Nod-factor Attachment, Calcium-fluxes, and Lipidtransfer Proteins in Symbiotic Signal Transduction

Abstract

Temporal and spatial observations on the attachment of Nod-factors were made by biotinylating the reducing terminus of the lipo-chitooligosaccharides of the broad host-range Rhizobium sp. NGR234 with the fluorescent reagent 2-amino-(6-amidobiotinyl)pyridine. Complex formation between the biotinylated Nod-factors and fluorescent streptavidin allowed localisation of the binding-site. At concentrations of > 10^-7 M, these fluorescently tagged moleculs bound rapidly and asymmetrically (≅ 1 min) to nodulation competent root-hairs but they did not bind to root-hairs of the non-host Arabidopsis thaliana. Early cellular events within the root-hairs were studied by loading root-segments with the calcium indicators Fura-2 and Fluo-3. Fluorescence ratio imaging showed that addition of NodNGR factors provoked almost immediate, plateau-like increases in intra-cellular free calcium {[Ca²⁺]_i} in root-hairs and epidermal cells. Confocal laser scanning microscopy (CLSM) revealed that calcium accumulation was concentrated at the tips and the sides of the responsive root-hairs. A gene encoding a lipid transfer-like protein (LPT2) was isolated from a Vigna unguiculata root-hair cDNA bank. Levels of the LTP2-transcript increased in root-hairs 24 h after treatment with NGR234, or its Nod-factors. The LTP2-gene was cloned into the pMal^TM expression vector and LTP2 purified by affinity chromatography. It was unable to transfer phospholipids between liposomes and mitochondria. Anti-sense analysis in which the LTP2 coding region was cloned between the 35S promoter and terminator sequences reduced nodulation when transformed into V. unguiculata.