WO2002034925A1

WO2002034925A1 - Use of bacterial acetate kinase and their genes for protection of plants against different pathogens

Info

Publication number: WO2002034925A1
Application number: PCT/US2000/029015
Authority: WO
Inventors: Arthur G. Hunt; Glenn B. Collins; Christopher Lawrence; Qingshun Li; Santanu Dasgupta
Original assignee: University Of Kentucky Research Foundation
Priority date: 2000-10-20
Filing date: 2000-10-20
Publication date: 2002-05-02
Also published as: AU1338301A; AU2001213383B2; BR0017359A; EP1328648A1

Abstract

An isolated gene fragment that encodes for acetate kinase, which confers disease resistance in plants is disclosed. The gene can be cloned into an expression vector to produce a recombinant DNA expression system suitable for insertion into cells to form a transgenic plant transformed with the gene fragment. A method for conferring disease resistance in plants that consists of growing plant host cells transformed with the expression system and expressing the gene conferring disease resistance to impart such resistance to host cells is also disclosed.

Description

USE OF BACTERIAL ACETATE KINASE AND THEIR GENES FOR PROTECTION OF PLANTS AGAINST DIFFERENT PATHOGENS

TECHNICAL FIELD

The present invention generally relates to an isolated gene fragment that encodes for acetate kinase, which confers disease resistance in plants. The gene can be cloned into an expression vector to produce a recombinant DNA expression system suitable for insertion into cells to create a transgenic plant containing the gene fragment. The present invention also relates to a method for conferring disease resistance in plants that consists of growing plant host cells transformed with the expression system and expressing the gene to impart such resistance to host cells. More particularly, the present invention relates to the introduction of bacterial acetate kinase (ack) into plants to induce systemic acquired resistance; a chimeric gene construct containing the ack sequence; an expression vector containing the chimeric gene construct; a plant cell transformed with the chimeric gene construct; a plant tissue transformed with the chimeric gene construct; and a pathogen resistant or disease resistant transgenic plant transformed with the chimeric gene construct.

BACKGROUND ART

Pathogen defense in plants is a varied and complicated process, involving a host of local and systemic events geared towards arresting growth of a pathogen. The cascade of events that lead to pathogen resistance is usually triggered by recognition of any of a range of pathogen-produced molecules (or elicitors (1-3)), and involves rapid local responses (such as ion fluxes and production of reactive oxygen species at the site of ingress (4-6)) and more prolonged local and systemic events (localized cell death, production of poorly understood translocated messengers, and induction of the expression of genes whose products act to limit the spread of pathogens (7,8). These recognition events are often mediated by specific pairs of molecules - pathogen-derived elicitors and cognate plant-encoded receptors (9-15). These receptors, when "activated" by their respective ligands, trigger a large number of subsequent events that are themselves mediated by a number of signaling pathways.

Interestingly, plants undergo similar responses when challenged by pathogenic organisms and by organisms that do not cause disease. Thus, in both instances, systemic induction of so-called defense genes occurs. However, in the case where resistance is to be manifest, a rapid response is apparent and can include localized cell death referred to as the hypersensitive response (HR), while a slower response occurs in cases where disease is the ultimate result. These observations serve to emphasize the fact that the timing of a response is an important factor in disease resistance in plants (16,17). Thus, while much is known about the signaling events involving early (local) and late (systemic) responses to pathogens, much remains to be learned regarding the multiplicity of signaling pathways and the integration of multiple pathways.

SUMMARY OF THE INVENTION

The present invention is directed to a pathogen or disease resistant transgenic plant that is produced by the introduction of a gene encoding acetate kinase into plant cells and plants to increase their resistance to bacterial and fungal pathogens.

The invention provides novel chimeric gene constructs that contain an ack coding sequence. The invention also provides transformed plant cells and transgenic plants transformed with novel chimeric gene constructs that contain the ack coding sequence.

The invention thus provides a method for the expression of the ack gene in the cytoplasm of plant cells and plants.

