Pathogen resistant citrus compositions, organisms, systems, and methods

Abstract

The present disclosure relates, according to some embodiments, to pathogen resistant citrus compositions, organisms, systems, and methods. For example, a composition may comprise a peptide (e.g., a defensin peptide) and/or a nucleic acid (e.g., a defensin nucleic acid). A pathogen resistant citrus plant may comprise, in some embodiments, a defensin peptide and/or an expressable nucleic acid encoding a defensin peptide.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims priority to U.S. Provisional Patent Application 61 / 591,680, filed on Jan. 27, 2012 and U.S. Provisional Patent Application 61 / 641,641, filed on May 2, 2012, both of which applications are incorporated herein by reference.FIELD OF THE DISCLOSURE[0002]The present disclosure relates, in some embodiments, to pathogen resistant citrus compositions, organisms, systems, and methods.BACKGROUND OF THE DISCLOSURE[0003]At present, there are no Citrus cultivars resistant to bacterial canker (Xanthomonas axonopodis pv. citri) (Xac), and / or citrus Huanglongbing (ex greening) caused by Candidatus Liberibacter asiaticus (Las). Indeed, no genetic resistance to these microbial pathogens has ever been found within the Citrus genus. Conventional cross-breeding efforts to produce resistant cultivars have been hindered by the complex reproductive biology and long life cycle of Citrus spp.SUMMARY[0004]Accordingly, a need has arisen for p...

AI Technical Summary

Benefits of technology

[0006]The present disclosure relates, in some embodiments, to defensin expression vectors operable in citrus. For example, an expression vector may comprise, in a 5′ to 3′ direction, (a) an expression control sequence; (b) an expressable nucleic acid (e.g., a nucleic acid encoding an exogenous polypeptide) operably linked to the expression control sequence; and (c) a 3′ termination sequence operably linked to the expressable nucleic acid. In some embodiments, an exogenous nucleic acid may comprise a nucleic acid sequence having at least about 75% identity (e.g., at least about 98% identity) to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 29. An expression vector may be located in a bacterial cell or a plant cell according to some embodiments. An expression vector may comprise, in some embodiments, the nucleotide sequence AACAATGG at positions −4 to 4 relative to a coding sequence (e.g., encoded by an exogenous nucleic acid sequence). According to some embodiments, an expression vector may comprise a linker (e.g., 3′ of the expression control sequence and / or 5′ of the nucleic acid (e.g., a nucleic acid encoding an exogenous polypeptide) having a length of from about 1 to about 200 nucleotides.

[0007]The present disclosure relates, in some embodiments, to a bacterial cell comprising an expression vector. For example, a bacterial cell may comprise an expression vector comprising, in a 5′ to 3′ direction, (a) an expression control sequence; (b) an expressable nucleic acid (e.g., a nucleic acid encoding an exogenous polypeptide) operably linked to the expression control sequence; and (c) a 3′ termination sequence operably linked to the expressable nucleic acid. A bacterial cell may comprise, for example, an expression vector comprising, in a 5′ to 3′ direction, (a) an expression control sequence; (b) an exogenous nucleic acid operably linked to the expression control sequence; and / or (c) a 3′ termination sequence operably linked to the exogenous nucleic acid, wherein the exogenous nucleic acid comprises a nucleic acid sequence having at least about 98% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12.

[0008]The present disclosure relates, in some embodiments, to a plant cell (e.g., a citrus plant cell) comprising an expression vector. For example, a plant cell (e.g., a citrus plant cell) may comprise an expression vector comprising, in a 5′ to 3′ direction, (a) an expression control sequence; (b) an expressable nucleic acid (e.g., a nucleic acid encoding an exogenous polypeptide) operably linked to the expression control sequence; and (c) a 3′ termination sequence operably linked to the expressable nucleic acid. A plant cell (e.g., a citrus plant cell) may comprise, for example, an expression vector comprising, in a 5′ to 3′ direction, (a) an expression control sequence; (b) an exogenous nucleic acid operably linked to the expression control sequence; and / or (c) a 3′ termination sequence operably linked to the exogenous nucleic acid, wherein the exogenous nucleic acid comprises a nucleic acid sequence having at least about 98% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 29. A plant cell (e.g., a citrus plant cell) may be located in a plant (e.g., a citrus plant) according to some embodiments. Examples of citrus plants include, without limitation, orange and grapefruit. A plant cell may comprise a defensin peptide. A defensin peptide may have, in some embodiments, an amino acid sequence having at least about 99% identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 28 (e.g., encoded by and / or expressed from an expression vector nucleic acid) according to some embodiments.

