AU726513B2 - Fumonisin-detoxifying enzymes - Google Patents

Description

S F Ref: 366749D1

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT 0* 00 0 0 0 0 0

ORIGINAL

S 0 0 0 *0 0 Name and Address of Applicant: Actual Inventor(s): Address for Service: Invention Title: Pioneer Hi-Bred International, Inc.

700 Capital Square 400 Locust Street Des Moines Iowa 50309 UNITED STATES OF AMERICA Jonathan Duvick and Tracy A. Rood Spruson Ferguson, Patent Attorneys Level 33 St Martins Tower, 31 Market Street Sydney, New South Wales, 2000, Australia Fumonisin-Detoxifying Enzymes 00 0 0

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The following statement is a best method of performing it full description known to me/us:of this invention, including the 5845 FUMONIS IN-OETOXI FYING ENZYMES.

Technical Field The present invention relates generally to the detection and isolation of fumnorism resistant organismrs and to compositions and methods for the in vivo detoxification or degradation of fumonisin This method has broad appiication in agricultural biotechnology and crop agriculture and in the improvement of food grain quality.

on f h neto Fungal diseases are common ),oblems in crop agriculture Many strides have been made against plant diseases as exemplfied by the use of hvbnrd plants. pesticides and improved @0e Sagnicultural practices However, as any grower or home gardener can atiest the problems **fungzal plant disease continue to cause difficulties in plant cultivation. I hus, t-here is a S. i' coninuig ned fr nw methods and materials for solving the problems caus-ed b\ i'nri *diseases of plants I'hese problems ca-n be met through a variet.: of appioache, I of exumii-Ce the infecious organisms can be controlled through the use of agents that a-re s-clectively biocidal for the pathogens Another method is interference with the mechanism by w hich the pathogen invades the host crop plant Yet a-nother method, in the case of pathogens that c-ause 520 crop losses, is interference with the mechanism by which the pathogen causes InjurN, to the S*host crop plant. Still another method, in the case of pathogens that produce toxins that a-re undesirable to mammals or other animals that feed on the crop plants, is interference with 0 toxin production, storage, or activity. This invention falls into the latter two categories, Since their discovery and structural elucidation in 1988 XBezuidenhout S, Gelderblom W, Gost-AllanC, Horak R, Marasas W, Spitefler B, Vleggaar R (1988) "Structure elucidation of the flunronisins, mycotoxins from Fusarium moni/iforme." Jourrnal Chemn Soc, Chem Commun 1988: 743-745), fumonisins have been recognized as a potentially serious problem in maize-fed livestock. They are liniked to several animal toxicoses including leukoencepha omaca Narsa WFO, Keflerman TS, Gelderblom WCA, Coetzer JAW, Thiel P (1988) "l-eukoenceph~lonirajacia, in a horse induced by fiorusin B3-1I isolated from Fusantian moniiorne." Onder-stepooz-t Journal of Veterinary Research 55: 197-204, Wilson TM, Ledet AE, Owens DI Rice LG, Nelson HA (1990) "Experimental liver disease in Ponies associated with the ingestion of a corn-based ration naturall contaminated withtbumonisin American Association of Veterinary. Laboratory Diagnosticians: Abstracts 33rd Annual Meeting, Denver. Colorado. October 7-9, 1990. Madison, Wisconin, USA) and porcine pulmonary edema 4 Colvin BM. Harrison LR "Fumonisin- nduced Pumonaryv Edema and Hydrothorax in Swvine." Mycopathologia 117. 79-82). and are also suspected carcinogens.(Geary W 1971 Coord Chem Rev 7, 8 1, Gelderblom WCA, lKrick NPJ, Niarasas WFO, Thiel PG (1991) "Toxicity and Ca-rcinogenmcty of the Fusanum-Moniliformc Metabolite. Fumnonjsin-B 1 in Rats Carcinogenesis 12. 1247- 125 1. Gelderblom kVCA Sempl E. Marasas WFO. Farber

L

(1992) 'The Cancer- Intiating Potential of the Fumonjsmn-B Nlvcotoxlns Carcinogenesis 13 433-437) -usarium isolates in section Liseola produce tionisins in culture at levels from 2 0**to >409 ppm .ALesh-- J, Plattner R_ Deslardins A, Klittich (1992) [Tumronisin 3L, prouctio, stains frm diflerent maing populations of(ihhLrc11a fuikorvi (IunsariJ,, section L.iewa) PhN opathology 82 34 1-345) .Isolates from maze (predominant!% mtine Spopulation.- A are among the highest prod, cers .(Leslie eta/ suprak umonisin levels detected in fleid-qi-own maize have fluctuated widelv dependinig on location and g~rmow 1 1

L

*season, but both preharNvest and postharvest survevs of field maize have indicated that the potential rFor hjigh levels of lbimorusins exists Murphy P. Rice L6, Ross P- I Ym3) "Fumomisin-Bl1 Fumnorisin-B2 and F umonisin-B3 content of- Iowa, \Visconsin, and Illinois 20 corn and corn screenings." J Agr Food Chem 41: 263-266) Surveys of tood ana teed products have also dtce uouiIHocmM.T mpnCr Hankinsl~ LIW193) "Analysis of fimonisin B3-I1 in rodent feed by gradient elution HPLC using precolumit derivation with FMOC and fluorescence detection." J Agr Food Chem 41: 704- 767, Hopmans EC, Mu-phyPA (1993) "Detection of FumonisinB(l),Fumorusin..B(2) and Fuxnonisin.13(3) and hydrolyzedj Fumorusin-B( in Corn-Containing foods." J Agr Food Chem 41: 1655-1658; Sydenhain EW, Shepha-d GS, Thiel PG, Marasas

WFO,

S Stockenstrom S (1991) "Fumonisin Contamination of Commercial Corn-Based Human Foodstuffs J Agr Food Chemn 39: 2014-2018). The etiology of 1kusarium ear mold is porl uZnderstood, alhuhpysical damage tothe ear and certain environmntal conditons an onribte o ts ccurece( lsn P (992) "Taxonomy and Biology of kusarlum maize, even when no visible mold is present. The relationship between seedling infection and 06 .0 6 6 6 66 6 6 666 66 6 6 6 66 6* 6 0.6 6666 96 66 6 69 6. 66 6666 6 66 60 6 66 6 66 66 66666.

6 stalk and ear diseases caused by /'usarlium is not clear Genetic resistance to visible kernel mold has been identified lGendlofl. Rossman L. Casale W. Isicib I Hart 1P 198(j) "Components of resisance to I-usar,m ear rot in field corn -Phytopathology 76: 684-688, Holley RN, Hamilton PB. Goodman MM (1989) "Evaluation of tropical maize germpla 5 s 1 for resistance to kernel colonization by usarium momluifo. "1plant Dis 73: 5 78-580L but the relationship to visible mold to fi'monjsin production has Vet to be elucidated Fumonisins have been shown in in virv manunaian cell studies to inhibit sphingolipid biosynthesis through inhibition of the enzyme sphinganine acyl transferase .(Nored "TWP.ang E, Yoo H, Riley RT. Merrill All 1992) vIrrv toxIcologY of f-umrnosins and the mechanistic implications Mycopathologia 117: 73-78, Wang Norred kk. Bacon CRilev R- Mlerrill A Jr (19)91) inhibition of sphlngolipid biosynthesis by tilmonisins implicaionst or diseases associated 'Aithl-usarlum momhifrrme J L'iol Chemi 266: l448(.)i'oolUS. .\orrcd WVP. \kang fr* Meml AR. Riley RI 1992) furnonusi Inhibition ot dec SI Sphi nwl ipid BiosYnthesis and Cvtotoxicirx Are Correlated in LL('-IK I Cells oxicol Appl Pharmacol v 114: 15 L resulting in the accumlulation of the precursor sphingzanine it is likek' that inhibition of this pathwaN accounts t for at least somne of fumonisin s twoctit and suppori for this comes from measures 01 sphunswanine sphingosine ratios in animals t'ed puritrled tuillrlisiri IWang L. Ross Wilson FNM. Rilev R 1. Mernil All 1992) -increases in Ser-um Sphingosine and Sphinganine and Decreases in Complex Sphingolipids in Ponies Given Feed 20 Containing Fumonisins, Mycotoxons Produced bv I'Usariurn inmiforme' J Nutr 122: 1 7uo- 1 716) Fumorusins also affect plant cell growth X.Abbas H-K, IBovmte CD 992) "Phytotoxicity of fiumonjsin 1 on weed and crop species Weed rechnol 6: 548-552, Vanasch MA), Rijkenberg FR)J, Coutinho TA (1992) "Phytotoxicity of fiumonisin

B,

moniliforrnin, and t-2 toxin to corn callus cultures." Phytopathology 82: 1330-1332, Vesonder RE, Peterson RE, Labeda D. Abbas H-K (1992) "Comparative phvtotoxicity of the furnonisins, AAL-Toxin and yeast Sphingolipids in Lemna minor L. ([uckweed)." Arch Environ Contain Toicoll 23: 464-46 Kutj et al. "Effect of fumonismn B I on virulence of' Fusarium species isolated fi-om tomato plants." (Abstract, Annual Meeting American Phytopathological Societ, Memphis, TN: APS Press 1993) reported on the ability of exogenously added fiimoriis to accelerate disease development and increase sporulation of' 1I ,usarium monlhfo-,,e and E oxysporum on tomato.

The toxicity of fumonisins and their potential widespread occurrence in food and makes it imperative to find detoxification or elimination strategies to remove the compounds from the food chain.

Disclosure of the Invention The present invention is based on the discovery of organisms with the ability to degrade the mycotoxin fumonisin. In a search for a biological means of detoxifying fumonisins, we have isolated from field-grown maize kernels several dematiaceous hyphomycetes capable of growing on fumonisin B I or B 2

(FB

1 or FB 2 as a sole carbon source, degrading it partially or completely in the process. One species, identified as lo Exophiala spinifera, a "black yeast", was recovered from maize seed from diverse S* locations in the southeastern and south central U.S. A related species, Rhinocladiella atrovirens, was isolated from seed originating in both Iowa and Georgia. We also isolated a bacterium, believed to be a Xanthomonas or Sphingomonas species, designated isolate 2412.1, from a field-grown maize stalk sample from Johnston, Iowa. This 15 bacterium also showed growth on FB 1 as a sole carbon source, and since its taxonomy is not certain we have deposited the strain with the American Type Culture Collection (ATCC) on 18 February 1994 and it is referred to herein by its ATCC deposit number, 55552. We have also deposited on 18 February 1994 enzyme-active strains of Exophiala spinifera (ATCC 74269) and Rhinocladiella atrovirens (ATCC 74270).

All isolates showed the capability to degrade FB 1 in liquid culture. By "degrade" is S: simply meant that the enzyme is capable of using fumonisin as a substrate and converting it to a different chemical structure. However, our studies indicate that the resulting compounds are less toxic than the fumonisins themselves.

Overall, only 16 of 70 independent seed samples tested yielded degraders.

25 However, several E. spinifera isolates, collected outside the U.S. from non-maize sources, were also found to metabolise fumonisins. Representative isolates of other Exophiala species tested jeanselmi, E. salmonis, E. piscifera) did not degrade fumonisins, nor did non-maize Rhinocladiella isolates, including R. atrovirens and R.

anceps, nor fungi associated with ear molds including Fusarium moniliforme,

F.

graminearum, Aspergillus flavus and Diplodia maydis. Fumonisin-metabolising black yeasts were found to possess an inducible hydrolase activity that cleaves the tricarballylate esters of FB 1 as monitored by C1i-thin layer chromatography (TLC) and fluorescence detection of amines. The identity of the resulting amino alcohol compound, designated API, was verified by FAB-mass spectroscopy. The latter [N:\LIBffJ01174:AJS compound has utility as a chemical indicator of fumonisin metabolism. E. spinifera cultures further metabolized AP 1 to compounds of unknown identity that were not detectable by amine reagents on TLC. In sealed culture chambers, E. spinifera grown on 14 C FB, as a sole carbon source, released 1 4

CO

2 as detected in 1N KOH-saturated filter paper strips, totaling percent of added label in 48 hours. Heat-killed cultures similarly incubated did not release appreciable 1 4 CO2. Thus, at least a portion of the fumonisin is fully metabolized by this fungus. Crude, cell-free culture filtrates of the E. spinifera isolate designated 2141.10 contained a heat-labile, protease-sensitive hydrolase activity attributed to an enzyme tentatively characterized as an esterase with specificity for tricarballylate esters of fumonisins and similar molecules such as AAL-toxin from Alternaria alternata lycopersici. This purified esterase is believed to be a new chemical entity, since no commercially available esterases tested were able to hydrolyze the Stricarballylate ester of FB 1 suggesting a novel enzyme specificity produced by these fungi. Cell-free extracts of E. spinifera isolate 2141.10 also contain an AP1 catabolase capable of converting AP1 to a compound lacking a free amine group, possibly an aldehyde. These enzymes and genes coding for these enzymes, being involved in Sfumonisin degradation, have utility in detoxification of maize seed pre- or post-harvest.

According to a first embodiment of the invention, there is provided a method of detoxifying a fumonisin, or a structurally related mycotoxin, the method comprising contacting the fumonisin with an esterase enzyme as produced by Exophiala spinifera, S9* ATCC 74269, Rhinocladiella atrovirens, ATCC 74270, or the bacterium of ATCC 55552.

According to a second embodiment of the invention, there is provided an isolated DNA polynucleotide comprising a member selected from the group consisting of: 25 a) a polynucleotide encoding a polypeptide comprising amino acids 1 to 525 of SEQ ID NO: *6S b) a polynucleotide which is complementary to the polynucleotide of and c) a polynucleotide comprising at least 130 bases of the polynucleotide of or f(b); d) a polynucleotide comprising bases 1 to 26 of or According to a third embodiment of the invention, there is provided an isolated DNA polynucleotide comprising a member selected from the group consisting of: a) a polynucleotide encoding a polypeptide comprising amino acids 1 to 525 of Figure 2; b) a polynucleotide which is complementary to the polynucleotide of and c) a polynucleotide comprising at least 15 bases of the polynucleotide of or d) a polynucleotide comprising bases 1 to 37 of or [N:\L1BFF]O303:MCC 0 *0 0 0 000 9 00 0r 0 0 tO 00 0i According to a fourth embodiment of the invention, there is provided a transformed plant cell comprising the coding sequence of Figure 1 or variations thereof permitted by the degeneracy of the genetic code.

