MXPA98007696A - Fungal gene that confirms fitotoxin resistance cercospor - Google Patents

Abstract

The present invention is directed to sequences of nucleic acids and amino acids that are responsible for moving the fungal toxin cercosporin through the plasma membrane of living cells, the DNA can be introduced to a plant using conventional transformation methods in order to confer to the plants resistance to cercospori

Description

SEN FUNGAL THAT CONFIRMS RESISTANCE TO FITOTQXIN CERCOSPORINE BACKGROUND ^ INQUIRY FIELD OF THE INVENTION This invention relates to nucleic acid and amino acid sequences that regulate the cercosporin polypeptide toxin, gene constructs and methods related thereto. It also relates to the use of a nucleic acid to manipulate plants by genetic engineering techniques to obtain resistance to the toxin.

DESCRIPTION OF THE PREVIOUS TECHNIQUE Fungi of the genus Cercospora are economically important widespread pathogens of a diverse range of crop plants including. for example, banana, sugar beet, coffee »tobacco, corn, sorghum. peanut and soybean (Citrus, Plant Pathology, 3rd ed., Academic Press, San Diego, Calif., 356-357, 1988). Phytosis resistance to cercospor a »produced by many species of phytopathogenic fungi of the genus Cercospora. It has not been found in all commercial crops. Cercosporin is the main disease factor in the purple spot of the seeds of Soy and other crop diseases caused by this fungus. Corn producers in the United States have experienced increasingly serious outbreaks of the gray leaf spot due to Cercospora zeae-maydis. The lack of cultivars of highly resistant plants is crucial. particularly early maturing corn, in the central states of the United States. With a total value in the United States of more than 16,000 million dollars "corn producers, state economies and commercial hybrid seed producers. face potentially dramatic economic losses due to gray leaf spot. The situation with the solas, although significant, is less dramatic. The losses of soybean production due to the purple spot of seeds have averaged approximately 1 to 254 or less in recent years. However, »soybean cultivation in the United States is now estimated at more than 11,000 million dollars. The presence of more than 554 bean seeds that exhibit purple spot on the market "results in significantly lower producer prices due to lower bean quality and the additional processing required to treat bean seeds that show the illness. Further. Substantial crop losses have been attributed to the foliar phase of the disease in the southern states of the United States when weather conditions favor disease. A new approach to the management of disease in crops »is the use of genes for resistance derivatives of the pathogen. Scientists have traditionally used cross-breeding and hybridization techniques to provide plants that have particular desired characteristics such as "better resistance", nutritional value, "taste," appearance, and disease resistance, etc., "but these techniques are at best durable procedures that consume time and that do not necessarily result in the achievement of a particular goal. In the case of soybean, an oilseed of great importance to the world economy, the search for improved or durable resistance to Cercospora is complicated by the fact that the susceptibility of the cultivar to foliar and seed infections is not close. relationship. In addition »some cultivars show resistance to seed stain and susceptibility to leaf blight. The arrival of genetic engineering provides the opportunity to introduce genetic material directly into a plant "that after being expressed in it" would result in a plant with resistance to cercosporia. Polids such as cercosporin, for example, are products of secondary metabolism in bacteria, fungi and plants. This group of compounds includes important bacterial and fungal antibiotics »plant flavonoids and roicotoxins and phytotoxins of fungal origin (Hopwood and others» Annual Review of Genetics »Volume 24» 37-66 »1990). Many phytopathogenic fungi of the genus Cercospora produce the Polypeptide of red toxin cercosporin (Daub »Phytopathology» Volume 72 .. 370-374, 1982 »Lynch et al., Trans. Br. Mycol. Soc., Volume 69» 496-498, 1977) which was isolated for the first time from C K Kuchi («uyama and others» J. Am. Chem. Soc. »Volume 79. 5725-5762» 1957). The structure of the cercosporin a »a derivative of the red peronenoane Cl» 12-bis (2-hydroxypropyl 1) -2 »ll dimethoxy-6,7-methyndiioxy-4.9-dihydroxyperi leno-3» 10-qui ona »Molecular weight: 534], was determined by Lousberg et al. (J. Chem. Soc. Chem. Co mun., 1971: 1463-1464» 1971) and YamazaKi et al. (Agrie. Biol. Chem. Volume. 36, 1707-1718, 1972). Cercospor a is a non-specific host toxin that, in the presence of light, interacts with molecular oxygen to produce superoxide radicals and individual band oxygen (Daub et al. »Plant Physiol.» Volume 73 »855-857» 1983). These activated oxygen species cause lipid peroxidation of the cell membrane, which results in electrolyte leakage »decreased membrane fluidity» and cell death (Daub »ACS Sy p. Ser., Volume 339. 271- 280. 1987). Several lines of evidence indicate that cercosporin plays an essential role in the pathogenesis of Cercospora: absolutely high intensity of light is required for the development of both the disease (Calpouzos »Ann. Rev. Phytopathology, Volume 4. 369-390» 1966 Calpouzos et al., Phytopathology »Volume 57, 799-800, 1967) as well as the action of the toxin (Daub, 1982 cited above); the toxin can be isolate from naturally infected tissues (Fajóla, Physiol, Plant Pathol., Volume 13, 157-164, 1978, Upchurch et al., Appl. Env, Microbiol., Volume 57 (10), 2940-2945, 1991); the application of the toxin alone can produce symptoms of disease in host plants (Bal s and others »Phytopathology» Volume 61, 1477-1484, 1971, Fajóla, 1978 »cited above)» and the mutants of Cercospora KiKuchi i that do not produce the toxin they do not induce symptoms of disease in soybean plants (Upchurch »1991» cited above). Although little is known about the biosynthesis of cercosporin. the results of nuclear magnetic resonance and mass spectrometry analysis have indicated a pathway for the synthesis of the polyketide "and an unstable intermediate of polyketone or has been proposed" but has not been isolated (Okubo et al., Agrie. Biol. Chem. , Volume 39. 1173-1175, 1975). No enzyme or chemical intermediate in the biosynthetic pathway of cercosporin has been identified. The identification and isolation of a gene that confers an important level of resistance to cercosporin-producing microorganisms would allow the development of crops resistant to fungal diseases caused by this toxin. Although there are no reports of resistance to cercosporin in crop plants. Batchvarova et al. (Phytopathol .. Volume 82 »1477-1484» 1992) describe a type of resistance to cercosporin in a common weed. The annual weed »Louisiana red rice» is resistant to all known breeds of Cercospora oryzae. and exhibits resistance to cercosporin. In sensitive rice plants, it was shown that cercosporin accumulates in the plant tissue, a phenomenon that has been observed in soybeans. It was assumed that the resistance observed in Louisia red rice a »is due to a combined effect of the active efflux of the toxin from resistant cells» possibly associated with the degradation of cercosporin or the action of carotenoids in the extinction of oxygen species active The Patent of E.U.A. No. 5,262,303 (Robeson et al.) Describes bacteria resistant to cercosporin that have the ability to degrade it. The patent also refers to the fact that the gene responsible for this characteristic of degradation of the cercosporin can be isolated and cloned into an appropriate vector and can be inserted into a plant. To date, no culture plants resistant to cercosporin have been discovered. Therefore, the development of varieties of transgenic plants resistant to cercosporin would be a useful approach to the control of plant diseases induced by Cercospora. The present invention "described below" provides a direct means for manipulating plants by genetic engineering techniques and exhibiting resistance to this universally toxic polyketide "cercosporin" which is different from the prior art.

