Jens Stougaard

Publications of Jens Stougaard

• Genome-wide LORE1 retrotransposon mutagenesis and high-throughput insertion detection in Lotus japonicus.

  Authors: Dorian Fabian Urbański, Anna Małolepszy, Jens Stougaard, Stig Uggerhøj Andersen

  The Plant journal : for cell and molecular biology. 02/2012; 69(4):731-41.

  Use of insertion mutants facilitates functional analysis of genes, but it has been difficult to identify a suitable mutagen and to establish large populations for reverse genetics in most plantUse of insertion mutants facilitates functional analysis of genes, but it has been difficult to identify a suitable mutagen and to establish large populations for reverse genetics in most plant species. The main challenge is developing efficient high-throughput procedures for both mutagenesis and identification of insertion sites. To date, only floral-dip T-DNA transformation of Arabidopsis has produced independent germinal insertions, thereby allowing generation of mutant populations from seeds of single plants. In addition, advances in insertion detection have been hampered by a lack of protocols, including software for automated data analysis, that take full advantage of high-throughput next-generation sequencing. We have addressed these challenges by developing the FSTpoolit protocol and software package, and here we demonstrate its efficacy by detecting 8935 LORE1 insertions in 3744 Lotus japonicus plants. The identified insertions show that the endogenous LORE1 retrotransposon is well suited for insertion mutagenesis due to homogenous gene targeting and exonic insertion preference. As LORE1 transposition occurs in the germline, harvesting seeds from a single founder line and cultivating progeny generates a complete mutant population. This ease of LORE1 mutagenesis, combined with the efficient FSTpoolit protocol, which exploits 2D pooling, Illumina sequencing and automated data analysis, allows highly cost-efficient development of a comprehensive reverse genetic resource.

• The integral membrane protein SEN1 is required for symbiotic nitrogen fixation in Lotus japonicus nodules.

  Authors: Tsuneo Hakoyama, Kaori Niimi, Takeshi Yamamoto, Sawa Isobe, Shusei Sato, Yasukazu Nakamura, Satoshi Tabata, Hirotaka Kumagai, Yosuke Umehara, Katja Brossuleit, Thomas R Petersen, Niels Sandal, Jens Stougaard, Michael K Udvardi, Masanori Tamaoki, Masayoshi Kawaguchi, Hiroshi Kouchi, Norio Suganuma

  Plant & cell physiology. 11/2011; 53(1):225-36.

  Legume plants establish a symbiotic association with bacteria called rhizobia, resulting in the formation of nitrogen-fixing root nodules. A Lotus japonicus symbiotic mutant, sen1, forms nodules thatLegume plants establish a symbiotic association with bacteria called rhizobia, resulting in the formation of nitrogen-fixing root nodules. A Lotus japonicus symbiotic mutant, sen1, forms nodules that are infected by rhizobia but that do not fix nitrogen. Here, we report molecular identification of the causal gene, SEN1, by map-based cloning. The SEN1 gene encodes an integral membrane protein homologous to Glycine max nodulin-21, and also to CCC1, a vacuolar iron/manganese transporter of Saccharomyces cerevisiae, and VIT1, a vacuolar iron transporter of Arabidopsis thaliana. Expression of the SEN1 gene was detected exclusively in nodule-infected cells and increased during nodule development. Nif gene expression as well as the presence of nitrogenase proteins was detected in rhizobia from sen1 nodules, although the levels of expression were low compared with those from wild-type nodules. Microscopic observations revealed that symbiosome and/or bacteroid differentiation are impaired in the sen1 nodules even at a very early stage of nodule development. Phylogenetic analysis indicated that SEN1 belongs to a protein clade specific to legumes. These results indicate that SEN1 is essential for nitrogen fixation activity and symbiosome/bacteroid differentiation in legume nodules.

• Cytokinin induction of root nodule primordia in Lotus japonicus is regulated by a mechanism operating in the root cortex.

  Authors: Anne Birgitte Heckmann, Niels Sandal, Anita Søndergaard Bek, Lene Heegaard Madsen, Anna Jurkiewicz, Mette Wibroe Nielsen, Leila Tirichine, Jens Stougaard

  Molecular plant-microbe interactions : MPMI. 07/2011; 24(11):1385-95.

