Studying Early Nodulin GeneENOD40Expression and Induction by Nodulation Factor and Cytokinin in Transgenic Alfalfa1

Abstract

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 isolated two MsENOD40 clones, designated MsENOD40-1 and MsENOD40-2, with distinct promoters from an alfalfa (Medicago sativa cv Chief) genomic library. The promoters were fused to the reporter gene uidA (gus), and the constructs were introduced into alfalfa. We observed that the MsENOD40-1 construct was expressed almost exclusively under symbiotic conditions. The MsENOD40-2 construct was transcribed under both symbiotic and nonsymbiotic conditions and in nonnodular and nodular tissues. Both MsENOD40 promoter-gus constructs were similarly expressed as nodules developed, and both were expressed in roots treated with 6-benzylaminopurine 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 MsENOD40-2 promoter construct stained blue. A 616-bp region at the distal 5 end of the promoter is important for proper spatial expression of MsENOD40 in nodules and also for Nod-factor and cytokinin-induced expression.

Flavonoids and Auxin Transport Inhibitors Rescue Symbiotic Nodulation in the Medicago truncatula Cytokinin Perception Mutant cre1

“…The expression of a cytokinin synthesis gene in Sinorhizobium meliloti nodulation mutants was sufficient to induce cortical cell divisions and expression of nodulation genes in alfalfa (Cooper and Long, 1994). Application of cytokinin can induce cortical cell divisions and expression of nodulation genes in alfalfa Fang and Hirsch, 1998), white clover (Trifolium repens) (Mathesius et al, 2000), and Lotus japonicus (Heckmann et al, 2011), and pseudonodules on the actinorhizal plant Alnus glutinosa (Rodriguez-Barrueco and Bermudez de Castro, 1973). Legume mutants impaired in cytokinin perception typically fail to initiate nodules (Murray et al, 2007;Plet et al, 2011), while constitutive cytokinin signaling was found to cause spontaneous nodule formation in the absence of rhizobia (Tirichine et al, 2007).…”

Medicago truncatula plants overexpressing the early nodulin gene enod40 exhibit accelerated mycorrhizal colonization and enhanced formation of arbuscules

“…Alternatively, enod40 transcripts might stimulate fungal growth in the root cortex by affecting targets that interfere with plant hormone action and unknown cell-to-cell communication processes between vascular and cortical tissues. It is worth noting in this context that the amount of enod40 transcripts in M. sativa can be enhanced by treatment of roots with cytokinins and rhizobial Nod factors as well (9,11,27). It is possible therefore that enod40 in mycorrhizal roots is activated via hormonal imbalances.…”

ENOD40Gene Expression and Cytokinin Responses in the Nonnodulating, Nonmycorrhizal (Nod−Myc−) Mutant,Masym3, ofMelilotus albaDesr.

“…14 ENOD40 is a nodulin gene that is expressed within hours in response to Rhizobium inoculation and has been correlated with nodule development. 27,28 ENOD40 transcripts have been detected by in situ hybridization analysis in the dividing inner cortical cells, the origin of the nodule primordium, and the meristem and the infection zone of established indeterminate nodules, 29,30 making ENOD40 useful as a marker for assessing the progress of the symbiosis. ENOD40 has been detected in a broad range of indeterminate-nodule forming legumes (pea, vetch, Medicago truncatula, Trifolium repens), in determinate-nodule forming legumes (soybean, lotus, Sesbania rostrata), and also in nonlegumes (tomato, rice).…”