Patent Application
20180153124
The wheat (Triticum aestivum) Fusarium head blight (FHB), is mostly caused by Fusarium graminearum and has resulted in losses of $3 billion/year in North America. This pathogen not only reduces the crop yield, but also contains deoxynivalenol (DON), a mycotoxin that is harmful to the human and animal health. Several reports confirm the resistance of Chinese wheat lines to FHB: Suami 3 and Wangshuibai. However, strategies to transfer the FHB resistance genes into economically important wheat via conventional breeding have not been successful, due in part because resistance to the FHB pathogen is a complex trait and breeding of these two genotypes with agronomically important wheat lines is very difficult as. It has been reported that a few genes including the Thaumatin-Like Protein1 (tlp1) and the gene coding for the involvement of the coronatine insensitive 1-like protein (coi1) receptor are important in response to infection by the FHB pathogen.
Described herein are expression systems that provide resistance to Fusarium head blight (FHB). Wheat plants that include such expression systems are at least 2-fold to 5-fold more resistant to FHB than wild type or parent plant lines that do not have the expression systems.
For example, the expression systems described herein can have an expression cassette comprising at least one promoter operably linked to a nucleic acid that encodes a COI1 protein, a Tlp1 protein, or a combination thereof. In some cases, the expression system can hare two expression cassettes, a first expression cassette comprising a first promoter operably linked to a nucleic acid that encodes a COI1 protein, and a second promoter operably linked to a nucleic acid that encodes a Tlp1 protein. The expression systems provide enhanced levels of COI1 and/or Tlp1 proteins, which provides plants with improved resistance to Fusarium head blight (FHB).
Also described herein are plants, plant cells, and plant seeds that have the expression systems, as well as methods of making FHB-resistant plant cells, plants, and plant seeds.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Described herein are expression cassettes, plant cells, plants, and plant seeds that include heterologous nucleic acids that encode polypeptides that confer resistance to Fusarium head blight (FHB). As shown herein, enhanced expression of CORONATINE INSENSITIVE 1 (COI1) protein and Thaumatin-Like Protein (tlp1) reduces the rate of FHB disease by at least two-fold to five-fold.
COI1 is an F-box protein that can mediate jasmonate signaling by promoting hormone-dependent ubiquitylation and degradation of transcriptional repressor JASMONATE ZIM DOMAIN (JAZ) proteins. JAZ proteins are repressors of the jasmonic acid signaling pathway. COI1 proteins can form a co-receptor with one or more JAZ transcriptional repressor protein that can bind jasmonate. Formation, or lack of formation, of jasmonate/COI1/JAZ complexes can regulate the sophisticated, multilayered immune signaling network present in plants.
The stress hormone jasmonate (JA) plays a central role in regulating plant defenses against a variety of chewing insects and necrotrophic pathogens. Salicylic acid (SA) is another plant hormone that can be employed for plant defense against biotrophic or hemibiotrophic pathogens. During host-pathogen coevolution, however, many successful plant pathogens developed mechanisms to attack or hijack components of the plant immune signaling network as part of their pathogenesis strategies. As a result, the plant immune system, although powerful, is often fallible in the face of highly evolved pathogens.
The COI1 protein expressed by the expression cassette, plant cells, plants, and plant seeds can have a variety of sequences. An example of a COI1 protein from Triticum aestivum (wheat) with NCBI accession number ADK66973.1 has the following sequence (SE ID NO:1).
An example of a nucleotide (cDNA) sequence that encodes the SEQ ID NO:1 COI1 protein (NCBI accession number HM447645.1) is shown below as SEQ ID NO:2.
Another example of a COI1 protein from Triticum aestivum (wheat) has NCBI accession number ADK66974.1 (GI:301318118), with the following sequence (SEQ ID NO:3).
An example of a nucleotide (cDNA) sequence that encodes the second Triticum aestivum (wheat) SEQ ID NO:3 wheat COI1 protein (NCBI accession number HM447646.1) is shown below as SEQ ID NO:4.
A comparison of the Triticum aestivum (wheat) COI1 SEQ ID NO:1 sequence and the Triticum aestivum (wheat) COI1 SEQ ID NO:3 sequence is shown below, illustrating that the two proteins have at least 79% sequence identity.
In another example, an Oryza sativa Indica Group COI1 protein with a sequence provided by the NCBI database as accession number EAY98249.1, is shown below as SEQ ID NO:5.
A comparison of the Triticum aestivum (wheat) COI1 SEQ ID NO:1 sequence and the Oryza sativa Indica Group COI1 protein COI1 SEQ ID NO:5 sequence is shown below, illustrating that the two proteins have at least 86% sequence identity.
In another example, a Hordeum vulgare subsp. vulgare (domesticated barley) COI1 protein with a sequence provided by the NCBI database as accession number BAJ94334.1, is shown below as SEQ ID NO:6.
An example of a nucleotide (cDNA) sequence that encodes the SEQ ID NO:6 Hordeum vulgare subsp. vulgare COI1 protein (NCBI accession number AK363130.1) is shown below as SEQ ID NO:7.
A comparison of the Triticum aestivum (wheat) COI11 SEQ ID NO:1 sequence and the Hordeum vulgare subsp. vulgare COI1 protein with SEQ ID NO:6 sequence is shown below, illustrating that the two proteins have at least 94% sequence identity.
In another example, a second Hordeum vulgare subsp. vulgare (domesticated barley) COI1 protein with a sequence provided by the NCBI database as accession number BAJ90363.1, is shown below as SEQ ID NO:8.
An example of a nucleotide (cDNA) sequence that encodes the SEQ ID NO:8 Hordeum vulgare subsp. vulgare COI1 protein (NCBI accession number AK359152.1) is shown below as SEQ ID NO:9.
A comparison of the Triticum aestivum (wheat) COI1 SEQ ID NO:1 sequence and the second Hordeum vulgare subsp. vulgare COI1 protein with SEQ ID NO:8 sequence is shown below, illustrating that the two proteins have at least 93% sequence identity.
In another example, a Sorghum bicolor (sorghum) COI1 protein with a sequence provided by the NCBI database as accession number XP_002439888.1, is shown below as SEQ ID NO:10.
An example of a nucleotide (cDNA) sequence that encodes the SEQ ID NO:10 Sorghum bicolor COI1 protein (NCBI accession number XM_002439843.1) is shown below as SEQ ID NO:11.
A comparison of the Triticum aestivum (wheat) COI1 SEQ ID NO:1 sequence and the Sorghum bicolor COI1 protein with SEQ ID NO:10 sequence is shown below, illustrating that the two proteins have at least 84% sequence identity.
In another example, an Arabidopsis thaliana COI1 protein with a sequence provided by the NCBI database as accession number 004197.1 (GI:59797640) is shown below as SEQ ID NO:12.
An example of a nucleotide (cDNA) sequence that encodes the SEQ ID NO:12 COI1 protein (NCBI accession number NM_129552.4 (GI: 1063702813)) is shown below as SEQ ID NO:13.
A comparison of the Triticum aestivum (wheat) COI11 SEQ ID NO:1 sequence and the Arabidopsis thaliana COI1 protein COI1 SEQ ID NO:12 sequence is shown below, illustrating that the two proteins have at least 56% sequence identity.
An example of a COT 1 protein from Brassica rapa (turnip) with NCBI accession number XP_009133392.1 (GI:685284974) has the following sequence (SEQ ID NO:14).
An example of a nucleotide (cDNA) sequence that encodes the SEQ ID NO:14 Brassica rapa (turnip) COI1 protein (NCBI cDNA accession number XM_009135144.1 (GI:685284973)) is shown below as SEQ ID NO:15.
A comparison of the Triticum aestivum (wheat) COI1 SEQ ID NO:1 sequence and the Brassica rapa (turnip) COI1 protein SEQ ID NO:14 sequence is shown below, illustrating that the two proteins have at least 56% sequence identity.
An example of a COI1 protein from Brassica napus (rapeseed) with NCBI accession number CDY60996.1 (GI:674872982) has the following sequence (SEQ ID NO:16).