BRIEF DESCRIPTION OF THE INVENTION Therefore, an object of the present invention is to provide DNA capable of conferring resistance to cercosporin in plants. Another objective of the invention is to provide DNA for the expression of Cercospora KiKuchi i cercosporin membrane pump protein. Another objective of the present invention is to provide a protein capable of conferring resistance to cercosporin in plants. A further object of the invention is to provide a protein that is a protein of the membrane pump of the cercospor. A further object of the present invention is to provide a hybrid plant resistant to cercosporin that produces progeny with resistance to cercosporin. Another objective of the present invention is to provide a vector containing a DNA sequence capable of conferring resistance to cercosporin in plants. Yet another objective of the present invention is to provide a vector containing a DNA sequence for the expression of a protein that is a protein of the cercosporin membrane pump. Another objective of the present invention is to provide a transformed prokaryote containing a vector with a DNA sequence for the expression of a membrane pump protein from the cercospori a. Yet another object of the present invention is to provide a method for conferring resistance to cercosporin in plants using a plant transformation vector containing a DNA sequence for the expression of a protein from the cercosporin membrane pump. Other objects and advantages of the present invention will become apparent from the following description.

DEPOSIT OF MICROORGANISMS The cDNA LE6 (cfP) of the present invention, known as plasmid cLE6-cfp »was deposited in accordance with the provisions of the Budapest Treaty with the American Type Culture Collection» 12301 Par lawn Drive »RocKville» Maryland 20852 on March 15, 1996. The access number is 97482 from ATCC.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a Northern hybridization analysis of PR of C. kiKuchii wild type. Two micrograms of poly-A- * RNA "from PD broth cultures grown in the presence of light (L) and in darkness (D) per band" were subjected to electrophoresis through a denaturing agarose gel at 1. 254 »and were transferred to nitrocellulose. Duplicate blots were treated with DNA of insertions marked with a8P from each of the cLEs (cLEl cLE3 and cLE7). Figure 2 is the genomic DNA sequence »the cDNA sequence and the amino acid sequence for LE6-cfp. Figure 3 is an amino acid sequence for LE6-cfp showing a translation generated by MacVector of the open reading frame. The double sequences of underlined amino acids are helical alpha transmembrane hydrophobic regions »determined by Tmpred prediction of transmembrane regions and orientations. The lowercase letters in bold represent the 19 amino acids comprising the motif associated with all known transporters of drug resistance by efflux. Figure 4 shows a comparative amino acid homology of the region around the 19 amino acid motif generated by searching the BLAST database for peptide sequence. CmcT = cefamycin exit protein from Norcardia lactarodurans »M rB = resistance protein to meti lenom ciña, from Streptomyces coelicolor; TETr = Aeromonas tetracycline resistance protein »MMrS = another resistance protein to methy lenomycin identical to MMrB; ToxA »HC = protein form of resistance to toxin, of Cochl iobolus carbonum; SSE1 = violet crystal resistance protein »of Saccharomyces cerevisiae» CFP = cercosporin-facilitating protein (the protein of the present invention); ActVA-l, ORF-1 = transmembrane protein from the group of actinorrodine genes »of Streptomyces coel color; LmrA = protein of resistance to linco icina »of Escherichia coli; QacA = antiseptic resistance protein »from Staphylococcus aureus; TcmA = protein of resistance to tetracene ic a »of Streptomyces glaucescens. Figure 5 shows a phylogenetic tree showing the relative relationship between LE6 protein and other drug transporting proteins. Figure 6 is a graph of the hydrophilic character of the LE6 protein from MacVector showing the position of the 19 amino acid motif with respect to the entire sequence. Figure 7 shows the plant expression vector pB35S-LE6CFP, which includes a 2.1 Kb BamHI / Kpnl fragment comprising the full length of the LE6-cfp cDNA. Figure Ba is a graph showing the time course analysis of the accumulation of cercosporin in dry weight, and the stable state levels of RNA corresponding to cLE6 and cLE7 of improved cDNA molecules in the presence of light in the PR strain of wild type. Figure 8b is an autoradiograph showing the sotlot blot analysis of total RNA extracted from the samples used to generate the results shown in Figure 7a. Figure 9a shows two different interrupted versions of the LE6 protein constructed and transformed into PR from C. kikuch i. as well as genomic LE6 (gLE6).

Figure 9b shows an autoradiogram of three switches containing a single copy of an interrupted version of the LE6 (cfp) gene. Band 1 is the wild-type PR strain. band 2 is Barí, band 3 is Bar2, and band 4 is Gus3. Figure 10 is a graph showing the production of cercosporin by the BarI »Bar2 and Gus3» transforming transformants compared to Cercospora KiKuchi i wild-type PR developed in the presence of continuous light (PRLt) or continuous dark (PRDk). Figure 11 is a graph showing the sensitivity to cercosporin of BarI, Bar2 and Gus3 switch transformants »compared to wild-type Cercospora KiKuchi i PR. All develop in the presence of continuous light or continuous darkness "and the levels are shown as a percentage of radial growth control.

