AU697810B2 - Genes regulating the response of (zea mays) to water deficit - Google Patents

Description

1- 9 r- WO 951/30005 PCTIUS95/05366 -1-

DESCRIPTION

GENES REGULATING THE RESPONSE OF ZEA MAYS TO WATER DEFICIT BACKGROUND OF THE INVENTION A. Field of the Invention The present invention relates generally to the field of maize growth and the response to varying conditions of water availability, the use of water, and resistance to water deficit. The invention also concerns the genes involved in plant resistance to water deficit and availability of water. More particularly, it concerns the identification and various uses of genes involved in the process of wilting during periods of drought.

B. Description of the Related Art Drought is a significant problem in agriculture today. Over the last 40 years, drought accounted for 74% of the total U.S. crop losses of corn (Agriculture, 1990). It is not only important for plants to withstand water deficit conditions, but also to grow and yield under these conditions. Thus, it is important to understand the fundamental process by which plants respond and adapt to water deficits. Identifying key genes in this process will permit a detailed characterization of this process at the cellular and whole plant level. This basic knowledge would allow informed strategies to be developed which potentially would enable plants to .adapt to less than ideal environmental conditions.

An important agricultural problem is the protection of plants from water deficits. When the rate of transpiratior. exceeds that of absorption, a water deficit I B I L WO 95/30005 PCT/US95/05366 2 occurs and wilting symptoms appear. The responses of plants to water deficits include leaf shedding, stomata closure, leaf temperature increases, and leaf rolling and wilting. Cells of a wilted leaf have reduced turgor pressure and plant growth is impaired. Metabolism is also profoundly affected. General protein synthesis is inhibited and significant increases in certain amino acid pools, such as proline, become apparent (Barnett Naylor 1966). During these water deficit periods, photosynthetic rate decreases with the ultimate result of loss in yield (Boyer, 1976). If carried to an extreme, severe water deficits result in death of the plant.

Many studies have attempted to unravel the complex array of physiological and morphological changes that occur during water deficits. Abscisic acid (ABA) is known to regulate stomata opening and perhaps signal other responses (Milborr-w, 1981). Comparison of drought-resistant and drought-sensitive lines of Zea mays indicate that higher ABA levels are correlated with resistance. In addition, ABA-insensitive mutants (Koorneef et al., 1984; Finkelstein Somerville 1990) and ABA-deficient mutants (Koorneef et al., 1982) of Arabidopsis are prone to wilting. Other biochemical differences have been implicated in drought tolerance.

Quaternary ammonium compounds, such as glycine-betaine, have been implicated (McCue Hanson, 1990), as has proline which is thought to play an important role in osmoregulation in plants (Delauney Verma, 1993).

Moreover, drought-resistant maize lines have been shown to contain thicker veins, larger epidermal cells, and a greater vessel cross sectional area per vascular bundle than drought sensitive lines (Ristic Cass, 1991).

These differences at the cellular level may indicate that resistant plants have a greater potential to transport water which may allow faster adaptation to, and recovery from, water deficits.

i i j i i E:i i i, :i WO 95/30005 PCT/US95/05366 3 The molecular biology of osmoregulation is less understood. Genes involved in seed desiccation have been implicated in this process. For instance, LEA (late embryogenic abundant) and RAB (responsive to abscisic acid) genes are induced by water stress or by exogenous application of abscisic acid (ABA) (Dure et al., 1989; Skriver Mundy, 1990). The LEA and RAB proteins are highly conserved and have been found in several plant species. These proteins contain conserved amino acid sequence motifs that are thought to form amphiphilic alpha helical structures which may have a functional role in preventing cellular damage during desiccation.

Cis-acting sequence motifs, ABREs (ABA responsive elements) have been identified in RAB promoters which are involved in transcriptional activation in response to ABA (Mundy et al., 1990). While these studies suggest an association of specific genes with drought and desiccation, further direct evidence is needed.

A genetic approach to dissecting developmental and physiological process is beginning to revolutionize our understanding of plant biology. Using mutagens, genes required for plant processes can be identified and their function inferred by phenotype. A good example of this application is the current understanding of floral development made possible by mutational analysis of floral homeotic genes in Arabidopsis and Antirrhinum (Coen Meyerowitz, 1991; Coen, 1991). This general approach is now being extended to studies of many different plant processes including environmental signal transduction pathways.

A mutational approach has a distinct advantage over conventional physiological and molecular studies. Rather than a descriptive approach, mutations are used to define genes that are functionally implicated in this process based on mutant phenotypes. By relying on phenotypes, 1 l

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t^ WO 95/30005 PCTUS95/05366 characterizing genes or gene products that are simply correlated with the process can be avoided. Moreover, by characterizing mutant phenotypes at the physiological and cellular levels, it is possible select those that are potentially more interesting. This provides a direct link between genes and biological responses at the cellular and whole plant levels.

Three mutants have been identified in maize which demonstrate the wilt phenotype. Plants demonstrating the wilt phenotype show loss of turgidity in leaf tissues, accompanied by drooping and sometimes shrivelling of the leaf. wil is a recessive mutant mapped to chromosome 6L and characterized by chronic wilting (Postlethwait Nelson, 1957). Leaves are not as cool as normal and there is delayed differentiation of the metaxylem vessels. Wi2 and Wi3 are dominant mutations that demonstrate wilting of the top leaves under moisture and temperature stress (Neuffer, 1989; Neuffer, 1990). Wi2 maps to chromosome 3. The Wi3 mutation is nonallelic to Wi2 and probably unlinked, but is not mapped.

Mutations, however, are only one component of an approach to understanding wilt-related genes. Besides the requisite physiological and biochemical techniques, cloned genes are needed for functional studies of genes and the products they encode. In complex genomes, such as maize, chemical mutagens can be effective at defining loci but these alleles have little value in obtaining molecular probes. Nevertheless, both goals (mutant alleles and cloned genes) can be achieved using non-conventional mutagenesis techniques such as gene tagging methods. Maize, in particular, is well suited for such studies given the dearth of well-characterized transposable element systems and genetic strategies for gene tagging (Chomet, 1994; Dellaporta Moreno, 1994; Cone, 1994). Transposable elements have enabled the I r l i: WO 95/30005 PCTIUS95/05366 isolation and characterization of numerous genes in several biosynthetic pathways, including, anthocyanins (Fedoroff et al., 1984; Cone et al., 1986; Dellaporta et al., 1988; O'Reilly et al., 1985; Wienand et al., 1986; McLaughlin Walbot, 1987), carotenoids (Buckner et al., 1990), carbohydrate metabolism (Sullivan et al., 1991), and the regulation seed storage protein synthesis (Schmidt et al., 1987) to name a few. Moreover, genes involved in disease resistance (Johal Briggs, 1992) and in plant development (Hake et al., 1989; Martienssen et al., 1989; DeLong et al., 1993) have also been isolated by gene tagging strategies in maize.

Maize is an ideal organism for these studies for many reasons. Maize is a model genetic system for plant studies because of well developed technologies for formal genetic and molecular analyses (Freeling Walbot, 1994).

Because maize is an important agricultural plant, the results obtained will have direct relevance to an important agricultural problem.

The identification and characterization of plants that are resistance to water deprivation has long been sought. However, the identification of genes that are directly involved in resistance to water stress has been lacking. The isolation and characterization of maize genes that provide for an improved resistance to water stress have the potential for the long term improvement in and sustainability of agriculture world-wide.

SUMMARY OF THE INVENTXON The present invention, in a general and overall sense, concerns plant genes, and particularly maize genes, that are involved in the maintenance of water relations in a plant. Such genes are herein termed wilt llC91i ii-_-C1~ -6genes (wilt), due to the fact that disruption of these genes impairs a plant's normal balance of turgor potential and hence causes the plant to wilt.

A wilt gene in accordance with the invention is a gene that, when present within a plant, allows the plant to maintain proper internal water balance under normal conditions or under low water availability or contributes to such a response and a drought-resistance phenotype. It is envisioned that a wilt gene will encode one or more proteins or polypeptides that function in the mechanisms of drought-resistance or that stimulate the production of, or regulate, other proteins involved in drought-resistance. A ready means to assess whether a gene or cDNA falls within the wilt gene group is to disrupt, inactivate or otherwise mutate the gene, and to assess whether plants bearing such a disrupted gene wilt under normal water level conditions. The precise S mechanisms by which a wilt gene achieves its phenotypic effect are not S relevant to the practice of the invention, given that the isolation and various c.1 b uses of are wilt genes is disclosed herein.

The invention provides, in certain embodiments, a purified maize wilt gene DNA segment, wherein said wilt gene is defined as a gene that, when

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present in a plant, allows the plant to maintain proper internal water balance under normal water conditions or under low water availability or contributes to such a response and a drought-resistance phenotype and is a wild type allele of wilt-1256.

C As used herein, the term "DNA segment" refers to a DNA molecule that has been isolated free of total genomic DNA of a given plant species, particularly maize. Therefore, a purified maize wilt gene DNA segment refers to a DNA segment that contains maize wilt gene coding sequences yet is isolated away from, or purified free from, total genomic DNA of Zea mays. Included within the term "DNA segment", are DNA segments and smaller fragments of such segments, and also recombinant vectors, including, for example, plasmids, cosmids, phage, viruses, and the like.

S10o/7/98LP8963.SPE,6 WO 95/30005 PCTUS95/05366 7 The term "gene" is used herein for simplicity to refer to DNA coding unit that encodes a functional protein, polypeptide or peptide, which coding unit is isolated substantially away from other genes or protein encoding sequences. As will be urderstood by those in the art, this functional term includes both genomic sequences and cDNA sequences. "Isolated substantially away from other coding sequences" means that the gene of interest, in this case a wilt gene, forms the significant part of the coding region of the DNA segment, and that the DNA segment does not contain large portions around 40-50 kb or larger) of naturally-occurring coding DNA, such as large chromosomal fragments or other functional genes or cDNA coding regions. Of course, this refers to the DNA segment as originally isolated, and does not exclude genes or coding regions later added to the segment by the hand of man.

A particular example of a DNA segment of the invention is that termed wilt-1256. Wilt-1256 is a 18 kilobase Sal I fragment isolatable by the methods disclosed herein in Examples I through III.

Recombinant vectors that include a wilt gene DNA segment form important further aspects of the present invention. Particularly useful vectors are contemplated to be those vectors in which the wilt DNA segment is positioned under the control of a promoter. In preferred embodiments, the promoter will be one that is operable in maize plants. As used herein, the'term "operable in maize plants" is used to describe a promoter that is capable of directing the expression of a gene or transcription unit in a maize cell or maize plant.

However, the use of other promoters not operable in maize is also contemplated, such as may be employed in directing the expression of a recombinant protein in a bacterial host cell, as is commonly performed in the art..

i) WO 95/30005 PCTUS9505366 -8- A preferred embodiment of the present invention is the use of a promoter that is operable in maize plants, with the promoter that is naturally associated with a wilt gene in Zea mays being particularly preferred. One may obtain such a promoter by isolating the 5' non-coding a sequences located upstream of a transcribed coding segment or exon, for example, using recombinant cloning and/or PCR technology, in connection with the compositions disclosed herein, such as the 18 kb Sal I fragment including wilt-1256. Also encompassed within naturally occurring promoters are regions such as enhancers, silencers and transcriptional activation sequences that may also be used, and are encompassed by the present invention.

