Towards synthetic nitrogen-fixing symbioses in grasses

Transcription

1 Towards synthetic nitrogen-fixing symbioses in grasses Michael Udvardi, Evangelia Kouri, John Peters, Amaya Garcia Costas, Florence Mus, Jean-Michel Ane, Kevin Garcia, Chris Voigt, Min-Hyung Ryu, Giles Oldroyd, Ponraj Paramasivian, Ramakrishnan Karunakaran, Barney Geddes, and Philip Poole. 1

2 Problems: Over-use in many regions, under-use in others 5

3 The Vision Endophyte / Epiphyte Plant 1. ATP N 2 NH 4 + NH 4 + O Aminoacids 6. 3

4 Model and crop systems for modifying associative interactions Setaria viridis Barley Maize Pseudomonas florescens Pf5: epiphyte Rhizobium sp. IRBG74: endophyte 7

5 The Vision Endophyte / Epiphyte Plant 1. ATP N 2 NH 4 + NH 4 + O Aminoacids 6. 5

6 1. Colonization of various cereals by Rhizobium sp. IRBG74 Rhizobium sp. IRBG74 is an efficient colonizer of Setaria and rice Setaria viridis Oryza sativa Rhizobium sp. IRBG74 is also an efficient colonizer of Hordeum vulgare Ané Lab. Kevin Garcia Log10(CFU/mg of FW) Total colonization Internal colonization Detachment 6

7 Colonization of Zea mays by Rhizobium sp. IRBG74 Rhizobium sp. IRBG74 does not seem to be an efficient colonizer of B73 Zea mays But B73 Zea mays can be colonized by several nitrogen-fixing bacteria GFP BF Other Zea mays germplasm (H99 and T43) can be colonized by Rhizobium sp. IRBG74 LR H99 LR T43 7 Ané Lab. Kevin Garcia

8 Identification of genes regulated in response to Rhizobium sp. IRBG74 Incubation of rice and maize (B73) seedlings with diffusible microbial signals, including those from IRBG74 4 plants per condition Maize / Rice seedlings H 2 O GSE S-Myc NS-Myc IRBG74 CO4 CO8 24h of incubation 4 biological replicates Root RNA extraction RNAseq analysis 8 Ané Lab. Kevin Garcia

9 The Vision Endophyte / Epiphyte Plant 1. ATP N 2 NH 4 + NH 4 + O Aminoacids 6. 9

10 synthesis Literature pathway of rhizopine synthesis MosB MosA MosC myo-inositol scyllo-inosamine 3-O-methyl scyllo-inosamine 13 Poole Lab. Barney Geddes

11 Proposed novel pathway of rhizopine synthesis IdhA MosB MosC Inositol dehydrogenase Aminotransferase myo-inositol scyllo-inosose scyllo-inosamine MosDEF MosB MosC ononitol Ononitol dehydrogenase keto-ononitol Aminotransferase 3-O-methyl scyllo-inosamine 11 Poole Lab. Barney Geddes

12 Tobacco transient transformation IdhA MosB Inositol dehydrogenase Aminotransferase myo-inositol scyllo-inosose scyllo-inosamine scyllo-inosamine Ion count Retention time (min) 12 Oldroyd Lab. Ponraj Paramasivan

13 Optimize carbon supply from roots to bacteria Identify and quantify metabolites in S. viridis and maize roots and root exudates by GC-MS Among the metabolites identified were sugars, organic and amino acids, sugar alcohols, etc. TIC Maize root metabolites Maize root exudate metabolites Mannose Sucrose Fructose Glucose Phosphoric acid Myo-inositol Trehalose 13 Retention time Udvardi Lab. Evangelia Kouri

14 Novel rhizopine biosensor demonstrates rhizopine secretion M. sativa nodulated by rhizopine-producing S. meliloti L5-30. biosensor on root surface 14 Poole Lab. Barney Geddes