DETAILED DESCRIPTION OF THE INVENTION The isolation and cloning of a cDNA and its genomic DNA that is involved in the regulation of the cercosporin according to the present invention allows the production of plant varieties resistant to cercosporin and cercospora strains susceptible to cercospor. Cercosporin allows the fungus to colonize and extract the nutrients necessary for growth and sporulation in tissues infected plants. Environmental conditions such as light intensity, temperature and nutrient ratios affect the production of cercosporin in culture. While the temperature and composition of the growth medium affect the amount of toxin produced, light appears to be the dominant regulatory factor (Jenns et al., Phytopathology, Volume 79, 213, 219, 1989, Lynch et al., Trans. Br. Mycol. Soc., Volume 73 »311-327» 1979, incorporated herein by reference). This means that light must regulate certain genes that are involved in the metabolism of cercosporin. Light induction was used to isolate improved cDNA molecules in the presence of light. by means of a subtractive hybridization technique (Maniatis and others »Molecular Cloning: A Laboratory Manual» pages 8.49 and 10.40-10.43 »1989, incorporated herein by reference). A cDNA library was constructed with the bacteriophage lambda lambda-ZAPII vector (Stratogene Cloning Systems »La Jolla» Calif.) Using wild-type poly-A - * - RNA from Cercospora KiKuchi i PR developed in the presence of light. The library is maintained as a bacteriophage supply material and is infected in XLl-blue cells of Escherichia coli (Stratogene Cloning Systems, La Jolla, Calif.) For selection. To isolate cDNA molecules from this library regulated in the presence of light. a subtracted probe is obtained from RNA poly-A - * - of C. KiKuchi developed in the presence of light »and used for treat with high density plate elevations probe. Individual hybridizing plates are isolated and converted into plasmids using an auxiliary bacteriophage and a plasmid excision technique in vivo (Stratogene). The insertions of these plasmids are then used to probe Northern blots (RNA) containing poly-A * RNA from cultures of C. KiKuchi i developed in the presence of light and dark. Six improved cDNA clones are identified in the presence of light. cLEs, each of which hybridizes to a distinct individual mRNA band in Northern blots (Figure 1). One of these cDNA molecules »LE6» shows improved accumulation of the transcript 20 times greater in the presence of light »and is correlated with the accumulation of cercosporin in culture» and is of almost complete length at 2.1 Kb. The sequence of this cDNA (Figure 2) contains a putative 1818 base pair open reading frame (ORF) coding for a predominantly hydrophobic and cysteine-rich protein of 606 amino acids (pLE6) (Figure 2) with a molecular weight of 65424 and an isoelectric point of 5.08. The translation of the ORF generated by the MacVector »shows sequences that are hydrophobic and alpha helical transmembrane regions, determined by the Tmpred prediction of regions and transmembrane orientation, as seen in Figure 3 (Hofmann et al., Chem. Hoppe-Sey er, Volume 347, 166-176, 1993). The Kyte-Dool ittle analysis of the protein »indicates that pLE6 contains 12 to 13 helical alpha transmembrane regions. A search for amino acid sequence homology »has allowed the identification of two regions of homology. GPVLGG and SWRWCLYINLPIG. for efflux-mediated resistance determinants »a subfamily of the main facilitator superfamily (figure 4). A comparative amino acid homology of the region around this 19 amino acid motif was generated by a search of the BLAST database for peptide sequence (Figure 4). Homology was found in the following efflux drug resistance transporters: C cT. cefamycin exit protein »from Nocard to lactamdurans (Coque et al.» EMBO J. »Volume 12» 631-639 »1993); M rB. protein form of resistance to methi lenomicin. of Streptomyces coelicolor (Neal et al., Gene. Volume 58. 229-241 »1987); TETr, tetracycline resistance protein »by Aeromonas (Varela and others» Antimicrob Agents Chemother. »Volume 37» 1253-125B, 1993); MmrS, another meti lenomicin resistance protein identical to MMrV; ToxA »HC» toxin transport protein »from Cochliobolus carbonum (J. Pitkin» Michigan State University »personal communication); SGE1 »violet crystal resistance protein» from Saccharomyces cereviseae (Eherhofer-Murray et al., Mol.Genet.Genet. »Volume 244, 287-294, 1994); CFP (LE6), protein facilitator of cercospor a; ActVA-1 »ORF-1» transmembrane protein a from the group of genes of actinorrodine »by Strepto yces coel i col or (Cavallero et al.» Mol.Ge.Genet. »Volume 230» 401-412 »1991); LmrA »resistance protein linco ici a. from Streptomyces l ncolnensis (Zhang et al. »Molec.Microbiol .. Volume 6» 2147-2157, 1992); EMRB, multiple drug resistance protein »from Escherichia coli (Lomovskaya et al.» Proc. Nati. Acad. Sci. »USA» Volume 89 »8938-8942» 1992); QaCA »antiseptic resistance protein, from Staphyl ococcus aureus (Tennent et al.» J. Gen. Microbiol. »Volume 135. 1-10» 1989); and TcmA, tetracene icine resistance protein from Streptomyces galucescens (Guilfoile et al., J. Bacterio! .. Volume 174. 3651-365B, 1992). Figure 5 shows a phylogenetic tree showing the relative relationship between pLE6 and other drug transporting proteins. The tree was constructed using the group method of non-evaluated pairs provided by software, nucleic acid / protein analysis. of GeneWorks (Intell iGenetics Inc .. Mountain View, Calif.). A plot of MacVector's hydrophilic character (the inverse of hydropotia) indicates the position of the 19 amino acid motif with respect to the entire sequence, being between amino acid 210 and amino acid 237. as shown in Figure 6. These data suggest that LE6 is responsible for the movement of cercosporin through the plasma membrane. Subsequent analysis shows that this hydrophobic protein contains a region with significant amino acid sequence homology for both prokaryotic and yeast proteins. that intervene in antibiotic resistance. Southern genomic analysis shows that LE6cfp exists in the genomes of other species fi upapogens of Cercospora such as »for example» Cercospora beti col a (sugar beet) »Cercospora nicotianae (tobacco) and Cercospora zeae-maydis (maize). Gene disruption of LE6 results in dramatically reduced cercosporin production in C. ki uchi i developed in the presence of continuous light »loss of pathogenesis of C. kikuchi i in soy» decreased accumulation of transcription of other improved cDNA in presence of light »an altered profile of pigment accumulation» and substantial loss of self-resistance to cercosporin. All this evidence indicates that pLE6 is responsible for the movement of cercosporin through the cell membrane, that is, it is a membrane pump, which confers resistance to the fungus. This further indicates that it can be inserted into plant cells to obtain transgenic plants that are resistant to cercosporin. A transgenic plant resistant to cercosporin "is any transgenic plant that exhibits some level of resistance to it" compared to the non-transformed plant. The DNA construct can be introduced into a plant using any method that provides efficient transformation. Several methods for transformation into plants include the use of Ti or Ri plasmids »bombardment of DNA particles» microinjection »electroporation, fusion of harsh as »DNA bombardment» etc .. See »for example» Gordon-Kam and others »The Plant Cell» Volume 2, 603-618, 1990; and Lowe and others »BIO / TECHNOLOGY, Volume 13, 677-682 »1995; which are incorporated herein by reference. The tissue of the plant is manipulated by genetic engineering techniques with cDNA (LEGcfp) isolated from PR of C. ki uchi i, and inserted into an expression vector in plants such as, for example. pBIN / 35S (Bevan, Nuc Acids Res. »Volume 12» 8711-8721) A 2.1 kb BamHI / Kpnl fragment comprising the full length of the LE6 cDNA (cfp) (Figure 2) was cloned into the polylinker site of the expression vector .sn plants pBIN / 35S "as described by Bevan (cited above) (figure 7) The following examples are intended only to illustrate the invention" and are not intended to limit the scope of the invention " it is described by the rei vindi caei ones.

EJ? M Q 1 PR was isolated from Cercospora Kikuchi de soyas in Puerto Rico, and was provided by J. B. Siclair. University of Illinois »Urban» IL. Fungi are grown in potato dextrose medium (Difco Laboratories, Detroit, Michigan) in liquid form (PD) or solidified with agar (PDA). Fungi grown in liquid medium are grown in 50 ml volumes in 125 ml Erlenmeyer flasks on a rotary shaker (180 rpm) at 20 ° C under continuous white fluorescent light (approximately 15 microeinsteins or continuous darkness.

EXAMPLE 2 In order to isolate the DNAs induced in the light, PR cultures of C. kikuchi i are produced either in continuous white fluorescent light of approximately 15 m croeinstei ns _ * s_i or in continuous darkness »or in each dextrose of» papa (PD) (Difco Laboratories »Detroit» MI) or in complete medium (MC) containing salts, yeast extract and inoculum acids (Jenns et al. »Above). Cultures are grown in shaking culture (200 rpm) until the early stationary phase. 5 mm aliquots of culture (mycelium plus medium) are mixed for 30 seconds in a Waring blender with 5 ml of distilled water. The resulting aqueous mixture is used to determine the concentration of cercosporin as described by Jenns et al. (Phytopathology, Volume 79, 213-219, 19B9, above). The dry weights are determined after the operation.

