AU743027B2 - Transgenic tomato plants with altered polygalacturonase isoforms - Google Patents

Description

S F Ref: 239630D2

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIRCATlON FOR A STANDARD PATENT

ORIGINAL

Name and Address of Applicant: Actual Inventor(s): Address for Service: Invention Title: The Arizona Board of Regents on Behalf of the University of Arizona 1430 East Fort Lowell Road Suite 200 Tucson Arizona 85719 UNITED STATES OF AMERICA Dean DellaPenna Spruson Ferguson, Patent Attorneys Level 33 St Martins Tower, 31 Market Street Sydney, New South Wales, 2000, Australia Transgenic Tomato Plants with Altered Polygalacturonase Isoforms ft u *ft The following statement is a full description of this invention, including the best method of performing it known to me/us:- 5845

'I

-1- TRANSGENIC TOMATO PLANTS WITH ALTERED POLYGALACTURONASE

ISOFORMS

Field Of The Invention The present invention relates generally to transgenic plants. Specifically, the present invention relates to a 15 transgenic tomato plant with altered levels of isoforms of S. the enzyme polygalacturonase.

Background Of The Invention Polyqalacturonase In recent years Lycopersicon esculentum, the 20 cultivated tomato, has become a popular system for studying fruit ripening. Tomato fruit ripening is characterized by a series of coordinated biochemical and physiological changes within the various subcellular compartments of the fruit tissue. These changes collectively contribute to the overall quality of the ripe fruit. The most obvious of the changes are alterations in fruit color, flavor, texture and resistance to certain pathogens.

One biochemical change in ripening fruit is the depolymerisation and solubilization of cell wall polyuronides by the ripening-induced cell wall degrading enzyme, polygalacturonase PG activity increases dramatically during the ripening of many fruits, including tomato, and is the primary enzymic activity responsible for cell wall polyuronide degradation during fruit ripening. Reviewed in Giovannoni, et al, 1991 Ann. Rev.

Hortic Sci: 67-103.

-2- PG activity isolated from ripe tomato fruit is due to the presence of three structurally and immunologicallyrelated isoforms of PG. These isoforms are termed PG1, PG2A and PG2B. (Ali, et al. Aust. J. Plant Physiol.

9:171, 1982). The PG2A and PG2B isoforms (45 and 46 kDa, respectively) appear well after the onset of ripening and are each composed of a single catalytic PG polypeptide differing only in degree of glycosylation. Because of the physical and biochemical similarity of PG2A and PG2B, the two isoforms shall be treated herein as a single isoform activity (the PG2 activity).

The PG 1 isoform (approximately 100 kDa) is the first isoform to appear, at the onset of ripening, and is a heterodimer composed of the single catalytic PG2 S 15 polypeptide (either PG2A or PG2B) tightly associated with an ancillary cell wall glycoprotein, the PG beta-subunit.

The formation of PG1 by association of the PG2 polypeptide with the PG beta-subunit protein alters both the biochemical and enzymic properties of the associated catalytic PG2 protein. The isoelectric point and pH optimum of PG1 are both a full unit lower than those of PG2. PG1 is more thermo-stable than PG2. PG1 retains complete activity after heating for 5 minutes at 65 0 C, a treatment that completely inactivates PG2.

25 In recent years, cDNA clones for the catalytic PG2 polypeptide have been identified and used to examine in detail the regulation of PG gene expression in wild-type and mutant tomato fruit (DellaPenna, et al, Proc. Natl.

Acad. Sci. USA 83:6420 (1986). Analysis of PG2 genomic and cDNA clones has revealed that the catalytic

PG

polypeptide is encoded by a single gene which is transcriptionally activated at the onset of wild-type fruit ripening (DellaPenna et al, Plant Physiol. 90:1372 (1989). PG2 mRNA is synthesized de novo during the ripening of wild-type fruit and accumulates to high levels, accounting for greater than 1% of the mRNA mass.

Ripening-impaired mutants of tomato, which are inhibited in many ripening processes including PG2 expression, have greatly reduced levels of PG2 mRNA. The severe reduction -3in steady-state PG mRNA levels in the mutant genotypes is due to greatly reduced transcriptional activity of the PG gene (DellaPenna, et al, 1989, supra).

The PG beta-subunit has also been studied. The levels of PG beta-subunit increase approximately 4-fold during fruit ripening (Pressey, Eur. J. Biochem.

144:217-221 (1984)) and apparently determine the amount of PGl produced during tomato ripening. Therefore, as PG beta-subunit levels are depleted (by formation of PGl), the timing of appearance of the PG2 isoform is also controlled.

While it is clear from in vitro studies that PGI and S. PG2 differ in their biochemical properties, the physiological significance of the isoforms and the role of the S. 15 PG beta-subunit protein remains uncertain. From a physiological point of view, it seems likely that a cell wall enzyme like PG might be localized or its activity restricted to specific regions of the cell wall by association with an adhesion or localizing factor, such as 20 the PG beta-subunit protein. Recent results in transgenic systems have also suggested that PGI may be the physiologically active isoform in vivo with regard to pectin degradation, presumably due to its association with the PG beta-subunit protein.

