WO1994024295A1

WO1994024295A1 - Exogenous regulation of gene expression in plants

Info

Publication number: WO1994024295A1
Application number: PCT/US1994/003933
Authority: WO
Inventors: John A. Ryals; Leslie B. Friedrich; Eric R. Ward; Scott J. Uknes; Thomas D. Gaffney; Helmut Kessmann; Bernard Vernooij
Original assignee: Ciba-Geigy Ag
Priority date: 1993-04-12
Filing date: 1994-04-11
Publication date: 1994-10-27
Also published as: AU6557494A

Abstract

Disclosed is a method for the exogenous regulation of gene expression in plants, which comprises obtaining a plant incapable of regulating at least one gene or gene family, or at least one heterologous gene, due to the deactivation of at least one endogenous signal transduction cascade which regulates the gene in the plant, and applying a chemical regulator to the plant at a time when expression of the gene is desired. Also disclosed is a method of assaying for chemicals capable of exogenously regulating gene expression, as well as modified plants, plant tissue and plant cells incapable of regulating at least one gene, gene family or heterologous gene due to at least one deactivated signal transduction cascade, per se.

Description

EXOGENOUS REGULATION OF GENE EXPRESSION IN PLANTS

The present invention relates to the regulation of gene expression in plants, particularly to exogenous control of such gene regulation.

Advances in the understanding of gene regulation have made it possible to characterize the elements which control gene expression. In particular, the analysis of promoters has demonstrated that gene expression is often restricted to certain cell types or organs, and is often developmentally regulated according to the cues and requirements imposed by the physiological changes which occur over time, and the specialized function that cells assume. In plants, for example, genes have been characterized which are specifically expressed in roots, e.g. HRGPnt3 (Keller and Lamb, Genes Dev. 3:1639-1646 (1990)); tubers, e.g., patatin (Mignery et al., Gene 62:27-64 (1988)); nodules, e.g., leghemaglobin (Stougaard and Marcker, Nature 321:669-674 (1986)); flowers, e.g., PAL (Liang et al., J. Biol. Chem. 264: 14486-14492 (1989)); and seeds, e.g., HMW glutenin (Robert et al., Plant Cell 1 :569-578 (1989)). Gene expression can also be induced in response to physiological conditions such as anaerobiosis, e.g., ADH1 (Ellis et al., EMBO J. 6:11-16 (1987)) and sucrose accumulation, e.g., patatin (Wenzler et al., Plant Mol. Biol. 13:347-354 (1989)). Progress also has been made towards the goal of developing systems which allow the external manipulation of gene expression by the application of chemical inducers which are active on specific promoters and thus have the capability to turn on gene expression. See, Ward et al., Plant Cell 3:1085-1094 (1991); Williams et al., Bio/Technology 10:540-543 (1992) and Uknes et al., Plant Cell 5:159-169 (1993). These systems are limited, however, in terms of the uncontrollable induction of these genes by endogenous metabolites and cell signals at a time when the chemical inducer has not been applied and the expression of the gene is undesirable.

The present invention is drawn to a method for the exogenous regulation of gene expression in a plant, comprising the steps of:

* obtaining a plant incapable of regulating expression of at least one gene, family of genes, or at least one heterologous gene due to deactivation of at least one corresponding endogenous signal transduction cascade which produces a signal that regulates the gene in the plant; and

* applying a chemical regulator to the thus-obtained plant at a predetermined time when expression of the gene is desired. Thus the method involves altering a plant to inactivate a predetermined signal transduction cascade, and subsequently treating the thus-modified plant with a chemical regulator that is capable of inducing expression of the gene or genes which is regulated by the native, non-modified signal transduction cascade. The resultant plant which as its progeny, propagule or seed is another object of the present invention may be further altered by transformation with a heterologous gene of interest which is expressed upon treatment of the plant with the chemical regulator. The method is useful in controlling or altering traits such as height, shape, development, male sterility, and female sterility, and the ability of a plant to withstand cold, salt, heat, drought, disease or pest infestation. The method is especially useful when constitutive expression of gene(s) involved in manifestation of these traits might be deleterious to the growth or health of the plant. The method has further usefulness in rendering plants capable of functioning as bioreactors for the production of industrial or pharmaceutical biomaterials and precursors thereof. In the alternative, the altered plant containing the inactivated signal transduction cascade can be used in an assay to identify downstream-acting chemical regulators. That is, the chemical is not dependent upon the signal cascade, and is capable of regulating, e.g., inducing expression of the gene or genes regulated endogenously by the native, functional cascade.

The present invention provides a method for exogenous regulation of gene expression in plants wherein the corresponding, native, endogenous regulation mechanism of the genes in the plant is rendered non-functional. In general, the method is applicable to any plant capable of being altered in a manner described herein, and is particularly applicable to agronomically important plants such as maize, wheat, soybean, cotton, rapeseed, barley, rice, sorgum, sunflower, bean, beet and tobacco.

Certain genes in plants are regulated endogenously by at least one corresponding signal transduction cascade (pathway), that is, the production in the plant cell of various regulating chemicals, e.g., signal molecules. These molecules often are produced via a biosynthetic pathway in response to an external stimulus such as, for example, a necrotizing pathogen. In turn, these signal molecules regulate, i.e., induce or repress, the expression of various genes in the plant. For instance, treatment of a plant such as tobacco by a necrogenic pathogen, e.g., TMV, or salicylic acid or 2-chloroethylphosphonic acid (Ethephon, Sigma Chemicals, St. Louis, MO) initiates a process that leads to the accumulation of high concentrations of salicylic acid (SA) in other, non-infected parts of the plant. SA is bound by receptors in or on the target cells. The signal is transduced intra-cellularly. SA then activates the coordinate induction of the expression of a set of at least nine systemic acquired resistance (SAR) gene families, which include the ten pathogeneisis-related (PR) proteins of tobacco. The expression products of these gene families causes the plant target cells to become resistant to attack by a wide variety of agronomically important bacterial, fungal and viral pathogens. For example, transgenic tobacco expressing high levels of PR- la have reduced disease symptoms following infection by oomycete fungi, including Peronospora tabacina (downy mildew) and Phytopthora parasitica (black shank disease) (examples 12-15).