A comparison of the Triticum aestivum (wheat) COI1 SEQ ID NO:1 sequence and the Brassica napus (rapeseed) COI1 protein SEQ ID NO:16 sequence is shown below, illustrating that the two proteins have at least 56% sequence identity.
An example of a COI1 protein from Brassica oleracea (cabbage, Brussel sprouts, kale, cauliflower, etc.) with NCBI accession number XP_013628733.1 (GI:922451771) has the following sequence (SEQ ID NO:17).
An example of a nucleotide (cDNA) sequence that encodes the Brassica oleracea SEQ ID NO:17 COI1 protein (NCBI cDNA accession number XM_013773279.1 (GI:922451770)) is shown below as SEQ ID NO:18.
A comparison of the Triticum aestivum (wheat) COI1 SEQ ID NO:1 sequence and the Brassica oleracea (cabbage, Brussel sprouts, kale, cauliflower, etc.) COI1 protein SEQ ID NO:17 sequence is shown below, illustrating that the two proteins have at least 56% sequence identity.
An example of a COI1 protein from Theobroma cacao (cocoa) with NCBI accession number XP_007009091.2 (GI: 1063526274) has the following sequence (SEQ ID NO:19).
An example of a nucleotide (cDNA) sequence that encodes the Theobroma cacao SEQ ID NO:19 COI1 protein (NCBI cDNA accession number XM_007009029.2 GI:1063526273)) is shown below as SEQ ID NO:20.
A comparison of the Triticum aestivum (wheat) COI1 SEQ ID NO:1 sequence and the Theobroma cacao (cocoa) COI1 protein SEQ ID NO:19 sequence is shown below, illustrating that the two proteins have at least 61% sequence identity.
An example of a COI1 protein from Glycine max (soybean) with NCBI accession number NP_001238590.1 (GI:351724347) has the following sequence (SEQ ID NO:21).
An example of a nucleotide (cDNA) sequence that encodes the Glycine max SEQ ID NO:21 COI1 protein (NCBI cDNA accession number NM_001251661.1 (GI:351724346)) is shown below as SEQ ID NO:22.
A comparison of the Triticum aestivum (wheat) COI1 SEQ ID NO:1 sequence and the Glycine max (soybean) COI1 protein SEQ ID NO:21 sequence is shown below, illustrating that the two proteins have at least 60% sequence identity.
An example of a COI1 protein from Zea mays (corn) with NCBI accession number NP_001150429.1 (GI:226503785) has the following sequence (SEQ ID NO:23).
An example of a nucleotide (cDNA) sequence that encodes the Zea mays SEQ ID NO:27 COI1 protein (with NCBI cDNA accession number NM_001156957.1, GI:226503784)) is shown below as SEQ ID NO:24.
A comparison of the Triticum aestivum (wheat) COI1 SEQ ID NO:1 sequence and the Zea mays (corn) COI1 protein SEQ ID NO:23 sequence is shown below, illustrating that the two proteins have at least 60% sequence identity.
An example of a COI1 protein from Arachis hypogaeal (peanut) with NCBI accession number AGH62009.1 (GI:469609864) has the following sequence (SEQ ID NO:25).
An example of a nucleotide (cDNA) sequence that encodes the Arachis hypogaeal (peanut) SEQ ID NO:38 COI1 protein (with NCBI cDNA accession number KC355791.1 (GI:469609863)) is shown below as SEQ ID NO:26.
A comparison of the Triticum aestivum (wheat) COI1 SEQ ID NO:11 sequence and the Arachis hypogaeal (peanut) COI1 protein SEQ ID NO:25 sequence is shown below, illustrating that the two proteins have at least 33% sequence identity.
In some cases, the COI1 protein can have a sequence related to SEQ ID NO:1, 2, 5, 8, 10, 13, 16, 19, 22, 25, 28, 31, 33, 36, 39, 42, 45, or 48. However, the modified COI1 protein can have some sequence variation relative to SEQ ID NO:1, 2, 5, 8, 10, 13, 16, 19, 22, 25, 28, 31, 33, 36, 39, 42, 45, or 48. For example, a modified COI1 protein can have an amino acid sequence that has at least 90%, or at least 95%, or at least 96%, or at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:1, 2, 5, 8, 10, 13, 16, 19, 22, 25, 28, 31, 33, 36, 39, 42, 45, or 48.
The Tlp1 protein expressed by the expression cassette, plant cells, plants, and plant seeds can have a variety of sequences. An example of a Tlp1 protein from Triticum aestivum (wheat) with NCBI accession number CAA41283.1 has the following sequence (SEQ ID NO:27).
An example of a nucleotide (cDNA) sequence that encodes the Triticum aestivum (wheat) SEQ ID NO:27 Tlp1 protein (with NCBI cDNA accession number X58394.1) is shown below as SEQ ID NO:28.
A second example of a Tlp1 protein from Triticum aestivum (wheat) with NCBI accession number AAK60568.1 has the following sequence (SEQ ID NO:29).
An example of a nucleotide (cDNA) sequence that encodes the Triticum aestivum (wheat) SEQ ID NO:29 Tlp1 protein (with NCBI cDNA accession number AF384146.1) is shown below as SEQ ID NO:30.
A comparison of the Triticum aestivum (wheat) Tlp1 SEQ ID NO:27 sequence and the Triticum aestivum (wheat) Tlp1 protein SEQ ID NO:29 sequence is shown below, illustrating that the two proteins have at least 95% sequence identity.
An example of a Hordeum vulgare (domesticated barley) Tlp1 protein with a sequence provided by the NCBI database as accession number P32938.1, is shown below as SEQ ID NO:31.
A comparison of the Triticum aestivum (wheat) Tlp1 SEQ ID NO:27 sequence and the Hordeum vulgare (domesticated barley) Tlp1 protein SEQ ID NO:31 sequence is shown below, illustrating that the two proteins have at least 97% sequence identity.
An example of a Triticum urartu (red wild einkorn) Tip 1 protein with a sequence provided by the NCBI database as accession number EMS68875.1, is shown below as SEQ ID NO:32.
A comparison of the Triticum aestivum (wheat) Tlp1 SEQ ID NO:27 sequence and the Triticum urartu (red wild einkorn) Tlp1 protein SEQ ID NO:32 sequence is shown below, illustrating that the two proteins have at least 96% sequence identity.
An example of an Aegilops tauschii (goat grass) Tlp1 protein with a sequence provided by the NCBI database as accession number EMT13094.1, is shown below as SEQ ID NO:33.
A comparison of the Triticum aestivum (wheat) Tlp1 SEQ ID NO:27 sequence and the Aegilops tauschii (goat grass) Tlp1 protein SEQ ID NO:33 sequence is shown below, illustrating that the two proteins have at least 96% sequence identity.
An example of a Secale cereale (rye) Tlp1 protein with a sequence provided by the NCBI database as accession number AAC67259.1, is shown below as SEQ ID NO:34.
An example of a nucleotide (cDNA) sequence that encodes the Secale cereale (rye) SEQ ID NO:34 Tlp1 protein (with NCBI cDNA accession number AF096927.1) is shown below as SEQ ID NO:35.
A comparison of the Triticum aestivum (wheat) Tlp1 SEQ ID NO:27 sequence and the Secale cereale (rye) Tlp1 protein SEQ ID NO:34 sequence is shown below, illustrating that the two proteins have at least 94% sequence identity.
An example of an Avena sativa (oat) Tlp1 protein with a sequence provided by the NCBI database as accession number P50695.1, is shown below as SEQ ID NO:36.
A comparison of the Triticum aestivum (wheat) Tlp1 SEQ ID NO:27 sequence and the Avena sativa (oat) Tlp1 protein SEQ ID NO:36 sequence is shown below, illustrating that the two proteins have at least 80% sequence identity.
A third example of a Tlp1 protein from Triticum aestivum (wheat) with NCBI accession number AIG62904.1 has the following sequence (SEQ ID NO:37).