S 25 Pectolytic enzymes, such as PG, may have a role in o- plant pathogen interactions. Pathogen-derived pectolytic enzymes are thought to be important components of the mechanism by which pathogens penetrate and colonize plant tissues. Preliminary results from recently completed experiments have suggested that PG induction in transgenic mutant fruit increases colonization of the fruit by Alternaria alternata, a common late-season pathogen of wild-type tomato fruit to which mutant fruit are normally resistant. The apparent conferral of pathogen sensitivity to mutant fruit by the specific induction of PG expression suggests that increasing PG activity during fruit ripening may play an important role in altering the susceptibility of the fruit to pathogens.

-4- In addition, antisense inhibition of PG expression in wild-type tomato has been correlated with a decrease in "fieldrot" during later stages of tomato ripening in the field. (Kramer, et al, 1990, Horticultural Biotechnoloqy, pp 347-355, Wiley-Liss Inc.). Although the mechanism of PG associated alterations in pathogen susceptibility is not known, these results strongly suggest a role for PG in postharvest pathogenesis.

Tomato Processing An important determinant of many processed tomato fruit products, including sauce, paste and catsup, is the viscosity thickness) of the final product. One of the primary determinants of high viscosity is the presence of large, unmodified pectin molecules. Pectin is a 15 naturally occurring plant cell wall carbohydrate polymer So that is composed primarily of polygalacturonic acid residues. Maintenance of pectin integrity during tomato processing is an extremely important part of the commercial process.

An important factor in loss of pectin integrity (decrease in the polymer size and subsequent loss of ~viscosity) during commercial processing of tomatoes is enzymatic degradation of pectin by PG. Although some modification of pectins by PG occurs naturally during the 25 ripening process (DellaPenna, et al, Plant Physioloy 94:1882-86, (1990)), by far the most dramatic and commercially damaging action of PG on pectins and, hence, viscosity occurs when the tomato fruit is homogenized for processing. The PG enzyme present in the fruit has the potential to act in an uncontrolled fashion in homogenized fruit tissues and can rapidly degrade pectin polymers.

A rapid, high-temperature heat treatment is used in commercial tomato processing to destroy PG enzyme activity and thereby maintain a higher viscosity in the final product. This treatment often comprises a process known as "hot break" and is performed by the rapid heating of the tomato product to near boiling point, to inactivate the PG enzyme as rapidly as possible. The annual cost associated with the input of large amounts of energy to bring millions of tons of tomatoes to the temperature needed to rapidly inactivate PG represents a significant cost to tomato processing industries. Annual tomato production in the US is approximately 6 million metric tons representing approximately 10% of worldwide production (1980 figures).

It follows that any process that would allow less energy to be used to inactivate PG in tomato products would result in substantial savings to the industry.

A

process that would decrease the thermal stability of the PG isoforms would therefore decrease the minimum temperature needed to heat-inactivate PG during processing. All commercially useful, non-genetically 15 engineered tomato varieties currently on the market contain both PG1 and PG2 isoforms. Generally, 10-30% of total PG activity is PG1 in a ripe fruit. One way to decrease the thermal stability of the PG isoforms would be to inactivate or lessen the amount of PG1, the more thermo-stable PG isoform.

Antisense

RNA

It has been found in both procaryotes and eukaryotes, that the production of specific endogenous proteins can be inhibited by use of an antisense RNA. An "antisense

RNA"

S 25 is a complementary version of a naturally occurring or endogenously produced RNA. Because of its complementary sequence, the antisense RNA will hybridize to the mRNA of the protein sought to be inhibited under physiological conditions. This hybridization prevents translation and, therefore, protein production. The duplex RNA complex thus formed is eventually degraded by appropriate cellular mechanisms, without resulting in expression of a protein.

An antisense RNA can conveniently be formed for a known protein coding region by reversing the orientation of the protein coding region so that the end that is normally transcribed last is now transcribed first.

Investigators have inhibited production of the catalytic PG2 polypeptide by antisense RNA technology and -6have shown a greater than 92% reduction of total PG activity relative to wild-type activity levels (Kramer, et al., Horticultural Biotechnology, 1990, pp. 347-355, Wiley-Liss, Inc.). This reduction had significant effects on processed tomato product viscosity when the product was subjected to the normal "hot break methods. These investigators did not, however, determine which PG isoforms were produced in the transgenic fruit. One would expect that the PG2 polypeptide levels were greatly reduced and that all of the PG activity formed would be in the PG1 (heat-stable) isoform due to the presence of existing beta-subunit protein, which would not have been affected by antisense inhibition of PG2 protein.

Inhibiting the level of PG2 expression is not the 15 equivalent of lowered levels of PG1. The reduction of catalytic PG2 polypeptide levels results in the lowering of total PG activity levels and PG2 protein levels, without affecting the formation of the heat stable PG1 isoform directly except by reducing the amount of PG2 20 available to form PG1. Because 100% inhibition of PG2 production has not been reported, any residual PG2 produced in PG2 antisense plants would associate with the existing beta-subunit protein to produce PG1. Heat inactivation of this remaining PG1 should still require a S 25 significant energy input.

