Immunogold localization studies of two nodulation gene products of Rhizobium meliloti

Monospecific, polyclonal antibodies to the nodA and nodC gene products of R. meliloti were used in combination with immunogold labeling and transmission electron microscopy to localize the NodA and Node proteins in cultures of R. meliloti and in mature nodule tissue of alfalfa and soybean plants. Both NodC and NodA were detected in the cytoplasm and cell envelope in thin sections of free-living rhizobia treated with luteolin, a known inducer of nod gene expression. Only NodC was detected on the surfaces of cells when immunolabeling was performed with intact induced cells. In view of biochemical data characterizing Node as an outer membrane protein, the pattern of immunolabeling on thin sections suggests that NodC is produced on free cytoplasmic ribosomes prior to assembly in the membrane. NodA and NodC were both detected in infection threads of mature alfalfa nodules. In similar experiments with nodule tissue from soybean, these proteins were localized in bacteroids; however, quantitation of labeling showed that peroxisomes in uninfected cells were the most heavily labeled structures in thin sections probed for NodC. Evidence was also presented to demonstrate detection of NodA in peroxisomes of soybean nodules.