Submitted by ETC Staff on
The Novartis patents comprise a wide-ranging and inter-related group directed toward controlled expression of desired traits in plants, these resulting from introduction of DNA constructs. While a principal focus of the work is to regulate gene expression by exposure of the plant to chemicals that can activate the promoter regions of introduced genes, the work also includes constructs that confer the constitutive expression (i.e. maintained in the absence of external stimulus) of introduced traits. In addition, considerable effort has been devoted to isolating the gene products which are produced as a stress response by the plants. Isolation of the genes that code for these proteins, and of the promoter sequences which regulate their expression, provide the basis for much of the work. Several of the claims are methodological, defining ways of isolating desired genes and the promoters that control them, but essentially they are variants of themes already well-developed in biotechnology. Similarly, while there is considerable space given in the discussion to the methods of transforming the plants through introduction of DNA sequences, the approaches used are standard for the industry and not relevant to understanding the result.
The analysis of this group has been rendered more difficult by the method of presentation. Readers who download a given patent will be confronted with a 700 KB file, containing the theoretical background and the experiments for the entire group, while the individual patents differ in the particular claims registered as part of the whole. This discussion will focus on the principles of the claims, without necessarily specifying which experimental plant species has been used as a demonstration of success. Where species are mentioned, they are not meant to include all which may have been used to make the point.
OBJECTIVES
Two principal objectives underly the set of patents. The first involves the establishment of chemically regulatable DNA sequences, and the identification of the chemicals which regulate them. What is meant here is a promoter, i.e. a DNA sequence some hundreds or thousands of nucleotides long that, when joined in the same piece of DNA to a second sequence that codes for a protein, can regulate the synthesis of this protein. (Throughout the document there are references to 'coding' and 'non-coding' DNA sequences - the coding sequences invariably specify the sequence of amino acids in a protein, while the non-coding sequences virtually all refer to the promoter elements which regulate the expression of the coding sequences). In the Novartis patents, promoters of interest are those which can be chemically regulated, to permit the gene's expression to be turned on in response to exogenous application of the inducer. As the method in principle does not depend on the nature of the coding sequence being regulated, or on the promoter sequence doing the regulating, the full panoply of traits that might be of interest to the producer (or seed seller) can be made subject to this control. 'The chimeric genes described above embrace a variety of possible constructions. A chemically regulatable non-coding sequence can be associated with a gene controlling flowering or fruit ripening; a gene effecting tolerance or resistance to herbicides or to many types of pests, for example fungi, viruses, bacteria, insects, nematodes, or arachnids; a gene controlling production of enzymes or secondary metabolites; male or female sterility; dwarfness; flavor; nutritional qualities; and the like'.
The second major objective is the isolation (and patenting) of the genes which code for proteins that form part of the natural capacity of the plant to resist pathogens, whether these be insects, viruses, fungi, or bacteria. Here the object is straightforward, to lay private claim to these natural immune capabilities, existent for millions of years, and by subsequent bioengineering, to incorporate them in one way or another into proprietary crop varieties. Because the mode of this incorporation will almost certainly involve a promoter-gene construct, with the promoters also proprietary and chemically inducible, several levels of control and profit-taking will be available to the patent-holder.
In this connection, it is worth noting that these patents, and presumably a host of others to follow, can be expected to follow the same logic as expressed here and in similar documents produced by firms such as Monsanto and AstraZeneca. This is because the concept of control of gene expression by promoters located adjacent to them is absolutely fundamental, and the mechanisms for turning them on or off are likewise of a few types. Thus we can expect to see variations on a few basic themes. Gene expression can be turned on by ratcheting up a positively acting element, it can be turned down by ratcheting it down or by increasing production of some inhibitor; increases in expression can also be effected by producing an inhibitor of the inhibitor, and so forth. Combinations of promoter-coding sequences, when introduced into the same cell, can ultimately provide much more control of the desired end point, but the basic element of any given component will consist of a promoter driving a coding sequence. What will vary will be the combinations of elements, and the precise identity of the promoters and coding sequences involved.
STRATEGY FOR ISOLATION OF CHEMICALLY INDUCIBLE PROMOTERS
Unlike the some other chemically-induced systems, involving a totally foreign promoter system relying for control on a drug such as tetracycline, the Novartis patents focus primarily on innate responses of plants to stress, in particular a process known as 'systemic acquired resistance' (SAR). This process was first described as a kind of immune response (resistance to infection caused by prior infection) to tobacco mosaic virus (TMV). Interestingly, the plants showed resistance not only to infection by TMV, but to other viruses and bacteria as well. Subsequent work showed that the immune response was accompanied by the production of so-called 'pathogenesis-related' (PR) proteins, together with the accumulation of salicylic acid (a close relative of aspirin) and related compounds. It is believed that salicylic acid induces the accumulation of the PR proteins. Salicylic acid, and a variety of other organic compounds, can in fact induce the appearance of the PR proteins when applied to the plant. The patents mention the existance of nine SAR gene families, including ten different PR proteins in tobacco, whose accumulation can be so induced.