An example of a nucleotide (cDNA) sequence that encodes the Triticum aestivum (wheat) SEQ ID NO:37 Tlp1 protein (with NCBI cDNA accession number KJ764822.1) is shown below as SEQ ID NO:38.
A comparison of the Triticum aestivum (wheat) Tlp1 SEQ ID NO:27 sequence and the third Triticum aestivum (wheat) Tlp1 protein SEQ ID NO:37 sequence is shown below, illustrating that the two proteins have at least 92% sequence identity.
An example of a Tlp1 protein from Zea mays (maize) with NCBI accession number NP_001141293.1 has the following sequence (SEQ ID NO:39).
An example of a nucleotide (cDNA) sequence that encodes the Zea mays (maize) SEQ ID NO:39 Tlp1 protein (with NCBI cDNA accession number NM_001147821.2) is shown below as SEQ ID NO:40.
A comparison of the Triticum aestivum (wheat) Tlp1 SEQ ID NO:27 sequence and the Zea mays (maize) Tlp1 protein SEQ ID NO:39 sequence is shown below, illustrating that the two proteins have at least 68% sequence identity.
An example of a Tip 1 protein from Sorghum bicolor (sorghum) with NCBI accession number XP_002443621.1 has the following sequence (SEQ ID NO:41).
An example of a nucleotide (cDNA) sequence that encodes the Sorghum bicolor (sorghum) SEQ ID NO:41 Tlp1 protein (with NCBI cDNA accession number XM_002443576.1) is shown below as SEQ ID NO:42.
A comparison of the Triticum aestivum (wheat) Tlp1 SEQ ID NO:27 sequence and the Sorghum bicolor (sorghum) Tlp1 protein SEQ ID NO:41 sequence is shown below, illustrating that the two proteins have at least 69% sequence identity.
An example of a Tlp1 protein from Oryza sativa Indica Group (rice) with NCBI accession number EAY83985.1 has the following sequence (SEQ ID NO:43).
A comparison of the Triticum aestivum (wheat) Tlp1 SEQ ID NO:27 sequence and the Oryza sativa Indica Group (rice) Tlp1 protein SEQ ID NO:43 sequence is shown below, illustrating that the two proteins have at least 65% sequence identity.
An example of a Tlp1 protein from Setaria italica (foxtail millet) with NCBI accession number XP_004963268.1 has the following sequence (SEQ ID NO:44).
An example of a nucleotide (cDNA) sequence that encodes the Setaria italica (foxtail millet) SEQ ID NO:45 Tlp1 protein (with NCBI cDNA accession number XM_004963211.1) is shown below as SEQ ID NO:45.
A comparison of the Triticum aestivum (wheat) Tlp1 SEQ ID NO:27 sequence and the Setaria italica (foxtail millet) Tlp1 protein SEQ ID NO:44 sequence is shown below, illustrating that the two proteins have at least 65% sequence identity.
Plant cells can be modified to include expression cassettes or transgenes that can express any of the COI1 and/or Tlp1 proteins described herein. Such an expression cassette or transgene can include a promoter operably linked to a nucleic acid segment that encodes any of the COI1 and/or Tlp1 proteins described herein.
Promoters provide for expression of mRNA from the COI1 nucleic acids. In some cases the promoter can be a COI1 and/or Tlp1 native promoter. However, the promoter can in some cases be heterologous to the COI1 nucleic acid segment. In other words, such a heterologous promoter may not be naturally linked to such a COI1 nucleic acid segment. Instead, some expression cassettes and expression vectors can be recombinantly engineered to include a COI1 and/or Tlp1 nucleic acid segment operably linked to a heterologous promoter. A COI1 and/or Tlp1 nucleic acid is operably linked to the promoter, for example, when it is located downstream from the promoter.
A variety of promoters can be included in the expression cassettes and/or expression vectors. In some cases, the endogenous COI1 and/or Tlp1 promoter can be employed. Promoter regions are typically found in the flanking DNA upstream from the coding sequence in both prokaryotic and eukaryotic cells. A promoter sequence provides for regulation of transcription of the downstream gene sequence and typically includes from about 50 to about 2,000 nucleotide base pairs. Promoter sequences can also contain regulatory sequences such as enhancer sequences that can influence the level of gene expression. Some isolated promoter sequences can provide for gene expression of heterologous DNAs, that is a DNA different from the native or homologous DNA.
Promoters can be strong or weak, or inducible. A strong promoter provides for a high level of gene expression, whereas a weak promoter provides for a very low level of gene expression. An inducible promoter is a promoter that provides for the turning on and off of gene expression in response to an exogenously added agent, or to an environmental or developmental stimulus. For example, a bacterial promoter such as the Ptac promoter can be induced to vary levels of gene expression depending on the level of isothiopropylgalactoside added to the transformed cells. Promoters can also provide for tissue specific or developmental regulation. A strong promoter for heterologous DNAs can be advantageous because it provides for a sufficient level of gene expression for easy detection and selection of transformed cells and provides for a high level of gene expression when desired. In some cases, the promoter within such expression cassettes/vectors can be functional during plant development or growth.
Expression cassettes/vectors can include, but are not limited to, a promoter such as the rice actin1 (Act1) promoter. Other examples of promoters that can be used include the CaMV 35S promoter (Odell et al., Nature. 313:810-812 (1985)), or others such as CaMV 19S (Lawton et al., Plant Molecular Biology. 9:315-324 (1987)), nos (Ebert et al., Proc. Natl. Acad. Sci. USA. 84:5745-5749 (1987)). Adh1 (Walker et al., Proc. Natl. Acad. Sci. USA. 84:6624-6628 (1987)), sucrose synthase (Yang et al., Proc. Natl. Acad. Sci. USA. 87:4144-4148 (1990)), α-tubulin, ubiquitin, actin (Wang et al., Mol. Cell. Biol. 12:3399 (1992)), cab (Sullivan et al., Mol. Gen. Genet. 215:431 (1989)), PEPCase (Hudspeth et al., Plant Molecular Biology. 12:579-589 (1989)) or those associated with the R gene complex (Chandler et al., The Plant Cell. 1:1175-1183 (1989)). Further suitable promoters include the poplar xylem-specific secondary cell wall specific cellulose synthase 8 promoter, cauliflower mosaic virus promoter, the Z10 promoter from a gene encoding a 10 kD zein protein, a Z27 promoter from a gene encoding a 27 kD zein protein, inducible promoters, such as the light inducible promoter derived from the pea rbcS gene (Coruzzi et al., EMBO J. 3:1671 (1971)) and the actin promoter from rice (McElroy et al., The Plant Cell. 2:163-171 (1990)). Seed specific promoters, such as the phaseolin promoter from beans, may also be used (Sengupta-Gopalan, Proc. Natl. Acad. Sci. USA. 83:3320-3324 (1985). Other promoters useful in the practice of the invention are available to those of skill in the art.
Alternatively, novel tissue specific promoter sequences may be employed in the practice of the present invention. cDNA clones from a particular tissue can be isolated and those clones which are expressed specifically in that tissue are identified, for example, using Northern blotting. Preferably, the gene isolated is not present in a high copy number, but is relatively abundant in specific tissues. The promoter and control elements of corresponding genomic clones can then be localized using techniques well known to those of skill in the art.
Another regulatory element that the expression cassettes can have is a termination signal. Efficient expression of recombinant DNA sequences in eukaryotic cells can be enhanced by use of signals directing the efficient termination and polyadenylation of the resulting transcript. Transcription termination signals are generally found downstream of the polyadenylation signal and are a few hundred nucleotides in length. The term “poly(A) site” or “poly(A) sequence” as used herein denotes a DNA sequence which directs both the termination and polyadenylation of the nascent RNA transcript. Efficient polyadenylation of the recombinant transcript is desirable, as transcripts lacking a poly(A) tail are unstable and are rapidly degraded. The poly(A) signal utilized in an expression vector may be “heterologous” or “endogenous.” An endogenous poly(A) signal is one that is found naturally at the 3′ end of the coding region of a given gene in the genome. A heterologous poly(A) signal is one which has been isolated from one gene and positioned 3′ to another gene. A commonly used heterologous poly(A) signal is the SV40 poly(A) signal. The SV40 poly(A) signal is contained on a 237 bp BamHI-BclI restriction fragment and directs both termination and polyadenylation (Sambrook, supra, at 16.6-16.7). An example of such a termination signal is a potato protease II terminator.