According to a fifth embodiment of the invention, there is provided a plant having stably incorporated within its genome a nucleic acid sequence comprising the coding sequence of Figure 1 or variation thereof permitted by the degeneracy of the genetic code.

According to a sixth embodiment of the invention, there is provided a polypeptide having fumonisin esterase activity comprising the two amino acid domains ATLM and

TNI.

1o According to a seventh embodiment of the invention, there is provided a polynucleotide sequence encoding an enzyme having fumonisin esterase activity comprising the two amino acid domains ATLM and TNI.

According to an eighth embodiment of the invention, there is provided fungal isolates, their components and mutants thereof, selected from the group consisting of Exophiala spinifera, ATCC 74269 and Rhinocladiella atrovirens, ATCC 74270.

According to a ninth embodiment of the invention, there is provided a bacterial isolate, its components and mutants thereof of isolate 2412.1, ATCC 55552.

According to a tenth embodiment of the invention, there is provided a method of detoxifying a fumonisin, a structurally related mycotoxin, a fumonisin hydrolysis product, 20 or a hydrolysis product of a structurally related mycotoxin present in harvested grain, the method comprising reacting the hydrolysis product with an AP 1 catabolase produced by Exophiala spinifera, ATCC 74269, Rhinocladiella atrovirens, ATCC 74270, or the bacterium of ATCC 55552.

According to an eleventh embodiment of the invention, there is provided a method for detoxifying, in harvested grain, a fumonisin or a structurally related mycotoxin and for detoxifying a fumonisin hydrolysis product or a hydrolysis product of a structurally related mycotoxin, the method comprising reacting the fumonisin with an enzyme having S the structure of the fumonisin degradative enzyme produced by Exophiala spinifera, ATCC 74269, Rhinocladiella atrovirens, ATCC 74270, or the bacterium of ATCC 55552, and reacting the hydrolysis product with an AP 1 catabolase produced by Exophiala spinifera, ATCC 74269, Rhinocladiella atrovirens, ATCC 74270, or the bacterium of ATCC 55552.

According to a twelfth embodiment of the invention, there is provided a method of producing the fumonisin degradative enzyme produced by Exophiala spinifera,

ATCC

74269, Rhinocladiella atrovirens, ATCC 74270, or the bacterium of ATCC 55552, the method comprising producing a transgenic plant which expresses one or more of said fumonisin degradative enzymes and isolating and purifying the enzymes from the plant tissues expressing the enzymes.

[N:\LIBFFIO303:MCC According to a thirteenth embodiment of the invention, there is provided a genetically engineered ruminal microorganism comprising an expression vector capable of expressing proteins in microorganisms, said vector comprising a nucleotide sequence encoding the fumonisin degradative enzyme produced by Exophiala spinifera, ATCC 74269, Rhinocladiella atrovirens, ATCC 74270, or the bacterium of ATCC 55552.

According to a fourteenth embodiment of the invention, there is provided a genetically engineered microorganism comprising an expression vector capable of expressing proteins in microorganisms, said vector comprising a nucleotide sequence encoding the fumonisin degradative enzyme produced by Exophiala spinifera, ATCC 74269, Rhinocladiella 0i alrovirens, ATCC 74270, or the bacterium of ATCC 55552.

According to a fifteenth embodiment of the invention, there is provided a method of identifying transformed plant cells using a Fusarium species or the toxin produced by Fusar.ium as a phytotoxic marker, comprising the steps of: a) culturing cells or tissues from a selected target plant in a culture medium; I b) introducing into cells of the culture at least one copy of an expression cassette comprising a coding region that codes for a fumonisin degradative enzyme produced by Exophiala spinifera, ATCC 74269, Rhinocladiella alrovirens, ATCC 74270, or the bacterium of ATCC 55522, operatively linked to an upstream transcription initiation sequence and a downstream polyadenylation sequence causing expression of the enzyme in 0 the cells; c) introducing Fusarium or a fumonisin into the culture medium; and d) identifying transformed cells as the surviving cells in the culture.

According to a sixteenth embodiment of the invention, there is provided a method of reducing pathogenicity of a fungus which produces a fumonisin or structurally related 2 5 mycotoxin, the method comprising expressing, in a host plant susceptible to infestation by lhe fungus, a fumonisin degradative enzyme produced by Exophiala spinifera,

ATCC

74269, Rhinocladiella alrovirens, ATCC 74270, or the bacterium of ATCC 55522.

According to a seventeenth embodiment of the invention, there is provided a method of degrading a fumonisin, or a structurally related mycotoxin, the method comprising contacting the fumonisin with a fumonisin degradative enzyme produced by Exophiala siinifera, ATCC 74269, Rhinocladiella atrovirens, ATCC 74270, or the bacterium of ATCC 55522, wherein the degradation reaction occurs in processed grain which is to be used as a human food product.

IN:\LIBFF]0303a:gcc Brief Description of the Drawings FIGURE 1 is the nucleotide sequence including the open reading frame coding for the bacterial esterase gene (bases 94 to 1683).

FIGURE 2 is a hypothetical amino acid sequence of the polypeptide encoded by bases 94 to 1683 of the nucleotide sequence of FIGURE 1. Residues 1-38 represent the putative signal sequence. The polypeptide including the signal sequence has a calculated molecular weight of 55,026.68 (529 residues), with a calculated pI of 8.70. The polypeptide without the putative signal sequence has a calculated molecular weight of 51,495.63 (491 residues), with a calculated pl of 8.19.

II Detailed Description of the Invention This invention provides newly discovered enzymes capable of degrading and detoxifying fumonisins, produced by fermentation of one or more of Exophiala spinifera, ATCC 74269, Rhinocladiella atrovirens, ATCC 74270, or the bacterium of ATCC 55552.

The invention further comprises methods for making enzymes that are capable of detoxifying fumonisins, comprising the step of growing one or more of Exophiala spinifera, ATCC 74269, Rhinocladiella atrovirens, ATCC 74270, or the bacterium ATCC 55552 in the presence of a

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[N:\LIBFF]0303a:gcc tijnOnisinl or the metabolite produced by action of the enzYme on a tilmorusin This invention tfnher Provides methods of detoxifying flimonsins comprising the step of reacting them with an enzyme derived from Exophlala spillfera, ATCC 7426-9, RIhlnocladie//a atrvirens,, AIcc- 74270, or the bacterium of AJCC 55552.

We have isolated and sequenced the gene that codes for the fiumonisin.derding enzyme from one of these organisms and provide the amino acid sequence of the enzyme here. It Is known that genes encoding desired proteins can be identified. isolated, cloned and expressed in transgeic organisrm, including several important crop plants, One cormmornly used method of gene transfer in plants involves the use of a disarmed form of the Ti plasmiud of the sol bacterium A grobacterium lumefaclenzs A. fumnefaciens is a plant pathogen that causes crown- Votoo gall tumors in infected plants. Large plasmnids. termed Ti- or tumor-inducing plasmuds, a-re *responsible for the on o e i ivof the bacte rum as well as f r h r n f ro f r i n D A t the plant. Sirrularly, A. rhiz-ogenes contai ns or root-jnducin'ilsrid htinuero 0 0growth Both pla-srrud types include a vir or vii ujence region that must be tunctionali n order to transform wvild-type cells to tumor cells 040 1 ransformation rslsi h nerto fnte plaso-ud portion, termed the I. o transf'er-DNA- irnto tenuclear gzenome of the transformed cells Ki and Iij plasmnics can bc maniupulated to allow insertion of fore:gn DNA, -ncoding a desired protein, into the I region The foreign DNA can be transferred either via a vector bearing both the irir gene and 020 the fioreign gene or by a binary vector system consisting of two plasmids. one contairnn the @0S0 vir gene and the other carrying the foreign gene. See, U S Pat No 4.05 .8,O2 p .0Transformed plant cels can then be regenerated to produce varieti es bear-ng the inserted gene Theprouctonof transgenic. fmonisin-reisant patwilrodea useful and novel approach for the control of Fusanum-inuc1j plantdies.

This invention also provides a mechanism for selection of transformants. growth of plant cells in the Presenlce of a Fusar-ium or its mycotoxin favors the survival .of plant cells that 0*00~~have been transformed to express the coding sequence that codes for the enzyme of this invention and degrade the toxin. Thus, the coding sequence that codes for the enzyme of this invention can itself be used as a selectable marker, or as a scorable marker by measuring formation of the amino alcohol metabolite.

Another embodimnent of the present invention is directed to a DNA construct comprising an expression cassette comprised of a) a DNA coding sequence for a Polypeptide capable of degrading fumionisin. and b) control sequences that are operably linked to the coding sequence whereby the coding sequence can be transcribed and translated in a host cell. and at least one of the DNA coding sequences or control sequences is heterologous to the host cell.

Preferred embodiments of the subject invention include a host cell stably transformed by a DNA construct as described above-, and a method of producing a polypeptide of a recombjpmt gene comprising.

a) providing a population of these host cells, and b) growing the population of cells under conditions whereby the polypeptide encoded Io bv the coding sequence of the expression cassette is expressed In yet another embodiment. the present invention is directed to a transgentc plant capable of degrading fiimonasin In another embodiment, the transgzenic plant is a maize plant capable ot'degrading tijmontsin 00 Another embodiment of the subject invention comises a tnethod of confhmnQ furioisn-rsitaecto a plant sbtni.1 without sucfi resistance compnsing rnl-c~nzt *000 the plant an expressible gene encoding a polypeptide capable of degrading fumronisinl 0 Thus. DNA encoding a protein able to inactivate furmonisin can be isolated and clonied in an appropriate vector and inserted into an organism normally sensitive to the Iua ozn i its toxin. Orgarusms expressing the gene can be easily identified by their ability to deizrade 0020 fumonisin The protein capable of degrading fiimonisin can be isolated and chara'-terized usngtchiqe well known in the art. Furthermore. the gene imparting fumnonisin-rusistance can be transferred into a suitable plasmid, such as into the I-DNA region of the Ti or Ri plasmid ofthe soil bacteria Agrobacterium lumefaciens or Agrobacterium rhzogenes, 0 respectively. Plant tissue can be inoculated with the transformed bacteria. Additionally' plant 23 tissues which have been co-cultivated with Agrobacterium spp. can be incubated in the presence of ilinonisi to select for fumonisin..degraing transgenic plants, ie., the gene for fimnisin degradation ca ev sa selectable marker. Thus, the inoculated tissue is ~mii.4regenerated to produce fiimonindegading transgenic plants.

Additionally, the present i~avention relates to ruminaj microorganist-is that have been genetically engineered with the genes imparting flimonisin resistance. These engineered runinal mnicroorganisms can then be added to feed for consumption by animals susceptible to fumonismn and structurally related mycotoxins.

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IndlUstrial Arwlicabulity The practice of the present invention will employ, urless otherwise indicated.

conventional techniques of botany, microbiology, tissue culture. molecular biology, chemist -y, biochemistryv and recombinant DNA technology, which are within the skill of the art. Such techniques are explained fully in the literature. See, J. H. Langenheirn and K V Thimann.

Botany,: Plant Biology and Its Relation to Human Affairs (1982) John Wiley, Cell Culture and Somatic Cell Genetics of Plants, Vol. I (L K. Vasil, ed 1984), R V Stanier, J. L. Ingraham, NI L Wheelis, and P. R Painter, T1he Microbial World. (1986) 5th Ed, Prentice-Hall, 0 D Dhringra and J B3 Siniclair, Basic Plant Pathology Methods, (1985) Il0 CRC Press, Nianiatis, Fritsrh Sambrook, Molecular Cloning: A Laboratory M~anual 1982), DNA Cloning, Vols I and 11 N Clover ed 1985), Oligonucleotide Synthesis J Gait ed 1984), Nucleic Acid Hybridization D Flames S J f iiggins eds 1984).

a-nd the senes Nlethods in [nay mology (S C'oloxvick and N Kaplan- eds A\cademic Plress.

Inc) 5In descnhiing the present invention, the tibllowing terms %vill be cminlo cc, and are intended to be detined as indicated bejowk liv "microbe" is meznt any microorganism (incliding both euicaryoti and procar-,otirmcroorganisms), such as fungi, yeasts, bacteria, actinomycetes. algae and protozoa, as vk ell as other unicellular structures capable of growth in culture.

20 A "furnonisin-producing microbe" is any microbe capable of producing the mycotoxin fumonisin or analogs thereof Such microbes are generally members of the finizal genus I-usvarium, as well as recombinantly derived organiisms which have been genetically altered to enable them to produce fumonisin or analogues thereof Byi"degrading furnonisin" is meant any modification to the fumorusin molecule v.tuch causes a decrease or loss in its toxic activity. Such a change can comprise cleavage of any of' the various bonds, oxidation, reduction, the addition or deletion of a chemical moiety, or any other change that affects the activity of the molecule. In a preferred embodiment, the modification includes hydrolysis of the ester linkage in the: molecule as a first step.