To a biotechnologist, the production of a given protein, in response to a chemical stimulus, means that the associated promoter for the gene that codes for this protein has been induced by the stimulus. And because the promoter is located adjacent to the gene for the protein, methods for isolating the protein gene will allow finding its location in the plant genome, and thereby give access to the promoter sequence, usually some thousands of bases long. Once in hand, this promoter sequence can be used to regulate other genes for chemically-induced expression in plants (see above) ; also, by coupling the promoter to a 'reporter gene' i.e. a gene which codes for a protein that easy to detect, one can test new compounds for their ability to act as chemical inducers for this promoter.
Thus Novartis has adapted existing molecular biology protocols to isolate the coding sequences for several of the PR proteins, both to manipulate their expression in plants, and to isolate their associated promoters for use as described above. But there is one other component to this set which requires particular comment. While the major focus is ostensibly on manipulating resistance genes to generate increased resistance, there is one explicit component of the patent series which is devoted to the opposite (see 'Enhanced Exogenous Regulation via Inactivation of Endogenous Regulation'). Specifically, one of the patented approaches involves transfecting plants with a gene which codes for an enzyme that metabolizes salicylic acid (which, we have seen, is a mediator in the SAR response), thereby reducing the plant's natural capacity for disease resistance. This is presented both as a technique to allow transient expression of a trait, when its continued expression might be harmful, but primarily as a methodological aid in the search for inducers that can regulate the disease response. Since the plant has a natural capacity for response, it interferes with testing of the efficacy of resistance-enhancing DNA constructs and the inducers which regulate them. So by knocking out this inherent resistance, the bioengineer has an easier time in testing the efficacy of the introduced genes, or so it is stated.
But in the further discussion in this section, they point out that this approach can be applied not only to signalling pathways for resistance genes, but for a variety of other metabolic pathways that regulate other plant functions, and whose components have been, or are being, worked out. Then, as they state, 'Once inactivation of the cell signal is achieved, the genes which are natively regulated can be regulated exclusively by the application to the plant of a chemical regulator'. In short, from a program devoted to enhancing the capabilities of varieties, we move to a bioengineered strains which are rendered absolutely dependent on the application of the exogenous regulator for maintenance of key metabolic pathways, pathogen-resistance being one of them. And as more research proceeds into the basic mechanisms regulating plant physiology, still more avenues for interference will present themselves, whether the trait be flowering, fruiting, germination or what have you.
In short, all the issues that have evoked such controversy regarding the Monsanto and Zeneca inducible-promoter patents and germination are brought forward front and centre here, but with a greater level of generality. Here, any and all traits are being viewed openly as possible candidates for creation of dependency on external chemicals. As with the other patents, the key to profitability would have to be the coupling of these dependency-creating traits with other characteristics that create demand for the variety. In principle, this presents no theoretical problem since multiply-transformed plants can be created simply through proceeding one step at a time. It thus seems no accident that at the precise moment that Novartis is laying patent claims in order to exploit innate positive plant characteristics (e,g. resistance to pathogens), they are coupling this initiative to approaches which would make the plants helpless in other respects without the administration of some proprietary inducer.
LIST OF PATENTS AND COMMENTS ON CLAIMS
Inasmuch as the set of Novartis patents have the same overall text associated with each, differing only in the claims, the following comprises a series of comments on the individual sets of claims; readers wishing to download the entire text from the Web can proceed to http://www.uspto.gov/patft/index.html, and search by patent number. The order of presentation is chosen to give a certain logical progression in development of the work, inasmuch as this is possible. For reference, the claims for the respective patents are appended at the end of this document in the order discussed in these Comments.
1. US 5,777,200 July 7, 1998 Chemically regulatable and antipathogenic DNA sequences and uses thereof
Comments : This is the method underlying cloning of the DNA that codes for the PR proteins, as well as the promoters that regulate these coding sequences. As mentioned above it is a variant of so-called differential screening, where the workers isolate coding DNA1s from plants subjected to infection or induction by some of the known chemical inducers, and screen them against cDNA1s from plants which have not been infected or induced. Essentially this involves working with the messenger RNAs that are produced as intermediates when a given gene is activated - with this RNA, we can work backwards to the original DNA coding sequence, termed 'cDNA' , where the 'c' stands for 'complementary'. The basic idea is that the cDNA1s that result from response to the noxious stimulus will be made in addition to the normal housekeeping ones of the untreated plant; by subtracting the normal ones, seen in the untreated plant, from the total in the treated plant, we will see the new species that are specific for making the PR proteins. Of the chemical inducers used, salicylic acid (claim #10) is a physiological mediator of the PR response, which also can induce it if added exogenously, while the other compounds (claims 5-9) have been found empirically to work as well.
There is little in this set of claims that requires specific comment - we must assume that the work was done as described.
2. US 5,847,258 December 8, 1998 DNA encoding beta 1,3 glucanases
Comments : Claims 1-13 here involve patenting genes for the beta-glucanase family which are part of the group of PR proteins which have been cloned using the methodology given above. Claims 14-21 involve using these coding sequences, in conjunction either with constitutive promoters (always turned on) or with chemically inducible promoters, to create transgenic plants, and seed from same, which will show an enhanced response to pathogens. Here, and elsewhere, 'Chimeric gene' refers to coupling sequences that are not found in nature, in this case the promoter - coding gene combination.'
