Transgenic plants and fungi capable of metabolizing phosphite as a source of phosphorus

Abstract

System, including methods and compositions, for making and using transgenic plants and/or transgenic fungi that metabolize phosphite as a source of phosphorus for supporting growth.

1 . A nucleic acid, comprising:
 a chimeric gene including (a) a coding region that encodes a phosphite dehydrogenase enzyme and (b) a transcription promoter operatively linked to the coding region,   wherein the promoter is heterologous with respect to the coding region and is functional in plants, fungi, or both, and   wherein the chimeric gene provides sufficient expression of the enzyme, in a plant or fungal cell containing the chimeric gene, to confer an ability on the cell to metabolize phosphite (Phi) as a phosphorus source for supporting growth, thereby enabling growth of the cell without an external source of phosphate (Pi).   
     
     
         2 . The nucleic acid of  claim 1 , wherein the phosphite dehydrogenase enzyme is of bacterial origin. 
     
     
         3 . The nucleic acid of  claim 2 , wherein the phosphite dehydrogenase enzyme is PtxD of  Pseudomonas stutzeri  (SEQ ID NO:1), an analog or derivative of the PtxD of SEQ ID NO:1, or a PtxD-like homolog from another bacterial species. 
     
     
         4 . The nucleic acid of  claim 2 , wherein the bacterial phosphite dehydrogenase enzyme has an amino acid sequence with at least 80% sequence identity to at least one of SEQ ID NOS:1-14. 
     
     
         5 . The nucleic acid of  claim 1 , wherein the phosphite dehydrogenase enzyme has an amino acid sequence including a first sequence region having an NAD-binding motif with sequence similarity or identity to VGILGMGAIG (SEQ ID NO:15), a second sequence region having sequence similarity or identity to XPGALLVNPCRGSVVD (SEQ ID NO:16), where X is K or R, and a third sequence region having sequence similarity or identity to GWX1PX2X3YX4X5GL (SEQ ID NO.19), where X1 is R, Q, T, or K, X2 is A, V, Q, R, K, H, or E, X3 is L or F, X4 is G, F, or S, and X5 is T, R, M, L, A, or S. 
     
     
         6 . The nucleic acid of  claim 1 , wherein the phosphite dehydrogenase enzyme has an amino acid sequence that is at least 90% identical to PtxD from  Pseudomonas stutzeri  (SEQ ID NO:1). 
     
     
         7 . The nucleic acid of  claim 1 , wherein the chimeric gene further includes a transcription terminator that is operatively linked to the coding region and heterologous with respect to the coding region. 
     
     
         8 . The nucleic acid of  claim 1 , wherein the promoter is a plant promoter or a viral promoter of a plant virus and is capable of promoting the sufficient expression of the enzyme in a plant cell. 
     
     
         9 . The nucleic acid of  claim 8 , wherein the promoter corresponds to the 35S promoter of Cauliflower Mosaic Virus. 
     
     
         10 . The nucleic acid of  claim 8 , wherein the promoter is inducible by low phosphate availability. 
     
     
         11 . The nucleic acid of  claim 8 , wherein the promoter corresponds to a promoter of the PLDZ2 gene of  Arabidopsis thaliana.    
     
     
         12 . The nucleic acid of  claim 1 , wherein the chimeric gene is capable of promoting the sufficient expression of the enzyme both in a plant cell and in a fungal cell each containing the chimeric gene. 
     
     
         13 . The nucleic acid of  claim 1 , wherein the promoter is a fungal promoter capable of promoting the sufficient expression of the enzyme in a fungal cell. 
     
     
         14 . A plant cell comprising the nucleic acid of any one of  claims 1 - 12  and capable of metabolizing phosphite as a source of phosphorus for supporting growth. 
     
     
         15 . The plant cell of  claim 14 , further comprising a nucleic acid that expresses a bacterial hypophosphite dehydrogenase enzyme in the plant cell. 
     
     
         16 . The plant cell of  claim 15 , wherein the nucleic acids collectively confer an ability on the cell to metabolize hypophosphite (Hphi) as a phosphorus source for supporting growth. 
     
     
         17 . The plant cell of  claim 16 , wherein expression of the phosphite dehydrogenase enzyme, the bacterial hypophosphite dehydrogenase enzyme, or both are controlled by a root-specific promoter. 
     
     
         18 . The plant cell of  claim 14 , wherein the plant cell is a eukaryotic algal cell. 
     
     
         19 . The plant cell of  claim 18 , wherein the algal cell is a Chlamydomonas cell. 
     
     
         20 . The plant cell of  claim 14 , wherein the plant cell is from a species of vascular plant. 
     
     
         21 . A plant composed of a plurality of plant cells according to  claim 14 . 
     
     
         22 . A plant composed of a plurality of plant cells according to  claim 20 . 
     
     
         23 . The plant of  claim 22 , wherein the plant is a species of crop plant. 
     
     
         24 . The plant of  claim 23 , wherein the species of crop plant is selected from the group consisting of maize, soybean, rice, potatoes, tomatoes, sugarcane, and wheat. 
     
     
         25 . A fungal cell comprising the nucleic acid of any one of  claims 1 - 7 ,  12 , and  13  and capable of metabolizing phosphite as a source of phosphorus for supporting growth. 
     
     
         26 . The fungal cell of  claim 25 , further comprising a nucleic acid that expresses a bacterial hypophosphite dehydrogenase enzyme in the fungal cell. 
     
     
         27 . The fungal cell of  claim 26 , wherein the nucleic acids collectively confer an ability on the cell to metabolize hypophosphite (Hphi) as a phosphorus source for supporting growth. 
     