Applicants have discovered that inactivating an endogenous signal transduction cascade such that the expression of the target gene(s) is effectively eliminated affords the exclusive exogenous control of these genes. For example, Applicants, having confirmed that SA is the. endogenous signal molecule that mediates SAR in plants such as Arabidopsis, tobacco and cucumber, have discovered that this signal cascade can be controlled, i.e., inactivated, disarmed or rendered disfunctional, such that the induction of the target genes by SA is essentially eliminated. That is, the resultant concentration of the signal molecule in the plant cell is insuffient to activate the promoters of the signal-regulated genes. In turn, they have discovered that expression of the target genes can be induced by exogenous application of a chemical which acts downstream of the signal transduction cascade, or otherwise acts independently of the SA pathway.

The signal cascade can be rendered non-functional in a number of ways. First, the plant cell can be stably transformed with a recombinant DNA molecule comprising a promoter capable of functioning in plant cells operably linked to a structural gene encoding an enzyme that degrades the signal, a metabolic precursor thereof, or any necessary component of the cascade. Thus said enzyme is capable of metabolizing or inactivating the plant cell signal. The gene encoding such an enzyme may be derived from any organism, e.g., microbe, plant or animal, or may be a truncated or synthetic gene, provided, however, that the gene is functional in plants. The gene can be linked to a promoter functional in plants and which allows expression at high levels in those cell types in which the subsequent exogenous chemical regulation is intended to be effected. In the alternative, a promoter may be used which drives expression at high levels in all or nearly all cell types. The promoter must be capable of functioning independently of the signal, i.e., espression of the operably linked gene(s) does not depend on the signal, and the exogenous chemical. Examples of suitable promoters include constitutive promoters such as the CaMV 35 S promoter, small subunit of RUBISCO, an enhanced 35S promoter such as that described in Kay et al., Science 236:1299-1302 (1987), a double 35S promoter such as that cloned into pCGN2113 (ATCC 40587) and disclosed in the co-pending applications set forth above, and any other constitutive promoter capable of functioning in the plant tissue of interest. In a preferred embodiment, a plant is transformed with nahG, a gene which encodes salicylate hydroxylase (SH) and renders the signal salicylic acid deactivated. The plant is incapable of producing salicylic acid in an amount sufficient to regulate the genes in the plant regulated thereby. Preferably the nahG gene is linked to a constitutive promoter such as the CaMV 35S promoter in a chimaeric DNA molecule. SH (E.C. 1.14.13.1) catalyzes the conversion of salicylate to catechol. Yamamoto et al., J. Biol. Chem. 240(8):3408-3413 (1965). This gene can be obtained from any soil microbe capable of growth on salicylate as sole carbon source. Examples include Pseudomonas sp., e.g., ATCC 29351 and 29352, Pseudomonas cepacia and Trichosporon cutaneum (Einarsdottir et al., Biochemistry 27:3277-3285 (1988)). A preferred source is Pseudomonas putida PpG7 (ATCC 17485), wherein nahG is located on the 83 kilobase plasmid NAH7 in one of two operons involved in the conversion of naphthalene to pyruvate and acetaldehyde (Yen et al., Proc. Natl. Acad. Sci. USA 79:874-878 (1982)). The 1305 base pair nucleotide sequence of the nahG coding region and approximately 850 base pairs of the 3' flanking sequence have been determined (You et al., Biochemistry 30:1635-1641 (1991)). Approximately 200 base pairs of the 5' flanking sequence also have been determined. See, Schell, Proc. Natl. Acad. Sci. USA 83:369-373 (1986). Methods of transforming plants are known, and are disclosed in EP-478 502, the relevant disclosure of which is incorporated herein by reference. Those skilled in the an could select an appropriate transformation method depending upon the type of target plant.

Those skilled in the art will appreciate that other means can be employed to achieve the same effect. For instance, a second method involves the expression or overexpression in a transformed plant of a gene encoding an enzyme which catalyzes the modification, e.g., degradation, of a metabolic precursor of the signal molecule so that the plant is rendered incapable of producing the signal molecule. A third method involves the external application to the plant of antagonists of the target cell signal. Such antagonists compete with the cell signal for the cell signal target site, but do not activate the response generated by the cell signal. Instead, inhibition of the cell signal response is effected. In the case of salicylic acid, o-trimethylsilyl benzoic acid exhibits such an antagonistic effect when applied exogenously to a plant. Further, aminoethoxyvinyl glycine and aminooxyacetic acid have been found to inhibit the ethylene cascade in plants, and that the ethylene response was restored upon subsequent, exogenous application of ethylene . See, Yang and Hoffman, Ann. Rev. Plant Physiol. 35: 155-189 (1984). Yet a fourth method involves the selection of plant mutants which are non-responsive to exogenously applied signal and thus fail to respond to the selected cell signal, but which are responsive to the predetermined exogenous chemical regulator. Methods of selecting mutants for a predetermined trait are known in the art. These include EMS, gamma-rays, T-DNA transposon insertion, and the like. A fifth method involves the expression of antisense RNA to any gene encoding a protein of the signal transduction cascade. This may include the expression of antisense RNA to a gene involved in the biochemical pathway leading to the synthesis of the cell signal, or in the alternative, to a gene encoding a receptor or other component of the pathway. See, Oeller et al. Science 254:437-439 (1991). In either case, the cell signal is rendered non-functional, or its efficacy in regulating particular genes or sets of genes is significantly reduced. The cell signal also can be effectively rendered non-functional by overexpressing sense transcripts of any gene involved in the transduction cascade (pathway) utilizing a promoter functional in plant cells. This strategy is based on the observation that the attempted overexpression of a gene in transgenic plants or plant cells can lead to a down-regulation of the homologous gene in the host plant as well as the transgene. See, van der Krol et al., Plant Cell 2:291-299 (1990).

In cases where two or more endogenous signal transduction cascades can regulate at least one gene or gene family of interest, techniques to deactivate all cascades can be used, thereby rendering the genes of interest regulatable only by exogenous application of a chemical regulator.

There are several signal transduction cascades known in plants. Representative examples include the phytohormones such as ethylene which affects fruit ripening and other responses (Guzman and Ecker, Plant Cell 2:513-523 (1990); light (Chong et al., Cell 58:991-999 (1990); touch (Braam and Davis, Cell 60:357 (1990); and gravity (Okada et al., Cell 70:369-372 (1992).