In other embodiments, it is contemplated that certain advantages will be gained by positioning the coding DNA segment under the control of a recombinant, or heterologous, promoter. As used herein, a recombinant or heterologous promoter is intended to refer to a promoter that is not normally associated with a wilt gene in its natural environment. Such promoters may include promoters normally associated with other maize genes, and/or promoters isolated from any other bacterial, viral, eukaryotic, or even a mammalian cell.

Naturally, it will be important to employ a promoter that effectively directs the expression of the DNA segment in the cell type chosen for expression. In terms of expression in maize cells or plants, one may mention, by way of example, promoters such as the Cauliflower Mosaic Virus 355, rice actin, rbcS, c-tubulin and ocs promoters. The use of promoter and cell type combinations for protein expression is generally known to those of skill in the art of molecular biology, for example, see Sambrook et al. (1989). The promoters employed may be constitutive, or inducible, and can be -t WO 95/30005 PCTJUS95/05366 -9used under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous for inducing an increased resistance to water deprivation in a plant or simply to provide high level production of recombinant protein.

Further embodiments of the invention concern methods for preparing a wilt gene DNA segment. Certain preferred methods are those involving a technique known in the art as transposon tagging. To obtain a wilt gene DNA segment in this manner one would generally, first, obtain a collection of maize plants bearing transposon induced mutations and screen the group of plants to identify one or more plants that exhibit a wilt phenotype. The process of obtaining or preparing a collection of maize plants bearing transposon induced mutations involved identifying plants that harbor an active transposon system. With some transposon systems activator) it is possible to select individual plants in which the transposon has moved to different locations in the genome (Chomet, 1994; Dellaporta Moreno, 1994, Cone, 1994, incorporated herein by reference). Such plants are then either selfed or crossed to plants carrying a previously identified wilt mutation. The selfed progeny or test crossed progeny are then screened for the wilt phenotype.

Plants that exhibit the wilt phenotype are readily identifiable by simply growing the collection, or population, of plants under normal water levels and detecting those plants that exhibit decreased turgor, visibly wilt, leaf roll, or droop, under such conditions or by identifying those plants that show significantly decreased turgor in comparison to other plants in the collection. The precise mechanisms by which the mutations that affect water balance achieves the identified phenotypic effects are not relevant to the practice of the invention, it is clear, however, that i WO 95/30005 PCTUS95/05366 10 leaf roll precedes wilting. Leaf roll was effective in providing an early indication of wilting, for example, as early as the third leaf stage.

After identifying one or more plants that exhibit a wilt phenotype one would next prepare a genomic library from such a plant or plants. That is, one would obtain DNA from the nuclei of cells from such a plant and formulate the DNA so that it contains smaller, distinct pieces of DNA that can identified as distinct from other pieces using any one of a variety of structural and functional characteristics. The library of DNA thus formed would than be screened to identify a genomic clone that comprises the wilt gene in association with a transposon. This screening process will generally involve using a probe directed to a transposon sequence to identify the transposon in combination with the wilt gene. A number of transposons that induce mutations may be used with the present invention, with the Ac and Ds transposons being particularly preferred.

Further advantageous procedures that may be used in connection with the preparation of wilt gene DNA segments are those that concern isolating a wilt gene cDNA clone.

Such methods generally comprise identifying a DNA sequence that flanks the transposable element located within the genomic clone first isolated and using the flanking sequence in another round of molecular screening. In particular, the flanking sequence would be employed to screen a cDNA library prepared from a wild type plant and, by identifying sequences that hybridize, to thus identify one or cDNA clones that include a wilt gene. Suitable methods for preparing and screening cDNA libraries will be generally known to those of skill in the art of molecular biology in light of the present disclosure and published references such as, for example, Sambrook et al. (1989; incorporated herein by reference).

WO 95/30005 PCTIUS95/05366 11 Yet further aspects of the present invention concern plants, particularly maize plants, that have been stably transformed with a purified wilt gene DNA segment, such as the wilt-1256 18 kb DNA segment. In preferred embodiments, the stably transformed plants will include a recombinant vector that comprises a wilt gene that is positioned under the control of a promoter that is operable in maize plants and will thus direct the expression of the wilt gene in the plant. Exemplary promoters that may be used in this regard include the Cauliflower Mosaic Virus 35S, rice actin, rbcS, a-tubulin and ocs promoters.

The present invention thus also provides methods for preparing plants, particularly maize plants, that have increased turgor, exhibit an increased resistance to water deprivation or drought. Such methods involve stably introducing a purified wilt gene DNA segment, such as the 18 kb wilt-1256 DNA segment, into the genome of a host plant in a manner %.ffective to result in expression of the wilt gene. In general, the gene will be introduced in the form of a recombinant vector and will be under the control of a promoter operable in maize plants. Any means of introducing the DNA segment into the host plant may be employed including, for example, electroporation and microprojectile bombardment.

Another embodiment of the present invention is a method for the selective breeding of plants that are resistant to water deprivation. The method includes, generally, obtaining a collection of maize plants and screening for increased expression, copy number, or message stability of a wilt gene DNA segment. Those isolates having a favorable genotype, that is an increase in expression, copy number or message stability are tested for increased resistance to water deprivation followed by breeding of those isolates having increased i 1 le Commissioner or raents WO 95/30005 PCT/US95/05366 12 resistance to water deprivation. The progeny are then screened with a wilt gene DNA segment in accordance with the present invention, and the favorable genotypes are tested resistance to water deprivation and once again bred.

The DNA segments of the present invention encompass biologically functional equivalent wilt genes and their corresponding proteins and peptides. Such sequences may arise as a consequence of codon redundancy and functional equivalency that are known to occur naturally within nucleic acid sequences and the proteins thus encoded.

Alternatively, functionally equivalent proteins or peptides may be created via the application of recombinant DNA technology, in which changes in the protein structure may be engineered, based on considerations of the properties of the amino acids-being exchanged. Changes designed by man may be introduced through the application of site-directed mutagenesis techniques, to introduce improvements to the stability or specific expression of the resultant protein or to test wilt mutants in order to examine the mechanisms underlying wilting activity at the molecular level.

BRIEF DESCRIPTION OF THE DRAWINGS The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIG. 1. Restriction enzyme digestion map of X5Sal clone containing the Ac transposable element inserted in the WO 95/30005 PCT/US95/05366 13 wilt-1256 gene. The activator is indicated by the crosshatched line. The hybridization probes for (0.9H/E) and flanking genomic DNA probes (0.5 and 0.4B/S) are shown. A portion of the DNA sequence of the genomic DNA flanking the Ac insertion in wilt-1256 has been determined. This sequence is a 247 base pair open reading frame which has shown homology to 2 rice cDNA sequences of unknown function. S=SalI, B=BamHI, E=EcoRI, H=HindIII, P=PstI.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS A. Screening for Ac-induced mutations Transposon tagging has several benefits over conventional gene cloning strategies. No prior knowledge of the gene product or even previous identification of the gene is a prerequisite for transposon tagging. It can be used to clone and study genes whose products are rarely expressed. Insertional mutations often completely or partially block gene function, resulting in phenotypic alterations. This phenotypic alteration often implicates the gene or its product in a particular developmental or biochemical pathway. Transposon tagging strategies can operate efficiently in organisms with large genomes, even with great amounts of repetitive DNA. Genes for disease resistance, nutritional qualities, and developmental processes can thus be tagged and cloned. Mutant alleles can also serve as the basis for saturation mutagenesis and fine structure analysis of complex loci.

An essential feature for developing an efficient transposon tagging system is the ability to efficiently recover transposition events. Two genetic features of Ac transposition were exploited to develop an efficient recovery strategy. First, because transposition is a j WO 95/30005 PCTIUS95/05366 14 coupled to DNA replication, transposed Ac elements are recovered within cell lineages that contain an both empty and filled donor site (see Chen et al., 1990, 1992). The inventors' strategy relies on detecting the lineage containing the filled donor site and tr-Ac element. This lineage contains two Ac elements (the original donor Ac and the transposed Ac element). The increase in Ac copy number results in a dosage effect transposition is developmentally delayed. We exploited this second feature (Ac dosage effect) by using a Ds-induced reporter allele (r-sc:m3). In practice, any Ds reporter allele will suffice, however. By monitoring the developmental pattern of Ds transposition early verses late), we have the ability to select progeny that contain more than one Ac. Since the parent line carries only one Ac (donor element), progeny kernels showing a late transposition pattern will carry not only the original Ac element but also a transposed element.

In these studies, Ac elements located at two chromosomal positions were used to assess the feasibility of our strategy to recover transpositions. The donor Ac elements inserted into the P gene on chromosome IS and the liguleless-l gene on chromosome 2 were employed for these purposes. Females homozygous for the Ac-induced mutations were crossed to males homozygous for the Ds reporter allele, r-sc:m3. All Fl progeny are expected to receive a single Ac (donor element) and the r-sc:m3 reporter allele except in cases where transposition of Ac had occurred. These kernels of the wilt-1270 mutation isolated from Ac-Ds tagging lines show the expected coarse variegated aleurone pattern typical of endosperms that contain two maternally-transmitted Ac elements. In cases where Ac transposition had occurred, two types of Fl kernels are expected: i) in cases where the donor element was lost, as in the wilt-1256 mutation isolated i from Ac-Ds tagging lines, a colorK"cv- aleurone phenotype i1 WO 95/30005 PCTUS95/05366 15 is observed, ii) in cases where the donor and transposed element is recovered, as in the wilt-1269 mutation isolated from Ac-Ds tagging lines, the increase in Ac dosage results in very late transactivation of the rsc:m3 reporter allele which gives a near colorless or very finely spotted aleurone. The inventors recovered approximately 10,000 kernels containing tr-Ac elements (Ml kernels) by using the Ds reporter system to recover high dose Ac kernel progeny. The efficiency of recover, based on F2 and testcross data, was greater than 75%. In control populations (coarse variegated sib kernels) the efficiency of transposed Ac recover was less than This indicates that the genetic strategy for recovering transposed Ac elements greatly improves the frequency of recovering transposed elements over non-selected methods.

The clear advantage of this method over other schemes is that this strategy can be used with any Ac element in the maize genome regardless of its chromosomal position.

The Ml kernel population was screened four times.

First, M1 kernel populations and control siblings were field grown and screened for dominant mutations.

Mutations involved in the flowering process such as early or late flowering phenotypes, floral morphological transformations, male and female sterility and mutations affecting vegetative characters such as ligule and leaf development, or tillering were sought. All M1 and control plants were self-pollinated to generate M2 and F2 control progeny populations. Mutants were also testcrossed to Ac tester stocks to identify Ac-linked phenotypes.

