WO1998041612A1

WO1998041612A1 - Cercosporin-degrading bacteria

Info

Publication number: WO1998041612A1
Application number: PCT/US1998/005279
Authority: WO
Inventors: Margaret E. Daub; Thomas K. Mitchell
Original assignee: North Carolina State University
Priority date: 1997-03-17
Filing date: 1998-03-13
Publication date: 1998-09-24
Also published as: AU6470898A; EP1003838A1; ZA982196B; EP1003838A4

Abstract

A method of degrading cercosporin comprises contacting a cercosporin-degrading bacteria to a composition containing cercosporin for a time sufficient to degrade the cercosporin. The cercosporin-degrading bacteria is preferably selected from the group consisting of Xanthomonas campestris pv. pruni, Xanthomonas campestris pv. zinniae, Mycobacterium smegatis, and bacteria able to utilize propane but not propylene as a carbon source. A cercosporin breakdown product is also disclosed.

Description

CERCOSPORIN-DEGRADING BACTERIA

Field of the Invention

The present invention relates to the identification of cercosporin- degrading bacteria, methods of use thereof, and the identification of a cercosporin breakdown product. Cercosporin-degrading bacteria are useful in producing cercosporin-resistant plant varieties.

Background of the Invention Cercosporin is a small (534 mw) lipid-soluble polyketide secreted by many species of Cercospora. While its synthesis is unknown but hypothesized to proceed through the polyketide pathway, much has been learned about its activation and toxicity. Siimilar to other naturally occuring perylenequinones, cercosporin is a photosensitizer. Upon illumination, cercosporin is converted to an electronically activated state. In this state, it reacts with molecular oxygen to produce extremely toxic molecules such as singlet oxygen ('O^. Singlet oxygen has the ability to interact with and destroy cellular membranes (lipids), proteins, and nucleic acids. In plants, the primary site of localization appears to be in the cell and organelle membranes where it peroxidizes lipids. Following peroxidation, a breakdown of the membrane occurs leading to electrolyte leakage, loss of organelle structure, and cell death.

Cercospora species infect tobacco, soybean, corn, coffee, sugar beat, and other crops. Studies utilizing toxin-deficient mutants and studies investigating the effects of light on disease development demonstrate that cercosporin-producing fungi require it for pathogenicity.

Robeson (U.S. Patent No. 5,262,306) describes a method by which organisms able to degrade cercosporin should be identified. Through an extensive screen, we have tested the method of Robeson et al. and have identified additional bacteria able to degrade cercosporin.

Summary of the Invention

A first aspect of the present invention is a method of degrading cercosporin, comprising contacting a cercosporin-degrading bacteria to a composition containing cercosporin for a time sufficient to degrade the cercosporin. The cercosporin-degrading bacteria is selected from the group consisting of Xanthomonas campestris pv. pruni, Xanthomonas campestris pv. zinniae, Mycobacterium smegatis, and bacteria able to utilize propane but not propylene as a carbon source. The contacting step is preferably carried out in the dark, and preferably by combining the bacteria with a cercosporin-containing bacterial growth medium.

A second aspect of the present invention is a cercosporin breakdown product that has a green hue in bacterial culture medium, is soluble in organic solvents at a pH below 3.0, has an absorbance spectrum with dual peaks at about 443 ran and 565 nm, and has a molecular weight of 517 or 518 as determined by fast atom bombardment. In addition to being useful as simply a green pigment, this compound is useful as a marker for detecting the presence or absence of a DNA as described below in transformed cells, and is useful in confirming the identity of DN As as DNAs encoding a cercosporin-degrading protein (by examining the structure of the encoded protein and comparing regions of expected enzyme activity with the activity indicated by the change in structure from cercosporin to the cercosporin breakdown product).

A third aspect of the present invention is an isolated DNA encoding a cercosporm-degrading protein. Preferably, the DNA encodes a protein that converts (in a single step or participates as one of multiples steps) cercosporin to a cercosporin breakdown product as described above. In one embodiment, the DNA is: (a) DNA from a bacteria selected from the group consisting of Xanthomonas campestris pv. pruni, Xanthomonas campestris pv. zinniae, Mycobacterium smegatis, and bacteria able to utilize propane but not propylene as a carbon source; (b) DNA that hybridizes to DNA of (a) above and encodes a cercosporin-degrading protein; and (c) DNA that differs from the DNA of (a) and (b) above due to the degeneracy of the genetic code, and encodes a cercosporin-degrading protein encoded by DNA of (a) or (b) above.