Once inactivation of the cell signal is achieved, the genes which are natively regulated by the signal can be regulated exclusively by the exogenous application of a gene-regulating effective amount of a chemical regulator to the plant. In general, the chemical regulator, which can be a naturally or non-naturally occurring in plants, functions "downstream" of the signal in the transduction pathway, or functions completely independently, e.g., is not involved in the pathway. In the case of the SA pathway, for example, representative chemical regulators capable to inducing expression of PR genes include benzo- 1 ,2,3-thiodiazole-7-carboxylic acid, methyl benzo- 1 ,2,3-benzothiodiazole-7-carboxylate, n-propyl benzo- 1 ,2,3-benzothiodiazole-7-carboxylate, benzyl benzo- l,2,3-benzothiodiazole-7-carboxylate, and benzo- 1 ,2,3-benzothiodiazole-7-carboxylic acid N-secbutylhydrazide.

In nature these compounds do not occur in plants.

Other chemicals encompassed by the present invention can be determined by a method for the identification of chemicals capable of regulating plant genes comprising the steps of:

* obtaining a plant, plant tissue or plant cells incapable of regulating expression of at least one gene or family of genes due to deactivation of at least one corresponding endogenous signal transduction cascade which produces a signal that natively regulates the gene in the plant;

* applying a chemical suspected of having the capability of regulating the gene to the thus-obtained plant, plant tissue or plant cells; and

* 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 the endogenous signal transduction cascade.

Assaying the test chemical can be done in the presence of a plant modified in a manner described above, which plant also contains an endogenous or heterologous reporter gene operably linked to a promoter regulatable by the signal molecule. Since the modified plant is incapable of producing the signal molecule in sufficient amounts to induce expression of the reporter gene, no difference will be observed upon application to the plant with the chemical unless the chemical is capable of regulating expression of the reporter gene. Examples of reporter genes include luciferase (LUX), chloramphenicol acetyltransferase (CAT), neomycin phosphotransferase (NPT), nopaline synthase (NOS), octopine synthase (OCS), beta-l,3-glucuronidase (GUS), acetohydroxyacid synthase (AHAS) and Bacillus thuringiensis endotoxin (Bt) (Williams et al., Bio/Technology, 10:540-543 (1992). The assay can be performed using whole plants or with plant tissue in an in vitro assay. In certain situations, it would be desirable to regulate the expression of various heterologous genes (transgenes) in transgenic plants. For example, the effectiveness of disease- or insect resistance in transgenic plants transformed with genes encoding disease- or insect-resistant proteins, respectively, could be enhanced if the timing of the expression could be controlled. See, e.g., Uknes, Plant Cell, 4:645-656 (1992); Ward et al., Plant Cell 3: 1085-1094 (1991); Gould, Bioscence 38:26-33 (1988); and Gould, TIBTECH 6:S 15-S18 (1988). Also, the chemical regulation of developmental processes such as homeosis, germination, tillering, sprouting, flowering, anthesis, fruit ripening, and abscission offers several advantages such as the facilitated production of hybrid seed, greater reduction of crop loss, and more generally, control of the growth and development of the plant by the farmer. Thus, the present invention applies equally to transgenic plants containing heterologous genes, e.g., disease resistance genes including PR and SAR genes, pest resistance genes, insect resistance genes such as Bt genes, herbicide resistance genes such as altered acetohydroxyacid synthase (AHAS; US-4,761,373), mutant glutamine synthase (GS; US-4,975,374), mutant acetolacate synthase (US-5,013,659), altered acetyl coenzyme carboxylase (US-5, 162,602), and imidazoleglycerol phosphate dehydratase (IGPD), and genes involved in developmental processes such as those described above. It also includes genes encoding industrial or pharmaceutical biomaterials such as plastics and precursors thereof, perfumes, additives, enzymes and other proteins, and pharmaceuticals, wherein the plant effectively would be used as a bioreactor, e.g., the two genes encoding production of polyhydroxybutyrate, a thermoplastic (Poirer et al., Science 256:520-523 (1992). To practice this embodiment of the present invention, the heterologous gene of interest should be fused to a promoter capable of being regulated by the exogenous chemical regulator, which promoter is not necessarily regulatable by the endogenous signal. In other words, the promoter can be regulatable by the endogenous signal, provided that it can be regulated by a chemical regulator in the absence of a functional, endogenous signal. Examples include the PR- la promoter such as those disclosed in Williams et al., Bio/Technology 10:540-543 (1992); Uknes et al., The Plant Cell 5:159-169 (1993); Van de Rhee et al, Plant Cell 2:357-366 (1990); and EP-0 332 104, herein incorporated by reference in their entireties, and the promoters of other tobacco PR protein genes such as PR- lb, PR-lc, PR-1 ', PR-Q, PR-R, PR-S, cucumber chitinase and the basic and acidic tobacco β-l,3-glucanase genes isolated from chemically regulated plant genes such as those described in beforementioned EP-0 332 104, and in Payne et al., Plant Mol. Biol. 11:89-94 (1988).

Apart from their utility in chemical regulation, plants which are disrupted in the signal transduction cascade leading to the expression of PR-proteins and therefore systemic acquired resistance, have further utility for disease testing. Plants incapable of expressing PR proteins do not develop the systemic acquired restistance response and thus develop larger lesions more quickly when challenged with pathogens. These plants are useful as "universal disease susceptible" (UDS) plants by virtue of their being susceptible to many strains and pathotypes of pathogens of the host plant and also to pathogens which do not normally infect the host plant, but which infect other hosts. They provide useful indicators of evaluation of disease pressure in field pathogenesis tests where the natural resistance phenotype of so-called wilde-type (i.e. non-transgenic) plants may vary and therefore not provide a reliable standard of susceptibility. Furthermore, these plants have additional utility for the testing of candidate disease resistance transgenes. Using a nahG-expressing stock line as a recipient for transgenes, the contribution of the transgene to disease resistance is directly assessable over a base level of susceptibility. Thus the present invention can be used in a sensitive method of assaying a DNA molecule for the ability to confer resistance to a plant pathogen comprising the steps of:

(a) transforming said DNA molecule into a plant, plant tissue or plant cells incapable of regulating expression of at least one gene or family of genes due to deactivation of at least one corresponding endogenous signal transduction cascade which produces a signal that natively regulates the gene in the plant;

(b) challenging the transformed plant, plant tissue or plant cells of step (a) with said plant pathogen under conditions that cause disease in said plant, plant tissue or plant cell which are not transformed; and

(c) assaying said transformed plant, plant tissue or plant cells for the development of disease symptoms associated with said pathogen.