A COI1 and/or Tlp1 nucleic acid can be combined with the promoter by standard methods to yield an expression cassette or transgene, for example, as described in Sambrook et al. (M
In some embodiments, a cDNA clone encoding a COI1 and/or Tlp1 protein is synthesized, isolated, and/or obtained from a selected cell. In other embodiments, cDNA clones from other species (that encode a COI1 and/or Tlp1 protein) are isolated from selected plant tissues. For example, the nucleic acid encoding a COI1 protein can be any nucleic acid with a coding region that hybridizes to SEQ ID NO:2 and that has COI1 activity. For example, the nucleic acid encoding a Tlp1 protein can be any nucleic acid with a coding region that hybridizes to SEQ ID NO:28 and that has Tlp1 activity. In another example, the COI1 nucleic acid can encode a COI1 protein with an amino acid sequence that has at least 90%, or at least 95%, or at least 96%, or at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:1, 3, 5, 6, 8, 10, 12, 14, 16, 17, 19, 21, 23, or 25. In another example, the Tlp1 nucleic acid can encode a Tlp1 protein with an amino acid sequence that has at least 90%, or at least 95%, or at least 96%, or at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:27, 29, 31, 32, 33, 34, 36, 37, 39, 41, or 43. Using restriction endonucleases, the entire coding sequence for the COI1 and/or Tlp1 nucleic acid is subcloned downstream of the promoter in a 5′ to 3′ sense orientation.
In some cases, an endogenous COI1 and/or Tlp1 gene can be modified to generate plant cells and plants that can express increased levels of COI1 and/or Tlp1 protein(s). Mutations can be introduced into promoter regions of COI1 and/or Tlp1 loci within plant genomes by introducing targeting vectors, T-DNA, transposons, nucleic acids encoding TALENS, CRISPR, or ZFN nucleases, and combinations thereof into a recipient plant cell to create a transformed cell.
The frequency of occurrence of cells taking up exogenous (foreign) DNA can sometimes be low. However, certain cells from virtually any dicot or monocot species can be stably transformed, and these cells can be regenerated into transgenic plants, through the application of the techniques disclosed herein. The plant cells, plants, and seeds can therefore be monocotyledons or dicotyledons.
The cell(s) that undergo transformation may be in a suspension cell culture or may be in an intact plant part, such as an immature embryo, or in a specialized plant tissue, such as callus, such as Type I or Type II callus.
Transformation of the cells of the plant tissue source can be conducted by any one of a number of methods available to those of skill in the art. Examples are: Transformation by direct DNA transfer into plant cells by electroporation (U.S. Pat. No. 5,384,253 and U.S. Pat. No. 5,472,869, Dekeyser et al., The Plant Cell. 2:591 602 (1990)); direct DNA transfer to plant cells by PEG precipitation (Hayashimoto et al., Plant Physiol. 93:857 863 (1990)); direct DNA transfer to plant cells by microprojectile bombardment (McCabe et al., Bio/Technology. 6:923 926 (1988); Gordon Kamm et al., The Plant Cell. 2:603 618 (1990); U.S. Pat. No. 5,489,520; U.S. Pat. No. 5,538,877; and U.S. Pat. No. 5,538,880) and DNA transfer to plant cells via infection with Agrobacterium. Methods such as microprojectile bombardment or electroporation can be carried out with “naked” DNA where the expression cassette may be simply carried on any E. coli derived plasmid cloning vector. In the case of viral vectors, it is desirable that the system retain replication functions, but lack functions for disease induction.
One method for dicot transformation, for example, involves infection of plant cells with Agrobacterium tumefaciens using the leaf disk protocol (Horsch et al., Science 227:1229 1231 (1985). Monocots such as Zea mays can be transformed via microprojectile bombardment of embryogenic callus tissue or immature embryos, or by electroporation following partial enzymatic degradation of the cell wall with a pectinase containing enzyme (U.S. Pat. No. 5,384,253; and U.S. Pat. No. 5,472,869). For example, embryogenic cell lines derived from immature wheat embryos can be transformed by accelerated particle treatment as described by Gordon Kamm et al. (The Plant Cell. 2:603 618 (1990)) or U.S. Pat. No. 5,489,520; U.S. Pat. No. 5,538,877 and U.S. Pat. No. 5,538,880, cited above. Excised immature embryos can also be used as the target for transformation prior to tissue culture induction, selection and regeneration as described in U.S. application Ser. No. 08/112,245 and PCT publication WO 95/06128. Furthermore, methods for transformation of monocotyledonous plants utilizing Agrobacterium tumefaciens have been described by Hiei et al. (European Patent 0 604 662, 1994) and Saito et al. (European Patent 0 672 752, 1995).
Methods such as microprojectile bombardment or electroporation are carried out with “naked” DNA where the expression cassette may be simply carried on any plasmid cloning vector. In the case of viral vectors, it is desirable that the system retain replication functions, but lack functions for disease induction.
The choice of plant tissue source for transformation will depend on the nature of the host plant and the transformation protocol. Useful tissue sources include callus, suspension culture cells, protoplasts, leaf segments, stem segments, tassels, pollen, embryos, hypocotyls, tuber segments, meristematic regions, and the like. The tissue source is selected and transformed so that it retains the ability to regenerate whole, fertile plants following transformation, i.e., contains totipotent cells. Selection of tissue sources for transformation of monocots is described in detail in U.S. application Ser. No. 08/112,245 and PCT publication WO 95/06128.
The transformation is carried out under conditions directed to the plant tissue of choice. The plant cells or tissue are exposed to the DNA or RNA carrying the targeting vector and/or other nucleic acids for an effective period of time. This may range from a less than one second pulse of electricity for electroporation to a 2-3 day co cultivation in the presence of plasmid bearing Agrobacterium cells. Buffers and media used will also vary with the plant tissue source and transformation protocol. Many transformation protocols employ a feeder layer of suspended culture cells (tobacco or Black Mexican Sweet corn, for example) on the surface of solid media plates, separated by a sterile filter paper disk from the plant cells or tissues being transformed.
Where one wishes to introduce DNA by means of electroporation, it is contemplated that the method of Krzyzek et al. (U.S. Pat. No. 5,384,253) may be advantageous. In this method, certain cell wall degrading enzymes, such as pectin degrading enzymes, are employed to render the target recipient cells more susceptible to transformation by electroporation than untreated cells. Alternatively, recipient cells can be made more susceptible to transformation, by mechanical wounding.
To effect transformation by electroporation, one may employ either friable tissues such as a suspension cell cultures, or embryogenic callus, or alternatively, one may transform immature embryos or other organized tissues directly. The cell walls of the preselected cells or organs can be partially degraded by exposing them to pectin degrading enzymes (pectinases or pectolyases) or mechanically wounding them in a controlled manner. Such cells would then be receptive to DNA uptake by electroporation, which may be carried out at this stage, and transformed cells then identified by a suitable selection or screening protocol dependent on the nature of the newly incorporated DNA.
A further advantageous method for delivering transforming DNA segments to plant cells is microprojectile bombardment. In this method, microparticles may be coated with DNA and delivered into cells by a propelling force. Exemplary particles include those comprised of tungsten, gold, platinum, and the like.
It is contemplated that in some instances DNA precipitation onto metal particles would not be necessary for DNA delivery to a recipient cell using microprojectile bombardment. In an illustrative embodiment, non-embryogenic cells were bombarded with intact cells of the bacteria E. coli or Agrobacterium tumefaciens containing plasmids with either the β-glucuronidase or bar gene engineered for expression in maize. Bacteria were inactivated by ethanol dehydration prior to bombardment. A low level of transient expression of the β-glucuronidase gene was observed 24-48 hours following DNA delivery. In addition, stable transformants containing the bar gene were recovered following bombardment with either E. coli or Agrobacterium tumefaciens cells. It is contemplated that particles may contain DNA rather than be coated with DNA. Hence it is proposed that particles may increase the level of DNA delivery but are not, in and of themselves, necessary to introduce DNA into plant cells.
