SHORT-ROOT paralogs mediate feedforward regulation of D-type cyclin to promote nodule formation in soybean

Nitrogen fixation in soybean takes place in root nodules that arise from de novo cell divisions in the root cortex. Although several early nodulin genes have been identified, the mechanism behind the stimulation of cortical cell division during nodulation has not been fully resolved. Here we provide evidence that two paralogs of soybean SHORT-ROOT (GmSHR) play vital roles in soybean nodulation. Expression of GmSHR4 and GmSHR5 (GmSHR4/5) is induced in cortical cells at the beginning of nodulation, when the first cell divisions occur. The expression level of GmSHR4/5 is positively associated with cortical cell division and nodulation. Knockdown of GmSHR5 inhibits cell division in outer cortical layers during nodulation. Knockdown of both paralogs disrupts the cell division throughout the cortex, resulting in poorly organized nodule primordia with delayed vascular tissue formation. GmSHR4/5 function by enhancing cytokinin signaling and activating early nodulin genes. Interestingly, D-type cyclins act downstream of GmSHR4/5, and GmSHR4/5 form a feedforward loop regulating D-type cyclins. Overexpression of D-type cyclins in soybean roots also enhanced nodulation. Collectively, we conclude that the GmSHR4/5-mediated pathway represents a vital module that triggers cytokinin signaling and activates D-type cyclins during nodulation in soybean.

Analysis of Promoter Activity of the Early Nodulin Enod40 in Lotus japonicus

Our comparative studies on the promoter (pr) activity of Enod40 in the model legume Lotus japonicus in stably transformed GusA reporter lines and in hairy roots of L. japonicus demonstrate a stringent regulation of the Enod40 promoter in the root cortex and root hairs in response to Nod factors. Interestingly, the L. japonicus Enod40-2 promoter fragment also shows symbiotic activity in the reverse orientation. Deletion analyses of the Glycine max (Gm) Enod40 promoter revealed the presence of a minimal region -185 bp upstream of the transcription start. Stable transgenic L. japonicus reporter lines were used in bioassays to test the effect of different compounds on early symbiotic signaling. The responses of prGmEnod40 reporter lines were compared with the responses of L. japonicus (Lj) reporter lines based on the LjNin promoter. Both reporter lines show very early activity postinoculation in root hairs of the responsive zone of the root and later in the dividing cells of nodule primordia. The LjNin promoter was found to be more responsive than the GmEnod40 promoter to Nod factors and related compounds. The use of prGmEnod40 reporter lines to analyze the effect of nodulin genes on the GmEnod40 promoter activity indicates that LJNIN has a positive effect on the regulation of the Enod40 promoter, whereas the latter is not influenced by ectopic overexpression of its own gene product. In addition to pointing to a difference in the regulation of the two nodulin genes Enod40 and Nin during early time points of symbiosis, the bioassays revealed a difference in the response to the synthetic cytokinin 6-benzylaminopurine (BAP) between alfalfa and clover and L. japonicus. In alfalfa and clover, Enod40 expression was induced upon BAP treatment, whereas this seems not to be the case in L. japonicus; these results correlate with effects at the cellular level because BAP can induce pseudonodules in alfalfa and clover but not in L. japonicus. In conclusion, we demonstrate the applicability of the described L. japonicus reporter lines in analyses of the specificity of compounds related to nodulation as well as for the dissection of the interplay between different nodulin genes.

Development of Ectopic Roots from Abortive Nodule Primordia

The symbiotic phenotype of five Tn5-induced mutants of Rhizobium etli affected in different anabolic pathways (namely, gluconeogenesis and biosynthesis of lysine, purine, or pyrimidine) was analyzed. These mutants induced, on the root of Phaseolus vulgaris, a normal early sequence of morphogenetics events, including root hair deformation and development of nodule primordia. Later on, however, from the resulting root outgrowths, instead of nodules, one or more ectopic roots (spaced closely related and agravitropic) emerged. Therefore, this group of mutant was collectively called “root inducer” (RIND). It was observed that the RIND-induced infection threads aborted early inside the invaded root hair, and that the resulting abortive nodules lack induction of late nodulin genes. Moreover, experiments performed using a conditional mutant (a methionine-requiring invader) revealed that bacterial invasion plays a key role in the maintenance of the program of nodule development and, in particular, in the differentiation of the most specific symbiotic tissue of globose nodules, the central tissue. These data indicate that, in P. vulgaris, the nodule primordium is a root-specified pro-meristematic tissue.

Cytological, Genetic, and Molecular Analysis to Characterize Compatible and Incompatible Interactions Between Medicago truncatula and Colletotrichum trifolii

In this study, a new pathosystem was established using the model plant Medicago truncatula and Colletotrichum trifolii, the causal agent of anthracnose on Medicago sativa. Screening of a few M. truncatula lines identified Jemalong and F83005.5 as resistant and susceptible to Colletotrichum trifolii race 1, respectively. Symptom analysis and cytological studies indicated that resistance of Jemalong was associated with a hypersensitive response of the plant. The two selected lines were crossed, and inoculations with C. trifolii were performed on the resulting F1 and F2 progenies. Examination of the disease phenotypes indicated that resistance was dominant and was probably due to a major resistance gene. Molecular components of the resistance were analyzed through macroarray experiments. Expression profiling of 126 expressed sequence tags corresponding to 92 genes, which were selected for their putative functions in plant defense or signal transduction, were compared in Jemalong and F83005.5 lines. A strong correlation was observed between the number of up-regulated genes and the resistance phenotype. Large differences appeared at 48 h postinoculation; more than 40% of the tested genes were up-regulated in the Jemalong line compared with only 10% in the susceptible line. Interestingly, some nodulin genes were also induced in the resistant line upon inoculation with C. trifolii.