A further utility of for nahG-expressing plants is as tool in the understanding of plant-pathogen interactions. NahG-expressing host plants do not mount a systemic response to pathogen attack, and an unabated development of the pathogen is an ideal system in which to study its biological interaction with the host. As nahG-expressing host plants may also be susceptible to pathogens outside of whose host range they normally fall, these plants also have significant utility in the molecular, genetic, and biological study of host-pathogen interactions. Furthermore, the UDS phenotype of the nahG-expressing plants also renders them of utility for fungicide screening. Plants expressing nahG in a particular host have considerable utility for the screeing of fungicides using that host and pathogens of the host. The advantage lies in the UDS phenotype of the nahG-expressing host which circumvents the problems encountered by hosts being differentially susceptible to different pathogens and pathotypes, or even resistant to some pathogens or pathotypes. nahG-expressing plants have further utility for the screeing of fungicides against a range of pathogens and pathotypes using a heterologous host i.e. a host which may not normally be within the host species range of particular pathogens. Thus the susceptibility of nahG-expressing host plants such as Arabidopsis, which are easily manipulable and have limited space requirements, to pathogens of other species (e.g. crop plant species) would facilitate efficacious fungicide screening procedures for compounds against important pathogens of crop plants. Thus the present inventions provides a sensitive method of assaying a putative fungicide for the ability to confer resistance to a fungus comprising the steps of: (a) treating a plant, plant tissue or plant cells incapable of regulating expression of at least one gene or family of genes due to deactivation of at least one corresponding endogenous signal transduction cascade which produces a signal that natively regulates the gene in the plant with said fungicide;

(b) challenging the plant, plant tissue or plant cells of step (a) with said fungus under conditions that cause fungal infection in said plant, plant tissue or plant cells which have not been treated; and

(c) assaying said plant, plant tissue or plant cells for the development of infection by said fungus.

In a similar method the present invention can be used in a sensitive method for detecting the presence of a plant pathogen in an environment comprising:

(a) culturing a plant, plant tissue or plant cells incapable of regulating expression of at least one gene or family of genes due to deactivation of at least one corresponding endogenous signal transduction cascade which produces a signal that natively regulates the gene in the plant in said environment; and

(b) assaying said plant, plant tissue or plant cells for the development of disease symptoms associated with said pathogen.

In the situations described avove the nahG gene can be expressed behind promoters which are expressible in plants using gene cloning techniques which are well known in the art. A preferred promoter would express nahG at the time of disease challange. A particularly preferred promoter would be expressed constitutively e.g. the Cauliflower Mosaic Virus 35S promoter.

The following examples are offered by way of illustration and not by way of limitation.

EXAMPLES

1. Cloning of a DNA fragment encoding the Pseudomonas putida nahG gene into the plant expression vector pCIB200.

Genes encoding enzymes involved in the metabolic pathway which converts naphthalene to pyruvate and acetaldehyde in the bacterium Pseudomonas putida PpG7 are organized in two operons on the plasmid NAH7. Salicylate hydroxylase, which catalyzes the conversion of salicylate to catechol, is encoded by nahG. pSR20, a plasmid NAH subclone obtained from M. Schell (University of Georgia), was digested with Sspl and Hpal to obtain a ca 1.5 kb restriction fragment containing nahG. This Sspl-Hpal fragment was ligated to EcoRI linkers, digested with EcoRI, and cloned into the EcoRI site of pCGN1761 (EP-0 392 225-A3), a derivative of ρCGN2113 (ATCC 40587) to add plant-recognized regulatory sequences. pCGN1761 is prepared by digesting pCGN2113 with EcoRI and ligating the plasmid in the presence of a synthetic DNA adaptor containing an Xbal site and a BamHI site. The adaptor contained EcoRI sticky ends on either end, but the adjacent bases were such that an EcoRI site was not reconstructed at this location. pCGN1761 contains a double CaMV 35 S promoter and the tml-3' region with an EcoRI site between contained in a pUC-derived plasmid backbone. The promoter-EcoRI-3' processing site cassette is bordered by multiple restriction sites for easy removal.

A pCGN1761 derivative was identified with the nahG gene oriented 5' to 3' behind a double 35S promoter from the Cauliflower Mosaic Virus, and followed by the efficient 3' terminator tml. This plasmid was digested with Xbal to release a 4.7 kb restriction fragment containing the nahG construct, which was subsequently cloned into the Xbal site of the plant transformation vector pCIB200. See, Uknes et al.. Plant Cell 5:159-169 (1993).

2. Transformation of the nahG containing plant expression vector pCIB200/nahG into Nicotiana tabacum cv. Xanthi-nc.

pCIB200/nahG was first transferred from E. coli to Agrobacterium tumefaciens strain CIB542 (Uknes et al., supra.) using the elctroporation method described by Wenjun and Forde, Nucleic Acids Res. 17:8385 (1989)). Transformed Agrobacterium colonies were selected on 25 ug/ml kanamycin.

Agrobacterium tumefaciens-mediated transformation of Nicotiana tabacum cv Xanthi-nc was undertaken essentially as described by Horsch et al., Science 227:1229-1231 (1985). Leaf disks of Nicotiana tabacum cv Xanthi-nc were infected with the Agrobacterium tumefaciens strain carrying pCIB200/nahG and sleeted for callus growth on kanamycin. A single shoot was regenerated (Tl generation) from each leaf disk and grown in soil until seed set. Seed resulting from self-pollination (T2 generation ) of the regenerated transformants was scored for antibiotic resistance on MS medium (Murashige and Skoog, Physiol. Plant:473 (1962) containing 150 ug/ml kanamycin. Lines homozygous for the transgene were identified by allowing ten kanamycin resistant T2 progeny from each independent transformant to self-pollinate and set seed and screening for plants whose seed (T3 generation) were 100% kanamycin resistant.

Plants expressing nahG at high levels were not visually distinct from wild-type plants. They flowered and set seed normally. 3. Analysis of transformants expressing the nahG gene.

