Methods and compositions for improving plant traits
Disclosed herein are methods of increasing nitrogen fixation in a non-leguminous plant. The methods can comprise exposing the plant to a plurality of bacteria. Each member of the plurality comprises one or more genetic variations introduced into one or more genes or non-coding polynucleotides of the bacteria's nitrogen fixation or assimilation genetic regulatory network, such that the bacteria are capable of fixing atmospheric nitrogen in the presence of exogenous nitrogen. The bacteria are not intergeneric microorganisms. Additionally, the bacteria, in planta, produce 1% or more of the fixed nitrogen in the plant.
1. A method of increasing nitrogen fixation in a plant, said method comprising exposing said plant to a plurality of engineered, non-intergeneric diazotrophs, wherein said engineered, non-intergeneric diazotrophs comprise at least one genetic variation within one or more genes for regulating nitrogen fixation or assimilation, said genetic variation comprising genetic material that originates from an organism of the same genus as said engineered, non-intergeneric diazotrophs, and wherein said diazotrophs, in planta, produce 1% or more of the fixed nitrogen in the plant.
2. The method of claim 1 , wherein said plant is an agricultural crop plant.
3. The method of claim 1 , wherein said plant is selected from the group consisting of corn, wheat, barley, rice, sorghum, and soybean.
4. The method of claim 1 , wherein said plant is in a field.
5. The method of claim 1 , wherein said engineered, non-intergeneric diazotrophs, in planta, produce 5% or more of the fixed nitrogen in said plant.
6. The method of claim 1 , wherein said engineered, non-intergeneric diazotrophs, in planta, produce 10% or more of the fixed nitrogen in said plant.
7. The method of claim 1 , wherein said engineered, non-intergeneric diazotrophs are bacteria.
8. The method of claim 7 , wherein said bacteria are proteobacteria.
9. The method of claim 7 , wherein said bacteria are in the genus Enterobacter.
10. The method of claim 7 , wherein said bacteria are in the genus Rahnella.
11. The method of claim 7 , wherein said plurality of engineered, non-intergeneric diazotrophs comprise two different taxa of bacteria, wherein bacteria in each taxa comprise at least one genetic variation within one or more genes for regulating nitrogen fixation or assimilation, said genetic variation comprising genetic material that originates from an organism of the same genus.
12. The method of claim 1 , wherein said genetic variation is within a gene in the nitrogen fixation genetic regulatory pathway.
13. The method of claim 1 , wherein said genetic variation within the one or more genes for regulating nitrogen fixation or assimilation alters the activity or expression of a gene selected from the group consisting of NifA, NifL, and NifH.
14. The method of claim 1 , wherein said genetic variation within the one or more genes for regulating nitrogen fixation or assimilation alters the activity or expression of a gene selected from the group consisting of GlnE, AmtB, ntrB, ntrC, polynucleotide encoding glutamine synthetase, glnA, glnB, glnK, drat, polynucleotide encoding glutaminase, and glnD.
15. The method of claim 1 , wherein said genetic material comprises a regulatory element.
16. The method of claim 1 , wherein said plurality of engineered, non-intergeneric diazotrophs are applied into furrows in which seeds of said plant are planted.
17. The method of claim 1 , wherein said plurality of engineered, non-intergeneric diazotrophs are coated onto a seed of said plant.
18. The method of claim 1 , wherein said plurality of engineered, non-intergeneric diazotrophs colonize at least a root of said plant such that said plurality of engineered, non-intergeneric diazotrophs are present in said plant in an amount of at least 10 5 colony forming units per gram fresh weight of tissue.