An advantage of microprojectile bombardment, in addition to being an effective means of reproducibly stably transforming monocots, is that the isolation of protoplasts (Christou et al., PNAS. 84:3962 3966 (1987)), the formation of partially degraded cells, or the susceptibility to Agrobacterium infection is not required. An illustrative embodiment of a method for delivering DNA into maize cells by acceleration is a Biolistics Particle Delivery System, which can be used to propel particles coated with DNA or cells through a screen, such as a stainless steel or Nytex screen, onto a filter surface covered with plant cells cultured in suspension (Gordon Kamm et al., The Plant Cell. 2:603 618 (1990)). The screen disperses the particles so that they are not delivered to the recipient cells in large aggregates. It is believed that a screen intervening between the projectile apparatus and the cells to be bombarded reduces the size of projectile aggregate and may contribute to a higher frequency of transformation, by reducing damage inflicted on the recipient cells by an aggregated projectile.
For bombardment, cells in suspension are preferably concentrated on filters or solid culture medium. Alternatively, immature embryos or other target cells may be arranged on solid culture medium. The cells to be bombarded are positioned at an appropriate distance below the macroprojectile stopping plate. If desired, one or more screens are also positioned between the acceleration device and the cells to be bombarded. Through the use of techniques set forth here in one may obtain up to 1000 or more foci of cells transiently expressing a marker gene. The number of cells in a focus which express the exogenous gene product 48 hours post bombardment often range from about 1 to 10 and average about 1 to 3.
In bombardment transformation, one may optimize the prebombardment culturing conditions and the bombardment parameters to yield the maximum numbers of stable transformants. Both the physical and biological parameters for bombardment can influence transformation frequency. Physical factors are those that involve manipulating the DNA/microprojectile precipitate or those that affect the path and velocity of either the macroprojectiles or microprojectiles. Biological factors include all steps involved in manipulation of cells before and immediately after bombardment, the osmotic adjustment of target cells to help alleviate the trauma associated with bombardment, and also the nature of the transforming DNA, such as linearized DNA or intact supercoiled plasmid DNA.
One may wish to adjust various bombardment parameters in small scale studies to fully optimize the conditions and/or to adjust physical parameters such as gap distance, flight distance, tissue distance, and helium pressure. One may also minimize the trauma reduction factors (TRFs) by modifying conditions which influence the physiological state of the recipient cells and which may therefore influence transformation and integration efficiencies. For example, the osmotic state, tissue hydration and the subculture stage or cell cycle of the recipient cells may be adjusted for optimum transformation. Execution of such routine adjustments will be known to those of skill in the art.
Examples of plants, plant seeds, and/or plant cells that can have the expression systems described herein include wheat, rye, maize, millet, red wild einkorn, amaranth, bulgur, farro, maize, oats, rice, sorghum, spelt, barley, alfalfa (e.g., forage legume alfalfa), algae, apple, avocado, balsam, barley, broccoli. Brussels sprouts, cabbage, canola, cassava, cauliflower, cocoa, cole vegetables, collards, corn, cottonwood, crucifers, earthmoss, grain legumes, grasses (e.g., forage grasses), jatropa, kale, kohlrabi, maize, miscanthus, moss, mustards, nut, nut sedge, oats, oil firewood trees, oilseeds, peach, peanut, poplar, potato, radish, rape, rapeseed, rice, rutabaga, sorghum, soybean, sugar beets, sugarcane, sunflower, switchgrass, tobacco, tomato, turnips, and wheat. In some embodiments, the plant is a grain producing species. In some embodiments, the plant, plant seed, or plant cell can be a wheat plant, wheat seed, or wheat cell.
An exemplary embodiment of methods for identifying transformed cells involves exposing the bombarded cultures to a selective agent, such as an infectious agent (e.g., the causative agent of Fusarium Head Blight (FHB)), a metabolic inhibitor, an antibiotic, herbicide or the like. Cells which have been transformed and have stably integrated a marker gene conferring resistance to the selective agent used, will grow and divide in culture. Sensitive cells will not be amenable to further culturing.
To use the bar-bialaphos or the EPSPS-glyphosate selective system, bombarded tissue is cultured for about 0-28 days on nonselective medium and subsequently transferred to medium containing from about 1-3 mg/l bialaphos or about 1-3 mM glyphosate, as appropriate. While ranges of about 1-3 mg/l bialaphos or about 1-3 mM glyphosate can be employed, it is proposed that ranges of at least about 0.1-50 mg/l bialaphos or at least about 0.1-50 mM glyphosate will find utility in the practice of the invention. Tissue can be placed on any porous, inert, solid or semi-solid support for bombardment, including but not limited to filters and solid culture medium. Bialaphos and glyphosate are provided as examples of agents suitable for selection of transformants, but the technique of this invention is not limited to them.
An example of a screenable marker trait is the red pigment produced under the control of the R-locus in maize. This pigment may be detected by culturing cells on a solid support containing nutrient media capable of supporting growth at this stage and selecting cells from colonies (visible aggregates of cells) that are pigmented. These cells may be cultured further, either in suspension or on solid media. The R-locus is useful for selection of transformants from bombarded immature embryos. In a similar fashion, the introduction of the C1 and B genes will result in pigmented cells and/or tissues.
The enzyme luciferase is also useful as a screenable marker in the context of the present invention. In the presence of the substrate luciferin, cells expressing luciferase emit light which can be detected on photographic or X-ray film, in a luminometer (or liquid scintillation counter), by devices that enhance night vision, or by a highly light sensitive video camera, such as a photon counting camera. All of these assays are nondestructive and transformed cells may be cultured further following identification. The photon counting camera is especially valuable as it allows one to identify specific cells or groups of cells which are expressing luciferase and manipulate those in real time.
It is further contemplated that combinations of screenable and selectable markers may be useful for identification of transformed cells. For example, selection with a growth inhibiting compound, such as bialaphos or glyphosate at concentrations below those that cause 100% inhibition followed by screening of growing tissue for expression of a screenable marker gene such as luciferase would allow one to recover transformants from cell or tissue types that are not amenable to selection alone. Slowly growing tissue was subsequently screened for expression of the luciferase gene and transformants can be identified.
Cells that survive the exposure to the selective agent, or cells that have been scored positive in a screening assay, are cultured in media that supports regeneration of plants. One example of a growth regulator that can be used for such purposes is dicamba or 2,4-D. However, other growth regulators may be employed, including NAA, NAA+2,4-D or perhaps even picloram. Media improvement in these and like ways can facilitate the growth of cells at specific developmental stages. Tissue can be maintained on a basic media with growth regulators until sufficient tissue is available to begin plant regeneration efforts, or following repeated rounds of manual selection, until the morphology of the tissue is suitable for regeneration, at least two weeks, then transferred to media conducive to maturation of embryoids. Cultures are typically transferred every two weeks on this medium. Shoot development signals the time to transfer to medium lacking growth regulators.
The transformed cells, identified by selection or screening and cultured in an appropriate medium that supports regeneration, can then be allowed to mature into plants. Developing plantlets are transferred to soilless plant growth mix, and hardened, e.g., in an environmentally controlled chamber at about 85% relative humidity, about 600 ppm CO2, and at about 25-250 microeinsteins/sec·m2 of light. Plants can be matured either in a growth chamber or greenhouse. Plants are regenerated from about 6 weeks to 10 months after a transformant is identified, depending on the initial tissue. During regeneration, cells are grown on solid media in tissue culture vessels. Illustrative embodiments of such vessels are petri dishes and Plant Con™. Regenerating plants can be grown at about 19° C. to 28° C. After the regenerating plants have reached the stage of shoot and root development, they may be transferred to a greenhouse for further growth and testing.