  Cytokinin plays a central role in the formation of nitrogen-fixing root nodules following inoculation with rhizobia. We show that exogenous cytokinin induces formation of discrete and easily visibleCytokinin plays a central role in the formation of nitrogen-fixing root nodules following inoculation with rhizobia. We show that exogenous cytokinin induces formation of discrete and easily visible nodule primordia in Lotus japonicus roots. The expression of nodulin genes was up-regulated upon cytokinin treatment, suggesting that the genuine nodulation program was indeed activated. This offers a simple approach for dissecting the underlying mechanism. Cytokinin-induced nodule primordia formation was unperturbed in several loss-of-function mutants impaired in epidermal responses to either rhizobial infection, Nod factor application, or both. However, absence of primordia in nsp1, nsp2, and nin mutants showed the requirement for these transcriptional regulators in the cytokinin-mediated activation of the root cortex. Distinguishing the epidermal and cortical responses further, we found that external cytokinin application induced expression of the Nin::GUS reporter gene within the root cortex but not in the root epidermis. Using L. japonicus lhk1-1 and har1 mutants, we demonstrate that discrete activation of root cortical cells by cytokinin depends on the LHK1 cytokinin receptor and is subjected to HAR1-mediated autoregulation.

• Five phosphonate operon gene products as components of a multi-subunit complex of the carbon-phosphorus lyase pathway.

  Authors: Bjarne Jochimsen, Signe Lolle, Fern R McSorley, Mariah Nabi, Jens Stougaard, David L Zechel, Bjarne Hove-Jensen

  Proceedings of the National Academy of Sciences of the United States of America. 06/2011; 108(28):11393-8.

  Organophosphonate utilization by Escherichia coli requires the 14 cistrons of the phnCDEFGHIJKLMNOP operon, of which the carbon-phosphorus lyase has been postulated to consist of the sevenOrganophosphonate utilization by Escherichia coli requires the 14 cistrons of the phnCDEFGHIJKLMNOP operon, of which the carbon-phosphorus lyase has been postulated to consist of the seven polypeptides specified by phnG to phnM. A 5,660-bp DNA fragment encompassing phnGHIJKLM is cloned, followed by expression in E. coli and purification of Phn-polypeptides. PhnG, PhnH, PhnI, PhnJ, and PhnK copurify as a protein complex by ion-exchange, size-exclusion, and affinity chromatography. The five polypeptides also comigrate in native-PAGE. Cross-linking of the purified protein complex reveals a close proximity of PhnG, PhnI, PhnJ, and PhnK, as these subunits disappear concomitant with the formation of large cross-linked protein complexes. Two molecular forms are identified, a major form of molecular mass of approximately 260 kDa, a minor form of approximately 640 kDa. The stoichiometry of the protein complex is suggested to be PhnG(4)H(2)I(2)J(2)K. Deletion of individual phn genes reveals that a strain harboring plasmid-borne phnGHIJ produces a protein complex consisting of PhnG, PhnH, PhnI, and PhnJ, whereas a strain harboring plasmid-borne phnGIJK produces a protein complex consisting of PhnG and PhnI. We conclude that phnGHIJK specify a soluble multisubunit protein complex essential for organophosphonate utilization.

• Autophosphorylation is essential for the in vivo function of the Lotus japonicus Nod factor receptor 1 and receptor-mediated signalling in cooperation with Nod factor receptor 5.

  Authors: Esben B Madsen, Meritxell Antolín-Llovera, Christina Grossmann, Juanying Ye, Syndi Vieweg, Angelique Broghammer, Lene Krusell, Simona Radutoiu, Ole N Jensen, Jens Stougaard, Martin Parniske

  The Plant journal : for cell and molecular biology. 02/2011; 65(3):404-17.

  Soil-living rhizobia secrete lipochitin oligosaccharides known as Nod factors, which in Lotus japonicus are perceived by at least two Nod-factor receptors, NFR1 and NFR5. Despite progress inSoil-living rhizobia secrete lipochitin oligosaccharides known as Nod factors, which in Lotus japonicus are perceived by at least two Nod-factor receptors, NFR1 and NFR5. Despite progress in identifying molecular components critical for initial legume host recognition of the microsymbiont and cloning of downstream components, little is known about the activation and signalling mechanisms of the Nod-factor receptors themselves. Here we show that both receptor proteins localize to the plasma membrane, and present evidence for heterocomplex formation initiating downstream signalling. Expression of NFR1 and NFR5 in Nicotiana benthamiana and Allium ampeloprasum (leek) cells caused a rapid cell-death response. The signalling leading to cell death was abrogated using a kinase-inactive variant of NFR1. In these surviving cells, a clear interaction between NFR1 and NFR5 was detected in vivo through bimolecular fluorescence complementation (BiFC). To analyse the inter- and intramolecular phosphorylation events of the kinase complex, the cytoplasmic part of NFR1 was assayed for in vitro kinase activity, and autophosphorylation on 24 amino acid residues, including three tyrosine residues, was found by mass spectrometry. Substitution of the phosphorylated amino acids of NFR1 identified a single phosphorylation site to be essential for NFR1 Nod-factor signalling in vivo and kinase activity in vitro. In contrast to NFR1, no in vitro kinase activity of the cytoplasmic domain of NFR5 was detected. This is further supported by the fact that a mutagenized NFR5 construct, substituting an amino acid essential for ATP binding, restored nodulation of nfr5 mutant roots.

• The Clavata2 genes of pea and Lotus japonicus affect autoregulation of nodulation.

  Authors: Lene Krusell, Naoto Sato, Izumi Fukuhara, Bjørn E V Koch, Christina Grossmann, Satoru Okamoto, Erika Oka-Kira, Yoko Otsubo, Grégoire Aubert, Tomomi Nakagawa, Shusei Sato, Satoshi Tabata, Gerard Duc, Martin Parniske, Trevor L Wang, Masayoshi Kawaguchi, Jens Stougaard

  The Plant journal : for cell and molecular biology. 12/2010; 65(6):861-71.

  The number of root nodules developing on legume roots after rhizobial infection is controlled by the plant shoot through autoregulation and mutational inactivation of this mechanism leads toThe number of root nodules developing on legume roots after rhizobial infection is controlled by the plant shoot through autoregulation and mutational inactivation of this mechanism leads to hypernodulation. We have characterised the Pisum sativum (pea) Sym28 locus involved in autoregulation and shown that it encodes a protein similar to the Arabidopsis CLAVATA2 (CLV2) protein. Inactivation of the PsClv2 gene in four independent sym28 mutant alleles, carrying premature stop codons, results in hypernodulation of the root and changes to the shoot architecture. In the reproductive phase sym28 shoots develops additional flowers, the stem fasciates, and the normal phyllotaxis is perturbed. Mutational substitution of an amino acid in one leucine rich repeat of the corresponding Lotus japonicus LjCLV2 protein results in increased nodulation. Similarly, down-regulation of the Lotus Clv2 gene by RNAi mediated reduction of the transcript level also resulted in increased nodulation. Gene expression analysis of LjClv2 and Lotus hypernodulation aberrant root formation Har1 (previously shown to regulate nodule numbers) indicated they have overlapping organ expression patterns. However, we were unable to demonstrate a direct protein-protein interaction between LjCLV2 and LjHAR1 proteins in contrast to the situation between equivalent proteins in Arabidopsis. LjHAR1 was localised to the plasma membrane using a YFP fusion whereas LjCLV2-YFP localised to the endoplasmic reticulum when transiently expressed in Nicotiana benthamiana leaves. This finding is the most likely explanation for the lack of interaction between these two proteins.

• Proteome analysis of pod and seed development in the model legume Lotus japonicus.

  Authors: Gitte Nautrup-Pedersen, Svend Dam, Brian S Laursen, Astrid L Siegumfeldt, Kasper Nielsen, Nicolas Goffard, Hans Henrik Stærfeldt, Carsten Friis, Shusei Sato, Satoshi Tabata, Andrea Lorentzen, Peter Roepstorff, Jens Stougaard

  Journal of proteome research. 11/2010; 9(11):5715-26.

  Legume pods serve important functions during seed development and are themselves sources of food and feed. Compared to seeds, the metabolism and development of pods are not well-defined. The presentLegume pods serve important functions during seed development and are themselves sources of food and feed. Compared to seeds, the metabolism and development of pods are not well-defined. The present characterization of pods from the model legume Lotus japonicus, together with the detailed analyses of the pod and seed proteomes in five developmental stages, paves the way for comparative pathway analysis and provides new metabolic information. Proteins were analyzed by two-dimensional gel electrophoresis and tandem-mass spectrometry. These analyses lead to the identification of 604 pod proteins and 965 seed proteins, including 263 proteins distinguishing the pod. The complete data set is publicly available at http://www.cbs.dtu.dk/cgi-bin/lotus/db.cgi , where spots in a reference map are linked to experimental data, such as matched peptides, quantification values, and gene accessions. Identified pod proteins represented enzymes from 85 different metabolic pathways, including storage globulins and a late embryogenesis abundant protein. In contrast to seed maturation, pod maturation was associated with decreasing total protein content, especially proteins involved in protein biosynthesis and photosynthesis. Proteins detected only in pods included three enzymes participating in the urea cycle and four in nitrogen and amino group metabolism, highlighting the importance of nitrogen metabolism during pod development. Additionally, five legume seed proteins previously unassigned in the glutamate metabolism pathway were identified.

• Common and not so common symbiotic entry.

  Authors: Mark Held, Md Shakhawat Hossain, Keisuke Yokota, Paola Bonfante, Jens Stougaard, Krzysztof Szczyglowski

  Trends in plant science. 10/2010; 15(10):540-5.

  Great advances have been made in our understanding of the host plant's common symbiosis functions, which in legumes mediate intracellular accommodation of both nitrogen-fixing bacteria and arbuscularGreat advances have been made in our understanding of the host plant's common symbiosis functions, which in legumes mediate intracellular accommodation of both nitrogen-fixing bacteria and arbuscular mycorrhiza (AM) fungi. However, it has become apparent that additional plant genes are required specifically for bacterial entry inside the host root. In this opinion article, we consider Lotus japonicus nap1 and pir1 symbiotic mutants within the context of other deleterious mutations that impair an intracellular accommodation of bacteria but have no impact on the colonization of roots by AM fungi. We highlight a clear delineation of early signaling events during bacterial versus AM symbioses while suggesting a more intricate origin of the plant's ability for intracellular accommodation of bacteria.