EXAMPLE 3 In order to extract RNA and build a cDNA library, the mycelia of the liquid cultures described in Example 2 above are harvested by vacuum filtration through Mira cloth. They are frozen in liquid nitrogen and lyophilized. The lyophilized tissue is frozen again in liquid nitrogen and crushed to a powder with mortar and grinder » previously cooled with liquid nitrogen. RNA is extracted as described by Williamson et al. (Plant Physiol. »Volume 88» 1002-1007 »1998, incorporated herein by reference.) Poly i (A) * RNA is extracted from total RNA by chromatography with oligo- (dt) -cellulose as described by Maniatis et al. (formerly »pages 8.49 and 10.40-10.43» incorporated herein by reference.) The RNA extracted from the cultures produced in the light »described in Example 1. are used to construct a gene library. CDNA with the lambda vector ZAPII (Stratagene) of bacteriophage lambda using Poly (A) - *? RN isolated from PR of C. Kikuchi i produced in the light.It remains as a bacteriophage strain and is infected in blue cells Escherichia coli XLl (Stratagene) to sift.

EJ PL 4 To obtain cDNA clones from the cDNA library constructed from Pol i (A) - * - from C. kikuchii. produced in light, the hybridization technique is used as described by Maniatis and others (pages 8.49 and 10.40-10.43, above). The cDNA of the first chain is synthesized from Pol i (A ^ AR of C. kikuchi i produced in the light using _a-3ap-? DCTP.The Pol (A) *? RN of C. kikuchii produced in the dark biotinylated and hybridized to the improved cDNA in the light of the first strand and subtracted in the hybrids and the Pol i (A) * C. ki uch RNA produced in the unhybridized dark of the mixture with estrepavidi a as described by S ve and others (Nuclei Acids Res. »Volume 16» 10937, 1998; incorporated herein by reference ». The procedure is repeated twice. After two subtraction series, the improved cDNA is labeled in the excess light with C-35Bp_dCTP by random initiation with hexane as described by Feinberg et al. (Anal. Biochem., Volume 132, 6-13, 1983; present by reference) and this is used to probe duplicate plant surveys. as described by Maniatis and others (pages 10.40-10.43, supra), which contain the improved cDNA library in light. The hybridizing areas containing multiple plaques are subsequently purified from the plates and the cloned DNA is converted into the DNA in the plasmid using an auxiliary bacteriophage (Stratagene) and the plasmid excision technique in vivo (Stratagene, incorporated herein by reference). > EXAMPLE 5 To isolate the specific clones for which the corresponding mRNAs exhibit enhanced light accumulation, two μg of glyoxylated poly i (A) * RNA samples under denaturing conditions are subjected to electrophoresis through a 1.254 agarose gel as described Maniatis and others (previous) and are transferred to nitrocellulose. For the slit stain analysis, either 10 or 20 μg of samples are applied to the nitrocellulose with a Schleicher & Schuell Minifold II. Hybridizations are performed in a formamide pH regulator at 5054 at 42 ° C with ADn inserted from the plasmids described above in Example 4 labeled at high specific activity with _a-3a! P_dCTP by randomly labeling with hexamer as described by Feinberg and others (previous). The probe is removed from the slotted spots after hybridization by washing in 0.1 X SSC (1 X SSC is 0.15 M NaCl plus 0.15 M sodium citrate) -odium sodium diet at 0.154 at 95 ° C. The spots are then hybridized with Neurospora crassa probe consisting of rDNA (rRNA encoding genes) as described by Russell et al (Mol.Gen.Gen., Volume 196. 275-282, 1984; incorporated herein by reference). . These control hybridizations verify that all samples within a slot spot are equally loaded. To quantify the intensity of the hybridization within the slot stain, the autoradiographs are analyzed by laser scanning densitometry. Six light-enhanced cDNA clones (cLEs), cLEl and cLE3-cLE7, each of which hybridize to a unique distinct mRNA band on Northern blots, were identified (Figure 1). The lengths of these transcripts vary from about 0.8 to 6.0 kb, while the lengths of the cDNA inserts vary from about 0.8 to 2.4 kb. Two of the cDNA clones are almost full length, while the others vary from approximately 32 to 7954 of the lengths of their respective mRNA. They do not detect transcripts for cLE6 in pol (A) * RNA from C. kikuchii produced in the dark. The RNAs for the other sequences enhanced in the light are detected at several levels in the poly (A) * RNA of C_¡_ ki uchi i. These six cloned DNAs are converted to plasmid DNA using a helper bacteriophage and an i.n. live plasmid excision technique (Stratogene "above) to form pcLEl and pcLE3-pcLE7 from plasmid, especially CLE6-CFP, which are then infected in Blue Escherichia col i XLl (Stratagene) to maintain.

EXAMPLE 6 To determine the relationship between synthesis and toxin expression of enhanced cDNAs in light in PR »of C. Kikuchi. the kinetics of the transcript and the accumulation of toxin are compared. The samples are collected at intervals of 24 hours of a single large culture of PD broth. Aliquots are used for toxin analysis as described by Jenns and others (above), and the remaining culture is recovered and lyophilized for dry weight determination and RNA extraction. Accumulation of the transcript is assessed by stain analysis with total RNA slot of all samples except day one sample, which produced very little tissue for RNA extraction. The signal intensities are quantified by laser scanning densitometry.

The onset of toxin accumulation occurs drastically between days 2 and 3 »leaving behind the onset of logarithmic growth for at least about 24 hours (Figure 8a). The transcripts both for cLE6 and cLE7 exhibit accumulation kinetics identical to that of the toxin (Example 8e), that is, each also increased drastically between days 2 and 3. Between days 2 and 3 »cercosporin levels increased from undetectable at approximately 29 nmol / mg of fungal tissue (dry weight), while steady state RNA levels for cLE6 and cLE7 increase approximately 16 and 4 times, respectively. The transcripts for the other cLEs are already present at higher levels than those for cLE6 and cLE7 on day 2. They did not show, however, any noticeable change in the stable level in the course of the experiment (data not shown). In the six cLEs, cLE6 exhibits the most surprising relationship with the production of cercosporin. In PR, the accumulation of cLE6 transcripts in the light is increased by approximately 20 times over the accumulation of transcript in the dark.EXAMPLE 7 The fungal transformations were conducted as described by Upchurch and others (Applied and Environmental Microbiology, Volume 60 (12), 4592-4595-1994; incorporated in the present by reference). The transformants were produced on plates containing regeneration medium supplemented with 10 μM of bialaphos. This allows the selection of fungal colonies that have integrated a copy of the mud gene of Streptomyces hygroscopicus into their genome. The putative transformants were transferred to media containing bialaphos for several generations to ensure marker stability.