[0009]In some embodiments, the present disclosure relates to a citrus plant (e.g., orange and / or grapefruit) comprising an expression vector. A citrus plant may comprise an expression vector in a single cell, a plurality of cells (e.g., mosaic), or in all cells. A mosaic plant may arise from a graft in some embodiments. For example, a citrus plant may comprise a graft of a transgenic plant having an expression vector in all cells (e.g., scion) and a plant having a different expression vector or no expression vector in its cells (e.g., rootstock). A citrus plant may comprise, in some embodiments, in a single cell, a plurality of cells (e.g., mosaic), or in all cells a first expression vector (e.g., encoding a first defensin peptide) and in a single cell, a plurality of cells (e.g., mosaic), or in all cells a second expression vector (e.g., encoding a second defensin peptide). For example, a citrus plant cell may comprise (a) a first expression vector, the first expression vector comprising, in a 5′ to 3′ direction, (i) a first expression control sequence; (ii) a first exogenous nucleic acid operably linked to the first expression control sequence; and (iii) a first 3′ termination sequence operably linked to the first exogenous nucleic acid, wherein the first exogenous nucleic acid comprises a nucleic acid sequence having at least about 98% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 9, and SEQ ID NO: 11; and (b) a second expression vector, the second expression vector comprising, in a 5′ to 3′ direction, (iv) a second expression control sequence; (v) a second exogenous nucleic acid operably linked to the second expression control sequence; and (vi) a second 3′ termination sequence operably linked to the second exogenous nucleic acid, wherein the second exogenous nucleic acid comprises a nucleic acid sequence having at least about 98% identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 10, and SEQ ID NO: 12. According to some embodiments, a citrus plant may comprise in a single cell, a plurality of cells (e.g., mosaic), or in all cells an expression vector comprising a first nucleic acid sequence encoding a first defensin peptide (e.g., SoD2) and a second nucleic acid sequence encoding a second defensin peptide (e.g., SoD7). In some embodiments, a citrus plant may comprise a defensin peptide in a single cell, a plurality of cells (e.g., mosaic), or in all cells. A citrus plant may comprise in a single cell, a plurality of cells (e.g., mosaic), or in all cells a first defensin peptide (e.g., a peptide having at least about 99% identity to SEQ ID NO: 1 or SEQ ID NO: 7) and in a single cell, a plurality of cells (e.g., mosaic), or in all cells a second defensin peptide (e.g., a peptide having at least about 99% identity to SEQ ID NO: 2 or SEQ ID NO: 8).

[0010]The present disclosure relates, in some embodiments, to methods of expressing in a citrus plant an exogenous nucleic acid comprising a nucleic acid sequence encoding an expressed peptide (e.g., a defensin peptide). For example, a method may comprise contacting an expression cassette comprising an exogenous nucleic acid or an expression vector comprising an exogenous nucleic acid with the cytosol of a cell of a citrus plant under conditions that permit expression of the exogenous nucleic acid and formation of the expressed peptide. In some embodiments, an exogenous nucleic acid may comprise a nucleic acid sequence having at least 98% identity to a nucleic acid sequence selected from SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and / or SEQ ID NO: 29. In some embodiments, an expression vector and / or an expression cassette may comprise, in a 5′ to 3′ direction, an expression control sequence, the exogenous nucleic acid operably linked to the expression control sequence, and a 3′ termination sequence operably linked to the exogenous nucleic acid. An expressed peptide may comprise an amino acid sequence having at least 99% identity to an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 7, SEQ ID NO: 8, and / or SEQ ID NO: 28 according to some embodiments. Contacting an expression vector or cassette may further comprise, in some embodiments, co-cultivating the cell with an Agrobacterium cell comprising the expression vector or expression cassette to form a co-cultivated plant cell. According to some embodiments, a plant may be regenerated from a co-cultivated plant cell.

[0011]The present disclosure relates, in some embodiments, to methods for treating a citrus plant having and / or at risk of having a microbial infection (e.g., bacterial canker (Xanthomonas axonopodis pv. citri) (Xac), and / or citrus Huanglongbing (ex greening) caused by Candidatus Liberibacter asiaticus (Las)). For example, a method may comprise forming in the citrus plant at least one defensin peptide. Forming in the citrus plant at least one defensin peptide may comprise, in some embodiments, grafting the citrus plant to cutting (e.g., a scion or a rootstock) from a second citrus plant, the second citrus plant comprising an expression vector and / or an expression cassetted comprising, in a 5′ to 3′ direction, an expression control sequence, a defensin nucleic acid operably linked to the expression control sequence, and a 3′ termination sequence operably linked to the defensin nucleic acid, wherein the defensin nucleic acid comprises a nucleic acid sequence encoding an amino acid sequence having at least 99% identity to an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 7, SEQ ID NO: 8, and / or SEQ ID NO: 28 under conditions that permit expression of the defensin nucleic acid.

Problems solved by technology

Indeed, no genetic resistance to these microbial pathogens has ever been found within the Citrus genus.

Conventional cross-breeding efforts to produce resistant cultivars have been hindered by the complex reproductive biology and long life cycle of Citrus spp.

Images