Further aspects of the present invention include vectors and expression cassettes (or chimeric genes) containing the aforesaid DNA, methods of transforming plants therewith, and transformed plants exhibiting a cercosporin- resistant phenotype produced therewith.

Brief Description of the Drawings Figure 1 illustrates the concentration of cercosporin present in cercosporin-cσntaining medium (control) and medium inoculated with XCP-76 (non- degrading isolate) and XCP-77 and XCZ-1 (degrading isolates) at various times after inoculation. Cercosporin concentrations were determined by extraction of cercosporin and quantifying by measuring absorbance of the orgamc extract at 471 nm.

Figure 2 illustrates the absorbance of cercosporin and the cercosporin breakdown product over time. Both compounds were extracted from liquid media containing cercosporin inoculated with isolate XCZ-1 (cercosporin degrader).

Absorbances were measured of the organic extract at 471 nm and 443 nm for cercosporin and the cercosporin breakdown product respectively.

Detailed Description of the Invention

The present invention now will be described more fully hereinafter with reference to the accompanying Figures, in which certain embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. The present invention is drawn to compositions and methods for providing or increasing cellular resistance to cercosporin in organisms that are sensitive to cercosporin. The compositions are proteins and the genes encoding them, which act to provide or increase cellular resistance to cercosporin in such organisms.

The proteins and the genes encoding resistance to cercosporin, and the methods described herein that utilize these compounds, are useful in providing cellular resistance to pathogens that produce cercosporin, and survival of these cells, particularly after pathogen attack. These same compositions and methods also provide or increase resistance to singlet oxygen itself.

One aspect of the invention is drawn to proteins which are involved in conferring resistance to cercosporin. The proteins function to inhibit the spread of infection caused by pathogenic fungi that encode cercosporin, and control resistance to such compounds in a number of organisms, including plants, bacteria, insects and animals. Therefore, the proteins are useful in a variety of settings involving the control of disease and toxicity resistance in plants and other organisms. Modifications of such proteins are also encompassed by the present invention. Such modifications include substitution of amino acid residues, deletions, additions, and the like.

Accordingly, the proteins of the invention include naturally occurring proteins and modifications thereof. Such proteins fmd use in preventing or increasing resistance to cercosporin. The proteins are also particularly useful in protecting organisms against pathogenic infection. In this manner, the organism is transformed with a nucleotide sequence encoding the protein. According to the present invention, organisms transformed in this manner may be plants, bacteria, fungi, and animals, with plants being preferred. The expression of the protein in the organism prevents toxicity and injury caused by singlet oxygen-generating cercosporin, and confers resistance to infection by fungal pathogens.

Selection of Strains Amenable to Molecular Genetic Studies. Of the bacteria that degraded cercosporin, the Xanthomonas isolates are the most attractive for molecular genetic studies. Xanthomonas-compett,-t cloning vectors, transposon- carrying suicide vectors, and the methodologies for manipulating them, are available.

Numerous genes have been cloned from members of this genus following two basic experimental designs. The first is to generate a genomic library to be maintained in a laboratory strain of Escherichia coli, then screen that library for isolates that exhibit the phenotype of interest (in our case the ability to degrade cercosporin). The second strategy is to mobilize a suicide vector carrying the TN5 transposon into the

Xanthomonad of interest and screen for a loss of function or a change in phenotype (in our case, the loss of the ability to degrade cercosporin). Dr. Lindgren has donated the cloning vector pLAFR-6 and transposon-carrying suicide vectors pGS-9, pTN4431, and pSUP-2021. Conjugation experiments were conducted following standard protocols in which the pLAFR-6 carrying E. coli strain was mated to Xanthomonas isolates XCZ-1, XCZ-3, XCP-77, and XCP-76. pLAFR-6 was only able to mobilize into and be maintained in XCZ-1 and XCZ-3, with a frequency of approximately 10^"4 per recipient. In similar experiments, only suicide vector pGS-9 was able to mobilize into and function in these same isolates; it had a significantly low3er frequency of 10^"9 resulting in a few hundred transconjugants per experiment. Subsequent experiments have shown that the TN5 transposon will transpose from - pGS-9 into the recipient genome randomly. Based on this information, XCZ-3 and vector pLAFR-6 and pGS-9 have been chosen to be used in studies aimed at elucidating the genes and mechanisms involved in the bacterial degradation of cercosporin.

Methods are readily available in the art for the hybridization of nucleic acid sequences. Coding sequences from other species may be isolated according to well known techniques based on their sequence homology to the coding sequences of interest. In these techniques, all or part of the known coding sequence is used as a probe which selectively hybridizes to other cercosporin resistance coding sequences present in a population of cloned genomic DNA fragments or cDNA fragments (i.e. genomic or cDNA libraries) from a chosen organism.

