STUDYING EARLY NODULIN GENE ENOD40 EXPRESSION AND INDUCTION BY NODULATION FACTOR AND CYTOKININ IN TRANSGENIC ALFALFA

ENOD40, an early nodulin gene, is expressed following inoculation with Rhizobium meliloti or by adding R. meliloti-produced nodulation (Nod) factors or the plant hormone cytokinin to uninoculated roots. We isol ated two MsENOD40 clones, designated MsENOD40-1 and MsENOD40-2, with d istinct promoters from an alfalfa (Medicago sativa cv Chief) genomic l ibrary. The promoters were fused to the reporter gene uidA (gus), and the constructs were introduced into alfalfa. We observed that the MsEN OD40-1 construct was expressed almost exclusively under symbiotic cond itions. The MsENOD40-2 construct was transcribed under both symbiotic and nonsymbiotic conditions and in nonnodular and nodular tissues. Bot h MsENOD40 promoter-gus constructs were similarly expressed as nodules developed, and both were expressed in roots treated with 6-benzylamin opurine or purified Nod factor. However, no blue color was detected in nodule-like structures induced by the auxin transport inhibitor N-1-( naphthyl)phthalamic acid on roots of plants containing the MsENOD40-1 promoter construct, whereas pseudonodules from plants containing the M sENOD40-2 promoter construct stained blue. A 616-bp region at the dist al 5' end of the promoter is important for proper spatial expression o f MsENOD40 in nodules and also for Nod-factor and cytokinin-induced ex pression.