D. Lohar

Transcript Analysis of Early Nodulation Events in Medicago truncatula

Within the first 72 h of the interaction between rhizobia and their host plants, nodule primordium induction and infection occur. We predicted that transcription profiling of early stages of the symbiosis between Medicago truncatula roots and Sinorhizobium meliloti would identify regulated plant genes that likely condition key events in nodule initiation. Therefore, using a microarray with about 6,000 cDNAs, we compared transcripts from inoculated and uninoculated roots corresponding to defined stages between 1 and 72 h post inoculation (hpi). Hundreds of genes of both known and unknown function were significantly regulated at these time points. Four stages of the interaction were recognized based on gene expression profiles, and potential marker genes for these stages were identified. Some genes that were regulated differentially during stages I (1 hpi) and II (6–12 hpi) of the interaction belong to families encoding proteins involved in calcium transport and binding, reactive oxygen metabolism, and cytoskeleton and cell wall functions. Genes involved in cell proliferation were found to be up-regulated during stages III (24–48 hpi) and IV (72 hpi). Many genes that are homologs of defense response genes were up-regulated during stage I but down-regulated later, likely facilitating infection thread progression into the root cortex. Additionally, genes putatively involved in signal transduction and transcriptional regulation were found to be differentially regulated in the inoculated roots at each time point. The findings shed light on the complexity of coordinated gene regulation and will be useful for continued dissection of the early steps in symbiosis.

RNA Interference Identifies a Calcium-Dependent Protein Kinase Involved in Medicago truncatula Root Development

Changes in cellular or subcellular Ca2+ concentrations play essential roles in plant development and in the responses of plants to their environment. However, the mechanisms through which Ca2+ acts, the downstream signaling components, as well as the relationships among the various Ca2+-dependent processes remain largely unknown. Using an RNA interference–based screen for gene function in Medicago truncatula, we identified a gene that is involved in root development. Silencing Ca2+-dependent protein kinase1 (CDPK1), which is predicted to encode a Ca2+-dependent protein kinase, resulted in significantly reduced root hair and root cell lengths. Inactivation of CDPK1 is also associated with significant diminution of both rhizobial and mycorrhizal symbiotic colonization. Additionally, microarray analysis revealed that silencing CDPK1 alters cell wall and defense-related gene expression. We propose that M. truncatula CDPK1 is a key component of one or more signaling pathways that directly or indirectly modulates cell expansion or cell wall synthesis, possibly altering defense gene expression and symbiotic interactions.

Differential response of the plant Medicago truncatula to its symbiont Sinorhizobium meliloti or an exopolysaccharide-deficient mutant

Sinorhizobium meliloti forms symbiotic, nitrogen-fixing nodules on the roots of Medicago truncatula. The bacteria invade and colonize the roots through structures called infection threads. S. meliloti unable to produce the exopolysaccharide succinoglycan are unable to establish a symbiosis because they are defective in initiating the production of infection threads and in invading the plant. Here, we use microarrays representing 16,000 M. truncatula genes to compare the differential transcriptional responses of this host plant to wild-type and succinoglycan-deficient S. meliloti at the early time point of 3 days postinoculation. This report describes an early divergence in global plant gene expression responses caused by a rhizobial defect in succinoglycan production, rather than in Nod factor production. The microarray data show that M. truncatula inoculated with wild-type, succinoglycan-producing S. meliloti more strongly express genes encoding translation components, protein degradation machinery, and some nodulins than plants inoculated with succinoglycan-deficient bacteria. This finding is consistent with wild-type-inoculated plants having received a signal, distinct from the well characterized Nod factor, to alter their metabolic activity and prepare for invasion. In contrast, M. truncatula inoculated with succinoglycan-deficient S. meliloti more strongly express an unexpectedly large number of genes in two categories: plant defense responses and unknown functions. One model consistent with our results is that appropriate symbiotically active exopolysaccharides act as signals to plant hosts to initiate infection thread formation and that, in the absence of this signal, plants terminate the infection process, perhaps via a defense response.

