Niels Sandal

Publications of Niels Sandal

• 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.

• Interplay of flg22-induced defence responses and nodulation in Lotus japonicus.

  Authors: Miguel Lopez-Gomez, Niels Sandal, Jens Stougaard, Thomas Boller

  Journal of experimental botany. 09/2011; 63(1):393-401.

  In this study the interplay between the symbiotic and defence signalling pathways in Lotus japonicus was investigated by comparing the responses to Mesorhizobium loti, the symbiotic partner of L.In this study the interplay between the symbiotic and defence signalling pathways in Lotus japonicus was investigated by comparing the responses to Mesorhizobium loti, the symbiotic partner of L. japonicus, and the elicitor flg22, a conserved peptide motif present in flagellar protein of a wide range of bacteria. It was found that defence and symbiotic pathways overlap in the interaction between L. japonicus and M. loti since similar responses were induced by the mutualistic bacteria and flg22. However, purified flagellin from M. loti did not induce any response in L. japonicus, which suggests the production of other elicitors by the symbiotic bacteria. Defence responses induced by flg22 caused inhibition of rhizobial infection and delay in nodule organogenesis, as demonstrated by the negative effect of flg22 in the formation of spontaneous nodules in the snf1 L. japonicus mutant, and the inhibition of NSP1 and NSP2 genes. This indicates the antagonistic effect of the defence pathway on the nodule formation in the initial rhizobium-legume interaction. However, the fact that flg22 did not affect the formation of new nodules once the symbiosis was established indicates that after the colonization of the host plant by the symbiotic partner, the symbiotic pathway has prevalence over the defensive response. This result is also supported by the down-regulation of the expression levels of the flg22 receptor FLS2 in the nodular tissue.

• 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.

• 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.

• 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.

• 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.

• 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.

• Legume anchor markers link syntenic regions between Phaseolus vulgaris, Lotus japonicus, Medicago truncatula and arachis.

  Authors: Birgit Kristine Hougaard, Lene Heegaard Madsen, Niels Sandal, Marcio de Carvalho Moretzsohn, Jakob Fredslund, Leif Schauser, Anna Marie Nielsen, Trine Rohde, Shusei Sato, Satoshi Tabata, David John Bertioli, Jens Stougaard

  Genetics. 09/2008; 179(4):2299-312.

  We have previously described a bioinformatics pipeline identifying comparative anchor-tagged sequence (CATS) loci, combined with design of intron-spanning primers. The derived anchor markers definingWe have previously described a bioinformatics pipeline identifying comparative anchor-tagged sequence (CATS) loci, combined with design of intron-spanning primers. The derived anchor markers defining the linkage position of homologous genes are essential for evaluating genome conservation among related species and facilitate transfer of genetic and genome information between species. Here we validate this global approach in the common bean and in the AA genome complement of the allotetraploid peanut. We present the successful conversion of approximately 50% of the bioinformatics-defined primers into legume anchor markers in bean and diploid Arachis species. One hundred and four new loci representing single-copy genes were added to the existing bean map. These new legume anchor-marker loci enabled the alignment of genetic linkage maps through corresponding genes and provided an estimate of the extent of synteny and collinearity. Extensive macrosynteny between Lotus and bean was uncovered on 8 of the 11 bean chromosomes and large blocks of macrosynteny were also found between bean and Medicago. This suggests that anchor markers can facilitate a better understanding of the genes and genetics of important traits in crops with largely uncharacterized genomes using genetic and genome information from related model plants.

• A gain-of-function mutation in a cytokinin receptor triggers spontaneous root nodule organogenesis.

  Authors: Leïla Tirichine, Niels Sandal, Lene H Madsen, Simona Radutoiu, Anita S Albrektsen, Shusei Sato, Erika Asamizu, Satoshi Tabata, Jens Stougaard

  Science (New York, N.Y.). 02/2007; 315(5808):104-7.