     
         28 . The fungal cell of  claim 25 , wherein the fungal cell is from a species of  Trichoderma.    
     
     
         29 . A method of reducing fungal infections in plants, the method comprising:
 applying a plurality of the fungal cells of  claim 25  to a seed form of plants, the plants themselves, soil in which the plants are or will be disposed, or a combination thereof.   
     
     
         30 . The fungal cell of  claim 25 , wherein the fungal cell is a member of a species of mycorrhizal fungus capable of forming a symbiotic relationship with a plant. 
     
     
         31 . A plant associated with a plurality of fungal cells according to  claim 30  to form mycorrhizae. 
     
     
         32 . The plant of  claim 31 , wherein the fungal cells render the plant capable of growing on a medium containing phosphite (Phi) as a phosphorus source for supporting growth. 
     
     
         33 . A method of fertilizing a crop plant using hypophosphite and/or phosphite as a phosphorus source for supporting growth, the crop plant (a) including a plurality of cells comprising the nucleic acid of  claim 1 , (b) forming mycorrhizae with a mycorrhizal fungus comprising the nucleic of  claim 1 , and/or (c) being associated with a Trichoderma fungus comprising the nucleic acid of  claim 1 , the method comprising:
 applying at least one reduced form of phosphorus to the plant and/or to soil adjacent the plant, such that the reduced form is metabolized to phosphate by the plant and/or the fungus to support growth and productivity of the plant.   
     
     
         34 . A method of fertilizing the plant of  claim 22 , the method comprising:
 applying at least one reduced form of phosphorus to the plant and/or to soil adjacent the plant, such that the reduced form is metabolized to phosphate by the plant to support growth and productivity of the plant.   
     
     
         35 . A method of treating liquid waste to lower its content of reduced phosphorus, the method comprising:
 contacting (i) water containing hypophosphite and/or phosphite as a contaminant and (ii) a plurality of the plant cells and/or fungal cells comprising the nucleic of  claim 1 , such that at least a portion of the hypophosphite and/or phosphite is oxidized to phosphite and/or phosphate.   
     
     
         36 . The method of  claim 35 , wherein the step of contacting includes a step of contacting the water and a plurality of vascular plants composed of plant cells comprising the nucleic acid of  claim 1 . 
     
     
         37 . A method of isolating transformed cells containing the nucleic acid of  claim 1 , the method comprising:
 contacting cells selected from plant cells and fungal cells with a composition comprising the nucleic acid of  claim 1 ; and   selectively proliferating one or more of the cells that have been transformed by the nucleic acid by culturing the cells in a medium containing phosphite and lacking sufficient phosphate to support growth.   
     
     
         38 . A method of utilizing the nucleic acid of  claim 1  for production of a transgenic plant, the method comprising:
 selecting for growth of plant cells comprising the nucleic acid of  claim 1  as a selectable marker during production of a transgenic plant. 
 
     
     
         39 . A method of obtaining a plant transformed with a nucleic acid encoding a phosphite dehydrogenase enzyme that is expressible from the nucleic acid as a selectable marker, comprising:
 contacting plant cells and a composition including the nucleic acid under conditions that promote introduction of the nucleic acid into at least a subset of the plant cells;   culturing the plant cells in a medium containing phosphite as a primary or exclusive phosphorus source for growth;   selecting transformed plant cells produced by the steps of contacting and culturing, and expressing the phosphite dehydrogenase enzyme as evidenced by growth in the medium; and   regenerating at least a portion of the transformed plant cells into a transgenic plant.   
     
     
         40 . The method of  claim 39 , wherein the composition includes Agrobacterium cells that supply the nucleic acid during the step of contacting. 
     
     
         41 . The method of  claim 39 , wherein the composition includes projectiles that are fired at the plant cells in the step of contacting. 
     
     
         42 . A plant, comprising:
 a nucleic acid including a chimeric gene expressing a phosphite dehydrogenase enzyme such that the plant is capable of metabolizing phosphite (Phi) as a phosphorus source for supporting growth, thereby enabling growth of the plant without an external source of phosphate (Pi).   
     
     
         43 . The plant of  claim 42 , wherein the nucleic acid is stably integrated into the genome of the plant. 
     
     
         44 . The plant of  claim 42 , wherein the plant is a vascular plant. 
     
     
         45 . The plant of  claim 42 , wherein the plant is a species of algae. 
     
     
         46 . A fungus, comprising:
 a nucleic acid including a chimeric gene expressing a phosphite dehydrogenase enzyme such that the fungus is capable of metabolizing phosphite (Phi) as a phosphorus source for supporting growth, thereby enabling growth of the fungus without an external source of phosphate (Pi).   
     
     
         47 . The fungus of  claim 46 , wherein the nucleic acid is stably integrated into the genome of the fungus. 
     
     
         48 . The fungus of  claim 46 , wherein the fungus is a species of  Trichoderma.    
     
     
         49 . The fungus of  claim 46 , wherein the fungus is a mycorrhizal species capable of forming a symbiotic relationship with a plant. 
     
     
         50 . The fungus of  claim 46 , further comprising a plant associated with the fungus to form mycorrhizae. 
     
     
         51 . A nucleic acid, comprising:
 a chimeric gene including (a) a coding region that encodes an enzyme that catalyzes oxidation of phosphite to phosphate and (b) a transcription promoter operatively linked to the coding region,   wherein the promoter is heterologous with respect to the coding region and is functional in a plant cell, and   wherein the chimeric gene provides sufficient expression of the enzyme, in a plant cell containing the chimeric gene, to confer an ability on the plant cell to metabolize phosphite (Phi) as a phosphorus source for supporting growth.