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To elucidate the function of the ndx homeobox genes during the Rhizobium-legume symbiosis, two Lotus japonicus ndr genes were expressed in the antisense orientation under the control of the nodule-expressed promoter Psenod12 in transgenic Lotus japonicus plants. Many of the transformants obtained segregated into plants that failed to sustain proper development and maintenance of root nodules concomitant with down-regulation of the two ndx genes. The root nodules were actively fixing nitrogen 3 weeks after inoculation, but the plants exhibited a stunted growth phenotype. The nodules on such antisense plants had under-developed vasculature and lenticels when grown on medium lacking nitrogen sources. These nodules furthermore entered senescence earlier than the wild-type nodules. Normal plant growth was resumed upon external addition of nitrogen. This suggests that assimilated nitrogen is not properly supplied to the plants in which the two ndx genes are down-regulated. The results presented here, indicate that the ndx genes play a role in the development of structural nodule features, required for proper gas diffusion into the nodule and/or transport of the assimilated nitrogen to the plant.

A DNA-binding protein, VsENBP1, previously isolated from Vicia sativa was shown to bind in a sequence-specific manner to the early nodulin ENOD12 gene promoter from Pisum sativum. Here, the functional importance of the VsENBP1 binding sites on the PsENOD12B promoter has been studied in vivo. A promoter-gusA fusion in which a mutation was introduced at the putative target sequence, AATAA, was inactive in nodules of transgenic Vicia hirsuta roots. Gel retardation assays showed that VsENBP1 does not bind to the mutated promoter segment, suggesting that VsENBP1 activates the PsENOD12B expression in nodules through its interaction with its target sequence. In the presence of the 35S enhancer, an ENOD12 promoter-GUS construct gave expression in root vascular tissue in addition to the root nodules. Overexpression of Vsenbp1 in transgenic V. hirsuta roots reduced the leaky expression in root vascular tissue in contrast to nodules in which a small increase in GUS expression was observed. The results indicate that VsENBP1 acts as a repressor of ENOD12 expression in root tissue.

A cDNA containing a homeobox sequence was isolated from a soybean nodule-specific expression library. This homeobox cDNA, Ndx (nodulin homeobox), represents a small gene family with at least two members in soybean (Glycine max) and three in Lotus japonicus. One complete 3304 bp Ndx cDNA from L. japonicus encodes a protein, NDX, of 958 amino acids. An unusual type of homeodomain that differs in two of the most conserved amino acid positions in the consensus sequence is located close to the C-terminal and appears to be the only DNA-binding domain. Weak Ndx gene expression in the root increases very shortly after infection with Rhizobium and remains throughout nodule development. In situ hybridizations show cell-specific expression patterns that suggest developmentally separate regions in maturing determinate nodules. Thus in the maturing nodule Ndx and leghemoglobin genes are expressed in a mutually exclusive fashion. The Ndx transcript is also detectable in the young nodule primordium. Ndx expression is not confined to the root nodule since Ndx is also expressed in shoot and root meristems, indicating that the Ndx gene products might also be involved in developmental processes in other plant tissues.

The pea genes PsENOD12A and PsENOD12B are expressed in the root hairs shortly after infection with the nitrogen-fixing bacterium Rhizobium leguminosarum bv. viciae or after application of purified Nod factors. A 199 bp promoter fragment of the PsENOD12B gene contains sufficient information for Nod factor-induced tissue-specific expression. We have isolated a Vicia sativa cDNA encoding a 1641 amino acid protein, ENBP1, that interacts with the 199 bp ENOD12 promoter. Two different DNA-binding domains were identified in ENBP1. A domain containing six AT-hooks interacts specifically with an AT-rich sequence located between positions -95 and -77 in the PsENOD12B promoter. A second domain in ENBP1 is a cysteine-rich region that binds to the ENOD12 promoter in a sequence non-specific but metal-dependent way. ENBP1 is expressed in the same cell types as ENOD12. However, additional expression is observed in the nodule parenchyma and meristem. The presence of three small overlapping ORFs in the 5'-untranslated region of the ENBP1 cDNA indicates that ENBP1 expression might be regulated at the translational level. The interaction of ENBP1 with a conserved AT-rich element within the ENOD12 promoter and the presence of the ENBP1 transcript in cells expressing ENOD12 strongly suggest that ENBP1 is a transcription factor involved in the regulation of ENOD12. Finally, the C-terminal region of ENBP1 shows strong homology to a protein from rat that is specifically expressed in testis tissue.