Phytohormones are well-known regulators of the symbiotic Rhizobium-legume association in the plant host. The enod40 nodulin gene is associated with the earliest phases of the nodule organogenesis programme in the legume host and modifying its expression resulted in perturbations of nodule development in Medicago truncatula. Therefore in our pursuit to mimic the initial signal transduction steps of legume nodulation in the alien physiological set-up of a rice plant, we have expressed the Mtenod40 gene in rice. Molecular data confirm the stable integration, inheritance and transcription of the foreign gene in this non-legume. We have compared the phytohormonal responses of Mtenod40-overexpressing and control plants in a homologous legume background (M. truncatula) and in the non-legume rice. An enod40-mediated root growth response, induced by inhibition of ethylene biosynthesis, was observed in both plants. On the other hand, a significant differential effect of cytokinins was observed only in rice plants. This suggests that ethylene inhibits enod40 action both in legumes and non-legumes and reinforces that some of the early signal transduction steps of the nodule developmental programme may function in rice.

A diversity of mRNAs containing only short open reading frames (sORF-RNAs; encoding less than 30 amino acids) have been shown to be induced in growth and differentiation processes. The early nodulin gene enod40, coding for a 0.7-kb sORF-RNA, is expressed in the nodule primordium developing in the root cortex of leguminous plants after infection by symbiotic bacteria. Ballistic microtargeting of this gene into Medicago roots induced division of cortical cells. Translation of two sORFs (I and II, 13 and 27 amino acids, respectively) present in the conserved 5' and 3' regions of enod40 was required for this biological activity. These sORFs may be translated in roots via a reinitiation mechanism. In vitro translation products starting from the ATG of sORF I were detectable by mutating enod40 to yield peptides larger than 38 amino acids. Deletion of a Medicago truncatula enod40 region between the sORFs, spanning a predicted RNA structure, did not affect their translation but resulted in significantly decreased biological activity. Our data reveal a complex regulation of enod40 action, pointing to a role of sORF-encoded peptides and structured RNA signals in developmental processes involving sORF-RNAs.

The mutualistic symbiosis between flowering plants and arbuscular mycorrhizal fungi is extremely abundant in terrestrial ecosystems. In this symbiosis, obligately biotrophic fungi colonize the root of the host plants, which can benefit from these fungi by enhanced access to mineral nutrients in the soil, especially phosphorus. One of the main goals of research on this symbiosis is to find plant genes that control fungal development in the host plant. In this work, we show that mycorrhizal colonization is regulated by enod40 , an early nodulin gene known to be involved in the nodule symbiosis of legumes with nitrogen-fixing bacteria. Medicago truncatula plants overexpressing enod40 exhibited stimulated mycorrhizal colonization in comparison with control plants. Overexpression of enod40 promoted fungal growth in the root cortex and increased the frequency of arbuscule formation. Transgenic lines with suppressed levels of enod40 transcripts, likely via a cosuppression phenomenon induced by the transgene, exhibited reduced mycorrhizal colonization. Hence, enod40 might be a plant regulatory gene involved in the control of the mycorrhizal symbiosis.

Systematic sequencing of expressed sequence tags (ESTs) can give a global picture of the assembly of genes involved in the development and function of organs. Indeterminate nodules representing different stages of the developmental program are especially suited to the study of organogenesis. With the vector lambdaHybriZAP, a cDNA library was constructed from emerging nodules of Medicago truncatula induced by Sinorhizobium meliloti. The 5' ends of 389 cDNA clones were sequenced, then these ESTs were analyzed both by sequence homology search and by studying their expression in roots and nodules. Two hundred fifty-six ESTs exhibited significant similarities to characterized data base entries and 40 of them represented 26 nodulin genes, while 133 had no similarity to sequences with known function. Only 60 out of the 389 cDNA clones corresponded to previously submitted M. truncatula EST sequences. For 117 cDNAs, reverse Northern (RNA) hybridization with root and nodule RNA probes revealed enhanced expression in the nodule, 48 clones are likely to code for novel nodulins, 33 cDNAs are clones of already known nodulin genes, and 36 clones exhibit similarity to other characterized genes. Thus, systematic analysis of the EST sequences and their expression patterns is a powerful way to identify nodule-specific and nodulation-related genes.

We examined the timing and location of several early root responses to Rhizobium leguminosarum bv. trifolii infection, compared with a localized addition of cytokinin in white clover, to study the role of cytokinin in early signaling during nodule initiation. Induction of ENOD40 expression by either rhizobia or cytokinin was similar in timing and location and occurred in nodule progenitor cells in the inner cortex. Inoculation of rhizobia in the mature root failed to induce ENOD40 expression and cortical cell divisions (ccd). Nitrate addition at levels repressing nodule formation inhibited ENOD40 induction by rhizobia but not by cytokinin. ENOD40 expression was not induced by auxin, an auxin transport inhibitor, or an ethylene precursor. In contrast to rhizobia, cytokinin addition was not sufficient to induce a modulation of the auxin flow, the induction of specific chalcone synthase genes, and the accumulation of fluorescent compounds associated with nodule initiation. However, cytokinin addition was sufficient for the localized induction of auxin-induced GH3 gene expression and the initiation of ccd. Our results suggest that rhizobia induce cytokinin-mediated events in parallel to changes in auxin-related responses during nodule initiation and support a role of ENOD40 in regulating ccd. We propose a model for the interactions of cytokinin with auxin, ENOD40, flavonoids, and nitrate during nodulation.

