Submitted to: Molecular Plant-Microbe Interactions
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/26/2003
Publication Date: 7/1/2003
Citation: KRISHNAN, H.B., LORIO, J., KIM, W.S., JIANG, G., KIM, K.Y., DEBOER, M., PUEPPKE, S.G. EXTRACELLULAR PROTEINS INVOLVED IN SOYBEAN CULTIVAR-SPECIFIC NODULATION ARE ASSOCIATED WITH PILUS-LIKE SURFACE APPENDANGES AND EXPORTED BY A TYPE III PROTEIN SECRETION SYSTEM IN SINORHIZOBIUM FREDII USDA257. MOLECULAR PLANT-MICROBE INTERACTIONS. 2003. v. 16. p. 617-625. Interpretive Summary: Sinorhizobium fredii USDA257, a soil-bacterium, forms nodules on the roots of primitive soybean plants. The nodules are specialized structures where atmospheric nitrogen is fixed by the bacterium, which in turn, is utilized by soybean plants for growth and development. This process is termed biological nitrogen fixation and it enables soybean plants to grow in nitrogen-poor soils. Sinorhizobium fredii USDA257 secretes proteins into the rhizosphere when they come into contact with soybean root exudates. Some of these proteins are involved in regulating nodulation on soybean plants. Currently, very little is known about how these proteins are exported to the rhizosphere. Such information is required for designing strategies targeted toward improving biological nitrogen fixation. We have identified and sequenced a 31.2 kb DNA region of S. fredii USDA257 that contains a specialized protein secretion system. Sinorhizobium fredii USDA257 uses this transport system to secrete proteins into the rhizosphere. Some of these proteins either promote nodulation or reduce nodulation in host-dependent manner. Information obtained from this basic study will help to better understand the factors that limit the formation of nitrogen-fixing nodules on North American soybean cultivars. Such an understanding should enable scientists to manipulate biological nitrogen fixation so that farmers can increase the soybean yields with minimal use of nitrogen fertilizers.
Technical Abstract: Several Gram-negative plant and animal pathogenic bacteria have evolved a type III secretion system (TTSS) to deliver effector proteins directly into the host cell cytosol. Sinorhizobium fredii USDA257, a symbiont of soybean and many other legumes, secretes signal-responsive proteins (SR proteins) into the extracellular environment upon flavonoid induction. Mutation analysis and the nucleotide sequence of a 31.2 kb symbiosis (sym) plasmid DNA region of USDA257 revealed the existence of a TTSS locus in this symbiotic bacterium. This locus includes rhc (rhizobia conserved) genes that encode components of a TTSS and proteins that are secreted into the environment. The genomic organization of the TTSS locus of USDA257 is remarkably similar to that of another broad host range symbiont, Rhizobium sp. NGR234. We demonstrate that the secretion of NolX, SR-3, and SR-5 is controlled by the TTSS. Flavonoids that activate the transcription of the nod genes of USDA257 also stimulate the production of novel filamentous appendages known as pili. Electron microscope examination of purified pili reveals needle-like filaments of 6-8 nm in diameter. The production of the pili is completely dependent on a functional nodD1 gene and the presence of a nod-gene-inducing compound. Mutations in several of the TTSS genes negate the ability of USDA257 to elaborate pili. Western blot analysis using antibodies raised against purified NolX, SR-3, and SR-5 reveals that these proteins were intimately associated with the pili. Immunocytochemical localization studies also demonstrate that NolX and SR-3 are localized along the entire length of the pili. Polar mutations in rhcN, rhcJ, rhcC1, and y4xI alter the ability of USDA257 to form nodules on Glycine max and Macroptilium atropurpureum.