EXAMPLE 8 The LE6cfp gene is contained within the genic clone gLE6. This fragment of EcoRI genomic DNA of 6.5 kb is used to construct deletion clones. The plasmid BAR pCFP was constructed after its pressure of a Cla / HindlII fragment of 2.5 kb of gLE6 followed by the ligation of the "expression unit" of the Cla / HindIII bar of 3.1 kb subcloned from the pNBl plasmid (Upchurch » 1994 »incorporated herein by reference). The bar gene confers resistance to the bialaphos herbicide (Straubinger and others »Fungal Genet, Newsl.» Volume 39 »82-83» 1992 »incorporated herein by reference. The pCFP GUS plasmid was constructed by excising the 6.5 kb EcoRI fragment. of the gLE6 plasmid The purified insert DNA was diluted substantially and exposed to T4 DNA ligase allowing it to circulate in a head-to-tail orientation, immediately after the heat inactivation of the ligase was restricted in the sample with H ndlII. thus causing the gLE6 insert DNA to remain in the head-to-tail orientation with the H-iindIII ends. The head-to-tail construct of appropriate size was gel purified and ligated into the 7.1 kb pN0M102 plasmid digested by Hind111 (Roberts et al., Curr., Volume 15, 177-180, 1989, incorporated herein by reference). . Transformations were performed as described above in Example 7 and transformants were selected on bialaphos modified regeneration media. The Southern analysis used genomic DNA digested by EcoRI and insert DNA in radiolabelled gLE6 as well as the bar and uiDA genes to determine the number and locations of the insertion events. Two different disintegrated versions of the LE6cfp gene were constructed and transformed into PR of C. kikuchi as described above and represented in Figure 9a. The number and position of the insertion events were evaluated by Southern hybridization analysis of twelve bialaphos resistant transformants of each disintegrator type. Of these analogous colonies, it was shown that three contained a single copy of a disintegrated version of the LE6cfp gene (Figure 9b). Disintegrants containing PpLE6CFPDBar are indicated »Barí and Bar2 and the one containing a single copy of PLE6CFP GUS is labeled Gue3. Northern analysis was performed to ensure that the disintegration of the native LEcfp gene had blocked its transcription. None was shown Hybridization when the total RNA of Bari and Bar2 was probed with LE6cfp. but a minimal signal was detected in the RNA in steady state isolated from Gus3 (data not shown).

EXAMPLE 9 Cercosporin levels produced by disintegrating transgenic C. ki uchi i were determined by taking 10 ml of aliquots of fungal liquid cultures (mycelium plus medium). The aliquots are mixed in an aring mixer and treated with a volume of 5N KOH as described by Jenns and others (above) and clarified by centrifugation. The concentrations of cercosporin are determined spectrophotometrically from the A_ »ßo and the molar extinction coefficient of 23,300 for the cercosporin in base (Jenns and others, above). Freeze-dried mycelial samples are evaluated in order to express the cercosporin concentration as nmol / mg dry weight. Disintegrating transformants Bari, Bar2 and Gus3 are tan to red rather than bright red, when they are produced in PD medium. The levels of cercosporin in the transforming fungi are shown in Figure 10. The levels of production of cercospor a in Barí. Bar2 and Gus3 accumulated approximately 554 of the PR levels produced in the light. The levels are significantly lower than the values obtained routinely in PR crops produced in the dark. These measurements are consistent with the levels observed by thin layer chromatographic separation (data not shown).

EXAMPLE 10 To determine the self-resistance of transgenic fungi to cercosporin, the PR of C. kikuchii and the disintegrating transformants are tested for inhibition of radial growth on agar. Fungus plugs (5 mm) are inoculated on Petri dishes divided with the PDA medium on a medium of each cassette modified by the addition of a cercosporin bank solution to give a final concentration of 10 mM as described by Daub et al. (Phytopathology Volume 7 »1515-1520» 1987, incorporated herein by reference). Since the cercosporin bank is prepared in acetone, the second half of each box is modified with an equal volume of acetone. The boxes are maintained at 25 ° C in continuous fluorescent light (80 ml croeinsteins m-2 s-3-). T radial growth is measured 3 and 4 days after inoculation. Cercospora kikuchi wild type shows only a slight inhibition in growth when it occurs in the presence of cercosporin (Daub et al., In R. Heitz et al. (Ed.) »Light activated Pesticides» American Chemical Society »Washington DC» pages 271 -280, 1987, incorporated herein by reference). The relative growth of C¿_ Kikuchi i of wild type and disintegrant is evaluated in the presence of cercosporin. The Bari and Bar2 disintegrants exhibited the highest sensitivity to cercosporin "with inhibitions in growth of average percentage of approximately 48 and 52" respectively (Figure 11). The other transformant »Gus3» also has growth inhibition of the mean percentage of 48. The disintegrating strains of LE6cfp are drastically more sensitive to cercosporin than the PR of type 1 vestre.

EXAMPLE The aggressiveness of the transformants of C. KiKuchii in the plants is determined by the inoculation of leaf of plants of Lee-68 cultivar of soybean of 5 weeks of age cultivated in greenhouse (61ye ne max L.). The fungal inoculum is prepared by mixing approximately 0.5 g (fresh weight) of dark mycelia produced in the dark of each strain in 20 ml of sterile water in a sterile Wari mixer as described by Upchurch et al. (Appl. Environ. , Volume 57 »2940-2945» 1991 »incorporated herein by reference). Mycelial suspensions are then sprayed on the lower part of the leaves until the inoculum is spilled. The plants are covered with plastic bags for an initial period of 48 hours in reduced light to maintain moisture for infection. The information of injury during a period of 14 days. Five plants are inoculated with each fungal strain tested and the entire experiment is repeated. Thirty panels of trifoliate soy leaves (90 separate leaves) were examined in two inoculation experiments. Lesions extending 2-8 mm formed regularly with necrotic centers were observed on the upper surfaces of Lee-68 cultivars 7 days after inoculation with isolated wild-type PR. Only spots similar to pinheads were observed and frequently on leaves inoculated with a disintegrant of LE6 Bari, Bar2 and Gus3 indicating the diminished aggressiveness of these mutants in comparison with the svest type. No lesions were detected on the plants that were falsely inoculated with a water control. This decrease in aggressiveness in the disintegrating fungi seems to be caused by the decrease in the production of cercosporin in these fungi. It has been shown that cercosporin is a critical pathogenicity factor in soybean infection by C. Kikuch? I (Upchurch, 1991, above). Since the experiments show that the concentration of cercosporina produced by these fungal strains is much lower than the wild type levels, it would be expected that these fungi would be less aggressive. In addition to decreased aggressiveness, these disintegrating fungi may have a decreased activity in their natural environment since they have an increased sensitivity to their own toxin, even if environmental fitness is not tested. The detailed description above is for the purpose of illustration. Such detail is for that purpose only and those skilled in the art can make variations therein without deviating from the spirit and scope of the invention.

