Reporter constructs for compound screening
The instant description provides reporter constructs, transgenic cells, and transgenic organisms and methods for identifying agents that can regulate gene expression and improve plant performance and yield. Compounds that increase plant performance or yield are identified by contacting a test compound with a plant cell that comprises a target promoter sequence operably linked to a polynucleotide sequence encoding a DNA sequence-specific transactivator, and a reporter polynucleotide that is operably linked to a promoter sequence that is recognized by the DNA sequence-specific transactivator. The target promoter sequence can be recognized by a transcriptional regulatory polypeptide capable of modulating specific signaling pathways that enhance plant performance or yield.
1. A method of screening for a hit compound that increases a plant performance-related or yield-related trait in a plant, said method comprising the steps of:
(a) contacting at least one test compound with a transgenic plant cell comprising: (i) a target inducible promoter sequence that is operably linked to a polynucleotide sequence that encodes a DNA sequence-specific transactivator; and (ii) a reporter polynucleotide that is operably linked to a promoter sequence that can be recognized by the DNA sequence-specific transactivator;
wherein the target inducible promoter sequence can be recognized by a transcriptional regulatory polypeptide that modulates a signaling pathway and said modulation results in increased plant performance or increased yield in a plant;
(b) identifying a test compound that alters the reporter gene activity relative to controls and selecting a hit compound so identified; and
(c) in a secondary screen, said hit compound is applied to the plant and said hit compound increases the plant performance or yield of the plant.
2. The method of claim 1 , wherein the DNA sequence-specific transactivator is a translational fusion of a DNA binding domain (DBD) and a transcriptional activation domain (AD);
wherein the AD comprises SEQ ID NO: 20; and
wherein the DBD comprises SEQ ID NO: 16.
3. The method of claim 1 , wherein the reporter polynucleotide encodes green fluorescent protein.
4. The method of claim 1 , wherein the target inducible promoter sequence is a desiccation- or drought-inducible promoter located in the promoter region of AT1G71520, At5g52310, At5g52300, AT1G16850, At3g46230, AT1G52690, At2g37870, AT5G43840, At5g66780, At3g17520, or At4g09600 (SEQ ID NO: 48, 38, 30, 29, 31, 32, 33, 34, 35, 36, or 37, respectively), or a functional part thereof having a promoter function.
5. The method of claim 1 , wherein the transgenic cell further comprises a polynucleotide sequence that encodes an additional DNA sequence-specific transactivator,
wherein transcription of the polynucleotide sequence encoding the additional DNA sequence-specific transactivator is under the control of a promoter sequence that is recognized by the first DNA sequence-specific transactivator; and wherein the additional DNA sequence-specific transactivator regulates transcription of the reporter gene through binding of its cognate sequence that is operably linked to the reporter polynucleotide.
6. The method of claim 1 , further comprising the step of:
(d) contacting a plant with the hit compound and detecting the increased performance or yield in the plant relative to controls.
7. The method of claim 1 , wherein the at least one test compound is obtained from a combinatorial chemical library, a natural product library, an exudate of a microbial culture, an extract of a microbial culture, or a microbially-derived natural product.
8. A method for increasing a performance-related or yield-related trait of a plant, said method comprising the steps of:
(a) contacting at least one test compound with a transgenic plant cell comprising: (i) a target inducible promoter sequence that is operably linked to a polynucleotide sequence that encodes a DNA sequence-specific transactivator; and (ii) a reporter polynucleotide that is operably linked to a promoter sequence that can be recognized by the DNA sequence-specific transactivator;
wherein the target inducible promoter sequence can be recognized by a transcriptional regulatory polypeptide that modulates specific signaling pathways and said modulation results in increased plant performance or yield in the plant;
(b) identifying a test compound that produces a level of induction of the reporter polynucleotide greater than the level of induction in a control plant and selecting a hit compound so identified; and
(c) in a secondary screen, contacting the plant with the hit compound and detecting increased performance or yield in the plant relative to a control plant.
9. The method of claim 8 , wherein the level of induction of the reporter polynucleotide is at least 2.5 fold.
10. The method of claim 8 , wherein the DNA sequence-specific transactivator is a translational fusion of a DBD comprising a sequence encoded by SEQ ID NO: 1 and an AD comprising SEQ ID NO: 20.
11. The method of claim 8 , wherein the reporter polynucleotide encodes green fluorescent protein.
12. The method of claim 8 , wherein the target inducible promoter sequence is a desiccation- or drought-inducible promoter located in the promoter region of AT1G71520, At5g52310, At5g52300, AT1G16850, At3g46230, AT1G52690, At2g37870, AT5G43840, At5g66780, At3g17520, or At4g09600 (SEQ ID NO: 48, 38, 30, 29, 31, 32, 33, 34, 35, 36, or 37, respectively), or a functional part thereof having a promoter function.
13. The method of claim 8 , wherein the at least one test compound is obtained from a combinatorial chemical library, a natural product library, an exudate of a microbial culture, an extract of a microbial culture, or a microbially-derived natural product.
14. A method of screening for a hit compound that increases a plant performance-related or yield-related trait in a plant, said method comprising the steps of:
(a) contacting at least one test compound with a transgenic plant cell comprising: (i) a target inducible promoter sequence that is operably linked to a polynucleotide sequence that encodes a DNA sequence-specific transactivator; and (ii) a reporter polynucleotide that is operably linked to a promoter sequence that can be recognized by the DNA sequence-specific transactivator;
wherein said at least one test compound is obtained from an exudate of a microbial culture, an extract of a microbial culture, or a microbially-derived natural product;
wherein the target inducible promoter sequence can be recognized by a transcriptional regulatory polypeptide that modulates a signaling pathway and said modulation results in increased plant performance or increased yield in a plant;
(b) identifying a test compound that alters the reporter gene activity relative to controls and selecting a hit compound so identified; and
(c) in a secondary screen, said hit compound is applied to the plant and said hit compound increases the plant performance or yield of the plant.
15. The method of claim 1 , wherein the target inducible promoter sequence is a disease-inducible promoter located in the promoter region of AT1G15125 or AT5G44420 (SEQ ID NO: 44 or 46, respectively), or a functional part thereof having a promoter function.
16. The method of claim 1 , wherein the target inducible promoter sequence is a promoter inducible by a change of nitrogen status located in the promoter region of AT1G13300, AT2G48080, AT3G25790, AT5G10210, AT5G19970, AT5G44420, or AT5G24770 (SEQ ID NO: 39, 40, 41, 42, 43, 46, or 47, respectively), or a functional part thereof having a promoter function.
17. The method of claim 1 , wherein the target inducible promoter sequence is a promoter inducible by low phosphate availability located in the promoter region of At2g38940 (SEQ ID NO: 45), or a functional part thereof having a promoter function.
18. The method of claim 8 , wherein the target inducible promoter sequence is a disease-inducible promoter located in the promoter region of AT1G15125 or AT5G44420 (SEQ ID NO: 44 or 46, respectively), or a functional part thereof having a promoter function.
19. The method of claim 8 , wherein the target inducible promoter sequence is a promoter inducible by a change of nitrogen status located in the promoter region of AT1G13300, AT2G48080, AT3G25790, AT5G10210, AT5G19970, AT5G44420, or AT5G24770 (SEQ ID NO: 39, 40, 41, 42, 43, 46, or 47, respectively), or a functional part thereof having a promoter function.
20. The method of claim 8 , wherein the target inducible promoter sequence is a promoter inducible by low phosphate availability located in the promoter region of At2g38940 (SEQ ID NO: 45), or a functional part thereof having a promoter function.