The application generally relates to chloroplast targeting of nuclear-encoded proteins of interest in microalgae. Provided herein are expression cassettes comprising a nucleotide sequence encoding a chloroplast targeting peptide operably linked to a nucleotide sequence encoding a protein of interest, wherein said chloroplast targeting peptide comprises the bipartite targeting sequence of the phosphoribulokinase of Nannochloropsis gaditana (NgPRK BTS). The invention further provides vectors comprising the expression cassettes, and microalgae having stably incorporated or transiently expressed into their nuclear genomes an expression cassette described above. Methods are also provided for the production of a protein of interest in the chloroplast of a microalga, as well as methods for modulating chloroplast pathways.

Disclosed herein are a suite of genetic tools suitable for engineering both the nucleus and chloroplast in diverse microalgae.

Compositions for chemical and/or genetic modification of chloroplasts of plants include a functionalized nanoparticle composition linked to a chloroplast-targeting peptide and a functionalized single walled carbon nanotube (SWNT) composition complexed with a nucleic acid cassette encoding a plastid-specific ribosomal RNA operon (prrn). Methods for chemically and/or genetically modifying chloroplasts of plants include administering these chloroplast-targeted compositions to the leaves of live plants.

This invention relates to systems, methods, and devices for inducing and/or repressing the expression of proteins. More particularly, the invention relates to systems, methods, and devices for inducing and/or repressing the expression of proteins in plastids. An exemplary embodiment involves the regulation of the expression of proteins involved in hydrogen production to stimulate the production of hydrogen gas using the methods, systems, and devices described herein.

The application generally relates to chloroplast targeting of nuclear-encoded proteins of interest in microalgae. Provided herein are expression cassettes comprising a nucleotide sequence encoding a chloroplast targeting peptide operably linked to a nucleotide sequence encoding a protein of interest, wherein said chloroplast targeting peptide comprises the bipartite targeting sequence of the phosphoribulokinase of Nannochloropsis gaditana (NgPRK BTS). The invention further provides vectors comprising the expression cassettes, and microalgae having stably incorporated or transiently expressed into their nuclear genomes an expression cassette described above. Methods are also provided for the production of a protein of interest in the chloroplast of a microalga, as well as methods for modulating chloroplast pathways.

The disclosure provides a method of expressing an agronomically beneficial trait in a plant plastid comprising expressing an exogenous nucleic acid in the plant to produce non-photosynthetic mutant plants, and using callus grown from the mutant plants as recipients for introduction of a construct having a functional copy of the mutated gene and a gene conferring an agronomically beneficial trait. Embodiments provide for mutations in chloroplast-encoded genes, as well as mutations in nuclear-encoded genes targeted to the chloroplast that are required for photosynthesis. The disclosure also provides plants and plant parts produced from such methods, as well as kits for performing the methods as described.

A method for controlling expression of a nuclear-encoded gene in a plant comprising expressing a chloroplast-encoded dsRNA that silences an endogenous nuclear-encoded gene in the plant to produce a transformed plant line expressing the selected trait.

A method of transforming a mitochondrion of a plant cell to express a nitrogenase enzyme includes exposing the plant cell to a mitochondrial-targeting nanocarrier polypeptide and one or more nucleic acids encoding the nitrogenase enzyme. The one or more genes encoding the nitrogenase enzyme can be one or more Klebsiella nif genes. The method can be used to generate plants which have the capability of fixing atmospheric elemental nitrogen.

Compositions and methods for improving plastid transformation in difficult to transform plants are disclosed.

The present invention provides a method for selecting a promoter that functions in an organelle, the method comprising: (1) the step of preparing sequence information obtained by RNA sequencing analysis; (2) the step of mapping the sequence information prepared in the step (1) onto a sequence of organellar DNA; (3) the step of calculating the amount of change in RNA expression in each region based on the mapping information obtained in the step (2); (4) the step of selecting regions in which the amount of change obtained in the step (3) is within a range of preset reference values; and (5) the step of identifying a region, among the regions selected, as a promoter functioning in an organelle.