Additional advantages of the present invention will be set forth in the description and examples that follow, or may be learned from practicing the invention. These and other advantages may be realized and attained by means of the features, instrumentalities and/or combinations particularly described herein. It is also to be understood that the foregoing general description and the following detailed description are only exemplary and explanatory and are not to be viewed as limiting or restricting the invention as claimed.

The invention itself, together with further advantages, will best be understood by reference to the following detailed description, taken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows resistance of AK tobacco plants to wildfire (a bacterial pathogen). (See legends to Figures 2 and 3 for methods.)

Figure 2 shows assembly of the constructs used in plant transformation: cytoplasmic vs. chloroplast-localized AK, untransformed controls.

Figure 3 shows the results of Peronospora parasitica (a fungal pathogen) tests: (A) infected control; (B) spores on infected control; and (C) autofluorescence.

Figure 4 shows resistance of AK plants to Pseudomonas syringe pv. maculicola ES 4326 (a bacterial pathogen). Figure 5 shows the level of PR gene expression in uninoculated AK plants untransformed control lines before inoculation (Figure 5A) and after inoculation (Figure 5B).

DETAILED DESCRIPTION OF THE INVENTION

All patents, patent applications and literatures that may be cited in this description are incorporated herein by reference in their entirety.

In the course of studies dealing with strategies for expressing multiple genes in plants (18), it was observed that tobacco plants that express the E. coli acetate kinase (ack) gene (in the cytoplasm) seemed to retain a healthy appearance in the greenhouse after other neighboring plants (not possessing this gene) had begun to show signs of disease and/or senescence. For this reason, a limited battery of pathogenicity tests was conducted. Specifically, αcfc-transformed tobacco were tested for their responses to Pseudomonas syringe pv. tabaci WF4, a bacterial pathogen of tobacco. The results of these tests were dramatic, in that plants with cytoplasmically localized acetate kinase (AK) were significantly resistant to this pathogen (Figure 1). In contrast, untransformed plants as well as plants expressing a chloroplast-targeted form of AK were susceptible to infection by this pathogen.

To explore this phenomenon in more detail, we elected to express the ack gene in Arabidopsis. For this, two constructs were used - one in which the AK would be localized to the cytoplasm (Figure 2), and one in which the acetate kinase would reside in the chloroplast (termed here as TP-AK (Figure 2)). The results of this process were a number of transgenic Arabidopsis lines that carried the AK and the TP-AK gene. Plants that expressed detectable quantities of the AK gene product were identified by immunoblot analysis (not shown) and several of these selected for further study. The AK-containing Arabidopsis plants thrived and were fertile.

AK Arabidopsis plants proved to be highly resistant to an isolate of Per onospor a parasitica capable of causing disease on the untransformed parent and on TP-AK-containing plants. On untransformed control plants and TP-AK plants, spores were able to germinate and grow throughout infected leaves, and eventually into other parts of the plant (Figure 3 A). In contrast, on AK plants, spores rarely germinated, and those that did grew only a minimal distance (Figure 3B). In addition, spore germination and invasion on AK plants was accompanied by the appearance of localized autofluorescent material (Figure 3C) indicative of a strong hypersensitive response. The combination of limited germination and growth resulted in a virtual lack of disease symptoms in inoculated plants (not shown). This resistance corroborates the results obtained with the tobacco lines, and suggests that AK expressing Arabidopsis can respond very rapidly to limit the growth and spread of the oomycete pathogen. In addition, the lack of resistance that was observed in TP-AK plants indicates that a specific subcellular location is required for the effects of the ack gene.

Additional tests were conducted with a bacterial pathogen (Pseudomonas syringaepv maculicola ES 4326). As was observed with P. parasitica, AK-containing Arabidopsis were highly resistant to this pathogen. Specifically as seen in Figure 4, a minimum of bacterial growth was seen on the AK plants, in contrast to the extensive growth observed on the untransformed and TP-AK plants.

