Visualization of nodulation gene activity on the early stages of Rhizobium leguminosarum bv. viciae symbiosis

A technique was optimized for thein situ detection of nodulation (nod) gene activity inRhizobium leguminosarum bv.viciae symbiosis with compatible plant hostsVicia tetrasperma (L.)Schreb. andPisum sativum L. The transcription ofnodABC-lacZ fusion was visualized as β-galactosidase (β-Gal) activity after reaction with the chromogenic substrate X-Gal and subsequent light microscopy, while the back-ground of the indigenous β-Gal activity of rhizobia and the host plant was eliminated by glutaraldehyde treatment.V. tetrasperma was suggested as a suitable model plant for pea cross-inoculation group due to its advantages over the common model ofV. hirsuta (L.) S.F.Gray: compactness of the plant, extremely small seeds, fast development and stable nodulation under laboratory conditions. In the roots of both plants, a certain extent ofnod gene activity was detectable in all rhizobia colonizing the rhizoplane. In pea 1 d after inoculation (d.a.i.), the maximum was localized in the region of emerging root hairs (RH) later (3 and 6 d.a.i.) shifting upwards from the root tip. Nodulation genes sustained full expression even in the infection threads inside the RH and the root cortex, independently of their association with nodule primordia. Comparison of two pea symbiotic mutant lines, Risnod25 and Risnod27, with the wild type did not reveal any differences in the RH formation, RH curling response and rhizoplane colonization. Both mutants appeared to be blocked at the infection thread initiation stage and in nodule initiation, consistent with the phenotype caused by other mutant alleles in the peasym8 locus. Judging from thenod gene expression level and pattern in the rhizoplane, flavonoid response upon inoculation is preserved in both pea mutants, being independent of infection thread and nodule initiation.