Mature plants are then obtained from cell lines that have expression cassettes encoding COI1 and/or Tlp1 proteins. In some embodiments, the regenerated plants are self-pollinated. In addition, pollen obtained from the regenerated plants can be crossed to seed grown plants of agronomically important inbred lines. In some cases, pollen from plants of these inbred lines is used to pollinate regenerated plants. The trait is genetically characterized by evaluating the segregation of the trait in first and later generation progeny. The heritability and expression in plants of traits selected in tissue culture can be useful if the traits are to be commercially useful.
Regenerated plants can be repeatedly crossed to inbred plants in order to introgress the mutations into the genome of the inbred plants. This process is referred to as backcross conversion. When a sufficient number of crosses to the recurrent inbred parent have been completed in order to produce a product of the backcross conversion process that is substantially isogenic with the recurrent inbred parent except for the presence of the introduced expression cassette encoding a COI1 or Tlp1 protein, the plant can be self-pollinated at least once in order to produce a homozygous backcross converted inbred containing the mutations. Progeny of these plants are in many cases true breeding.
Alternatively, seed from transformed mutant plant lines regenerated from transformed tissue cultures is grown in the field and self-pollinated to generate true breeding plants.
Seed from the fertile transgenic plants can then be evaluated for the presence of the desired COI1 and/or Tlp1 expression cassette(s), and/or the expression of the desired levels of COI1 and/or Tlp1 protein. Transgenic plant and/or seed tissue can be analyzed using standard methods such as SDS polyacrylamide gel electrophoresis, liquid chromatography (e.g., HPLC) or other means of detecting a mutation.
Once a transgenic plant with COI1 and/or Tlp1 expression cassette(s) and having pathogen resistance is identified, seeds from such plants can be used to develop true breeding plants. The true breeding plants are used to develop a line of plants with improved pathogen resistance relative to wild type, while still maintaining other desirable functional agronomic traits. Adding the mutation to other plants can be accomplished by back-crossing with this trait and with plants that do not exhibit this trait and studying the pattern of inheritance in segregating generations. Those plants expressing the target trait (e.g., pathogen resistance, good growth, good seed/kernel yield) in a dominant fashion are preferably selected. Back-crossing is carried out by crossing the original fertile transgenic plants with a plant from an inbred line exhibiting desirable functional agronomic characteristics while not necessarily expressing the trait of increased pathogen resistance and good plant growth. The resulting progeny are then crossed back to the parent that expresses the increased pathogen resistance and good plant growth. The progeny from this cross will also segregate so that some of the progeny carry the trait and some do not. This back-crossing is repeated until an inbred line with the desirable functional agronomic traits, and with expression of the trait involving an increase in pathogen resistance. Such pathogen resistance can be expressed in a dominant fashion.
The new transgenic plants can also be evaluated for a battery of functional agronomic characteristics such as growth, lodging, kernel hardness, yield, resistance to disease, resistance to insect pests, drought resistance, and/or herbicide resistance.
Plants that may be improved by these methods include but are not limited to agricultural plants of all types. Examples include grains (maize, wheat, barley, oats, rice, sorghum, amaranth, bulgur, red wild einkorn, farro, spelt, millet and rye), oil and/or starch plants (canola, potatoes, lupins, sunflower and cottonseed), forage plants (alfalfa, clover and fescue), grasses (switchgrass, prairie grass, wheat grass, sudangrass, sorghum, straw-producing plants), softwood, hardwood and other woody plants (e.g., those used for paper production such as poplar species, pine species, and eucalyptus). Plants useful for making biofuels and ethanol include corn, grasses (e.g., miscanthus, switchgrass, and the like), as well as trees such as poplar, aspen, willow, and the like. Plants useful for generating dairy forage include legumes such as alfalfa, as well as forage grasses such as bromegrass, and bluestem. In some embodiments the plant is a gymnosperm. Examples of plants useful for grain production, include wheat, rye, maize, millet, red wild einkorn, amaranth, bulgur, farro, maize, oats, rice, sorghum, spelt, and barley.
To confirm the presence of COI1, Tlp1 and/or expression cassettes encoding COI1 and/or Tlp1 proteins in the regenerating plants, or seeds or progeny derived from the regenerated plant, a variety of assays may be performed. Such assays include, for example, molecular biological assays available to those of skill in the art, such as Southern and Northern blotting and PCR; biochemical assays, such as detecting the presence of a protein product, e.g., by immunological means (ELISAs and Western blots) or by enzymatic function; plant part assays, such as leaf, seed or root assays; and also, by analyzing the phenotype of the whole regenerated plant.
Whereas DNA analysis techniques may be conducted using DNA isolated from any part of a plant, RNA may only be expressed in particular cells or tissue types and so RNA for analysis can be obtained from those tissues. PCR techniques may also be used for detection and quantification of RNA produced from introduced expression cassettes encoding COI1 and/or Tlp1 proteins. For example, PCR also be used to reverse transcribe RNA into DNA, using enzymes such as reverse transcriptase, and then this DNA can be amplified through the use of conventional PCR techniques.
For example, if no amplification of COI1 and/or Tlp1 mRNAs is observed, then an expression cassette encoding COI1 protein and/or Tlp1 protein may not have been successfully introduced. Information about introduced expression cassettes can also be obtained by primer extension or single nucleotide polymorphism (SNP) analysis.
Further information about the nature of the RNA product may be obtained by Northern blotting. This technique will demonstrate the presence of an RNA species and give information about the integrity of that RNA. The presence or absence of an RNA species (e.g., COI1 and/or Tlp1 RNA) can also be determined using dot or slot blot Northern hybridizations. These techniques are modifications of Northern blotting and also demonstrate the presence or absence of an RNA species.
While Southern blotting and PCR may be used to detect the expression cassettes encoding COI1 and/or Tlp1 proteins, they do not provide information as to whether the preselected DNA segment is being expressed. Expression may be evaluated by specifically identifying the protein products of the introduced COI1 and/or Tlp1 expression cassette, by detecting expression of the COI1 and/or Tlp1 proteins, or evaluating the phenotypic changes brought about by introduction of such proteins.
Assays for the production and identification of specific proteins may make use of physical-chemical structural, functional, or other properties of the proteins. Unique physical-chemical or structural properties allow the proteins to be separated and identified by electrophoretic procedures, such as native or denaturing gel electrophoresis or isoelectric focusing, or by chromatographic techniques such as ion exchange, liquid chromatography or gel exclusion chromatography. The unique structures of individual proteins offer opportunities for use of specific antibodies to detect their presence in formats such as an ELISA assay. Combinations of approaches may be employed with even greater specificity such as Western blotting in which antibodies are used to locate individual gene products, or the absence thereof, that have been separated by electrophoretic techniques. Additional techniques may be employed to absolutely confirm COI1 and/or Tlp1 mRNA or protein expression. Amino acid sequencing following purification can also be employed. The Examples of this application also provide assay procedures for detecting and quantifying infection and plant growth. Other procedures may be additionally used.
The expression of a gene product can also be determined by evaluating the phenotypic results of its expression. These assays also may take many forms including but not limited to analyzing changes in the resistance to infection, resistance to herbicides, growth characteristics, or other physiological properties of the plant. Expression of selected DNA segments encoding different amino acids or having different sequences and may be detected by amino acid analysis or sequencing.
The term “heterologous” when used in reference to a nucleic acid or protein refers to a nucleic acid or protein that has been manipulated in some way. For example, a heterologous nucleic acid includes a nucleic acid from one species introduced into another species. A heterologous nucleic acid also includes a nucleic acid that is native to an organism that has been altered in some way (e.g., mutated, added in multiple copies, present in a locus within the genome, expressed from an autonomously replicating vector, linked to a non-native promoter, linked to a mutated promoter, or linked to an enhancer sequence, etc.). Heterologous nucleic acids may comprise plant gene sequences that comprise cDNA forms of a plant gene; the cDNA sequences may be expressed in either a sense (to produce mRNA) or anti-sense orientation (to produce an anti-sense RNA transcript that is complementary to the mRNA transcript). In some cases, heterologous nucleic acids are distinguished from endogenous plant genes in that the heterologous nucleic acids can be joined to nucleotide sequences comprising regulatory elements such as promoters that are not found naturally associated with the nucleic acid. In another example, the heterologous nucleic acids are associated with portions of the chromosome not found in nature (e.g., genes expressed in loci where the gene is not normally expressed).
