A nopA deletion mutant of Sinorhizobium fredii USDA257, a soybean symbiont, is impaired in nodulation

Authors: Kim, Wonseok; Krishnan, Hari

Submitted to: Current Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/27/2013
Publication Date: 10/10/2013
Publication URL: http://handle.nal.usda.gov/10113/58763
Citation: Kim, W., Krishnan, H.B. 2013. A nopA deletion mutant of Sinorhizobium fredii USDA257, a soybean symbiont, is impaired in nodulation. Current Microbiology. 68:239-246.

Interpretive Summary: Rhizobia are soil-dwelling bacteria that form nodules on the roots of legume plants. The nodules are specialized structures where atmospheric nitrogen is fixed by the bacterium, which in turn, is utilized by legumes for growth and development. This process is termed biological nitrogen fixation and it enables legumes to grow in nitrogen-poor soils. Sinorhizobium fredii USDA257 forms nodules on primitive soybean cultivars such as “Peking” but fails to induce nodules on agronomically improved cultivars. USDA257 secretes proteins into the rhizosphere and these proteins are believed to play an important role in regulating nodulation on soybean and other legumes. Currently, very little is known about these extracellular proteins. In this study we have characterized NopA, an extracellular protein which is the major component of USDA257 pili. Pili are surface appendages that deliver proteins into soybean roots. We have demonstrated that deletion of nopA drastically lowered the number of nodules formed by USDA257 on soybean cultivars. Information obtained from this basic study will help scientists to better understand the factors that limit the formation of nitrogen-fixing nodules on legumes. Such an understanding should enable scientists to manipulate biological nitrogen fixation so that farmers can increase yields with minimal use of nitrogen fertilizers.

Technical Abstract: Sinorhizobium fredii USDA257 employs type III secretion system (T3SS) to deliver effector proteins into the host cells through filamentous surface appendages, called pili. The NopA protein is the major component of USDA257 pili. The promoter region of USDA257 nopA posses a well conserved tts box. Serial deletion analysis revealed that the tts box is absolutely essential for flavonoid induction of nopA. We have expressed USDA257 NopA in Escherichia coli and raised polyclonal antibodies to the purified recombinant protein. SDS-PAGE analysis of extracellular proteins of a 'nopA mutant induced by flavonoids revealed the complete absence of Nop proteins, including NopX, NopP, NopL, NopB, NopC and NopA. Western blot analysis using antibodies raised against these Nop proteins failed to detect their accumulation both in the extracelluar media and the cell lysates. Interestingly, the nopA mutant was also severely affected in flagellar production. Introduction of a plasmid containing the wild-type nopA fragment into the mutant restored the production of flagellum but not the Nops. Electron microscopy observation revealed that the USDA257 'nopA was compromised in both flagellar and pili production. Deletion of nopA drastically lowered the number of nodules formed by USDA257 on cowpea and soybean cultivar Peking. In contrast to the parental strain, the USDA257 'nopA was able to form few nodules on North American soybean cultivars McCall and Williams 82. Light and transmission electron microscopy examination of these nodules revealed numerous starch grains both in the infected and uninfected cells.