Furthermore, chemically altered fu'nonisin can be isolated from cultures of mnicrobes that produce an enzyme of this invention, such as by growing the organisms on media containing radioactively-labeled filmonisin, tracing the label, and isolating the degraded toxin for further study. The degraded fulmonisin can be compared to the active compound for its r* 4 *e

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W d .s aC 9e phytotoxicity or mammalian toxicity in known sensitive species, such as porcmes and eques Such toxicity assays are known in the art. For example, in plants a whole leaf bioassav can be used in which solutions of the active and inactive compound are applied to the leaves oft sensitive plants. The leaves may be treated Mi su or, alternatively excised leaves may be used The relative toxicity of the compounds can be estimated by grading the ensuing damage to the plant tissues and by measuring the size of lesions formed within a given time period Other known assays can be performed at the cellular level, employing standard tissue culture methodologies using cell suspension cultures By "structurally related mycotoxin' is meant any mycotoxin having a chemical to structure related to a fimonisin such as fumorusin BI for example AAL toxin, fumonrusn 132 fumorusin B3 tumorsin B4, fumorusin C. fumorusin Al and A2 and their analogs, as well other mvcotoxns having similar chermcal structures that would be expected to be detoxitied by acti\-vr ot the fumorusin dePradative enrzvmes elaborated by troplnala .pinnera A .I( 74209, fRhnolad,./la arrovirens. ATCC 74270. or the bactena of ATCC 55552 i 8v "harvested grain" is meant any form of ain r h uch has been somehok remi ved trom the environment in which it was wrown For example, harvested grain could consiiuic ear cor or corn kernels remoed from the ear, or cut wheat stalks, or barley or rice kernel.

or the like liarvested grain may be in storage or may be being processed "Processed cran is grai that has been through some form of processing and xi'l be used in the production of 2 lfood otr human consumption or will be used as arumal feed By "transgenic plant" is meant any plant or plant cell that has become transformed by the introduction, stable and heritable incorporatio, into the subject plant or plant cell of foreign DNA, i.e. DNA encoding for a protein not normally found within that plant species "Plantlet" refers to a plant sufficiently developed to have a shoot and a root that is asexually reproduced by cell culture.

"Explant" refers to a section or piece of tissue from any part of a plant for culturing.

By "hormone" is meant any plant growth regulator that affects the growth or differentiation of plant cells. Such hormones include cytokinins, auxins, and gibberellins, as well as other substances capable of affecting plant cells.

The term "callus" and its plural "calli", refer to an unorganized group of cells formed in response to cutting, severing, or other injury inflicted on plant tissue. Excised pieces of plant tissue and isolated cells can be induced to form callus under the appropriate culture conditions Callus can be maintained in culture for a considerable time by transfernng or subcultunng pans of the callus to fresh medium at regular intervals. The transfer of callus to liquid medium leads to dispersion of the tissue and the formation of a plant cell suspension culture. Callus can be induced to undergo organized development to form shoots and roots.

"Embryoid" refers to a structure similar in appearance to a plant zygotic embryo "Somatic hybrid" and "somatic hybridization" refers generally to stable combination of cellular material, be it protoplast/protoplast or protoplast/cytoplast combinations, and includes cybrids and cybndization.

A "replicon" is any genetic element (eg. plasmid, chromosome, virus) that tunctions as an autonomous unit of DNA replication in vivo, i capable of replication under its own control A "vector" is a replicon. such as a plasrmd. phage. or cosrmd. to which another DNA ,segment mav be attached so as to bnnng about the repication of the attached seement As used herein, the term "nucleotide sequence" means a DNA or RNA. molecule or 15 sequence. and can include, for example, a cDNA. genomic DNA. or a svntheutc DNA sequence, a structural gene or a fragment thereof. or an mRNA sequence that encodes an active or tunctional polypeptide A DNA "coding sequence" is a DNA sequence which is transcnbed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences The 20 boundaries of the coding sequence are determined by a start codon at the (aimno) terminus and a translation stop codon at the 3' (carboxy) terminus. A coding sequence can include, but Is not limited to, procaryotic sequences, cDNA from eucarvotic mRNA, genonuc DNA sequences from eucaryotic mammalian) DNA, and even synthetic DNA sequences. A polyadenylation signal and transcription termination sequence will usually be located 3' to the %j 4 25 coding sequence.

A "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream direction) coding sequence. For purposes of defining the present invention, the promoter sequence is bound at its 3' terminus by the translation start codon (ATG) of coding sequence and extends upstream direction) to include the minimum number of bases or elements necessary to initiate transcription. Within the promoter sequence will be found a transcription initiation site, as well as protein binding domains responsible for the binding of RNA polymerase.

Eucarvotic promoters will often, but not alwavs, contain "TATA" boxes and "CAT" boxes Procarvotic promoters contain Shine-Dalgarno sequences DNA "control sequences" refers collectively !o promoter sequences, ribosome binding~ sites, polvadenylation signals, transcription termination sequences. upstream regulatory' domains, enhancers, and the like, which collectively provide for the transcription and translatieni of a coding sequence in a host cell.

A coding sequence is "operably linked to" or "under the control of' control sequences in a cell when RNA polvmerase will bind the promoter sequence and transcribe the coding seq uence into rnRNA. which is then translated into the polvpeptide encoded bY the coding sequence A4 "host cell" is a cell wich has been transf-ormed. or is capable of undergoine .transformistion, by an exogenous DNA sequcrnce A cell has been "transformed" b% xo ous DNA- w hen such e\oeenous ha *00been introduced inside the cell membrane Exogzenous DNA may or niav no-,t tic interaied inito (coaletklined o)chromosomal DN A making up the 2enome of the transicrnied In S procarvotes and \easts. tbor examiple, the exogenous DNA may be maintained on m,i ',omaijI lemicni, such as a plasmid With respect to eucanotic cells, a stably iransl'omieji ce! s n inl which the exogenous DNA has become inteizrated into the chromosome so that !t is innented bdaughter cells through chromosome replication. This stability is demonstrated by the abdit *020 of- the eucaryotic cell to establish cell lines or clones comprised of a population of daughier Os:. *cells containing the exo~genous

DNA.

A "clone" is a population of cells derived from a singic cell or common ancestor by *moss A "cell line" is a clone of a primary cell that ib capable of stable growt-h in vitro for many generations.

Two DNA, RNA or polyetide sequences are "substantially homologous" when at least about 85% (preferably at least about 901/6, and most preferably at least about 95%) of the **too nucleotides or amino acids match over a defined length of the molecule. DNA sequences that are substantially homologous can be identified in a Southern hybridizati on experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, Maniatis et al., supra. DNA Cloning, Vols. I 11. supra; Nucleic Acid Hybri ization, supra A "heterologous' region of a DNA construct is an Identifiable segment of'DNA wthin or attached to another DNA molecule that is not found in association with the other molecule in nature. Thus, wvhen the heterologous region encodes a bacterial gene. the gene Will usually.

be flanked by DNA that does not flank the bacteri gene In the genome of the source bacterium. Another example of a heterologous coding sequence is a construct where the codingsqeceisl s o on in nature synthetic sequences ha,.:ng codons different from the native gene) "Heterologous" DNA also refers to DNA not found wkithin the host cell in nature. Allelic variation or naturally occurring mutational eventf do not give rise to a heterologzous revion of DN.A. as these terms are used herein The term "polvpcptide" as used herein is used in its broadest sense. e ,any polner oft amino acids (dipeptide or greater) lin-ked througzh peptide bonds Thus, the term "polvpepie includes proteins. oligopeptidles. protein fragzments. analop~es, muteiris. fusion ')rotein~s arid S. the like The term also encompasses arruno acid polvmers as described abo\-- thai inciude additional noni-amno acid moieties Thus, the term "polvpepide' includes ilccpicien, 5 lipoproteins, phosphoproteins. metalloproitis, nucleoproteins. as w ell as other olcr :s ~prote~ns I he term "polvpeptidec contemplates polx-peptides as defined abo\ e that are robinantly produced. isoiated tromn an appropnate sourc, or svnthesizek This invention can be better undcrstood rcfterencc to the iollo\ 11 inc On-11i~liIT examples It A1ll be appreciated by those skiled in the arti that other embodirments ol ih invention may be practiced without departing from the spirit and the scope of the in\vention as herein disclosed and claimed Example I :Chemicals and reagents. All chemicals we-e re-agent grade or better uniess otherwise indicated. Fumnonisin B, and B 2 were obtained from Sigma Chemical Co Partiall\y purified 025 fimonisis (eluate from C8 column) were obtained from Dr- Pat Murphy (Iowa State University). AAL-toxi (TA isomer) was a gift of Dr. David Gilchrist (University of Caifornia-Davis).

Plant tissue samples. Mature, field-grown maize seed was obtained from maize breeding locations of Pioneer Hi-Bred internationaL, ic. in the Southeast. Midwest and South Central regons of the U.S. Seed was stored at room temperature in individual packets Fungal and bacterial isolates. Exophiala and Rhiinocladuiela isolates from maize were isolated as described below. Other isolates were obtained from Dr. CiJ Wangc (Syracuse, NY), Dr Michael McGinnis (Case -stern Reserve University Cleveland, 011). and from the American Type Culture Collection (Bethesda, MD) pusarium granu,,ea,,,, Ij61bbe'relhi( zeac (Schw) Petsch], LDiplodia mmidis, and Pusarium moni/iforme Sheld., were obtained from the microbial culture collection of Pioneer Hi-Bred International. Inc.. Ap~g1a iv.

(Schw.) Petsch, isolate CP22. was obtained from Don Sumrner at the University of Georgia (Tifton- GA) Xainihomonas sp. 24 12. 1 was isolated fTom maize stak tissue as described below.

Isolation methods Individual kernels. etther intact or split in two kkith a sterile ra-zor blade, wvere rinsed for I hr in S, ml sterile wvater %kith acitation F-romn I to 5 pti oftihc rinse fluid wa5 .dded to 100 4iL of sterile. carbon-fice nenral salts medium FBI

I

giliter NHS0 4 I wliter K 2 HP110 I g'liter NaCI. 0 2 wtliter NMgSO 4 7Hu p1 7o ontairnn FB 3Stigma Chemical Co )at u 5 to 1 0 mo-'mfl Ihe pHi ot the mediumn Wajs lr prk)\ tile: addition of EB, -Vter Ito 2 weeks incubation at 28(C in the dark- sena liolj ~.:ere made in stenile dll..O. and aliquots were plated onto I 2% Bacio-apar cont airink:e V\east exutac:. I'o Bacto-peptone and 0 l'o dextrose iP aar) I unizal and 0eacteti ~Co Ionics that appeared on a 2ar k er e transf'erred onto fresh l ates.. I d Jih. MCI I k\di evaluated for frirnonisin metanoltzinkz abilitv by inoeulatinQ them Into fresh NIS-t BH tfrimonisin from the medium was monitored periodica-ily hY sporting (I5 to I tuicoic e .uklio: of culture supernatant on Cis silica g.el plates that were then air-dried and developed as l ecie eo (see Analvsis of fumonisins and metabolism products i Direct isolation of black yeasts from seed was accomplished by plating I w microlers ot seed wash fluid onto YPD or Sabow-aud agar augmnented with cycl ohexirtude (500 miwliter) :and chloramphenicol (50 mgi/Per). Plates were incubated at room temperature for 7-14 days.

and individual pigmented colonies that arose were counted and cultured for analysis of' fumonisin-degracling ability as described above.

For stalk isolations, mature stalk samples 0 5 x 0.5 x 2 cmweetknto Suhetype maize tnbredr grown in Johnston. Iowa by Pioneer Hi-Bred Internationa) Inc a seed company, in 1993. One-inch sections of the center (pith) or the outside of non- surfacesterilized stalk were cut and placed in 10 mld. sterile water in a small, sterilized tube ['he tubes 3U were shaken for I hour. and then 2 p1 of washate were AthdraNn and used to inoculate 100 p1 of MIS-FB, 1in ;1 microtiter plate. Subsequent steps were as above .Analysis of funionisins and metabolism products. Analytical thin-layer chromatography was carried out on 100%/ silanized Civ silica plates (SigmaTM #T-7020. cm; 0. 1 mm thick) by a modification of the published method of Rottinghaus. Sample lanes were pre-wet with methanlol to facilitate sample tapplication. After application of from 0. 1 to 2 l.i of aqueous sample, the plates were air-dried and developed in MeOH:4% KCI or MeOH:0.2 M KOH and then sprayed successively with 0. 1 M sodium borate (pH and fluorescamine (0.4 mg/mil in acetonitrile). Plates were air-dried and viewed under long wave UV Alkaline hydrolysis of FBI to API. FBI or crude flimonisin Cx material was suspended in water at 10- 100 mg/mId and added to an equal volume of 4 N NaOH in a screwcap tube The tube was sealed and incubated at 60'C1 for I hr The hvdrolysate was cooled to RI and mixed with an equal volume of ethyl acetate. centrtuv~ed at 1000 ((CF for 5 mninute **and the organi-c (upper) layer recovered The pooled ethyl acetate laYers from t10o succeSSI\C extractions were dried under N, and resuspended in dH 2 0 The resulting material (the 1; aminopentol of FBI or -iAP') was analyzed by TLC Soo.. Tables I and 2 illustrate the results o 'etibris to isolate a 1TurnuJnisidegradin cnzvnie rom a w-ide assortment of sources. As is noted. L. vspinifcru isolates frTom maize seed fromi various locations were always able to produce a tumorusin-degradini enzrYme when grow n on fumonisin as a sole carbon source (Table I as were spinifera isolates from other sources from around the world (Table Some samples of Rh iur 2/adiella arrovirens. from maize seed were also able to produce this enzyme (Table I Other species ot'Lxophiala and other CC C sources and species of Rhinocladiefla were routinely unable to produce the enzyme, even :when isolated fr-om plant-related sources (Table 2).