The second screen was for mutations affecting seed characteristics such as endosperm morphology and defective kernels. This screen was done directly on the M2 and F2 ears. For instance, mutants in starch, protein, and carotenoid pathways were detected in

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nf WO 95/30005 PCTJUS95/05366 16 endosperm cells. From this screen, mutations affecting kernel development (defective and embryoless kernels) were recovered. Several mutations affecting starch biosynthesis (shrunken (sh) and brittle were also recovered.

The third screen involved the growing the first seed set in sand benches to score for percent germination and aberrant seedling phenotypes such as chlorophyll pigmentation changes and disease susceptibility. Two sets of twenty kernels were selected randomly from each M2 and F2 family. From this screen many mutations affecting seedling development (lethals, pale-greens, albinos, yellow-greens, dwarfs, high chlorophyll fluorescence, disease lesion mimics, and the like) were recovered.

The fourth screen involved growing the second seed set in field plots and scoring for segregation of vegetative and floral phenotypes. From this screen mutations affecting leaf morphology (liguleless, wilts, knotted, etc.) and inflorescence morphology (tasselseeds) were recovered.

Selected mutations were studied by genetic and molecular analysis. For example, some M1 and M2 mutants were testcrossed to a set of Ac tester lines and a genetic segregation analysis between the mutant phenotype and the transposed Ac element was determined. These tests identified which mutants were segregating with tr-Ac activity and putatively Ac-induced. Southern blot analysis was performed on selected mutant families to identify tr-Ac elements for cloning purposes.

EL i- ui e h i- -CtCrS 1J II i i WO 95/30005 PCTUS95/05366 17 B. Transposon Tagging with Mutator Transposable elements have been useful in the identification and cloning of genes that were previously inaccessible by other cloning methods. In maize, three transposable element systems have been utilized for transposon tagging: Activator/Dissociation, Suppressormutator (Enhancer/Inhibitor), and Robertson's Mutator.

Each system has inherent advantages and disadvantages,and the choice of system depends on the tagging approach taken, the genomic position and the expression of the gene sought, as well as the stocks available for tagging.

The Mutator transposable element family was originally identified in lines that exhibited an unusually high frequency of forward mutation (Robertson, 1978). Extensive genetic and molecular analyses have demonstrated that the increase in mutation frequency is caused by a family of transposable elements, designated Mutator (Mu) elements. The Mutator system consists of more than eight different classes of Mu transposable elements each of which can be found in multiple copies.

Each element class is defined by a unique internal sequence flanked by inverted repeats about 200 bp long common to all Mu elements. Germinal transposition (and presumably forward mutation) and somatic excision of the non-autonomous Mu elements are under the control of the autonomous MuRI element (Chomet et al., 1991), now designated MuDR-1. Other Mu elements have been cloned that are similar or identical to MuDR-1 in sequence; these were called MuA2 (Qin et al., 1991) and Mu9 (Hershberger et al., 1991), and are now called MuDR transposons. Lines harboring the autonomous element(s) are referred to as Active Mutator lines. The Mu system has been useful in cloning a number of genes, including al (O'Reilly et al., 1985), bz2 (McLaughlin Walbot, seuec flne byivre rpasaot 0 pln commo toalM lmns emnltasoiin(n presumably___. fowr uain)adsmtcexiino h I WO 95/30005 PCT/US95/05366 18 1987), vpl (McCarty et al., 1989), hcfl06 (Martienssen et al., 1989), yl (Buckner et al., 1990), and hml.

C. The Mutator System for Tagging One of the advantages of Mu over the Ac/Ds and the Spm families lies in its non-localized mutagenic action.

Ac and Spm often transpose to linked regions of the chromosome (Greenblatt Brink, 1962; Dooner Belachew, 1989; Nowick Peterson, 1981). Mu lines exhibit an increase in mutation frequency for all loci examined (Robertson, 1978; 1983). This does not prove that Mu elements readily transpose to unliked sites although recent evidence suggests this is the case (Lioch, Choret Freeling, 1995).

D. Targeted and Nontargeted Approaches to Tagging Two approaches to obtain mutations can be pursued dependent on the needs of the investigator. With the targeted (or directed) method the goal is to recover insertions in a previously identified locus. This method presumes a mutant alleged of the target locus is available in a tester line. In such a case, homozygous wild-type Mutator stocks for the target gene are crossed to homozygous tester lines. The F, progeny are then screened for the mutant phenotype. The reported Sfrequency of mutation for such an approach varies from 3 to 104 (Robertson, 1985; Walbot et al., 1986; Patterson et al., 1991; Brown et al., 1989). Therefore, if possible, at least l05 gametes should be screened for a directed tagging study. Furthermore, it is important to have prior knowledge of the spontaneous mutation rate for the targeted locus. Some loci can be highly unstable in the absence of a transposable element system (Pryor, 1987; Stadler, 1948).

I i 1 i WO 95/30005 PCT/US95/05366 19 In the case of directed tagging of dominant loci, it is possible to "knock out" the dominant mutant allele with Mu (Hake, 1992). In this case, a Mutator line homozygous for the dominant allele is crossed (as a female) to a wild-type tester. The F, is screened for phenotypically wild-type plants. This presumes a deletion heterozygote for the given locus is phenotypically wild-type. Such an assumption may not always be the case and tests should be undertaken to determine the phenotype of the hemizygote before proceeding with the screening of the Fi population.

The nontargeted approach allows the investigator to identify new, uncharacterized mutations as well as to identify new mutations with previously characterized phenotypes. Active Mutator lines should be crossed by a non-Mutator line (inbred) or a tester line carrying a Muinduced unstable marker allele. (See non-autonomous genetic marker, below). The FI seed is selfed to produce

F

2 populations; 20-40 seeds from each F 2 ear are then screened for the mutant phenotype(s). In this case, lethal or sterile phenotypes can be identified as homozygotes and recovered as heterozygotes in the population.

E. Monitoring Mutator Activity Before making the first cross to generate either an F, population or the selfed populations, it is best to determine which plants in the Mutator stock carry Mutator activity and are mutagenic. This information can be obtained wholly or partially by a number of tests outlined below. Because each assay measures a different aspect of Mu transposition, each test does not necessarily match results from another test for Mutator activity.

1 I WO 95/30005 PCT/US95/05366 20 F. Robertson's Mutator Test Robertson devised an assay for Mutator activity that give an estimate of the forward mutation frequency of Mutator plants (Robertson, 1978). The test is performed by selfing and crossing the Mutator plant as a male to a hybrid non-Mutator line to produce FI seed. The second ear on the non-Mutator plant is selfed as well. F, seed, from plants which did not segregate for a visible mutant (as determined by the parental selfs), are sown and plants are selfed to produce F 2 ears. Kernels from these

F

2 ears are planted in a sand bench and observed for new seedling traits albino, yellow, yellow-green, etc.). With active Mutator lines, Robertson reported approximately 10% of the F 2 families exhibited a new seedling mutation (1980).

G. Non-autonomous Genetic Marker(s) Non-autonomous Mu insertion alleles report on the presence or absence of MuDR, the regulator element i (Chomet et al., 1991). The presence or absence of spotting of a Mu-induced insertion allele reports only on the somatic reversion of one Mu element (such as Mul as bzlmum9, or Mul at al-mum2). This is in contrast to the Robertson test, which can measure th forward mutation or insertion of diverse Mu into many different loci. Since these two assays do not measure the same event, use of one assay is not always a substitute for another.

However, there is a correlation between an increase in forward mutation rate with lines containing multiple regulator elements that exhibit a high frequency of spotting. Plants that segregate for one MuDR-1 (position 1) usually do not exhibit a high forward mutation rate (Robertson Stinard, 1989). When using a mutable Mu allele as a marker for MU activity, it is best to propagate the high spotting pattern-. This will likely I i I 1 WO 95/30005 PCT/US95/05366 21 select for an increased number of MuDR-1 elements in the stock (Chomet et al., 1991).

H. Molecular Markers A number of studies have demonstrated a correlation between loss of Mutator activity and methylation of Mul at a number of methylation-sensitive restriction enzyme sites including Hinfl (Chandler Walbot, 1986; Bennetzen, 1987). Hinfl sites are located in the ends of Mul and produce a 1.3-kb fragment upon cleavage. To determine the status of Mul methylation and the associated Mutator activity, the DNA from Mu lines is cleaved with Hinfl and hybridized to a Mul internal probe by Southern blot analysis. The predominance of a 1.3-kb fragment indicates cleavage, whereas the presence of a ladder of fragments (and a reduced amount or lack of a 1.3-kb fragment) indicates Hinfl sites in the ends of Mul are methylated and Mu activity is probably absent. It should be noted that this correlation is not absolute (Bennetzen, 1987; Bennetzen et al., 1988).

The generation of unique Mul-homologous fragments, not detected in parent plants, has been correlated with Mutator activity (Alleman Freeling, 1986; Bennetzen et al., 1987). To detect new Mul elements, DNA from parent and progeny of a Mutator lineage should be cleaved with an enzyme that does not cut within Mul such as SEcoRl, BamH1, or HindIII. Southern blot analysis with the Mul probe will reveal a series of Mul-hybridizing fragments that represent Mul at unique genomic locations.

Mul fragments not found int he parent most likely represent new insertion events, indicating the presence of Mutator activity.

N I 'ii WO 95/30005 PCT/US95/05366 22 I. Crosses with Newly Arisen Mutants Once a mutant is iden.tified, it is useful to have crosses to a number of different lines. Therefore, it is wise to plant a number of tester lines along with the population to be screened.

1. If possible, selfing, to recover homozygotes, is important. A homozygous stock can be utilized to screen for germinal reversion events; revertants are a useful tool in molecular identification of a tagged locus.

2. Outcrossing the mutation to a few different non- Mutator (inbred) lines will be useful for subsequent molecular analyses. Identifying the original, tagged locus of interest will be important and can be accomplished because RFLPs associated with the locus can be associated within a given line.

Crossing to different lines will also allow the production of F 2 populations segregating for the mutant allele. These populations are necessary for subsequent molecular analyses. Furthermore, introduction of the mutant allele into a number of different lines will also insure expression and subsequent recovery of the mutant in later generations because expressivity or penetrance of the mutant can be affected by genetic background.

3. If the Mutator line is recessive for Al, Cl, Bzl or Bz2 (or other anthocyanin loci), it is advantageous to outcross the mutant to a line lacking Mutator activity and homozygous for a Mu-induced allele of the same gene (such as bz-mum9, or aal-mum2). This allows selection in the F 1 generation of seed that carried or lacked Mutator activity. These seed can then be grown, selfed, and screened for the mutant WO 95/30005 PCTIUS95/05366 23 phenotype. Production of populations segregating for the mutation of interest and lacking Mutator activity facilitates molecular analyses. New Mu fragments are not generated, and existing, unliked Mu fragments segregate out of the line with subsequent outcrosses. It is important to point out that some Mu-induced alleles are suppressible; that is, in the absence of Mu activity the phenotype is wild type (Martienssen et al., 1990). In such a case, selection of inactivity selects for phenotypically wild-type plants.

