A critical evaluation of differential display as a tool to identify genes involved in legume nodulation: looking back and looking forward.

Screening for differentially expressed genes is a straightforward approach to study the molecular basis of a biological system. In the last 10 years, differential screening technology has evolved rapidly and currently high-throughput tools for genome-wide transcript profiling, such as expressed sequence tags and microarray analysis, are becoming widely available. Here, an overview of this (r)evolution is given with emphasis on the differential display method, which for many years has been the preferred technique of scientists in diverse fields of research. Differential display has also been the method of choice for the identification of genes involved in the symbiotic interaction between Azorhizobium caulinodans and Sesbania rostrata. The advantages with respect to tissue specificity of this particular model system for legume nodulation and the results of a screening for early nodulation-related genes have been considered in the context of transcriptome analyses in other rhizobium-legume interactions.

Expression of cell cycle genes during Sesbania rostrata stem nodule development.

Upon infection of Sesbania rostrata with Azorhizobium caulinodans, nodules are formed on roots and stems. Stem nodules develop from abundantly distributed dormant root primordia. To acquire more insight into the meristem organization during stem nodule development, the expression patterns of a mitotic B1-type cyclin gene (Sesro; CycB1;1), a cyclin-dependent kinase gene (Cdc-2-1Sr), and a histone H4 gene (H4-1Sr) of S. rostrata were followed by in situ hybridization. Cdc2-1Sr transcripts were found in all cells of uninfected and infected root primordia. In uninfected root primordia, Sesro;CycB1;1 transcripts were detected in a few cells of the apical root meristem whereas H4-1Sr transcripts were abundant in this region. Interestingly, after inoculation with A. caulinodans, H4-1Sr transcripts disappeared in the root meristem and a patchy pattern of Sesro;CycB1;1 and H4-1Sr expression appeared in the cortex of the root primordium, reflecting the formation of globular nodule primordia. When bacterial invasion started, a distal nodule meristem was delimited wherein Sesro;CycB1;1 and H4-1Sr expression was concentrated. Approximately 1 week after inoculation, meristem activity ceased, indicated by the loss of Sesro;CycB1;1 and H4-1Sr expression.

Use of differential display to identify novel Sesbania rostrata genes enhanced by Azorhizobium caulinodans infection.

Upon infection of the tropical legume Sesbania rostrata with Azorhizobium caulinodans ORS571, nodules are formed on the roots as well as on the stems. Stem nodules appear at multiple predetermined sites consisting of dormant root primordia, which are positioned in vertical rows along the stem of the plant. We used the differential display method to isolate and characterize three cDNA clones (differential display; didi-2, didi-13, and didi-20), corresponding to genes whose expression is enhanced in the dormant root primordia after inoculation. Database searches revealed that the deduced (partial) didi-2 gene product shares significant similarity with hydroxyproline-rich cell wall proteins. The (partial) didi-13 and didi-20 products are similar to chitinases and chalcone reductases, respectively. Transcripts corresponding to the cDNA clones didi-2 and didi-13 were first detectable 1 day after inoculation. In contrast, didi-20 transcripts were found at low levels in uninfected root primordia and were enhanced significantly around 3 days after inoculation. In addition, a cDNA was isolated (didi-42) that corresponds to the previously identified leghemoglobin gene Srlb6. These studies show that differential display is a useful method for the isolation of infection-related genes.

Nodule development on the tropical legume Sesbania virgata under flooded and non-flooded conditions.

The interaction between the Brazilian pioneer legume Sesbania virgata and its microsymbiont Azorhizobium doebereinerae leads to the formation of nitrogen-fixing nodules on roots that grow either in well-aerated soils or in wetlands. We studied the initiation and development of nodules under these alternative conditions. To this end, light and fluorescence microscopy were used to follow the bacterial colonisation and invasion into the host and, by means of transmission electron microscopy, we could observe the intracellular entry. Under hydroponic conditions, intercellular invasion took place at lateral root bases and mature nodules were round and determinate. However, on roots grown in vermiculite that allows aerated growth, bacteria also entered via root hair invasion and nodules were both of the determinate and indeterminate type. Such versatility in entry and developmental plasticity, as previously described in Sesbania rostrata, enables efficient nodulation in both dry and wet environments and are an important adaptive feature of this group of semi-tropical plants that grow in temporarily flooded habitats.

Patterns of ENOD40 gene expression in stem-borne nodules of Sesbania rostrata.

