VsENBP1 regulates the expression of the early nodulin PsENOD12B

A DNA-binding protein, VsENBP1, previously isolated from Vicia sativa was s hown to bind in a sequence-specific manner to the early nodulin ENOD12 gene promoter from Pisum sativum. Here, the functional importance of the VsENBP 1 binding sites on the PsENOD12B promoter has been studied in vivo. A promo ter-gusA fusion in which a mutation was introduced at the putative target s equence, AATAA, was inactive in nodules of transgenic Vicia hirsuta roots. Gel retardation assays showed that VsENBP1 does not bind to the mutated pro moter segment, suggesting that VsENBP1 activates the PsENOD12B expression i n nodules through its interaction with its target sequence. In the presence of the 35S enhancer, an ENOD12 promoter-GUS construct gave expression in r oot vascular tissue in addition to the root nodules. Overexpression of Vsen bp1 in transgenic V. hirsuta roots reduced the leaky expression in root vas cular tissue in contrast to nodules in which a small increase in GUS expres sion was observed. The results indicate that VsENBP1 acts as a repressor of ENOD12 expression in root tissue.