J. DNA Analysis Identifying the gene responsible for the mutant phenotype involves screening for a Mu-homologous fragment that cosegregates with the mutant phenotype. This is done by examining the DNA of the segregating population(s) (as produced above) by Southern blot analyses. The preliminary screen is expedited by examining a small population first. As many different outcrossed segregating lines should be examined as possible (See, Walbot, 1992). It is also useful to examine the population utilizing a number of different restriction enzymes, since segregating fragments may be obscured by other Mu homologous bands. The population should also be screened with probes to all known Mu elements. Inclusion of DNA from t he parent lines on these blots is also important. A cosegregating fragment should not be present in the parental plant. Once a cosegregating fragment is identified, additional analyses with different populations and a larger population set should be performed. Furthermore, as noted above, Muinduced suppressible alleles can confound the cosegregation analysis (Martienssen et al., 1989; 1990).

For this reason, it is important to emphasize linkage of WO 95/30005 PCTIUS95/05366 24 a fragment with mutant individuals may be genotypically mutant.

Once a cosegregating band is identified, cloning or PCR is used to obtain a flanking, unique sequence. This flanking probe is then used to prove the locus is responsible for the mutant phenotype. This can be accomplished in a variety of ways: 1. Identification of DNA rearrangements, insertions, or deletions at the locus of independently generated alleles (O'Reilly et al., 1985) demonstrated the clone is (or is nearby) the locus of interest.

Multiple alleles generated within the same tagging study can be used for this purpose.

2. The correlation of the loss of the transposon from the locus in garminal revertant alleles or within somatic sectors of revertant tissue also indicates the cloned locus is responsible for the phenotype.

The generation of stocks homozygous for the mutation and subsequent analyses (see above) is useful here.

Mu elements transpose germinally from a locus at a very low frequency as compared to other transposon systems. It may soon be possible to utilize an early reverting line identified by V. Walbot (19I1; 1992) to increase the frequency of germinal reversion. It has been shown that a high frequency of germinal revertants of bz2-Pu2 are recovered in this line, but work with other Mu-ii.duced alleles is necessary before its generalized use can be established.

3. Differential RNA hybridization can facilitate identification of the correct clone in special cases where the expression of the locus is well understood, as was done for Bz2 (McLaughlin Sj t f i__I iA l, i 7 "1 WO 95/30005 PCT/US95/05366 25 Walbot, 1987). Hybridization were performed with the putative clones to RNA isolated from various tissues or allelic variants that showed a predicted pattern of expression.

The overall goal of these studies was to understand the fundamental mechanisms by which plants respond to and protect themselves from water deficits. The approach was to dissect the components of this response process by isolating mutations that fail to respond normally and which showed spontaneous symptoms of water deficit (classified as wilt mutations). Several wilt genes were identified and one was cloned to provide molecular probes for functional studies. A formal genetic characterization of these mutations, the molecular biology of the genes and products they encode, and their regulation and expression is currently underway. These studies provide important information concerning the biochemistry, cell biology, genetics, and physiology of water stress in plants.

Even though the invention has been described with a certain degree of particularity, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art in light of the foregoing disclosure. Accordingly, it is intended that all such alternatives, modifications, and variations which fall within the spirit and the scope of the invention be embraced by the defined claims.

3 0 The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus 'I can be considered to constitute preferred modes for its WO 95/30005 PCTUS95/05366 26 practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLE I PRIMARY CHARACTERIZATION OF WILT MUTANTS Independent transpositions of Ac from a donor locus are selected by excision of Ac and its reinsertion elsewhere in the genome. Recovery of the transposed Ac element in the heterozygous condition is possible in kernel progeny using Ds-induced reporter genes. Using this strategy, and a slightly modified version, over 10,000 independent transpositions of Ac have been recovered and screened for mutations. Plants carrying transposed Ac elements are field-grown, screened for dominant mutations, and self-pollinated to uncover recessive Ac-induced mutations. The F2 progeny are screened for recessive mutations. By this approach, several hundred mutations have been identified in transposed Ac families.

In particular, four recessive mutations displaying a wilt phenotypt were identified under normal water deficit conditions (Table In addition to these four mutations, a fifth spontaneous allele (wiltl-l) is currently under study.

I ,j WO 95/30005 PCT/US95/05366 27 Table 1. Preliminary Characterizations of wilt Mutations ALLELE MAP DESIG. SOURCE POSITION PHENOTYPE wilt-1256 Ac-Ds iS early wilting, viable wilt-1269 Ac-Ds late wilting, viable wilt-1270, Ac-Ds IS early wilting, viable wilt-6945 wilt-113 Mutator early wilting, viable wilt-129 Mutator early wilting, viable wilt-222 Mutator early wilting, viable wilt-i spontaneous 6L late wilting, viable Two of the mutants, wilt-1256 and wilt-6945 were shown to be linked to the donor P locus (source of Ac) on chromosome 1S. Linkage of the wilt mutants to the donor P locus is expected, in some cases, since Ac usually transposes to nearby chromosomal sites. The genetic location of the other two mutations is unknown.

One of the more interesting aspects of the wilting phenotype is its developmental onset. For instance, the wilt-1256 mutant showed the first signs of leaf roll at the third leaf stage. Young expanding leaves were severely leaf rolled, yet older leaves appeared to recover and support growth of the plant. In contrast, wilt-1269 mutants did not show any visible signs of leaf roll until just prior to flowering. The wilt-i mutant (obtainable from the Maize Genetics Cooperation Stock Center, E.B. Patterson, S-118 Turner Hall, Agronomy Department, University of Illinois, 1102 S. Goodwin Avenue, Urbana, IL 61801) exhibited wilting in late vegetative growth. Plants homozygous for wilt-i reached maturity and produced seed, although plants were generally reduced in stature. This previously identified mutation (Postlethwait Nelson, 1957) has been mapped to chromosome 6L.

1 WO 95130005 PCT/US95/05366 28 Besides onset of the wilting phenotype, as identified by leaf roll, the mutations also differ in whether wilting was lethal to the plant. For example, wilt-1270 and wilt-6945 are seedling lethals, i.e., mutant plants grew to the 4-5 leaf stage, leaves wilted, growth ceased, and cell necrosis eventually killed the plant. However, this characteristic is not linked to the early wilting effect. For example, wilt-1270 and wilt- 694w were seedling lethals while wilt-1256 wilted during this same developmental period, yet recovered. It appeared that lethality was related to the inability of the plant to recover from the early onset of wilting. To date, both late wilting alleles (wiltl-l and wilt-1269) were viable mutations. Viable wilt mutants grew to maturity, flower, and were fertile, although yields were substantially reduced. The mutations were generally deleterious, mutant plants were smaller than wildtype and exhibited poor leaf expansion.

Anatomical studies of wilt-6945 leaves revealed no structural difference between mutant plants and normal siblings. The leaf turgor potential of wilt-6945 mutants was near zero. Thus, although the cellular morphology of wilt-6945 appears normal, it was unable to maintain sufficient leaf turgor. This aspect of the mutant appearz.d to be the primary lesion which was responsible for its lethal phenotype. The cellular characterization of other wilt mutations is currently under study.

K. Identification of additional mutations The identification of additional wilt mutations may be employed to define additional genes involved in the water stress response pathway. Fifteen additional wilt mutations were identified in Ac-Ds and Robertson's Mutator screens. All mutants are regrown to confirm Mendelian inheritance patterns and dominant, codominant,

I

ei WO 95/30005 PCTIUS95/05366 29 recessive relationships to wild-type. Pairwise complementation tests between each mutations is performed. In addition, a directed tagging study of wiltl-l using the transposable element Mutator is performed. The testcross population of over 100,000 kernels is screened to identify wilt-1 alleles.

Several criteria are established to decide the priority given to each mutation for characterization.

For example, exogenous ABA application may be used to identify mutants that can be rescued or identify mutants that show differential sensitivity. Standard genetic tests can be used to define potential mutants that are linked to transposed Ac or Mutator transposable elements (Dellaporta Moreno, 1994). Complementation analysis determines the number of independent loci that mutate to give the wilt phenotype. Based on this information, selected mutations are characterized further and cloned.

For mutations that do not show linkage to transposable elements, the loci are mapped by bulk segregant analysis (Michelmore et al., 1991).

Wilt-1270, another Ac-derived wilt phenotype mutant is mapped in this manner. Briefly, the mutant is outcrossed to a different genetic background B73) and Fl plants are self-pollinated. DNA from normal and mutant F2 plants are pooled and bulked DNA is hybridized with probes from known linkage groups. Unlinked probes hybridize with equal intensity (linkage equilibrium) to both bulked DNA samples while probes linked to the mutation show differential hybridization signals (linkage disequilibrium). These studies help map genes on the maize genome involved in the response to water stress which may be important for future studies.

(I

WO 95/30005 PCT/US95/05366 30 EXAMPLE II MOLECULAR CHARACTERIZATION OF WILT MUTATIONS Although transposed Ac elements were selected in this program to isolate wilt mutations, the mutations were not always Ac-induced. Many of the mutations were shown to be caused by lesions other than Ac. Therefore, genetic and molecular tests were necessary to determine which mutations are Ac-induced to define those alleles useful for gene cloning. Confirmation of the linkage of Ac and wilt mutations is provided by reversion analysis.

wilt plants are screened for wild type, non-wilting, revertants. A reversion corresponding to excision of Ac from the wilt gene confirms linkage of Ac and wilt. Four recessive wilt mutations were screened using Southern hybridization for linkage of the wilt phenotype with a transposed Ac element. In the case of wilt-1256, linkage between the mutant allele and Ac is complete. DNA was isolated from sibling plants obtained from a selfed wilt- 1256 heterozygote and digested with Eco RI or separately with Sal I for Southern Blot analysis. In each analysis, this digested DNA from wilted plants and wild-type siblings was probed with an 0.4 Kb Bam HI-Sal I wilt gene fragment or separately with an Ac 0.9 Kb EcoRI-HindIII 25 fragment. The data from the analysis using an Ac 0.9 Kb EcoRI-HindIII probe showed that the wilt-1256 fragment co-segregated with an 18 kb Sal I fragment and 15 kb EcoRI restriction fragment which hybridized to an Ac probe. Both bands represented the Ac elements found in this line. These bands appeared in all of the mutants (total of 74 DNAs analyzed to date) and segregated, as expected, in wild-type siblings (2/3 of the wild type plants were expected to be heterozygous for the mutation and to carry these fragments).

