NolX of Sinorhizobium fredii USDA257, a Type III-Secreted Protein Involved in Host Range Determination, Is Localized in the Infection Threads of Cowpea (Vigna unguiculata [L.] Walp) and Soybean (Glycine max [L.] Merr.) Nodules

Sinorhizobium fredii USDA257 forms nitrogen-fixing nodules on soybean (Glycine max [L.] Merr.) in a cultivar-specific manner. This strain forms nodules on primitive soybean cultivars but fails to nodulate agronomically improved North American cultivars. Soybean cultivar specificity is regulated by the nolXWBTUV locus, which encodes part of a type III secretion system (TTSS). NolX, a soybean cultivar specificity protein, is secreted by TTSS and shows homology to HrpF of the plant pathogen Xanthomonas campestris pv. vesicatoria. It is not known whether NolX functions at the bacterium-plant interface or acts inside the host cell. Antibodies raised against S. fredii USDA257 NolX were used in immunocytochemical studies to investigate the subcellular localization of this protein. Immunostaining of paraffin-embedded sections of developing soybean and cowpea (Vigna unguiculata [L.] Walp) nodules revealed localization of NolX in the infection threads. Protein A-gold immunocytochemical localization studies utilizing affinity-purified NolX antibodies revealed specific deposition of gold particles in the fibrillar material inside infection threads. Similar immunogold localization studies failed to detect NolX in thin sections of mature soybean and cowpea nodules. The results from this study indicate that NolX is expressed in planta only during the early stages of nodule development.