The Transcription Factor MtSERF1 of the ERF Subfamily Identified by Transcriptional Profiling Is Required for Somatic Embryogenesis Induced by Auxin Plus Cytokinin in Medicago truncatula

Transcriptional profiling of embryogenic callus produced from Medicago truncatula mesophyll protoplasts indicated up-regulation of ethylene biosynthesis and ethylene response genes. Using inhibitors of ethylene biosynthesis and perception, it was shown that ethylene was necessary for somatic embryogenesis (SE) in this model legume. We chose several genes involved in ethylene biosynthesis and response for subsequent molecular analyses. One of these genes is a gene encoding a transcription factor that belongs to the AP2/ERF superfamily and ERF subfamily of transcription factors. We demonstrate that this gene, designated M. truncatula SOMATIC EMBRYO RELATED FACTOR1 (MtSERF1), is induced by ethylene and is expressed in embryogenic calli. MtSERF1 is strongly expressed in the globular somatic embryo and there is high expression in a small group of cells in the developing shoot meristem of the heart-stage embryo. RNA interference knockdown of this gene causes strong inhibition of SE. We also provide evidence that MtSERF1 is expressed in zygotic embryos. MtSERF1 appears to be essential for SE and may enable a connection between stress and development.

Population density-dependent regulation of the Bradyrhizobium japonicum nodulation genes.

The nodulation genes of Bradyrhizobium japonicum are essential for infection and establishment of a nitrogen‐fixing symbiosis. Here, we demonstrate that plant‐produced isoflavones induce nodulation gene expression in a population density‐dependent fashion. Nodulation gene induction is highest at a low population density and significantly reduced in more dense cultures. A quorum signal molecule in the conditioned medium of B. japonicum cultures mediates this repression. Repression in response to the quorum signal results from the induction of NolA which, in turn, induces NodD2 leading to inhibition of nod gene expression. Consistent with this, nolA–lacZ and nodD2–lacZ expression increased with increasing population density. Unlike the wild type, the ability to induce nodY–lacZ expression did not decline with population density in a NolA mutant. Normally, nod gene expression is repressed in planta (i.e. within nodules). However, expression of a nodY–GUS fusion was not repressed in a NolA mutant, suggesting that quorum‐sensing control may mediate in planta repression of the nod genes. Addition of conditioned medium to cultures significantly reduced nod gene expression. Treatment of inoculant cultures with conditioned medium also reduced the ability of B. japonicum to nodulate soybean plants.

Genetic analysis of ethylene regulation of legume nodulation.

The gaseous hormone ethylene has multiple roles in plant development and responses to external cues. Among these is the regulation of ‘Rhizobium’-induced nodulation in legumes. Extensive descriptive literature exists, but has been expanded to allow more mechanistic analysis through the application of genetics. Both mutants and transgenics displaying ethylene insensitivity have now been described, suggesting an intimate interplay of ethylene response, plant development and nodulation.

A Two-Component Regulator Mediates Population-Density-Dependent Expression of the Bradyrhizobium japonicum Nodulation Genes

Bradyrhizobium japonicum nod gene expression was previously shown to be population density dependent. Induction of the nod genes is highest at low culture density and repressed at high population densities. This repression involves both NolA and NodD2 and is mediated by an extracellular factor found in B. japonicum conditioned medium. NolA and NodD2 expression is maximal at high population densities. We demonstrate here that a response regulator, encoded by nwsB, is required for the full expression of the B. japonicum nodYABC operon. In addition, NwsB is also required for the population-density-dependent expression of both nolA and nodD2. Expression of nolA and nodD2 in the nwsB mutant remained at a basal level, even at high culture densities. The nwsB defect could be complemented by overexpression of a second response regulator, NodW. Consistent with the fact that NolA and NodD2 repress nod gene expression, the expression of a nodY-lacZ fusion in the nwsB mutant was unaffected by culture density. In plant assays with GUS fusions, nodules infected with the wild type showed no nodY-GUS expression. In contrast, nodY-GUS expression was not repressed in nodules infected with the nwsB mutant. Nodule competition assays between the wild type and the nwsB mutant revealed that the addition of conditioned medium resulted in a competitive advantage for the nwsB mutant.

