Michael John

Organization, structure and symbiotic function of Rhizobium meliloti nodulation genes determining host specificity for alfalfa.

In R. meliloti we have identified four nodulation genes determining plant host-range specificity and have designated them hsnABC and D. The genes code for 9.7, 41.7, 26.7, and 28.6 kd proteins, respectively, and are organized into two transcriptional units. Mutations in these genes affect nodulation of their natural plant hosts Medicago sativa and Melilotus albus to different extents and hsnD mutants have an altered host-range. These Nod- mutations are not complementable by nodulation genes of other Rhizobium species such as R. leguminosarum. The hsn genes determine plant-specific infection through root hairs: hsnD is required for host-specific root hair curling and nodule initiation while the hsnABC genes control infection thread growth from the root hairs.

Soybean ENOD40 encodes two peptides that bind to sucrose synthase

ENOD40 is expressed at an early stage in root nodule organogenesis in legumes. Identification of ENOD40 homologs in nonleguminous plants suggests that this gene may have a more general biological function. In vitro translation of soybean ENOD40 mRNA in wheat germ extracts revealed that the conserved nucleotide sequence at the 5′ end (region I) encodes two peptides of 12 and 24 aa residues (peptides A and B). These peptides are synthesized de novo from very short, overlapping ORFs. Appropriate ORFs are present in all legume ENOD40s studied thus far. In this case small peptides are directly translated from polycistronic eukaryotic mRNA. The 24-aa peptide B was detected in nodules by Western blotting. Both peptides specifically bind to the same 93-kDa protein, which was affinity purified from soybean nodules. Using peptide mass fingerprinting, we identified this binding protein as nodulin 100, which is a subunit of sucrose synthase. Based on our data we suggest that ENOD40 peptides are involved in the control of sucrose use in nitrogen-fixing nodules.

Cell signalling by oligosaccharides

Only recently has it begun to be understood how plants use the structural diversity of oligosaccharides to regulate important cellular processes such as growth, development and defence. A characteristic feature of these regulatory molecules is that they are biologically active at extremely low concentrations. A central remaining question is how cells perceive and transduce oligosaccharide signals, and current research is aimed at providing the answer.

Growth of Tobacco Protoplasts Stimulated by Synthetic Lipo-Chitooligosaccharides

Nodulation (Nod) factors are lipo-chitooligosaccharides (LCOs) secreted by rhizobia to trigger the early steps of nodule organogenesis in leguminous plants. A method to synthesize LCOs in vitro was developed. Synthetic LCOs alleviated the requirement for auxin and cytokinin to sustain growth of cultured tobacco protoplasts. LCOs containing C18:1 trans—fatty acyl substituents were more effective than those containing cis—fatty acids in promoting cell division as well as in activating an auxin-responsive promoter and the expression of a gene implicated in auxin action. These data indicate that LCOs redirect plant growth also in nonlegumes by activating developmental pathways also targeted by phytohormones.

STIMULATION OF INDOLE-3-ACETIC ACID PRODUCTION IN RHIZOBIUM BY FLAVONOIDS

Flavonoids activate nod gene expression in Rhizobium resulting in the synthesis of Nod signals which trigger organogenesis in the host plant. This paper shows that nod‐inducers also stimulate the production of the phytohormone IAA (indole‐3‐acetic acid).

The Role of Nodulation Genes in Bacterium-Plant Communication

Bacteria belonging to the genera Rhizobimn, Bradyrhizobium and Azorhizobium have the ability to establish nitrogen-fixing symbiosis with leguminous plants. The development of symbiosis takes place in several stages, determined by genes in both partners. A particular rhizobial partner induces root or/and stem nodules on the appropriate plant hosts. The first observable signs of interaction are the curling of root hairs and induction of meristematic cell division leading to nodule development. Then, the bacteria infect the root hairs and within a cellulose tube, the infection thread, they are transported into the developing nodule tissue. Many of the nodule cells are invaded by the nitrogen-fixing form of rhizobia, the bacteroids. Further nodule development culminates in symbiotic nitrogen fixation. The produced ammonia is then utilized by the plant as the major nitrogen source (1,2).

Identification And Organization Of Rhizobium Meliloti Genes Relevant To The Initiation And Development Of Nodules

The bacterium species Rhizobium meliloti induces nitrogen fixing nodules on the roots of its host plant Medicago sativa (alfalfa). The majority of symbiotic genes of R. meliloti are located on a megaplasmid, including genes coding for early functions in nodulation (nod), the nitrogenase genes (nif) and other genes required for nitrogen fixation (fix) (Banfalvi et al. 1981; Rosenberg et al. 1981). When this megaplasmid was transferred into other Rhizobium species or into Agrobacterium tumefaciens, these latter bacteria became able to induce ineffective nodules on alfalfa, indicating that the essential genes coding for nodule initiation and development are carried by this megaplasmid (Kondorosi et al. 1982). The development of these nodules, however, halted at an early stage: infection threads did not form and neither bacteria nor bacteroids were found in the inner nodule tissue (Wong et al. 1983).

Common and Host Specific Nodulation Genes in Rhizobium Meliloti and Their Conservation in Other Rhizobia

Recognition of the appropiate legume host and nodule induction are controlled by two sets of Rhizobium genes, common nodulation (nod) and host-specific nodulation (hsn) genes. These genes have been identified in several Rhizobium species, including R.meliloti, the symbiotic partner of alfalfa (Medicago). Here we present our studies on the organization and regulation of R.meliloti nodulation genes. Moreover, these genes were used to identify and analyse genes of similar function in other rhizobia which may help us to elucidate the basis of host-specificity of Rhizobium-legume interaction and the genetic control of these processes.

RECENT PROGRESS IN THE ELUCIDATION OF THE FUNCTION OF nodA, B, AND C GENES OF RHIZOBIUM MELILOTI

ABSTRACT The common nodulation genes nodA, B, and C are highly conserved between different Rhizobium species and are required for the nodulation of legumes and non-legumes. The expression of these genes in Rhizobium meliloti is under both positive and negative control. Monospecific polyclonal antibodies were used to localize the NodA and NodB proteins in the cytosol of R. meliloti. These proteins are involved in the generation of small heat-stable compounds that stimulate the mitosis of different plant protoplasts. Our experiments suggest that the NodC transmembrane protein is not involved in the synthesis of these factors. Gene fusion experiments were used to define the membrane-anchor domain which is necessary for the insertion of the NodC protein into the membrane. A highly hydrophobic transmembrane-anchor domain was found near the carboxyl terminus, separating a large extracellular domain which contains a cystein-rich cluster from a short putative intracellular domain. The domain structure of the dime...

Lipo-Chitooligosaccharides (LCO’s) as Growth Hormones

Lipo-chitooligosaccharides (LCO’s) have been known for some time as the Nod factors that are released by nitrogen-fixing Rhizobia and are essential for their capacity to induce nodules on leguminous plants. The structure of these LCO’s was solved by Lerouge in 1990 (P. Lerouge et al., 1990). The question we want to address is whether LCO’s are specific signals evolved by Rhizobia to induce nodulation or whether LCO’s are playing a more general role in plant development and have been adapted by Rhizobia to play a crucial role in nitrogen fixation by being essential for nodule formation.