Disease resistance in transgenic plants is often associated with elevated and constitutive expression of a range of genes associated with systemic acquired resistance (19). Accordingly, the levels of expression of so-called PR genes (20) were determined in various of these lines. In uninoculated AK plants, a low but significant level of PR gene expression was observed (Figure 5). In contrast, with untransformed control lines, no detectable PR gene expression was apparent in the absence of inoculation with pathogens (Figure 5). After inoculation with P. parasitica, PR gene expression was dramatically induced in the AK plants (Figure 5), beginning in two days and reaching the highest levels after 4 days. In contrast, for as much as 2 days, PR gene expression remained very low in control plants that had been inoculated with P. parasitica (Figure 5). Interestingly, at later times after inoculation, PR gene expression in control plants rose to levels that exceeded those seen in AK plants, even though the control plants developed disease symptoms.

The presence of a low level of PR gene expression in uninoculated AK plants suggests that the expression of the ack gene in Arabidopsis induces constitutive systemic acquired resistance (SAR), perhaps analogous to that seen in several Arabidopsis mutants (19, 24-29). However, the very low PR gene expression in these plants, and the rapid increase in these levels upon challenge with pathogens, is distinctive. In addition, the deposition of autofluorescent material near the sites of germinating spores (Figure 3C), along with the altered kinetics of response of PR gene expression and SA synthesis in the AK plants, suggests that the expression of the ack gene in Arabidopsis has in some way conditioned these plants for a rapid response to what is otherwise a virulent pathogen. In other systems, the difference between rapid response (and subsequent HR and resistance) and slow response (thereby permitting disease development) has been linked to the ability of the host plant to recognize specific pathogen-derived signals (elicitors (30)). One might hypothesize that the expression of the ack gene in Arabidopsis in some way mimics this recognition process, perhaps by elevating the expression of resident (unexpressed) R genes in Arabidopsis. However, other properties of the AK plants argue against this. For example, tomato plants that express the Pto gene in a constitutive manner are resistant to a range of pathogens not normally dependent upon Pto-mediated elicitor recognition, but these plants also possess micro,-HR lesions and high constitute SA and PR gene expression (31). Likewise, plants that express various R-gene-independent harpin genes (such as hrmA) are also resistant to a broad range of pathogens, and possess elevated SA and PR gene expression (32,33). In contrast, uninoculated AK plants lack detectable microlesions and have normally modest S A (not shown) and PR gene expression levels.

In bacteria, the expected metabolic product of the expression of the ack gene (acetyl phosphate) can gratuitously phosphorylate two-component receiver modules in bacteria, and such modifications may play important roles in the regulation of responses to osmotic changes or phosphate status (34-36). Moreover, phosphorylated acetate kinase can itself transfer this phosphate to enzyme I of the phosphoenolpyruvate:glucose phosphotransferase system (PTS; 37). Given these precedents, our results suggest an involvement of a two-component-like signaling pathway in the development of the hypersensitive response in plants. The effects of the ack gene in Arabidopsis would thus be explained by analogy with prior studies in bacteria - one (or more) of the components of this hypothetical pathway would be gratuitously phosphorylated (by phosphorylated acetate kinase, as is seen with enzyme I of the PTS (37), or by acetyl phosphate (34-36), thereby altering the activity status of the rest of the pathway, resulting in an ability to respond rapidly and effectively to otherwise pathogenic microorganisms. This eventuality would insert an additional layer of complexity in the defense signaling network in plants. In particular, it would imply a separate signaling pathway that functions to facilitate rapid local and systemic defense responses.

At this time, however, an indirect effect is equally likely. As is to be expected, defense responses in plant are integrated with many other signaling pathways; these include pathways that mediate the actions of ethylene, cytokinin, jasmonate. Interestingly, two- component-like factors have been implicated in ethylene (38) and cytokinin signaling (39-41). The general appearance of the AK plants is relatively normal and does not suggest a dramatic and general effect on ethylene- or cytokinin- related responses. However, given the multiplicity of histidine-kinase-related ethylene receptors in Arabidopsis (38,42), and a similar multiplicity of cytokinin-inducible response regulators (39-41), it remains possible that selective modification of a small subset of these can lead to the pre-conditioning for rapid defense responses that is seen in AK plants. This model is intriguing in that it suggests that rapid defense responses might be affected, or conditioned, by factors in addition to pathogen recognition.