At the base of adventitious root primordia, located on the stem of the tropical legume Sesbania rostrata, nitrogen-fixing nodules are formed upon inoculation with the microsymbiont Azorhizobium caulinodans. This pattern of nodule development presents features of indeterminate and determinate nodules in early and later stages, respectively. A S. rostrata cDNA clone homologous to early nodulin ENOD40 genes was isolated from a cDNA library of developing stem nodules. SrENOD40-1 contained the conserved regions I and II of other ENOD40 genes. By reverse transcriptase PCR, enhanced SrENOD40-1 expression was observed in the adventitious root primordia between 4 and 8 h after inoculation with A. caulinodans. In situ hybridization showed that SrENOD40-1 transcripts, present around the central vascular bundle of the uninfected root primordia, were strongly enhanced upon induction of nodule development. De novo SrENOD40-1 expression was observed in the initiating and growing nodule primordia and around vascular bundles. When cell type specification sets in, the expression became pronounced in cells derived from the meristematic regions. In other parts of the plant, weak SrENOD40-1 expression was associated with vascular bundles and was observed in leaf and stipule primordia.

Srchi13, a novel early nodulin from Sesbania rostrata, is related to acidic class III chitinases.

On the tropical legume Sesbania rostrata, stem-borne nodules develop after inoculation of adventitious root primordia with the microsymbiont Azorhizobium caulinodans. A cDNA clone, Srchi13, with homology to acidic class III chitinase genes, corresponds to an early nodulin gene with transiently induced expression during nodule ontogeny. Srchi13 transcripts accumulated strongly 2 days after inoculation, decreased from day 7 onward, and disappeared in mature nodules. Induction was dependent on Nod factor-producing bacteria. Srchi13 was expressed around infection pockets, in infection centra, around the developing nodule and its vascular bundles, and in uninfected cells of the central tissue. The specific and transient transcript accumulation together with the lipochitooligosaccharide degradation activity of the recombinant protein hint at a role of Srchi13 in normal nodule ontogeny by limiting the action of Nod factors.

Ethylene-mediated phenotypic plasticity in root nodule development on Sesbania rostrata.

Leguminous plants in symbiosis with rhizobia form either indeterminate nodules with a persistent meristem or determinate nodules with a transient meristematic region. Sesbania rostrata was thought to possess determinate stem and root nodules. However, the nature of nodule development is hybrid, and the early stages resemble those of indeterminate nodules. Here we show that, depending on the environmental conditions, mature root nodules can be of the indeterminate type. In situ hybridizations with molecular markers for plant cell division, as well as the patterns of bacterial nod and nif gene expression, confirmed the indeterminate nature of 30-day-old functional root nodules. Experimental data provide evidence that the switch in nodule type is mediated by the plant hormone ethylene.

Chalcone reductase-homologous transcripts accumulate during development of stem-borne nodules on the tropical legume Sesbania rostrata.

During a search for genes with induced or enhanced expression in the early stages of development of stem-borne nodules on Sesbania rostrata, a cDNA with homology to chalcone reductase (CHR) genes was isolated. Here, we describe the characterization of a full-length CHR cDNA (Srchr1) and the pattern of CHR transcript accumulation in stem-borne nodules. Expression was correlated with both nodule development and bacterial invasion. In young nodules, CHR transcripts were observed in cells of the parenchyma, in cells around the nodule vascular bundles, and in the uninfected cells of the central tissue.

Identification of nodSUIJ genes in Nod locus 1 of Azorhizobium caulinodans: evidence that nodS encodes a methyltransferase involved in Nod factor modification.

The Azorhizobium caulinodans strain ORS571 nodulation genes nodSUIJ were located downstream from nodABC. Complementation data and transcriptional analysis suggest that nodABCSUIJ form a single operon. Mutants with Tn5 insertions in the genes nodS, nodU, and nodJ were delayed in nodulation of Sesbania rostrata roots and stems. The NodS amino acid sequences of ORS571, Bradyrhizobium japonicum, and Rhizobium sp. strain NGR234, contain a consensus with similarity to S-adenosylmethionine (SAM)-utilizing methyltransferases. A naringenin-inducible nodS-dependent protein of approximately 25 kDa could be cross-linked to radiolabelled SAM. By applying L-[methyl-3H]-methionine in vivo, Nod factors of ORS571, known to be N-methylated, could be labelled in wild type and nodU mutants but not in nodS mutants. Therefore, we propose that NodS is a SAM-utilizing methyltransferase involved in Nod factor synthesis.

Srchi24, a chitinase homolog lacking an essential glutamic acid residue for hydrolytic activity, is induced during nodule development on Sesbania rostrata.

The interaction between the tropical legume Sesbania rostrata and the bacterium Azorhizobium caulinodans results in the formation of nodules on both stem and roots. Stem nodulation was used as a model system to isolate early markers by differential display. One of them, Srchi24 is a novel early nodulin whose transcript level increased already 4 h after inoculation. This enhancement depended on Nod factor-producing bacteria. Srchi24 transcript levels were induced also by exogenous cytokinins. In situ hybridization and immunolocalization experiments showed that Srchi24 transcripts and proteins were present in the outermost cortical cell layers of the developing nodules. Sequence analyses revealed that Srchi24 is similar to class III chitinases, but lacks an important catalytic glutamate residue. A fusion between a maltose-binding protein and Srchi24 had no detectable hydrolytic activity. A function in nodulation is proposed for the Srchi24 protein.