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 composition comprising:
an engineered diazotroph comprising at least one genetic variation introduced in a nitrogen fixation genetic regulatory network, whereby the engineered diazotroph comprises increased expression or activity of nifH, increased expression or activity of nifA, and decreased expression or activity of nifL, wherein the at least one genetic variation comprises genetic material that originates from the same genus as said engineered diazotroph, whereby the engineered diazotroph has increased nitrogen fixation activity as compared to an unmodified organism of the same species as the engineered diazotroph; and
a plant seed.
2. The composition of claim 1 , wherein said plant seed is a non-leguminous plant seed.
3. The composition of claim 1 , wherein said plant seed is a cereal plant seed.
4. The composition of claim 1 , wherein said plant seed is a seed of a plant selected from the group consisting of corn, wheat, rice, and sorghum.
5. The composition of claim 1 , wherein said engineered diazotroph is present as a coating on said plant seed.
6. The composition of claim 5 , wherein said engineered diazotroph is within a seed coating that is coated on a portion of said plant seed.
7. The composition of claim 1 , wherein said engineered diazotroph is an engineered bacterium.
8. The composition of claim 1 , wherein said engineered diazotroph further comprises a genetic variation introduced within a nitrogen assimilation genetic regulatory network.
9. The composition of claim 8 , wherein said genetic variation alters activity or expression of glnE.
10. The composition of claim 1 , wherein said engineered diazotroph further comprises a genetic variation that alters activity or expression of a gene selected from the group consisting of amtB, ntrB, ntrC, polynucleotide encoding glutamine synthetase, glnA, glnB, glnK, drat, polynucleotide encoding glutaminase, and glnD.
11. The composition of claim 1 , wherein said genetic material that originates from said same genus as said engineered diazotroph comprises a regulatory element.
12. The composition of claim 11 , wherein said regulatory element is a promoter.
13. The composition of claim 12 , wherein said promoter is active in the presence of exogenous nitrogen.
14. The composition of claim 1 , wherein said engineered diazotroph has increased nitrogen fixation activity in the presence of at least 0.01 mM fixed nitrogen as compared to an unmodified diazotroph of said same species as said engineered diazotroph in the presence of the same concentration of fixed nitrogen.
15. The composition of claim 1 , wherein said engineered diazotroph has at least two-fold increased nitrogen fixation activity in a field comprising exogenous nitrogen, as compared to an unmodified diazotroph of said same species as said engineered diazotroph in said field.
16. The composition of claim 15 , wherein said field has a fixed nitrogen concentration of at least 0.01 mM.
17. The composition of claim 1 , wherein said engineered diazotroph is an epiphyte.
18. The composition of claim 1 , wherein said engineered diazotroph is an endophyte.
19. The composition of claim 1 , wherein said engineered diazotroph is a rhizophyte.
20. The composition of claim 1 , wherein said genetic material of said engineered diazotroph does not comprise genetic material that originates from a different genus than said engineered diazotroph.
21. The composition of claim 1 , wherein said genetic material of said engineered diazotroph does not comprise genetic material that originates from a different species than said engineered diazotroph.
22. The composition of claim 1 , wherein the engineered diazotroph is of a genus selected from the group consisting of Enterobacter, Rahnella, Kosakonia, Burkholderia , and Klebsiella.