We would like to acknowledge the Max Planck Society and the Alexander von Humboldt Foundation for generous support.

Section 4: Signal Transduction



School of Molecular Biosciences, Institute of Biological Chemistry Washington State University, Pullman, WA 99164-6340, USA

Nitrogen fixation that occurs in the context of a symbiotic relationship requires a multilevel exchange of signals between the two symbionts to elicit an appropriate developmental response. Establishing how this signal exchange takes place has been difficult, despite the fact that many clues have been available from the host-specific nature of the interaction. Landmarks along the way have included: (1) recognition that there was a correlation between surface antigens on the bacteria and lectins present in the host (Dazzo, Hubbell 1975); (2) realization that host defenses were activated even during successful nodulation (Bhuvaneswari et al. 1980); (3) that the defense response was systemic (Kosslak, Bohlool 1984); and (4) that it could be eliminated by mutation (Olsson et al.

1989); (5) identification of bacterial genes necessary for nodulation (Long et al. 1982); (6) that expression of these genes was induced by specific small molecules produced by the host (Peters et al. 1986); and (7) characterization of the Nod factor signals produced by these genes (Lerouge et al.

1990) and an appreciation that there were both specific and non-specific effects of applying these Nod factors to roots.

It has been over ten years since the first Nod factor was isolated and shown to initiate nodule formation on specific hosts in a way that correlated with the Nod factor's structure. Because Nod factors are diverse compounds based on modifying a lipochito-oligosaccharide backbone and are active at low concentrations they were hailed as a new type of plant signal molecule that behaved more like some of the animal hormones than did the recognized plant hormones, such as an auxin or cytokinin. The discovery of Nod factors implied that there should be a Nod factor receptor, which would presumably start a signal cascade leading to the cell division and other changes in root development characteristic of nodule formation. Finding this receptor has been very hard work. Nod factors have hydrophobic and hydrophilic domains and, like the classic detergents, they stick to many cellular components. The target tissue where they specifically act is not abundant and the observation that Nod factors will stimulate responses in non-host plant cells calls almost any assay other than nodulation into question. The report at this meeting by M. Etzler following on earlier work by her group (Etzler et al. 1999) strengthens the case that a lectin nucleotide phosphohydrolase (LNP) is a candidate for a Nod factor receptor. Information presented at the meeting showed that when substrates known to bind LNP are added to roots, they induce root deformation, that expression of LNP in Arabidopsis makes the transgenic roots sensitive to root hair curling by substrate or bacteria and that plants with antisense constructs of LNP do not show normal root hair curling. However, the affinity of isolated LNP for Nod factor is relatively low, suggesting that there is more to Nod factor binding than is currently appreciated.

LNP is not the only plant protein that binds Nod factor and it may be that there are several levels at which Nod factor acts. This was certainly suggested by results from A. Downie's group, who showed that some early responses to Nod factors do not require the fully decorated molecule and that the nodO protein, which is in no way related to Nod factor synthesis, is able to potentiate the activity of an incompletely decorated Nod factor. This suggests that there may be more than one pathway to some of Nod factor's effects. In that light, G. Stacey's description of the complex pattern of regulation of the Bradyrhizobium nodulation genes reinforces the idea that communication between bacteria and host can be subtle and is probably sensitive to many inputs, perhaps including some that we are not aware of yet.

One possible gene in the plant signal cascade was identified by G. Kiss and his group who identified the lesion in a classical alfalfa non-nodulating mutant, MN-108, as a change in the sequence of a potential receptor kinase gene. The approach taken was a technical tour de force of map-based cloning and sequencing which was capped by the finding that a similar lesion was present in a pea mutant also defective in nodulation. In addition to linking a point mutation to a function involved in nodule formation, the ability to cross species boundaries for confirmation gives hope that mutants in legume species with difficult molecular genetics will be able to contribute to the overall picture. While the evidence is fairly strong that a change in this receptor kinase is important in the mutant phenotype, the observation by Karlowski and Hirsch that expression of a zinc-finger binding protein is also altered in the mutant is intriguing. Changes in expression of this protein may also play a role in disconnecting the Nod signal from nodulation. Taken together with the isolation of a transposon-tagged Lotus japonicus nodulation gene (Schauser el al. 1999), the field appears to be moving a step at a time into the difficult developmental territory between infection and fixation.

At the other end of the signal cascade, various groups are examining the changes that occur in the plant cells within nodules. In particular, proteomics, EST analysis and mRNA profiling approaches are identifying large numbers of changes in expression that need to be integrated with models of how these changes relate to alterations in the cell cycle of nodule cells. Some changes are likely to result from blocking cell division, and would occur in any tissue where division was blocked but others are potentially nodule-specific. Working back from the mature nodules to see how the cells developed is potentially a very powerful approach to understanding nodule organogenesis. In these investigations, the ability to generate transgenic plants that have altered expression of particular proteins is turning into a powerful technique, though it is necessary to examine closely other potential phenotypes of the transgenics that might alter nodule maturation indirectly.

These advances, together with advances in mutant isolation in the two model legume species, Lotus japonicus and Medicago truncatula, indicate that a description of the plant response pathway downstream of Nod factor activation is on the verge of becoming a reality. With strong candidate genes in hand, techniques like two-hybrid analysis may be able to identify additional factors that are connected to proteins in the signal cascades. As patterns of gene expression in the pathway become more established, the genomic tools presented at the meeting will come to play a larger and larger role in discovering novel proteins that also fit the patterns.

While the last 20 years could be characterized as gene-by-gene discovery of elements involved in the legume-rhizobial conversation, we can expect that the future will be more concerned with assembling the flood of genetic information into a coherent picture of relationships. There is a strong temptation to view transduction as a linear device for attaching a signal to a response since it is often a single assay that establishes new connections. However, there are already many distinct mutants that affect nodulation, suggesting that there are many connections to be made. Signals are often transduced through a series of proteins as a way of bringing several inputs into the ultimate decision process. So, for example, legumes growing in sufficient nitrogen do not respond to bacteria in the same way as when they are nitrogen-stressed and bacteria arriving late at the root hair will not find the same welcoming reception as those that arrived earlier. How Nod factor signal transduction is connected to the perception of other signals, like nitrogen or infection status, are only two of the many questions that will hopefully be answered in the years ahead.


Bhuvaneswari TV, Turgeon BG, Bauer WD (1980) Plant Physiol. 66, 1027-1031 Dazzo FB, Hubbell DH (1975) Appl. Microbiol. 30, 1017-33

Etzler ME, Kalsi G, Ewing NN, Roberts NJ, Day RB, Murphy JB (1999) Proc. Natl. Acad. Sei.

USA 96, 5856-61 Kosslak RM, Bohlool BB (1984) Plant Physiol. 75, 125-130

Lerouge P, Roche P, Faucher C, Maillet F, Truchet G, Prome J, Denarie J (1990) Nature 344, 781-4

Long SR, Buikema WE, Ausubel FM (1982) Nature 298, 485-488

Olsson JE, Nakao P, Bohlool BB, Gresshoff PM (1989) Plant Physiol. 90, 1347-1352

Peters NK, Frost JW, Long SR (1986) Science 233, 977-80

Schauser L, Roussis A, Stiller J, Stougaard J (1999) Nature 402, 191-195

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