The term “nucleic acid,” “nucleic acid segment” or “nucleic acid of interest” refers to any RNA or DNA, where the manipulation of which may be deemed desirable for any reason (e.g., treat or reduce the incidence of disease, confer improved qualities, etc.), by one of ordinary skill in the art. Such nucleic acids include, but are not limited to, coding sequences of structural genes (e.g., disease resistance genes, reporter genes, selection marker genes, oncogenes, drug resistance genes, growth factors, etc.), and noncoding regulatory sequences which do not encode an mRNA or protein product (e.g., promoter sequence, polyadenylation sequence, termination sequence, enhancer sequence, etc.).
The term “hybridization” refers to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is impacted by such factors as the degree of complementary between the nucleic acids, stringency of the conditions involved, the Tm of the formed hybrid, and the G:C ratio within the nucleic acids. A single molecule that contains pairing of complementary nucleic acids within its structure is said to be “self-hybridized.”
The term “Tm” refers to the “melting temperature” of a nucleic acid. The melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands. The equation for calculating the Tm of nucleic acids is available to those of skill in the art. As indicated by standard references, a simple estimate of the Tm value may be calculated by the equation: Tm=81.5+0.41 (% G+C), when a nucleic acid is in aqueous solution at 1 M NaCl (See e.g., Anderson and Young, Quantitative Filter Hybridization, in Nucleic Acid Hybridization (1985)). Other references include more sophisticated computations that take structural as well as sequence characteristics into account for the calculation of Tm.
The term “stringency” refers to the conditions of temperature, ionic strength, and the presence of other compounds such as organic solvents, under which nucleic acid hybridizations are conducted. With “high stringency” conditions, nucleic acid base pairing will occur between nucleic acid fragments that have a high frequency of complementary base sequences. Thus, conditions of “low” stringency are often required with nucleic acids that are derived from organisms that are genetically diverse, as the frequency of complementary sequences is usually less. Stringency conditions are substantially determined by wash conditions (and not by hybridization conditions).
“Low stringency conditions” when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 42° C. in a solution consisting of 5×SSPE (43.8 g/l NaCL 6.9 g/l NaH2PO4H2O and 1.85 g/l EDTA. pH adjusted to 7.4 with NaOH), 0.1% SDS, 5×Denhardt's reagent (50×Denhardt's contains per 500 ml: 5 g Ficoll (Type 400, Pharmacia), 5 g BSA (Fraction V; Sigma)) and 100 μg/ml denatured salmon sperm DNA. Washing conditions that substantially determine whether “low stringency” hybridization occurs, include washing in a solution comprising 5×SSPE, 0.1% SDS at 42° C., for example, when a probe of about 500 nucleotides in length is employed.
“Medium stringency conditions” when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 42° C. in a solution consisting of 5×SSPE (43.8 g/l NaCl, 6.9 g/l NaH2PO4H2O and 1.85 g/l EDTA. pH adjusted to 7.4 with NaOH), 0.5% SDS, 5×Denhardt's reagent and 100/μg/ml denatured salmon sperm DNA. Washing conditions that substantially determine whether “medium stringency” hybridization occurs, include washing in a solution comprising 1.0×SSPE, 1.0% SDS at 50° C. when a probe of about 500 nucleotides in length is employed.
“High stringency conditions” when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 42° C. in a solution consisting of 5×SSPE (43.8 g/l NaCl, 6.9 g/l NaH2PO4H2O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS, 5×Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA. Washing conditions that substantially determine whether “high stringency” hybridization occurs include washing in a solution comprising 0.1×SSPE, 1.0% SDS at 60° C. when a probe of about 500 nucleotides in length is employed.
The term “expression” or “express” when used in reference to a nucleic acid, refers to the process of converting genetic information encoded in a nucleic acid into RNA (e.g., mRNA, rRNA, tRNA, or snRNA) through “transcription” of the nucleic acid (i.e., via the enzymatic action of an RNA polymerase). In some cases, “expression” or “express” can include translation into protein (as when a gene encodes a protein), through “translation” of mRNA. Expression can be regulated at many stages in the process. “Up-regulation” or “activation” refers to regulation that increases the production of nucleic acid expression products (i.e., RNA or protein), while “down-regulation” or “repression” refers to regulation that decrease production. Molecules (e.g., transcription factors) that are involved in up-regulation or down-regulation are often called “activators” and “repressors,” respectively.
The term “operably linked” refers to the linkage of nucleic acid segments in such a manner that a regulatory nucleic acid segment capable of directing the transcription of a given nucleic acid segment (e.g., a coding region) and/or the synthesis of a desired protein molecule is produced. The term also refers to the linkage of amino acid sequences in such a manner so that a functional protein is produced.
The term “regulatory element” refers to a genetic element that controls some aspect of nucleic acid expression. For example, a promoter is a regulatory element that facilitates the initiation of transcription of an operably linked coding region. Other regulatory elements are splicing signals, polyadenylation signals, termination signals, etc.
Transcriptional control signals in eukaryotes comprise “promoter” and “enhancer” elements. Promoters and enhancers consist of short nucleic acid segments that interact specifically with cellular proteins involved in transcription (Maniatis. et al., Science 236:1237 (1987), herein incorporated by reference). Promoter and enhancer elements have been isolated from a variety of prokaryotic (e.g., bacterial) and eukaryotic sources. Eukaryotic promoter and enhancer elements can be obtained, for example, from genes in yeast, insect, mammalian and plant cells. Promoter and enhancer elements have also been isolated from viruses and analogous control elements, such as promoters, are also found in prokaryotes. The selection of a particular promoter and enhancer depends on the cell type used to express the protein of interest. Some eukaryotic promoters and enhancers have a broad host range while others are functional in a limited subset of cell types (for review, see Maniatis, et al., supra (1987), herein incorporated by reference). The terms “promoter element.” “promoter,” or “promoter sequence” refer to a nucleic acid segments that are generally located at the 5′ end (i.e. precedes) of the coding region of nucleic acid. The location of most promoters known in nature precedes the transcribed region. The promoter functions as a switch, activating the expression of an encoded product (e.g., an RNA). If a gene or expression cassette is activated, it can be transcribed, or participate in transcription. Transcription involves the synthesis of RNA from the gene. The promoter, therefore, serves as a transcriptional regulatory element and also provides a site for initiation of transcription of the gene into mRNA.
Promoters may be tissue specific or cell specific. The term “tissue specific” as it applies to a promoter refers to a promoter that is capable of directing selective expression of a nucleotide sequence of interest to a specific type of tissue (e.g., seeds) in the relative absence of expression of the same nucleotide sequence of interest in a different type of tissue (e.g., leaves). Tissue specificity of a promoter may be evaluated by; for example, operably linking a reporter gene to the promoter sequence to generate a reporter construct, introducing the reporter construct into the genome of a plant such that the reporter construct is integrated into every tissue of the resulting transgenic plant, and detecting the expression of the reporter gene (e.g., detecting mRNA, protein, or the activity of a protein encoded by the reporter gene) in different tissues of the transgenic plant. The detection of a greater level of expression of the reporter gene in one or more tissues relative to the level of expression of the reporter gene in other tissues shows that the promoter is specific for the tissues in which greater levels of expression are detected.
The term “cell type specific” as applied to a promoter refers to a promoter that is capable of directing selective expression of a nucleotide sequence of interest in a specific type of cell in the relative absence of expression of the same nucleotide sequence of interest in a different type of cell within the same tissue. The term “cell type specific” when applied to a promoter also means a promoter capable of promoting selective expression of a nucleotide sequence of interest in a region within a single tissue. Cell type specificity of a promoter may be assessed using methods well known in the art, e.g., immunohistochemical staining. Briefly, tissue sections are embedded in paraffin, and paraffin sections are reacted with a primary antibody that is specific for the polypeptide product encoded by the nucleotide sequence of interest whose expression is controlled by the promoter. A labeled (e.g., peroxidase conjugated) secondary antibody that is specific for the primary antibody is allowed to bind to the sectioned tissue and specific binding detected (e.g., with avidin/biotin) by microscopy.