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: United States of America, represented by the Secretary of Agriculture and the State University of North Carolina (ii) TITLE OF THE INVENTION: Fungus Gene Coding Resistance to F totoxin Cercosporin ( iii) SEQUENCE NUMBER: 3 (iv) ADDRESS FOR CORRESPONDENCE: (A) RECIPIENT: USDA-ARS-OTT < B) STREET: Romm 407, BLDG. 005, BARC-W < C) CITY: Beltsville < D) STATE: Mary1 and < E) COUNTRY: E.U.A. < F) POSTAL CODE: 20705 (V) COMPUTER LEADABLE FORM: < A): TYPE OF MEDIUM: Flexible disk < B > COMPUTER: IBM compatible PC < C) OPERATING SYSTEM *. PC-DOS / MS-DOS < D) SOFTWARE: Patentln Reléase ttl.O, Version ttl.25 (vi) PREVIOUS INFORMATION OF THE APPLICATION: (A) NUMBER OF APPLICATION: IB) DATE OF PRESENTATION: IC) CLAIFICATION: (Viii) INFORMATION ON THE POWDER / AGENT (A) ) NAME: Gail E. PoulOS IB) REGISTRATION NUMBER: 36,327 IC) REFERENCE NUMBER / RECORD: 0045.96 (ix) TELECOMMUNICATIONS INFORMATION: (A) TELEPHONE: 301-504-5302 (B9 TELEFAX: 301-504-5060 ( 2) INFORMATION FOR SEQ ID NO: i: (i) CHARACTERISTICS OF THE SEQUENCE: ÍA) LENGTH: 2192 base pairs B) TYPE: nucleic acid ÍC) TYPE OF CHAIN: individual ID) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (A) DESCRIPTION: desc = "synthetic DNA" (i) HYPOTHETIC: NO (IV) ANTI-SENSE: NO (Vi) ORIGINAL SOURCE: (A) ORGANISM: cercospora kikuchii (B) CEPA: PR (ix) ARACTERISTIC: (A) NAME / KEY: intron (B) LOCATIONS: 746..798 (ix) CHARACTERISTICS: (A) NAME / KEY: intron (B) LOCATIONS: 1199..1248 (ix) CHARACTERISTICS: (A) NAME / KEY: intron (B) LOCATIONS: 1459..1507 (x) SEQUENCE DESCRIPTION: SEQ ID N?: I: GCCGTCGAAT GAGTTCCCTC ATAGCAGTCT GGACCGGCTA CTTTCCATAC ATTGACAATG 60 ACGAGCCCAG CGCGAtCAAC GCATACTGAT ACAGAGTCTC ACGACGTCGT AAAGAGCGAC 120 TCGGAATCGA AACTGGAACT GGAGCACAGC GATTCGGATA ATCAAGATGA GAAGTCCAAC 180 GCTAAGTTGG CGGAACGTCC TGAAGCCAAG CCAGAAGAAG ATGAAGAACT CAATGATCAA 240 GGCGAGAGGT ACATCTGCGG CTGGCCTCTG GTATTTCTCT TGTTAGCCAT GGTCTCCACA 300 GTCTTCATTG TCGCTTTGAG CAACACCATC ATCAGCACAG CAATCCCGGC CATCACAACA 360 GCGTTCAATA GTACCCGAGA TATTGGCTGG TACAACTCTG GAGAAGCTCT TGCAGCCACT 420 GCCTTCCAAC TACCTTTCGG GCGAGCGTAT CTCTTGATGG ACCTGAAGTG GACTTTCCTC 480 GTCTCACTGG CCTTATATCT GATCGGCAGC CTGATCTGTG GTGTGGCAAA CTCTTCTGAG 540 CTTCTCATTT TTGGCCGATC GATTGCAGGA GTTGGCAACG CTGGCGTCTT CGCTGGCGTG 600 TTCATCATTA TTGCTCGAAA CGTTCCTCTG CGGAAACGCA CTTTATGCTG GATTGGTTGG 660 AGCGACTTTT GCCATTGCTG CTGTGCTGGA CCTGTCCTGG GTGGTATCTT TACTGACCGT 720 ATTAGCTGGA GGTGGTGTTT GTACAGTAAG TCTCTAGAAC CCGTGCACTT TATTCCGTTC 780 ATTGACACTT TTCAACAGTT AACCTGCCTA TCGGAGCTGT ACGTGTCGCA ATCATCATAT 840 TCCTCCTTCC ATCTCGTCCT GGCGAAAAGG CAGCAGAAGT CAAGGACCTG TCCTGGTGGC 900 AGTTCTTCCT AAAGCTCAAT CCTTTTGGGT CGGCTCTCCT ACTCGGTTCC CTGACGTGCT 960 TTTTCCTCGC CCTACAGTGG GGCGGCGGCG AATACCGTTG GAGTGCTGGT CGTGTCGTTG 1020 CTGTACTGGT GGTCTTCGCC GTCAGCTTCA TCGGATGGCT GGTTCTGCAA TACTTCCAAG 1080 GCGACGAAGC CACACTGCCA TTTAACGTTG CAAAACAGCG TACCGTTGGT GGTGCCTCTA 1140 TCTACACTCT GCATCTGAGC GCCGCATTTG GACTCGTCAT ATACTATCTG CCTCTCTGGT 1200 GAGTTGATTC ATGAGCATGC ACTGGGCTCA CGAACTGACA TTATGAAGGT TTCAAGCAGT 1260 ACGATCTGAC AGTGCCGAAG CTGCTGGTCT CAAGCAACTT GGCATCGTCA TCTCGCTCAC 1320 TCTCTCGTCA ATTGCAGCTG GCGGTGCTGT TGTAAAAATA GGATATTACT ATCCTTTCAT 1380 TTACGCCGGA ACGGTCTTAT GCAGCATCGG CTCTGGCTTG CTTTACACGA TCACACTCGA 1440 TACACCGCAA TGGGATATGT AAGTAATCGA GCTCCGACTG AATTTGAACA TTTCTAACGC 1500 ATGACAGTAT CGGTTATTCG ATCGTATTCG CCATTGGAAT CGGCGTCAGT CTCGAGCAAT 1560TGTCCAGACT GTCCTGCCCG ATGCTCAGAT ACCAGCAGGA ACAAGCTTGG 1620 TTCTGTTCGT CCGACTACTT GGATCAGCAA TCCCCGGACC CATCGGACAG AGTGTACTCC 1680 AGACAACACT TGCCAGTAGG CTAGGGACTG AGGTCGCAGA GCAAGCATAT GGTGGTACCG 1740 GAGCAACTGA AATCCGCTCA AAGCTCGACA ACATTTTTGG AGCTGGCACA CCTGAAGCTC 1800 GAGATGCCCT TGACGCTTTC AACGATTCTG TGACGAAGAT CTTCATGGTC GCAATCATAG_1860_TCTCATGTCT GAGTGCGCTG CCTCTTCCCC TCATCGAGCT CAAGAGCGTC AAGCGTGAGA 1920 AACGAGACAA CGAAGACGCC AAAGAAGGCA AGAAAACTAA TGGGACGACG CGTGAGATAG_1980_AAGATCCAGA GAAGGGGCAG AGTGCAGAGA TCGTGGGCAA AGCAGTGTGA GATGTGGCAT 2040 CAGACCGAGC GACGATTTTA TAGACATTGT AGCGAGCTGT TACGACTAAC GCATGTACCC 2100 AACAGAGTGT GTGGCTCAGA GGCAATAGAG CTTTGACGAC ATAATAAACC AAGAATTTTA 2160 ATGGCTACGA GTCCTCTCAA AACCTCGCCG GA 2192 (2) INFORMATION FOR SEQ ID NO. 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1893 base pairs (B) TYPE: nucleic acid (OR TYPE OF CHAIN: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: CATACATTGA CAATGACGAG CCCAGCGCGA TCAACGCATA CTGATACAGA GTCTCACGAC 60 GTCGTAAAGA GCGACTCGGA ATCGAAACTG GAACTGGAGC ACAGCGATTC GGATAATCAA 120 GATGAGAAGT CCAACGCTAA GTTGGCGGAA CGTCCTGAAG CCAAGCCAGA AGAAGATGAA 180 GAACTCAATG ATCAAGGCGA GAGGTACATC TGCGGCTGGC CTCTGGTATT TCTCTTGTTA 240 GCCATGGTCT CCACAGTCTT CATTGTCGCT TTGAGCAACA CCATCATCAG CACAGCAATC 300 CCGGCCATCA CAACAGCGTT CAATAGTACC CGAGATATTG GCTGGTACAA CTCTGGAGAA 360 , GCTCTTGCAG CCACTGCCTT CCAACTACCT TTCGGGCGAG CGTATCTCTT GATGGACCTG 420 AAGTGGACTT TCCTCGTCTC ACTGGCCTTA TATCTGATCG GCAGCCTGAT CTGTGGTGTG 480 GCAAACTCTT CTGAGCTTCT CATTTTTGGC CGATCGATTG CAGGAGTTGG CAACGCTGGC 540 GTCTTCGCTG GCGTGTTCAT CATTATTGCT CGAAACGTTC CTCTGCGGAA ACGCACTTTA 600 TGCTGGATTG GTTGGAGCGA CTTTTGCCAT TGCTGCTGTG CTGGACCTGT