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-10934226-B2
Publication Date
2021-03-02
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 invention relates to an engineered, non-intergeneric diazotroph having increased nitrogen fixation activity as compared to an unmodified organism of the same species. The engineered non-intergeneric diazotroph comprises a genetic variation within one or more genes for regulating nitrogen fixation or assimilation, and the genetic variation comprises genetic material that originates from at least one organism of the same species as the engineered, non-intergeneric diazotroph.
The invention further addresses increased nitrogen fixation activity under nitrogen-exposed conditions by configuring the genetic variation in genes for regulating nitrogen fixation or assimilation. The genetic variation produces increased expression or activity of nitrogen-regulation targets while changing the expression or activity of shutoff and assimilation-regulation targets, including NifA and related nitrogen fixation regulation factors, and targets associated with glutaminase and PII signaling cascade components.
The disclosure also encompasses embodiments in which the engineered non-intergeneric diazotroph is used in association with plants, including compositions comprising the engineered non-intergeneric diazotroph and a plant seed. The claimed subject matter supports comparisons of increased nitrogen fixation activity versus an unmodified same-species diazotroph, including in a field context and under specified fixed-nitrogen concentrations.
Claims Coverage
The independent claim is directed to an engineered, non-intergeneric diazotroph with increased nitrogen fixation activity achieved by introducing same-species-origin genetic variation in nitrogen fixation or assimilation regulatory genes. The coverage is further refined by dependent claims that add activity comparisons under fixed-nitrogen concentrations, field use comparisons, specified loci, and downstream expression/activity changes within nitrogen regulation pathways.
Engineered non-intergeneric diazotroph with increased nitrogen fixation activity
An engineered, non-intergeneric diazotroph having increased nitrogen fixation activity as compared to an unmodified organism of the same species.
Genetic variation in genes regulating nitrogen fixation or assimilation
The engineered, non-intergeneric diazotroph comprises a genetic variation within one or more genes for regulating nitrogen fixation or assimilation.
Same-species origin of genetic material in the variation
The genetic variation comprises genetic material that originates from at least one organism of the same species as the engineered, non-intergeneric diazotroph.
Increased nitrogen fixation activity at fixed nitrogen presence
The engineered non-intergeneric diazotroph has increased nitrogen fixation activity when fixed nitrogen is present at at least 0.01 mM, compared with an unmodified diazotroph of the same species under the same fixed-nitrogen concentration.
Field-based increased activity comparison
The engineered non-intergeneric diazotroph has increased nitrogen fixation activity in a field compared with an unmodified diazotroph of the same species in the same field.
Regulatory genes specified for the nitrogen-fixation or assimilation genetic variation
The genetic variation comprises one or more nitrogen-fixation or nitrogen-assimilation regulatory genes selected from a specified list including nifA, nifL, ntrB, ntrC, glnA, glnB, glnK, draT, amtB, glnD, glnE, nifH, nifJ, nifD, nifK, nifY, nifE, nifN, nifU, nifS, nitV, nifW, nifZ, nifM, nifF, nifB, and nifQ.
Regulatory downstream expression and activity changes to nitrogen regulation targets
The genetic variation results in one or more of increased expression or activity of NifA or glutaminase, decreased expression or activity of NifL, NtrB, glutamine synthetase, GlnB, GlnK, DraT, or AmtB, decreased adenylyl-removing activity of GlnE, or decreased uridylyl-removing activity of GlnD.
Plant-seed composition including the engineered diazotroph
A composition comprises the engineered non-intergeneric diazotroph of claim 1 and a plant seed.
Overall, the claim set centers on engineering a non-intergeneric diazotroph by inserting same-species-origin genetic variation into nitrogen fixation or nitrogen assimilation regulatory genes, yielding increased nitrogen fixation activity. Dependent claims further constrain the conditions and contexts for demonstrating increased activity, specify regulatory gene loci, and require particular downstream changes in expression or activity of nitrogen-regulation targets, with embodiments extending to compositions that include a plant seed.
Stated Advantages
Increased nitrogen fixation activity as compared to an unmodified organism of the same species.
Increased nitrogen fixation activity under fixed nitrogen presence (at least 0.01 mM).
Increased nitrogen fixation activity in a field compared with an unmodified diazotroph in the same field.
Documented Applications
Use of the engineered non-intergeneric diazotroph in a composition comprising the engineered non-intergeneric diazotroph and a plant seed.
Field context comparison demonstrating increased nitrogen fixation activity versus an unmodified diazotroph of the same species in the same field.
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