FIG 2.

Experimental design and execution of tobacco NifB libraries. Two libraries of 30 ts-nifB genes with targeting sequences for chloroplasts or mitochondria were constructed and cloned in binary plasmids. The TS-NifB proteins were transiently expressed in A. tumefaciens-infiltrated tobacco leaves together with NifU, NifS, FdxN, GFP, and p19. NifB expression (i.e., NifB present in total protein extracts) and solubility (i.e., NifB present in soluble fraction of protein extracts) were determined by immunoblot analysis of the protein extracted from the homogenized leaf tissue. The genes encoding soluble NifB variants (for mitochondria and chloroplasts, respectively) were assembled with nifU, nifS, and fdxN in multigenic vectors. Large-scale A. tumefaciens infiltrations were performed to obtain sufficient leaf material for NifB isolation by STAC.