For example, the entire sequence of interest, or portions thereof may be used as probes capable of specifically hybridizing to corresponding coding sequences and messenger RNAs. To achieve specific hybridization under a variety of conditions, such probes include sequences that are unique among coding sequences that encode resistance to cercosporin (hereinafter resistance coding sequences), and are preferably at least about 10 nucleotides in length, and most preferably at least about 20 nucleotides in length. Such probes may be used to amplify resistance coding sequences from a chosen organism by the well-know process of polymerase chain reaction (PCR). This technique may be used to isolate additional resistance coding sequences from a desired organism or as a diagnostic assay to determine the presence of resistance coding sequences in an organism. Such techniques include hybridization screening of plated DNA libraries (either plaques or colonies; see, e.g.. Sambrook et al. , Molecular Cloning, eds. , Cold Spring Harbor Laboratory Press (1989)) and amplification by PCR using oligonucleotide primers corresponding to sequence domains conserved among the amino acid sequences (see, e.g. Innis et al. , PCR Protocols, a Guide to Methods and

Applications, eds. , Academic Press (1990)).

For example, hybridization of such sequences may be carried out under conditions of reduced stringency, medium stringency or even stringent conditions (e.g. , conditions represented by a wash stringency of 35-40% Formamide with 5x- Denhardt's solution, 0.5 % SDS and lx SSPE at 37° C; conditions represented by a wash stringency of 40-45 % Formamide with 5x Denhardt's solution, 0.5% SDS, and lx SSPE at 42° C; and conditions represented by a wash stringency of 50% Formamide with 5x Denhardt's solution, 0.5% SDS and lx SSPE at 42° C, respectively), to DNA encoding resistance to cercosporin disclosed herein in a standard hybridization assay. See J. Sambrook et al. , Molecular Cloning, A

Laboratory Manual 2d Ed. (1989) Cold Spring Harbor Laboratory. In general, sequences which code for a cercosporin resistance protein and hybridize to the DNA of interest will be at least 50% homologous, 70% homologous, and even 85% homologous or more with that DNA. That is, the sequence similarity of sequences may range, sharing at least about 50%, about 70%, and even about 85% sequence similarity.

Also provided are mutant forms of the cercosporin resistance gene, and the proteins they encode. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel, T. (1985) Proc. Natl. Acad. Sci. USA 32:488-492; Kunkel et al. (1987) Methods in Enzymol.

154:361-382; US Patent No. 4,873,192; Walker and Gaastra (eds.) Techniques in Molecular Biology, MacMillan Publishing Company, NY (1983) and the references cited therein. Thus, the genes and nucleotide sequences of the invention include both the naturally occurring sequences as well as mutant forms. Likewise, the proteins of the invention encompass both naturally occurring proteins as well as variations and modified forms thereof. The nucleotide sequences encoding the proteins or polypeptides of the invention are useful in the genetic mampulation of organisms, including bacteria, fungi, plants and animals. This aspect of the invention is illustrated herein with respect to the genetic manipulation of plants. In this manner, the nucleotide sequences of the present invention are provided in expression cassettes for expression in the plant of interest. The cassette will include 5' and 3' regulatory sequences operably linked to the gene of interest. The term "operably linked," as used herein, refers to DNA sequences on a single DNA molecule which are associated so that the function of one is affected by the other. Thus, a promoter is operatively associated with a gene of the present invention when it is capable of affecting the expression of the gene of the present invention (i.e. , the gene is under the transcriptional control of the promoter). The promoter is said to be "upstream" from the gene, which is in turn said to be "downstream" from the promoter.

Expression cassettes of the present invention include, 5 '-3' in the direction of transcription, a promoter as discussed above, a gene of the present invention operatively associated with the promoter, and, optionally, a termination sequence including stop signal for RNA polymerase and a polyadenylation signal for polyadenylase (e.g., the nos terminator). All of these regulatory regions should be capable of operating in the cells of the tissue to be transformed. The 3' termination region may be derived from the same gene as the transcriptional initiation region or may be derived from a different gene.

The cassette may additionally contain at least one additional gene to be cotransformed into the organism. Alternatively, the gene(s) of interest can be provided on another expression cassette. Where appropriate, the gene(s) may be optimized for increased expression in the transformed plant.

The expression cassettes may additionally contain 5' leader sequences in the expression cassette construct. Such leader sequences can act to enhance translation. Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5' noncoding region) (Elroy-Stein, O. , Fuerst, T.R. , and Moss, B. (1989) PNAS USA, 56:6126-6130); poty virus leaders, for example, TEV leader (Tobacco Etch Virus) (Allison et al. (1986); MDMV leader (Maize Dwarf Mosaic Virus); Virology, 154:9-20), and human immunoglobulin heavy-chain binding protein (BiP), (Macejak, D.G. , and P. Sarnow (1991) Nature, 555:90-94; untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4), (Jobling, S.A. , and Gehrke, L. , (1987) Nature, 525:622-625; tobacco mosaic virus leader (TMV), (Gallie, D.R. et al. (1989) Molecular Biology of RNA, pages 237-256; and maize chlorotic mottle virus leader

(MCMV) (Lommel, S.A. et al. (1991) Virology, 81:382-385). See also, Della-Cioppa et al. (1981) Plant Physiology, 84:965-968. Other methods known to enhance translation can also be utilized, for example, introns, and the like.

In preparing the expression cassette, the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. Towards this end, adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resection, ligation, PCR, or the like may be employed, where insertions, deletions or substitutions, e.g. transitions and trans versions, may be involved.

The compositions and methods of the present invention can be used to transform any plant, or any portion of a plant thereof. In this manner, genetically modified plants, plant cells, plant tissue, seed, and the like can be obtained.

Transformation protocols may vary depending on the type of plant or plant cell, i.e. monocot or dicot, targeted for transformation. Suitable methods of transforming plant cells include microinjection (Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA, 55:5602-5606, Agrobacterium mediated transformation (Hinchee et al. (1988) Biotechnology,

6:915-921), direct gene transfer (Paszkowski et al. (1984) EMBO J., 5:2717-2722), and ballistic particle acceleration (see, for example, Sanford et al. , U.S. Patent 4,945,050; WO91/10725 and McCabe et αZ. (1988) Biotechnology, 6:923-926). Also see, Weissinger et al. (1988) Annual Rev. Genet., 22:421-477; Sanford et al. (1987) Paniculate Science and Technology, 5:27-37 (onion); Christou et al. (1988) Plant

Physiol. 87: 67 l-674(soybean); McCabe et al. (1988) Bio /Technology, 6:923-926 (soybean); Datta et al. (1990) Biotechnology, S:736-740(rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA, S5:4305-4309(maize); Klein et al. (1988) Biotechnology , 6:559-563 (maize); WO91/10725 (maize); Klein et al. (1988) Plant Physiol, 91 :440-444(maize); Fromm et al. (1990) Biotechnology, 8: 833-839; and Gordon-Kamm et al. (1990) Plant Cell, 2:603-618 (maize); Hooydaas-Van Slogteren & Hooykaas (1984) Nature (London), 311:163-164; Bytebier et al. (1987) Proc. Natl.

Acad. Sci. USA, §4:5345-5349 (Liliaceae); De Wet et al. (1985) In The Experimental Manipulation of Ovule Tissues, ed. G.P. Chapman et al. , pp. 197-209. Longman, NY (pollen); Kaeppler et al. (1990) Plant Cell Repons, 9:415-418; and Kaeppler et al. (1992) Theor. Appl. Genet., 84:560-566 (whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell, 4:1495-1505 (electroporation); Li et al. (1993)

Plant Cell Repons, 2:250-255 and Christou and Ford (1995) Annals of Botany, 75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology, 14:145-150 (maize via Agrobacterium tumefaciens); all of which are herein incorporated by reference.

Plant species may be transformed with the DNA construct of the present invention by the DNA-mediated transformation of plant cell protoplasts and subsequent regeneration of the plant from the transformed protoplasts in accordance with procedures well known in the art.

Any plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with a vector of the present invention. The term "organogenesis," as used herein, means a process by which shoots and roots are developed sequentially from meristematic centers; the term "embryogenesis, " as used herein, means a process by which shoots and roots develop together in a concerted fashion (not sequentially), whether from somatic cells or gametes. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed.

Exemplary tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g. , apical meristems, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem). Plants of the present invention may take a variety of forms. The plants may be chimeras of transformed cells and non-transformed cells; the plants may be clonal transformants (e.g. , all cells transformed to contain the expression cassette); the plants may comprise grafts of transformed and untransformed tissues (e.g. , a transformed root stock grafted to an untransformed scion in citrus species). Plants which may be employed in practicing the present invention include (but are not limited to) tobacco (Nicotiana tabacum), potato (Solanum tuber osum), soybean (glycine max), peanuts (Arachis hypogaea), cotton (Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta) , coffee (Cofea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), Avocado (Persea americana), Fig (Ficus casica), Guava (Psidium guajava), Mango (Mangifera indica), Olive (Olea europaea), papaya (Carica papaya), Cashew

(Anacardium occidentale) , Macadamia (Macadamia integrifolia) , Almond (Prunus amygdalus), sugar beets (Beta vulgaris), corn (Zea mays), wheat, oats, rye, barley, rice, vegetables, ornamentals, and conifers. Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g. , Lactuea sativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathy rus spp.) and members of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis) , and musk melon (C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis) , roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petnunias (Petunia hybrida), carnation (dianthus caryophyllus) , poinsettia (Euphorbia pulcherima), and chyrsanthemum.

Conifers which may be employed in practicing the present invention include, for example, pines such as loblolly pine (Pinus^~taeda) , slash pine (Pinus elliotiϊ), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contona), and Monterey pine (Pinus radiata); Douglas-fir (Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood (Sequoia sempervirens); true firs such as silver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedars such as Western red cedar (Thuja plicatά) and Alaska yellow-cedar (Chamaecyparis nootkatensis) .

The cells which have been transformed may be grown into plants in accordance with conventional ways. See, for example, McCormick et al. (1986)

Plant Cell Repo s, 5:81-84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting hybrid having - lithe desired phenotypic characteristic identified. Two or more generations may be grown to ensure that the subject phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure the desired phenotype or other property has been achieved. When a gene encoding resistance to a cercosporin is included in an expression cassette, the gene may be used in combination with a marker gene, which may be useful in one or more hosts, or different markers for individual hosts. That is, one marker may be employed for selection in a prokaryotic host, while another marker may be employed for selection in a eukaryotic host, particularly the plant host. The markers may be protection against a biocide, such as antibiotics, toxins, heavy metals, or the like; provide complementation, by imparting prototrophy to an auxotrophic host: or provide a visible phenotype through the production of a novel compound in the plant. Exemplary genes which may be employed include neomycin phosphotransferase (NPTII), hygromycin phosphotransferase (HPT), chloramphenicol acetyltransf erase (CAT), nitrilase, and the gentamicin resistance gene. For plant host selection, non-limiting examples of suitable markers are beta-glucuronidase, providing indigo production, luciferase, providing visible light production, NPTII, providing kanamycin resistance or G418 resistance, HPT, providing hygromycin resistance, and the mutated aroA gene, providing glyphosate resistance. Selectable marker genes and reporter genes are known in the art. See generally, G. T. Yarranton (1992) Curr.

Opin. Biotech., 5:506-511; Christopherson et al. (1992) Proc. Natl. Acad. Sci. USA, 59:6314-6318; Yao et al. (1992) Cell, 71:63.-12; W. S. Reznikoff (1992) Mol. Microbiol, 6:2419-2422; Barkley et al. (1980) 77ze Operon, pp. 177-220; Hu et al. (1987) Cell, 45:555-566; Brown et al. (1987) Cell, 49:603-612; Figge et al. (1988) Cell, 52:713-722; and, Deuschle et al. (1989) Proc. Natl. Acad. Aci. USA, 56:5400-

5404. Other genes of interest may additionally be included. The respective genes may be contained in a single expression cassette, or alternatively in separate cassettes. Methods for construction of the cassettes and transformation methods have been described above. As discussed, the genes of the invention can be manipulated to enhance disease resistance in plants. In this manner, the expression or activity of the gene encoding resistance to cercosporin is altered. Such means for alteration of the gene include co-suppression, antisense, mutagenesis, alteration of the sub-cellular localization of the protein, etc. In some instances, it may be beneficial to express the gene from an inducible promoter, particularly from a pathogen inducible promoter. Such promoters include those from pathogenesis-related proteins (PR proteins) which are induced following infection by a pathogen; e.g. , PR proteins, SAR proteins, beta-

1,3-glucanase, chitinase, etc. See, for example, Redolfi et al. (1983) Neth. J. Plant Pathol. 59:245-254; Uknes et al. (1992) 77ze Plant Cell 4:645-656; and Van Loon (1985) Plant Mol. Virol. 4: 111-116.

The present invention is explained in the following non-limiting Examples.

EXAMPLE 1

Screen for Bacteria with

Cercosporin-degrading Ability

To identify additional bacteria with comparable abilities, a screen of 160 isolates, representing over 11 genera containing 19 different species, was conducted (see Table 1 below). Isolates were obtained from collections maintained by Prof. David F. Ritchie and Prof. Peter B. Lindgren of the North Carolina State University (NCSU) Department of Plant Pathology in Raleigh, North Carolina (USA), by Prof. Jerome J. Perry of the NCSU Department of Microbiology, and other faculty of the NCSU Departments of Crop Science and Microbiology. The screen was conducted by stabbing each isolate onto NACE medium (nutrient agar containing 50 μg/ml cercosporin, U.S. Patent No. 5,262,306) and incubated in the dark for ten days at 28 °C. Most isolates in Table 1 were screened a minimum of two times with the first screen resulting in a visual ranking from 1-4 based on the degree of cercosporin decolorization (1 being minimal and 4 being extensive). In the second screen, the medium directly below each colony was removed with a #3 cork borer, gently crushed, and extracted in 250 μ\ of acetone at room temperature in the dark for twelve hours. Remaining agar particles were removed by centrifugation, and the supernatant was assayed by spectrophotometry to determine the concentration of cercosporin. It was determined that this method removes greater than 89% of the cercosporin from the sample. TABLE 1. Bacteria isolates tested for the ability to generate clear zones in cercosporin-containing medium.

As described in U.S. Patent No. 5,262,306, an initial transient discoloration of the medium immediately around the colony was observed for most isolates; this is hypothesized to be due to cercosporin diffusing into the bacterial cells. A few isolates established a permanent and expanded clear zone in the medium indicating the degradation of the cercosporin in that area. Analysis of these areas revealed a loss of over 85 % of the beginning cercosporin, thus giving strong evidence that the bacteria were actively degrading the toxin. Bacterial colonies unable to degrade cercosporin turned purple while those able to degrade did not. Isolates with the greatest ability to degrade cercosporin, as defined by greater than 85 % cercosporin removal, were: Xanthomonas campestris pv. pruni strain XCP-77, 32 strains of X. campestris pv. zinniae (strains XCZ1 through XCZ32), Mycobacterium smegatis strain Sp7EIBW, and unidentified bacteria able to utilize propane as a carbon source. Three isolates able to rapidly degrade cercosporin (XCP-77, XCZ-1 and XCZ-3) were chosen for further investigations, as well as an isolate unable to degrade (x. campestris pv. pruni strain SCP-76), which was used as a negative control. It should be noted that while able to degrade cercosporin in the dark, these isolates did not grow on cercosporin containing medium in the light.

EXAMPLE 2 Time Course of Cercosporin Degradation A series of experiments wer conducted to examine the temporal degradation of cercosporin. In these experiments 1 ml of log phase liquid cultures of XCP-76, XCP-77, and XCZ-1 were inoculated into flasks of LB broth containing 50 μM cercosporin and incubated in the dark at 28°C. Every 12 hours over a 110 hour period, 5 ml of the growing culture was removed and stored at -20 °C until analyzed. Each sample was extracted with 2 ml acetone and 2 ml chloroform. To determine the amount of cercosporin in each sample, the organic phase was assayed with a spectrophotometer and the absorbance was determined at 471 nm. this experiment was performed three separate times with each experiment showing the same results. Results from one of these experiments is shown in Figure 1. Cercosporin is stable in liquid medium as shown by the lack of change in cercosporin levels in control medium. Isolate XCP-76, not a cercosporin degrader, served as a negative control and also caused no loss of cercosprin. XCP-77 and XCZ-1 both decreased the cercosporin in the medium to the point that no detectable levels of cercosprin remained. A difference was observed between these two isolates in all experiments in that XCZ-1 initiated cercosporin breakdown earlier. This may be a function of growth rate differences between the two isolates. Once started, the continued rate and the extent of the degradation was similar with the two isolates.

EXAMPLE 3 Isolation of the Cercosporin Breakdown Product

- As the bacteria begin to degrade cercosporin, there is the emergence of a green hue in the medium as the read disappears (Figure 2). The color was believed to be due to the breakdown product of cercosporin. In attempts to extract, purify, and characterize this compound, it was found that the green compound was water soluble. If the pH of the medium was dropped below 3.0, the compound became soluble in organic solvents, suggesting that it contains phenolic or carboxylic acid groups. Once extracted and concentrated, thin layer chromatography (TLC) experiments were conducted using a solvent system developed by Maura Mead for purifying cercosporin (12:3:3:2:8 ethyl acetate : methanol : acetic acid : water : hexane). The unidentified compound separated into two spots, one running just ahead of the cercosporin front and one significantly below. No other spots were found that differed from thos from extracts of XCP-76 (non-degrader) grown in cercosporin. these two spots were scraped from TLC plates and the compound analyzed by spectrophotometry, fast atom bombardment (FAB), and separated again on TLC. Compounds from both spots have the same absorbance spectrum with dual peaks at approximately 443 nm and 565 nm. When run a second time on TLC, the eluted compounds migrated to the same point above the cercosporin front. This is strong evidence that the two spots represent the same compound. Fast atom bombardment data shows two major peaks at molecular weights 517 and 518 for both spots. The appearance of two peaks one mass unit apart may be a result of certain anomalies associated with the FAB procedure. A molecular weight of 518 is 16 mass units less than that of cercosporin (534). It can be hypothesized that this indicates a loss of a single oxygen molecule possibly through an initial reduction and then the removal of water. The color and spectrum indicate that this compound has the same chromaphore as reduced cercosporin giving the idea that a reduction event is the first step in the degradation process.

EXAMPLE 4 Confirmation of Detoxification

To confirm that the degradation of cercosporin resulted in a product that was not toxic, a cercosprin-sensitive mutant of Cercospora nicotianae (CS-8) was grown on medium that was previously inhabited by XCZ-3. Mutant CS-8 was developed in -our laboratory by ultraviolet irradiation. XCZ-3 was grown in cercosporin containing malt medium for eight days. The bacteria was removed by centrifugation. Mycelial plugs of CS-8 were inoculated into the cleared media and assayed for growth compared to controls in cercosporin containing medium with no prior bacteria and in control medium. In the medium containing degraded cercosporin, CS-8 grew to equivalent levels as cultures growing in medium unamended with cercosporin. In medium with undegraded cercosporin (no XCZ-3), there was no growth. Our conclusion is that the breakdown of cercosporin by XCZ-3 results in a product that is not toxic.

EXAMPLE 5 Isolation of genes necessary for cercosporin breakdown with pLAFR-6 It has been established that the ^"promoters from many X. campestris genes will function normally in E. coli. We will exploit this fact by generating a genomic library of isolate XCZ-3 in pLAFR-6 to be maintained in e. coli strain DH5- a, and plating this library on medium containing cercosporin. We will consider 1,500 individual clones a complete representation of the bacterial genome (C-H. Liao et al., MPMI 9: 14-21 (1996)). Plasmid inserts that confer the ability to degrade cercosporin to E. coli will be isolated and the corresponding gene located. This will be accomplished by first restriction mapping the plasmid harboring the gene of interest. Next, the genomic DNA insert will be digested with various enzymes (as dictated by restriction enzyme sites available), and the resulting fragments of DNA will be sub-cloned into pLAFR-6. Each sub-clone will be tested for the ability to confer the cercosporin-degrading phenotype.

Once the smallest segment of DNA able to confer this phenotype is identified, it will be labeled using the Erase-A-Base kit (Promega Biotech) to produce nested deletions to be sent to The Iowa State Nucleic Acid Facility for sequencing.

The nucleotide and derived amino acid sequence will be analyzed for homology to other sequences using the National Center for Biotechnology Institute BLAST network service. The expression patterns of this DNA sequence will be assayed by first identifying promoter sequences and subsequently cloning them in front of the reporter gene sequences for -gaucuronidase (GUS). The bacterial cells will then be assayed for enzyme activity over time after incubation in cercosporin containing medium following established protocols (S. Fenselau and U. Bonas, MPMI 8: 845-854 (1995)). Further characterization may include mobilizing the gene coding for cercosporin degradation (under the control of fungal promoters and terminators currently used in our laboratory) into cercosporin sensitive fungi and determining if transformants are no longer sensitive to the toxin.

EXAMPLE 6 Isolation of genes necessary for cercosporin breakdown with pGS-9

The distinct possibility exists that screening the library on cercosporin containing medium will not reveal any clones with the desired phenotype.

Concurrently, we will be amassing XCZ-3 mutants through transposon mutagenesis utilizing suicide vector pGS-9. We expect to screen over thirty-five hundred exconjugants in order to find one that is unable to degrade cercosporin (V. Waney et al. MPMI 4: 623-627 (1991)). The loss of this ability will be assayed by stabbing the mutants onto solid medium containing cercosporin and screen for the loss of decolorizing ability. Transposon mediated mutants unable to degrade cercosporin will be characterized for any other phenotypic differences from that of the parent strain.

DNA will be extracted from each mutant for inverted PCR experiments. Inverted

PCR will result in DNA fragments containing part of the Tn5 transposon plus flanking DNA representing sequences of the loci that the transposon inserted into (J.