The wilt-1256 gene is cloned according to the procedures disclosed by Federoff (Federoff et al., 1984; i. -B c"

L

jl

II

WO 95/30005 PCT/US95/05366 31 Federoff, 1988; both references incorporated herein by reference). A Sal I genomic library was constructed in EMBL3 using DNA isolated from a mutant plant. One clone was isolated which contained the Ac-hybridizing 18 kb Sal I genomic fragment (X5Sal, FIG. This clone was shown by restriction mapping to contain a transposed Ac element. Unique probes were isolated from DNA flanking the Ac element of this clone (shown in FIG. 1 as 0.4 B/S) and used to probe genomic blots. These probes detected only the expected 15 kb EcoRI fragment and 18 kb Sal I fragment in mutant plants. As expected, some normal plants also contained the mutant band and a wild type allele, while some have only wild-type alleles. The flanking DNA detects a transcript in maize shoots and leaves. This probe was used to isolate seven cDNA clones from a cDNA library constructed from shoot mRNA. These cDNA clones have been named XW1, XW4, XW7, XW8, XW9, and XW11. cDNA clones are verified to contain the wilt-1256 gene by hybridization to X5Sal. Only those clones containing the wilt gene will hybridize to In summary, the wilt-1256 mutation was tightly linked to a transposed Ac element and a genomic clone, X5Sal (FIG.

was obtained that contained flanking DNA that represented unique transcribed sequences.

EXAMPLE III WILT GENE CHARACTERIZATION A. Genetic and molecular confirmation of cloned sequences Further genetic and molecular evidence is obtained to demonstrate that cloned sequences do indeed correspond to the wilt-1256 locus. Complementation studies with existing wilt mutations are used to determine whether additional wilt-1256 alleles have been generated. Since all of the mutants are generated in near isogenic Y* L.

WO 95/30005 PCT/US95/05366 -32backgrounds, progenitor wild-type alleles are available for analysis. Progenitor and mutant alleles are analyzed by Southern hybridization using wilt-1256 sequences. If these probes detect additional rearrangements (e.g.

insertions) this provides further evidence that cloned sequences represent wilt-1256 DNA. The second criteria used to establish identity is the analysis of revertant alleles detected as Ac excision events from the wilt-1256 allele. Phenotypic reversion is accompanied by excision of Ac if the cloned sequence is indeed the wilt-1256 gene. Either criteria provides the necessary data to confirm the identity of the clone.

B. Sequence analysis cDNA clones of the wilt-1256 locus are analyzed.

Clones that hybridize to DNA probes adjacent to the Ac insertion are isolated from a cDNA library made from maize shoot apices (leaves and shoots). These clones are restriction mapped and hybridized back to genomic DNA to confirm their identity as wilt-1256 sequences. Part of this genomic DNA has been sequenced and determined to be an open reading frame as shown below: GANTCGATGG TGTNCCTGGT TCANTCGGTG ATCCCCGACA GGGGGCAGAA GACGCTGACN AAGTTCGTCA ACGGCGGGTC CANCAACGTN TTCTACGCGC ACGAGTACAA CGCGACGGTG GAGTTCTACT GGGCGCCCTT CCTGGTGGAG TCCAACTCCG ACAACCCCAA GGTTCACANC GTCCCCGACC GGATCATCCA GTGGCACGCC ATCNCCAANC ACGCGCNCAA CTGGATCGGC GTCGACA The position of this sequence is indicated in FIG.

1. The largest cDNA clones are sequenced and the predicted protein product determined. Sequence data of I

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pr;, WO 95/30005 PCT/US' .05366 I 33 the wilt-1256 gene and its predicted protein product are analyzed via computer algorithms such as BLAST (Altschul et al., 1990) and BLOCKS (Henikoff Henikoff, 1991) to search for the presence of functional domains in the wilt-1256 protein. This information may be useful in the design of future biochemical and physiological studies.

For example, it is contemplated that this analysis may suggest a regulatory DNA binding protein) or biochemical enzymatic activity) role of the wilt-1256 protein by its similarity to known proteins.

The wilt-1256 coding region is subcloned into E.

coli expression vectors for production of protein. The protein is tagged with a peptide (polyhistidine or maltose binding protein) for affinity purification.

Protein is purified by metal chelation chromatography using commercially available resins (Le Grice Grueninger-Leitch, 1990) or by maltose affinity techniques (Kellermann Ferenci, 1982; Guan et al., 1988). Polyclonal antibodies to the fusion protein are produced and used in immunolocalization studies.

C. Expression and regulation of the wilt-1256 gene The temporal and spatial expression pattern of the wilt-1256 gene is important in understanding the function of this gene. It is contemplated that one may want to know whether gene expression is constitutive or induced in leaf cells. By Northern hybridization analysis (Ausebel et al., 1992), the tissue-specific expression of mRNA (leaf, root, stem, inflorescence, etc.) is investigated. Polyclonal antibodies and Western analysis (Harlow Lane, 1988) are used to determine protein expression patterns. Both water-stressed, unstressed, and ABA-treated tissue are analyzed by Northern hybridization and Western analysis to determine whether induction occurs (RNA or protein). Detailed

I

ii__llL_ h L I i r I/ ~Lti-; -iir l ii L* WO 95/30005 PCT/US95/05366 34 characterization of the regulation of expression of wilt-1256 regulation is the result of these studies. For example, it is contemplated that Northern analysis will indicate whether steady-state mRNA levels are affected by water stress, yet Western analysis may indicate water deficits affect protein levels. This result would suggest regulation is post-transcriptional and would be further investigated. It is further contemplated that regulation of wilt-1256 activity may be at the level of RNA transcription or protein processing.

More detailed expression patterns are obtained by in situ hybridization techniques (Jackson, 1992; Freeling Walbot, 1994). Both riboprobes (sense and antisense) and antibodies (pre-immune and post-immune) are used to localize mRNA and proteins, respectively, in particular cell types. For example, if Northern and Western results indicate that wilt-1256 is only expressed in leaves, the cell-type specificity (mesophyll, bundle sheath, epidermal, veins, etc.) of wilt-1256 mRNA and protein localization are determined.

In sum, the molecular characterization of the wilt-1256 gene and its expression, regulation, and localization patterns provide important information regarding the function of this gene. As additional tagged mutants become available, similar studies are undertaken to clone and characterize selected mutations.

EXAMPLE IV MOLECULAR MND GENETIC EPISTASIS AND COMPLEMENTATION STUDIES The availability of multiple wilt mutations allows i the water stress response pathway to be defined using formal genetic studies. For example, complementation cC- LWIIII,,,,Y U WO 95/30005 PCT/US95/05366 35 studies with existing and forthcoming wilt mutants determines the number of genes that contribute to the wilt phenotype. Each available wilt mutant is crossed systematically to all other mutants. For viable mutants, homozygous lines are crossed reciprocally. For lethal mutations, heterozygous lines are employed.

Complementation groups are assigned. Double mutant lines are constructed with complementing mutations to determine genetic epistasis. To follow each allele, maize RFLPs or microsatellite markers are employed for genotyping purposes (Hosington, 1987). Homozygous double mutants are examined to determine whether an interaction between gene exists. For example, it is contemplated that mutations may be additive, synergistic, antagonistic, or epistatic to one another. Mutations that define independent pathways are additive while those that lie in a shared signal transduction pathway show epistasis.

The similarity between some mutant phenotypes may preclude a formal epistasis analysis. Nevertheless, single and double mutant lines are useful for defining molecular interactions. For example, it is contemplated that one can determine whether gene or protein expression is affected by mutation(s) at other loci using wilt-1256 probes. This result indicates that certain genes lie upstream regulate) of wilt-1256 function.

A. Morphology and Physiology of Mutant Phenotype The molecular characterization of wilt-1256 will determine the temporal and spatial pattern of gene expression. This information indicates which tissues and cells contain the primary defect. To further investigate the nature of the mutant, the tissue and cellular morphology of wilt-1256 mutants and wild type siblings under normal and water deficit conditions are examined.

Histological analysis of stressed and unstressed tissue j i- I- 1 Ii ~I L~ WO 95/30005 PCTIUS95/05366 36 from mutant and wild-type plants are performed (Sylvester Ruzin, 1994). Cross sections through the appropriate tissues leaves or roots) reveal any structural aberrations. Transmission and scanning electron microscopy are used to characterize mutant and wild-type cell structure and epidermal surfaces. Leaf surfaces are also examined for normal stomate structure and density using epidermal peels (Ristic Cass, 1991).

The relationship between the wilt phenotype and the phytohormone ABA is investigated. Mutant rescue studies using ABA are performed on wilt-1256 mutants to determine whether ABA can rescue the phenotype (wilting and/or lethality) or whether the mutant is hypo- or hyper-sensitive to ABA when compared to its normal sibs.

If wilt-1256 reveals an increased or decreased ABA sensitivity, this suggests that an important step in ABA synthesis or reception is affected. To further investigate this relationship, the ABA content of seedling leaves and roots is measured using a radioimmuno-based assay (Quarrie et al., 1988). This assay helps to identify whether the defect is in ABA synthesis or reception. For example mutations in the ABA biosynthetic pathway are expected to have reduced levels of ABA in the leaves and/or roots, whereas, ABA receptor mutants have normal or elevated levels.

EXAMPLE V TRANSGENIC ANALYSIS OF GENE FUNCTION A. Vector Construction Vectors are constructed that drive expression of a wilt gene in Zea mays cells. A vector is constructed to direct constitutive expression. For example, the Cauliflower Mosaic Virus 35S promoter (Odell et al., t l s 1: I WO 95/30005 PCTUS95/05366 37 1985) is placed 5' of the wilt gene. Alternatively the rice actin gene promoter (Wang et al., 1992) is placed of the wilt gene. It is anticipated that all promoters which direct constitutive gene expression in maize are useful when operably linked to a wilt gene. Sequences which direct polyadenylation are linked 3' to the wilt gene. These sequences included, but are not limited to, DNA sequences isolated from the 3' region of Agrobacterium tumefaciens nopaline synthase, octopine synthase or transcript 7, or potato proteinase inhibitor II genes. It is anticipated that constitutive expression of the wilt gene in all tissues of a maize plant will enhance the ability of the plant to maintain water turgor under conditions of decreased water availability.

It is further contemplated that tissue specific expression of a wilt gene will enhance the agronomic performance of a maize plant. Vectors for use in tissuespecific targeting of wilt genes in transgenic plants will typically include tissue-specific promoters and may also include other tissue-specific control elements such as enhancer sequences. Promoters which direct specific or enhanced expression in certain plant tissues will be known to those of skill in the art in light of the present disclosure. These include, for example, the rbcS promoter, specific for green tissue; the ocs, nos and mas promoters which have higher activity in roots or wounded leaf tissue; a truncated (-90 to 35S promoter which directs enhanced expression in roots, an a-tubulin gene that directs expression in roots and promoters derived from zein storage protein genes which direct expression in endosperm. It is particularly contemplated that one may advantageously use the 16 bp ocs enhancer element from the octopie synthase (ocs) gene (Ellis et al., 1987; Bouchez et al., 1989), especially when present in multiple copies, to achieve enhanced expression in roots.

also il oh t us i c as enacrsqecs.Pooeswihietseii or enane exrsini eti ln ise ilb WO 95/30005 PCTUS95/05366 38 Expression of wilt genes in transgenic plants may be desired under specified conditions. For example, the expression of wilt genes may be desired only under actual stress conditions. It is known that a large number of genes exist that respond to the environment. For example, expression of some genes such as rbcS, encoding the small subunit of ribulose bisphosphate carboxylase, is regulated by light as mediated through phytochrome.