Independently transformed plants were screened by RNA blot analysis (Ausubel et al., Current Protocols in Molecular Biology, Vol. 1, Wiley & Sons, New York (1987)) for nahG mRNA accumulation and several transformants were selected (NahG-1, -2, -3, -8, -9 and -10). These plants were allowed to set seed and homozygous T3 seed for further analysis was generated as described above.

4. Effect of nahG expression on the accumulation of salicylic acid.

To determine the effect of the expression of the nahG gene on the accumulation of salicylic acid, several plants from each of the lines selected above were inoculated with TMV. After 7 days, when lesions had formed on the infected leaves, leaf tissue was harvested and assayed for nahG mRNA, the salicylate hydroxylase protein, and salicylic acid. Assessment of mRNA abundance was undertaken by RNA blot analysis as described above.

Salicylate hydroxylase protein was determined by Western analysis using standard techniques. See, Pratt et al., Modern Methods Plant Anal. New Series 4:51 (1986). Salicylate hydroxylase was purified from E. coli after expression of the nahG gene in the E. coli expression vector pGEX-2Tb (AMRAD Corporation Limited), and the purified enzyme was used to raise antibodies in rabbits.

Salicylic acid concentration was determined after extraction from leaf tissue using the technique essentially as described by Yalpani et al., Plant Cell 3:809-818 (1991), except that samples were not allowed to overdry, and that the final samples were resuspended in 500ul of 20% methanol. 10-100 ul were injected onto a Dynamax 60A, 8 urn, C-18 (4.6 mm x 25 cm) column with guard column (Rainin Instruments Co., Emeryville, CA) maintained at 40 C. Isocratic separation was performed at 1 ml/min using 20% (v/v) methanol in 20 mM sodium acetate, pH 5.0. Fluorescence detection was done using a Model 980 detector (ABI/Kratos analytical, Forster City, CA) with a 5 ul flowcell, deuterium lamp with a 295 nm excitation setting and a 370 nm cutoff emission filter. The limit of detection was 500 pg SA in 50 ul. Quantification was determined versus a linear range (10-1000 ng/ml) of calibration standards for sodium salicylate. The nahG-3, -8 and -10 lines expressed high levels of nahG mRNA and salicylate hydroxylase protein. These lines accumulated about 100 ng/g SA following TMV treatment (see Table 1, below) which represented approximately a 2-3 fold increase above the concentration in buffer-treated control plants. Lines nahG-1 and -2 expressed intermediate levels of mRNA and barely detectable levels of salicylate hydroxylase protein, but accumulated 2824 and 979 ng/g SA, respectively, following TMV treatment, representing an 80 and a 30-fold induction, respectively. Line nahG-9 did not have detectable levels of either nahG mRNA or salicylate hydroxylase protein and accumulated 6334 ng/g SA. Similarly, the accumulation of SA in the non-transformed control line was 5937 ng/g, representing a 180-fold induction.

These results showed a tight inverse correlation between expression of the nahG transgene and accumulation of SA. The presence of high levels of nahG mRNA and salicylate hydroxylase protein resulted in a significant block in SA accumulation in TMV-treated transgenic plants.

TABLE 1

Salicylic Acid Levels (ng/g tissue)±standard deviation

Line Buffer TMV induction

Xanthi 32.1±2.3 593711011 185

NahGl 35.8±2.2 282411461 79

NahG2 38.8±10.2 979+113 25

NahG3 41.3±5.6 107145 3

NahG8 36.2±2.7 81122 2

NahG9 35.6±3.0 63341765 179

NahG 10 33.9±3.5 11214 3

All results were the average • standard deviation, after the results were corrected for recovery (57.1%). All values were based on triplicate assays, except for nahG/buffer, whose value was based on duplicate assay.

5. Effects of nahG expression on systemic acquired resistance (SAR)

To determine the effects of the reduced accumulation of SA on SAR, transgenic lines were challenge-inoculated with TMV. Lesion size was scored 7 days later and compared to lesion size on buffer treated controls (see Table 2, below). Control non-transgenic lines showed a reduction of lesion size of 63% relative to buffer-treated plants, which is typical of the SAR response to TMV. In the lines expressing high levels of nahG mRNA and salicylate hydroxylase protein, which were shown above not to accumulate SA, lesion size was reduced by only 5-9% (nahG-3, -8 and -10). Lines expressing intermediate levels of nahG mRNA and salicylate hydroxylase protein showed an intermediate reduction in lesion size (nahG-1, and -2). The nahG-9 line, which did not express detectable levels of nahG mRNA or salicylate hydroxylase protein, showed a 66% reduction in lesion size. These results clearly demonstrate that SA is required for the onset of SAR and is a cell signal in the SAR transduction pathway which can be effectively eliminated.

TABLE 2

Lesion Size (average standard deviation )/mm

Line Buffer TMV n %reduction

Xanthi 3.510.4 (d) 3 1.310.5 (d) 5 63

NahGl 4.110.4 (b) 3 2.710.6 (c) 5 34

NahG2 4.410.4 (a) 3 3.810.5 (b) 5 14

NahG3 4.410.4 (a) 2 4.010.5 (b) 5 9

NahG8 4.210.4 (b) 2 4.110.7 (b) 5 5

NahG9 3.810.4 (c) 3 1.310.7 (d) 5 66

NahG 10 4.510.4 (a) 3 4.210.8 (a) 5 7

Three to five plants were analyzed per sample as indicated. Per plant 10 lesions were measured on 3 leaves. The data were analyzed statistically by ANOVA II, followed by a Tukey-Kramer test. Within each treatment, statistically equivalent groups (p=0.05) are shown (a-d).

6. Benzo- l,2,3-thiodiazole-7-carboxylic acid induction of SAR in plants expressing nahG.

Three lines, nahG-3, -8 and -10, were shown above to express high levels of nahG mRNA and salicylate hydroxylase protein, and to be blocked in their SAR response to disease infection. Plants of these lines were treated with the inducing chemical benzo- l,2,3-thiodiazole-7-carboxylic acid, and were shown to possess the SAR response. This result demonstrated the position of benzo- l,2,3-thiodiazole-7-carboxylic acid downstream relative to SA in the SAR signal transduction pathway. 7. Chemical regulation of gene expression in nahG-expressing plants.