Rich and D. Willis, Nuc. Acids Resistance 18: 6673-6676 (1990)). In this procedure, the DNA is first cut with the restriction enzyme Eco Rl (this enzyme will not cut in the Tn5 transposon), and then circularized by ligating diluted DNA. The resulting circular DNA fragments are subjected to PCR cycling reactions following standard protocols using primers designed complementary to and extending outward from the Tn5 transposon (Rich and Willis 1990). The amplified DNA will represent sequences into which the transposon inserted. Next, the genomic library will be screened for clones carrying the loci of interest by hybridization of the PCR generated DNA fragment. Positively hybridizing clones will be isolated and the gene(s) conferring the cercosporin-degrading phenotype will be identified and characterized as described above. .

In the event that there is more than one gene controlling the bacteria's ability to degrade cercosporin, transposon mutagenesis experiments will yield non- degrading mutants with different transposon insertion sites. Each gene subsequently identified following the experimental design above, will functionally complement only the mutant strain from which it was initially identified. If this scenario occurs, we will isolate the different genes necessary for cercosporin degradation and dissect the cercosporin degradation pathway while exploring methods to mobilize these into plants.

EXAMPLE 7 Plant Transformation

The gene(s) that confer the cercosporin degrading phenotype are mobilized into a plant expression cassette maintained in Agrobacterium tumefaciens. We will use plasmid vectors that are currently in use in the laboratory (M. Daub et al. Tob. Sci. 38: 51-54 (1994), M. Ni et al., Plant J. 7: 661-676 (1995)). These vectors contain eukaryotic promoter (CaMV 35S and A. tumefaciens octopine and mannopine synthase promoters) and terminator (nopoline synthase) sequences allowing for expression of bacterial genes in plants. In initial experiments, we will transform plants of tobacco cultivar "Burley 21'. Transformed plants will be assayed for expression of the gene(s). Expressing plants will then be assayed for cercosporin resistance and resistance to Cercospora nicotianae. Cercosporin resistance will be assayed by an electrolyte leakage assay of leaf disks as previously described (M. Daub, Plant Physiol. 69: 1361-1364 (1982)). Plants that express detectable levels of cercosporin resistance will be assayed for disease resistance by inoculation with conidia of C. nicotianae (A. Jenns and M. Daub, Phytopathology 85: 906-912 (1995)).

The foregoing examples are illustrative of the present invention, and are not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.

Claims

THAT WHICH IS CLAIMED IS:

1. A method of degrading cercosporin, comprising: contacting a cercosporin-degrading bacteria to a composition containing cercosporin for a time sufficient to degrade said cercosporin; wherein said cercosporin-degrading bacteria is selected from the group consisting of Xanthomonas campestris pv. pruni, Xanthomonas campestris pv. zinniae, Mycobacterium smegatis, and bacteria able to utilize propane but not propylene as a carbon source.

.

2. A method according to claim 1, wherein said contacting step is carried out in the dark.

3. A method according to claim 1 , wherein said contacting step is carried out by combining said bacteria with a cercosporin-containing bacterial growth medium.

4. An isolated DNA encoding a cercosporin-degrading protein.

5. An isolated DNA according to claim 4, wherein said protein converts cercosporin to a compound that: has a green hue in bacterial culture medium; is soluble in organic solvents at. a pH below 3.0; has an absorbance spectrum with dual peaks at about 443 nm and 565 nm; and has a molecular weight of 517 or 518 as determined by fast atom bombardment.

6. An isolated DNA according to claim 4, wherein said DNA is: (a) DNA from a bacteria selected from the group consisting of Xanthomonas campestris pv. pruni, Xanthomonas campestris pv. zinniae,

Mycobacterium smegatis, and bacteria able to utilize propane but not propylene as a carbon source; (b) DNA that hybridizes to DNA of (a) above and encodes a cercosporin-degrading protein; and

(c) DNA that differs from the DNA of (a) and (b) above due to the degeneracy of the genetic code, and encodes a cercosporin-degrading protein encoded by DNA of (a) or (b) above.

7. An expression cassette comprising a chimeric gene, said gene comprising a nucleotide sequence according to Claim 4 operably linked with a promoter.

8. A transformed cell comprising a chimeric gene, said gene comprising a nucleotide sequence according to Claim 4 operably linked with a promoter.

9. A transformed cell according to Claim 8, wherein said organism is a plant cell.

10. A transformed plant comprising a chimeric gene, said gene comprising a nucleotide sequence according to Claim 4 operably linked with a promoter.

11. A method of increasing resistance in a cell to a pathogen, wherein the pathogen produces cercosporin, the method comprising transforming said cell with an isolated nucleic acid molecule of Claim 4.

12. A method according to Claim 16, wherein said cell is a plant cell.