Interactions between legumes and rhizobia are controlled by the sequential exchange of symbiotic signals. Two different techniques, 2D-PAGE electrophoresis and differential display were used to study the effects of rhizobial signals on legume development. Application of variously substituted lipo-oligo-saccharidic Nod-factors to roots of Vigna unguiculata resulted in changes in the phosphorylation patterns of microsomal proteins. Reliable amino-acid sequences were obtained for one Nod-factor enhanced protein which was highly homologous to the 57-kDa subunit from Arabidopsis thaliana vacuolar membrane H(+)-ATPase. Immuno-blotting techniques demonstrated that Nod-factors cause rapid and massive increases of this enzyme in treated roots, suggesting that H(+)-ATPases play symbiotic roles. Concomitantly, we used differential display (DD) techniques on mRNA isolated from root-hairs to analyse early root responses to NGR234. Significant matches of several DD clones to known sequences were found. Clone D2.62 was homologous to a multitude of receptor kinases including S receptor-like kinases of A. thaliana and clone D4.1 showed similarities to Lotus japonicus phosphatidylinositol transfer-like protein III and late nodulin 16. Independent confirmatory analyses of these differentially expressed clones indicated expression at very low levels.

Rhizobium sp. NGR234 nodulates many plants, some of which react to proteins secreted via a type three secretion system (T3SS) in a positive- (Flemingia congesta, Tephrosia vogelii) or negative- (Crotalaria juncea, Pachyrhizus tuberosus) manner. T3SSs are devices that Gram-negative bacteria use to inject effector proteins into the cytoplasm of eukaryotic cells. The only two rhizobial T3SS effector proteins characterized to date are NopL and NopP of NGR234. NopL can be phosphorylated by plant kinases and we show this to be true for NopP as well. Mutation of nopP leads to a dramatic reduction in nodule numbers on F. congesta and T. vogelii. Concomitant mutation of nopL and nopP further diminishes nodulation capacity to levels that, on T. vogelii, are lower than those produced by the T3SS null mutant NGR(Omega)rhcN. We also show that the T3SS of NGR234 secretes at least one additional effector, which remains to be identified. In other words, NGR234 secretes a cocktail of effectors, some of which have positive effects on nodulation of certain plants while others are perceived negatively and block nodulation. NopL and NopP are two components of this mix that extend the ability of NGR234 to nodulate certain legumes.

Bacterial effector proteins delivered into eukaryotic cells via bacterial type III secretion systems are important virulence factors in plant-pathogen interactions. Type III secretion systems have been found in Rhizobium species that form symbiotic, nitrogen-fixing associations with legumes. One such bacterium, Rhizobium sp. NGR234, secretes a number of type III effectors, including nodulation outer protein L (NopL, formerly y4xL). Here, we show that expression of nopL in tobacco (Nicotiana tabacum) prevents full induction of pathogenesis-related (PR) defense proteins. Transgenic tobacco plants that express nopL and were infected with potato virus Y (necrotic strain 605) exhibited only very low levels of chitinase (class I) and beta-1,3-glucanase (classes I and III) proteins. Northern-blot analysis indicated that expression of nopL in plant cells suppresses transcription of PR genes. Treatment with ethylene counteracted the effect of NopL on chitinase (class I). Transgenic Lotus japonicus plants that expressed nopL exhibited delayed development and low chitinase levels. In vitro experiments showed that NopL is a substrate for plant protein kinases. Together, these data suggest that NopL, when delivered into the plant cell, modulates the activity of signal transduction pathways that culminate in activation of PR proteins.

Bacterial pathogens use type III secretion systems (TTSSs) to deliver virulence factors into eukaryotic cells. These effectors perturb host-defence responses, especially signal transduction pathways. A functional TTSS was identified in the symbiotic, nitrogen-fixing bacterium Rhizobium sp. NGR234. NopL (formerly y4xL) of NGR234 is a putative symbiotic effector that modulates nodulation in legumes. To test whether NopL could interact with plant proteins, in vitro phosphorylation experiments were performed using recombinant nopL protein purified from Escherichia coli as well as protein extracts from Lotus japonicus and tobacco plants. NopL serves as a substrate for plant protein kinases as well as purified protein kinase A. Phosphorylation of NopL was inhibited by the Ser/Thr kinase inhibitor K252a as well as by PD98059, a mitogen-activated protein (MAP) kinase kinase inhibitor. It thus seems likely that, after delivery into the plant cell, NopL modulates MAP kinase pathways.

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 pMalTM 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.

Rhizobia excrete variously substituted lipo-oligosaccha-ride Nod factors into the legume rhizosphere. Homologous legumes respond to these signals through deformation of the root hairs and the development of nodulation foci in the root cortex. Cellular events in root hairs from the susceptible zone of nearly mature root hairs were studied in root segments loaded with the calcium indicators Fura-2 or Fluo-3. Application of 10-9 M Nod factors of the broad-host-range Rhizobium sp. NGR234 to the homologous legume Vigna unguiculata resulted, within seconds, in plateau-like increases in intracellular free calcium ([Ca2+]i) in the root hairs and root epidermal cells. Nod factors of R. meliloti at 10-9 M caused equally rapid increases in [Ca2+]i in the root hairs and epidermal cells of the nonhost V. unguiculata, and also induced root-hair deformation. The chitin tetramer, N-N′-N″-N′″-tetracetylchitotetraose, which represents the backbone of Nod factors, induced neither root-hair deformation nor changes in [Ca2+]i in V. unguiculata. Root hairs and epidermal cells of the nonlegume non-host Arabidopsis thaliana showed neither [Ca2+]i increases nor root-hair deformation in response to both factors.