3. US 5,767,369 June 16, 1998 DNA sequences encoding SAR 8.2 proteins and uses thereof
Comments: This essentially is a reprise of the patent immediately preceding, involving another set of PR proteins, the SAR 8.2 group, whose coding DNA Novartis has cloned as well. Again, they make chimaeric constructs, involving a promoter and the DNA coding sequences, and transfect them into plants to give enhanced levels of the expressed clone. Here they refer only to use of a constitutive promoter, known as CaMV 35S, which is known to be active in plants; there is no specific reference in this set of claims to linking the SAR 8.2 proteins to inducible promoters. Claim 13, dealing with coding sequences that 'hybridize' to SAR 8.2 sequences refers to a technique for extracting sequences that are related to the ones initially found in the screen, and which code for related PR proteins. Novartis here is laying claim not only to the clones already established, but also to related ones that could be pulled out using this technology. Using one sequence to pull out other related ones, whether these latter occur in the same or other organisms, is a basic technique in molecular biology.
4. US 5,689,044 November 18, 1997 Chemically inducible promotion of a plant PR-1 gene
Comments : The promoters described here will have been isolated after the cloning of the coding sequence for the respective PR-1 genes from tobacco or Arabidopsis (a small plant which is becoming a model plant organism, just as fruit flies, mice, a small roundworm, and frogs are used as model animal species), using the differential screening technique described above. Once the coding sequence is in hand, it is straightforward to use it to isolate the adjacent promoter region from the plant1s DNA. (The promoter region is generally up to several thousand nucleotides long). This promoter can then be coupled to some other coding sequence for transfer into a plant (or plant tissue growing in a bioreactor) to confer a chemically inducible characteristic. The inducers mentioned will of course be of the same group that was used in the original screen that allowed isolation of the DNA for the PR proteins. This then becomes one of the many chemically inducible promoters in this set of patents. The coding sequence that is being driven by the promoter is selectable by the bioengineer and may be completed unrelated to the gene that was originally regulated.
5. US 5,654,414 August 5, 1997 Chemically inducible promoter of a cucumber chitinase/lysozyme gene
6. US 5,789,214 August 4, 1998 Method of Inducing Gene Transcription in a Plant
Comments: The material in these patents is similar to that immediately preceding, with a focus on the promoters that drive the chitinase/lysozyme gene, another of the family of proteins which mediate the plant1s response to pathogens (#5,654,414) or the PR-1 genes of tobacco and Arabidopsis (# 5,789,214). Again, the promoters have been coupled to a coding sequence, and the whole construct used to make a genetically modified plant which expresses the trait upon addition of the inducer. In the work with the PR-1 promoters, they use the full range of chemical inducers, including the physiological compound, salicylic acid, to serve as chemical regulators.
7. US 5,614,395 MARCH 25, 1997 Chemically regulatable and anti-pathogenic DNA sequences and uses thereof
Comments: This patent is involves using the promoter sequences isolated in the previous patents to facilitate the search for more compounds which can serve as their chemical regulators. The promoter sequences are coupled to reporter genes whose expression and consequent appearance of the protein that they code for (see claim # 11 for the list of reporters used here) can be readily observed, and then these constructs are used to transform plants. To test whether a given chemical can activate the promoter under examination, one simply applies the chemical and looks for a response. They seem to have used the full range of expected chemical regulators on the full range of promoter sequences, both for purposes of comparing how the various combinations work, and for testing out the system. Presumably they will now proceed further, to test out yet more variants of these inducers, to have more proprietary controls in hand.
8. US 5,650,505 July 22, 1997 Chemically regulatable and anti-pathogenic DNA sequences and uses thereof
Comments : At first sight this is simply another example of cloning the DNA that codes for a PR protein, linking it to promoters which are either constitutively active or chemically inducible, and then producing a transformed plant cell where the PR protein is produced in higher amounts, with a corresponding increase in pathogenesis resistance. The complication arises from claim 4, in which the promoter(s) drive the gene in the anti-sense direction. This impression is further reinforced on re-reading the list of components of the patents -see INTRODUCTION, point l. ' Transgenic plants constitutively transcribing sense or anti-sense mRNA strands of DNA sequences encoding plant pathogenesis-related proteins, or transcribing sense or anti-sense mRNA strands of DNA substantially homologous to genomic or cDNA sequences encoding plant pathogenesis-related proteins, such transgenic plants thus having an enhanced disease-resistant phenotype with respect to wild-type plants'
Now in all the other claims for the other patents above, the promoter drives a PR coding sequence in the 'sense direction' in order to increase protein production, and in turn increase plant resistance (as one would intuitively expect would be the aim). Using the promoters to drive the coding sequence in the antisense direction, however, can only serve to inhibit production of this same protein. Essentially what happens in the antisense case is that the messenger RNA intermediate, which is made whenever a coding sequence is being read, is now composed of nucleotides in a complementary sequence to 'sense' RNA strands in the cell, and will thus bind to them, making them unavailable to the cell for translation into protein. In fact, there is a whole mini-discipline in cell biology devoted to developing antisense RNA as a means for producing specific inhibition of cellular processes. The inclusion of antisense and sense in the same statement dealing with increased production of PR proteins is difficult to rationalize (it's rather like using white paint and black paint together to lighten a paint mixture).