The present invention demonstrates that the expression of the E. coli acetate kinase gene in Arabidopsis can pre-condition Arabidopsis for rapid and effective defense responses against otherwise pathogenic microorganisms. This invention indicates that rapidity of response can be manipulated in novel ways, without grossly changing the health of the plant. It also shows that rapidity of response is a viable target for modification as it relates to improving the disease defense characteristics of crop plants. Finally, the invention suggests that two-component signaling systems may be directly or indirectly involved in determining the rapidity with which Arabidopsis responds to challenge by pathogens.

Bacterial Acetate Kinase Gene

By bacterial acetate kinase or "aclt" gene or "AK" protein, any gene encoding acetate kinase that is derived from a bacteria is meant. Preferably, the bacteria may be Klebsiella, Propionibacterium, Corynebacterium, Aerobacter, Alcaligenes, Micrococcus, and Escherichia. More preferably, the bacteria is Escherichia coli.

Alternatively, the acetate kinase gene may be a polynucleotide sequence which hybridizes to the polynucleotide of the E. coli acetate kinase gene under 5X SSC and 42°C wash conditions.

Sequence Identity

Two sequences are said to be "identical" if the sequence of residues is the same when aligned for maximum correspondence as described below. The term "complementary" applies to nucleic acid sequences and is used herein to mean that the sequence is complementary to all or a portion of a reference polynucleotide sequence.

Optimal alignment of sequences for comparison can be conducted by the local homology algorithm of Smith and Waterman, Add. Appl. Math. , 2:482 (1981), by the homology alignment method of Needleman and Wunsch, J Mol. Biol, 48:443 (1970), by the search for similarity method of Pearson and Lippman, Proc. Natl. Acad. Sci. USA, 85:2444 (1988), or the like. Computer implementations of the above algorithms are known as part of the Genetics Computer Group (GCG) Wisconsin Genetics Software Package (GAP, BESTFIT, BLASTA, FASTA and TFASTA), 575 Science Drive, Madison, WI. "Percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the sequence in the comparison window may comprise additions or deletions (i.e. "gaps") as compared to the reference sequence for optimal alignment of the two sequences being compared. The percentage identity is calculated by determining the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window and multiplying the result by 100 to yield the percentage of sequence identity. Total identity is then determined as the average identity over all of the windows that cover the complete query sequence.

Recombinant DNA Constructs

Recombinant DNA constructs comprising one or more of the DNA or RNA sequences described herein and an additional DNA and or R A sequence are also included within the scope of this invention. These recombinant DNA constructs have sequences which do not occur in nature or exist in a form that does not occur in nature or exist in association with other materials that do not occur in nature. The DNA and or RNA sequences described hereinabove are "operably linked" with other DNA and/or RNA sequences. DNA regions are operably linked when they are functionally related to each other. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein which participates in the secretion of the polypeptide; a promoter is operably linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation. Generally, operably linked means contiguous (or in close proximity to) and, in the case of secretory leaders, contiguous and in reading phase.

Vectors

The invention is further directed to a replicable vector containing the ack gene sequence and cDNA which may code for a polypeptide and which is capable of expressing the polypeptide under the transcriptional control of a promoter. The vector is transferable to the host organism. Preferably, the host organism is a plant or plant cell. The vector may be an integrating or non-integrating vector and is conveniently a plasmid.

Transformed Cells

The invention further relates to a transformed cell or microorganism containing cDNA or a vector which codes for the polypeptide or a fragment or variant thereof and which is capable of expressing the polypeptide.

Plant Cell Expression Systems

Plant Vectors h plants, transformation vectors capable of introducing DNAs containing the ack gene are easily designed, and generally contain one or more DNA coding sequences of interest under

the transcriptional control of 5' and 3' regulatory sequences. Such vectors generally comprise, operatively linked in sequence in the 5' to 3' direction, a promoter sequence that directs the transcription of the ack gene in a plant; optionally a 5' non-translated leader sequence; a nucleotide sequence that encodes a protein of interest; and a 3' non-translated region that encodes a polyadenylation signal which functions in plant cells to cause the termination of transcription and the addition of polyadenylate nucleotides to the 3' end of the niRNA encoding said protein. Plant transformation vectors also generally contain a selectable marker. Typical 5'-3' regulatory sequences include a transcription initiation start site, a ribosome bmding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal. Vectors for plant transformation are described (Schardl et al., Gene 61, 1-14, (1987); Plant Mol Biol., 25:989-994 (1994)). Particularly useful vectors for this invention include, but are not limited to (pKYLX71, pPZP family, pKYLX71-35S2).

Plant Transformation and Regeneration

A variety of different methods can be employed to introduce such vectors into plant trichome, protoplasts, cells, callus tissue, leaf discs, meristems, etc., to generate transgenic plants, including ylgrob ctert^'ttm-mediated transformation, particle gun delivery, microinjection, electroporation, polyethylene glycol-mediated protoplast transformation, liposome-mediated transformation, etc. In general, transgenic plants comprising cells containing and expressing DNAs encoding various enzymes can be produced by transforming plant cells with a DNA construct as described above via any of the foregoing methods; selecting plant cells that have been transformed on a selective medium; regenerating plant cells that have been transformed to produce differentiated plants; and selecting a transformed plant which expresses the enzyme-encoding nucleotide sequence.

The DNAs can be introduced either in a single transformation event (all necessary DNAs present on the same vector), a co-transformation event (all necessary DNAs present on separate vectors that are introduced into plants or plant cells simultaneously), or by independent transformation events (all necessary DNAs present on separate vectors that are introduced into plants or plant cells independently). Traditional breeding methods can subsequently be used to incorporate the entire pathway into a single plant. Specific methods for transforming a wide variety of dicots and obtaining transgenic plants are well documented in the literature.

Successful transformation and plant regeneration have been achieved in the monocots as follows: asparagus (Asparagus officinalis; Bytebier et al, Proc. Natl. Acad. Sci. USA, 84:5345 (1987)); barley (Hordeum vulgarae; Wan and Lemaux, Plant Physiol., 104:37 (1994)); maize (Zea mays; Rhodes et al, Science, 240:204 (1988); Gordon-Kamm et al, Plant Cell, 2:603 (1990); Fromm et al, Bio/Technology, 8:833 (1990); Koziel et al, Bio/Technology, 11:194 (1993)); oats (Avena saliva; Somers et al, Bio/Technology, 10:1589 (1992)); orchardgrass (Dactylic glomerata; Horn et al, Plant Cell Rep., 7:469 (1988)); rice (Oryza saliva, including indica and japonica varieties; Toriyama et al, Bio/Technology, 6:10 (1988); Zhang et al, Plant Cell Rep., 7:379 (1988); Luo and Wu, Plant Mol. Biol. Rep., 6:165 (1988); Zhang and Wu, Theor. Appl. Genet, 76:835 (1988); Christou et al, Bio/Technology, 9:957 (1991)); rye (Secale cereale; De la Pena et al, Nature, 325:274 (1987)); sorghum (Sorghum bicolor; Cassas et al, Proc. Natl. Acad. Sci. USA; 90:11212 (1993)); sugar cane (Saccharum spp.; Bower and Birch, Plant J., 2:409 (1992)); tall fescue (Festuca arundinacea; Wang et al, Bio/Technology, 10:691 (1992)); turfgrass (Agrostis palustris; Zhong et al, Plant Cell Rep., 13:1 (1993)); and wheat (Triticum aestinum; Nasil et al, Bio/Technology, 10:667 (1992); Weeks et al, Plant Physiol., 102:1077 (1993); Becker et al, Plant J., 5:299 (1994)).

Relevant Plants

Particularly useful plants for insertion of the ack gene include plant and ferns of the genus: Populus, Ermophilia, Lycopersicon, Νicotiana, Cannabis, Pharbitis, Apteria, Psychotria, Mercurialis, Chrysanthemum, Polypodium, Pelargonium, Polytrichiales, Mimulus, Chamomile, Monarda, Solanum, Achillea, Naleriana, Ocimum, Medicago, Aesculus, Newcastelia, Plumbago, Pityrogramma, Phacelia, Avicennia, Tamarix, Frankenia, Limonium, Foeniculum, Thymus, Salvia, Kadsura, Beyeria, Humulus, Mentha, Artemisia, Nepta, Geraea, Pogogstemon, Majorana, Cleome, Cnicus, Parthenium, Ricinocarpos, Parthenium, Hymenaea. Larrea, Primula, Phacelia, Dryopteris, Plectranthus, Cypripedium, Petunia, Datura, Mucuna, Ricinus, Hypericum, Myoporum, Acacia, Diplopeltis, Dodonaea, Halgania, Cyanostegia, Prostanthera, Anthocercis, Olearia, Niscaria.

Production of Transgenic Plants Comprising a Gene or Multiple Genes of Interest Plant transformation vectors capable of delivering DΝA (genomic DΝAs, plasmid DΝAs, cDΝAs, or synthetic DΝAs) can be easily designed. Various strategies can be employed to introduce these DΝAs to produce transgenic plants capable of biosynthesizing high levels of

a gene product of interest including:

1. Transforming individual plants with an encoding DΝA of interest. Two or more transgenic plants, each containing one of these DΝAs, can then be grown and cross-pollinated so as to produce hybrid plants containing the two DΝAs. The hybrid can then be crossed with the remaining transgenic plants in order to obtain a hybrid plant containing all DΝAs of interest within its genome.

2. Sequentially transforming plants with plasmids containing each of the encoding

DΝAs of interest, respectively.

3. Simultaneously cotransforming plants with plasmids containing each of the encoding

DΝAs, respectively.

4. Transforming plants with a single plasmid containing two or more encoding DΝAs of interest. 5. Transforming plants by a combination of any of the foregoing techniques in order to obtain a plant that expresses a desired combination of encoding DNAs of interest.

Traditional breeding of transformed plants produced according to any one of the foregoing methods by successive rounds of crossing can then be carried out to incorporate all the desired encoding DNAs in a single homozygous plant line (Nawrath et al, 1994; PCT International Publication WO 93/02187).

The use of vectors containing different selectable marker genes to facilitate selection of plants containing two or more different-encoding DNAs is advantageous. Examples of useful selectable marker genes include those conferring resistance to kanamycin, hygromycin, sulphonamides, glyphosate, bialaphos, and phosphinothricin.

Stability of Transgene Expression

As several overexpressed enzymes may be required to produce optimal levels, the phenomenon of co-suppression may influence transgene expression in transformed plants. Several strategies can be employed to avoid this potential problem.

One commonly employed approach is to select and or screen for transgenic plants that contain a single intact copy of the transgene or other encoding DNA. Agrobacterium-mediated transformation technologies are preferred in this regard.

Inclusion of nuclear scaffold or matrix attachment regions (MAR) flanking a transgene has been shown to increase the level and reduce the variability associated with transgene expression in plants. Flanking a transgene or other encoding DNA with MAR elements may overcome problems associated with differential base composition between such transgenes or encoding DNAs and integration sites, and/or the detrimental effects of sequences adjacent to transgene integration sites.

The use of enhancers from tissue-specific or developmentally-regulated genes may ensure that expression of a linked transgene or other encoding DNA occurs in the appropriately

regulated manner.

The use of different combinations of promoters, plastid targeting sequences, and selectable markers in addition to the trichome-specific regulatory sequence, for introduced transgenes or other encoding DNAs can avoid potential problems due to trans-inactivation in

cases where pyramiding of different transgenes within a single plant is desired.

Finally, inactivation by co-suppression can be avoided by screening a number of

independent transgenic plants to identify those that consistently overexpress particular introduced encoding DNAs. Site-specific recombination in which the endogenous copy of a gene is replaced by the same gene, but with altered expression characteristics, should obviate

this problem.

Any of the foregoing methods, alone or in combination, can be employed in order to

insure the stability of transgene expression in transgenic plants of the present invention.

EXAMPLES

Methods

The AK coding sequence was isolated by PCR from E. coli genomic DNA as described by Dasgupta et al. (18). This gene was subcloned into pKYLX71 :35S (21) as an Xhol-Sacl fragment. The TP-AK gene was assembled basically as described by Dasgupta et al. (18) and also subcloned into pKYLX71:35S as an Xhol-Sacl fragment. The recombinant plasmids were mobilized into Agrobacterium tumefaciens C58C1 :pGV3850 (22) and transconjugants used to transform Arabidopsis (ecotype Columbia) by vacuum infiltration. Transformed plants were identified by selection on agarose media containing kanamycin (50 μg/ml). Expression of the transgene was assessed by immunoblot analysis. 500 mg of leaves from transgenic plants were homogenized in 1 ml of extraction buffer (0.0625 M Tris-HCl, pH 6.8/10% glycerol/2% SDS/10% 2-mercaptoethanol), boiled for 10 min and centrifuged at 12,000g for 10 min. 50 μg of total protein were separated on 12.5% polyacrylamide gel containing SDS, transferred to nitrocellulose membrane, and probed with the appropriate antisera (23). Antigen-antibody complexes were visualized using HRP conjugated anti-rabbit

IgG using a chemiluminesence kit (NEN Life Sciences).

Resistance to fungal infections was assessed using a compatible isolate Peronospora parasitica noco2. For inoculations, spores were collected from sporulating susceptible

arabidopsis plants, washed with deionized water, and suspended in water to a concentration of 50,000 spores/ml. Plants were sprayed with this suspension and kept in a high humidity growth chamber. Plants were examined visually and microscopically as shown. Autofluorescence was visualized using a fluorescent microscope under UV light.

Pseudomonas syringe pv. maculicola ES 4326 was grown in liquid Luria Bertani (LB)

medium with lOOmg/1 streptomycin at 28°C overnight, collected by centrifugation, and

resuspended in 10 mM MgCl₂, and adjusted to cell density of OD600=1-0- The OD600 ⁼ 1-0

bacterial suspension was diluted 250 times, then 10 μl were infiltrated to the leaves. After 5 days, leaves were collected, washed to remove incipient bacteria, and the bacteria counted by plating serial dilutions on media. PR gene expression was measured by northern blot analysis (23). Total RNA was isolated from the leaves of 2-3 week old plants, or the plants spread by E. parasitica , using the RNAqueous total RNA isolation Kit (Ambion Inc., Austin, Texas). Equal amounts of total RNA (10 μg) were resolved on a 1.2% (w/v) agarose/formaldehyde gel containing 1 X 3-(N-morpholino-)propanesulfonic acid (MOPS) buffer, and transferred to a Νytran membrane (Schleicher & Schuell, Keene, ΝH). Membranes were hybridized with a

32 P-labeled DΝA probe generated using the Prime-it II Random Primer Labeling kit (Stratagene, La Jolla, Ca), washed using standard protocols (23), and visualized by autoradiography on X-ray film.

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Claims

WHAT IS CLAIMED IS:

1. An isolated gene fragment conferring bacterial and viral pathogen resistance to plants by responding to an avirulence gene in bacterial and fungal plant pathogens, wherein said gene fragment encodes for acetate kinase.

2. A chimeric gene construct comprising: a promoter; and the isolated gene fragment of claim 1.

3. A recombinant DNA expression system comprising: an expression vector; and the isolated gene fragment of claim 1.

4. A plant cell transformed with the chimeric gene construct according to claim

2.

5. A transgenic plant transformed with the chimeric gene construct according to claim 2, wherein said transgenic plant is resistant to pathogen.

6. A method of conferring disease resistance to plants comprising: growing plant cells transformed with the recombinant DNA expression system according to claim 3; and expressing the DNA in said plant cells to confer disease resistance therein.

7. A vector comprising the chimeric gene construct according to claim 2.

8. A method of making a pathogen-resistant plant, comprising: growing plant cells transformed with the recombinant DNA expression system according to claim 3; and expressing the DNA in said plant cells to confer disease resistance therein.

9. The method according to claim 8, comprising testing the plant for pathogen resistance.

10. The method according to claim 9, wherein the acetate kinase is bacterial acetate kinase.