CCTGGGTGGT 660 ATCTTTACTG ACCGTATTAG CTGGAGGTGG TGTTTGTACA TTAACCTGCC TATCGGAGCT 720 GTACGTGTCG CAATCATCAT ATTCCTCCTT CCATCTCGTC CTGGCGAAAA GGCAGCAGAA 780 GTCAAGGACC TGTCCTGGTG GCAGTTCTTC CTAAAGCTCA ATCCTTTTGG GTCGGCTCTC 840 CTACTCGGTT CCCTGACGTG CTTTTTTCCTC GCCCTACAGT GGGGCGGCGG CGAATACCGT 900 TGGAGTGCTG GTCGTGTCGT TGCTGTACTG GTGGTCTTCG CCGTCAGCTT CATCGGATGG 960 CTGGTTCTGC AATACTTCCA AGGCGACGAA GCCACACTGC CATTTAACGT TGCAAAACAG 1020 CGTACCGTTG GTGGTGCCTC TATCTACACT CTGCATCTGA GCGCCGCATT TGGACTCGTC 1080 ATATACTATC TGCCTCTCT GGTTTCAAGC AGTACGATCT GACAGTGCCG AAGCTGCTGGT 1140 CTCAAGCAA CTTGGCATCG TCATCTCGCT CACTCTCTCG TCAATTGCAG CTGGCGGTGCT 1200 GTTGTAAAA ATAGGATATT ACTATCCTTT CATTTACGCC GGAACGGTCT TATGCAGCATC 1260 GGCTCTGGC TTGCTTTACA CGATCACACT CGATACACCG CAATGGGATA TTATCGGTTAT 1320 TCGATCGTA TTCGCCATTG GAATCGGCGT CAGTCTCGAG CAATCCAACG TTGCTGTCCAG 1380 ACTGTCCTG CCCGATGCTC AGATACCAGC AGGAACAAGC TTGGTTCTGT TCGTCCGACTA 1440 CTTGGATCA GCAATCCCCG GACCCATCGG ACAGAGTGTA CTCCAGACAA CACTTGCCAGT 1500 AGGCTAGGG ACTGíAGGTCG CAGAGCAAGC ATATGGTGGT ACCGGAGCAA CTGAAATCCGC 1560 TACAAGCTC GACAACATTT TTGGAGCTGG CACACCTGAA GCTCGAGATG CCCTTGACGCT 1620 TTCAACGAT TCTGTGACGA AGATCTTCAT GGTCGCAATC ATAGTCTCAT GTCTGAGTGCG 1680 CTGCCTCTT CCCCTCATCG AGCTCAAGAG CGTCAAGCGT GAGAAACGAG ACAACGAAGAC 1740 GCCAAAGAA GGC-7-AGAAAA CTAATGGGAC GACGCGTGAG ATAGAAGATC CAGAGAAGGGG 1800 CAGAGTGCA GAGATCGTGG GCAAAGCAGT GTGAGATGTG GCATCAGACC GAGCGACGATT 1860 TTATAGACA TTGTAGCGAG CTGTTACGAC TAA 1892 (2) INFORMATION FOR SEQ ID NO. 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 606 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: l neal (i) TYPE OF MOLECULE: protein (iii) HYPOTHETICAL : YES (iv) AMENITIES: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: Met Thr Ser Pro Ala Arg Ser Thr His Thr Asp Thr Glu Ser His Asp 1 5 10 15 Val Val Lys Ser Asp Ser Glu Ser Lys Leu Glu Leu Glu His Ser Asp 20 25 3rd Ser Asp Asn Gln Asp Glu Lys Ser Asn Ala Lys Leu Ala Glu Ara Pro 35 40 45 Glu Ala Lys Pro Glu Glu Asp Glu Glu Leu Asn Asp Gln Gly Glu Arg 55 60 Tyr He Cys Gly Trp Pro Leu Val Phe Leu Leu Leu Ala Met Val Ser 65 70 75 80 Thr Val Phe Val Phe He Val Ala Leu Ser Asn Thr He He Ser Thr 85 90 95 Ala He Pro Ala He Thr Thr Ala Phe Asn Arg Asp He Gly Trp Tyr 100 105 110 Asn Ser Gly Glu Ala Leu Ala Ala Thr Ala Phe Gln Leu Pro Phe Gly 115 120 125 Arg Ala Tyr Leu Leu Met Asp Leu Lys Trp Thr Phe Leu Val Ser Leu 130 135 140 Ala Leu Tyr Leu He Gly Ser Leu He Cys Gly Val Ala Asn Ser Ser 145 150 155 160 Glu Leu Leu He Phe Gly Arg Ser He Wing Gly Val Gly Asn Wing Gly 165 170 175 Val Phe Wing Gly Val Phe He He Wing Wing Arg Asn Val Pro Leu Arg 180 185 190 Lys Arg Thr Leu Cys Trp He Gly Trp Ser Asp Phe Cys His Cys Cys 195 200 205 Cys Wing Gly Pro Val Leu Gly Gly He Phe Thr Asp Arg He Ser Trp 210 215 220 Arg Trp Cys Leu Tyr He Asn Leu Pro He Gly Wing Val Arg Val Wing 225 230 235 240 He He He He Phe Leu Leu Pro Being Arg Pro Gly Glu Lys Ala Wing Glu 245 250 255 Val Lys Asp Leu Ser Trp Trp Gln Phe Phe Leu Lys Leu Asn Pro Phe 260 265 270 Gly Be Ala Leu Leu Leu Gly Ser Leu Thr Cys Phe Phe Leu Ala Leu 275 280 285 Gln Trp Gly Gly Gly Glu Tyr Arg Trp Ser Wing Gly Arg Val Val Wing 290 295 300 Val Leu Val Val Phe Wing Val Ser Phe He Gly Trp Leu Val Leu Gln 305 310 315 320 Tyr Phe Gln Gly Asp Glu Wing Thr Leu Pro Phe Asn Val Wing Ala Lys Gln 325 330 335 Arg Thr Val Gly Gly Wing Be He Tyr Thr Leu His Leu Ser Wing Wing 340 345 350 Phe Gly Leu Val He Tyr Tyr Leu Pro Leu Trp Phe Gln Wing Val Arg 355 360 365 Ser Asp Being Wing Glu Wing Wing Gly Leu Lys Gln Leu Gly He Val He 370 375 380 Ser Leu Thr Leu Ser Ser Wing Wing Wing Gly Gly Wing Val Val Lys He 385 390 395 400 Gly Tyr Tyr Tyr Pro Phe He Tyr Wing Gly Thr Val Leu Cys Ser He 405 410 415 Gly Ser Gly Leu Leu Tyr Thr He Thr Leu Asp Thr Pro Gln Trp Asp 420 425 430 He He Gly Tyr Ser He Val Phe Wing He Gly He Gly Val Ser Leu 435 440 445 Glu Gln Ser Asn Val Wing Val Gln Thr Val Leu Pro Asp Ala Gln He 450 455 460 Pro Wing Gly Thr Ser Leu Val Leu Phe Val Arg Leu Leu Gly Ser Wing 465 470 475 480 He Pro Gly Ero He Gly Gln Ser Val Leu Gln Thr Thr Leu Ala Ser 485 490 495 Arg Leu Gly Thr Glu Val Wing Glu Gln Wing Tyr Gly Gly Thr Gly Wing 500 505 510 Thr Glu He Arg Ser Lys Leu Asp Asn He Phe Gly Wing Gly Thr Pro 515 520 525 Glu Wing Arg Asp Wing Leu Asp Wing Phe Asn Asp Being Val Thr Lys He 530 535 540 Phe Met Val Wing He He Val Val Cys Leu Ser Ala Leu Pro Leu Pro 545 550 555 560 Leu He Glu Leu Lys Ser Val Lys Arg Glu Lys Arg Asp Asn Glu Asp 565 570 575 Wing Lys Glu Gly Lys Lys Thr Asn Gly Thr Thr Arg Glu He Glu Asp 580 585 590 Pro Glu Lys Gly Gln Ser Wing Glu He Val Gly Lys Wing Val 595 600 605 097/35001 PCT / US97 / 04603 INDICATIONS RELATED TO A DEPOSITED MICROORGANISM (PCT Rule I3bis) A. The indications made below relate to the microorganism referred to in the description on page 5 1 line 9-14 B. IDENTIFICATION OF THE DEPOSIT additional deposits are identified on an additional sheet p Name of the depository institution American Type Culture Collection Address of depository institution (including zip code and country 12301 Par lawun Drive Rockv lle, Mary1 and 20852 E.U.A.

