Nitrogen fixation using refactored nif clusters

Abstract

The invention relates to methods for promoting fixed nitrogen from atmospheric nitrogen, and related products. Endophytic bacteria having an exogenous nif cluster promote fixed nitrogen for cereal plants.

1 - 12 . (canceled) 
     
     
         13 . A method, comprising delivering a modified non-pathogenic bacteria having exogenous genes for enabling plant endosymbiosis to a cereal plant, or to soil where a cereal plant or seed is growing or is to be planted. 
     
     
         14 . The method of  claim 13 , wherein the non-pathogenic bacteria is  E. coli.    
     
     
         15 . The method of  claim 14 , wherein the genes encode effectors or apparatus for a secretion system. 
     
     
         16 . The method of  claim 15 , wherein the apparatus for a secretion system is type 3 secretion system (T3SS). 
     
     
         17 . The method of  claim 13 , wherein the exogenous gene includes a controller. 
     
     
         18 . The method of  claim 17 , wherein the controller is a nucleic acid encoding an IPTG inducible T7 RNA polymerase. 
     
     
         19 . The method of  claim 17 , wherein the controller is a partitioning system encoded by the two par operons (parCBA and parDE). 
     
     
         20 . The method of  claim 17 , wherein the partitioning system is a RK2 par system. 
     
     
         21 . A composition, comprising: (a) an agriculturally suitable carrier; and (b) a gamma-proteobacteria having an exogenous nif cluster present on or in the agriculturally suitable carrier. 
     
     
         22 . The composition of  claim 21 , wherein the gamma-proteobacteria is a  Salmonella typhimurium  or  E. coli.    
     
     
         23 . The composition of  claim 21 , wherein the nif cluster is a native nif cluster. 
     
     
         24 . The composition of  claim 21 , wherein the nif cluster is a refactored nif cluster. 
     
     
         25 . The composition of  claim 21 , further comprising an exogenous gene encoding a plant growth-stimulating peptide. 
     
     
         26 . The composition of  claim 21 , wherein the agriculturally suitable carrier is selected from the group consisting of seeds, seed coats, granular carriers, soil, solid carriers, liquid slurry carriers, and liquid suspension carriers. 
     
     
         27 . The composition of  claim 21 , wherein the agriculturally suitable carrier includes a wetting agents, a synthetic surfactant, a water-in-oil emulsion, a wettable powder, granules, gels, agar strips or pellets, thickeners, microencapsulated particles, or liquids such as aqueous flowables or aqueous suspensions. 
     
     
         28 . The composition of  claim 21 , wherein the exogenous nif cluster includes a controller. 
     
     
         29 . The composition of  claim 28 , wherein the controller is a nucleic acid encoding an IPTG inducible T7 RNA polymerase. 
     
     
         30 . The composition of  claim 28 , wherein the controller is a partitioning system encoded by the two par operons (parCBA and parDE). 
     
     
         31 . The composition of  claim 28 , wherein the partitioning system is a RK2 par system. 
     
     
         32 . A seed or seedling of a cereal plant having a modified bacteria associated with an external surface of the seed or seedling; wherein said modified bacteria comprises a refactored exogenous nif cluster. 
     
     
         33 . (canceled) 
     
     
         34 . The seed or seedling of  claim 32 , wherein the nif cluster is a native nif cluster. 
     
     
         35 . The seed or seedling of  claim 32 , wherein the nif cluster is a refactored nif cluster. 
     
     
         36 . The seed or seedling of  claim 32 , wherein the modified bacteria is a gamma-proteobacteria. 
     
     
         37 . The seed or seedling of  claim 36 , wherein the gamma-proteobacteria is a  Salmonella typhimurium.    
     
     
         38 . The seed or seedling of  claim 37 , wherein the  Salmonella typhimurium  strain is selected from SL1344, LT2, and DW01. 
     
     
         39 . The seed or seedling of  claim 32 , wherein the modified bacteria is a  E. coli , optionally of strain H7:0157. 
     
     
         40 . The seed or seedling of  claim 32 , wherein the nif cluster is a  Klebsiella  wild-type nif cluster, a  Pseudomonas  Stutzi nif cluster, or a  Paenibacillus  cluster. 
     
     
         41 . The seed or seedling of  claim 32 , wherein the cereal plant is selected from wheat, rye, barley, triticale, oats, millet, sorghum, teff, fonio, buckwheat,  quinoa , corn and rice. 
     
     
         42 . The seed or seedling of  claim 32 , further comprising an exogenous gene encoding a plant growth-stimulating peptide. 
     
     
         43 . The seed or seedling of  claim 42 , wherein the exogenous gene encoding the plant growth-stimulating peptide is regulated by a type 3 secretion system (T3SS). 
     
     
         44 . A cereal plant having a modified bacteria in the plant, wherein the modified bacteria has an exogenous nif cluster. 
     
     
         45 . The cereal plant of  claim 44 , wherein the nif cluster is a refactored nif cluster. 
     
     
         46 . The cereal plant of  claim 44 , further comprising an exogenous gene encoding a plant growth-stimulating peptide. 
     
     
         47 . The cereal plant of  claim 46 , wherein the exogenous gene encoding the plant growth-stimulating peptide is regulated by a type 3 secretion system (T3SS). 
     
     
         48 . The cereal plant of  claim 46 , wherein the exogenous gene encodes effectors or apparatus for a secretion system. 
     
     
         49 . The cereal plant of  claim 47 , wherein the exogenous gene is in root or stem tissues of the plant. 
     
     
         50 . A method of providing a corn plant with fixed nitrogen from atmospheric nitrogen, comprising: delivering a transgenic bacteria having a refactored exogenous nif cluster to a corn plant, or to soil where a corn plant or seed is growing or is to be planted. 
     
     
         51 . The method of  claim 37 , wherein a non-transgenic bacteria having a same species as said transgenic bacteria lacks a nif cluster.