• The molecular network governing nodule organogenesis and infection in the model legume Lotus japonicus.

  Authors: Lene H Madsen, Leïla Tirichine, Anna Jurkiewicz, John T Sullivan, Anne B Heckmann, Anita S Bek, Clive W Ronson, Euan K James, Jens Stougaard

  Nature communications. 04/2010; 1:10.

  Bacterial infection of interior tissues of legume root nodules is controlled at the epidermal cell layer and is closely coordinated with progressing organ development. Using spontaneous nodulatingBacterial infection of interior tissues of legume root nodules is controlled at the epidermal cell layer and is closely coordinated with progressing organ development. Using spontaneous nodulating Lotus japonicus plant mutants to uncouple nodule organogenesis from infection, we have determined the role of 16 genes in these two developmental processes. We show that host-encoded mechanisms control three alternative entry processes operating in the epidermis, the root cortex and at the single cell level. Single cell infection did not involve the formation of trans-cellular infection threads and was independent of host Nod-factor receptors and bacterial Nod-factor signals. In contrast, Nod-factor perception was required for epidermal root hair infection threads, whereas primary signal transduction genes preceding the secondary Ca2+ oscillations have an indirect role. We provide support for the origin of rhizobial infection through direct intercellular epidermal invasion and subsequent evolution of crack entry and root hair invasions observed in most extant legumes.

• Evolution and regulation of the Lotus japonicus LysM receptor gene family.

  Authors: Gitte Vestergaard Lohmann, Yoshikazu Shimoda, Mette Wibroe Nielsen, Frank Grønlund Jørgensen, Christina Grossmann, Niels Sandal, Kirsten Sørensen, Søren Thirup, Lene Heegaard Madsen, Satoshi Tabata, Shusei Sato, Jens Stougaard, Simona Radutoiu

  Molecular plant-microbe interactions : MPMI. 04/2010; 23(4):510-21.

  LysM receptor kinases were identified as receptors of acylated chitin (Nod factors) or chitin produced by plant-interacting microbes. Here, we present the identification and characterization of theLysM receptor kinases were identified as receptors of acylated chitin (Nod factors) or chitin produced by plant-interacting microbes. Here, we present the identification and characterization of the LysM receptor kinase gene (Lys) family (17 members) in Lotus japonicus. Comprehensive phylogenetic analysis revealed a correlation between Lys gene structure and phylogeny. Further mapping coupled with sequence-based anchoring on the genome showed that the family has probably expanded by a combination of tandem and segmental duplication events. Using a sliding-window approach, we identified distinct regions in the LysM and kinase domains of recently diverged Lys genes where positive selection may have shaped ligand interaction. Interestingly, in the case of NFR5 and its closest paralog, LYS11, one of these regions coincides with the predicted Nod-factor binding groove and the suggested specificity determining area of the second LysM domain. One hypothesis for the evolutionary diversification of this receptor family in legumes is their unique capacity to decipher various structures of chitin-derived molecules produced by an extended spectrum of interacting organisms: symbiotic, associative, endophytic, and parasitic. In a detailed expression analysis, we found several Lotus Lys genes regulated not only during the symbiotic association with Mesorhizobium loti but also in response to chitin treatment.

• Improved Characterization of Nod Factors and Genetically Based Variation in LysM Receptor Domains Identify Amino Acids Expendable for Nod Factor Recognition in Lotus spp.

  Authors: Anita S Bek, Jørgen Sauer, Mikkel B Thygesen, Jens Ø Duus, Bent O Petersen, Søren Thirup, Euan James, Knud J Jensen, Jens Stougaard, Simona Radutoiu

  Molecular plant-microbe interactions : MPMI. 01/2010; 23(1):58-66.

  Formation of functional nodules is a complex process depending on host-microsymbiont compatibility in all developmental stages. This report uses the contrasting symbiotic phenotypes of LotusFormation of functional nodules is a complex process depending on host-microsymbiont compatibility in all developmental stages. This report uses the contrasting symbiotic phenotypes of Lotus japonicus and L. pedunculatus, inoculated with Mesorhizobium loti or the Bradyrhizobium sp. (Lotus), to investigate the role of Nod factor structure and Nod factor receptors (NFR) for rhizobial recognition, infection thread progression, and bacterial persistence within nodule cells. A key contribution was the use of 800 MHz nuclear magnetic resonance spectroscopy and ultrahigh-performance liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry for Nod factor analysis. The Nod factor decorations at the nonreducing end differ between Bradyrhizobium sp. (Lotus) and M. loti, and the NFR1/NFR5 extracellular regions of L. pedunculatus and L. japonicus were found to vary in amino acid composition. Genetic transformation experiments using chimeric and wild-type receptors showed that both receptor variants recognize the structurally different Nod factors but the later symbiotic phenotype remained unchanged. These results highlight the importance of additional checkpoints during nitrogen-fixing symbiosis and define several amino acids in the LysM domains as expendable for perception of the two differentially carbamoylated Nod factors.

