Adam Kondorosi

The mitotic inhibitor ccs52 is required for endoreduplication and ploidy‐dependent cell enlargement in plants

Plant organs develop mostly post‐embryonically from persistent or newly formed meristems. After cell division arrest, differentiation frequently involves endoreduplication and cell enlargement. Factors controlling transition from mitotic cycles to differentiation programmes have not been identified yet in plants. Here we describe ccs52, a plant homologue of APC activators involved in mitotic cyclin degradation. The ccs52 cDNA clones were isolated from Medicago sativa root nodules, which exhibit the highest degree of endopolyploidy in this plant. ccs52 represents a small multigenic family and appears to be conserved in plants. Overexpression of ccs52 in yeast triggered mitotic cyclin degradation, cell division arrest, endoreduplication and cell enlargement. In Medicago, enhanced expression of ccs52 was found in differentiating cells undergoing endoreduplication. In transgenic M.truncatula plants, overexpression of the ccs52 gene in the antisense orientation resulted in partial suppression of ccs52 expression and decreased the number of endocycles and the volume of the largest cells. Thus, the ccs52 product may switch proliferating cells to differentiation programmes which, in the case of endocycles, result in cell size increments.

A Novel Family in Medicago truncatula Consisting of More Than 300 Nodule-Specific Genes Coding for Small, Secreted Polypeptides with Conserved Cysteine Motifs

Transcriptome analysis of Medicago truncatula nodules has led to the discovery of a gene family named NCR (nodule-specific cysteine rich) with more than 300 members. The encoded polypeptides were short (60–90 amino acids), carried a conserved signal peptide, and, except for a conserved cysteine motif, displayed otherwise extensive sequence divergence. Family members were found in pea (Pisum sativum), broad bean (Vicia faba), white clover (Trifolium repens), and Galega orientalis but not in other plants, including other legumes, suggesting that the family might be specific for galegoid legumes forming indeterminate nodules. Gene expression of all family members was restricted to nodules except for two, also expressed in mycorrhizal roots. NCR genes exhibited distinct temporal and spatial expression patterns in nodules and, thus, were coupled to different stages of development. The signal peptide targeted the polypeptides in the secretory pathway, as shown by green fluorescent protein fusions expressed in onion (Allium cepa) epidermal cells. Coregulation of certain NCR genes with genes coding for a potentially secreted calmodulin-like protein and for a signal peptide peptidase suggests a concerted action in nodule development. Potential functions of the NCR polypeptides in cell-to-cell signaling and creation of a defense system are discussed.

enod40, a gene expressed during nodule organogenesis, codes for a non-translatable RNA involved in plant growth

Rhizobium meliloti can interact symbiotically with Medicago plants, thereby inducing root nodules. However, certain Medicago plants can form nodules spontaneously, in the absence of rhizobia. A differential screening was performed using spontaneous nodule versus root cDNAs from Medicago sativa ssp. varia. Transcripts of a differentially expressed clone, Msenod40, were detected in all differentiating cells of nodule primordia and spontaneous nodules, but were absent in fully differentiated cells. Msenod40 showed homology to a soybean early nodulin gene, Gmenod40, although no significant open reading frame (ORF) or coding capacity was found in the Medicago sequence. Furthermore, in the sequences of cDNAs and a genomic clone (Mtenod40) isolated from Medicago truncatula, a species containing a unique copy of this gene, no ORFs were found either. In vitro translation of purified Mtenod40 transcripts did not reveal any protein product. Evaluation of the RNA secondary structure indicated that both msenod40 and Gmenod40 transcripts showed a high degree of stability, a property shared with known non‐coding RNAs. The Mtenod40 RNA was localized in the cytoplasm of cells in the nodule primordium. Infection with Agrobacterium tumefaciens strains bearing antisense constructs of Mtenod40 arrested callus growth of Medicago explants, while overexpressing Mtenod40 embryos developed into teratomas. These data suggest that the enod40 genes might have a role in plant development, acting as ‘riboregulators’, a novel class of untranslated RNAs associated with growth control and differentiation.