Table 1: Dematiaceous fungi isolated from maize weed that degrade fuinonisia Modification Gt substrates Isolate#o Speckm Locatio of origin Isoixted trum Appear- FBI

AP,

_ance 2369.E7 Eropiuala Tifton. GA Maize seed clean 2369.G5 Erophiala T ifton. GA Maize seed clean s/xmjer (3329) 27.4 Eohca Tfo-GA Maize seed Moldy' Modification of efbstrates Isote# specie Loction of origin Iolated frm Appear. FB, AP, 66 66 6 6@ .6 *6 *6@ *666 6 66 9* 0* *6 6 66666t 2369.F7 Erophiala Spinifera 2369.H9 Erophiala spinifera 2141.10 Exophialo Spintfera 2174.C6 Rhinoclatela 01Mviftns 21 70.2 Eixop ilala .piflJera 2174 A4 EXophIala spinifrra 22 19.W 1-op 1 iaia spi lhcra 2363 1 Erophiala 23633 Erophiala 2363.3 Exophlola spInijera 2363.8 Erophialo spinifera 2363.10 Exophiala spimfera 2369.F11 Rhinodadlla afrobm'ns Winterville. NC Winteville NC Wintcrvile NC Winici-ilke NC Union Cit. TN Union CaN. TN Wcsaco, TX Wcslaco. TX Weslaco. TX Weslaco. TX Weslaco. TX Johnston LA (3170) Maize seed (3379) Maize seed (unk) Maize seed (Unk) Maize seed (inbred) Maize seed (Inbred) Maze seed (Inbred) MNauc seed (Inbred) Maizc seed (inbred) Maize seed (Inbred) Maize seed (inbred) Maize seed (inbred) Maize seed (inbred) moldy moldy moldN a-BceI Winterville. NC Maize seed moldy moldy mold\' mold\ mold mold moldN moldh moldy nt clean implies vise dioloion okr pencar.p cracking or spliwng; "clean" implies no visible signs of info uon on the kernel 'Evaluaed by TLC analvsis of cultre superatans as dcnbed herein nt not testd Table 2: Other fungal isolates tested for degradation of fumonisin BI in liquid culture Modification of substrates Isolate Species Source Location of Isolated from FB, AP, Origin -Black Yeate Ftangi 9 99 9 *9 9 9999 99 99 9 #9r '9 9 9 99 99 .9 0 26089 Erophiala spini era 26090 Exophtala spinifera 26091 lxophiala .vpiera 26092 P-ophiala .spini]ero 4?173 1-rophiala .spinijera 5o567 1Exophwala spi ni/ra 182 18 txop/ ala sptntera 5"092 Lrophia/a spinijera 66T75 Erophiala montleae 32288 Eop/iala salmonis 26438 Erwphiala pisciphila 26272 Erophiala jeanselmi P-154 Rhinclaoella atovrnS P-330 Rhinocladeidla atrevirens P-646 Rhinocladiella afrovirens P-1492 Rhinocladzela atroirens ATCC Urgua Palm tree frut ATCC Uruguay Bird's nest Birds nest

ATCC

Arcc

AJTCC

ATCC

ATCC

ATCC

Uruguay ATCC Uruguay Palm tnmk Nasal Granuloma Nasal Granulon-a Huuman ATCC Unknown Leaf Lirtcr ATCC Austraha Wheal rhizosple're ATCC Canada Activated sludge C.J. Cbcst NJ Sothr pine Wang pole C.J. Binghamn. South pine Wang NY pole C.J. Viginia Southern pine Wang pole C.I. Chester, NJ Southern pine Wang pole it species Source Location of Origin Modifica~ion of substrates Lsolated from FB, AP, ED-43 Rhinocladzdja Otrovirens ED-1 24 Rhinodoa~dzella atrovrrns 28220 Rhinacladzella anceps

C..

Wang

C.J.

ATCC

Unknown Douglas-fir pole Unknown Douglas-fir pole Maryland Grass lt n t t FMO001 Fusanum morn iforme FGRcKo I P-usan, urn -Ear mold fungi MH Unknown PHI Unkcnovai MaizeC Maize Maize Maize 00 *0 *0 0 0 00 0 000 0 9. 0 90

S.

00 0 000 *000 00 00 0 00 00 0 0* 0000 00 *0 0 00 0 0 0 00 00000.

0 000000

S

'4 CP22 DMAtxH ,graminearun: Diplodia rnavuis UnkcnoAn Unknown *Tested bth %Nith KB and as a sole carbon souroc and with FBI amnended with 1% sucro,,c Table 3: Frequency Of iso;atioe nf fumonisin-degradinz black yeast isolates from m~aize seed Location of origin samples N Samples containing Species identified tested positive FBI-degradiag black yeast Weslaco. TX 8 6 75.0 Erop/n ala spzrnyera Winterville. NC 19 4 47.5 Erophiala spinifera, Rhinocladjella arrovi Tens Ttftmn GA 8 3 37.5 Axopluala spiifera Union City. TN 7 2 28.2 Erophi ala spinifera Johnston, IA 7 1 14.3 Rhinocdaikella arrayrens Shelbyville, IL 3 0 0 none MacomnbIL 4 0 0 Champaign, IL 3 0 0 Yale. IN 3 0 0 California 8 0 0 Total 70 16 22.8 Organisms can be screened for their ability to degrade fumonisin using the present meth~ods. In this way, plant, soil, marine and fresh water samples can be screened and organisms isolated therefrom that are able to degrade flimonisin. Alternatively, already isolated microbial strains that are suspected of possessing this capability can be screened.

Putative fumonisin-resistant bacteria include bacteria associated with plant species susceptible to Fusanum infection. For instanc.e, bacteria associated with Fusaium-infected tomnato and pepper as well as other susceptible plant species, might be expected to degrade fumonisin.

Furthermore, members of bacterial genera known to be versatile in their catabolism of complex organic molecules, such as members of the genus Pseudomonas, rnight degrade fumonisin.

Generally, media used to culture the above miucrobes will contain a known amount of fumorusin- ic. from 0. I to 3 mg of fumronisin per ml of media- more usuallv from 25 to 2 miz 6per nil of media, and preferably from 0.5 to I mg of fumonisin per nil of media .A further study was perform-d to determine if colonv morphology could be used to determiune Nvhich strains of these species would produce a fumonisin-degradinge enizymie I he V, results as shownr in Table 4 indicated that E spnif era and X arrovir'flA colonies having different morphologies could nevertheless produce the fumionisin-degrading enzyme Table 4: Black vexa recovered from a single kernel by di rrci platin~ seed ashite onto N*PD cyclobeximide cblora~mpbcnicol' Isolate Colony 1ype on YPD Species colontce S FB' dc 2r agar *6 *0 0~ a a. *6 S a a.

*SSCS*

a 2403.5 Light brown, slun. Lxophi ala spinijera 3133 2403.25 Dark brownm. hiy Era pluola spinifera 2403.12 Brown. velvety Rinoclacbefla 4 4 alrovirens 2403.2 Grey, wvvty, Rinocldiela

I

atmvrens TotaLs 39 3T9 20 Kernel source: Tifton, Georgia. Seed was split, washed in. 5 ml sterile water and then 100 ul was plated onto YPD agar containing cyclohexirnide (500 mg/I) and chloramphenicol mgfL).

From these results it was concluded that growth on flimonisin as the sole carbon source is the most reliable indicator of the ability to produce the fumionisin-degrading esterase.

The esterase isolated from L spinffera was then subjected to other treatments, including proteases, to determine whether and how the enzyme would function in various envirornents. The results are indicated in Table Table 5: Effect of various tratmnenUon modificational'

FB,

Treatment Conditions FB, Hydrula-W activtV*t Control 16 hr. 3 7 C, pH 5.2 Baihing watcr ba.th 1000r C, 3 0 mntL pH 5. 2 Protease K 0.0 1 mg/nil, 16 hr, 3 70 C, pH 5.2 Pronasc E 0.0 1 mg/nd. 16 hr. 370C. pH- 5.2 ChNvmomrpsin 0.01 mg/rn!. 10 hr. 3 7- C. pH 5.2 Trypsti 0,01 mg/mi. 16 hr. 37" C. pH 5.2 EDTA DTT *Ca" 50 nLM Ng *S0PMSF 10 11M I lO-tbld concentrated, I I to 15 day culture filtrates treated as descnbed and then inCuoa-1,ttC( 0S 0with

FBI

1 (0 5 mgiri final cone) overnight at 37'C Analysis by, -I'Lc~tluorescrne spraN tbollowing overnight Incubation at 371C with I mg/rnJ flimonuisn no hydrolysis trace amount of hydrolysis incomplete hydrolysis icomplete hydrolysis @000.:Next, the pH range of activity of the fumonismn esterase was evaluated by measuring 15i ftimonisui degradation in the presence of citrate and citrate- phosphate buffers at varying pH levels, Results are shown in Table 6. From this, it was concluded that the pH range of the enzyme was quite wide, and that the enzyme would function at the internal pH of plants and a plant cells.

Table 6: Effect of buffer pH on hydrolysis of fumonisin B, by E spinifera culture filtrate Buffer PH FBi Hydrolase activtity- U. IM citrate 0.1 M citrate-phosphate 0.1 M citrate-phosph~ate 0. 1 M cilrate-phosphate 0. 1 M phosphate 0. 1 M phosphate Sreactions were carried out at 37'C overnight and then assayed by TLC Analysis by Cig TLC/fluorescamine spray following oernight incubation at 37 with meirn] fumonisin no hydrolysis trace amount of hydrolysis ~-incomplete hydrolysis 0 t -10 incomplete hydrolysis complete hydrolysis.

The fumorusin esterase isolated from spnlera and R t?rovircn.\ 'ka> comlpale xit other known esterases from various sources as supplied by commercial vendors l'hc results shown in Table 7 indicate that the flimonisin esterase is a unique enzyme that is highly specific 15I in its activity and does not have a generalized esterase activity comparable to that ol'any% of the known enzymes tested.

Table 7: Hydrolysis of fumonizin B, by commertiai exterasa3 and hYdrolAs3es .Enzyme Code Source, Purity Units/r UnLit3 Amay FB, Prot per rxs PH hydrolysis Esterase, EC 3. 1. 1.1 Rabbit 100 nonspecific *Esteruse, EC Porcine liver 200 ~Onspcfic Lipasc EC 3.1.1.3 Candida 35 7.7- Cbolnesterase. EC 3.1.1.8 Horse serum. 500 15 Enzyme Code Source, purity Units/rnt g Prot.

Units per i-in Amy FB, pHl hydrolysis Cholinestcrase.

acetyl Cholesterol esterast Cholesterol esterase Cholsterol esteraNC Cholesterol cstcrase.

Acmol .serase Pecuncsterase Pcctunasc Pccu nasc EC 3.1.1. Bovine_ parmall%.

03.33 0.15

EC

3.1.1.13

EC

3.1.1.13

EC

EC

31 1 13 EC 3.1,1.6

EC

S1.1 11

EC

321

EC

3.2. 1. 15 pure Bomie. partially pure Porcine, partially purr I'seudomonoas ]Iuorrscens I'seudomonas sp Orange Peel partially purc Orange Peel.

partially purc Rhizopus Cna&c Partially purc Exopiala spinifera. crude 0.5 0.15 0 15 8.0 12 1.5 200 15 o 015 4) o 3 I 4 (1 unk unk ;2 0@

S

SO@

S

*5 0

*OS

005s S S *0 0 Fumonisin esterase

S.

S *5 *0 0~ *0 0 S. S S 6S .5*.O

S

55 *Analysis by Cis TLC/fiuorescami~ne spray following overnight incubation at 37' C with I mg/nil fumnonisin.

no hydrolysis trace amount of hydrolysis 5 incomplete hydrolysis incomplete hydrolysis i complete hydrolysis The enzyme of this invention was evaluated for inducibility by growing an Exophiala culture on various carbon sources of varying degrees of structural similarity to fumomsin. The results, shown in Table 8, ilustrae that both the original form of fumonisin and its metabolite are capable of inducing enzyme production. but thaL induibiityv of the enzyme is also quite specific.

0@ 0 0 0 00 0 000 0 0* S 06 0S 66 0 0** *6S0 0* S. W6 6 0 Og 000t 0* 00 00 0*

S.

S

000000

S.

W O 96M'O 17 CT U 9S 1 0 8 Table 8: Ability of various carbon sources to support growth and/or induction of FBI hydrolytic actfh.itv Exophiala culturc actiitv Carbon source Con:entrton Grosi-tb FBI hydrolw, actiiv FB, 0.1% Alkaline hvdrol%-zed FBI (API) 0.1% Na+ TricartJllatc 01 Sphingosmnc 1 Phylosph~ngosinc 0.1% Na+ Cucrate 0.1% Sucrose 0 1% Glucose V1% The ability of the fimoniisin esterase to cleave other organc carboxyjesters was also evaluated in comparison to its ability to hydroiyse turmonisin The results. shown In I able S.

5 also illustrate that the enzyme hydrolvzed the tricarballylates of other related arrunoalcohols such as FB, and AAL toxin Table 9: II' drjl'si% Of Organic cirboindessers bi Lroph iala crude concentratedj culture filtrate Substrate Conditions Assay method Hyvdrolysis bN Lxophiala culture filtrate FpH 5.2. 3 7- C. I Cis f-lC hr fluorcscamunc FE12 pH- 5.2. 3 7-C, I Cis TLC: hr fluorescarmnc AAL-ioxan pH 5.2. 3 7- C. I Cis TLC: hr fluorescamine Enzyme activity of culture filtrate and mycelium. Exrophiala spinifera isolIate 214 1. 10 was grown on YPD agar for I week, and conidia were har~csted, suspended in sterile water, and used at 105 conidia per n-l to inoculate sterile Fries .tineral salts medium containing 1 mg/rn! purified FB 1 (Sigma Chemical After 2 weeks incubation at 28 0 C in the dark, cultures were filtered through 0.45 micron cellulose acetate filters and rinsed with Fries mineral salts. Fungal myceliurn was suspended in 15 mL of 0. 1 MC-FB I, pH 5.21- I mM EDTA 3 v.g/mL Pepstaiin A 1.5 4g/naL Leupeptin and disrupted in a Bead Beater T m using 0.5 mm beads and one minute pulses, with ice cooling. H-yphal pieces were collected by tilterintt through Spin X (0.22 pim). and both mycelial supernatant and original culture filtrates were assayed for flimonisin modification by methods outlined above.