The PR-la promoter (Uknes et al., Plant Cell 5:159-169 (1993) is chemically regulated by exogenously applied benzo- l,2,3-thiodiazole-7-carboxylic acid and derivatives thereof, as well as by SA. Plants possessing the PR- la promoter fused to the GUS reporter gene are crossed to nahG-expressing lines nahG-3, -8 and -10. Progeny lines carrying both transgene constructions are found to express GUS when induced by benzo- l,2,3-thiodiazole-7-carboxylic acid, but not when treated with SA. Further, there is no GUS expression in response to fluctuating endogenous levels of SA as would occur in plants not expressing the nahG gene prior to and during flowering, for example. Consequently, the chemical regulation of the PR- la gene promoter can be utilized without the activation of the endogenous cell signal SA.

8. . Chemical regulation of a gene encoding the delta-endotoxin of Bacillus thuringiensis in nahG-expressing plants.

Plants possessing the PR- la promoter fused to a gene encoding the delta-endotoxin of Bacillus thuringiensis (Williams et al., Bio/Technology 10:540-543 (1992) are. crossed to nahG-expressing lines nahG-3, -8 and -10. Progeny lines carrying both transgene constructions are found to express the endotoxin gene when induced by benzo- l,2,3-thiadiazole-7-carboxylic acid, but not when treated with SA. Further, there is no endotoxin gene expression in response to fluctuating endogenous levels of SA as would occur in plants not expressing the nahG gene prior to and during flowering, for example. Consequently, the chemical regulation of the PR- la gene promoter can be utilized without the activation of the endogenous cell signal SA.

All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are incorporated herein by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims. 9. Use of nahG-Expressing Plant Lines in Disease Testing

Tobacco and Arabidopsis plants expressing the nahG gene constitutively, were challenged with numerous pathogens and found to develop larger lesions more quickly than wild-type plants. This phenotype is referred to as UDS (i.e. universal disease susceptibility) and is a result of the plants being unable to express SAR genes to effect the plant defence against pathogens by virtue of the expression of salicylate hydroxylase protein, and the inactivation of salicylic acid as an endogenous cell signal in systemic acquired resistance. Table 2 (in example 5) shows a comparison of lesion size in plants expressing nahG and wild-type plants and table 3 (below) shows a comparison of the development of lesions over an eight-day period in wild-type tobacco and nahG-expressing tobacco.

TABLE 3

Lesion Growth over a period of eight Days in NahG-transgenic and non-transgenic Tobacco

Xanthi NahG- 10 mean SD mean SD DPI

50.8 12.4 47.5 12.6 2

102.2 19.3 113.4 26.7 3

138.0 25.6 186.4 29.0 4

170.8 29.7 227.3 27.1 5

199.0 34.7 276.5 35.3 6

220.9 39.8 332.4 38.0 7

234.2 47.5 376.4 42.5 8

Plants of nahG-expressing line NahG- 10 (see tables 1 and 2) and wild-type tobacco cultivar Xanthi were inoculated with TMV and lesion size was monitored over a period of 8 days post inoculation (DPI). Mean values for lesion size are reported in 1/1000 inch with standard deviation (SD). The nahG-expressing line develops larger lesions more quickly than does the wild-type line Xanthi.

The UDS phenotype of these nahG expressing plants renders them useful as control plants for the evaluation of disease symptoms in experimental lines in field pathogenesis tests where the natural resistance phenotype of so-called wild-type lines may vary (i.e. to different pathogens and different pathotypes of the same pathogen). Thus, in a field environment where natural infection by pathogens is being relied upon to assess the resistance of experimental lines, the incorporation into the experiment of nahG expressing lines of the appropriate crop plant species would enable an assessment of the true level and spectrum of pathogen pressure, without the variation inherent in the use of non-experimental lines.

10. Assessment of the Utility of Transgenes for the Purposes of Disease Resistance

Plants constitutively expressing nahG are used as host plants for the transformation of transgenes to facilitate their assessment for use in disease resistance. A stock of Arabidopsis or tobacco plants is created which express the nahG gene. This stock is used for subsequent transformations with candidate genes for disease resistance thus enabling an assessment of the contribution of an individual gene to resistance against the basal level of the UDS nahG expressing plants.

1 1. NahG-expressing Plants as a Tool in Understanding Plant-Pathogen Interactions

Plants expressing nahG are useful for the understanding of plant pathogen interactions, and in particular for the understanding of the processes utilized by the pathogen for the invasion of plant cells. This is so because nahG-expressing host plants do not mount a systemic response to pathogen attack, and the unabated development of the pathogen is an ideal scenario in which to study its biological interaction with the host.

Of further significance is the observation that a host species expressing nahG may be susceptible to pathogens not normally associated with that particular host, but instead associated with a different host. Arabidopsis plants were transformed with nahG and those expressing the gene were characterized by the UDS phenotype. These plants are challenged with a number of pathogens which normally only infect tobacco, and found to be susceptible. Thus, the expression of nahG in a host plant and the accompanying UDS phenotype leads to a modification of pathogen-range susceptibility and this has significant utility in the molecular, genetic a d biochemical analysis of host-pathogen interaction. 12. NahG-expressing Plants for Use in Fungicide Screening

Plants expressing nahG are particularly useful in the screening of new chemical compounds for fungicide activity. The advantage lies in the UDS phenoytpe of the nahG-expressing host plant which circumvents the problems encountered by the host being differentially susceptible to different pathogens and pathotypes, or even resistant to some pathogens or pathotypes. By way of example transgenic wheat expressing nahG could be effectively used to screen for fungicides to a wide range of wheat pathogens and pathotypes as the nahG-expressing line would not mount a resistance response to the introduced pathogen and would not display differential resistance to different pathotypes which might otherwise require the use of multiple wheat lines, each adequately susceptible to a particular test pathogen. Wheat pathogens of particular interest include (but are not limited to) Erisyphe graminis (the causative agent of powdery mildew), Rhizoctonia solani (the causative agent of sharp eyespot), Pseudocercosporella herpotrichoides (the causative agent of eyespot), Puccinia spp. (the causative agents of rusts), and Septoria nodorum. Similarly, corn plants or tobacco plants expressing nahG would be highly susceptible to their respective pathogens and would therefore be useful in the screening for fungicides.

