Molecular genetics of Rhizobium Meliloti symbiotic nitrogen fixation

The application of recombinant DNA techniques to the study of symbiotic nitrogen fixation has yielded a growing list of Rhizobium meliloti genes involved in the processes of nodulation, infection thread formation and nitrogenase activity in nodules on the roots of the host plant, Medicago sativa (alfalfa). Interaction with the plant is initiated by genes encoding sensing and motility systems by which the bacteria recognizes and approaches the root. Signal molecules, such as flavonoids, mediate a complex interplay of bacterial and plant nodulation genes leading to entry of the bacteria through a root hair. As the nodule develops, the bacteria proceed inward towards the cortex within infection threads, the formation of which depends on bacterial genes involved in polysaccharide synthesis. Within the cortex, the bacteria enter host cells and differentiate into forms known as bacteroids. Genes which encode and regulate nitrogenase enzyme are expressed in the mature nodule, together with other genes required for import and metabolism of carbon and energy sources offered by the plant.     

Expression profile of maize (Zea mays) scutellar epithelium during imbibition

The scutellum is a shield-shaped structure surrounding the embryo axis in grass species. The scutellar epithelium (Sep) is a monolayer of cells in contact with the endosperm. The Sep plays an important role during seed germination in the secretion of gibberellins and hydrolytic enzymes and in the transport of the hydrolized products to the growing embryo. We identified 30 genes predominantly expressed after imbibition in the Sep as compared to other parts of the scutellum. A high proportion of these genes is involved in metabolic processes. Some other identified genes are involved in the synthesis or modification of cell walls, which may be reflected in the changes of cell shape and cell wall composition that can be observed during imbibition. One of the genes encodes a proteinase that belongs to a proteinase family typical of carnivorous plants. Almost nothing is known about their role in other plants or organs, but the scutellar presence may point to a "digestive" function during germination. Genes involved in the production of energy and the transport of peptides were also identified.     

Homology of Rhizobium meliloti NodC to polysaccharide polymerizing enzymes.

Rhizobium bacteria form nitrogen-fixing nodules on legume roots. As part of the nodulation process, they secrete Nod factors that are beta-1,4-linked oligomers of N-acetylglucosamine. These factors depend on nodulation (nod) genes, but most aspects of factor synthesis are not yet known. We show here that one gene, nodC, shows striking similarity to genes encoding proteins known to be involved in polysaccharide synthesis in yeast and bacteria, specifically chitin and cellulose synthases, as well as a protein with unknown function in Xenopus embryos, DG42. This similarity is consistent with a role for the NodC protein in the formation of the beta-1,4-linkage in Nod factors.     

Induction of thioredoxin is required for nodule development to reduce reactive oxygen species levels in soybean roots

Nodules are formed on legume roots as a result of signaling between symbiotic partners and in response to the activities of numerous genes. We cloned fragments of differentially expressed genes in spot-inoculated soybean (Glycine max) roots. Many of the induced clones were similar to known genes related to oxidative stress, such as thioredoxin and beta-carotene hydroxylase. The deduced amino acid sequences of full-length soybean cDNAs for thioredoxin and beta-carotene hydroxylase were similar to those in other species. In situ RNA hybridization revealed that the thioredoxin gene is expressed on the pericycle of 2-d-old nodules and in the infected cells of mature nodules, suggesting that thioredoxin is involved in nodule development. The thioredoxin promoter was found to contain a sequence resembling an antioxidant responsive element. When a thioredoxin mutant of yeast was transformed with the soybean thioredoxin gene it became hydrogen peroxide tolerant. These observations prompted us to measure reactive oxygen species levels. These were decreased by 3- to 5-fold in 7-d-old and 27-d-old nodules, coincident with increases in the expression of thioredoxin and beta-carotene hydroxylase genes. Hydrogen peroxide-producing regions identified with cerium chloride were found in uninoculated roots and 2-d-old nodules, but not in 7-d-old and 27-d-old nodules. RNA interference-mediated repression of the thioredoxin gene severely impaired nodule development. These data indicate that antioxidants such as thioredoxin are essential to lower reactive oxygen species levels during nodule development.     

Role of trehalose transport and utilization in Sinorhizobium meliloti--alfalfa interactions

Genes thuA and thuB in Sinorhizobium meliloti Rm1021 code for a major pathway for trehalose catabolism and are induced by trehalose but not by related structurally similar disaccharides like sucrose or maltose. S. meliloti strains mutated in either of these two genes were severely impaired in their ability to grow on trehalose as the sole source of carbon. ThuA and ThuB show no homology to any known enzymes in trehalose utilization. ThuA has similarity to proteins of unknown function in Mesorhizobium loti, Agrobacterium tumefaciens, and Brucella melitensis, and ThuB possesses homology to dehydrogenases containing the consensus motif AGKHVXCEKP. thuAB genes are expressed in bacteria growing on the root surface and in the infection threads but not in the symbiotic zone of the nodules. Even though thuA and thuB mutants were impaired in competitive colonization of Medicago sativa roots, these strains were more competitive than the wild-type Rml021 in infecting alfalfa roots and forming nitrogen-fixing nodules. Possible reasons for their increased competitiveness are discussed.