Preparation of crude culture filtrate_ Agar cultures grown as above were used to inoculate YPD broth cultures (500 ni) in conical flasks at a final concentration of 105 cells per culture. Cultures were incubated 5 days at 28-C without agitation and mycelia harvested by filtration through 0.45 micron filters under vacuum. The filtrate was discarded and the mycelial mat was washed and resuspended in sterile carbo-a-firee, low miineral salts medium (1 glliter NHNO 4 I g/liter NaH 2 PO., 0.5 g/liter MgCI 2 0 1 g/liter NaCI. 0 13 2./iter CaCl., 0 02 it/liter FeSO, 7H 2 0, pH 4 5) containing 0.5 rn/mI alkaline hydrolyzed crude FBI After 1 days at 28'C in the dark with no agitation the cultures were filtered through low protein binding- 0 45 nicron filters to recover the culture filtrate Phen vlniet hYI sulf-onvl Iluoride PMSF) was added to a concentration of2.5 rm\1 and the culture tiltrate was concentrated 0usine an AmiconiM YNI 10 membrane in a stirred cell at room temperature, and resuspended iin to. 50 rU in sodium acetate. pH 5 2 containing 10 rmN (iaCl. The crude culture filtrate (ap pro\ 0. 0-tl Ito concentrated) was stored at 1 o obtain preparative amounts of en7zsme-hvdro1\vzd fIIO M10I 01 Y *(Sitim.3, i as dissolved in 20 mL of 50 mMN sodium acetate at p11 I I U tni and U 25 mL of 200x concentrated crude culture filtrate of 2! 4 1 10 wkas added ]Vhe solution wA.as incubated at 37'C f'or 14 hours, and then cooled to room temperature The reaction mixture was brought to approx. pH 9.5 by addition of 0.4 ml- of 4 N KOK. and the mixtiure was 'A extracted twice \kith 10 miL ethyl acetate. The combined organic layers were dried under LN., *0 and resuspended in dH- 2 0. 2.5 mniigams of organic extracted material were analyzed by Fast :Atom Bombardment (FAB) mass spectrometry, The resulting mass spectrum showed a major ion at M/2=406 mass units, indicating the major product of enz-ymtatic hydrolysis was A.

25 which has a calculated molecular weight of 406.63.

N o -IAdditional characterization of fuionisin esterases from Exophiala spinifera and Gram-negative bacterium species. Crude, concentrated culture filtrates (induced for FBI esterase activity) from E- spinifera isolate 214 1. 10 and Aznthomonas sp. 2412.1 were chromatographed on a Pharmacia® Superdex 75 size exclusion column and eluted with mM sodium phosphate, pH 6.0 containing 0.2 M NaCI. One-ni fractions were collected and assayed for FBI esterase activity by methods described above. The retention times for the ta 0 0 a.

it a a .4 a a a .t *0 a.

a a a a. a *0 a a 4 de a. a.

a.

a. a. a a 4 a.

a a a

N.

a ~a.

214.1 10 and 2412.1 FBI esterases resulted in estimated molecular weights of 44,5 and 28 7 kilodatons. respectively.

Similarly, crude concentrated culture filtrates in 1 7 %1 ammonium sulfate were injected onto a Pharmaciact Phenyl Sepharose FPLC column equilibrated wthI7Marmnu sulfate in 50 mMN sodium phosphate pH 0 0 (Bufler A) A 30 mL. linear gradient of Bufler A to distilled water was applied, followed by a wash with 0 1 Triton X- W0() in. 50 mM1 sodium phosphate, pH b 0 One-mt. fractions were collected and assayed for both FBI esterase and f -or nonspecific esterase (as measured bv napthyl acetate hvdrolvsis using the method ot'Dar, et a] 1990) -Microplaie adaptation of Gomon's assay for quantitative deten-unationto Jora8fEoomcEtmlg 3: _'187-2 192 Fizure 2a and b sho%,s the retention times for the specific (I e FBI) versus nonspecific (I c naphthvi acetate esterase) activities B~oth tunizal and oac~enal FBI esterase acti, -tN e luted at approx u -4 N1 ammonium sulfate Naphthv acetate esterase activit-v, was detected in both flnual and bactcnai culiures but this acti-ir,. did not co-elute A~ith the FBI esterase acti~ity Vhu s trie fun~zai and bacteriai [H1 1csteiiases are not the same as nonspecific esterases detectable in tme culture nmraic 01 theicc microbes Example 2 Cloning of genes coding for fumonisin esterase Microorgantisms demonstrating tumomisin- resistance can be used to create a szenornic 20 library using standard techniques, well known in the ant. Thus, restriction enzyNmes can be used to render DNA fragments which can in tum be inserted into any number of'suitable cloning vectors. Numerous cloning vectors are known to those of skill in the art, and the selection of an appropriate cloning vector is a matter of choice. The clornig vector need only be capable of transforming a host cell incapable of fumonisin degradation. Examples of recombinant DNA vectors for cloning and host cells which they can transform, shown in parentheses, include the bacteriophage lambda coil), pBR322 co/i), pACYC 177 (E.

cob), pKT23O (gram-negative bacteria), pGVI 1106 (gram-negative bacteria), pLAFRI (gramnegative bacteria), pME290 (non-E. co/i gram-negative bacteria), plJ6 I (Strc'pzornces), pUC6 (Streptomy-ces), YlpS (Sacchcirom)yces), and YCp 19 (Saccharomvces). See, generally DNA Cloning, Vols. I and 11, supra, and Maniatis et al., supra. Particularly useflil is a cloning vector able to transform K colt.

Once the cloning vector has been inserted into an appropnate hiost cell, the cells are grown on fumonisin containing media and screened for their ability to degrade fimonisin as previously described. Plasmid DNA inserts from colonies that degrade fumonisin are characterized by subcloning, transposon tagging, and DNA sequence analysis, all well within the skill in the art (see. Napoli, and Staskawicz, B (1987) J. Bact 169.572-578) Once a coding sequence is determined, rec:.-binant protein molecules able to derade fumonisin can be produced according to the present invention by constructing an expression cassette and transforming a host cell therewith to provide a cell line or culture capable of expressing the desired protein which is encoded within the expression cassette Sequences encoding the fumonisin deg.adation enzyme can be either prepared directly by synthetic methods based on the determined sequence, or by using the sequence to design S oligonucleotide probes to clone the native coding sequence using known techniques The oilgonucleotide probes can be prepared and used to screen a DNA lbrary from an orcanism able to degrade furnonisin as detemnined above The basic stratesues for prepann 1I oligonucleotide probes and DNA libranes, as well as their screening hv nacleic acid hybndization are weU known to those ofordinary skill in the ar. Se. c DNA (lonine Vol 1. supra. Nucleic Acid Hybridization, supra. Oligonucleoide Synthesis. sura.

daruaus et supra The coding sequence can be comprised entirely of the coding sequence so denved. or 20 such sequences can be fused to other sequences leader sequences) so that a tusion protein is encoded See. eg U S. Patents Nos .443 1.730. 4.425.437 and 4.338.30, the disclosures of which are hereby incorporated by reference Once an appropnate coding sequence for the fumonisin-degrading enzyme has been prepared or isolated, it can be cloned into any suitable vector or replicon, known in the art These vectors are descnbed above, with coli being the host bacterium particularly preferred To complete construction of the expression cassettes, the coding sequence is then operably linked to control sequences such as a promoter, nbosome binding site (for bactenal expression) and, optionally, an operator, so that the DNA sequence encoding the protein is transcribed into messenger RNA in the host cell transformed by the vector containing the expression construction. It is within the skill of the an to operably link the fumonisindegrading enzyme coding sequence to appropriate control sequences in order to bring about transcription and translation. In general, the coding sequence will be downstream from the promoter sequence and any expression regulatory. regions. such as znhancers or operator sequences. If the coding sequence is linked to a heterologous codingt sequence or start codon.

then it is Important to place the coding sequence in reading &ame with the latter If the intended expression host is procarvotic. then it wiall also be necessarv to include a -ibosome binding site among the upstream control sequences. Downstream operably linked control sequences will usually comprise a transcription :erimunation sequence.

The construct can then be inserted into an appropriate expression vector. A number of procarvotic and eucaryotic expression vectors are known in the art Preferred vectors are procaryotic expression vectors A particularly preferred host for such v-ectors IS L CON Fhe furnonisin-degradinig enzyme is then produced by growing the host cells transformed by tht4 expression cassette under conditions which cause the expression of the biologicdly active protein, as indicated by the host cells ability to degr~ade tilmonisin in the medium on %k hich it is, grown. as described above The protein can be isolated from the host cells and punfie"i tor furhestd If the protein is not secreted, it may be necess-ary tdirupttehs ei 11 Purify the protein from the cellular lvsate Various punitiation tectntques, such as [tPt f sie-xcuso chromatographN, ciectrophoresis, and irnmunoar-init% -t1FrniatoQ aPfl .i CC known, and the selection of the appropriate purifcation and recover\ method is wkithin 1r ,n siwl of the art Similarly-, the gene can- be inserted into the T-DNA region of a I I or Ri plasmic derived from A. lumelaciens or A. rhizogenes, respectively Thus, expression cassettes can he constructed as above, using these plasrmdis Many control sequences are knowni '.ch \Y hen coupled to a heterologous coding sequence and transformed into a host organim show. fidelitN in gene expression with respect to tissue/organ specificity of the original coding sequence a See, eg., Beniey, P. and Chua, N. H. (1989) Science 244 174-181I Particularly suitable control sequences for use I these plasmnids are promoters for constitutive leaf-specific a expression of the gene in the various target plants. Other useful control sequences include a ~j promoter and terminator from the nopahne synthase gene (NOS) The NOS promoter and temntrare present in the plasnud pARC2, available from the American TyeCutr Collection and designated ATCC 67238. If such a system is used, the %irulence (vir) g~ene from either the Ti or Ri plasmid must also be present, either along with the T-DNA port on. or viaa binaryv system where the Wr gene is present on a stparate vector. Such systems, vectors for use theetin, and methods of transformning plant cells are described in U.S. Pat No 4.(558,082. L.S application Ser. No 913.914, filed Oct 1. 1 98O. as referenced in L S Patent 5.262.30b. issued November lb. 1993 to Robeson- et al and Simpson, R B3 et al 198o) Plant Mol. Biol. 0 403-4 15 (also referenced in the '30o patent), all incorporated by reterence in their entirety Once constructed, these plasmids can be placed into q. rhizogenes or A. tumc'tucien.

and these vectors used to transform cells of plant species which are ordinarily susceptible to Susarium or Aizerniaira infection For example. non-resistant varieties of tomato (Lvopersicon esculnturn) are oten plagued w~ith such infection and new- resistant varietes could be developed to withstand A/temrna-induced disease in emerging tom-ato seed jines, In althoutzh it should be noted that fujsarium vilt in tomato is thoutzht to be aused by orY'soporum, not monicltortuni. and 1. oxvsoporum apparently does not produce Pumonisin Several other transgeic plants are also contemplated by the present in'. e-ii\ n ncludiniv but not limited to soybean. ccrn. sorhum. alfalfa. nc. clover. cabbaLc ,nn Se cortle. celery, tobacco, cowpea. cotton. melon and pepper I he selection of either 4 a. IIImCAICIL'n' or A1 rtrI:ugCItA ilfl de-pend on the plant beinc: tirstbrnice t heret-. cenci a! -i rmetacietL is the preferred orizanisrn tIr transtormation Miost Liicot'.ieuonis, ,orme izx-rno sperms, and a t6\\ monocotvledons (e S certain members of theI.:zcai are susceptible to inf'ection ruth.t zmejaciens .A rhtzom,,',1' aiso has a '.vide ntos, ranve, embracing most dicots and some g)rcsperms, which includes members of the It~'d~u'a (ompo~sia and (henopodiaceae AlUternative techniques w hich have proven to *r'e etfec itin vz.-neticalv transforrrung plants include particle bombardment and clectroporation S"'eL Rhodes, C A et a] 1988) Science 240, 204-207, Shiszekaw'a, K and Dower. W J (1988) BioTechniques 6, 742-75 1 Sanford, 1. et al 1987) Particulate Science Technology 5,527-37. and McCabe, D. E. (1988) BioTechnology 6 923-926) Once transformed, these cells can be used to regenerate trasgeni'c plants. capable of e~g degradin tiunonisin. For example, whole plants can be infiected with these vectors bv wounding the plant and then introducing the vector into the wound site Any part of the plait can be wounded, including leaves, stems and roots. Alternatively, plant tissue. in the form of' an exp nt, such as cot yledonary tissue or leaf disks, can be inoculated with these vectors arid cultured un-der conditions Ahiich promote plant regeneration Roots or shoots transformed by inoculation of plant tissue with A. rhizogenes or A. iui'eam ,coiigte reue coing for~~ th imnsndgradlation enzyme, can be used as a source of plant tise torgenerate fumonisin-resistant transgenic plants, either via somatic embryogenesis or organogenesis Examples of such methods for regenerating plant tissue are disclosed in Shahin, E. A. (1985) Theor. Appl. Genet. 69:235-240; U.S. Pat. No. 4,658,082; Simpson. R. et al (1986) Plant MoL Biol. 6: 403-415; and U.S. patent applications Ser. Nos. 913,913 and 913,914, both filed Oct. 1, 1986, as referenced in U.S. Patent 5,262,306, issued November lo, 1993 to Robeson, et al., the entire disclosures therein incorporated herein by reference Such transformed cells can also be used to regenerate transgenic plants capable of expressing, in specific tissues or constitiuatively, depending upon the type of promoter utilized, either the fumonisin degrading enzymes elaborated by Exophiala spinefera. ATCC 74269, Rhmocladiella atrovirens, ATCC 74270, or the bacterium of ATCC 55552. or the API catabolase elaborated by those strains Such transgenic plants can be harvested, and the appropriate tissues (seed, for example, if a seed specific promoter were used) can be subiected to large scale protein extraction and punrification techruques, and the frmonn c aegradation enzymes or AP, catabolases can be isolated for use in tumonsin and fumorunin hvdrT,ivsis 15 prcduct detoxitfication processes Certain ester.ses fall into a familv that is related by pnma.n scouence and .er~structure (Cygier N. Schrag JD. Sussman JL, Harel M, Silmanl. entr, MK. iocior H' i993) "Relationship between sequence conservation and 3-Dimensional structure in a large family of esterases, lipases, and related proteins Protein Sci 2: 366-382.). PCR pnmers were 20 designed based on highly conserved regions of this esterase family and using these pnmers, a 1 cDNA clone from Erophiala spinifera isolate 2141 10 was obtained that showed signiicant homology to known esterases, ard was specifically induced by fumonisin and other inducers This esterase can be expressed in E. colt and its enzyme activity can be measured by means of the TLC assay described above. If no activity is obtained in E. coll then expression can be measured in yeast or another eukaryotic system.