Physical and genetic analysis of a symbiotic region of Rhizobium meliloti: Identification of nodulation genes

SummaryA 135 kb long segment of the symbiotic region of the Rhizobium meliloti megaplasmid was mapped with the help of a Rhizobium meliloti gene library, made in the cosmid vehicle pJB8. A set of overlapping cosmid clones was used to identify the inserts in R-primes carrying megaplasmid sections, and to map 20 deletion mutations and 24 insertion mutations with Nod- or Fix- phenotypes. This led to the identification of DNA regions carrying nod or fix (nif) genes. The results of this study correlate well with transcription data of nodule-specific expression of plasmid sequences. The nod mutations were localized in two groups. Using directed Tn5 mutagenesis, correlated physical-genetic maps for these regions were established. One nod gene cluster is about 2.5–3.0 kb in size and carries genes involved in root hair curling, a very early step in nodule formation. Mutations in these genes can be complemented by sym plasmids of other Rhizobium species, such as Rhizobium leguminosarum. We designate these genes as “common” nod genes because mutations in them can be complemented by plasmids derived from different Rhizobium strains. The other nod gene cluster consists of a 2 kb and a 1 kb long DNA segment, separated by a 1 kb region nonessential for nodulation. These nod genes are probably involved in the host specificity of nodulation.

Location of nodulation and nitrogen fixation genes on a high molecular weight plasmid of R. meliloti

SummaryR. meliloti strain 41 (Rm41) was shown to harbour two indigenous plasmids with molecular weights of 140 Mdal (pRmc41a) and more than 300 Mdal (pRme41b), respectively. Using a heat-treatment procedure, derivatives of Rm41 defective in nodulation (Nod-) or nitrogen fixation (Fix-) have been readily obtained. In some Nod- mutants the deletion of a segment of plasmid pRme41b was found.Based on the demonstrated homology between the nitrogen fixation (nif) genes of Klebsiella pneumoniae and of R. meliloti the Rhizobium nif region has been cloned into the cosmid vector pHC79, then recloned into pBR322 and the restriction map of the nif region has been determined. 32P-labelled nick-translated probe prepared from the cloned nif DNA fragment hybridized to pRme41b of Rm41 but for most Nod- mutants this hybridization was not detected. Hybridization of a cosmid containing Rm41 DNA to total DNA digests from the wild-type bacterium and from a series of Nod- mutants revealed that at least a 24 kb DNA fragment including the nif structural genes was missing from most of the Nod- mutants. These results, together with the genetic analyses of these symbiotic mutations suggest that some nod and fix genes are located on pRme41b.

Endoreduplication Mediated by the Anaphase-Promoting Complex Activator CCS52A Is Required for Symbiotic Cell Differentiation in Medicago truncatula Nodules

In Medicago nodules, endoreduplication cycles and ploidy-dependent cell enlargement occur during the differentiation of bacteroid-containing nitrogen-fixing symbiotic cells. These events are accompanied by the expression of ccs52A, a plant ortholog of the yeast and animal cdh1/srw1/fzr genes, acting as a substrate-specific activator of the anaphase-promoting complex (APC) ubiquitin ligase. Because CCS52A is involved in the transition of mitotic cycles to endoreduplication cycles, we investigated the importance of somatic endoploidy and the role of the M. truncatula ccs52A gene in symbiotic cell differentiation. Transcription analysis and ccs52A promoter–driven β-glucuronidase activity in transgenic plants showed that ccs52A was dispensable for the mitotic cycles and nodule primordium formation, whereas it was induced before nodule differentiation. The CCS52A protein was present in the nucleus of endoreduplication-competent cells, indicating that it may activate APC constitutively during the endoreduplication cycles. Downregulation of ccs52A in transgenic M. truncatula plants drastically affected nodule development, resulting in lower ploidy, reduced cell size, inefficient invasion, and the maturation of symbiotic cells, accompanied by early senescence and finally the death of both the bacterium and plant cells. Thus, ccs52A expression is essential for the formation of large highly polyploid symbiotic cells, and endoreduplication is an integral part of normal nodule development.

Rapid and efficient transformation of diploid Medicago truncatula and Medicago sativa ssp. falcata lines improved in somatic embryogenesis

Abstract We describe a simple and efficient protocol for regeneration-transformation of two diploid Medicago lines: the annual M. truncatula R108-1(c3) and the perennial M. sativa ssp. falcata (L.) Arcangeli PI.564263 selected previously as highly embryogenic genotypes. Here, embryo regeneration of R108-1 to complete plants was further improved by three successive in vitro regeneration cycles resulting in the line R108-1(c3). Agrobacterium tumefaciens-mediated transformation of leaf explants was carried out with promoter-gus constructs of two early nodulins (MsEnod12A and MsEnod12B) and one late nodulin (Srglb3). The transgenic plants thus produced on all explants within 3–4 months remained diploid and were fertile. This protocol appears to be the most efficient and fastest reported so far for leguminous plants.

Cell and Molecular Biology of Rhizobium-Plant

Publisher Summary This chapter discusses the Cell and molecular biology of Rhizobium -plant interactions. Soil bacteria, referred to as rhizobia belonging to the genera Rhizobium , Bradyrhizobium , and Azorhizobium , have the unique ability to induce nitrogen-fixing nodules on the roots or stems of leguminous plants. Nodule development consists of several stages determined by different sets of genes both in the host and symbiont. At least at the very early steps of symbiosis, the bacterial and plant genes are activated consecutively by signal exchanges between the symbiotic partners. First, flavonoid signal molecules exuded by the host plant root induce the expression of nodulation ( nod, nol ) genes in Rhizobium in conjunction with the bacterial activator NodD protein. Then, in the second step, lipooligosaccharide Nod factors with various host-specific structural modifications are produced by the bacterial Nod proteins. The Nod factors induce various plant reactions, such as root hair deformation, initiation of nodule meristems, and induction of early nodulin genes, leading to nodule formation. Other classes of bacterial genes are required for successful infection and for nitrogen fixation. This chapter includes only the early events of communication between rhizobia and their host plants, that is, the perception of flavonoid signals by the bacteria, the production of Nod signals by rhizobia, and the early plant responses to the bacteria.

Organization, structure and symbiotic function of Rhizobium meliloti nodulation genes determining host specificity for alfalfa.

In R. meliloti we have identified four nodulation genes determining plant host-range specificity and have designated them hsnABC and D. The genes code for 9.7, 41.7, 26.7, and 28.6 kd proteins, respectively, and are organized into two transcriptional units. Mutations in these genes affect nodulation of their natural plant hosts Medicago sativa and Melilotus albus to different extents and hsnD mutants have an altered host-range. These Nod- mutations are not complementable by nodulation genes of other Rhizobium species such as R. leguminosarum. The hsn genes determine plant-specific infection through root hairs: hsnD is required for host-specific root hair curling and nodule initiation while the hsnABC genes control infection thread growth from the root hairs.

Enod40, a short open reading frame-containing mRNA, induces cytoplasmic localization of a nuclear RNA binding protein in Medicago truncatula.

In eukaryotes, diverse mRNAs containing only short open reading frames (sORF-mRNAs) are induced at specific stages of development. Their mechanisms of action may involve the RNA itself and/or sORF-encoded oligopeptides. Enod40 genes code for highly structured plant sORF-mRNAs involved in root nodule organogenesis. A novel RNA binding protein interacting with the enod40 RNA, MtRBP1 (for Medicago truncatula RNA Binding Protein 1), was identified using a yeast three-hybrid screening. Immunolocalization studies and use of a MtRBP1-DsRed2 fluorescent protein fusion showed that MtRBP1 localized to nuclear speckles in plant cells but was exported into the cytoplasm during nodule development in enod40-expressing cells. Direct involvement of the enod40 RNA in MtRBP1 relocalization into cytoplasmic granules was shown using a transient expression assay. Using a (green fluorescent protein)/MS2 bacteriophage system to tag the enod40 RNA, we detected in vivo colocalization of the enod40 RNA and MtRBP1 in these granules. This in vivo approach to monitor RNA–protein interactions allowed us to demonstrate that cytoplasmic relocalization of nuclear proteins is an RNA-mediated cellular function of a sORF-mRNA.