Other genes are induced by secondary stimuli. For example, synthesis of abscisic acid (ABA) is induced by certain environmental factors, including but not limited to water stress. A number of genes have been shown to be induced by ABA (Skriver Mundy, 1990). Promoter regions that regulate expression of these genes will be useful when operably linked to a wilt gene.

Alternatively, one may wish to obtain novel tissuespecific promoter sequences for use in accordance with the present invention. To achieve this, one may first isolate cDNA clones from the tissue concerned and identify those clones which are expressed specifically in that tissue, for example, using Northern blotting.

Ideally, one would like to identify a gene that is not present in a high copy number, but which gene product is relatively abundant in specific tissues. The promoter and control elements of corresponding genomic clones may then be localized using the techniques of molecular biology known to those of skill in the art.

It is proposed that in some embodiments of the present invention expression of a wilt gene in a transgenic plant will be desired only in a certain time period during the development of the plant.

Developmental timing is frequently correlated with tissue specific gene expression. For example, expression of zein storage proteins is initiated in the endosperm about days after pollination.

WO 95/30005 PCT/US95/05366 39- B. Initiation and Maintenance of Recipient Cell Cultures Wilt genes may be introduced into maize cells including, but not limited to, cultured cells or immature embryos. Transformable cell lines of maize are developed using the protocols familiar to one of skill in the art, including, but not limited to the following. The composition of each culture medium is listed in Table 2.

C. Cell Line AT824 For development of the transformable cell line AT824, immature embryos (0.5 1.0mm) were excised from the B73-derived inbred line AT and cultured on N6 medium with 100 AM silver nitrate, 3.3 mg/L dicamba, 3% sucrose and 12 mM proline (2004, see Table Six months after I initiation type I callus was transferred to medium 2008 (see Table Two months later type I callus was transferred to a medium with a lower concentration of sucrose (279, see Table A sector of type II callus was identified 17 months later and was transferred to 279 (see Table 2) medium. This cell line is uniform in nature, unorganized, rapid growing, and embryogenic.

This culture is desirable in the context of this invention as it is easily adaptable to culture in liquid or on solid medium.

The first suspension cultures of AT824 were initiated 31 months after culture initiation. Suspension cultures were initiated in a variety of culture media including media containing 2,4-D as well as dicamba as the auxin source, media designated 210, 401, 409, 279 (see Table Cultures were maintained by transfer of approximately 2 ml packed cell volume to 20 ml fresh culture medium at 3 day intervals. AT824 can be routinely transferred between liquid and solid culture media witl no effect on growth or morphology.

I i r "";.Lrri Il~-L-f-*ti C 1~Y-llilii--il 11^;;11__1 WO 95/30005 PCT/US95/05366 40 Suspension cultures of AT824 were initially cryopreserved 33-37 months after culture initiation. The survival rate of this culture is improved when it is cryopreserved following three months in suspension culture. AT824 suspension cultures have been cryopreserved and re-initiated from cryopreserved cells at regular intervals since the initial date of freezing.

Repeated cycles of freezing have not affected the growth or transformabilicy of this culture.

Table 2: Illustrative Embodiments of Tissue Culture Media Which are Used for Type II Callus Development, Development of Suspension Cultures and Regeneration of Plant Cells (Specifically Maize Cells) MEDIA BASAL SUCROSE pH OTHER COMPONENTS" NO. MEDIUM (Amount/L) 101 MS 3% 6.0 MS vitamins 100mg myo-inositol _____Bactoagar 189 MS 5.8 3mg BAP .04mg NAA niacin 800mg L-asparagine 100mg casamino acids sorbitol 1.4g L-proline 100mg myo-inositol Gelgro 201 N6 2% 5.8 N6 vitamins 2 mg L-glycine 1 mg 2,4-D 100 mg casein hydrolysate 2.9 g L-proline Gelgro L i i WO 95/30005 WO 9530005PCTIUS9/05366 41 MEDIA BASAL SUCROSE pH OTHER COMPONENTS" NO. MEDIUM ____(Amount/L) 210 N6 3% 5.5 N6 vitamins 2 mg 2,4-D 250 mg Ca pantothenate 100 mg myo-inositol 790 mg L-asparagine 100 mg casein hydra lysate 1.4 g L-proline 2 mg glycine Hazelton agar 223 N6 2% 5.8 3.3 mg dicamba 1 mg thiamine mg niacin 800 mg L-asparagine 100 mg casein hydrolysate 100 mg myo-inositol 1.4 g praline Geigro mg bialaphas 279 N6 2% 5.8 3.3 mig dicamba 1 mg thiamine mg niacin 800 mg L-asparagine 100 mg casein hydrolysate 100 mg myo-inositol 1.4 g praline eigro 401 MS 3% 6.0 3.73 Dig Na2EDTA 0.25 mg thiamine 1 Dig 2,4-D 2 mg NAA 200 mg casein hydrolysate 500 Mg I24 4 00 mg KH 2

PO

4 100 mg myo-inosital- 409 MS 3% 6.0 3.73 mg Na 2

EDTA

0.25 mg thiamine 9.9 mg dicamba 200 mg casein hydrolysate 500 Mg K 2 S0 4 4 00 mg KH1 2 P0 4 1___100 mg myo-inositol Wo 95/30005 PCTJUS95/05366 42 MEDIA BAA URSE PH OTHER COMPONENTS" N0. MEDIUM ___(Amount/L) 425 MS 3% 6.0 3.73 mg Na 2

EDTA

0.25 mg thiamine 9.9 mg dicamba 200 mg casein hydrolysate 500 mg K 2 S0 4 4 00 mg KH2PO4 100 mg myo-inositol 3 mg bialaphos 501 Clark's 2% 5.7 Med ium* 607 0.5x MS 3% 5.8 0.5 mg thiamine mg niacin Geirite 734 N6 2% 5.8 N6 vitamins 2 mg L-glycine mg 2,4-D 14 g Fe sequestrene 200 mg casein hydrolysate 0.69 g L-proline Gelrite 735 N6 2% 5.8 1 mg 2,4-D mg niacin 0.91 g L-asparagine 100 mg myo-inositol 1 mg thiamine g MES 0.75 g MgCl 2 100 mg casein hydrolysate 0.69 g L-proline Geigro 739 N6 2% 5.8 1 mg 2,4-D mg niacin 0.91 g L-asparagine 100 mg myo-inositol 1 mg thiamine g MES 0.75 g MgCl 2 100 mg casein hydrolysate 0.69 g L-proline Geigro 1 mg bialaphos

I

r WO 95130005 WO 9530005PCTIUS95/05366 43 MEDIA BASAL SUCROSE PH OTHER COMPONENTS" NO. MEDIUM ____(Amount/L)_ 750 N6 5.8 1 mg 2,4-D mg niacin 0.91 g L-asparagine 100 mg myo-inositol 1 mg thiamine g MES 0.75 g MgC1 2 100 mg casein hydrolysate 0.69 g L-proline Geigro 0.2 M mannitol mg bialaphos 758 N6 2% 5.8 1 mg 2,4-D mg niacin 0.91 g L-asparagine 100 mg myo-inositol 1 mg thiamine g MES 0.75 g MgC1 2 100 mg casein hydrolysate 0.69 g L-proline Geigro mg bialaphos 2004 N6 3% 5.8 1 mg thiamine mg niacin 3.3 mg dicamba 17 mg AgNO3 1.4 g L-proline 0.8 g L-asparagine 100 mg casein hydrolysate 100 mg myo-inositol _______Geirite 2008 N6 3% 5.8 1 mg thiamine mg niacin 3.3 mg dicamba 1.4 g L-proline g L-asparagine Basic MS medium described in Murashige Skoog (1962)/. This medium is typically modified by decreasing the NH 4 N0 3 from 1.64 g/l to 1.55 g/l, and omitting the pyridoxine HC1, nicotinic acid, myo-inositol and glycine.

1:

I

WO 95/30005 PCT/US95/05366 44 N6 medium described in Chu et al., 1975.

NAA Napthol Acetic Acid IAA Indole Acetic Acid 2-IP 2, isopentyl adenine 2,4-D 2, 4-Dichlorophenoxyacetic Acid BAP 6-benzyl aminopurine ABA abscisic acid **Basic medium described in Clark (1982) D. Initiation of Cell Lines of the Hill Genotype Transformable cell cultures are routinely developed from the genotype Hi-II using the following protocol.

The Hi-II genotype of corn was developed from an A188 x B73 cross. This genotype was developed specifically for a high frequency of initiation of type II cultures (100% response rate, Armstrong et al., 1991). Immature embryos (8-12 days post-pollination, 1 to 1.2 mm) are excised and cultured embryonic axis down on N6 medium containing 1 mg/L 2,4-D, 25 mM L-proline (201, see Table 2) or N6 medium containing 1.5 mg/L 2,4-D, 6mM L-proline (734, see Table Type II callus can be initiated either with or without the presence of 100 gM AgNO 3 Cultures initiated in the presence of AgNo 3 are transferred to medium lacking this compound 14-28 days after culture initiation.

Callus cultures are incubated in the dark at 23-28 0 C and transferred to fresh culture medium at 14 day intervals.

SHi-II type II callus is maintained by manual selection of callus at each transfer. Alternatively, callus can be resuspended in liquid culture medium, passed through a 1.9 mm sieve and replated on solid culture medium at the time of transfer. It is believed that this sequence of manipulations is one way to enrich for recipient cell types. Regenerable type II callus that is suitable for transformation can be routinely developed from the Hi-II genotype and hence new cultures I. WO 95/30005 PCTUS95/05366 45 are developed every 6-9 months. Routine generation of new cultures reduces the period of time over which each culture is maintained and hence insures reproducible, highly regenerable, cultures that routinely produce fertile plants.

E. Microprojectile Bombardment DNA is introduced into cultured cells as follows.

Cultured cells are subcultured to fresh medium 409 (see Table 2) two days prior to particle bombardment. If grown in liquid medium cells are plated on solid 409 (Table 2) medium 16-24 hours before bombardment (about ml packed cell volume per filter). Tissue is treated with 409 (Table 2) medium containing 200 mOsm sorbitol (medium 431, see Table 2) for 1 hour prior to bombardment.

DNA is introduced into cells using the DuPont Biolistics PDS1000He particle bombardment device.

DNA is precipitated onto gold particles as follows.

A stock solution of gold particles is prepared by adding mg of 1 gm gold particles to 1000 Al absolute ethanol and incubating for at least 3 hours at room temperature followed by storage at -20°C. Twenty to thirty five pl sterile gold particles are centrifuged in a microcentrifuge for 1 min. The supernatant is removed and one ml sterile water is added to the tube, followed by centrifugation at 2000 rpm for 5 minutes.

Microprojectile particles are resuspended in 30 pl of DNA solution (30 Mg total DNA). The DNA solution contains a vector containing a chimeric wilt gene and a vector containing a selectable marker gene, the bar gene which is used for selection of transformants based on resistance to the herbicide bialaphos (Gordon-Kamm et al., 1990). Two hundred twenty microliters sterile I M -'--1111 I WO 95/30005 PCT/US95/05366 46 water, 250 ll 2.5 M CaCl 2 and 50 gl spermidine are added.