  Legume root nodules originate from differentiated cortical cells that reenter the cell cycle and form organ primordia. We show that perception of the phytohormone cytokinin is a key element in thisLegume root nodules originate from differentiated cortical cells that reenter the cell cycle and form organ primordia. We show that perception of the phytohormone cytokinin is a key element in this switch. Mutation of a Lotus japonicus cytokinin receptor gene leads to spontaneous development of root nodules in the absence of rhizobia or rhizobial signal molecules. The mutant histidine kinase receptor has cytokinin-independent activity and activates an Escherichia coli two-component phosphorelay system in vivo. Mutant analysis shows that cytokinin signaling is required for cell divisions that initiate nodule development and defines an autoregulated process where cytokinin induction of nodule stem cells is controlled by shoots.

• GeMprospector--online design of cross-species genetic marker candidates in legumes and grasses.

  Authors: Jakob Fredslund, Lene H Madsen, Birgit K Hougaard, Niels Sandal, Jens Stougaard, David Bertioli, Leif Schauser

  Nucleic acids research. 08/2006; 34(Web Server issue):W670-5.

  The web program GeMprospector (URL: http://cgi-www.daimi.au.dk/cgi-chili/GeMprospector/main) allows users to automatically design large sets of cross-species genetic marker candidates targetingThe web program GeMprospector (URL: http://cgi-www.daimi.au.dk/cgi-chili/GeMprospector/main) allows users to automatically design large sets of cross-species genetic marker candidates targeting either legumes or grasses. The user uploads a collection of ESTs from one or more legume or grass species, and they are compared with a database of clusters of homologous EST and genomic sequences from other legumes or grasses, respectively. Multiple sequence alignments between submitted ESTs and their homologues in the appropriate database form the basis of automated PCR primer design in conserved exons such that each primer set amplifies an intron. The only user input is a collection of ESTs, not necessarily from more than one species, and GeMprospector can boost the potential of such an EST collection by combining it with a large database to produce cross-species genetic marker candidates for legumes or grasses.

• Deregulation of a Ca2+/calmodulin-dependent kinase leads to spontaneous nodule development.

  Authors: Leïla Tirichine, Haruko Imaizumi-Anraku, Satoko Yoshida, Yasuhiro Murakami, Lene H Madsen, Hiroki Miwa, Tomomi Nakagawa, Niels Sandal, Anita S Albrektsen, Masayoshi Kawaguchi, Allan Downie, Shusei Sato, Satoshi Tabata, Hiroshi Kouchi, Martin Parniske, Shinji Kawasaki, Jens Stougaard

  Nature. 07/2006; 441(7097):1153-6.

  Induced development of a new plant organ in response to rhizobia is the most prominent manifestation of legume root-nodule symbiosis with nitrogen-fixing bacteria. Here we show that the complexInduced development of a new plant organ in response to rhizobia is the most prominent manifestation of legume root-nodule symbiosis with nitrogen-fixing bacteria. Here we show that the complex root-nodule organogenic programme can be genetically deregulated to trigger de novo nodule formation in the absence of rhizobia or exogenous rhizobial signals. In an ethylmethane sulphonate-induced snf1 (spontaneous nodule formation) mutant of Lotus japonicus, a single amino-acid replacement in a Ca2+/calmodulin-dependent protein kinase (CCaMK) is sufficient to turn fully differentiated root cortical cells into meristematic founder cells of root nodule primordia. These spontaneous nodules are genuine nodules with an ontogeny similar to that of rhizobial-induced root nodules, corroborating previous physiological studies. Using two receptor-deficient genetic backgrounds we provide evidence for a developmentally integrated spontaneous nodulation process that is independent of lipochitin-oligosaccharide signal perception and oscillations in Ca2+ second messenger levels. Our results reveal a key regulatory position of CCaMK upstream of all components required for cell-cycle activation, and a phenotypically divergent series of mutant alleles demonstrates positive and negative regulation of the process.

• Spontaneous root-nodule formation in the model legume Lotus japonicus: a novel class of mutants nodulates in the absence of rhizobia.

  Authors: Leïla Tirichine, Euan K James, Niels Sandal, Jens Stougaard

  Molecular plant-microbe interactions : MPMI. 05/2006; 19(4):373-82.