15 Engineering rhizopine catabolism MocB MocDEF MocAC Inositol catabolism 3-O-methyl scyllo-inosamine scyllo-inosamine myo-inositol Successfully transferred rhizopine catabolism to Rhizobium sp. IRBG74 15 Poole Lab. Barney Geddes

16 The Vision Endophyte / Epiphyte Plant 1. ATP N 2 NH 4 + NH 4 + O Aminoacids 6. 16

17 Creation of large part libraries for 10 microsymbionts IPTG (mm) atc (ng/ml) DAPG (m M) 3OC6 (nm) 10 1 ( Fluorescence in IRBG74 (au) Fluorescence in Pf-5 (au) IPTG (mm) atc (ng/ml) DAPG (m M) 3OC6 (nm) 10 1 OD 600( Inducible promoters Ara. (mm) atc (ug/ml) time DAPG (h) (mm) 3OC6 (nm) ( Phage polymerase system Fluorescence (au) teta Ara. (mm) atc (ug/ml) DAPG (mm) 3OC6 (nm) B( C( D( Terminator strength (Ts) orit tetr 10 1 Ribosome binding sites Reg trfa2 RBSs in IRBG74 Controller( trfa1 trba K2 rep E D pbbr1 C parb RBSs in Pf-5 Terminators para mrfp Terminator strength in Pf-5 Reporter( Gm R orit 10 1 [IPTG] R 2 = Terminators Terminator strength in IRBG74 Figure 1. Creation of large part libraries in the microsymbionts. The fundamental set of parts that provide precision expression control is shown (A), inducible promoters (B), ribosome binding sites (C), terminators (D), T7 RNAP-inducible systems. P T7 Terminators teta orit tetr trfa2 Reg trfa1 T7 RNAP Controller trba RK2 rep E D pbbr1 C Reporter parb para mrfp Gm R orit [IPTG] P T7 Fluorescence in IRBG74 (au) Fluorescence in Pf-5 (au) IPTG(mM) Ara. (m M) 17 Voigt Lab. Min-Hyung Ryu

18 Engineering nitrogen fixation clusters for microsymbionts Klebsiella oxytoca M5a1 (+NtrBC) Klebsiella oxytoca refactored cluster v1 Pseudomonas stutzeri A1501 Paenibacillus sp. WLY78 Azorhizobium caulinodans ORS571 N 2 fixation by nif expression Oxygen tolerance of N 2 fixation IPTG inducible N 2 fixation Ethylene production (au) E. coli Pf-5 M5a1 v1 A1501 WLY78 Nitrogenase activity (%) in E. coli M5a1 A Oxygen (%) Nitrogenase activity (%) in Pf Oxygen (%) Ethylene production (au) in A. caulinodans 10 5 Wild-type (100%) 10 0 IPTG (mm) 18 Voigt Lab. Min-Hyung Ryu

19 The Vision Endophyte / Epiphyte Plant 1. ATP N 2 NH 4 + NH 4 + O Aminoacids 6. 19

20 4. Forging the blueprint for engineering nitrogen fixation in aerobes How do nif complements differ in aerobes and anaerobes? nif operons differ in the number of nif genes they contain Work published in J. Bacteriol. 1) Many more nif genes in aerobes than anaerobes 2) Most extensive suite if nif genes are associated with one of our targets (Psuedomonads) 3) Nif genes not associated with specific modes of oxygen protection 4) Anaerobes energetically challenged and likely synthesize and turnover active nitrogenase slowly (postranslational regulation) 5) Aerobes turnover nitrogenase more rapidly (transcriptionally regulated) and have adapted more nif genes to a) improve kinetics of synthesis b) improve fidelity of synthesis and c) perhaps protect and repair oxygen sensitive elements 20 Peters Lab. Amaya Garcia Costas

