NOPB, A SOYBEAN CULTIVAR-SPECIFICITY PROTEIN FROM SINORHIZOBIUM FREDII USDA257, IS A TYPE III SECRETED PROTEIN

Authors: LORIO, JULIO - UNIVERSITY OF MISSOURI; KIM, WON SEOK - UNIVERSITY OF MISSOURI; Krishnan, Hari

Submitted to: Molecular Plant-Microbe Interactions
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/4/2004
Publication Date: 11/1/2004
Citation: Lorio, J.C., Kim, W., Krishnan, H.B. 2004. Nopb, a soybean cultivar-specificity protein from sinorhizobium fredii usda257, is a type iii secreted protein. Molecular Plant-Microbe Interactions. 17:1259-1268.

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 it comes 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 an 18-kDa protein of S. fredii USDA257 to be exported to soybean rhizosphere by a specialized transport system called by type III secretion system. This extracellular protein interacts with soybean roots to either promote 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: The type III secretion system (TTSS) of plant and animal pathogenic bacteria is involved in translocation of virulence factors into the host cell cytosol where they modulate cellular processes. Sinorhizobium fredii USDA257 is a gram-negative soil bacterium that forms nitrogen-fixing nodules on specific soybean cultivars (Glycine max [L.] Merr.). This microsymbiont is known to secrete at least five nodulation outer proteins (Nops) in response to flavonoid induction. Some of these Nops have been shown to be secreted by TTSS in this symbiotic bacterium. We have isolated and purified an 18-kDa extracellular protein from flavonoid-induced cultures of USDA257. The N-terminal amino acid sequence of this purified protein was identical to the published sequence of the soybean cultivar-specificity protein, NopB (formerly NolB). Inactivation of rhcN, which encodes an ATPase, abolished secretion of NopB. Similarly, a non-polar nopB deletion mutant was compromised in its ability to secrete several Nops. A construct containing the coding region of nopB under control of a T7 promoter was successfully expressed in Escherichia coli and subsequently, the recombinant NopB was purified by nickel-affinity column chromatography. Polyclonal antibodies raised against purified recombinant NopB were used in western blot analysis to demonstrate the association of NopB with pilus-like surface appendages. Deletion analysis indicated that the first 33 N-terminal residues of NopB were necessary and sufficient to mediate the secretion of a green fluorescent reporter protein. Introduction of plasmid-borne extra copies of nopB into USDA257 resulted in lower accumulation of native NopB. Reverse transcriptase-polymerase chain reaction analysis indicated that the observed reduction of native NopB accumulation presumably occurs at a post-transcriptional level. We also show that USDA 257 and its non-polar nopB deletion mutant exhibited discernible differences in their ability to nodulate their legume hosts.