  Root-nodule development in legumes is an inducible developmental process initially triggered by perception of lipochitin-oligosaccharide signals secreted by the bacterial microsymbiont. In nature,Root-nodule development in legumes is an inducible developmental process initially triggered by perception of lipochitin-oligosaccharide signals secreted by the bacterial microsymbiont. In nature, rhizobial colonization and invasion of the legume root is therefore a prerequisite for formation of nitrogen-fixing root nodules. Here, we report isolation and characterization of chemically induced spontaneously nodulating mutants in a model legume amenable to molecular genetics. Six mutant lines of Lotus japonicus were identified in a screen for spontaneous nodule development under axenic conditions, i.e., in the absence of rhizobia. Spontaneous nodules do not contain rhizobia, bacteroids, or infection threads. Phenotypically, they resemble ineffective white nodules formed by some bacterial mutants on wild-type plants or certain plant mutants inoculated with wild-type Mesorhizobium loti. Spontaneous nodules formed on mutant lines show the ontogeny and characteristic histological features described for rhizobia-induced nodules on wild-type plants. Physiological responses to nitrate and ethylene are also maintained, as elevated levels inhibit spontaneous nodulation. Activation of the nodule developmental program in spontaneous nodules was shown for the early nodulin genes Enod2 and Nin, which are both upregulated in spontaneous nodules as well as in rhizobial nodules. Both monogenic recessive and dominant spontaneous nodule formation (snf) mutations were isolated in this mutant screen, and map positions were determined for three loci. We suggest that future molecular characterization of these mutants will identify key plant determinants involved in regulating nodulation and provide new insight into plant organ development.

• Genetics of symbiosis in Lotus japonicus: recombinant inbred lines, comparative genetic maps, and map position of 35 symbiotic loci.

  Authors: Niels Sandal, Thomas Rørby Petersen, Jeremy Murray, Yosuke Umehara, Bogumil Karas, Koji Yano, Hirotaka Kumagai, Makoto Yoshikawa, Katsuharu Saito, Masaki Hayashi [......] Shingo Hata, Norio Suganuma, Hiroshi Kouchi, Shinji Kawasaki, Satoshi Tabata, Makoto Hayashi, Martin Parniske, Krzysztof Szczyglowski, Masayoshi Kawaguchi, Jens Stougaard

  Molecular plant-microbe interactions : MPMI. 02/2006; 19(1):80-91.

  Development of molecular tools for the analysis of the plant genetic contribution to rhizobial and mycorrhizal symbiosis has provided major advances in our understanding of plant-microbeDevelopment of molecular tools for the analysis of the plant genetic contribution to rhizobial and mycorrhizal symbiosis has provided major advances in our understanding of plant-microbe interactions, and several key symbiotic genes have been identified and characterized. In order to increase the efficiency of genetic analysis in the model legume Lotus japonicus, we present here a selection of improved genetic tools. The two genetic linkage maps previously developed from an interspecific cross between L. japonicus Gifu and L. filicaulis, and an intraspecific cross between the two ecotypes L. japonicus Gifu and L. japonicus MG-20, were aligned through a set of anchor markers. Regions of linkage groups, where genetic resolution is obtained preferentially using one or the other parental combination, are highlighted. Additional genetic resolution and stabilized mapping populations were obtained in recombinant inbred lines derived by a single seed descent from the two populations. For faster mapping of new loci, a selection of reliable markers spread over the chromosome arms provides a common framework for more efficient identification of new alleles and new symbiotic loci among uncharacterized mutant lines. Combining resources from the Lotus community, map positions of a large collection of symbiotic loci are provided together with alleles and closely linked molecular markers. Altogether, this establishes a common genetic resource for Lotus spp. A web-based version will enable this resource to be curated and updated regularly.

• A general pipeline for the development of anchor markers for comparative genomics in plants.

  Authors: Jakob Fredslund, Lene H Madsen, Birgit K Hougaard, Anna Marie Nielsen, David Bertioli, Niels Sandal, Jens Stougaard, Leif Schauser

  BMC genomics. 02/2006; 7:207.

