|KIM, WON-SEOK - University Of Missouri|
|LORIO, JULIO - University Of Missouri|
Submitted to: Nitrogen Fixation International Congress
Publication Type: Abstract Only
Publication Acceptance Date: 6/14/2009
Publication Date: N/A
Technical Abstract: Sinorhizobium fredii USDA257 is a Gram-negative bacterium that forms nitrogen-fixing nodules on diverse legumes, including soybeans. In spite of its broad host-range, USDA257 exhibits remarkable soybean cultivar specificity. USDA257 forms nitrogen-fixing nodules on primitive soybean cultivars such as 'Peking' but fails to nodulate agronomically improved North American cultivars. Our previous studies have demonstrated a key role for Type III secretion system (T3SS) in regulating soybean cultivar specificity of USDA257. T3SS of USDA257, like in few other rhizobia, is encoded by a family of conserved genes involved in the synthesis of the core secretion machinery. Here, we report the characterization of NopA, which we have earlier shown to be the major component of the surface appendages of USDA257. DNA sequence analysis indicated that nopC-nopA-y4yQ-rhcV-y4yS are possibly co-transcribed as an operon and that the promoter region in front of nopC contains a well conserved tts box. Deletion analysis revealed that the tts box is essential for flavonoid induction of nopC and nopA. SDS-PAGE analysis of extracellular proteins of USDA257 induced by flavonoids revealed several Nop proteins including NopX, NopP, NopL, NopB, NopC, and NopA. Western blot analysis utilizing antibodies raised against several Nop proteins revealed that a nonpolar delta nopA::nptII mutant failed to secrete these Nop proteins. RT-PCR analysis revealed the lack of expression of nopA in delta nopA::nptII mutant. Electron microscopy observation revealed that the nopA mutant, in contrast to the wild type strain, was unable to elaborate cell surface appendages. Unlike the parental strain, the delta nopA::nptII mutant was able to form nodules on the soybean cultivar 'McCall'. Examination, using light microscopy, of nodules initiated by USDA257 delta nopA::nptII mutants on the roots of soybean cultivar McCall revealed structural features typical of a determinate nodule. Ultrastructral analysis of the infected cells revealed the presence of numerous bacteroids that were enclosed in symbiosomes. Interestingly, copious starch grains were present in both the infected and uninfected cells.