Molecular mechanisms involved in the control of root nodule organogenesis in the plant host are poorly understood. One of the nodulin genes associated with the earliest phases of this developmental program is enod40. We show here that transgenic Medicago truncatula plants overexpressing enod40 exhibit accelerated nodulation induced by Sinorhizobium meliloti. This resulted from increased initiation of primordia, which was accompanied by a proliferation response of the region close to the root tip and enhanced root length. The root cortex of the enod40-transformed plants showed increased sensitivity to nodulation signals. T(1) and T(2) descendants of two transgenic lines with reduced amounts of enod40 transcripts (probably from cosuppression) formed only a few and modified nodulelike structures. Our results suggest that induction of enod40 is a limiting step in primordium formation, and its function is required for appropriate nodule development.

Nodule organogenesis starts with the dedifferentiation and division of root cortical cells. In these cells, the early nodulin gene enod40 which encodes an unusually small peptide (12 or 13 amino acids) is induced from the beginning of this process. To investigate the role of enod40 in nodule development we constructed transgenic Medicago truncatula plants overexpressing enod40. These plants exhibit extensive cortical cell division (ccd) in their roots in the absence of Rhizobium under nitrogen-limiting conditions (Charon et al., 1997, in press). In parallel, we developed a transient assay on Medicago roots using alfalfa plants carrying a fusion of the Msenod12A promoter to GUS. Msenod12A expression is detected in the initially dividing cortical cells induced either by R. meliloti or its Nod factor and these transgenic plants have been used to study the reprogramming of inner cortical cells for nodule initiation (Bauer et al., 1996). Particle bombardment with an enod40-expressing DNA cassette was assayed and shown to induce cortical cell division and Msenod12A expression. Even though the majority of the particles were found in the epidermis and outer cortex, this response was always detected in the inner cortical cells. This indicates that the enod40 gene product is able to signal to these inner cortical cells. Transient expression of either the enod40 region spanning the oligopeptide sequence or a 3′ enod40 region without this sequence induced this response (Charon et al., 1997). Hence, enod40 contains at least two biologically active regions, able to elicit a cell-specific growth response in Medicago roots. Using “biolistic” particle bombardment we could not detect any activity of the synthetic ENOD40 peptide.

Nodule organogenesis starts with the dedifferentiation and division of root cortical cells. These dividing cells develop into a primordium where an apical meristem is formed from which all tissues of the mature nodule originate. We have previously used differential screening procedures to identify alfalfa early nodulin genes and studied their expression pattern during nodule development (Crespi et al., 1994; Coba de la Pena et al., 1997; McKhann et al., 1997). Several early nodulin genes expressed in the primordium cells have been found: Msenod12A (coding for a cell wall protein, Bauer et al., 1996) Msenod40, Mscal and Msgbl. Mscal codes for a carbonic anhydrase, an enzyme catalyzing the hydration of CO2. Mscal function might possibly be related to the increased amyloplast deposition and activated carbon metabolism of the dividing cortical cells of the nodule primordium (Coba de la Pena et al., 1997). Msgbl codes for a new member of the RACK1 (=receptor of activated C kinase) subfamily of WD-repeat proteins (McKhann et al., 1997). This subfamily shows homology to the β-subunit of heterotrimeric G proteins. Besides RACK1, this subfamily contains several plant genes including the well characterized auxin-inducible arcA gene of tobacco.

We are interested in the molecular mechanisms involved in nodule initiation in the R. meliloti-Medicago symbiosis. To that end, early nodulin genes expressed in the initially dividing cortical cells of Medicago have been identified: Msenod40, Mscal and Msenodl2A. Using these molecular markers, evidence indicating that Nod factors and cytokinins may share certain common signalling elements will be presented. In addition, we propose that the induction of enod40 in the cortex may be involved in the elicitation of cortical cell divisions.

Under nitrogen-limiting conditions Rhizobium meliloti can establish symbiosis with Medicago plants to form nitrogen-fixing root nodules. Nodule organogenesis starts with the dedifferentiation and division of root cortical cells. In these cells the early nodulin gene enod40, which encodes an unusually small peptide (12 or 13 amino acids), is induced from the beginning of this process. Herein we show that enod40 expression evokes root nodule initiation. (i) Nitrogen-deprived transgenic Medicago truncatula plants overexpressing enod40 exhibit extensive cortical cell division in their roots in the absence of Rhizobium. (ii) Bombardment of Medicago roots with an enod40-expressing DNA cassette induces dedifferentiation and division of cortical cells and the expression of another early nodulin gene, Msenod12A. Moreover, transient expression of either the enod40 region spanning the oligopeptide sequence or only the downstream region without this sequence induces these responses. Our results suggest that the cell-specific growth response elicited by enod40 is involved in the initiation of root nodule organogenesis.