nahG-expressing plants have further utility for the screening of a wide range of pathogens and pathotypes in a heterologous host i.e. in a host which may not normally be within the host species range of a particular pathogen and which may be particularly easy to manipulate (such as Arabidopsis). By virtue of its UDS phenotype the heterologous host expressing nahG is susceptible to pathogens of other plant species, including economically important crop plant species. Thus, by way of example, the same Arabidopsis nahG-expressing line could be infected with a wheat pathogen such as Erisyphe graminis (the causative agent of powdery mildew) or a corn pathogen such as Helminthosporium maydis and used to test the efficacy of fungicide candidates. Such an approach has considerable improvements in efficiency over currently used procedures of screening individual crop plant species and different cultivars of species with different pathogens and pathotypes which may be differentially virulent on the different crop plant cultivars. Furthermore, the use of Arabidopsis has advantages because of its small size and the possibility of thereby undertaking more tests with limited resources of space. 13. Evaluation of Transgenic Tobacco Expressing PR-1 in a Sense Orientation for Disease Resistance

The seed lines 1755A-4-2 and 1755B-2-1 (EP-0 392 225, example 89) are analyzed for resistance to TMV. The results of these experiments are that there is no significant difference in lesion size or lesion number due to either elevated or depressed levels of PR-1 protein.

The seed lines 1755A-4-2 and 1755B-2-1 (EP-0 392 225, example 89) are analyzed for resistance to the fungal pathogen Peronospora tabacina (blue mold, or downy mildew) by spraying a spore suspension on the leaves of the plants and incubating under standard conditions for seven days. The plants are then scored for resistance to bluemold based on the percentage of leaf surface area infected by the pathogen. Six plants of the 1774A-10-1 line are showing 6.3 % +/- 11 % infected surface area. Six plants of the 1774B-3-2 line are showing 46 % +/- 10 % infected surface area. Six plants derived from untransformed Xanthi. nc tobacco are showing 55 % +/- 5 % infected surface area. This result indicates that the sense expression of PR- la results in a significant and valuable resistance to downy mildew in transgenic plants.

14. Analysis of Seed Lines Derived From Transformation of Tobacco With the PCGN1790 Plasmid Series (Double CAMV 35S Promoter/SAR 8.2)

A leaf tissue sample is taken from Tl plants transformed with either of the binary vectors pCGN1790C or pCGN1790D (EP-0 392 225, example 54). The SAR8.2 protein content is estimated by an immunoblot technique (Towbin, H., et al., Proc. Natl. Acad. Sci. USA 76: 4350-4354 (1979) as modified by Johnson, D., et al., Gene Anal. Tech. 1: 3-8 (1984)), following SDS-polyacrylamide gel electrophoresis (Laemmli, E., Nature, 227: 680-685 (1970)). The antibodies used are raised against the SAR 8.2 protein by standard methods and are specific for the SAR 8.2 protein. Tl plants with the pCGN1790C plasmid (containing the sense expression cassette) showing high levels of expression relative to control and anti-sense plants, are advanced to T3 seed lines. Homozygous T2 plants which yield these T3 seed continue to express the protein at high levels. Tl plants transformed by the pCGN1790D plasmid (containing the anti-sense expression cassette) which give low levels of expression are also advanced to T3 seed. 15. Evaluation of Transgenic Tobacco Expressing SAR8.2 in a Sense Orientation for Disease Resistance

The seed lines 1790C (sense orientation) (Example 15 above) is analyzed for resistance to black shank, a disease caused by the fungal pathogen Phytopthora.

Four control plants are transformed with an empty expression cassette and are severely affected by black shank disease.

Four plants are transformed with the chimeric double 35S-SAR8.2 construct and exhibit appreciable enhanced resistance to black shank disease.

While the present invention has been described with reference to specific embodiments thereof, it will be appreciated that numerous variations, modifications, and embodiments are possible, and accordingly, all such variations, modifications and embodiments are to be regarded as being within the spirit and scope of the present invention.

LIST OF REFERENCES

Ausubel et al., Current Protocols in Molecular Biology, Vol. 1, Wiley & Sons, New York

(1987)

Braam and Davis, Cell 60:357 (1990)

Chong et al., Cell 58:991-999 (1990)

Einarsdottir et al., Biochemistry 27:3277-3285 (1988)

Ellis et al., EMBO J. 6:11-16 (1987)

Gould, Bioscence 38:26-33 (1988)

Gould, TIBTECH 6:S15-S18 (1988)

Guzman and Ecker, Plant Cell 2:513-523 (1990)

Horsch et al., Science 227: 1229-1231 (1985)

Johnson, D., et al., Gene Anal. Tech. 1:3-8 (1984)

Kay et al., Science 236:1299-1302 (1987)

Keller and Lamb, Genes Dev. 3:1639-1646 (1990)

Laemmli, E., Nature, 227: 680-685 (1970)

Liang et al, J. Biol. Chem. 264:14486-14492 (1989)

Mignery et al., Gene 62:27-64 (1988)

Murashige and Skoog, Physiol. Plant:473 (1962) Oeller et al. Science 254:437-439 (1991)

Okada et al., Cell 70:369-372 (1992)

Payne et al., Plant Mol. Biol. 11:89-94 (1988)

Poirer et al., Science 256:520-523 (1992)

Pratt et al., Modern Methods Plant Anal. New Series 4:51 (1986)

Robert et al., Plant Cell 1:569-578 (1989)

Schell, Proc. Natl. Acad. Sci. USA 83:369-373 (1986)

Stougaard and Marcker, Nature 321:669-674 (1986)

Towbin, H., et al., Proc. Natl. Acad. Sci. USA 76:4350-4354, (1979)

Uknes, Plant Cell, 4:645-656 (1992)

Uknes et al., The Plant Cell 5:159-169 (1993) van der Krol et al., Plant Cell 2:291-299 (1990)

Van de Rhee et al., Plant Cell 2:357-366 (1990)

Ward et al., Plant Cell 3:1085-1094 (1991)

Wenjun and Forde, Nucleic Acids Res. 17:8385 (1989)

Wenzler et al., Plant Mol. Biol. 13:347-354 (1989)

Williams et al., Bio/Technology 10:540-543 (1992)

Yalpani et al., Plant Cell 3:809-818 (1991)

Yamamoto et al., J. Biol. Chem. 240(8):3408-3413 (1965)

Yang and Hoffman, Ann. Rev. Plant Physiol. 35:155-189 (1984)

Yen et al., Proc. Natl. Acad. Sci. USA 79:874-878 (1982)

You et al., Biochemistry 30:1635-1641 (1991)

EP-0 332 104 EP-0 392 225 EP-0478 502

US-4,761,373 US-4,975,374 US-5,013,659 US-5,162,602

Claims

WE CLAIM:

1. A method for the exogenous regulation of gene expression in a plant, comprising the steps of:

(a) obtaining a plant incapable of regulating expression of at least one gene, family of genes, or at least one heterologous gene due to deactivation of at least one corresponding endogenous signal transduction cascade which produces a signal that regulates the gene in the plant; and

(b) applying a chemical regulator to the thus-obtained plant at a predetermined time when expression of the gene is desired.

