Methods and compositions for improving plant traits
Inventors
Temme, Karsten • Tamsir, Alvin • BLOCH, Sarah • CLARK, Rosemary • TUNG, Emily
Assignees
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Publication Number
US-11739032-B2
Publication Date
2023-08-29
Expiration Date
Abstract
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.
Core Innovation
The disclosure relates to methods and compositions for increasing nitrogen fixation in a plant by exposing a plant to a plurality of engineered, non-intergeneric diazotrophs. The engineered, non-intergeneric diazotrophs comprise at least one genetic variation within one or more genes for regulating nitrogen fixation or assimilation, and the genetic variations include genetic material that originates from an organism of the same genus as the engineered, non-intergeneric diazotrophs.
The disclosure explains that the engineered diazotrophs, in planta, produce fixed nitrogen at levels of 1% or more, with embodiments including 5% or 10% thresholds. The disclosure further supports multiple genetic targets within nitrogen fixation and assimilation regulatory networks, including genes and polynucleotides associated with NifA/NifL/NifH regulation and other nitrogen-assimilation related genes such as ntrB/ntrC and glutamine metabolism genes.
The disclosure also describes a rationale for decoupling nitrogen sensing from nitrogenase activity and provides Host-Microbe Evolution (HoME). HoME is described as iterative isolation, mutagenesis, and selection to evolve regulatory networks that maintain nitrogenase and fixed-nitrogen output under fertilized field conditions, together with performance and colonization evaluation in planta.
Claims Coverage
The independent claim covers a method of increasing nitrogen fixation by exposing a plant to engineered, non-intergeneric diazotrophs carrying genetic variations in nitrogen fixation or assimilation regulatory genes, producing 1% or more fixed nitrogen in the plant. The coverage is further tightened by dependent claims that add quantitative fixed-nitrogen thresholds, colonization constraints, specific genetic regulatory targets, and application formats such as seed coating.
Exposing a plant to engineered non-intergeneric diazotrophs with regulatory genetic variations
A method of increasing nitrogen fixation in a plant by exposing the plant to a plurality of engineered, non-intergeneric diazotrophs, wherein the engineered, non-intergeneric diazotrophs comprise at least one genetic variation within one or more genes for regulating nitrogen fixation or assimilation.
Regulatory genetic material originates from the same genus
The genetic variation comprises genetic material that originates from an organism of the same genus as the engineered, non-intergeneric diazotrophs.
In planta fixed-nitrogen production at 1% or more
The diazotrophs, in planta, produce 1% or more of the fixed nitrogen in the plant.
Higher in planta fixed-nitrogen threshold
The engineered, non-intergeneric diazotrophs, in planta, produce 10% or more of the fixed nitrogen in the plant.
Root colonization with minimum CFU per gram fresh weight
The engineered, non-intergeneric diazotrophs colonize at least a plant root and are present in the plant at an amount of at least 10^5 colony forming units per gram fresh weight of tissue.
Altering NifA/NifL/NifH activity or expression
A genetic variation within one or more genes for regulating nitrogen fixation or assimilation alters the activity or expression of a gene selected from NifA, NifL, and NifH.
Altering nitrogen-assimilation gene activity or expression
A genetic variation within one or more genes for regulating nitrogen fixation or assimilation alters the activity or expression of at least one gene selected from GlnE, AmtB, ntrB, ntrC, polynucleotide encoding glutamine synthetase, glnA, glnB, glnK, drat, polynucleotide encoding glutaminase, and glnD.
Seed coating with the plurality of engineered diazotrophs
The plurality of engineered, non-intergeneric diazotrophs are coated onto a seed of the plant.
Overall, the claims are directed to increasing plant fixed nitrogen by using engineered, non-intergeneric diazotrophs that carry genus-matched genetic variations in nitrogen fixation or assimilation regulatory genes, achieving in planta fixed nitrogen production including specified 1% or more and 10% or more thresholds. Dependent claim coverage narrows the genetic targets to named regulatory genes and adds constraints on colonization level and application format, including seed coating.
Stated Advantages
Increasing nitrogen fixation in a plant by exposing the plant to engineered, non-intergeneric diazotrophs.
Achieving in planta production of fixed nitrogen at levels of 1% or more, including embodiments with 10% or more.
Documented Applications
Agricultural crop use, including exposing plants in a growth context such as agricultural field/furrow conditions.
Seed application, including coating the plurality of engineered, non-intergeneric diazotrophs onto a seed of the plant.
In planta colonization and performance evaluation, including colonization in plant root tissues and evaluation of nitrogen fixation output under fertilized field conditions.
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