• Derepression of the plant Chromovirus LORE1 induces germline transposition in regenerated plants.

  Authors: Eigo Fukai, Yosuke Umehara, Shusei Sato, Makoto Endo, Hiroshi Kouchi, Makoto Hayashi, Jens Stougaard, Hirohiko Hirochika

  PLoS genetics. 01/2010; 6(3):e1000868.

  Transposable elements represent a large proportion of the eukaryotic genomes. Long Terminal Repeat (LTR) retrotransposons are very abundant and constitute the predominant family of transposableTransposable elements represent a large proportion of the eukaryotic genomes. Long Terminal Repeat (LTR) retrotransposons are very abundant and constitute the predominant family of transposable elements in plants. Recent studies have identified chromoviruses to be a widely distributed lineage of Gypsy elements. These elements contain chromodomains in their integrases, which suggests a preference for insertion into heterochromatin. In turn, this preference might have contributed to the patterning of heterochromatin observed in host genomes. Despite their potential importance for our understanding of plant genome dynamics and evolution, the regulatory mechanisms governing the behavior of chromoviruses and their activities remain largely uncharacterized. Here, we report a detailed analysis of the spatio-temporal activity of a plant chromovirus in the endogenous host. We examined LORE1a, a member of the endogenous chromovirus LORE1 family from the model legume Lotus japonicus. We found that this chromovirus is stochastically de-repressed in plant populations regenerated from de-differentiated cells and that LORE1a transposes in the male germline. Bisulfite sequencing of the 5' LTR and its surrounding region suggests that tissue culture induces a loss of epigenetic silencing of LORE1a. Since LTR promoter activity is pollen specific, as shown by the analysis of transgenic plants containing an LTR::GUS fusion, we conclude that male germline-specific LORE1a transposition in pollen grains is controlled transcriptionally by its own cis-elements. New insertion sites of LORE1a copies were frequently found in genic regions and show no strong insertional preferences. These distinctive novel features of LORE1 indicate that this chromovirus has considerable potential for generating genetic and epigenetic diversity in the host plant population. Our results also define conditions for the use of LORE1a as a genetic tool.

• Nodulation Gene Mutants of Mesorhizobium loti R7A-nodZ and nolL Mutants Have Host-Specific Phenotypes on Lotus spp.

  Authors: Patsarin Rodpothong, John T Sullivan, Kriangsak Songsrirote, David Sumpton, Kenneth W J-T Cheung, Jane Thomas-Oates, Simona Radutoiu, Jens Stougaard, Clive W Ronson

  Molecular plant-microbe interactions : MPMI. 12/2009; 22(12):1546-54.

  Rhizobial Nod factors induce plant responses and facilitate bacterial infection, leading to the development of nitrogen-fixing root nodules on host legumes. Nodule initiation is highly dependent onRhizobial Nod factors induce plant responses and facilitate bacterial infection, leading to the development of nitrogen-fixing root nodules on host legumes. Nodule initiation is highly dependent on Nod-factor structure and, hence, on at least some of the nodulation genes that encode Nod-factor production. Here, we report the effects of mutations in Mesorhizobium loti R7A nodulation genes on nodulation of four Lotus spp. and on Nod-factor structure. Most mutants, including a DeltanodSDeltanolO double mutant that produced Nod factors lacking the carbamoyl and possibly N-methyl groups on the nonreducing terminal residue, were unaffected for nodulation. R7ADeltanodZ and R7ADeltanolL mutants that produced Nod factors without the (acetyl)fucose on the reducing terminal residue had a host-specific phenotype, forming mainly uninfected nodule primordia on Lotus filicaulis and L. corniculatus and effective nodules with a delay on L. japonicus. The mutants also showed significantly reduced infection thread formation and Nin gene induction. In planta complementation experiments further suggested that the acetylfucose was important for balanced signaling in response to Nod factor by the L. japonicus NFR1/NFR5 receptors. Overall the results reveal differences in the sensitivity of plant perception with respect to signaling leading to root hair deformation and nodule primordium development versus infection thread formation and rhizobial entry.

• CERBERUS, a novel U-box protein containing WD-40 repeats is required for infection thread formation and nodule development in the legume-Rhizobium symbiosis.

  Authors: Koji Yano, Satoshi Shibata, Wen-Li Chen, Shusei Sato, Takakazu Kaneko, Anna Jurkiewicz, Niels Sandal, Mari Banba, Haruko Imaizumi-Anraku, Tomoko Kojima, Ryo Ohtomo, Krzysztof Szczyglowski, Jens Stougaard, Satoshi Tabata, Makoto Hayashi, Hiroshi Kouchi, Yosuke Umehara

  The Plant journal : for cell and molecular biology. 07/2009;

  Endosymbiotic infection of legume plants by Rhizobium bacteria is initiated through infection threads (ITs) which are initiated within and penetrate from root hairs and deliver the endosymbionts intoEndosymbiotic infection of legume plants by Rhizobium bacteria is initiated through infection threads (ITs) which are initiated within and penetrate from root hairs and deliver the endosymbionts into nodule cells. Despite recent progress in understanding the mutual recognition and early symbiotic signaling cascades in host legumes, the molecular mechanisms underlying bacterial infection processes and successive nodule organogenesis are still poorly understood. We isolated a novel symbiotic mutant of Lotus japonicus, cerberus, which shows defects in IT formation and nodule organogenesis. Map-based cloning of the causal gene allowed us to identify the CERBERUS gene, which encodes a novel protein containing a U-box domain and WD-40 repeats. CERBERUS expression was detected in the roots and nodules, and enhanced after inoculation of Mesorhizobium loti. Strong expression was detected in developing nodule primordia and the infected zone of mature nodules. In cerberus mutants, Rhizobium colonized curled root hair tips, but hardly penetrated into root hair cells. The occasional ITs that were formed inside the root hair cells were mostly arrested within the epidermal cell layer. Nodule organogenesis was aborted prematurely, resulting in formation of a large number of small bumps which contained no endosymbiotic bacteria. These phenotypic and genetic analyses, together with comparisons to other legume mutants with defects in IT formation, indicate that CERBERUS plays a critical role in the very early steps of IT formation as well as for growth and differentiation of nodules.

• Dissection of symbiosis and organ development by integrated transcriptome analysis of lotus japonicus mutant and wild-type plants.

  Authors: Niels Høgslund, Simona Radutoiu, Lene Krusell, Vera Voroshilova, Matthew A Hannah, Nicolas Goffard, Diego H Sanchez, Felix Lippold, Thomas Ott, Shusei Sato, Satoshi Tabata, Poul Liboriussen, Gitte V Lohmann, Leif Schauser, Georg F Weiller, Michael K Udvardi, Jens Stougaard

  PloS one. 02/2009; 4(8):e6556.

  Genetic analyses of plant symbiotic mutants has led to the identification of key genes involved in Rhizobium-legume communication as well as in development and function of nitrogen fixing rootGenetic analyses of plant symbiotic mutants has led to the identification of key genes involved in Rhizobium-legume communication as well as in development and function of nitrogen fixing root nodules. However, the impact of these genes in coordinating the transcriptional programs of nodule development has only been studied in limited and isolated studies. Here, we present an integrated genome-wide analysis of transcriptome landscapes in Lotus japonicus wild-type and symbiotic mutant plants. Encompassing five different organs, five stages of the sequentially developed determinate Lotus root nodules, and eight mutants impaired at different stages of the symbiotic interaction, our data set integrates an unprecedented combination of organ- or tissue-specific profiles with mutant transcript profiles. In total, 38 different conditions sampled under the same well-defined growth regimes were included. This comprehensive analysis unravelled new and unexpected patterns of transcriptional regulation during symbiosis and organ development. Contrary to expectations, none of the previously characterized nodulins were among the 37 genes specifically expressed in nodules. Another surprise was the extensive transcriptional response in whole root compared to the susceptible root zone where the cellular response is most pronounced. A large number of transcripts predicted to encode transcriptional regulators, receptors and proteins involved in signal transduction, as well as many genes with unknown function, were found to be regulated during nodule organogenesis and rhizobial infection. Combining wild type and mutant profiles of these transcripts demonstrates the activation of a complex genetic program that delineates symbiotic nitrogen fixation. The complete data set was organized into an indexed expression directory that is accessible from a resource database, and here we present selected examples of biological questions that can be addressed with this comprehensive and powerful gene expression data set.

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• Rearrangement of Actin Cytoskeleton Mediates Invasion of Lotus japonicus Roots by Mesorhizobium loti.