The mixture is thoroughly mixed and placed on ice, followed by vortexing at 4 0 C for 10 minutes and centrifugation at 500 rpm for 5 minutes. The supernatant is removed and the pellet resuspended in 600 p1 absolute ethanol. Following centrifugation at 500 rpm for minutes the pellet is resuspended in 36 Al of absolute ethanol.

Ten Al of the particle preparation are dispensed on the surface of the flyer disk and the ethanol was allowed to dry completely. Particles are accelerated by a helium blast of approximately 1100 psi. One day following bombardment cells are transferred to liquid medium 409 (10 ml, see Table Tissue is subcultured twice per week. During the first week there is no selection pressure applied.

Alternatively immature embryos may be directly subject to microprojectile bombardment. Immature embryos (1.2 2.0 mm in length) are excised from surface-sterilized, greenhouse-grown ears of Hi-II 11-12 days post-pollination. The Hi-II genotype was developed from an A188 x B73 cross for high frequency development of type II callus from immature embryos (Armstrong et al., 1991). Approximately 30 embryos per petri dish are plated axis side down on a modified N6 medium containing 1 mg/l 2,4-D, 100 mg/l casein hydrolysate, 6 mM L-proline, 0.5 g/1 2-(N-morpholino)ethanesulfonic acid (MES), 0.75 g/1 MgCl 2 and 2% sucrose solidified with 2 g/l Gelgro, pH 5.8 (735, Table 2) Embryos are cultured in the dark for two days at 24 0

C.

Approximately four hours prior to bombardment, embryos are transferred to the above culture medium with the sucrose concentration increased from 3% to 12%. When embryos are transferred to the high osmoticum medium they LI 1 WO 95/30005 PCTIUS95/05366 47 are arranged in concentric circles on the plate, starting 2 cm from the center of the dish, positioned such that their coleorhizal end is orientated toward the center of the dish. Usually two concentric circles are formed with 25-35 embryos per plate. Preparation of gold particles carrying plasmid DNA is described above.

The plates containing embryos are placed on the third shelf from the bottom, 5 cm below the stopping screen. The 1100 psi rupture discs are used. Each plate of embryos is bombarded once. Embryos are allowed to recover overnight on high osmotic strength medium prior to initiation of selection.

Although this example describes the introduction of DNA using particle bombardment, it is contemplated that DNA will be introduced into cells using any one of a number of techniques from which it is possible to recover fertile transgenic plants. For example, fertile transgenic plants are produced following electroporation of cells as described in Krzyzek et al, incorporated herein by reference.

F. Selection of Transformants Transformants are selected based on resistance to a toxic compound for which the introduced gene confers resistance. For example cells transformed with a gene encoding a glyphosate resistant EPSPS protein are resistant to the herbicide glyphosate. Similarly, cells transformed with a gene encoding neomycin phosphotransferase II are resistant to the antibiotics kanamycin or G418. It is anticipated that other selectable marker genes will be useful for identification of transformants. In this example transformants are identified based on resistance to the herbicide bialaphos conferred by the introduced bar gene. Following one week i l i WO 95/30005 PCTIUS9S/05366 48 Sculture in liquid medium 409 (Table 2) without selection pressure, particle bombarded tissue is transferred to liquid medium 409 (Table 2) containing 1 mg/L bialaphos.

Cells are transferred twice per week into fresh medium containing 1 mg/L bialaphos for two weeks. Tissue is thin planted 3 weeks following bombardment at a concentration of 0.1 ml packed cell volume per petri dish containing medium 425 (Table Transformants are identified as discreet colonies 6 weeks following bombardment. It is the experience of the inventors that all cell lines that grow on 3 mg/L bialaphos contain the bar gene.

Alternatively, following particle bombardment cells remain on solid 279 (Table 2) medium in the absence of selection for one week. At this time cells are removed from solid medium, resuspended in liquid 279 medium (Table replated on Whatman filters at 0.5 ml PCV per filter, and transferred to 279 medium (Table 2) containing 1 mg/L bialaphos. Following one week, filters are transferred to 279 medium (Table 2) containing 3 mg/L bialaphos. One week later, cells are resuspended in liquid 279 medium and plated at 0.1 ml PCV on 279 medium (Table 2)containing 3 mg/L bialaphos. Transformants are identified about 7 weeks following bombardment.

Following particle bombardment of immature embryos, transformants are recovered, Embryos are allowed to recover on high osmoticum medium (735, 12% sucrose, see Table 2) overnight (16 24 hours) and are then transferred to selection medium containing 1 mg/l bialaphos (739 or 750, see Table Embryos are maintained in the dark at 240 C. After three to four weeks on the initial selection plates about 90% of the embryos form Type II callus and are transferred to selective medium containing 3 m/1 bialaphos (758, Table Responding tissue is subcultured about every two i y ll WO 95/30005 PCT[US95/05366 49 weeks onto fresh selection medium (758, Table 2).

Transformants are identified six to eight weeks after bombardment.

G. Plant Regeneration Plants are regenerated from transformants. The following protocol describes method for plant regeneration, but one skilled in the art will be familiar with other equally efficient protocols. For regeneration tissue is first transferred to solid medium 223 (Table 2) and incubated for two weeks. Transformants may be initially subcultured on any solid culture that supports callus growth. Subsequently transformants are subcultured one to three times, but usually twice on 189 medium (Table 2; first passage in the dark and second passage in low light) and once or twice on 101 medium (Table 2) in petri dishes before being transferred to 607 medium (Table 2) in Plant Cons©. Variations in the regeneration protocol are normal based on the progress of plant regeneration. Hence some of the transformants are first subcultured once on 425 medium (Table twice on 189 medium (Table once or twice on 101 medium (Table 2) followed by transfer to 501 medium (Table 2) in Plant Cons©. As shoots developed on 101 medium (Table the light intensity is increased by slowly adjusting the distance of the plates from the light source located overhead. All subculture intervals are for about 2 weeks at 24 0 C. Transformants that developed 3 shoots and 2-3 roots are transferred to soil.

Plantlets in soil are incubated in an illuminated growth chamber and conditions was slowly adjusted to adapt or condition the plantlets to the drier and more illuminated conditions of the greenhouse. After adaptation/conditioning in the growth chamber, plants are i transplanted individually to 5 gallon pots of soil in the Y i lh -I ii WO 95/30005 PCTUS95/05366 50 greenhouse. Transformed plants in soil are cultivated in the greenhouse following standard greenhouse protocols and pollinated using standard plant breeding techniques.

It is the experience of the inventors that seed is recovered from most transgenic plants generated in accordance with these procedures.

Progeny and subsequent generations are grown in the field and assayed for their performance under a range of water availability conditions. Both qualitative and quantitative measures of the plant's ability to withstand water stress are made. Seeds are germinated in the greenhouses, growth chambers and field conditions under ample water supply. At one or more times during the plant's life cycle, water availability is reduced in order to identify plants expressing the wilt gene(s).

Visual signs of wilting or the reduction of turgor are noted. In addition to the visual signs of wilting, which may only be observed under more pronounced drought stress, measures of plant water relations are made.

Total water potential, osmotic potential and turgor potential are quantitatively measured and detection of differences in turgor or the ability of the plants not to wilt can be made even when no signs of plant stress are visible to the eye. Plants expressing the most favorable water status result in superior growth under water stress. Different measures of growth are used to document this superior performance. Measures of cell and leaf area expansion are used to identify superior plant growth under stress.

The physiological and biochemical activity of the transformed plant tissue is indicative of its improved stress tolerance. Such screening of plants with the measurement of photosynthetic activity or transpirational activity are examples, but not all of the types of measurement that can be done to identify the superiority i:;

I

WO 95/30005 PCTIUS95/05366 51 of the wilt expressing plants compared to non-transformed plants. Measurements of reproductive capacity including, but not limited to the synchrony of pollen shed and silk emergence are indicators of improved stress tolerance when the wilt gene is expressed. In addition, it is anticipated that the expression of the wilt gene will minimize kernel abortion during times of stress thereby increasing the amount of grain to reach maturity. The expression of the wilt gene allows for superior late season plant health and development of full ears. It is contemplated that barrenness will not be a problem.

Once the initial breeding lines are selected by the above but not limited to, criteria, testcrosses are made and hybrid seed is produced. The testcross hybrids and breeding populations are planted in several different fashions in the field. One scheme of evaluation is to grow populations of hybrid plants containing the wilt gene in many different locations and measure the performance of the plants at these different locations.

Given the variability of rainfall distribution, the different locations receive different quantities of rainfall and in some locations, the plants will receive stress. Yield information as well as measures which quantify plant response to stress as described earlier, are made. The information regarding the performance of these hybrids along with that of the performance of nontransformed or non-wilt containing hybrids is compared.

j It is anticipated that the hybrids expressing the wilt gene will be higher in yield performance at a given level of water availability than the controls.

Where irrigation is available, more controlled comparisons are made through the establishment of differential irrigation treatments. The same entries of hybrids or lines are grown under contrasting irrigation treatments. Such an approach limits the number of ii 1: WO 95130005 PCT1US95/05366 52 variables at work in the evaluation. Aside from the same types of measurements as defined above, differential responses are calculated because of the contrast in the date. It is anticipated that wilt expressing hybrids will have less yield reduction when grown under irrigated versus non-irrigated conditions when compared to hybrids without the wilt gene.

Upon the identification of the superior performance of the wilt gene expressing plants, the parent selections are advanced and inbred lines are produced through i conventional breeding techniques. Hybrid plants having i one or more parents containing the wilt gene are tested in commercial testing and evaluation programs and performance documented. This testing include performances trials over a wide geographical area as well as dedicated trials where water availability is varied io reveal performance advantage and hence value.

An additional advantage of the expression of the wilt gene is the superior performance of the parental inbred lines in production of hybrids. Less stress related parent yield loss is associated with higher green seed yield and thereby higher economic margins.

It is anticipated that the performance advantage will not only be present under stress conditions. Given the overall role of water in determining yield, it is contemplated that corn plants expressing the wilt gene will utilize water more efficiently. This will improve overall performance even when soil water availability is not limiting. Through the introduction of the wilt gene(s) and the improved ability of corn to maximize water usage across a full range of conditions relating to water availability including normal and stressed conditions), yield stability or consistency of yield performance will be achieved.

I: /j :R _::ii V: -i 4_ i Ls:i_ h Y- WO 95/30005 PCTUS95/05366 53 A fundamental premise behind these studies is that genes defined by wilt mutations define important steps in the environmental signal transduction pathway that protects plants from water deficits. An test of this hypothesis involves assessing the effects of overexpression on water deficit tolerance. These studies are conducted in maize and heterologous dicot systems.

EXAMPLE VI USE OF WILT GENES AS PROBES IN MARKER ASSISTED BREEDING The identification of maize that are bred for resistance to adverse water conditions may be readily assisted by using the wilt-related gene presently disclosed, as well as genes identified using the methods of the present invention. To positively identify plants for further crossings nucleic acids are isolated from potential F1 progeny and screened for the overexpression of mRNA or protein, amplification of the gene locus or increased stability of the gene product.

Techniques for isolating nucleic acids and proteins are well known to those of skill in the art (Maniatis et al., 1991), and may be used in conjunction with the gene of the present invention to selectively segregate plants that have increased resistance to water deprivation, with or without, genetic alteration of the plant. Natural variants of maize may be isolated and selected for breeding following a screen for altered wilt resistance gene message, product or product stability.

Accordingly, it is contemplated that wilt genes will be useful as DNA probes for marker assisted breeding. In the process of marker assisted breeding DNA sequences are used to follow desirable agronomic traits (Tanksley et al., 1989) iii the process of plant breeding. It is ILL %L 4_ h LI h Y'" WO 95/30005 PCT/US95/05366 54 anticipated that a wilt gene probe will be useful for identification of plants with enhanced ability to utilize water resources. Furthermore, through the use of more than one wilt gene probe it will be possible to combine genes that enhance the ability to utilize water. It is contemplated that such a combination of genes would be difficult to identify without marker assisted breeding, unless the plant is grown under conditions of limited water availability.

Marker assisted breeding using the wilt gene is undertaken as follows. Seed of plants with the desired ability to utilize water resources are planted in soil in the greenhouse or in the field. Leaf tissue is harvested from the plant for preparation of DNA at any point in growth at which approximately one gram of leaf tissue can be removed from the plant without compromising the viability of the plant. Genomic DNA is isolated using a procedure modified from Shure et al. (1983).

Approximately one gram of leaf tissue from a seedling is lyophilized overnight in 15 ml polypropylene tubes.

Freeze-dried tissue is ground to a powder in the tube using a glass rod. Powdered tissue is mixed thoroughly with 3 ml extraction buffer (7.0 M urea, 0.35 M NaCI, 0.05 M Tris-HCI ph 8.0, 0.01 M EDTA, 1% sarcosine).

Tissue/buffer homogenate is extracted with 3 ml phenol/ chloroform. The aqueous phase is separated by centrifugation, and precipitated twice using 1/10 volume of 4.4 M ammonium acetate pH 5.2, and an equal volume of isopropanol. The precipitate is washed with 75% ethanol and resuspended in 100-500 pl TE (0.01 M Tris-HCI, 0.001 M EDTA, pH Genomic DNA is digested with a 3-fold excess of restriction enzymes, electrophoresed through 0.8% agarose (FMC), and transferred (Southern, 1975) to Nytran using 10X SCP (20X SCP: 2 M NaCI, 0.6 M disodium phosphate, 0.02 M disodium EDTA).

.B L -L .l ~-P~rprrrrrs*pr~ WO 95/30005 PCT/US95/05366 55 One of skill in the art will recognize that many different restriction enzymes will be useful and the choice of restriction enzyme will depend on the DNA sequence of the wilt gene that is used as a probe and the DNA sequence in the maize genome surrounding the wilt gene. One will select a restriction enzyme that produces a DNA fragment following hybridization that is identifiable as that wilt gene. It is anticipated that one or more restriction enzymes will be used to digest genomic DNA either singly or in combinations. Filters are prehybridized in 6X SCP, 10% dextran sulfate, 2% sarcosine, and 500 jg/ml denatured salmon sperm DNA and 32 P-labelled wilt gene probe generated by random priming (Feinberg Vogelstein, 1983; Boehringer-Mannhelm).

Hybridized filters are washed in 2X SCP, 1% SDS at 650 for 30 minutes and visualized by autoradiography using Kodak XAR5 film. Those of skill in the art will recognize that there are many different ways to isolate DNA from plant tissues and that there are many different protocols for Southern hybridization that will produce identical results. Those of skill in the art will recognize that a Southern blot can be stripped of radioactive probe following autoradiography and re-probed with a different wilt gene probe. In this manner one identifies each of the various wilt genes that is present in the plant.

Each lane of the Southern blot represents DNA isolated from one plant. Through the use of a multiplicity of wilt genes as probes on the same genomic DNA blot, the wilt gene composition of each plant is determined. Correlations are established between the contributions of particular wilt genes to the ability of the plant to adapt to conditions of decreased water availability. Only those plants that contain the desired combination of wilt genes are advanced to maturity and used for pollination. DNA probes corresponding to wilt L WO 95130005 PCTJUS95/05366 56 genes are useful markers during the course of plant breeding to identify and combine particular wilt genes without having to grow the plants under conditions of decreased water availability and assay the plants for agronomic performance under these stressed conditions.

All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure.

While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the composition, methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and %oncept of the invention as defined by the appended claims.

Ix WO 95/30005 PCT/US95/05366 57

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A

ML IC CP C p L~ WO 95/30005 PCTYUS95/05366 66 Walbot. 1991. Early event sin Mutator lines. I. Germanl revertants, IL somatic reversion, Maize Genetics Cooperation News Letter 65:95-96, cited by permission.

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Ii i i i WO 95/30005 PCT/US95/05366 67 SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: DEIALB GENETICS CORPORATION STREET: 62 MARITIME DRIVE CITY: MYSTIC STATE: CONNECTICUT COUNTRY: UNITED STATES OF AMERICA POSTAL (ZIP) CODE: 06355-1959 and NAME: YALE UNIVERSITY STREET: 451 COLLEGE STREET CITY: HEW HAVEN STATE: CONNECTICUT COUNTRY: UNITED STATES OF AMERICA POSTAL (ZIP) CODE: 06520 (ii) INVENTORS: CHOMET, Paul DELLAPORTA, Stephen L.

ORR, Peter KRUEGER, Roger LOWE, Brenda (iii) TITLE OF INVENTION: GENES REGULATING THE RESPONSE OF ZEA MAYS TO WATER DEFICIT (iv) NUMBER OF SEQUENCES: 1 CORRESPONDENCE ADDRESS: g ADDRESSEE: ARNOLD, WHITE DURKEE STREET: P.O. BOX 4433 CITY: HOUSTON STATE: TEXAS Ii mo WO 95/30005 PCT/US95/05366 68 COUNTRY: UNITED STATES OF AMERICA ZIP: 77210 (vi) COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS/ASCII SOFTWARE: PatentIn Release Version #1.30 (vii) CURRENT APPLICATION DATA: APPLICATION NUMBER: UNKNOWN FILING DATE: 27-APR-1995 CLASSIFICATION: UNKNOWN (viii) PRIOR APPLICATION DATA: APPLICATION NUMBER: USSN 08/235,060 FILING DATE: 29-APR-1994 CLASSIFICATION: UNKNOWN (ix) ATTORNEY/AGENT INFORMATION: NAME: HIGHLANDER, STEVEN L.

REGISTRATION NUMBER: 37,642 REFERENCE/DOCKET NUMBER: DEKM087P-- TELECOMMUNICATION INFORMATION: TELEPHONE: (512) 418-3000 TELEFAX: (713) 789-2679 0 TELEX: 79-0924 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 247 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "DNA" (ix) FEATURE: NAME/KEY: modified -base LOCATION: one-of(3, 14, 24, 60, 83, 90, 179, 214, 219, 227) OTHER INFORMATION: /mod-base= OTHER /note= 'IN A, G, C, or T"I (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: GANTCGATGG TGTNCCTGGT TCANTCGGTG ATCCCCGACA GGGGGCAGAA GACGCTGACN AAGTTCGTCA ACGGCGGGTC CANCAACGTN TTCTACGCGC ACGAGTACAA CGCGACGGTG 120 GAGTTCTACT GGGCGCCCTT CCTGGTGGAG TCCAACTCCG ACAACCCCAA GGTTCACANC 180 GTCCCCGACC GGATCATCCA GTGGCACGCC ATCNCCAANC ACGCGCNCAA CTGGATCGGC 240 GTCGACA 247 -69a- Where the terms "comprise", "comprises", "comprised" or "comprising" are used in this specification, they are to be interpreted as specifying the presence of the stated features, integers, steps or components referred to, but not to preclude the presence or addition of one or more other feature, integer, step, component or group thereof.

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Claims (17)

1. A purified maize wilt gene DNA segment, wherein said wilt gene is defined as a gene that, when present in a plant, allows the plant to maintain proper internal water balance under normal water conditions or under low water availability or contributes to such a response and a drought-resistance phenotype and is a wild type allele of wilt-1256 and wherein said segment is functionally distinctive of said wild type allele.

2. The DNA segment of claim 1, the DNA segment being further defined as a 18 kb Sal I-fragment obtainable by the methods of Examples I to Ill.

3. The DNA segment of claim 1 or claim 2, wherein the DNA segment comprises the nucleotide sequence SEQ ID NO: 1 or biologically functional equivalent sequences corresponding, in terms of translation into the corresponding proteins and peptides, with the sequence of SEQ ID NO: 1. en.

4. The DNA segment of any one of the preceding claims, wherein said wilt gene is under the control of a promoter that is operable in maize.

5. The DNA segment of claim 4, wherein said promoter is a wilt gene promoter, a Cauliflower Mosaic Virus 35S, rice actin, rbcS, o-tubulin, or ocs promoter. o *Q 0 0 :0 25

6. The DNA segment of any one of the preceding claims, further defined as a recombinant vector comprising the wilt gene. 0 0

7. A method for preparing a wilt gene DNA segment as defined in claim 1, the method comprising the steps of: obtaining a collection of maize plants bearing transposon-induced mutations; screening plants of said collection to identify a plant exhibiting a wilt phonetype; preparing a genomic library from such an identified plant; and screening said genomic library to identify a genomic clone comprising a wilt gene in association with a transposon.

8. The method of claim 7, wherein said wilt phenotype is identified by exhibition of decreased turgor. ,I w I^ -71-

9. The method of claim 7 or claim 8, wherein said transposon- associated genomic clone is identified by hybridization of a transposon-specific probe with said genomic library.

The method of any one of claims 7 to 9, further comprising the steps of: preparing a cDNA library from a wild-type plant; and screening said cDNA library to identify a cDNA clone comprising a wilt gene.

11. The method according to claim 10, wherein flanking sequences from said transposon-associated genomic clone are identified.

12. The method according to claim 10 or claim 11, wherein said cDNA clone is identified by hybridization of said flanking sequences with said wild-type cDNA library.

13. A DNA segment prepared in accordance with the method of a.1: any one of claims 7 through 12.

14. A maize plant stably transformed with the DNA segment of any one of claims 1 through 6 or 13.

15. A method of preparing a maize plant having increased turgor, S comprising stably introducing into the genome of said plant the DNA segment of any one of claims 1 through 6 or 13. r:

16. A purified maize wilt gene DNA segment of claim 1, S substantially as herein described with reference to any one of the Examples and/or accompanying Figure.

17. A method of claim 7 for preparing a wilt gene DNA segment which method is substantially as herein described with reference to any one of the Examples and/or accompanying Figure. DATED this 10th day of July, 1998. DEKALB GENETICS CORPORATION AND YALE UNIVERSITY By their Patent Attorneys: CALLINAN LAWRIE i 10798LP8963.SP,71 il 1 I II i Y.