^Other methods can also be used to clone the gene. Purification of the protein and Ntermuinal sequencing allow design f specific DNA probes; generation of antibodies from purified protein and screening an expresrion library; using RNA enrichment methods to obtain cDNAs specific to the induced culture. Once the gene has been confirmed as corr-esponding to fumonisin esterase, the cDNA clone can easily be ligated into appropriate expression vectors for exp.ession of the enzyme in maize tissue culture cells, transgenic maize, and also in Fusarium moniliforme itself, that is useful for studying the mechanisms of pathogenests associated with the fungus and its toxin. Transformed or transient-expressing maize tissue culture cells can then be evaluated for resistance to fumonisins relative to control transformed tissue, and in fact fumonisin can be used as a selection agent to isolate transformed cells from tissue culture.

Cloning ofXanthomonas/Sphinromonas Esterase Gene: The Xanthomonas esterase gene was cloned in a lambda ZAP express expression library from Sau3A partially digested bacterial DNA (4-8 kb size selected from ATCC 55552) Pools of lambda lysates were tested for fumonisin esterase assay by TLC using pure timonisin as a substrate, and positive pools were sub-sampled to enrich for positive clones Individual Io plaques were resuspended and activity assayed m the lysate One positive clone was purified.

phagemid excised and DNA prepared for sequencing. A 4 kilobase DNA fragment containing fumonisin esterase activity was sequenced and found to contain a 1589 base pair region containing a 529 armno acid open reading frame with high homology to members ofthe senne carboxvlesterase type B superfamily The open reading framecodes for a hypotheticai protein 15 (called BESTI) with a putative signal peptide from amino acid I to 38, giving a mature protein vith a calculated molecular weight of 51,495.63 daltons and a pi of 8 ,1 I his open readinQ frame showed 52.5% similanty and 34% identity with the amino acid sequence ot a rabbit cholinesterase (P37176) Other cholineserases showed similar homology scores I he BESTI sequence was also 53.0% similar and 36.4% identical to a B,.cillus subtiis pararutrobenzyl esterase (P04058) The open reading frame also showed 54.60 similantv and '0 34.9% identity with the Exophiala spinifera fumomnsin esterase (Espl) Aside from their 0 overall similarity with other type B carboxylesterases. Esp I and BESTI share two shon amino acid domains not found in other known esterases of this type: 00 00 ti Seauence Fron To ESPI ATLM 292 295 0 BESTI ATLM 286 289 ESPI TNI 175 177 BESTI TNI 172 174 These domains may be involved in the substrate specificity of these enzymes (Cygler Schrag, Sussman, J.L. Harel, Silman Gentry, M.K. Doctor, B.P (1993) Relationship between sequence conservation and 3 -Dimcnsional structure in a large family of esterases, lipases, and related proteins. Protein Sci. 2:366-3 82).

Example 3 Preparation of API-induced and non-induced mycelium.

Exophiala spinifera Isolate 2141 !0 was grown in YPD broth for 5 days at 280C, mycelium was harvested on 05. mnicron cellulose acetate filters and transferred to fresh medium consisting of Fries mnineral salts (Gilchrist DG, Grogan RG 1976) "Production and nature of a host-specific toxin from Aliernaria alternala f.sp ly-copersic,." Phvtopathology 66: 165-17 1) amended with hydrolyzed fumonisin B I (API1) (0.5 mg/mL) or delta- It) aminobutync acid (6-ABA) (I mg/mL) as the sole carbon source Cultures were ncubated in the dark for 48 hr at 28*C and culture supernatanhs removed bv filtrat ion through 0 5 micron cellulose acetate The remaining mycelial mat was washed wsith sterile Fries mineral salts and .then frozen in liquid niitrogen for storage 5 5 Examnle RNA isolation from Exophiala spinifera I-he mycelial mats described above I gram) were ground in liquid nitro-gr, in ai St. Smorta-r and pestle following addition of 10 mnL "TRIREAGENT' (Miolecular Research (enter- Inc. Cincinnati, OH-) in the presence of 0.2 volume chloroformn The gnindate was ceritritiged and the resulting supernatant precipitated with isopropanol The resulting pellet was cxlracted too 0, 20 wkith phenol, ethanol precipitated, and stored at -80" C.

*:.The RNA in water (0.4 mL) was enriched for rpoly-A-containing mRNA using biotinoligo(dT) and a streptavidin magnetic bead system (Promega) using the manufacturer's instructions. The polyA(-s--enric.4ed RNA was stored at -800 C.

First strand cDNA synthesis from polyA(+)-enched RNA was carried out using Mz, MV reverse transcriptase (370 C, I hr). The reaction mixtue was extracted with phenol and chloroform. Aliquots were taken for polymerase chain reaction (PCR) using the degenerate primers identified in SEQUENCE I.D. NOS. I through 4.

-ESPY-OL I GGGGAArrCGARGAYTGNYTNTAYNTNAAYRT (SEQUENCE I. D. NO I 3o ESPY-0L2 GGGGAATTCNICNGTNNTNVTNTGGiATNYYGGhI

SQEC

I.D. NO. 2) ESP3Y-OL I GGGAAGCTTGGRTYNCCNCCRANBNCDART (SEQUENCL

I.D.

NO. 3) ESP3-OL2 GGGAAGCTTCNCCNGCNSWYTCNCCAJDNTN

(SEQUTENCE

1.D. NO. 4) Most bases designated were inosines.

Thermocycler reaction conditions were: I 9 4 0 C 2min 2. 9 4 C 30 sec 3. 4 5'C 2min 4. 72' C I min repeat steps 2-4 for 35 X *6 7 2' C *sThe PCR reaction products were electrophoresed on horizontal agarose v-ei5 bands that werc present only in induced lanes were excised and the DNA v'as eluted r-he recovered is DNA was digested with Hindlil and EcoRI and ligated into pBluescnpt SlK> A recombinant clone from. products amplified using ESPS'-0L2 and ESP3'-OL2 (ILSil2o-

I

,wa-s recovered and sequenced. The cloned region contains an open re-ading framne with the partial protein or amino acid sequence SFHLYDGASFAANQDVIVVTINYRTNLGFPAJIQLPITQNGLQ-. Dk Q 5 0 20 NIAA1FGGDPRJ(VT FFGESAL.. (SEQUENCE I.D. No. The above deduced amino acid sequence from DNA fragment ESP26-1I showed significant homology to a famnily of proteins that includes cholinesterases, acetylcholinesteras carboxylesterases, and certain lipases (Cygler M, Schrag

JD,

S Sussman JL, Hared M, Silman 1, Gentry Mfl, Doctor BP (1993) "Relationship between sequence conservation and 3-Dimensional structure in a large family of esterases, lipases, and 05550.related proteins. "Protein Sci 2: 366-382.) Comparison of Deduced Amino Acid Sequence to Known Sequences In comparison with a sequence pubfished in Arpagaus, Chatonnet, Masson,

P.,

3o Newton, Vaughan Bartes, Nogueira, La Du, and Lockridge, 0. J.

BioL Chem. 266, 6966-6974 (1991), 43 of the 76 amnino acids in ESP26-1 were identical to a dog pancreatic cholinesterase.

32 In another comparison 32 of 62 amino acids from ESP26-1 were identical to a fungal lipase, as published by Lotti, Grandori, Fusetti, Longhi, Brocca, Tramontano, and Alberghina, Gene 124, 45-55 (1993).

Example 7 Northern blot analysis of induced, non-induced Exophiala spinifera: Total RNA extracted from Exophiala spinifera cultures as described in the preceding examples was electrophoresed on agarose gels containing formaldeyde, blotted to nitrocellulose, and probed with random-primed 32P-labelled ESP26-1 cDNA. The probe hybridized to an RNA of approximately 2.0 kilobases in size in the induced lane, but not in the non-induced lane.

Example 8 Isolation of full length cDNA of ESP26-1 from Exophiala spinifera.

To obtain 3'-end of the cDNA coding for the putative esterase, a 3'-rapid amplification of cDNA ends protocol (3'-RACE) was employed (Frohman, M.A. Dush, M.K. and Martin, G.R. 1988 "Rapid production of full-length cDNAs from rare transcripts: Amplification using a single gene-specific oligonucleotide primer" Proc. Natl.

Acad. Sci 85 8998-9002) 5 pg of total RNA isolated from AP1 induced Exophiala spinifera mycelia was used as template for reverse transcription reaction. The reverse transcription reaction and subsequent PCR amplification was performed with a 3'-RACE 20 kit (Gibco BRL). The gene-specific primer (ESP3'-1:

GCTAGTTTCGCAGCCAATCA-

GGA) (SEQUENCE ID NO. 6) was designed based on ESP26-1 sequence.

PCR reaction conditions were: S 1. 94°C 4 min 2. 94'C 45 sec 25 3. 60 0 C 25 sec 4. 72 0 C 3 min 5. repeat steps 2-4 for 40 X 6. 72 0 C 10 min A resulting 1.5 kb DNA fragment was blotted to nitrocellulose and hybridized with cDNA ESP26-1 under highly stringent hybridization and wash conditions (last wash: 0.1 X SSC, 0.5% SDS, 65°C for 30 min). The DNA fragment was gel-isolated, ligated into a pGEM-T vector (Promega), and transformed into DH5a (Gibco BRL). The resulting plasmid [N:\libff]01028:ANB DNA (p3RC-2) was sequenced using M 13 universalpnrimer Sequence comparison of 3RC-2 and ESP26-1 indicated the ESP26-1I overlapped 100/% with the 5' end of 3RC-2 sequence.

To obtain the amino-terninal sequence, a 5'-RACE strategy was employed (Frohman, et at, supra). 5 pg of total RNA isolated from API induced Exophiala spimlfera.mycelia was reverse transcribed with SuperScript I RNase H- reverse Transcriptase (Gibco BRL) using an antt-sense pnrimer constructed against ESP26-1 sequence (ESP5'- AAAGGCTGCGATGTTCCGCTGTA) (SEQUENCE I.D. NO. The cDNA was tailed with dATP using terminal transferase (Promega) and used as a template for nested amplification using a second gene-specific anti-sense pnmer (ESP5'-2: TCGCTGTGTTATTGGCAGCTGAG. (SEQUENCE I.D. NO. C was a silent mutation of A in order to create a Pvu 11 restriction site) and an end-blocked polyT primer (BamT I 7V CGCGGATCCGTTTTTTTTTITTTTTTTTV) (SEQUENCE LD NO 9) PCR reacution conditions were 9 *40 C 4 rnin 15 2 94 0 C 45 sec *04 3 40' C 45 sec 4 6 C 25 sec 7 2 0 C 3 min 6 repeat steps 2-5 for 41 X 7. 7 2 0 C 10 min 9The PCR products were fractionted on a 1.5% agarose gel The amplied product was gel-isolated, ligated into pGEM-T (Promega), and transformed into DH5 (Gibco

BRL)

.00 9 0 The resulting 5' RACE product was sequenced and shown to overlap as expected with the 3' *RACE product and to contain an open reading frame with significant homology to members of the serine esterase/lipase superfatmily described by Cygler et al. (supra). The overlapping 0 sequences obtained by 3' RACE and 5' RACE were combined to yield a cDNA sequence corresponding to the complete open reading frame. The full length, 1937 bp cDNA clone from Exophiala spinifera 2141.10 (abbreviated ESPI) contains an open reading frame of 537 amino acids as shown below (SEQUENCE I.D. NO.

MPSRYILSWLLTCFLGAFGSRCGSSAPTVKIDAGMVVGTTTTVPGTTATVSEFLGVPF

AASPTRFAPPTRPVPWSTPLQATAYGPACPQQFNYPEELREITMAWFNTPPPSAGESED

CLNLNIYVPGTENTNKAVMVWIYGGALEYGWNSFHLYDGASFAANQDVIVVTINYRT

AThMNTEQLGLGFEYTLDNVTVVYRSETA

GDIVPVVGTVGLFLE

DTQAYLE-EAPNQPDLYQTLLGAYPIGSPGIGSPQDQJTERQPAIADP

PRTF

This open reading fr-ame (ORF) shows some homology to members of the seine esteraseilipase superfamily described by Cygler et (supra). The most extensive homology is 35.9%/ identity in 320 amino acid overlap with butyrylcholinesterase from ')rvciolagus cuniculus (rabbit).

The deduced Esp I protein contains a putative signal peptide which is probably cleaved at position 26/27 yieiding a mature protein with a calculated MW of 54953 781 and cculated p1 of 4.5 These calculated values are consistent with the estimated MR and p1 of' the fumnoni'sin esterase activity described above A comparison of the Esp I open reading frame consensus regions in the esterase supe-ta-uly (Cygler et supra) reveals numerous conserved features indicating Esp I may code for a serine esterase The Esp protein has a potential serine active site consensus at 22.1- 228, a putative aspartate active site consensus at 335-341I that is typical of cholesterol .2o esterases and DrosophzIa 6 and P proteins the majority of members of this superfarrdly, including fungal lipases and carboxylesterases have glutamnate at the active site instead of' aspartate]; and a putative bistidine active site that is different from any members of the family, a *:containing additional amino acids between the G and H. The putative Esp mature protein has a tota of 6 cysteines, for 3 possible disuffide bridges, consistent with at least a subset of the esterases in the superfniily described by Cygier et al., supra Thus the Esp ORF has most of the hallmarks of a bona fide member of the lipase/esteruse superflumily, including a putative active site triad and -ther conseived amino acids. The regions of conservation are not consistent with any one substrate subgroup (i.e.

lpscholinesterase, earboxylesterase, or cholesterol esterase), but seem to be contain some features of several of these, and Esp appears to be unique among known esterases in its putative active site His consensus sequence.