It turns out, however, that using the coding sequence in this counter-intuitive way is exactly the intention for another component of the invention. As described in the overall comments (and treated below in the claims for the final patent), a major focus of the Novartis series is the creation of plant varieties whose resistance capabilities have been sharply reduced or eliminated, rendering them dependent on the addition of exogenous inducers to drive promoters that can replace the knocked out capabilities. The principal focus of the approach (see below) involves inactivation of a pathway that acts to prevent the accumulation of a variety of PR proteins. The antisense strategy, on the other hand, can only act against one protein, or very close variants of it. It does, however, offer an approach to inactivating the production of any protein for which the coding sequence is known, and so can serve as a recourse in any context, even when very little is known of the means of action of the protein. This is not to say that antisense-RNA production will always work as planned (all of these techniques have to be tried in practice), but that it is always there to be tried. Furthermore, as with all transgenic approaches, multiple sequential transformations can be made to inactivate a series of different targets.
9. US 5.804,693 September 8, 1998 Chemically regulatable and antipathogenic DNA sequences and uses thereof
Comments : This set of claims essentially deals with the other component of the chemical dependency strategy, as previously discussed in the overall comments. Here Novartis is generating transgenic plants with constructs that code for the enzyme salicylate hydroxylase, which breaks down salicylic acid, thereby preventing the accumulation of PR proteins and thus rendering the plants more susceptible to pathogens. As opposed to the antisense approach, this method can serve to inactivate a whole series of responses, mediated by a whole series of inducers, as they all are dependent on salicylic acid. Thus there is no need to create multiply-transformed varieties to deal with all the PR components, as would be required in the antisense approach. Finally, as the authors point out in the patent Discussion, extending this technique to other signalling pathways controlling other plant functions can render the plant even more dependent on external regulators (required for activating inducible promoters) to restore the missing functions. Of course the write-up presents these concepts in the best light possible (preventing expression of traits when they are harmful, or not needed but energetically expensive, etc), but, as discussed above, this is simply a Monsanto Terminator 2 patent writ on a much larger canvas. Beginning from a perspective of enhancing endogenous capabilities for pathogen resistance, we proceed to an image of a bionic plant, completely dependent on doses of administered chemicals for its existence.
PATENT CLAIMS
1. US 5,777,200 July 7, 1998 Chemically regulatable and antipathogenic DNA sequences and uses thereof
1.A method for differential screening and enrichment of cDNA populations from non-infected plant tissue or chemically-induced plant tissue, comprising: a) providing single-stranded cDNA from induced and uninduced populations, the single-stranded cDNA from the induced and uninduced populations having opposite DNA polarity, and the cDNA from the uninduced population having a biotin-affinity tag; b) hybridizing the single-stranded cDNA populations of step (a) with each other and c) separating the hybridization mixture of step b by biotin-avidin chromatography to enrich for single stranded cDNAs from the induced population which are not hybridized to the cDNA from the uninduced population
2.A method for cloning cDNA's encoding plant pathogenesis-related proteins comprising: a) preparing a plant biologically induced to a state of systemic acquired resistance by infecting a first region of the plant with a biological inducer of systemic acquired resistance, and b) producing cDNA clones encoding plant pathogenesis-related proteins from RNA isolated from a second, non-infected region of the plant.
3.A method for cloning cDNA's encoding plant pathogenesis-related proteins comprising: a) inducing systemic acquired resistance in a plant by applying a chemical inducer to said plant; b) producing cDNA clones encoding plant pathogenesis-related proteins from RNA isolated from said plant in which systemic acquired resistance has been induced.
4.The method of claim 3, wherein said chemical inducer is applied to one region of said plant, and wherein said cDNA clones are produced from RNA isolated from a second region of said plant.
5.The method of claim 3, wherein said chemical inducer is selected from the group consisting of: benzo-1,2,3-thiadiazoles, isonicotinic acid compounds, and salicylic acid compounds.
6.The method of claim 5, wherein said chemical inducer is a benzo-1,2,3-thiadiazole.
7.The method of claim 6, wherein said benzo-1,2,3-thiadiazole is selected from the group consisting of: benzo-1,2,3-thiadiazole-7-carboxylic acid, methyl benzo-1,2,3-thiadiazole-7-carboxylate, benzo-1,2,3-thiadiazole-7-carbothioic acid S-methyl ester, n-propyl benzo-1,2,3-thiadiazole-7-carboxylate, benzyl benzo-1,2,3-thiadiazole-7-carboxylate, and benzo-1,2,3-thiadiazole-7-carboxylic acid N-sec-butylhydrazide.
8.The method of claim 7, wherein said benzo-1,2,3-thiadiazole is benzo-1,2,3-thiadiazole-7-carbothioic acid S-methyl ester.
