Method for the transient expression of nucleic acids in plants
View Patent ↗Compositions and methods for transiently expressing proteins in a plant are provided. The compositions comprise plants, seeds, plant tissues, and plant parts expressing a protein, wherein the protein is expressed transiently and the transient expression of the protein can be used as a predictive model of how said protein will be expressed in stable transgenic plants in regards to qualitative and quantitative data. The predictive model may be used but is not limited to: promoter evaluation, evaluation of expression cassette construction for best performance (e.g. addition of enhancers or gene silencing suppressors), evaluation of best ways to express heterologous genes (e.g. point mutations, targeting), fast evaluation of endogenous gene knockout, evaluation of protein expression levels, cellular targeting, tissue targeting, transcriptional enhancers, translational enhancer protein toxicity and metabolic profiling. Further provided are methods of use.
1. A method of transiently expressing a nucleotide sequence in a plant part of a monocot in planta, wherein the monocot plant is selected from the group consisting of maize, wheat, sorghum , barley, millet, oat, sugar cane and rice, comprising the steps of:
a) producing a binary vector comprising at least one expression cassette wherein said expression cassette comprises at least one nucleotide sequence operably linked to a promoter;
b) agro-infiltrating at least 0.05 milliliters of a liquid comprising said binary vector into a plant part of a monocot plant in planta, wherein the plant part is an intact leaf of a plant that is no greater than 5, 6, 7, 8, 9 or 10 days old;
c) transiently expressing said at least one nucleotide sequence in the plant part;
wherein said binary vector does not comprise a virus or virus vector and wherein agro-infiltration of individual intact leaves is carried out and wherein said agro-infiltration is carried out using a needleless syringe.
2. The method of claim 1 , wherein the plant part is from a monocot plant at the two leaf developmental stage or the three leaf developmental stage.
3. The method of claim 1 , wherein the promoter is a constitutive or leaf preferred promoter.
4. The method of claim 1 , wherein the nucleotide sequence is transiently expressed for at least 5, 6, 7, 8, 9 or 10 days.
5. The method of claim 1 , wherein said expression cassette is greater than 1.5 kilobases in size.
6. The method of claim 1 , wherein the method comprises agro-infiltration of a binary vector.
7. The method of claim 1 , wherein agro-infiltration is carried out on the underside of said leaf.
8. A method of transiently expressing a nucleotide sequence of interest in a maize plant part in planta comprising the steps of:
a) producing a binary vector comprising at least one expression cassette, wherein said expression cassette comprises at least one nucleotide sequence operably linked to a promoter;
b) agro-infiltrating at least 0.05 milliliters of a liquid comprising said binary vector into a plant part of a maize plant in planta, wherein the plant part is an intact leaf of a plant that is no greater than 5, 6, 7, 8, 9 or 10 days old;
c) transiently expressing said at least one nucleotide sequence in the plant part;
wherein said binary vector does not comprise a virus or virus vector and wherein agro-infiltration of individual intact leaves is carried out and wherein said agro-infiltration is carried out using a needleless syringe.
9. The method of claim 8 , wherein the maize plant part is V2 or V3 maize leaf tissue.