ExA-m-Re 9 Effect of FBI and API on maize coleoptiles Maize coleopties from 4 day dark-grown germinated maize seeds were excised above the growing point and placed in 96-well rnici-otiter plates in the presence of 60 microliters of i sterile distilled water containing FBI or API at approximately equimolar concentrations of 1 .15_ 05_ 015_ 005_ 0015, or .0005 millimolar, along with water controls. After 2days in the dark at 280 C the coleoptiles were placed in the light and incubated another 3 days. Injury or lack thereof was evaluated as follows: necrotic discoloration of coleoptile__ 10 -=no symptoms (same as water control) S The results (see table above) indicate there is at least a 30-fold difference in toxicitN bewn B and AP, to maize coleoptiles of this genotype This is in general agreement kt S..other studies where the toxicity of the two compounds was compared for plant tissues In 5 Lemna tissues, API was approx 40-fold less toxic (Vesonder RE Peterson RE., Labeda 1), Abbas H-K (1992) "Comparative phytotoxicity of the fumonisins, AAL-TFoxmn and v'east sphingolipids in Lemna minor L (Duckweed). "Arch Environ Contain Toxicol 23: 4t)4- 467.). Studies with both AAL toxin and FBI in tomato also indicate the hydrolyzed version of the molecule is much less toxic (Gilchrist DG, Ward B, Moussato V, Nfirocha CJ (1992) "Genetic and Physiological Response to Fumonisin and AAL-Toxin by Intact Tissue of a 20 1-igher Plant." Mycopathologia 117: 5 In a recent report Laniprecht et al. also observed an approximate I 00-fold reduction in toxicity to tomato by AP 1 versus FBI (Lamprecht S, Marasas3 W, Alberts J, Cawood M, Gelderblom W, Shephard G, Thiel P.

Calitz J (1994) Phytotoxicity of flimonisins and TA-toxin to corn and tomato.

Phytopatholog 814: 383391.) LHEmI

Q

(~425 Effect of FBI and AP, on maize tissue cultured cells (Black Mexican Sweet, BMS) FBI or AP, at various concentrations was added to suspensions of BMS cells growing in liquid culture medium in 96-well polystrne plates. After I week the cell density in wells was observed under low power mnagnification and growth of toxin-treated wells was compared to control wells that received water. Growth of BMS cells was significantly inhibited at 0.4 micromolar FBI, but no inhibition was observed until 40 micromolar AP,. This represents an approximate 100-fold difference in toxicity to maize tissue cultured cells. Similarly Van Asch et al. (Vanasch MAJ. Rijkenberg FHJ, Coutinho TA (1992) "Phytotoxicity of fumonisin bl.

moniliformin, and t-2 toxin to corn callus cultures." Phytopathology 82: 1330-1332) observed significant inhibition of maize callus grown on solid medium at 1.4 micromolar

AP,

was not tested in that study, however Example II APi Catabolase Activity A cell-free extract that contains the catabolase activity was obtained by subiecting to substrate-induced Erophiala spmtfera cells to disruption using a bead beater in sodium acetate buffer, pH 5.2, and recovering the cell-free supernatant by centrifugation and 45 micron filtration. Catabolic activity is assayed by incubating extracts with API (hydrolvzed furnonisn B, backbone) or 14C-labeliedwith the extract and evaluating by TLC on (CI silica Ihe *product API-NI has a lower Rf than AP, and is detected either by radiolabel scan or by F :So, 1 spray/charring of the TLC plate AP,-N does not react with the amine reauent, f tluorescamine. that is routinely used to detect AP, on TLC plates, suggesting that the amine group is missing or chemically modified Activity is greater at 37'"C than at room temperature but following 30 mi at 65C or 100C (no AP, catabolic activity remained) \ctivv is maximal at pH to 9 At pH 9, complete conversion to Api-N, occurred in 3 nminutes S. 20 Activity is retained by 30,000 dalton molecular weight cutoff membrane, but only partiall\ retained by 100,000 dalton molecular weight cutoff membrane Other anmne-containmn substrates were tested for modification by the crude extract tumonisin (with tncarboxyhc acids attached) is not modified by the extract, indicating that hydrolysis must occur first tor the catabolase to be actve Other long-chain bases (sphingosine, sphinganine, phytosphingosine) are apparently not modified by the crude catabolase, sugg-sting the enzyme(s) is specific for the fumonisin backbone. Preparative amounts of the product, tentatively named AP,-N I, have 0 also been purified and analyzed by C13 nmr. The results indicate that API-N, lack an armno nitrogen, and that it probably contains a keto function This may point to either an amine oxidase or an ammonia lyase enzyme. The product of either enzyme would not be expected to display any significant toxicity (although this has not been tested).

Example 12 Demonstration of Functional Esterase Activity Demonstration of esterase activity was accomplished by expressing the E. spimfera cDNA in two heterologous systems (an insect cell/baculovirus expression system, and transgenic maize obtained by microprojectile bombardment) and subsequently detecting fumonisn esterase activity in transformed tissue. Forty-four maize calli (Hi type 11) were bombarded with the esterase gene ifsed to the ubiquitin promoter plus a selectable marker (PAT) plasmid Using thin layer chromatographic analysis, 38 lines were positive for fumonisin hydrolysis based upon the presence of a free tricarboxv!ic acid spot in the TLC plates Four negative control calli had no such spot Similar results were obtained with insect cells infected with a baculovirus expression vector contaning the esterase clone Esterase activitv was also detected in leaf tissue of regenerated ro maize plants len S leaf punches (4mm dia) were taken from the oth leaf of regenerated plants ot rouehlv the same Sage (7 leaf stage) representing four classes of transforrants selectable marker (Bar oni\ ioN 1 esterase medium esterase and high esterase expressors (based on callus datal Four plants, representing different events in each class, were sampled Ihe punches were suspended in 200 microliters of 50 mM sodium acetate buffer, pH 5 2. contann leupeptin and pepstatin to inhibit proteinases. and homogenized by rapid agitation with a steel bead. twice for 30 sec The homogenates were spun at 4000 rpm, 10 mi at 4C and 20 microliters supernatant recovered Samples were assayed for protein concentration (Bradftrd 00: assay) and adjusted to the same protein concentration and then arsayed tor tumonisin esterase activity using 14C-FBI as a substrate. After 30 min. or 4 hrs reactions were spotted on ILC( plates (C18) and developed with MeOH.4%KCl Plates were scanned with a radiometric scanner (AMBIS) and scored for tumonisi esterase activity on a product), (up to 50%/0 conversion to product) and (between 90-100% conversion to product) doo WO 96106175 PCr/US95/1028 4 Results were as follows:

SAMPLE

Al BI Control C1

DI

El Control Fl

GI

HI

A-)

B2 Control C-1 Control D2 E2 F2 G2 H2 CALLUS

SCORE

MIN)

30 MIN. LEAF

SCORE

4 HR. LEAF

SCORE

9 9.

9

S

*9 9

S

9.

S.

*9* S S 9. 9

S

OS

9 9 *9 *91* 9.

S. 9 *9 S 9 *9

S

959*9 06 a In summa-y, 8 or 12 callus expressors were positive tor leaf expression All nevatlve controls plu,. four callus expressors were negativc Of the four '--"callus expressors. onlk one (132) had the same high level (30 min. assay), but all were positive Example 13 Detoxification of Harvested Grain The present invention also relates to a method of detoxifying a fiumonisin or a structurally related rnycotoxin with an enzyme having the structure of the fumonisin degradative enzymes or the API catabolase elaborated by Erophiala spinifera, ATCC 74269, Rhinocladiella atravirens, ATCC 742 10, or the bacterium of ATCC 55552 during the processing of grain for animal or human food consumption. Since the atmospheric amrnoniation of corn has proven to be an ineffective method of detoxification (see B Fitch Haumann, "Eradicating Mycotoxin in Food and Feeds," INFORM 6:248-257 (1995)), such a methodology is particulairly critical where trnsgenic detoxification is not applicable In this embodiment, the fumonisin degradative enzyme and/or the AP, catabolase elaborated by Erpohitiala spin/era. ATCC 74269,RJhmoc~xladiella atral'irents ATCC 742 10. or the bacterium of ATCC 55552. are presented to grain during the processing procedure, at the appropriate stages of the procedure and in amounts effective for detoxification Of fumonisins and structurally related mycotoxins. Detoxification by this method can occur not on. durin_ the grain processing, but also any time prior to feeding of the grain to an animal or incorporation of the grain into a human food product.

The enzymes can be introduced during processing in appropriate manners, for example as a wash or spray. or in dried or lyophilized form or powered form, depending upon the nature of the miflling process and/or the stage of processing at which the enm-Nmatic treatment is earried out See generally, Hoseney. R C Principles of Cereal Science and 'echnolog3.

SAmerican Assn of Cereal Chemiusts. Inc.. 1990 (especially Chapters and Jones, J M S.Food Sal Eagan Press, St Paul, M.N. 1992 (especialk- Chapters 7 and and Jelen. P., Introduction to Food-!roc swg Restan Pubi. Co., Reston-. VA.. l98 Processed gzrain to he used for animna] feed can be treated %%ith an effiecti-ve amount of the eruNymes in the form ot an inoculant or probiotic additiv-e, for example, or in any, form recoszntzed bNw those skille, n. the art for use in amumaJ feed The en,-yrnes of the present invention are expected to be particularly useful in detoxification during processing and'or in animal feed prio- to its use, since the enzvmes display relatively broad ranges of p1-I acti%,ity The esterase from Lrophlha 20 spin/era. ATCC 74269, showed a range of activity from about pH 3 to about p 1 o, and the :esterase from the bacterium of ATCC 5555' showed a range ot activiINy rrn about pihobto about pH 9 ExAmple 14 5 Genetic Engineering of Rtzminall Microorganisms 4125 Ruinal mcroorgantismrs o.an be genet cally engineered to contain and express either the fumonisin degrading enzymes or the AP, catabolase elaborated by Lrophilia spin fera.

~4 ATCC 74269, Rh nodladella aravirens, ATCC 74270, or the bacterium of ATCC 55552, or a %w4 combination of the enzymes. The genetic engineering of microorganisms is now an art recognized techniique, and ruminal microorganisms so engineered can be added to feed in any art recognized manner, for example as a probiotic or inoculant. In addition, a.icroorganisms capable of functioning as bioreactors can be engineered so as to be capable of mass producing either the fumnonisin degrading enzymes or the AP, catabolase elaborated by Leophilha w4**

S

444440 4 4. 4 4 4 *4 *5 4. 4 4. 4 4 44 44 4 .4 U 4~ 4 4 0O 4. 0 4 4 44 *4* 54 44 44 44 44 )JiVso WflU~3eq ~qi JO OL~t'L ~lIajiwiw V/pfl,7l2pOUlljy ~69~tL JJJY nJa/ifzd~ 4 41 SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: Duvick, Jon Rood, Tracy A.

(ii) TITLE OF INVENTION: FUMONISIN DETOXIFICATION COMPOSITIONS AND METHODS (iii) NUMBER OF SEQUENCES: (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Pioneer Hi-Bred International STREET: 700 Capital Square, 400 Locust Street CITY: Des Moines STATE: IA COUNTRY: USA ZIP: 50309 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: MS-DOS SOFTWARE: Microsoft Windows 3.1 Notepad 20 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE:

CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION: 25 NAME: Roth, Michael J.

REGISTRATION NUMBER: 29,342 REFERENCE/DOCKET NUMBER: 0272 US (ix) TELECOMMUNICATION INFORMATION: A) TELEPHONE: 515-248-4895 TELEFAX: 515-248-4934 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 32 bases TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: probe (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: GGGGAATTCG ARGAYTGNYT NTAYNTNAAY RT 32 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 37 bases TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: probe (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: GGGGAATTCM CNGTNNTNVT NTGGATNYAY GGNGGNG 37 20 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 35 bases TYPE: nucleotide STRANDEDNESS: single 25 TOPOLOGY: linear (ii) MOLECULE TYPE: probe (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: GGGAAGCTTG GRTYNCCNCC RAANKBNGCD ATRTT INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 36 bases a.