9.The method of claim 5, wherein said chemical inducer is selected from the group consisting of 2,6-dichloroisonicotinic acid and methyl 2,6-dichloroisonicotinate.
10.The method of claim 5, wherein said chemical inducer is salicylic acid.
2. US 5,847,258 December 8, 1998 DNA encoding beta 1,3 glucanases
1.An isolated DNA molecule comprising a coding sequence that encodes a pathogenesis-related protein having .beta.-1,3-glucanase activity selected from the group consisting of: PR-2, PR-2', PR-2', PR-N, PR-O, and PR-O'.
2.The isolated DNA molecule of claim 1, wherein said pathogenesis-related protein is a PR-2 protein.
3.The isolated DNA molecule of claim 2, wherein said PR-2 protein comprises the amino acid sequence encoded by SEQ ID NO:21.
4.The isolated DNA molecule of claim 1, wherein said pathogenesis-related protein is a PR-2' protein.
5.The isolated DNA molecule of claim 4, wherein said PR-2' protein comprises the amino acid sequence encoded by SEQ ID NO:25.
6.The isolated DNA molecule of claim 1, wherein said pathogenesis-related protein is a PR-2' protein.
7.The isolated DNA molecule of claim 6, wherein said PR-2' protein comprises the amino acid sequence encoded by SEQ ID NO:26.
8.The isolated DNA molecule of claim 1, wherein said pathogenesis-related protein is a PR-N protein.
9.The isolated DNA molecule of claim 8, wherein said PR-N protein comprises the amino acid sequence encoded by SEQ ID NO:24.
10.The isolated DNA molecule of claim 1, wherein said pathogenesis-related protein is a PR-O protein.
11.The isolated DNA molecule of claim 10, wherein said PR-O protein comprises the amino acid sequence encoded by SEQ ID NO:23.
12.The isolated DNA molecule of claim 1, wherein said pathogenesis-related protein is a PR-O' protein.
13.The isolated DNA molecule of claim 12, wherein said PR-O' protein comprises the amino acid sequence encoded by SEQ ID NO:14.
14.A chimeric gene comprising a nucleic acid promoter sequence operatively linked to the isolated DNA molecule of claim 1, wherein the coding sequence of said isolated DNA molecule is not naturally associated with said promoter sequence.
15.The chimeric gene of claim 14, wherein said promoter sequence comprises a constitutive promoter.
16.The chimeric gene of claim 14, wherein said promoter sequence comprises a chemically inducible promoter.
17.A vector comprising the chimeric gene of claim 14.
18.A host cell comprising the chimeric gene of claim 14.
19.The host cell of claim 18, wherein said host cell is a plant cell.
20.A transgenic plant comprising the chimeric gene of claim 14. 21.Seed from the transgenic plant of claim 20.
3. US 5,767,369 June 16, 1998 DNA sequences encoding SAR 8.2 proteins and uses thereof
1. A method for producing a transgenic plant that expresses elevated levels of an SAR8.2a, SAR8.2b, SAR8.2c, SAR8.2d, or SAR8.2e protein relative to a non-transgenic plant, comprising transforming a plant with a chimeric DNA construct that comprises: (a) a promoter sequence that promotes in a plant the transcription of an associated coding sequence at elevated levels; and (b) a coding sequence that encodes an SAR8.2a, SAR8.2b, SAR8.2c, SAR8.2d. or SAR8.2e protein operatively linked to said promoter sequence.
2. The method according to claim 1, wherein said promoter sequence comprises a double CaMV 35S promoter.
3. The method according to claim 1, wherein said coding sequence is selected from the coding sequences of the following group: SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19.
4. A transgenic plant produced according to the method of claim 1.
5. An isolated DNA molecule encoding an SAR8.2a, SAR8.2b, SAR8.2c SAR8.2d, or SAR8.2e protein.
6. The isolated DNA molecule according to claim 5, wherein said DNA molecule has a sequence selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19.
7. A plant transformation vector comprising the DNA molecule of claim 5.
8. A chimeric DNA construct, comprising: (a) a promoter that promotes in a plant the transcription of an associated DNA molecule at elevated levels; and (b) the DNA molecule of claim 5 operatively linked to said promoter.
9. The chimeric DNA construct according to claim 8, wherein said promoter is a double CaMV 35S promoter.
10. The chimeric DNA construct according to claim 8, wherein said DNA molecule has a coding sequence selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19.
11. A plant transformation vector comprising the chimeric DNA construct of claim 8.
12. A transgenic plant transformed with the plant transformation vector of claim 11.'
13. An isolated DNA molecule that encodes a plant pathogenesis-related protein and that hybridizes to the DNA molecule of SEQ ID NO: 15, 16, 17, 18, or 19 under either of the following sets of conditions: hybridization at 42.degree. C. in 30% formamide, 5x SSC 0.1% SDS, 1 mM EDTA, 10X Denhardt's solution, 25 mM sodium phosphate pH 6.5, and 250 .mu.g/ml sheared salmon sperm DNA; washing at 42.degree. C. in 2x SSC, 0.1% SDS (where 1x SSC is 150 mM NaCl, 15 mM Na citrate); or : washing at 50.degree. C. in 0.125 mM NaCl, 1% SDS, 40 mM sodium phosphate (pH 7.2), 1 mM EDTA
14. A chimeric DNA construct, comprising: (a) a promoter that promotes in a plant the transcription of an associated DNA molecule at elevated levels; and (b) the DNA molecule of claim 13, operatively linked to said promoter.