Deposit Date Income Number March 15, 1996 ATCC 974829 C. ADDITIONAL INDICATIONS This information (leave blank is not applicable) continues on an additional sheet i-i A microorganism is described in the specification. With respect to those designations in which a European patent is desired, a sample of the deposited micro-organism shall be made available until the publication or mention of the European patent expiry or until the date on which the request was made. denied or withdrawn or considered to have been withdrawn, only the issuance of such sample to an expert appointed by the person requesting the sample Rule 28 (4) D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications are not for all designated states) E. SEPARATE INDICATION OF INDICATIONS (leave blank if it is not applicable) The indications listed below will be submitted later to "the International Bureau (specify the general nature of the indications v.gr .." Deposit Income Number " ) Only for the use of the Office Only for the use of the Recipient -International Office- p This sheet was received. -'This sheet was received by the International Bureau with the request in: i ternational SIGNED Cathe ine B. Williams Official Authorized Officer authorized SIGNED CA J.A PASCHE

Claims (1)

1. NEW PE t? IN E TION CLAIMS 1. - An isolated DNA sequence capable of conferring resistance to cercosporin on plants. ? ..- An isolated DNA sequence for the expression of a cercosporin membrane pump protein. 3.- A vector that contains a DNA sequence which confers a higher level of cercosporin resistance to Cercospora fungi that produce cercosporin. 4. A transformed cell comprising a region encoding the production of SEQ ID NO. 3. The transformed cell of claim 4, further characterized in that the cell is a prokaryotic cell. G.- An isolated protein capable of conferring resistance to cercosporin on plants. 7.- An isolated protein that confers a higher level of cercospo resistance to the Cercospora fungi that produce cercospori a. T.- A plant transformation vector that contains a DNA sequence for the expression of cercospori a. 9. A transgenic plant that comprises a genome that contains genetic material for the expression of a heterologous membrane pump protein from Cercospora kikuchi i. 10. An isolated DNA molecule comprising a sequence selected from the group consisting of: (a) SEQ ID NO. 1 AND SEC ID NO. 2; and (b) DNA sequences encoding a membrane pump protein having a sequence of SEQ ID NO.3 11. A DNA construct comprising an expression cassette "comprising said construction in the direction of 5" to 3 * a promoter operable in a plant cell and a DNA sequence according to claim 16 located toward the 3 'end. from said promoter and operatively associated therewith 12. The DNA construct of claim 11 carried by a plant transformation vector 13. A DNA construct according to claim 11, further characterized in that said The promoter is the 35S promoter of the Cauliflower Mosaic virus 14. A plant cell containing a DNA construct of claim 11. 15. A transgenic plant comprising plant cells according to claim 14. 16. A method for being a transgenic plant comprising a genome containing genetic material encoding a membrane protein pump of Cercospora kikuchii comprising: ( a) provide a plant cell; (b) transform said plant cell with a hexogenic DNA construct comprising »in the direction of 5 * to 3 '» a promoter operable in a plant cell and a DNA sequence encoding the protein of SEQ ID NO.3 »operatively linked said DNA sequence to said promoter. 17. The method of conformance with the rei indication 16. further characterized in that said promoter is the 35S promoter of the Cauliflower Mosaic virus. 18. The method according to the rei indication 16"further characterized in that said step of transformation is carried out by bombarding said plant cell with microparticles carrying said DNA construction. 19. The method according to claim 16 »further comprising regenerating a plant from said transformed plant cell. 20. A transformed plant produced by the method of claim 19. 21. Seed or progeny of a plant in accordance with claim 20, seed or progeny that has inherited said DNA sequence encoding a protein of the SEQ. ID NO.3. 22. A transformed plant produced by the method of claim 19. 23. A transgenic plant according to claim 22 »further characterized in that said promoter is the 35S promoter of the Cauliflower Mosaic virus. 24. Progeny or seed of a plant according to claim 22, further characterized in that said seed or progeny has inherited the DNA sequence encoding a protein of SEQ ID NO.3.