What is needed is a transgenic tomato with lowered levels of PG1, the most heat-stable PG isoform.

Summary of the Invention The present invention is a method of creating a transgenic tomato that contains a lowered level of polygalacturonase isoform 1. The method begins with the isolating of a DNA sequence that encodes at least a portion of the polygalacturonase beta-subunit. This portion is sufficient to create an antisense construct which will hybridize effectively to the mRNA for the polygalacturonase beta-subunit. A genetic construction is created from the DNA sequence in which the DNA sequence is positioned so that the antisense version of the PG beta-subunit message may be produced from the construction. This construction is then transformed into a tomato cell whereby the tomato cell produces a lowered level of polygalacturonase beta-subunit and, therefore, produces a lowered level of the PGI isoform.

Thus, the invention provides a method of creating a transgenic tomato containing a lowered level of polygalacturonase isoform 1, comprising the steps of a) providing a DNA sequence that is at least a fifteen nucleotide in length, the DNA sequence has substantial homology to a fragment of SEQ ID NO:6 of comparable length; to b) creating a genetic construction including, 5' to 3' a promoter effective in tomato cells, a coding sequence, and a transcriptional terminator, the coding region being derived from the DNA sequence, wherein the DNA sequence from step is positioned so that an antisense polygalacturonase beta-subunit RNA is produced by the construction and wherein the antisense RNA is capable of inhibiting the polygalacturonase betai5 subunit; c) transforming a tomato cell with the genetic construction, whereby the tomato cell produces a lowered level of polygalacturonase isoform 1; and d) regenerating a tomato plant from the transformed cell, wherein the tomato plant produces tomatoes.

20 In a preferred embodiment of the present invention, the tomato plant transformation is performed by an Agrobacterium-mediated technique and the antisense construct has substantial homology to SEQ ID NO:6.

The present invention is also a genetic construction comprised of a promoter capable of expressing a gene in a tomato plant, a sequence encoding at least a portion of the polygalacturonase beta-subunit in the antisense orientation, wherein the portion is capable of effectively hybridizing with the mRNA produced from the native gene encoding polygalacturonase beta-subunit; and a 3' termination sequence.

Thus, the invention also provides a genetic construction comprised of 5' to 3' in order: a) a promoter capable of expressing a downstream coding sequence in a tomato plant; b) a sequence encoding a RNA of at least fifteen nucleotides the complement of A iich has substantial homology to a fragment of the mRNA of tomato polygalacturonase Sbet-ubunit set forth in SEQ ID NO:6; and [R:\L18ZJ05593.doc: [am c) a 3' termination sequence.

The object of the present invention is to create a tomato plant with lowered levels of polygalacturonase isoform 1. Because polygalacturonase isoform 1 is a more heat stable form of polygalacturonase, if isoforn 1 is missing, the total polygalacturonase in the tomato would be comprised of only PG2 and would be inactivated at a lower temperature than would be sufficient with a wild-type tomato whose total PG activity would consist of both PG1 and PG2 isoforms.

It is another object of the present invention to create a tomato that is potentially less susceptible to pathogens.

The invention therefore further provides a transgenic tomato fruit, wherein said tomato has lowered levels of the polygalacturonase isoform PG1.

.t The invention still further provides a transgenic tomato fruit, wherein said tomato has lowered levels of polygalacturonase beta-subunit.

15 The invention even further provides a transgenic tomato plant comprising in its S.o. genome a foreign genetic construction comprising 5' to 3' a promoter effective in tomato, a coding region encoding a RNA of at least 15 nucleotides the complement of which has substantial homology to a fragment of the mRNA of the tomato polygalacturonase betasubunit gene as set forth in SEQ ID NO:6, and a transcriptional terminator, the genetic 20 construction effective in vivo in the tomato fruit to lower the level of production of the polygalacturonase beta-subunit protein.

An advantage of the present invention is that less energy is required to inactivate the total polygalacturonase activity of the transgenic tomato described here as compared to polygalacturonase activity from wild-type tomato.

Other objects, advantages and features of the present invention will become apparent after examination of the specification, drawings and claims.

[R:\LIBZ]05593.doc:lam -8- Brief Description of the Drawings Fig. 1 is a restriction enzyme map of polygalacturonase beta-subunit cDNA clones pBsub 2.2 and pBsub 1.7.

Brief Description of the Invention The general strategy of the present invention is to lessen or eliminate the production of the PG1 isoform of tomato fruit by lowering the level of the beta-subunit protein by recombinant DNA techniques. PG1, which is an apparently heterodimeric protein complex composed of a catalytic PG2 polypeptide and the PG beta-subunit protein, is more heat stable than the PG2 isoforms (PG2A and PG2B).

.The difference in heat stability between the two isoforms

(PG

1 and PG2) is at least 20 0 C. The approximate 15 temperature of half inactivation, 5 minutes at said temperature, is ~65 0 C for the PG2 isoforms and ~90°C for

PG

1 Therefore, inactivating the PG1 isoform is the ratelimiting step with regard to complete heat inactivation of total PG activity during tomato processing. Elimination of beta-subunit protein production during tomato fruit development will cause the fruit to accumulate only the more heat-labile PG2 isoforms. This process will result in a commercially useful line of tomato in which higher viscosity can be maintained during processing, while simultaneously applying less heat to the tomato extracts during processing.