15. A plant transformation vector comprising the chimeric DNA construct of claim 14. 16. A transgenic plant transformed with the plant transformation vector of claim 15.
4. US 5,689,044 November 18, 1997 Chemically inducible promotion of a plant PR-1 gene
1.A chemically inducible nucleic acid promoter fragment isolated from the 5' flanking region upstream of the coding region of a tobacco PR-1a gene, wherein said promoter fragment comprises a nucleotide fragment of at least 603-bp adjacent to the coding region of said tobacco PR-1a gene, wherein said promoter fragment is inducible by application of a benzo-1,2,3-thiadiazole, an isonicotinic acid compound, or a salicylic acid compound.
2.The chemically inducible promoter fragment of claim 1, wherein said promoter fragment is isolated from the region upstream of nucleotide number 932 of SEQ ID No.1.
3.A chemically inducible nucleic acid promoter fragment isolated from the 5' flanking region upstream of the coding region of an Arabidopsis PR-1 gene, wherein the coding region of said Arabidopsis PR-1 gene comprises the DNA sequence set forth in SEQ ID NO:33 or a DNA sequence which would encode the protein encoded by SEQ ID NO:33, wherein said promoter fragment is inducible by application of a benzo-1,2,3-thiadiazole, an isonicotinic acid compound, or a salicylic acid compound.
4.The chemically inducible promoter of claim 3, wherein said promoter fragment is comprised within plasmid pAtPR1-P (NRRL B-21169).
5.An isolated DNA molecule comprising the nucleic acid promoter fragment of claims 1 or 3 operatively linked to a coding sequence of interest.
6.A plant transformation vector comprising the DNA molecule of claim 5.
7.A transgenic plant or plant tissue, each transformed with the plant transformation vector of claim 6.
5. US 5,654,414 August 5, 1997 Chemically inducible promoter of a cucumber chitinase/lysozyme gene
1.A nucleic acid promoter fragment isolated from the 5' flanking region upstream of the coding region of a cucumber chitinase/lysozyme gene that is inducible by application of benzo-1,2,3-thiadiazoles.
2.The nucleic acid promoter fragment according to claim 1, wherein the coding region of said cucumber chitinase/lysozyme gene comprises the DNA sequence set forth in SEQ ID NO:3.
3.The nucleic acid promoter fragment according to claim 1, wherein said promoter fragment is isolated from the region upstream of nucleotide number 6037 of SEQ ID NO:36.
4.The nucleic acid promoter fragment according to claim 3, wherein said promoter fragment comprises an at least 1338-bp fragment upstream of nucleotide number 6037 of SEQ ID NO:36.
5.The nucleic acid promoter fragment according to claim 1, wherein said cucumber chitinase/lysozyme gene is inducible by application of methyl benzo-1,2,3-thiadiazole-7-carboxylate.
6.An isolated DNA molecule comprising the nucleic acid promoter fragment of claim 1 operatively linked to a coding sequence of interest.
7.The DNA molecule according to claim 6, wherein the coding sequence of interest is the coding sequence of said cucumber chitinase/lysozyme gene.
8.The DNA molecule according to claim 6, wherein the coding sequence of interest is the coding sequence of a reporter gene.
9.The DNA molecule according to claim 8, wherein said reporter gene is the beta-1,3-glucuronidase gene.
10.A plant transformation vector comprising the DNA molecule of claim 6.
11.The plant transformation vector according to claim 10, which is plasmid pBScucchrcht5.
12.The plant transformation vector according to claim 10, which is plasmid pCIB2001/BamChit.
13.The plant transformation vector according to claim 10, which is plasmid pCIB2001/SalChit.
14.A transgenic plant or plant part, each transformed with the plant transformation vector of claim 10.
6. US 5,789,214 August 4, 1998 Method of Inducing Gene Transcription in a Plant
1.A method of inducing gene transcription in a plant or plant tissue, comprising the steps of: (a) transforming said plant or plant tissue, each with a chimeric gene comprising: (i) a chemically inducible nucleic acid promoter fragment of at least 603-bp isolated from the 5' flanking region adjacent the coding region of a tobacco PR-1a gene, and (ii) a coding sequence of interest operatively linked to said promoter fragment; and (b) exposing said transgenic plant or plant tissue to a benzo-1,2,3-thiadiazole, an isonicotinic acid compound, or a salicyclic acid compound, whereby transcription of said coding sequence of interest is induced in said plant or plant tissue.
2.The method of claim 1, wherein said promoter fragment is isolated from the region upstream of nucleotide number 932 of SEQ ID NO:1.
3.The method of claim 1, wherein said transgenic plant or plant tissue is exposed to a benzo-1,2,3-thiadiazole.