  Authors: Keisuke Yokota, Eigo Fukai, Lene H Madsen, Anna Jurkiewicz, Paloma Rueda, Simona Radutoiu, Mark Held, Md Shakhawat Hossain, Krzysztof Szczyglowski, Giulia Morieri, Giles E D Oldroyd, J Allan Downie, Mette W. Nielsen, Anna Maria Rusek, Shusei Sato, Satoshi Tabata, Euan K James, Hiroshi Oyaizu, Niels Sandal, Jens Stougaard

  The Plant cell. 02/2009;

  Infection thread-dependent invasion of legume roots by rhizobia leads to internalization of bacteria into the plant cells, which is one of the salient features of root nodule symbiosis. We found thatInfection thread-dependent invasion of legume roots by rhizobia leads to internalization of bacteria into the plant cells, which is one of the salient features of root nodule symbiosis. We found that two genes, Nap1 (for Nck-associated protein 1) and Pir1 (for 121F-specific p53 inducible RNA), involved in actin rearrangements were essential for infection thread formation and colonization of Lotus japonicus roots by its natural microsymbiont, Mesorhizobium loti. nap1 and pir1 mutants developed an excess of uncolonized nodule primordia, indicating that these two genes were not essential for the initiation of nodule organogenesis per se. However, both the formation and subsequent progression of infection threads into the root cortex were significantly impaired in these mutants. We demonstrate that these infection defects were due to disturbed actin cytoskeleton organization. Short root hairs of the mutants had mostly transverse or web-like actin filaments, while bundles of actin filaments in wild-type root hairs were predominantly longitudinal. Corroborating these observations, temporal and spatial differences in actin filament organization between wild-type and mutant root hairs were also observed after Nod factor treatment, while calcium influx and spiking appeared unperturbed. Together with various effects on plant growth and seed formation, the nap1 and pir1 alleles also conferred a characteristic distorted trichome phenotype, suggesting a more general role for Nap1 and Pir1 in processes establishing cell polarity or polar growth in L. japonicus.

• The proteome of seed development in the model legume Lotus japonicus.

  Authors: Svend Dam, Brian S. Laursen, Jane H Ornfelt, Bjarne Jochimsen, Hans Henrik Stærfeldt, Carsten Friis, Kasper Nielsen, Nicolas Goffard, Soren Besenbacher, Lene Krusell, Shusei Sato, Satoshi Tabata, Ida B Thogersen, Jan J Enghild, Jens Stougaard

  Plant physiology. 02/2009;

  We have characterized the development of seeds in the model legume Lotus japonicus. Like soybean and pea, Lotus develops straight seed pods and each pod contains approximately 20 seeds that reachWe have characterized the development of seeds in the model legume Lotus japonicus. Like soybean and pea, Lotus develops straight seed pods and each pod contains approximately 20 seeds that reach maturity within 40 days. Histological sections show the characteristic three developmental phases of legume seeds and the presence of embryo, endosperm and seed coat in desiccated seeds. Furthermore, protein, oil, starch, phytic acid, and ash contents were determined and this indicates that the composition of mature Lotus seed is more similar to soybean than pea. In a first attempt to determine the seed proteome both a 2-D PAGE approach and a GeLC-MS/MS approach were used. Globulins were analyzed by 2-D PAGE and five legumins, LLP1-5, and two convicilins, LCP1-2, were identified by MALDI Q-TOF mass spectrometry. For two distinct developmental phases, seed filling and desiccation, a GeLC-MS/MS approach was used, and 665 and 181 unique proteins corresponding to gene accession numbers were identified for the two phases respectively. All the proteome data including the experimental data and MS spectra peaks were collected in a database that is available to the scientific community via a web-interface (http://www.cbs.dtu.dk/cgi-bin/lotus/db.cgi). This database establishes the basis for relating physiology, biochemistry and regulation of seed development in Lotus. Together with a new a web-interface (http://bioinfoserver.rsbs.anu.edu.au/utils/PathExpress4legumes/) collecting all protein identifications for Lotus, Medicago, and soybean seed proteomes, this database is a valuable resource for comparative seed proteomics and pathway analysis within and beyond the legume family.

• An analysis of synteny of Arachis with Lotus and Medicago sheds new light on the structure, stability and evolution of legume genomes.

  Authors: David Bertioli, Marcio Moretzsohn, Lene Madsen, Niels Sandal, Soraya Leal-Bertioli, Patricia Guimaraes, Birgit Hougaard, Jakob Fredslund, Leif Schauser, Anna Nielsen, Shusei Sato, Satoshi Tabata, Steven Cannon, Jens Stougaard

  BMC genomics. 02/2009; 10(1):45.