To lower the level of PG1 enzyme in the tomato fruit, an antisense strategy will be used. Once the DNA sequence of the protein coding region of the native gene is known, as it is here, it becomes possible to construct antisense DNA constructions which will produce antisense RNA in vivo in transgenic tomato to inhibit beta-subunit production.

Presented below as SEQ ID NO: 6 is the near complete cDNA sequence for the beta-subunit gene. From this cDNA sequence, part or all of the protein coding sequence can be reversed in orientation, inserted into expression plasmids and transformed into tomato plants. As long as the antisense RNA produced is of sufficient length -9bases), it will hybridize in vivo to the beta subunit mRNA, thus inhibiting beta subunit expression.

It is not necessary that the entire coding sequence of the PG beta-subunit be used in the antisense orientation. A smaller portion of the message in the antisense orientation is sufficient to hybridize to the beta-subunit mRNA and inhibit transcription. A message as small as fifteen nucleotides, if properly complementary, homologous, would be sufficient to hybridize to the gene.

A sequence of in excess of twenty-five nucleotides is preferred.

Standard molecular biological methods may be used to S" attach the antisense RNA coding sequence to suitable and 3' processing sequences. A suitable promoter will be effective to promote the transcription of the antisense RNA in tomato cells. Many suitable plant promoters are known. Suitable 3' processing sequences will be effective to terminate transcription of the antisense RNA at an appropriate point. Similarly, several such polyadenylation sequences are widely known and available.

The antisense RNA construction will then be placed in an appropriate expression vector for transformation into tomato plants. For an Aqrobacterium-mediated transformation method, the construction should be placed in a unitary or binary Ti plasmid vector. Other methods of transformation will require different vectors. For instance, if accelerated particle-mediated transformation is desired, a simpler plasmid vector, consisting simply of the antisense construct along, may be employed. The efficacy of an inserted foreign gene construct in transgenic plants, such as the antisense construct here, is independent of the method of transformation.

Once the vector containing the antisense construction is prepared, it is then necessary to obtain transgenic plants. There are many suitable methods currently used.

Kramer, et al. (supra) employed an Agrobacterium-mediated method. Another transformation method that has been proven effective in tomatoes is electroporation of protoplasts. Accelerated particle transformation of tomato is also possible.

The transformed plants must be screened for the presence of the antisense RNA and for reduction in PG1 isoform levels. These methods are discussed below.

Examples 1. In General We have purified the beta-subunit protein of PG1 from ripe tomato fruit and isolated cDNA clones encoding the protein. The identity of beta-subunit cDNA clones has been verified by a number of methods and the nucleotide sequence of beta-subunit cDNA has been determined. These

C

DNA clones will be used in the production of a genetically engineered line of tomato in which the 15 expression of the beta-subunit will be almost entirely inhibited by an antisense gene construct.

This genetically engineered plant will contain a DNA construction capable of expressing the antisense

RNA

version of the beta-subunit cDNA message. Fruit 20 expressing an antisense beta-subunit RNA would not produce the beta-subunit protein during fruit development because the antisense message would hybridize with the beta-subunit mRNA and prevent translation of the protein.

The plant would therefore only accumulate the more heatlabile PG2 isoform and would not accumulate the more heatstable PG1 isoform. Inhibiting the expression of the beta-subunit protein in tomato fruit by antisense

RNA

technology will provide a decrease in natural pectin degradation in vivo during ripening, and allow for inactivation of PG activity during commercial processing at a much lower temperature than is presently used, or much more rapidly at the temperatures presently used, thereby causing substantial savings in the cost of energy involved in production.

2. Creation of Beta-subunit cDNA Clones A cDNA library was prepared with RNA obtained from mature green tomato with a commercially available kit -11- (Stratagene, LaJolla, CA). Mature fruit tissue was collected from greenhouse grown tomato plants and frozen with liquid nitrogen. Total RNA was extracted via the following method: Ten grams of frozen tissue was ground to a powder in liquid nitrogen with a mortar and pestle and homogenized with a polytron in 20 ml of lysis buffer [8 M guanidine thiocyanate, 10 mM EDTA, 300 mM Tris-Hcl (pH 8% b-mercaptoethanol]. Following centrifugation at 3,000 g for 10 minutes, the supernatant was filtered through miracloth and extracted twice with phenol/chloroform and once with chloroform. RNA was ethanol precipitated and the resulting pellet was washed with 3.0 M sodium acetate (pH then dissolved in mM Tris, pH 7.6; 1 mM EDTA; 1% SDS and reprecipitated with 15 2.5 M Li C l (Sambook, et al, Molecular Cloning Manual, Cold Spring Harbor, 1989). Total RNA was subjected to poly A+ selection as described (DellaPenna, 1986 supra).

This cDNA library was screened with probes prepared from peptide fragments of PG. The fragments were obtained 20 in the following way: Ripe pericarp tissue was homogenized in ice-cold distilled

H

2 0 at a ratio of 1 kg fruit to 1 liter water and the resulting slurry adjusted to pH 3.0. Cell debris was pelleted by centrifugation at 10,000 g for 20 minutes, resuspended in one half volume of S 25 cold H 2 0 at pH 3.0 and repelleted. The cell debris pellet was resuspended in cold buffer contained 50 mM sodium acetate, 1.25 M NaCl (pH 6.0) and stirred for at least one hour at 4 0 C. The extract was centrifuged at 10,000 g for minutes and proteins in the supernatant were precipitated by the addition of ammonium sulfate to saturation. After centrifugation, the resulting protein pellet was resuspended in 0.125 M sodium acetate (pH and dialyzed extensively against the same buffer. The dialyzed extract was then clarified by centrifugation and applied to a CM-Sepharose column equilibrated with 0.125 M sodium acetate (pH Bound proteins were eluted by a two step gradient of 0.45 M sodium acetate (pH 6.0) and M sodium acetate (pH PG2 A and B eluted with -12- 0.45 M sodium acetate while PG1 eluted with 1.0 M sodium acetate.

The 1.0 M sodium acetate eluent was concentrated by ultrafiltration, dialyzed against Concanavalin A (Con-A) buffer [500 mM NaC1, 50 mM sodium acetate, 1 mM calcium acetate, 1 mM manganese sulfate (pH6.0)] and further purified by Con-A chromatography as previously described (DellaPenna, 1986 supra). PGl-containing fractions were concentrated by ultrafiltration, dialyzed against 50 mM phosphate, 200 mM NaC1, 0.1 mM DDT (pH6.0) and further purified by Mono S FPLC chromatography, Pogson et al, Aust. J. Plant Phys. 18:65-79 (1991).

The subunits of PG1 were separated and isolated as described (Pogson et al., 1991 supra). PG1 purification 15 and separation of PG2 and the beta-subunit protein were .followed by SDS-PAGE. Electrophoretic blotting, and detection methods for the catalytic PG polypeptide were performed as described previously (DellaPenna et al., 1986, supra). PG1 and PG2 levels during extraction and *S 20 purification were determined by heat inactivation (Tucker et al., Eur. J. Biochem. 115:87-90, 1981) and activity staining of protein extracts separated by non-denaturing 4 PAGE (DellaPenna, 1987 supra).

N-terminal sequence analysis of the purified 25 beta-subunit was performed with a Beckman 890M gas phase S* sequenator. Internal beta-subunit proteolytic fragments were generated by digestion with Lys-C and Glu-C endoproteases following instructions supplied by the manufacturer (Promega, Madison, Wisconsin). The resulting proteolytic fragments were resolved by SDS-PAGE, blotted to PVDF membranes and directly sequenced.

Two internal peptide fragments were of interest. The amino acid sequence of the Lys-C peptide was: NH2-Asn-Gly-Asn-Gly-Ala-Asn-Gly-Gln-[?]-Val (SEQ ID NO: The amino acid sequence of the Glu-C peptide was: NH2-Ala-Asn-Ala-Gly-Asp-Gln-Tyr (SEQ ID NO: The underlined portion of these sequences indicates the sequence from which a nucleotide primer was constructed.

-13- These nucleotide primers are presented at SEQ ID NO: 3 (for the Lys-C primer) and 4 (for the Glu-C primer).

These degenerate oligonucleotides were used for library screening and PCR-based MOPAC generation of cDNA probes. One microgram of poly (A)+RNA from immature green, mature green, turning and fully ripe tomato pericarp tissues was used in PCR-based MOPAC reactions.

A

Not I primer-adaptor (Promega, Madison, WI) was used as a primer for first strand cDNA synthesis. Subsequent

PCR

1. 0 amplification cycles utilized a Not I adaptor as the 3' primer and a degenerate 5' primer (SEQ ID NO: derived from the Glu-C beta-subunit protease fragment. Amplified products were electrophoresed, blotted to nylon membranes and probed with a second degenerate oligonucleotide

(SEQ

ID NO: 3) derived from the Lys-C beta-subunit protease fragment. The Lys-C primer recognized a 1.3 kb product generated in the MOPAC reactions. This 1.3 kb product was recovered and amplified by PCR using the Lys-C primer (5'-end) and the Not I adaptor This second 1.25 20 kb MOPAC-derived PCR product was used in conjunction with degenerate oligonucleotides for library screening and Northern analysis. Oligonucleotide 5'-end labelling and random primer DNA labelling were performed following the manufacturer's protocol (BRL, Gaithersburg,

MD).

The cDNA library contained 1.0 x 107 individual recombinants before amplification. For primary screening, replica nitrocellose filters (25,000 pfu/plate) were probed with the degenerate 17-mer Lys-C oligonucleotide described above. Prehybridization was carried out for 4 hours at 37 0 C in a solution of 6 X SSC; 1 X Denhardt's solution; 0.5% SDS; 0.05% sodium pyrophosphate; 100 ug/ml denatured .salmon sperm DNA. Hybridization was carried out overnight at 37 0 C in 6 X SSC; 1 X Denhardt's solution; ug/ml tRNA; 0.05% sodium pyrophosphate. Following hybridization, the filters were washed twice for 5 minutes at room temperature and twice at 37 0 C for 30 minutes in X SSC; 0.05% sodium pyrophosphate. A final wash was performed in 5 X SSC; 0.05% sodium pyrophosphate at 40 0

C

-14for 10 minutes. The filters were exposed overnight with intensifying screens at -80 0

C.

Further rounds of screening were performed at low density using the PCR-generated MOPAC cDNA fragment (described above), the Glu-C oligonucleotide and a degenerate N-terminal oligonucleotide [5'-AT(AG)

TCX

CC(AG) CT(GA) TG(CT) TT(CT) TC (SEQ ID NO: derived from the N-terminal protein sequence. Hybridization conditions for these oligonucleotides were as described 1. 0 above. Hybridization conditions used with the e MOPAC-generated cDNA fragments were as described by Sambook et al 1989, (supra). Following plaque purification, plasmids were rescued by in vivo excision, following the manufacture's protocol (Stratagene).

Double-stranded DNA sequencing was performed.

All the beta-subunit clones we obtained have the restriction pattern illustrated in Fig. 1. We have sequenced two beta-subunit cDNA clones and fragments of 11 others. SEQ ID NO: 6 is the consensus DNA sequence we 20 obtained from the clones. A poly-(A) tail of 35 residues was found at the end of the cDNA but has been removed from SEQ ID NO: 6.

3. Antisense Expression of the Beta-Subunit cDNA Expression of the beta-subunit protein will be modified by introducing Cauliflower Mosaic Virus (CaMV) promoter driven chimeric genes containing a fulllength beta-subunit cDNA in the antisense orientation into various tomato genotypes. This highly expressed constitutive promoter is widely available. Other promoters may also be utilized. The constitutive CaMV promoter will be initially used for the proposed experiments because this promoter has been shown to promote high levels of protein production in most plant organs, including tomato fruit. Kramer, et al., supra.

The feasibility of reducing the expression of tomato fruit genes by antisense RNA technology has been demonstrated by Kramer, et al. (supra). A similar strategy will be employed to inhibit expression of the beta-subunit protein in transgenic tomato plants.

Antisense repression of beta-subunit protein production will greatly decrease the amount of beta-subunit protein available for the formation of PG1, thereby resulting in accumulation of only the PG2 isoforms.

First, an antisense DNA construction will be created.

At a minimum, this DNA construction must contain a promoter effective to promote transcription in tomato plants, an antisense version of a cDNA clone encoding

PG

10 beta-subunit, and a sequence effective to terminate transcription. Via standard molecular biological methods, the CaMV35S promoter sequence will be attached to the beta-subunit cDNA insert. The cDNA insert will be in the antisense orientation. This orientation will be accomplished by attaching the 3' end of the cDNA insert to the promoter. Preferably, the poly-(A) tail will be removed for the antisense construction with the enzyme NruI which will leave a DNA fragment of approximately 1800 bp for antisense construction (approximately 400 bp of 3' 20 sequence plus the poly-A tail will be deleted by this method). Therefore, the antisense RNA strand will be the transcription product.

A suitable termination sequence, such as the nopaline synthase 3' terminator, will be placed downstream from the cDNA insert.

The DNA construction will be placed in an appropriate vector for plant transformation. For Aqrobacterium-mediated transformation, the promoter/cDNA/terminator construction will preferably be placed in a Ti-based plasmid, such as pBIl21, a standard binary vector.

In general, transformation will preferably be done with two standard Aqrobacterium binary vectors: pBIl21 (sold by Clontech Laboratories, Palo Alto CA) and pGA643 (developed by G. An at Washington State University).

pBIl21 contains a CAMV promoter and GUS reporter gene.

The GUS coding sequence will be removed by digesting with SstI and SmaI (blunt end). The beta-subunit DNA fragment to be used will be produced by digesting with SstI (sticky -16end) and NruI (blunt end). The sticky/blunt ends will allow for directional cloning into pBIl21 in the antisense orientation. Standard methods for cutting, ligating and E. coli transformation will be used.

For plant transformation we will follow, in general, the methods of McCormick (1986, Plant Cell Reporter 5:81-84) and Plant Tissue Culture Manual B6:1-9 (1991) Kluwer Academic Publishers. This later reference compiles/compares various procedures for 10 Aqrobacterium-mediated transformation of tomato.

The level of beta-subunit, individual PG isozymes and overall heat stability of total PG activity as a function of time and temperature in the tomato will then be analyzed. Fruit from transgenic antisense tomatoes can be processed and heat-treated to assess levels of PG1 activity. If as the tomato pulp is heated to between 65 0

C

and 90 0 C, and PG activity is halted more rapidly than wild-type fruit, the effort would have been successful.

INFORMATION FOR SEQ ID NO:1: SEQUENCE

CHARACTERISTICS:

LENGTH: 10 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE: NO FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: ORGANISM: Lycopersicon esculentum -17- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: Asn Gly Asn Gly Ala Asn Gly Gin Xaa Val 1 5 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide O (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE: NO FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: ORGANISM: Lycopersicon esculentum 6 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Ala Asn Ala Gly Asp Gin Tyr 1 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: -0 LENGTH: 17 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Oligonucleotide 16 (iii) HYPOTHETICAL:

YES

(iv) ANTI-SENSE:

NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: GGNAAYGGNG

CNAAYGG

INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 17 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Oligonucleotide (iii) HYPOTHETICAL:

YES

(iv) ANTI-SENSE:

NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: AAYGCNGGNG

AYCARTA

INFORMATION FOR SEQ ID SEQUENCE

CHARACTERISTICS:

LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Oligonucleotide (iii) HYPOTHETICAL:

YES

(iv) ANTI-SENSE:

YES

(xi) SEQUENCE DESCRIPTION: SEQ ID IS- ATRTCNCCRC

TRTGYTTYTC

INFORMTION FOR SEQ I D NO:6: SEQUENCE

CHARACTERISTICS:

LENGTH: 2192 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: No (iv) ANTI-SENSE:

NO

(vi) ORIG INAL SOURCE: ORGANISM: Lycopersicon esculentum (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: TCTCTCTCTT CATCTCTGTT TCACACCAAA, GAA.ATGCACA CTAA.AATTCA TCTTCCTCCC TGCATCTTAC TTCTTCTTCT GTTCTCACTA CCATCTTTCA ATGTTGTTGT AGGTGGAGAT 120 GGTGAATCTG GTAACCCATT TACACCCAAA GGTTATCTGA TTAGGTACTG GAAGAAACAA 180 SATCTCAAATG ACTTACAA GCCATGGTTT CTTCTGAACA AGGCATCTCC ATTGAATGCT 240 GCACAATATG CAACTTACAC TAAACTTGTT GCTGATCAAATGCACTCAC CACACAGCTC 300 a -19- CATACCTTTT GCTCTTCAGC AAATCTCATG TGTGCACCAG ATCTGTCAcc AAGTCTTGAA 360 a. a a.

a a

AAACACAGT

ACCAATGAA

CCTGTAAkAT

GGAGGTTCA

ACTTCC TAT'

GCCAATGCT(

GAATTCGTCa t 0 CAGACAGC-,'U

GAAAATCAT'

TACAGAGAT(

GGCGGTGAAC

AC TAG TTACC

AACACTTTCP,

TC CCAAGTT'r

GTTGAGCCCG

CCAGATATAA

TTACCATTTT

TCTCAGTG

GGTGAGACGA

TTGGGTCGA

ATGATTGGAT

CAAACGCTGT

GAAGCGGATA

CACGTGGATA

CC CGGGAAAA~

GATTGAGAAA

'G GAGATATCC C CTGGAATTG, T CTTTCAGGCi G ATGGCAATG' G GCAAATTCAC T CCGATCAAG( 3 GTGACCAGTJ k. GCTATGGAAJ k ATGGGGGAGI V' TCACCAACT;

-AATCGAATGI

CGAATTTCAC

GCAAAGGGGG

LAAGATTATGT

TAGCATCGTT

GAAAGTTTTT

AGGATAAGAT

CTACTTCAAA

AGAAGATGAT

AACGATGTGT

ACGTCGTCGT

CAGTCAAAGG

ACCCTTACTT

TTTTGGACCC

CATCTTCATG

TAGAAGTTTG

AAAAA.AAGAA

A TTTTGCCACT G AGTTAACACT 3 ATATGGTAGA r TATTGACC- 2AAATTACGCG 3AACAGGAGGTj k TTTCAAAAT k TGACACAAAT

SCCAA.AAATTC

TGGTGCTGGA

TGGAGATGAC

CAACTATGGT

TAATGACCCA

CAAAGATACT

GATGGAGGTC

CCGGGAGAAG

GCCTAAAAGG

AATTGCTGAG

CGGCGATGCA 9J TAATTCAGCT

C

TCGAACGACT

G

AATCAACGGT

G

ACTGTATTAC

T

GAATTCAAAG

G

GGGACCGAGTC

TCATTGGATC

T

ATGAAATAAT A

TAC.AGTGACA

TTCAAGAACT

GGTTCTCCCC

AGTTTCAATT

GCGAATGCCA

GTACAGAAAT

TACGG CAAAA

GTTATCGGCT

ACATCTTATG

GGTAATGGTC

ACATTCACTA

CAATCATTCA

CATATCAATT

GCTACATTTT

AACGGTGGTA

kiTGTTGAAGA

C

rCCTTTTTGC

C

'TGAAGAAAA Tj rTGAGTGAGT

G

;AAGATATGA

TI

;AGGATACAAA

~GAAAAGTTA

C

GTCATTCGG

T

TTAAGATCA

A

ACGGAGCGT

T

TTGAGAATG

A'