21 Forging the blueprint for engineering nitrogen fixation in aerobes Defining patterns of gene expression associated with diazotrophy in aerobes: What genes are involved/critical under diverse carbon and oxygen conditions? Use Azotobacter vinelandii as model organism: 1. Well studied AEROBIC diazotroph Genome sequenced, ease of genetic manipulation We used transcriptomics to study differential gene expression during diazotrophic growth in addition to nif encoded genes other important genes that support nitrogen fixation have been identified (rnf, fix, and genes encoding respiratory complexes important Hamilton et al,j Bact 2011 We have developed capability to culture A. vinelandii in bioreactors under various oxygen tensions and have submitted RNA from these cultures for RNAseq Samples taken for transcriptomic analyses 21 Peters Lab. Amaya Garcia Costas

22 The Vision Endophyte / Epiphyte Plant 1. ATP N 2 NH 4 + NH 4 + O Aminoacids 6. 22

23 Forging the blueprint for engineering nitrogen fixation in aerobes Strategies for engineering ammonia excretion ❶ glna deletion: glna deletion is lethal for A. vinelandii (confirmed experimentally) ❷ targeted mutagenesis on glna: point mutation Tyr-183 to Cys (in progress) Healy et al., 2003 : Tyr-183 to Cys mutation in GS of A. variabilis leads to lower GS activity and excretion of ammonium (0.5 mm) residues involved in formation of negatively charged ammonium-binding pocket molecular construct carrying the point mutation Tyr-183 to Cys in GS of A. vinelandii has been generated selection of low affinity GlnA mutants of A. vinelandii using GlnE knockout background is in progress (selection on higher concentrations of ammonia) 23 Peters Lab. Florence Mus

24 Mutants of amtb and gltb in IRBG74 and Pf-5 amtb and gltb in-frame deletions made in IRBG74 IRBG74 gltb requires glu for growth E. coli gdha rescues rhizobial gltb growth Pf-5 mutants in progress Controller (e.g. rhizopine induced) and output modules constructed for repression of gltb and induction of gdh (tested with Gfp and mcherry) e.g. SI, flavonoid IPTG GDH GOGAT 24 Poole Lab. KK/Barney Geddes

25 The Vision Endophyte / Epiphyte Plant 1. ATP N 2 NH 4 + NH 4 + O Aminoacids 6. 25

26 6. Characterize and optimize ammonium uptake Setaria viridis growth kinetics at different concentrations of ammonium Analyze AMT gene expression in S. viridis roots by quantitative RT-PCR 2 AMT1-type transporters SvAMT1A expressed in both shoots and roots SvAMT1B expressed mainly in roots Highly expressed under low ammonium concentrations 26 Udvardi Lab. Evangelia Kouri

27 Characterize and optimize ammonium uptake 6 AMT2-type transporters SvAMT2C expressed in both shoot and root, under normal N conditions. SvAMT2D expression increased substantially at low ammonium concentrations. Relative expression level 27 Udvardi Lab. Evangelia Kouri

28 The Vision Endophyte / Epiphyte Plant 1. ATP N 2 NH 4 + NH 4 + O Aminoacids 6. 28

29 Acknowledgements Ané Lab Kevin Garcia Oldroyd Lab Ponraj Paramasivan Peters Lab Amaya Garcia Costas Florence Mus Poole Lab Barney Geddes Ramakrishnan Karunakaran Alison East Udvardi Lab Evangelia Kouri Voigt Lab Min-Hyung Ryu 29

30 NSF Research Coordination Network Plant Nitrogen Network (PlaNNet): Coordinating research on plant nitrogen for sustainable and productive agriculture PlaNNet aims to coordinate research activities related to the supply, utilization and loss of N by plants with the long-term objective of enhancing the efficiency and sustainability of N-use in agriculture. Research coordination network (RCN) activities will include: (i) Development of a networking website that includes information about hundreds of researchers within the USA and around the world who are involved in plant N-related research, opportunities for collaboration, and educational resources. (ii) Annual Workshops-Without-Walls, virtual meetings that will involve hundreds of participants in presentations, discussions, and consensus-building related to plant-n research and development. (iii) Satellite workshops at major conferences focused on specific aspects of plant N and agriculture. Contacts: meleavitt@noble.org; mudvardi@noble.org; john.peters@chemistry.montana.edu Look for us on: