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Adam Kondorosi

Publications and source records attributed to Adam Kondorosi.

At least 19 recordsLinked to original sources
PubMed2006

Isolation of mtpim proves Tnt1 a useful reverse genetics tool in Medicago truncatula and uncovers new aspects of AP1-like functions in legumes.

Comparative studies help shed light on how the huge diversity in plant forms found in nature has been produced. We use legume species to study developmental differences in inflorescence architecture and flower ontogeny with classical models such as Arabidopsis thaliana or Antirrhinum majus. Whereas genetic control of these processes has been analyzed mostly in pea (Pisum sativum), Medicago truncatula is emerging as a promising alternative system for these studies due to the availability of a range of genetic tools. To assess the use of the retrotransposon Tnt1 for reverse genetics in M. truncatula, we screened a small Tnt1-mutagenized population using degenerate primers for MADS-box genes, known controllers of plant development. We describe here the characterization of mtpim, a new mutant caused by the insertion of Tnt1 in a homolog to the PROLIFERATING INFLORESCENCE MERISTEM (PIM)/APETALA1 (AP1)/SQUAMOSA genes. mtpim shows flower-to-inflorescence conversion and altered flowers with sepals transformed into leaves, indicating that MtPIM controls floral meristem identity and flower development. Although more extreme, this phenotype resembles the pea pim mutants, supporting the idea that M. truncatula could be used to complement analysis of reproductive development already initiated in pea. In fact, our study reveals aspects not shown by analysis of pea mutants: that the mutation in the AP1 homolog interferes with the specification of floral organs from common primordia and causes conversion of sepals into leaves, in addition to true conversion of flowers into inflorescences. The isolation of mtpim represents a proof of concept demonstrating that Tnt1 populations can be efficiently used in reverse genetics screenings in M. truncatula.

Amino Acid Sequence↗
PubMed2006

Eukaryotic control on bacterial cell cycle and differentiation in the Rhizobium-legume symbiosis.

Symbiosis between legumes and Rhizobium bacteria leads to the formation of root nodules where bacteria in the infected plant cells are converted into nitrogen-fixing bacteroids. Nodules with a persistent meristem are indeterminate, whereas nodules without meristem are determinate. The symbiotic plant cells in both nodule types are polyploid because of several cycles of endoreduplication (genome replication without mitosis and cytokinesis) and grow consequently to extreme sizes. Here we demonstrate that differentiation of bacteroids in indeterminate nodules of Medicago and related legumes from the galegoid clade shows remarkable similarity to host cell differentiation. During bacteroid maturation, repeated DNA replication without cytokinesis results in extensive amplification of the entire bacterial genome and elongation of bacteria. This finding reveals a positive correlation in prokaryotes between DNA content and cell size, similar to that in eukaryotes. These polyploid bacteroids are metabolically functional but display increased membrane permeability and are nonviable, because they lose their ability to resume growth. In contrast, bacteroids in determinate nodules of the nongalegoid legumes lotus and bean are comparable to free-living bacteria in their genomic DNA content, cell size, and viability. Using recombinant Rhizobium strains nodulating both legume types, we show that bacteroid differentiation is controlled by the host plant. Plant factors present in nodules of galegoid legumes but absent from nodules of nongalegoid legumes block bacterial cell division and trigger endoreduplication cycles, thereby forcing the endosymbionts toward a terminally differentiated state. Hence, Medicago and related legumes have evolved a mechanism to dominate the symbiosis.

Cell Cycle↗
PubMed2006

Medicago truncatula transformation using leaf explants.

Legumes have long been recalcitrant to efficient Agrobacterium tumefaciens-mediated transformation. The choice and use of model legume plants (Medicago truncatula and Lotus japonicus) for molecular studies has triggered extensive studies devoted to the development of efficient Agrobacterium-mediated transformation protocols for these two plants. In M. truncatula, transformation protocols rely on the use of highly regenerable lines obtained by recurrent in vitro culture selection. These protocols are based on Agrobacterium-mediated transformation of M. truncatula followed by somatic embryogenesis-mediated plant regeneration. We describe here the protocol developed for M. truncatula R108-1 (c3).

Agrobacterium tumefaciens↗
PubMed2005

Arabidopsis anaphase-promoting complexes: multiple activators and wide range of substrates might keep APC perpetually busy.

The anaphase-promoting complex (APC), a multisubunit E3 ubiquitin ligase, is an essential regulator of the cell cycle from metaphase until S phase in yeast and metazoans. APC mediates degradation of numerous cell cycle-related proteins, including mitotic cyclins and its activation and substrate-specificity are determined by two adaptor proteins, Cdc20 and Cdh1. Plants have multiple APC activators and the Cdh1-type proteins, in addition, are represented by two subclasses, known as Ccs52A and Ccs52B. The Arabidopsis genome contains five cdc20 genes as well as ccs52A1, ccs52A2 and ccs52B. In Schizosaccharomyces pombe, expression of the three Atccs52 genes elicited distinct phenotypes supporting nonredundant function of the AtCcs52 proteins. Consistent with these activities, the AtCcs52 proteins were able to bind both to the yeast and the Arabidopsis APCs. In synchronized Arabidopsis cell cultures the cdc20 transcripts were present from early G2 until the M-phase exit, ccs52B from G2/M to M while ccs52A1 and ccs52A2 were from late M until early G2, suggesting consecutive action of these APC activators in the plant cell cycle. The AtCcs52 proteins interacted with different subsets of mitotic cyclins, in accordance with their expression profiles, either in free- or CDK-bound forms. Expression of most APC subunits was constitutive, whereas cdc27a and cdc27b, corresponding to two forms of apc3, and ubc19 and ubc20 encoding E2-C type ubiquitin-conjugating enzymes displayed differences in their cell cycle regulation. These data indicate the existence of numerous APC(Cdc20/Ccs52/Cdc27) forms in Arabidopsis, which in conjunction with different E2 enzymes might have distinct or complementary functions at distinct stages of the cell cycle.

Anaphase-Promoting Complex-Cyclosome↗
PubMed2005

Functional genomic analysis of global regulator NolR in Sinorhizobium meliloti.

NolR is a regulator of nodulation genes present in species belonging to the genera Rhizobium and Sinorhizobium. The expression of the nolR gene in Sinorhizobium meliloti AK631 was investigated in relation to stage of growth, availability of nutrients, and different environmental stimuli using the nolR::lacZ fusion report system. It has been shown that the nolR gene is regulated in a population-density-dependent fashion and influenced by a number of environmental stimuli, including nutrients, pH, and oxygen. Exploration of the physiological functions of NolR under various laboratory conditions has shown that NolR is required for the optimal growth of the bacteria on solid media, optimal survival of the bacteria in carbon-starved minimal medium, and after heat shock challenge. NolR also is involved in recipient-induced conjugative transfer of a plasmid. Proteome analysis of strain AK631 and its Tn5-induced nolR-deficient mutant EK698 revealed that a functional NolR induced significant differences in the accumulation of 20 polypeptides in peptide mass fingerprinting early-log-phase cultures and 48 polypeptides in stationary-phase cultures. NolR acted mainly as a repressor in the early-log-phase cultures, whereas it acted as both repressor and activator in the stationary-phase cultures. The NolR protein and 59 NolR-associated proteins have been identified by peptide mass fingerprinting. The NolR protein was differentially expressed only in the NolR+ wild-type strain AK631 but not in its NolR- derivative EK698, confirming that no functional NolR was produced in the mutant. The NolR-associated proteins have diverse functions in amino acid metabolism, carbohydrate metabolism, lipid metabolism, nucleotide metabolism, energy metabolism, metabolism of Co-factors, and cellular adaptation and transportation. These results further support our previous proposal that the NolR is a global regulatory protein which is required for the optimization of nodulation, bacterial growth and survival, and conjugative transfer of a plasmid.

Bacterial Proteins↗
PubMed2004

Endoreduplication and activation of the anaphase-promoting complex during symbiotic cell development.

Postembryonic development of plant organs requires a constant interplay between the cell cycle and the developmental programs. Upon endo- and exogenous signals, plant cells can enter, exit or modify the cell cycle. Alteration of mitotic cycles to endoreduplication cycles, where the genome is duplicated without mitosis, is common in plants and may play a role in cell differentiation. The switch from the mitotic to endocycles is regulated by Ccs52A, a plant orthologue of the yeast and animal Cdhl proteins, acting as substrate-specific activator of the anaphase-promoting complex E3 ubiquitin ligase. Here, several aspects of endoreduplication are discussed with special attention on nitrogen-fixing nodule development where endoreduplication is an integral part of symbiotic cell differentiation.

Anaphase-Promoting Complex-Cyclosome↗
PubMed2004

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.

Amino Acid Sequence↗
PubMed2004

Two classes of the CDh1-type activators of the anaphase-promoting complex in plants: novel functional domains and distinct regulation.

The Cdc20 and Cdh1/Fzr proteins are the substrate-specific activators of the anaphase-promoting complex (APC). In Medicago truncatula, the MtCcs52A and MtCcs52B proteins represent two subgroups of the Cdh1-type activators, which display differences in their cell cycle regulation, structure, and function. The ccs52A transcripts are present in all phases of the cell cycle. By contrast, expression of ccs52B is restricted to late G2-phase and M-phase, and its induced overexpression in BY2 cells inhibited mitosis. MtCcs52A is active in Schizosaccharomyces pombe and binds to the S. pombe APC, whereas MtCcs52B does not because of differences in the N-terminal region. We identified a new functional domain, the Cdh1-specific motif conserved in the Cdh1 proteins that, in addition to the C-box and the terminal Ile and Arg residues, was essential for the activity and required for efficient binding to the APC. Moreover, we demonstrate that cyclin-dependent kinase phosphorylation sites adjacent to the C-box may regulate the interaction with the APC. In the different plant organs, the expression of Mtccs52A and Mtccs52B displayed differences and indicated the involvement of the APC in differentiation processes.

Amino Acid Sequence↗
PubMed2004

Phytohormonal responses in enod40-overexpressing plants of Medicago truncatula and rice.

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.

Journal Article↗
PubMed2004

From pollen tubes to infection threads: recruitment of Medicago floral pectic genes for symbiosis.

While the biology of nitrogen-fixing root nodules has been extensively studied, little is known about the evolutionary events that predisposed legume plants to form symbiosis with rhizobia. We have studied the presence and the expression of two pectic gene families in Medicago, polygalacturonases (PGs) and pectin methyl esterases (PMEs) during the early steps of the Sinorhizobium meliloti-Medicago interaction and compared them with related pollen-specific genes. First, we have compared the expression of MsPG3, a PG gene specifically expressed during the symbiotic interaction, with the expression of MsPG11, a highly homologous pollen-specific gene, using promoter-gus fusions in transgenic M. truncatula and tobacco plants. These results demonstrated that the symbiotic promoter functions as a pollen-specific promoter in the non-legume host. Second, we have identified the presence of a gene family of at least eight differentially expressed PMEs in Medicago. One subfamily is represented by one symbiotic gene (MtPER) and two pollen-expressed genes (MtPEF1 and MtPEF2) that are clustered in the M. truncatula genome. The promoter-gus studies presented in this work and the homology between plant PGs, together with the analysis of the PME locus structure and MtPER expression studies, suggest that the symbiotic MsPG3 and MtPER could have as ancestors pollen-expressed genes involved in polar tip growth processes during pollen tube elongation. Moreover, they could have been recruited after gene duplication in the symbiotic interaction to facilitate polar tip growth during infection thread formation.

Carboxylic Ester Hydrolases↗
PubMed2003

Nodule initiation involves the creation of a new symplasmic field in specific root cells of medicago species.

The organogenesis of nitrogen-fixing nodules in legume plants is initiated in specific root cortical cells and regulated by long-distance signaling and carbon allocation. Here, we explore cell-to-cell communication processes that occur during nodule initiation in Medicago species and their functional relevance using a combination of fluorescent tracers, electron microscopy, and transgenic plants. Nodule initiation induced symplasmic continuity between the phloem and nodule initials. Macromolecules such as green fluorescent protein could traffic across short or long distances from the phloem into these primordial cells. The created symplasmic field was regulated throughout nodule development. Furthermore, Medicago truncatula transgenic plants expressing a viral movement protein showed increased nodulation. Hence, the establishment of this symplasmic field may be a critical element for the control of nodule organogenesis.

Biological Transport↗
PubMed2003

Ankyrin protein kinases: a novel type of plant kinase gene whose expression is induced by osmotic stress in alfalfa.

Interaction between Medicago spp. and Sinorhizobium meliloti leads to the development of a novel organ, the root nodule. A gene, Msapk1, encoding a novel type of plant protein kinase containing a N-terminal region with an ankyrin domain, was identified and shown to be expressed both in S. meliloti-infected and spontaneous nodules in alfalfa. This gene is not exclusively associated to nodulation since its expression was detected in other plant organs. Several genes coding for ankyrin protein kinases (APKs) were detected in various plants and animals. Three closest A. thaliana homologues of Msapk1 were identified in databases and two of them were shown to express differentially in various organs using gene-specific RT-PCR. In contrast, Southern analysis suggests that a single-copy gene exists in diploid M. truncatula. By screening a M. truncatula BAC library the Mtapk1 genomic region was isolated and sequenced. Two neighbouring genes showing homologies to previously identified sequences in data banks were detected in the vicinity of the Mtapk1 gene and compared to similar regions of the three Atapk genes. The distribution of exons/introns was the same for all expressed genes of both species although Mtapk1 contained larger introns. Upon osmotic stress Msapk1 expression was induced in roots of alfalfa starting from three hours up to two days of treatment. These data suggest that Msapk1, involved in alfalfa osmotic stress responses, belongs to a novel class of plant protein kinases.

