IN INOCULANTS Nodulaid - 17th International Nitrogen Fixation ...

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17 th International Congress on Nitrogen Fixation Fremantle, Western Australia 27 November – 1 December 2011 Title Functional Analysis of STM Mutants Concerning Amino Acid Metabolism of Mesorhizobium loti Authors Shigeyuki Tajima 1 , Mika Nomura 1 , Nanthipak Thapanapongworakul 2 , Ayao Enoki 1 and Hiroyuki Matsuura 1 Poster Board Number 35 1 Dept of Applied Life Sciences, Kagawa Universit 2 Dept of Entomology and Plant Pathology, Faculty of Agriculture, Chiang Mai University The soil bacterium Mesorhizobium loti is able to induce the formation of nitrogen-fixing nodules on the root of a determinate-type legume plant, Lotus japonicus. The research on various metabolites during symbiosis has been elucidated that several bacteroid metabolic pathways are essential for maintaining efficient symbiotic interaction and for enhancing the in vivo nitrogenase activity of the nodules. To elucidate the molecular mechanism of such metabolic bacteroid differentiation in determinate nodules, protein profiles and functions of Mesorhizobium loti for Lotus japonicus were compared between cultured bacteria and nodule bacteroids. The transposon insertion mutant strains of M. loti were generated using the signature-tagged mutagenesis (STM) technique. To determine the functions of the up-regulated proteins in the bacteroids, 130 STM mutants were inoculated with Lotus plants. We focused on the four STM mutants (STM5, 30, 42, 130) which were related to the amino acid metabolism. M. loti mutant (STM5) that was inserted a transposon in the PHGDH gene, mll3875, showed an absolute dependence on serine or glycine in the minimal medium for the growth. When L. japonicus plant was infected with STM5, the root formed nodules with comparable number with that of wild type M. loti. However, the nodules showed very low acetylene reduction activity and significant starch granule accumulation was observed in the uninfected cells. In addition, STM42 that was inserted in the amino acid transporter showed the 70 % acetylene reduction activity of wild type nodule. This amino acid transporter showed high homology to aapJ in R. leguminosarum. However, STM42 bacteroid showed lower concentration in some amino acids, especially Ala, Val, Leu, Lys, Arg, and Orn, but the concentrations of Glu and Asp did not change. This data suggested that this amino acid transporter was different from aap/bra amino acid transporter in R. leguminosarum. Other amino acid synthetic proteins, STM30 and STM130, were also discussed. 150 2011
17 th International Congress on Nitrogen Fixation Fremantle, Western Australia 27 November – 1 December 2011 Title Establishment of retrotransposon-mutagenized population of the model legume Lotus japonicus and high throughput, deep sequencing-based insertion site identification. Authors Dorian Fabian Urbański, Anna Małolepszy, Stig Uggerhøj Andersen, Jens Stougaard Poster Board Number 36 Centre for Carbohydrate Recognition and Signalling, Department of Molecular Biology, Aarhus University, 8000 Aarhus C, Denmark Targeted gene knockout by homologues recombination is not yet a standard technique for reverse genetics in plants. As an alternative we have taken advantage of inducible de-repression of Lotus retrotransposon 1 (LORE1), a member of small family belonging to the Ty3-Gypsy class of LTR retro-elements. LORE1 has a unique feature of being transcriptionally active in the male gametophyte 4, 5 , producing a small number of new insertions at random sites only during transition to a new generation. We are currently using a founder line of Lotus japonicus, with active LORE1 to generate a large, mutagenized population. For robust identification of novel insertion sites we are using a 2D pooling strategy and taking advantage of the known LORE1 LTR sequence to simultaneously amplify LORE1 flanking sequence tags (FSTs) in dozens of pooled plants. Illumina deep sequencing technology, an extremely high degree of multiplexing and custom-designed bioinformatics‟ pipeline led us to establishing high throughput detection method for robust identification of those insertions. Study on a test set of 3744 plants, enabled identification of app. 8000 novel insertions with an average of 2,4 per plant. Novel LORE1 copies do not show tendency to cluster and are evenly distributed along Lotus chromosomes. Moreover, genes were preferentially targeted and insertions in exons were 5.7 times more frequent than in intergenic regions (P
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INVENTING THE FUTURE IN INOCULANTS
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17 ICNF Organising Committee Graham
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16:30 - 16:50 Felix Dakora Tshwane
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