TGCAAAATT T( AAAACTTTAC CAATTATGGA 420 ACTCTGAAGG AGAAAACATC 480 GTGACAATAA ATTTGACAAT 540 CCTATAGC-AC AAGTACTGCT 600 ATGACCCCAA TCTGCATTTC 660 TCACAATATA CTCACAAGAA 720 ATGGGAATGG TGCTAATGGT 780 CA-ACATTTAC AAATTATGGT 840 GTTTCAACGG CAATGTTCCT 900 CATCTGAAAC TTTTAATAGT 960 CCTATGTTA-A GGATGCAAA 1020 !TGAAGGTAC TGATGTATTC 1080 rCAAAACTTA CGGAGTTAAc 1140 CCAATTACCA CAACAAAACT 1200 UiAAGGTGAA TAACCGGTGG 1260 TGGTACAAT CATGCCTATG 1320 CCGGGTGAT TGCTTCCAAA 1380 'CTTCCACGC CGGTGATGAG 1440 ~TGAAAGAGC ACCCAGCGCC 1500 'TGATTTCGC AACATCAGTG 1560 AGCATCAAA TGGGAATATC 1620 TAAATCAGT ATCATGTCAT 1680 TCCTAAAGT CCGGGTCTAC 1740 TCATGGTGT CGCGATTTGC 1800 TGTCGCACT CGGGTCGGGA 1860 rATGAcTTG GGCAATTGCT 1920 W~ GTGTGTTAA AGAAGAAGAA AAAAGGTACC ACTGGTTTGA CTTTTATAGT AATTA TTATT 2040 11, 1 4

Q-

ATTATACTCT TAATTTATAT TTTGAGTAAT TTTCGTGTAA GTTTCTCTTT GCCTTCATTA 2100 AGTATGAATG GCTATCAATT FACACTATTT GTTATGTAAT CATTTTATTG TTGACTCATA 2160 TTTGAGCAAG GTAATGTAGT TATTGCCAGA TG 2192

S

S

S

SS

S

SS S S S 5*

Claims (14)

1. A method of creating a transgenic tomato containing a lowered level of polygalacturonase isoform 1, comprising the steps of a) providing a DNA sequence that is at least a fifteen nucleotide in length, the DNA sequence having substantial homology to a fragment of SEQ ID NO:6 of comparable length; b) creating a genetic construction including, 5' to 3' a promoter effective in tomato cells, a coding sequence, and a transcriptional terminator, the coding region being derived from the DNA sequence, wherein the DNA sequence from step is positioned so that an antisense polygalacturonase beta-subunit RNA is produced by the construction and wherein the antisense RNA is capable of inhibiting the polygalacturonase beta- subunit; c) transforming a tomato cell with the genetic construction, whereby the tomato cell produces a lowered level of polygalacturonase isoform 1; and 1 5 d) regenerating a tomato plant from the transformed cell, wherein the tomato plant produces tomatoes.

2. The method of claim 1 wherein the transforming step is performed by Agrobacterium-mediated transformation.

3. The method of claim 1 wherein the DNA sequence is a fragment of SEQ ID NO:6.

4. A genetic construction comprised of 5' to 3' in order: a) a promoter capable of expressing a downstream coding sequence in a tomato plant; b) a sequence encoding a RNA of at least fifteen nucleotides the complement of which has substantial homology to a fragment of the mRNA of tomato polygalacturonase beta-subunit set forth in SEQ ID NO:6; and c) a 3' termination sequence. The construction of claim 4 wherein the RNA encoded by the sequence in b) is complementary to a fragment of the mRNA of tomato polygalacturonase beta- subunit set forth in SEQ ID NO:6.

6. A bacteria containing the construction of claim 4 or claim

7. A transgenic tomato fruit, wherein said tomato has lowered levels of the ,j polygalacturonase isoform PG1. [R:\LIBZ]05593.doc:lam 22

8. A transgenic tomato fruit, wherein said tomato has lowered levels of polygalacturonase beta-subunit.

9. A transgenic tomato containing the construction of claim 4 or claim A transgenic tomato plant comprising in its genome a foreign genetic construction comprising 5' to 3' a promoter effective in tomato, a coding region encoding a RNA of at least 15 nucleotides the complement of which has substantial homology to a fragment of the mRNA of the tomato polygalacturonase beta-subunit gene as set forth in SEQ ID NO:6, and a transcriptional terminator, the genetic construction effective in vivo in the tomato fruit to lower the level of production of the polygalacturonase beta-subunit protein.

11. A method of creating a transgenic tomato containing a lowered level of polygalacturonase isoform 1 substantially as hereinbefore described with reference to any one of the Examples. V

12. A transgenic tomato plant obtained by the method of any one of claims 15 1-3 or 11. o

13. Seed of the tomato plant of claim 10 or claim 12. '14. A genetic construction comprised of 5' to 3' in order: a) a promoter capable of expressing a downstream coding sequence in a tomato plant; 20 b) a sequence encoding an RNA of at least fifteen nucleotides the complement of which has substantial homology to a fragment of the mRNA of tomato polygalacturonase beta-subunit set forth in SEQ ID NO:6; and c) a 3' termination sequence substantially as hereinbefore described with reference to any one of the Examples.

15. A transgenic tomato fruit substantially as hereinbefore described with reference to any one of the Examples.

16. A transgenic tomato plant substantially as hereinbefore described with reference to any one of the Examples. Dated 12 November, 2001 The Arizona Board of Regents of Behalf of the University of Arizona Patent Attorneys for the Applicant/Nominated Person C SPRUSON FERGUSON F R: R L BZ

05593.doc:am C F: I