Amino Acid Sequence↗
PubMed2003

Efficient transposition of the Tnt1 tobacco retrotransposon in the model legume Medicago truncatula.

The tobacco element, Tnt1, is one of the few active retrotransposons in plants. Its transposition is activated during protoplast culture in tobacco and tissue culture in the heterologous host Arabidopsis thaliana. Here, we report its transposition in the R108 line of Medicago truncatula during the early steps of the in vitro transformation-regeneration process. Two hundred and twenty-five primary transformants containing Tnt1 were obtained. Among them, 11.2% contained only transposed copies of the element, indicating that Tnt1 transposed very early and efficiently during the in vitro transformation process, possibly even before the T-DNA integration. The average number of insertions per transgenic line was estimated to be about 15. These insertions were stable in the progeny and could be separated by segregation. Inspection of the sequences flanking the insertion sites revealed that Tnt1 had no insertion site specificity and often inserted in genes (one out of three insertions). Thus, our work demonstrates the functioning of an efficient transposable element in leguminous plants. These results indicate that Tnt1 can be used as a powerful tool for insertion mutagenesis in M. truncatula.

Culture Techniques↗
PubMed2003

Expression of MsPG3-GFP fusions in Medicago truncatula'hairy roots' reveals preferential tip localization of the protein in root hairs.

Tip growth is a specialized type of polar growth where new cell wall is deposited in a localized region of the cell, the growing tip. These cells show a characteristic zonation, with a high accumulation of secretory vesicles containing cell wall components at the tip, followed by an organelle-enriched zone. MsPG3 is a Medicago sativa polygalacturonase gene isolated in our laboratory, specifically expressed during the interaction of this plant with its symbiotic partner Sinorhizobium meliloti and which might participate in tip growth processes during symbiosis. We have used MsPG3-GFP fusions to study in vivo protein transport processes and localization during root hair growth. Different MsPG3-GFP fusions were expressed in Medicago truncatula'hairy roots' following a protocol developed for this study and also tested by transient expression in onion epidermal cells. Preferential accumulation of an MsPG3-GFP fusion protein in the tip of the growing root hair at different developmental stages was found, confirming the delivery of MsPG3 to the newly synthesized cell wall. This indicates that this protein may participate in tip growth processes during symbiosis and, in addition, that this fusion could be a useful tool to study this process in plants.

Gene Transfer Techniques↗
PubMed2003

The Medicago species A2-type cyclin is auxin regulated and involved in meristem formation but dispensable for endoreduplication-associated developmental programs.

Phytohormones as well as temporal and spatial regulation of the cell cycle play a key role in plant development. Here, we investigated the function and regulation of an alfalfa (Medicago sativa) A2-type cyclin in three distinct root developmental programs: in primary and secondary root development, nodule development, and nematode-elicited gall formation. Using transgenic plants carrying the Medsa;cycA2;2 promoter-beta-glucuronidase gene fusion, in combination with other techniques, cycA2;2 expression was localized in meristems and proliferating cells in the lateral root and nodule primordia. Rapid induction of cycA2;2 by Nod factors demonstrated that this gene is implicated in cell cycle activation of differentiated cells developing to nodule primordia. Surprisingly, cycA2;2 was repressed in the endoreduplicating, division-arrested cells both during nodule development and formation of giant cells in nematode-induced galls, indicating that CycA2;2 was dispensable for S-phase in endoreduplication cycles. Overexpression of cycA2;2 in transgenic plants corresponded to wild type protein levels and had no apparent phenotype. In contrast, antisense expression of cycA2;2 halted regeneration of somatic embryos, suggesting a role for CycA2;2 in the formation or activity of apical meristems. Expression of cycA2;2 was up-regulated by auxins, as expected from the presence of auxin response elements in the promoter. Moreover, auxin also affected the spatial expression pattern of this cyclin by shifting the cycA2;2 expression from the phloem to the xylem poles.

Animals↗
PubMed2003

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.

Amino Acid Sequence↗
PubMed2003

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 beta-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.

Anaphase-Promoting Complex-Cyclosome↗