A bacterial artificial chromosome library of Lotus japonicus constructed in an Agrobacterium tumefaciens-transformable vector.

We constructed a BAC library of the model legume Lotus japonicus with a 6-to 7-fold genome coverage. We used vector PCLD04541, which allows direct plant transformation by BACs. The average insert size is 94 kb. Clones were stable in Escherichia coli and Agrobacterium tumefaciens.

An integrated functional genomics and genetics approach for the plant's function in symbiotic nodulation.

Recent advances of high throughput DNA sequencing, bioinformatics robotics, BAC libraries, microarrays, insertional mutagenesis as well as promoter trapping open the opportunity for an integrated function and structure analysis of the genomes of soybean (Glycine max) and the model legume Lotus japonicus. We are specifically interested in the plant’s role during the establishment of nodule morphogenesis, and the genes shared during seemingly related developmental programs leading either to nodule or lateral root formation. Additionally our research seeks to elucidate plant genetic controls over plant-microbe interactions and symbiotic signaling of both Rhizobium and mycorrhizal symbioses. Nodulation in legumes involves the complex interaction of bacterial genes and their products with plant developmental processes governing mitogenic signal perception, signal transduction and morphogenesis. The challenge is to understand the plant’s genetic contribution to this symbiosis with the aim to improve natural associations of benefit for agriculture and the environment. Plant mutations were induced using EMS, fast neutron deletion as well as insertion mutagenesis. Single recessive loci were mapped using molecular markers, which were used to isolate soybean BAC clones to generate contigs spanning mutant deletions. Special emphasis was given to the Nts-1 locus of soybean that governs autoregulation of nodulation. If mutated, this locus leads to abundant nodulation (supernodulation) as well as nitrate tolerance in nodulation. Expression analysis of nodulation events using 4,200 micro-arrayed root ESTs was initiated to detect gene products temporarily expressed during early nodulation. Lotus japonicus as compared to soybean facilitates high throughput insertional mutagenesis and promoter trapping. Insertion of a promoter-less gus-reporter gene allowed the isolation of activated plant lines that showed development specific gus-gene expression. Isolation of flanking DNA sequences provided information of potential promoters and gene function as well as providing a link between structural and functional elements of nodulation-related genes. Evidence suggests that many nodule initiation functions evolved or are shared with lateral root related processes. The possibility exists that several non-legumes share such genes.

Functional Genomics and Genetic Analysis of Nodulation of Soybean and Lotus japonicus

Plant genes control nodulation and nitrogen fixation in all legume symbioses. The need discover the function and structure of these genes, their interplay, their “side-effects” on other morphogenetic steps, and their relationship to other genes involved in the metabolism, and signal transduction and cell division is becoming progressively and feasible in light of recent advances, commonly referred to as functional and structural genomics. Here we demonstrate how map-based cloning as well as promoter trapping have been applied to two legumes, namely soybean and model legume Lotus japonicus to discover genes involved in early nodulation responses. The results suggest that the developmental program underlying nodule initiation and pattern control “borrowed” many genes involved in lateral root formation and control. We focused initially on the supernodulation locus nts-1 (Kolchinsky et al, 1997) to develop strategies of map-based cloning. In parallel we initiated a large scale, Y-DNA-based transformation of the model legume Lotus japonicus (Jiang and Gresshoff, 1997; Handberg and Stougaard, 1992) to trap plant promoters and to possibly obtain flanking sequence as well as knock-out mutants (Stiller et al, 1997; Martirani et al, 1999).