Promoters may be “constitutive” or “inducible.” The term “constitutive” when made in reference to a promoter means that the promoter is capable of directing transcription of an operably linked nucleic acid in the absence of a stimulus (e.g., in the absence of heat shock, chemical inducers, light, etc.). Typically, constitutive promoters are capable of directing expression of a transgene in substantially any cell and any tissue. Exemplary constitutive plant promoters include, but are not limited to SD Cauliflower Mosaic Virus (CaMV SD; see e.g., U.S. Pat. No. 5,352,605, incorporated herein by reference), mannopine synthase, octopine synthase (ocs), superpromoter (see e.g., WO 95/14098, herein incorporated by reference), and ubi3 promoters (see e.g., Garbarino and Belknap, Plant Mol. Biol. 24:119-127 (1994), herein incorporated by reference). Such promoters have been used successfully to direct the expression of heterologous nucleic acid sequences in transformed plant tissue.
In contrast, an “inducible” promoter is one that is capable of directing a level of transcription of an operably linked nucleic acid sequence in the presence of a stimulus (e.g., heat shock, chemical inducers, light, etc.) that is different from the level of transcription of the operably linked nucleic acid sequence in the absence of the stimulus.
The following Examples describe some of the experiments performed in the development of the invention.
This Example describes some of the materials and methods employed in the development of the invention.
Construct pjBarTlp and pjBarCoi: The wheat 674 bp tlp1 GenBank accession number X58394 and wheat 2167 bp Coi1 GenBank accession number HM447645 genes were synthesized. The cDNAs encoding these Tlp1 and Coi1 proteins were cloned into the Pjs101 plasmid (Nguyen et al. 2013). The Pjs101 contains two linked cassettes, one containing the bacterial mannitol-1-phosphate dehydrogenase (mtlD) gene regulated by the rice actin promoter (Act1) and the potato protease inhibitor II terminator; and the other cassette containing the bar herbicide resistance selectable marker gene regulated by the 35S promoter and Nos terminator. A new construct was developed by eliminating the mtlD gene using Xba1 and inserting the synthesized tlp1 or coi1 respectively.
Wheat cv. Bobwhite plants were grown to maturity from seeds in greenhouses. Immature embryos were isolated and cultured in-vitro following Zhang et al. (2000).
transformants were transferred to the growth chamber and tested for herbicide resistance using leaf painting assay with a 0.1% aqueous Liberty™ solution containing 18.9% glufosinate ammonium.
Genomic DNAs were extracted from herbicide resistant plantlets, as well as the wild type control plants using the CTAB method (Xin and Chin 2012). PCR was performed by the amplifying of a part of the rice actin promoter and a part of tlp1 gene, using forward rice Actin primer and reverse primer of either tlp1 for detecting the pjBarTlp construct integration; or coi1 to detect pjBarCoi construct with expected size of (˜400 and 696 bp for Tlp1 and Coi1 respectively) as well as for the bar gene (400 bp) using specific primers (Table 1).
qPCR
Total RNA was extracted from transgenic as well as the control non-transgenic plants using total RNA isolation system (EZNA plant RNA Kit—Omega bio-tec, USA). Expression of the integrated transgene was tested on the RNA extracted from herbicide resistant T1 plants. First, the strand cDNA was synthesized using Goscript Reverse Transcriptase (Promega cat no. A5003)
SYBR Green Real-time RT-PCR was conducted on the resulting cDNA. The total reaction (20 μl) contained 10 ng cDNA, 0.3 μM of each primer, and 1× Fast SYBR green master Mix (Applied Biosystems, USA). Reactions were performed using ABI 7900 HT RT-PCR system (Applied Biosystems, USA) under the conditions of: 94° C. for 30 s, 40 cycles of 95° C. for 15 s, 60° C. for 60 s and 72° C. for 20 s to calculate cycle threshold (Ct) values. The ubiquitin-conjugating enzyme E2 gene was used as a housekeeping gene to standardize the gene expression. Relative Quantization of Gene Expression method ΔΔct was used to analyze the results using 2−ΔΔCT formula. The primer sequences employed are listed in Table 1.
The qPCR analysis showed that among forty-four first generation (T0) independent transgenic lines with the selectable marker bar gene and at least one of the two constructs, only six lines exhibited expression of both tlp1 and Coi1 genes. The level of expression of these two genes in these six independently transformed lines is shown in
The Example illustrates that over-expression of TLP1 and COI1 reduces the symptoms of FHB in transgenic wheat plant lines that over-express TLP1 and COI1.
Table 2 illustrates differences in the percentage of FHB infection after 21 days of point inoculation in non-transgenic Bobwhite wheat plants compared to six different independent transgenic plant lines that over-expressed of TLP1 and COI1.
Disease assessment was conducted at three different time points, at one week, two weeks, and three weeks. Disease progression was recorded for each inoculated spike by counting the healthy seeds in both sides (up and down) from the point of inoculation (see e.g.,
The AUDPC is graphically illustrated in
The Example illustrates that over-expression of TLP1 and COI1 does not adversely affect plant growth and seed production.
Transgenic wheat lines were generated containing the genes COI and TLP singly and in combination. Transgenic plants were generated from these plant lines. Transgenic plants were inoculated with Fusarium head blight (FHB) isolates, and the infected plants were maintained in greenhouse facilities for conducting disease assays and consultation.
Approximately 500 individual wheat heads and whole plants were evaluated, including thirty-one (31) lines with COI+TLP, seventeen (17) lines carrying COI, and one (1) line carrying TLP transgenes.
FHB resistance data was obtained for each line as illustrated in Table 4 below.
As illustrated, in this study overexpression of COI and COI+TLP provides greater resistance than overexpression of TLP alone.
All patents and publications referenced or mentioned herein are indicative of the levels of skill of those skilled in the art to which the invention pertains, and each such referenced patent or publication is hereby specifically incorporated by reference to the same extent as if it had been incorporated by reference in its entirety individually or set forth herein in its entirety. Applicants reserve the right to physically incorporate into this specification any and all materials and information from any such cited patents or publications.
The following statements are intended to describe and summarize various embodiments of the invention according to the foregoing description in the specification.
The specific products, compositions, and methods described herein are representative, exemplary and not intended as limitations on the scope of the invention. Other objects, aspects, and embodiments will occur to those skilled in the art upon consideration of this specification, and are encompassed within the spirit of the invention as defined by the scope of the claims. It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The terms and expressions that have been employed are used as terms of description and not of limitation, and there is no intent in the use of such terms and expressions to exclude any equivalent of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention as claimed. Thus, it will be understood that although the present invention has been specifically disclosed by embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims and statements of the invention.
The invention illustratively described herein may be practiced in the absence of any element or elements, or limitation or limitations, which is not specifically disclosed herein as essential. The methods and processes illustratively described herein may be practiced in differing orders of steps, and the methods and processes are not necessarily restricted to the orders of steps indicated herein or in the claims.
As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to “a plant” or “a seed” or “a cell” includes a plurality of such plants, seeds or cells, and so forth. In this document, the term “or” is used to refer to a nonexclusive or, such that “A or B” includes “A but not B,” “B but not A.” and “A and B,” unless otherwise indicated.
Under no circumstances may the patent be interpreted to be limited to the specific examples or embodiments or methods specifically disclosed herein. Under no circumstances may the patent be interpreted to be limited by any statement made by any Examiner or any other official or employee of the Patent and Trademark Office unless such statement is specifically and without qualification or reservation expressly adopted in a responsive writing by Applicants.
The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.
This application claims benefit of priority to the filing date of U.S. Provisional Application Ser. No. 62/428,841, filed Dec. 1, 2016, the contents of which are specifically incorporated herein by reference in their entity.
| Number | Date | Country | |
|---|---|---|---|
| 62428841 | Dec 2016 | US |