2. A method of claim 1, wherein the step of obtaining comprises stably transforming a plant with a recombinant DNA molecule comprising a promoter capable of functioning in plant cells operably linked to a structural gene encoding an enzyme that deactivates the signal, a metabolic precursor thereof, or any necessary component of the cascade.

3. A method of claim 2, wherein the structural gene is nahG which encodes salicylate hydrogenase, and the signal deactivated is salicylic acid.

4. A method of claim 1, wherein the step of obtaining comprises applying a cascade-deactivating effective amount of an antagonist of the cascade to the plant.

5. A method of claim 1, wherein the step of obtaining comprises selecting mutants non-responsive to exogenously applied signal.

6. A method of claim 1 , wherein the step of obtaining comprises stably transforming a plant with anti-sense RNA to a gene encoding a protein of the signal transduction cascade.

7. A method of claim 1, wherein the step of obtaining comprises preparing a plant incapable of producing salicylic acid in an amount sufficient to regulate the genes in the plant regulated thereby.

8. A method of claim 3, wherein the step of applying comprises applying a gene-regulating effective amount of benzo- l,2,3-thiodiazole-7-carboxylic acid, or a derivative therof, to the plant.

9. A plant or plant cell incapable of producing or responding to at least one endogenous cell signal in an amount sufficient to regulate expression of any endogenous gene regulated by the signal, or any heterologous gene linked to a promoter regulatable by the signal.

10. A plant or plant cell of claim 9, wherein the endogenous cell signal is salicylic acid.

11. A plant or plant cell of claim 10, stably transformed with a chimeric DNA molecule comprising a promoter functional in plant cells operably linked to a structural gene encoding an enzyme capable of metabolizing salicylic acid or a biochemical precursor thereof.

12. A plant or plant cell of claim 11, wherein the structural gene is nahG which encodes salicylate hydroxylase.

13. A plant or plant cell of claim 11, wherein the promoter is constitutive.

14. A plant or plant cell of claim 13, wherein the constitutive promoter is a CaMV 35S promoter.

15. A plant or plant cell of claim 9, comprising at least one recombinant DNA molecule comprising a promoter functional in plant cells and regulatable by the signal, operably linked to a heterologous structural gene of interest, wherein the gene is expressed by said plant only upon application of an exogenous chemical regulator to said plant.

16. A plant of plant cell of claim 15, wherein the endogenous signal is salicylic acid.

17. A plant or plant cell of claim 15, wherein the heterologous structural gene encodes a disease resistance protein, a pest resistance protein, an herbicide resistance protein, a plant developmental protein, or an industrial or pharmaceutical biomaterial or precursor thereof.

18. A plant or plant cell of claim 15, wherein the promoter is a PR- la promoter.

19. A progeny, propagule or seed of the plant of claim 9.

20. A progeny, propagule or seed of the plant of claim 10.

21. A progeny, propagule or seed of the plant of claim 15.

22. A method for the identification of chemicals capable of regulating plant genes comprising the steps of:

(a) obtaining a plant, plant tissue or plant cells incapable of regulating expression of at least one gene or family of genes due to deactivation of at least one corresponding endogenous signal transduction cascade which produces a signal that natively regulates the gene in the plant;

(b) applying a chemical suspected of having the capability of regulating the gene to the thus-obtained plant, plant tissue or plant cells; 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 the endogenous signal transduction cascade.

23. A method of claim 22, wherein the step of obtaining comprises stably transforming a plant with a recombinant DNA molecule comprising a promoter capable of functioning in plant cells operably linked to a structural gene encoding an enzyme that deactivates the signal, a metabolic precursor thereof, or any necessay component of the cascade.

24. A method of claim 23, wherein the structural gene is nahG which encodes salicylate hydrogenase, thus rendering the signal salicylic acid deactivated.

25. A method of claim 22, wherein the step of obtaining comprises applying a cascade-deactivating effective amount of an antagonist of the cascade to the plant.

26. A method of claim 22, wherein the step of obtaining comprises selecting mutants non-responsive to exogenously applied signal.

27. A method of claim 22, wherein the step of obtaining comprises stably transforming a plant with anti-sense RNA to a gene encoding a protein of the signal transduction cascade.

28. A method of claim 1, wherein the step of applying comprises applying a gene regulatory effective amount of a compound obtainable by a method according to any one of claims 22 to 27.

29. A sensitive method of assaying a DNA molecule for the ability to confer resistance to a plant pathogen comprising the steps of:

(a) transforming said DNA molecule into a plant, plant tissue or plant cells incapable of regulating expression of at least one gene or family of genes due to deactivation of at least one corresponding endogenous signal transduction cascade which produces a signal that natively regulates the gene in the plant; . (b) challenging the transformed plant, plant tissue or plant cells of step (a) with said plant pathogen under conditions that cause disease in said plant, plant tissue or plant cell which are not transformed; and

30. A sensitive method of assaying a putative fungicide for the ability to confer resistance to a fungus comprising the steps of:

(a) treating a plant, plant tissue or plant cells incapable of regulating expression of at least one gene or family of genes due to deactivation of at least one corresponding endogenous signal transduction cascade which produces a signal that natively regulates the gene in the plant with said fungicide;

31. A sensitive method for detecting the presence of a plant pathogen in an environment comprising:

(a) culturing a plant, plant tissue or plant cells incapable of regulating expression of at least one gene or family of genes due to deactivation of at least one corresponding endogenous signal transduction cascade which produces a signal that natively regulates the gene in the plant in said environment; and (b) assaying said plant, plant tissue or plant cells for the development of disease symptoms associated with said pathogen.