Expression of Genes Encoding Late Nodulins Characterized by a Putative Signal Peptide and Conserved Cysteine Residues Is Reduced in Ineffective Pea Nodules

Five nodulin genes, PsN1, PsN6, PsN314, PsN335, and PsN466, with reduced expression in ineffective nodules on the pea (Pisum sativum) mutant E135 (sym13) were characterized. They encode small polypeptides containing a putative signal peptide and conserved cysteine residues and show homology to the nodulins PsENOD3/14 and PsNOD6. For each gene, multiple bands were detected by genomic Southern analysis. Northern analysis showed that all five genes were expressed exclusively in nodules and that their temporal expression patterns were similar to that of the leghemoglobin (Lb) gene during nodule development. Their transcripts were localized predominantly from the interzone II–III to the distal part of nitrogen-fixing zone III in effective nodules, resembling the Lb gene. However, transcripts in ineffective E135 nodules were localized in narrower regions than those in the effective nodules. These results indicate that these nodulins are abundant in pea nodules and that their successive expression during nodule development is associated with nitrogen-fixing activity.

Analysis of Medicago truncatula Nodule Expressed Sequence Tags

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 λHybriZAP, 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.

Early Nodulin Genes are Not Markers of the Capacity of Woody Legumes to Nodulate

Knowing whether trees can develop nodules in which nitrogen is fixed is important for managing inputs during culture. Early nodulin genes, such as ENOD2 and ENOD12, could function as molecular markers for the capacity to nodulate if they are conserved only in species that nodulate. Several nodulating species are known to possess these genes; but, their occurrence has not been studied among non-nodulating taxa, including Cercis canadensis L. (redbud), Gleditsia triacanthos L. var. inermis Willd. (honey locust), and Gymnocladus dioica (L.) C. Koch (Kentucky coffee tree). Our objective was to determine the relationship between the capacity to nodulate and the occurrence of putative ENOD2 or ENOD12 genes by probing the genomes of these non-nodulating species and the genomes of two legumes that nodulate, Albizia julibrissin Durazz. (silk tree) and Laburnum alpinum (Mill.) Bercht. & J. Presl (Scotch laburnum). ENOD2 and ENOD12 cDNA clones from Glycine max (L.) Merill (soybean) and Pisum sativum L. (pea), respectively, and cloned ENOD2 PCR fragments from Maackia amurensis Rupr. & Maxim. (Amur maackia) and Styphnolobium japonicum (L.) Schott (Japanese pagodatree) were used as probes for Southern hybridizations. Sequences from genomes of silk tree, Scotch laburnum, honey locust, and Kentucky coffee tree hybridized to ENOD2 probes on Southern blots. Putative ENOD12 sequences were detected in the genomes of Scotch laburnum, redbud, and honey locust. Thus, we conclude that ENOD2 and ENOD12 can not be used as markers for the capacity to nodulate.

Symbiosis-Specific Expression of Two Medicago truncatula Nodulin Genes, MtN1 and MtN13, Encoding Products Homologous to Plant Defense Proteins

Two Medicago truncatula nodulin genes putatively encoding proteins structurally related to two classes of proteins commonly associated with plant defense reactions have been characterized. MtN1 is homologous to two small, cysteine-rich, pathogen-inducible proteins from pea (pI39 and pI230), whereas MtN13 is closely related to the PR10 family of pathogenesis-related proteins. We show that neither MtN1 nor MtN13 is induced in leaves in response to pathogenic bacteria, and that both are exclusively expressed during nodulation. In situ hybridization experiments as well as Northern (RNA) studies of interactions between M. truncatula and either wild-type Rhizobium meliloti or mutants deficient in infection establish that MtN1 is associated with the infection process, while MtN13 represents the first specific marker described for the nodule outer cortex. Possible roles for MtN1 and MtN13 are discussed. We also present the identification of another member of the PR10 family, designated as MtPR10-1, whose regulation is strikingly different from that observed for MtN13, being constitutively expressed in roots and pathogen-inducible in leaves.

Characterization of ENOD2 cDNAs in Maackia amurensis Rupr. & Maxim. (Amur Maackia)

ENOD2 and other early nodulin genes are conserved among legumes studied to date and might function as markers for the potential of legumes to nodulate. Early nodulin genes have been characterized only among herbaceous legumes. We are interested in understanding the nature of ENOD2 in a nodulating, woody legume. A 561-bp MaENOD2 PCR fragment was used as a probe to screen a cDNA library from nodules ≈1 mm in diameter on roots of Amur maackia, the only temperate and horticulturally desirable leguminous tree species known to nodulate. Five cDNAs were selected for nucleotide sequence analysis. Sequences were determined by using automated dideoxy sequencing and analyzed for identity to other genes with the Genetics Computer Group (GCG) program. The cDNA clones show 68% to 74% identity at the nucleic acid level with ENOD2 genes of Sesbania rostrata Brem. & Oberm., Glycine max (L.) Merrill, and Lupinus luteus L. Southern and northern analyses are being conducted to investigate the possibility of a gene family and to show differential and temporal production of transcripts, respectively. These studies provide new information about nodulins of woody legumes and are being used to facilitate related research on molecular barriers to nodulation in the closely related, non-nodulating tree species Cladrastis kentukea (Dum.-Cours.) Rudd (American yellowwood) and Sophora japonica L. (Japanese pagodatree).