  BACKGROUND: Complete or near-complete genomic sequence information is presently only available for a few plant species representing a large phylogenetic diversity among plants. In order toBACKGROUND: Complete or near-complete genomic sequence information is presently only available for a few plant species representing a large phylogenetic diversity among plants. In order to effectively transfer this information to species lacking sequence information, comparative genomic tools need to be developed. Molecular markers permitting cross-species mapping along co-linear genomic regions are central to comparative genomics. These "anchor" markers, defining unique loci in genetic linkage maps of multiple species, are gene-based and possess a number of features that make them relatively sparse. To identify potential anchor marker sequences more efficiently, we have established an automated bioinformatic pipeline that combines multi-species Expressed Sequence Tags (EST) and genome sequence data. RESULTS: Taking advantage of sequence data from related species, the pipeline identifies evolutionarily conserved sequences that are likely to define unique orthologous loci in most species of the same phylogenetic clade. The key features are the identification of evolutionarily conserved sequences followed by automated design of intron-flanking Polymerase Chain Reaction (PCR) primer pairs. Polymorphisms can subsequently be identified by size- or sequence variation of PCR products, amplified from mapping parents or populations. We illustrate our procedure in legumes and grasses and exemplify its application in legumes, where model plant studies and the genome- and EST-sequence data available have a potential impact on the breeding of crop species and on our understanding of the evolution of this large and diverse family. CONCLUSION: We provide a database of 459 candidate anchor loci which have the potential to serve as map anchors in more than 18,000 legume species, a number of which are of agricultural importance. For grasses, the database contains 1335 candidate anchor loci. Based on this database, we have evaluated 76 candidate anchor loci with respect to marker development in legume species with no sequence information available, demonstrating the validity of this approach.

• A nucleoporin is required for induction of Ca2+ spiking in legume nodule development and essential for rhizobial and fungal symbiosis.

  Authors: Norihito Kanamori, Lene Heegaard Madsen, Simona Radutoiu, Mirela Frantescu, Esben M H Quistgaard, Hiroki Miwa, J Allan Downie, Euan K James, Hubert H Felle, Line Lindegaard Haaning, Torben Heick Jensen, Shusei Sato, Yasukazu Nakamura, Satoshi Tabata, Niels Sandal, Jens Stougaard

  Proceedings of the National Academy of Sciences of the United States of America. 02/2006; 103(2):359-64.

  Nuclear-cytoplasmic partitioning and traffic between cytoplasmic and nuclear compartments are fundamental processes in eukaryotic cells. Nuclear pore complexes mediate transport of proteins, RNAs andNuclear-cytoplasmic partitioning and traffic between cytoplasmic and nuclear compartments are fundamental processes in eukaryotic cells. Nuclear pore complexes mediate transport of proteins, RNAs and ribonucleoprotein particles in and out of the nucleus. Here we present positional cloning of a plant nucleoporin gene, Nup133, essential for a symbiotic signal transduction pathway shared by Rhizobium bacteria and mycorrhizal fungi. Mutation of Nup133 results in a temperature sensitive nodulation deficient phenotype and absence of mycorrhizal colonization. Root nodules developing with reduced frequency at permissive temperatures are ineffective and electron microscopy show that Rhizobium bacteria are not released from infection threads. Measurement of ion fluxes using a calcium-sensitive dye show that Nup133 is required for the Ca2+ spiking normally detectable within minutes after application of purified rhizobial Nod-factor signal molecules to root hairs. Localization of NUP133 in the nuclear envelope of root cells and root hair cells shown with enhanced yellow fluorescent protein fusion proteins suggests a novel role for NUP133 nucleoporins in a rapid nuclear-cytoplasmic communication after host-plant recognition of symbiotic microbes. Our results identify a component of an intriguing signal process requiring interaction at the cell plasma membrane and at intracellular nuclear and plastid organelle-membranes to induce a second messenger.