4.The method of claim 3, wherein said benzo-1,2,3-thiadiazole is selected from the group consisting of benzo-1,2,3-thiadiazole-7-carboxylic acid, methyl benzo-1,2,3-thiadiazole-7-carboxylate, n-propyl benzo-1,2,3-thiadiazole-7-carboxylic, benzyl benzo-1,2,3-thiadiazole-7-carboxylate, and benzo-1,2,3-thiadiazole-7-carboxylic acid N-sec-butylhydrazide.
5.The method of claim 4, wherein said benzo-1,2,3-thiadiazole is methyl benzo-1,2,3-thiadiazole-7-carboxylate.
6.The method of claim 1, wherein said transgenic plant or plant tissue is exposed to an isonicotinic acid compound selected from the group consisting of 2,6-dichloroisoicotinic acid and methyl 2,6-dichloroisonicotinate.
7.The method of claim 1, wherein said transgenic plant or plant tissue is exposed to a salicylic acid compound.
8.A method of inducing gene transcription in a plant or plant tissue, comprising the steps of: (a) transforming said plant or plant tissue, each with a chimeric gene comprising: (i) a chemically inducible nucleic acid promoter fragment isolated from the 5' flanking region adjacent the coding region of an Arabidopsis PR-1 gene, wherein said Arabidopsis PR-1 gene comprises a DNA sequence that specifically hybridizes to SEQ ID NO:33 or wherein said Arabidopsis PR-1 gene comprises a DNA sequence that encodes the protein encoded by SEQ ID NO:33, and (ii) a coding sequence of interest operatively linked to said promoter fragment; and (b) exposing said transgenic plant or plant tissue to a benzo-1,2,3-thiadiazole, an isonicotinic acid compound, or a salicylic acid compound, whereby transcription of said coding sequence of interest is induced in said plant or plant tissue.
9.The method of claim 8, wherein said promoter fragment is comprised within plasmid pAtPR1-P (NRRL B-21169).
10.The method of claim 8, wherein said transgenic plant or plant tissue is exposed to a benzo-1,2,3-thiadiazole.
11.The method of claim 10, wherein said benzo-1,2,3-thiadiazole is selected from the group consisting of benzo-1,2,3-thiadiazole-7-carboxylic acid, methyl benzo-1,2,3-thiadiazole-7-carboxylate, n-propyl benzo-1,2,3-thiadiazole-7-carboxylate, benzyl benzo-1,2,3-thiadiazole-7-carboxylate, and benzo-1,2,3-thiadiazole-7-carboxylic acid N-sec-butylhydrazide.
12.The method of claim 11, wherein said benzo-1,2,3-thiadiazole is methyl benzo-1,2,3-thiadiazole-7-carboxylate.
13.The method of claim 8, wherein said transgenic plant or plant tissue is exposed to an isonicotinic acid compound selected from the group consisting of 2,6-dichloroisonicotinic acid and methyl 2,6-dichloroisonicotinate.
14.The method of claim 8, wherein said transgenic plant or plant tissue is exposed to a salicylic acid compound.
7. US 5,614,395 MARCH25, 1997 Chemically regulatable and anti-pathogenic DNA sequences and uses thereof
1.A method of screening for agrochemicals having the ability to induce SAR in plants, said method comprising. (a) transforming a chimeric DNA molecule into a plant or plant part, said chimeric DNA molecule comprising: (i) a nucleic acid promoter from the 5' flanking region of a plant pathogenesis-related protein gene inducible by a chemical regulator selected from the group consisting of: benzo-1,2,3-thiadiazoles, isonicotinic acid compounds, and salicylic acid compounds, and (ii) at least one reporter gene operatively linked to said promoter; (b) contacting the transformed plant or plant part with an agrochemical; and (c) assaying for the expression of said reporter gene wherein expression of the reporter gene indidcates that the agrochemical of step (b) has the ability to induce SAR.
2.The method according to claim 1 wherein said plant or plant part is selected from the group consisting of: a plant protoplast, a plant cell, plant tissue, a developing plantlet, an immature whole plant, and a mature whole plant.
3.The method according to claim 1 wherein said chemical regulator is selected from the group consisting of: benzo-1,2,3-thiadiazole-7-carboxylic acid, methyl benzo-1,2,3-thiadiazole-7-carboxylate, benzo-1,2,3-thiadiazole-7-carbothioic acid S-methyl ester, n-propyl benzo-1,2,3-thiadiazole-7-carboxylate, benzyl benzo-1,2,3-thiadiazole-7-carboxylate, and benzo-1,2,3-thiadiazole-7-carboxylic acid N-sec-butylhydrazide.
4.The method according to claim 3 wherein said chemical regulator is benzo-1,2,3-thiadiazole-7-carbothioic acid S-methyl ester.
5.The method according to claim 1 wherein said chemical regulator is selected from the group consisting of 2,6-dichloroisonicotinic acid and methyl 2,6-dichloroisonicotinate.
6.The method according to claim 1 wherein said chemical regulator is salicylic acid.
7.The method according to claim 1 wherein said promoter is isolated from a gene selected from the group consisting of: tobacco PR-1a, PR-1b, PR-1c, PR-1', PR-Q, PR-R, and PR-S genes, an Arabidopsis PR-1 gene, a cucumber chitinase gene, and basic and acidic .beta.-1,3-glucanase genes.