20 a 9* a a a 2 TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: probe (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: GGGAAGCTTC NCCNGCNSWY TCNCCRAANA DNGTNA INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 76 amino acid TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (partial) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID Ser Phe His Leu Tyr Asp Gly Ala Ser Phe Ala 5 10 Val Ile Val Val Thr Ile Asn Tyr Arg Thr Asn 25 Pro Ala Ala Pro Gin Leu Pro Ile Thr Gln Arg 35 40 Leu Asp Gin Arg Phe Ala Leu Asp Trp Val Gln 55 Ala Phe Gly Gly Asp Pro Arg Lys Val Thr Phe 70 Ala Asn Gin Asp Ile Leu Gly Phe Asn Leu Gly Phe Arg Asn Ile Ala Phe Gly Glu Ser Ala (2) INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 23 bases TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: probe (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE:

NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: GCTAGTTTCG CAGCCAATCA GGA 23 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 23 bases TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: probe (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: AAAGGCTGCG ATGTTCCGCT GTA 23 a INFORMATION FOR SEQ ID NO:8: .20 SEQUENCE CHARACTERISTICS: S(A) LENGTH: 23 bases TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear 25 (ii) MOLECULE TYPE: probe (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: TCGCTGTGTT ATTGGCAGCT GAG 23 INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 28 bases TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: probe (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: CGCGGATCCG TTTTTTTTTT TTTTTTTV INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 527 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID a a a a.

a a a...r a a a a a a a.

a Met Pro Ser Arg Tyr Ile Leu Ser Trp Leu 5 10 Gly Ile Ala Phe Gly Ser Arg Cys Gly Ser 20 25 Lys Ile Asp Ala Gly Met Val Val Gly Thr 35 40 Gly Thr Thr Ala Thr Val Ser Glu Phe Leu 25 50 55 Ala Ser Pro Thr Arg Phe Ala Pro Pro Thr 70 Ser Thr Pro Leu Gln Ala Thr Ala Tyr Gly 85 Gin Phe Asn Tyr Pro Glu Glu Leu Arg Glu 100 Phe Asn Thr Pro Pro Pro Ser Ala Gly Glu 110 115 Leu Thr Cys Phe Ser Ala Pro Thr Thr Thr Thr Val Gly Val Pro Phe Arg Pro Val Pro Pro Ala Cys Pro Ile Thr Met Ala Ser Glu Asp Cys Leu Val Pro Ala Trp Gln Trp 105 Leu 120 1 Asn Leu Asn Ile Val Met Val Trp Ser Phe His Leu Val Ile Val Val Pro Ala Ala Pro Leu Asp Gin Arg Ala Phe Gly Gly Ala Gly Gly Arg Asn Pro Pro Phe Tyr Asn Phe Pro Val Gin Ala Leu Met Arg Arg Val Tyr Val 125 Ile Tyr 140 Tyr Asp 155 Thr Ile 170 Gin Leu 185 Phe Ala 200 Asp Pro 215 Ser Val 230 Arg Ala 245 Lys Gly 260 Asn Cys 275 Asp Leu 290 Phe Glu 305 Thr Ala 320 Gly Thr 335 Asp Thr 350 46 Pro Gly Thr Glu Asn Thr Asn Lys 130 Gly Gly Ala Leu Glu Tyr Gly Trp 145 Gly Ala Ser Phe Ala Ala Asn Gin 160 Asn Tyr Arg Thr Asn Ile Leu Gly 175 Pro Ile Thr Gin Arg Asn Leu Gly 190 Leu Asp Trp Val Gin Arg Asn Ile 205 Arg Lys Val Thr Ile Phe Gly Gin 220 Asp Val Leu Leu Thr Ser Met Pro 235 Ala Ile Met Glu Ser Gly Val Ala 250 Asp Leu Ser Glu Pro Trp Asn Thr 265 Thr Thr Ser Ile Asp Ile Leu Ser 280 Ala Thr Leu Met Asn Thr Ile Glu 295 Tyr Thr Leu Asp Asn Val Thr Val 310 Arg Thr Thr Gly Asp Ile Ala Arg 325 Val Ala Asn Asp Gly Leu Leu Phe 340 Gin Ala Tyr Leu Glu Glu Ala Ile 355 Ala 135 Asn 150 Asp 165 Phe 180 Phe 195 Ala 210 Ser 225 His 240 Asn 255 Thr 270 Cys 285 Gin 300 Val 315 Val 330 Val 345 Pro 360 9 9 *9 99

C

9 9.

*9C Leu Gly Leu Gly Tyr Arg Ser Glu Pro Val Leu Val Leu Gly Glu Asn If 4-7 Thr Asn Gin Pro Asp Leu Tyr Gin Leu Leu Gly Ala Tyr Pro Ile Gly Ser Pro Giu Thr Giu Asp Ser Arg Ala Thr Phe His Ser Ser Ser Ala Thr Ala Trp Ala Lys Gin Val Ile Gin Val 365 Gly Ile 380 Val Arg 395 Asn Arg 410 Giu Asn 425 Glu Val 440 Ala Leu 455 Ala Phe 470 Pro Asn 485 Asp Val 500 Giy Ser Pro Phe Gin Cys Giy Ile Pro Leu Giu Leu Gly Met Val Glu Ala Gin Ala Lys Asn Val Ala Ala Ser Pro Ala 370 375 Gin Asp Gin Ile Ala-Ala Ile 385 390 Pro Ser Ala Ile Val Ala Gin 400 405 Ser Trp Arg Tyr Tyr Tyr Asn 415 420 Phe Pro Gly Ser Giu Val Tyr 430 435 Phe Gly Thr Tyr Pro Val Ala 445 450 Thr Ser Lys Tyr Met Gin Gly 460 465 Pro Met Asn Gly Pro Gly Trp 475 480 Leu Gly Ser Pro Gly Lys Ala 490 495 Thr Ile Asp Gin Arg Cys Ala 505 510 4*

S

S S 20 Leu Tyr Thr His Tyr 515 Tyr Thr Giu Leu Gly Thr Ile Ala Pro 520 Arg 525 Thr Phe

S

SS

SS

5555 *555

Claims (33)

1. A method of detoxifying a fumonisin, or a structurally related mycotoxin, the method comprising contacting the fumonisin with an esterase enzyme as produced by Exophiala spinifera, ATCC 74269, Rhinocladiella atrovirens, ATCC 74270, or the bacterium of ATCC 55552.

2. The method of claim 1 wherein the fumonisin or mycotoxin is present in plant tissue. 10 0 00 0 0 15 0 00 0 0* 0 0 0

3. consisting a) b) c) d)

4.

6. consisting An isolated polynucleotide comprising a member selected from the group of: a polynucleotide encoding a polypeptide comprising amino acids 1 to 525 of SEQ ID NO: a polynucleotide which is complementary to the polynucleotide of and a polynucleotide comprising at least 130 bases of the polynucleotide of or a polynucleotide comprising bases 1 to 26 of or The polynucleotide of claim 3 wherein the polynucleotide is DNA. The polynucleotide of claim 3 wherein the polynucleotide is RNA. An isolated polynucleotide comprising a member selected from the group of: a) a polynucleotide encoding a polypeptide comprising amino acids 1 to 525 of Figure 2; b) a polynucleotide which is complementary to the polynucleotide of and c) a polynucleotide comprising at least 15 bases of the polynucleotide of or SN 0000. S ci1 d) a polynucleotide comprising bases 1 to 37 of or S 7. The polynucleotide of claim 6 wherein the polynucleotide is DNA.

8. The polynucleotide of claim 6 wherein the polynucleotide is RNA.

9. The polynucleotide of claim 7 comprising nucleotide 1 to 1800 set forth in Figure 1. 30 10. The polynucleotide of claim 7 which encodes a polypeptide comprising amino acid 1 to 525 of Figure 2.

11. A vector for delivery of a nucleic acid sequence to a host cell, the vector comprising the nucleic acid sequence of claim 3 or claim 6.

12. A host cell containing the vector of claim 11.

13. A host cell including in its genome the nucleic acid sequence of claim 3 or claim 6.

14. consisting The host cell of claim 13, wherein the cell is a plant cell. The host cell of claim 14, wherein the plant is selected from the group of maize, sorghum, wheat, tomato, and rice. [N:\LIBFF]0303:MCC 49

16. A transformed plant cell comprising the coding sequence of Figure 1 or variations thereof permitted by the degeneracy of the genetic code.

17. A plant having stably incorporated within its genome a nucleic acid sequence comprising the coding sequence of Figure 1 or variation thereof permitted by the degeneracy of the genetic code.

18. A polypeptide having fumonisin esterase activity comprising the two amino acid domains ATLM and TNI.

19. A polynucleotide sequence encoding an enzyme having fumonisin esterase activity comprising the two amino acid domains ATLM and TNI.

20. A transformed plant cell comprising the polynucleotide sequence of claim 3 or claim 6.

21. A plant having stably incorporated within its genome a polynucleotide sequence comprising the nucleotide sequence of claim 3 or claim 6.

22. A secretory signal sequence comprising a polynucleotide from bases 94 to 204 15 of Figure 1.

23. A secretory signal sequence comprising a polypeptide from amino acid 1 to 37 of Figure 1 or from amino acid 1 to 26 of SEQ ID NO:

24. Fungal isolates and mutants thereof, selected from the group consisting of r* C 06 C. a a. C a *0 a cc .C Exophiala spinifera, ATCC 74269 and Rhinocladiella atrovirens, ATCC 74270. 20 25. A bacterial isolate and mutants thereof of isolate 2412.1, ATCC 55552.

26. The method of claim 1 wherein the detoxification reaction occurs during storage of the harvested grain.

27. The method of claim 1 wherein the detoxification reaction occurs during processing of the harvested grain. 25 28. The method of claim 1 wherein the detoxification reaction occurs in processed grain which is to be used as animal feed.

29. A method of detoxifying a fumonisin, a structurally related mycotoxin, a fumonisin hydrolysis product, or a hydrolysis product of a structurally related mycotoxin present in harvested grain, the method comprising reacting the hydrolysis product with an API catabolase produced by Exophiala spinifera, ATCC 74269, Rhinocladiella atrovirens, ATCC 74270, or the bacterium of ATCC 55552. The method of claim 29 wherein the detoxification reaction occurs during storage of the harvested grain.

31. The method of claim 29 wherein the detoxification reaction occurs during processing of the harvested grain.

32. The method of claim 29 wherein the detoxification reaction occurs in processed grain which is to be used as animal feed.

33. A method for detoxifying, in harvested grain, a fumonisin or a structurally related mycotoxin and for detoxifying a fumonisin hydrolysis product or a hydrolysis product of a structurally related mycotoxin, the method comprising reacting the fumonisin [N:\LIBFF10303:MCC with an enzyme having the structure of the fumonisin esterase enzyme produced by Exophiala spinifera, ATCC 74269, Rhinocladiella atrovirens, ATCC 74270, or the bacterium of ATCC 55552, and reacting the hydrolysis product with an AP 1 catabolase produced by Exophiala spinifera, ATCC 74269, Rhinocladiella atrovirens, ATCC 74270, or the bacterium of ATCC 55552.

34. The method of claim 33 wherein the detoxification reaction occurs during storage of the harvested grain. The method of claim 33 wherein the detoxification reaction occurs during processing of the harvested grain.

36. The method of claim 33 wherein the detoxification reaction occurs in processed grain which is to be used as animal feed.

37. A transgenic plant capable of expressing the fumonisin esterase enzyme produced by Exophiala spinifera, ATCC 74269, Rhinocladiella atrovirens, ATCC 74270, S" or the bacterium of ATCC 55552. 15 38. A method of producing the fumonisin esterase enzyme produced by Exophiala spinifera, ATCC 74269, Rhinocladiella atrovirens, ATCC 74270, or the bacterium of ATCC 55552, the method comprising producing a transgenic plant which expresses one or more of said fumonisin esterase enzymes and isolating and purifying the enzymes from the plant tissues expressing the enzymes. a ao 20 39. A genetically engineered ruminal microorganism comprising an expression *e ia vector capable of expressing proteins in microorganisms, said vector comprising a nucleotide sequence encoding the fumonisin esterase enzyme produced by Exophiala spinifera, ATCC 74269, Rhinocladiella atrovirens, ATCC 74270, or the bacterium of ATCC 55552.

40. A probiotic composition comprising the genetically engineered microorganism of claim 39.

41. A feed inoculant composition comprising the genetically engineered microorganism of claim 39.

42. A genetically engineered microorganism comprising an expression vector capable of expressing proteins in microorganisms, said vector comprising a nucleotide sequence encoding the fumonisin esterase enzyme produced by Exophiala spinifera, ATCC 74269, Rhinocladiella atrovirens, ATCC 74270, or the bacterium of ATCC

55552. 43. A probiotic composition comprising the genetically engineered microorganism of claim 42. [N:\LIBFF]O303:MCC 51 44. A feed inoculant composition comprising the genetically engineered microorganism of claim 42. A method of detoxifying a fumonisin, or a structurally related mycotoxin, substantially as hereinbefore described with reference to any one of the Examples. 46. A genetically engineered microorganism comprising an expression vector capable of expressing proteins in microorganisms, substantially as hereinbefore described with reference to any one of the Examples. 47. A method of identifying transformed plant cells using a Fusarium species or the toxin produced by Fusarium as a phytotoxic marker, comprising the steps of: 1i a) culturing cells or tissues from a selected target plant in a culture medium; b) introducing into cells of the culture at least one copy of an expression cassette comprising a coding region that codes for a fumonisin degradative enzyme produced by Exophiala spinifera, ATCC 74269, Rhinocladiella atrovirens, ATCC 74270, or the bacterium of ATCC 55522, operatively linked to an upstream transcription initiation is sequence and a downstream polyadenylation sequence causing expression of the enzyme in the cells; c) introducing Fusarium or a fumonisin into the culture medium; and d) identifying transformed cells as the surviving cells in the culture. 48. A method of reducing pathogenicity of a fungus which produces a fumonisin or 20 structurally related mycotoxin, the method comprising expressing, in a host plant susceptible to infestation by the fungus, a fumonisin degradative enzyme produced by Exophiala spinifera, ATCC 74269, Rhinocladiella atrovirens, ATCC 74270, or the bacterium of ATCC 55522. 49. The method of claim 48 wherein the fumonisin degradative enzyme comprises 25 the sequence of SEQ ID NO: 10 or Figure 2. 50. A method of degrading a fumonisin, or a structurally related mycotoxin, the Imethod comprising contacting the fumonisin with a fumonisin degradative enzyme produced by Exophiala spinifera, ATCC 74269, Rhinocladiella alrovirens, ATCC 74270, or the bacterium of ATCC 55522, wherein the degradation reaction occurs in processed grain 30 which is to be used as a human food product. o tR:\LIBFF]0303a.doc:gcc 52 51. A method of degrading a fumonisin, or a structurally related mycotoxin, .S Ubstanti ally as hereinbefore described with reference to any one of the examples. Dated 5 January, 2000 Pioneer Hi-Bred International, Inc. Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON [R\LI BF 1-03 03 a.doc:gcc