• A general pipeline for the development of anchor markers for comparative genomics in plants

  Authors: Jakob Fredslund, Lene Madsen, Birgit Hougaard, Anna Nielsen, David Bertioli, Niels Sandal, Jens Stougaard, Leif Schauser

  BMC Genomics. 01/2006;

  Abstract Background Complete or near-complete genomic sequence information is presently only available for a few plant species representing a large phylogenetic diversity among plants. In order toAbstract Background Complete or near-complete genomic sequence information is presently only available for a few plant species representing a large phylogenetic diversity among plants. In order to effectively transfer this information to species lacking sequence information, comparative genomic tools need to be developed. Molecular markers permitting cross-species mapping along co-linear genomic regions are central to comparative genomics. These "anchor" markers, defining unique loci in genetic linkage maps of multiple species, are gene-based and possess a number of features that make them relatively sparse. To identify potential anchor marker sequences more efficiently, we have established an automated bioinformatic pipeline that combines multi-species Expressed Sequence Tags (EST) and genome sequence data. Results Taking advantage of sequence data from related species, the pipeline identifies evolutionarily conserved sequences that are likely to define unique orthologous loci in most species of the same phylogenetic clade. The key features are the identification of evolutionarily conserved sequences followed by automated design of intron-flanking Polymerase Chain Reaction (PCR) primer pairs. Polymorphisms can subsequently be identified by size- or sequence variation of PCR products, amplified from mapping parents or populations. We illustrate our procedure in legumes and grasses and exemplify its application in legumes, where model plant studies and the genome- and EST-sequence data available have a potential impact on the breeding of crop species and on our understanding of the evolution of this large and diverse family. Conclusion We provide a database of 459 candidate anchor loci which have the potential to serve as map anchors in more than 18,000 legume species, a number of which are of agricultural importance. For grasses, the database contains 1335 candidate anchor loci. Based on this database, we have evaluated 76 candidate anchor loci with respect to marker development in legume species with no sequence information available, demonstrating the validity of this approach.

• LORE1, an active low-copy-number TY3-gypsy retrotransposon family in the model legume Lotus japonicus.

  Authors: Lene Heegaard Madsen, Eigo Fukai, Simona Radutoiu, Christopher Karl Yost, Niels Sandal, Leif Schauser, Jens Stougaard

  The Plant journal : for cell and molecular biology. 12/2005; 44(3):372-81.

  We have identified a low-copy-number retrotransposon family present in nine to 10 copies in the Lotus japonicus model legume genome, and characterized its activity. LORE1 (Lotus retrotransposon 1)We have identified a low-copy-number retrotransposon family present in nine to 10 copies in the Lotus japonicus model legume genome, and characterized its activity. LORE1 (Lotus retrotransposon 1) belongs to the Ty3-gypsy group of elements, and is a long terminal repeat (LTR) retrotransposon. Genetic mapping located LORE1 elements in gene-rich regions of Lotus chromosomes, and analysis of native as well as new insertion sites revealed integration outside the highly repetitive sequences of centromeres and telomeres. Sequencing of individual LORE1 family members identified several intact elements, and analysis of new insertions showed that at least one member is active and reinserts into functional genes, creating gene-disruption mutations. Southern blot analysis and SSAP on a selection of symbiotic mutants revealed up to 12 new insertion sites in individual mutant lines and a Mendelian segregation of new inserts. Expression analysis showed that LORE1 elements are transcribed in all organs analysed and, in contrast to other active retrotransposons, LORE1 appears not to be transcriptionally upregulated during in vitro tissue culture. Activity of LORE1 in callus and whole plants suggests that a simple insertion mutagenesis based on endogenous LORE1 elements can be established for Lotus.

• Seven Lotus japonicus genes required for transcriptional reprogramming of the root during fungal and bacterial symbiosis.

  Authors: Catherine Kistner, Thilo Winzer, Andrea Pitzschke, Lonneke Mulder, Shusei Sato, Takakazu Kaneko, Satoshi Tabata, Niels Sandal, Jens Stougaard, K. Judith Webb, Krzysztof Szczyglowski, Martin Parniske

  The Plant cell. 09/2005; 17(8):2217-29.

  A combined genetic and transcriptome analysis was performed to study the molecular basis of the arbuscular mycorrhiza (AM) symbiosis. By testing the AM phenotype of nodulation-impaired mutants andA combined genetic and transcriptome analysis was performed to study the molecular basis of the arbuscular mycorrhiza (AM) symbiosis. By testing the AM phenotype of nodulation-impaired mutants and complementation analysis, we defined seven Lotus japonicus common symbiosis genes (SYMRK, CASTOR, POLLUX, SYM3, SYM6, SYM15, and SYM24) that are required for both fungal and bacterial entry into root epidermal or cortical cells. To describe the phenotype of these mutants at the molecular level, we screened for differentiating transcriptional responses of mutant and wild-type roots by large-scale gene expression profiling using cDNA-amplified fragment length polymorphism. Two percent of root transcripts was found to increase in abundance during AM development, from which a set of AM-regulated marker genes was established. A Ser-protease (SbtS) and a Cys-protease (CysS) were also activated during root nodule development. AM-induced transcriptional activation was abolished in roots carrying mutations in common symbiosis genes, suggesting a central position of these genes in a pathway leading to the transcriptional activation of downstream genes. By contrast, AM fungus-induced gene repression appeared to be unaffected in mutant backgrounds, which indicates the presence of additional independent signaling pathways.

• PriFi: using a multiple alignment of related sequences to find primers for amplification of homologs.

  Authors: Jakob Fredslund, Leif Schauser, Lene H Madsen, Niels Sandal, Jens Stougaard

  Nucleic acids research. 08/2005; 33(Web Server issue):W516-20.

  Using a comparative approach, the web program PriFi (http://cgi-www.daimi.au.dk/cgi-chili/PriFi/main) designs pairs of primers useful for PCR amplification of genomic DNA in species where priorUsing a comparative approach, the web program PriFi (http://cgi-www.daimi.au.dk/cgi-chili/PriFi/main) designs pairs of primers useful for PCR amplification of genomic DNA in species where prior sequence information is not available. The program works with an alignment of DNA sequences from phylogenetically related species and outputs a list of possibly degenerate primer pairs fulfilling a number of criteria, such that the primers have a maximal probability of amplifying orthologous sequences in other phylogenetically related species. Operating on a genome-wide scale, PriFi automates the first steps of a procedure for developing general markers serving as common anchor loci across species. To accommodate users with special preferences, configuration settings and criteria can be customized.

• The sulfate transporter SST1 is crucial for symbiotic nitrogen fixation in Lotus japonicus root nodules.

  Authors: Lene Krusell, Katja Krause, Thomas Ott, Guilhem Desbrosses, Ute Krämer, Shusei Sato, Yasukazu Nakamura, Satoshi Tabata, Euan K James, Niels Sandal, Jens Stougaard, Masayoshi Kawaguchi, Ai Miyamoto, Norio Suganuma, Michael K Udvardi

  The Plant cell. 06/2005; 17(5):1625-36.

  Symbiotic nitrogen fixation (SNF) by intracellular rhizobia within legume root nodules requires the exchange of nutrients between host plant cells and their resident bacteria. Little is known at theSymbiotic nitrogen fixation (SNF) by intracellular rhizobia within legume root nodules requires the exchange of nutrients between host plant cells and their resident bacteria. Little is known at the molecular level about plant transporters that mediate such exchanges. Several mutants of the model legume Lotus japonicus have been identified that develop nodules with metabolic defects that cannot fix nitrogen efficiently and exhibit retarded growth under symbiotic conditions. Map-based cloning of defective genes in two such mutants, sst1-1 and sst1-2 (for symbiotic sulfate transporter), revealed two alleles of the same gene. The gene is expressed in a nodule-specific manner and encodes a protein homologous with eukaryotic sulfate transporters. Full-length cDNA of the gene complemented a yeast mutant defective in sulfate transport. Hence, the gene was named Sst1. The sst1-1 and sst1-2 mutants exhibited normal growth and development under nonsymbiotic growth conditions, a result consistent with the nodule-specific expression of Sst1. Data from a previous proteomic study indicate that SST1 is located on the symbiosome membrane in Lotus nodules. Together, these results suggest that SST1 transports sulfate from the plant cell cytoplasm to the intracellular rhizobia, where the nutrient is essential for protein and cofactor synthesis, including nitrogenase biosynthesis. This work shows the importance of plant sulfate transport in SNF and the specialization of a eukaryotic transporter gene for this purpose.