8.The method according to claim 7, wherein said promoter is isolated from a tobacco PR-1a gene.
9.The method according to claim 7, wherein said promoter is isolated from an Arabidopsis PR-1 gene.
10.The method according to claim 7, wherein said promoter is isolated from a basic .beta.-1,3-glucanase gene.
11.The method according to claim 1 wherein said reporter gene is selected from the group consisting of: luciferase, chloramphenicol acetyltransferase, neomycin phosphotransferase, nopaline synthase, octopine synthase, beta-1,3-glucuronidase, acetohydroxyacid synthase, and Bacillus thuringiensis endotoxin.
12.The method according to claim 11 wherein said reporter gene is beta-1,3-glucuronidase.
13.The method according to claim 11 wherein said reporter gene is luciferase.
8. US 5,650,505 July 22, 1997 Chemically regulatable and anti-pathogenic DNA sequences and uses thereof
1.An isolated DNA molecule that encodes PR-Q protein.
2.A chimeric DNA molecule comprising a promoter sequence operatively linked to a coding sequence that encodes PR-Q protein, wherein said coding sequence is not naturally associated with said promoter sequence.
3.The chimeric DNA molecule of claim 2, wherein said coding sequence is in a sense orientation with respect to said promoter sequence.
4.The chimeric DNA molecule of claim 2, wherein said coding sequence is in an anti-sense orientation with respect to said promoter sequence.
5.The chimeric DNA molecule of claim 2, wherein said promoter sequence comprises a constitutive promoter.
6.The chimeric DNA molecule of claim 5, wherein said promoter sequence comprises a CaMV 35S promoter.
7.The chimeric DNA molecule of claim 2, wherein said promoter sequence comprises a chemically inducible promoter.
8.A vector comprising the chimeric DNA molecule of claim 2.
9.A host cell comprising the chimeric DNA molecule of claim 2.
10.The host cell of claim 9, wherein said host cell is a plant cell.
11.A transgenic plant comprising the chimeric DNA molecule of claim 2.
9. US 5,804,693 September 8, 1998 Chemically regulatable and antipathogenic DNA sequences and uses thereof
1.A plant, plant cell, or plant tissue stably transformed with a chimeric DNA molecule comprising a promoter functional in plant cells operably linked to a coding sequence encoding an enzyme capable of metabolizing salicylic acid or a biochemical precursor thereof, whereby said transformed plant, plant cell, or plant tissue is rendered incapable of producing enough salicylic acid to regulate expression of a gene natively regulated by salicylic acid.
2.A plant, plant cell, or plant tissue according to claim 1, wherein said enzyme is salicylate hydroxylase.
3.A plant, plant cell, or plant tissue according to claim 2, wherein said coding sequence is nahG.
4.A plant, plant cell, or plant tissue according to claim 1, wherein said promoter is a constitutive promoter.
5.A progeny, propagule, or seed of the plant according to claim 1.
6.A method for identifying chemicals capable of regulating plant genes, comprising the steps of: (a) obtaining a plant, plant cell, or plant tissue according to claim 1; (b) applying to said plant, plant cell, or plant tissue a chemical suspected of having the capability of regulating the gene natively regulated by salicylic acid; and (c) determining whether the gene has been expressed as an indication of the capability of the chemical to regulate expression of the gene in the absence of salicylic acid.
7.A method according to claim 6, wherein said enzyme is salicylate hydroxylase.
8.A method according to claim 7, wherein said coding sequence is nahG.
9.A method for the exogenous regulation of gene expression in a plant, comprising the steps of: (a) obtaining a plant according to claim 1; (b) applying a chemical regulator to said plant at a predetermined time when expression of the gene natively regulated by salicylic acid is desired.
10.A method according to claim 9, wherein said enzyme is salicylate hydroxylase.
11.A method according to claim 10, wherein said coding sequence is nahG.
12.A method according to claim 9, wherein step (b) comprises applying a gene-regulating effective amount of a benzo- 1,2,3-thiadiazole, an isonicotinic acid compound, or a salicylic acid compound to the plant.
13.A method according to claim 12, wherein a benzo-1,2,3-thiadiazole is applied to the plant.
14.A method according to claim 13, wherein said benzo-1,2,3-thiadiazole is selected from the group consisting of benzo-1,2,3-thiadiazole-7-carboxylic acid, methyl benzo-1,2,3-thiadiazole-7-carboxylate, n-propyl benzo-1,2,3-thiadiazole-7-carboxylate, benzyl benzo-1,2,3-thiadiazole-7-carboxylate, and benzo-1,2,3-thiadiazole-7-carboxylic acid N-sec-butylhydrazide.
15.A method according to claim 14, wherein said benzo-1,2,3-thiadiazole is benzo-1,2,3-thiadiazole-7-carboxylic acid.
16.A method according to claim 12, wherein an isonicotinic acid compound selected from the group consisting of 2,6-dichloroisonicotinic acid and methyl 2,6-dichloroisonicotinate is applied to the plant.
17.A method according to claim 12, wherein a salicylic acid compound is applied to the plant.