  ABSTRACT: BACKGROUND: Most agriculturally important legumes fall within two sub-clades of the Papilionoid legumes: the Phaseoloids and Galegoids, which diverged about 50 Mya. The Phaseoloids areABSTRACT: BACKGROUND: Most agriculturally important legumes fall within two sub-clades of the Papilionoid legumes: the Phaseoloids and Galegoids, which diverged about 50 Mya. The Phaseoloids are mostly tropical and include crops such as common bean and soybean. The Galegoids are mostly temperate and include clover, fava bean and the model legumes Lotus and Medicago (both with substantially sequenced genomes). In contrast, peanut (Arachis hypogaea) falls in the Dalbergioid clade which is more basal in its divergence within the Papilionoids. The aim of this work was to integrate the genetic map of Arachis with Lotus and Medicago and improve our understanding of the Arachis genome and legume genomes in general. To do this we placed on the Arachis map comparative anchor markers defined using a previously described bioinformatics pipeline. Also we investigated the possible role of transposons in the patterns of synteny that were observed. RESULTS: The Arachis genetic map was substantially aligned with Lotus and Medicago with most synteny blocks presenting a single main affinity to each genome. This indicates that the last common whole genome duplication within the Papilionoid legumes predated the divergence of Arachis from the Galegoids and Phaseoloids sufficiently that the common ancestral genome was substantially diploidized. The Arachis and model legume genomes comparison made here, together with a previously published comparison of Lotus and Medicago allowed all possible Arachis-Lotus-Medicago species by species comparisons to be made and genome syntenies observed. Distinct conserved synteny blocks and non-conserved regions were present in all genome comparisons, implying that certain legume genomic regions are consistently more stable during evolution than others. We found that in Medicago and possibly also in Lotus, retrotransposons tend to be more frequent in the variable regions. Furthermore, while these variable regions generally have lower densities of single copy genes than the more conserved regions, some harbor high densities of the fast evolving disease resistance genes. CONCLUSIONS: We suggest that gene space in Papilionoids may be divided into two broadly defined components: more conserved regions which tend to have low retrotransposon densities and are relatively stable during evolution; and variable regions that tend to have high retrotransposon densities, and whose frequent restructuring may fuel the evolution of some gene families.

• The pea sym37 receptor kinase gene controls infection-thread initiation and nodule development.

  Authors: Vladimir Zhukov, Simona Radutoiu, Lene H Madsen, Tamara Rychagova, Evgenia Ovchinnikova, Alex Borisov, Igor Tikhonovich, Jens Stougaard

  Molecular plant-microbe interactions : MPMI. 01/2009; 21(12):1600-8.

  Phenotypic characterization of pea symbiotic mutants has provided a detailed description of the symbiosis with Rhizobium leguminosarum bv. viciae strains. We show here that two allelic non-nodulatingPhenotypic characterization of pea symbiotic mutants has provided a detailed description of the symbiosis with Rhizobium leguminosarum bv. viciae strains. We show here that two allelic non-nodulating pea mutants, RisNod4 and K24, are affected in the PsSym37 gene, encoding a LysM receptor kinase similar to Lotus japonicus NFR1 and Medicago truncatula LYK3. Phenotypic analysis of RisNod4 and K24 suggests a role for the SYM37 in regulation of infection-thread initiation and nodule development from cortical-cell division foci. We show that RisNod4 plants carrying an L to F substitution in the LysM1 domain display a restrictive symbiotic phenotype comparable to the PsSym2(A) lines that distinguish 'European' and 'Middle East' Rhizobium leguminosarum bv. viciae strains. RisNod4 mutants develop nodules only in the presence of a 'Middle East' Rhizobium strain producing O-acetylated Nod factors indicating the SYM37 involvement in Nod-factor recognition. Along with the PsSym37, a homologous LysM receptor kinase gene, PsK1, was isolated and characterized. We show that PsK1 and PsSym37 are genetically linked to each other and to the PsSym2 locus. Allelic complementation analyses and sequencing of the extracellular regions of PsSym37 and PsK1 in several 'European' and 'Afghan' pea cultivars point towards PsK1 as possible candidate for the elusive PsSym2 gene.

• Transposition of a 600 thousand-year-old LTR retrotransposon in the model legume Lotus japonicus.

  Authors: Eigo Fukai, Alicja Dobrowolska, Lene Madsen, Esben Madsen, Yosuke Umehara, Hiroshi Kouchi, Hirohiko Hirochika, Jens Stougaard

  Plant molecular biology. 10/2008;

  We have identified a new Ty3-gypsy retrotransposon family named LORE2 (Lotus retrotransposon 2) and documented its activity in the model legume Lotus japonicus. Three new LORE2 insertions were foundWe have identified a new Ty3-gypsy retrotransposon family named LORE2 (Lotus retrotransposon 2) and documented its activity in the model legume Lotus japonicus. Three new LORE2 insertions were found in symbiotic mutant alleles isolated from a plant population, established by tissue culture mediated transformation of the L. japonicus Gifu accession. Low transcriptional and transpositional activities of LORE2 in cultured cells suggested that the LORE2 transpositions identified in the three symbiotic mutants occurred in intact plants, not in callus. Tracing of the transpositional events identified two active LORE2 members in Gifu. One of them named LORE2A possesses a deletion in its coding region and polymorphisms between intraelemental LTRs. LORE2A is thus an aged element, estimated as 600 thousand years old. Our findings indicate that plant genomes carry more cryptic LTR retrotransposons, i.e., aged yet active, than estimated before, and that these cryptic elements may have contributed to plant genome dynamics, for example, the burst of transpositions reported in several plant species.