of molecular mobility and concentration of fluorescent Nod factors in living root hairs at a molecular level. This study is continued in chapter 5 in which also novel sulfated fluorescent Nod factors are used and characterized, enabling a direct comparison between sulfated and non-sulfated Nod factors on a host and non-host legume. Also, the origin of the molecular mobility of the Nod factors is studied in more detail. In chapter 6 a novel approach towards manipulating phospholipid second messengers in single cells with spatiotemporal control is presented. The synthesis of a fluorescent and caged derivative, NPE-phosphatidic acid, which releases phosphatidic acid upon exposure to UV is described. The release of phosphatidic acid from the caged compound is studied in vitro and in vivo. The use of photoreleasable phosphatidic acid for studying phospholipid signaling in vivo is evaluated. Chapter 7 summarizes the conclusions that can be drawn from the results described in this thesis. The implications for Nod factor secretion by the bacterium and subsequent perception by legume root hairs are discussed. Based on the results presented in this thesis, it is tempting to speculate that spatial restriction of signaling molecules in plants is achieved by immobilization in the cell wall. Subseqent perception of Nod factor takes place either in the plasma membrane or within the cell wall as is illustrated by two proposed modes of perception. The results of this thesis are discussed with respect to these two models.