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-9975817-B2
Publication Date
2018-05-22
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 methods and engineered diazotrophic bacteria for increasing the amount of atmospheric derived nitrogen in a corn plant in a field. The approach exposes the corn plant in the field to a plurality of engineered diazotrophic bacteria so that the atmospheric derived nitrogen within the corn plant is increased relative to a corn plant exposed to a same amount of a plurality of non-engineered diazotrophic bacteria of the same species.
The genetic variations in the nitrogen fixation genetic regulatory network alter the nitrogen fixation regulation through changes in NifA and NifL, including a mutation that results in increased expression or activity of NifA and/or decreased expression or activity of NifL. At least one genetic variation includes insertion of a heterologous sequence found within a genome of a bacteria of the genus of the engineered diazotrophic bacteria.
The engineered bacteria are characterized such that their genetic material consists essentially of genetic material from bacteria of the same genus. Nitrogen fixation activity of the engineered diazotrophic bacteria in non-nitrogen-limiting conditions is greater than nitrogen fixation activity of the same amount of the non-engineered diazotrophic bacteria in similar non-nitrogen-limiting conditions.
Claims Coverage
The independent claim is directed to a field method for increasing atmospheric derived nitrogen in corn by exposing the plants to engineered diazotrophic bacteria with engineered changes to a nitrogen fixation genetic regulatory network. The main inventive features in the independent claim comprise a comparative nitrogen increase outcome, specific engineered regulatory-network edits, and a constraint that the engineered bacteria’s genetic material consists essentially of genetic material from the bacterial genus.
Exposing corn in a field to engineered diazotrophic bacteria to increase atmospheric derived nitrogen
A method of increasing the amount of atmospheric derived nitrogen in a corn plant in a field by exposing the corn plant in the field to a plurality of engineered diazotrophic bacteria, thereby increasing an amount of atmospheric derived nitrogen within the corn plant relative to a corn plant exposed to a same amount of a plurality of non-engineered diazotrophic bacteria that are the same species as the engineered bacteria.
Nitrogen fixation genetic regulatory network variation with NifA/NifL expression or activity changes
A genetic variation introduced into a nitrogen fixation genetic regulatory network comprises a mutation that results in increased expression or activity of NifA, or decreased expression or activity of NifL.
Heterologous sequence insertion into the engineered nitrogen fixation regulatory network
A genetic variation introduced into the nitrogen fixation genetic regulatory network comprises insertion of a heterologous sequence found within a genome of a bacteria of the genus of the plurality of engineered diazotrophic bacteria.
Engineered bacterial genetic material consists essentially of genetic material from the same genus
The genetic material of the plurality of engineered diazotrophic bacteria consists essentially of genetic material from bacteria of the genus of the plurality of engineered diazotrophic bacteria.
Higher nitrogen fixation activity in non-nitrogen-limiting conditions versus non-engineered counterparts
Nitrogen fixation activity of the plurality of engineered diazotrophic bacteria in non-nitrogen-limiting conditions is greater than nitrogen fixation activity, in similar non-nitrogen-limiting conditions, of a same amount of the plurality of non-engineered diazotrophic bacteria that are the same species as the plurality of engineered diazotrophic bacteria.
Across the provided claim coverage, the inventive concept is a comparative field method for increasing atmospheric derived nitrogen in corn by using engineered diazotrophic bacteria whose nitrogen fixation genetic regulatory network is modified to increase nitrogen fixation via NifA/NifL regulatory changes and/or heterologous sequence insertion, while the engineered bacteria’s genetic material consists essentially of genetic material from the same genus, and the engineered bacteria demonstrate greater nitrogen fixation activity in non-nitrogen-limiting conditions.
Stated Advantages
Increasing the amount of atmospheric derived nitrogen within a corn plant relative to exposure to a same amount of non-engineered diazotrophic bacteria of the same species.
Greater nitrogen fixation activity of engineered diazotrophic bacteria in non-nitrogen-limiting conditions compared with non-engineered counterparts under similar conditions.
Documented Applications
Increasing atmospheric derived nitrogen in a corn plant in a field by exposing the plants to engineered diazotrophic bacteria.
Field-based use in contexts refined by dependent claims, including defining engineered bacteria colonization/presence and fixed-nitrogen measurement concepts such as fixed nitrogen in corn produced in fields treated with 15N fertilizer.
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