WO1997041237A1

WO1997041237A1 - Fungus resistant transgenic plants

Info

Publication number: WO1997041237A1
Application number: PCT/AU1997/000253
Authority: WO
Inventors: Kemal Kazan; Kenneth Clifford Goulter; John Michael Manners
Original assignee: COMMONWEALTH SCIENTIFIC AND INDUSTRIAL RESEARCH ORGANISATION (as a participant in the COOPERATIVE RESEARCH CENTRE FOR TROPICAL PLANT PATHOLOGY); THE STATE OF QUEENSLAND THROUGH ITS DEPARTMENT OF PRIMARY INDUSTRIES (as a participant in the COOPERATIVE RESEARCH CENTRE FOR TROPICAL PLANT PATHOLOGY); THE UNIVERSITY OF QUEENSLAND (as a participant in the COOPERATIVE RESEARCH CENTRE FOR TROPICAL PLANT PATHOLOGY); BUREAU OF SUGAR EXPERIMENT STATIONS (as a participant in the COOPERATIVE RESEARCH CENTRE FOR TROPICAL PLANT PATHOLOGY); QUEENSLAND UNIVERSITY OF TECHNOLOGY (as a participant in the COOPERATIVE RESEARCH CENTRE FOR TROPICAL PLANT PATHOLOGY); Grains Research & Development Corporation
Priority date: 1996-04-29
Filing date: 1997-04-29
Publication date: 1997-11-06
Also published as: CA2253245A1; AUPN953296A0

Abstract

A method of conferring fungal resistance on plants is described. In the method, a DNA construct which encodes a Stylosanthes humilis peroxidase isogene or the like is introduced into cells of the plant. The construct is stably incorporated into the plant genome.

Description

FUNGUS RESISTANT TRANSGENIC PLANTS TECHNICAL FIELD

This invention relates to transgenic plants with enhanced resistance to fungal pathogens According to the invention, a transgenic plant is prepared by the transfer of a DNA sequence into the plant, which DNA sequence encodes a particular peroxidase enzyme More specifically, the invention lelates to the use of a Stylosanthes humilis peroxidase isogene in tiansgenic plants foi the puipose of enhanced disease protection

BACKGROUND ART Diseases of ciop plants have a considerable impact on the agπcultuial industries causing millions of tons of crop losses every year Consequently, breeding resistant plant vaiieties using genes tiom compatible species has been the major objective of many plant bleeding piogiams With the advent ot lecombinant DNA techniques it has become possible to transfei genes between incompatible plant species to improve chaiacteπstics of a desned plant One appioach to pi otcuing plants against miciobes is to engineei the over-expiession of plant genes that play a l ole in plant defence

Plants can resist attack by a pathogen via a of complex network of defence mechanisms (Dixon and Hainson, Adv Genet 28 165-234 [1990]) Plants defence systems may include foimation of physical barriers (cutin, lignin, callose), the expression of low moleculai weight antibiotic compounds (phytoalexins) and anti-fungal proteins Ectopic ovei -expression ot anti-fungai pioteins such as chitinases and 3-1 ,3-glucanases and other plant proteins such as ribosome inactivating pioteins have shown to mediate increased protection against phytopathogens (Bioglie al Science 254 1 194 1 197 [1991 ], Jach et al , Plant J 8 97-109 [1995], Liu et al

1 3 686-691 [1994], Logeman et al Bio /Technology 10 305-308 [1992]) One class ot defence-i elated enzymes frequently hypothesised to have a role in defence are the peioxidases but to oui knowledge the genes encoding these enzymes have not been successfully used in transgenic plants to engineei disease resistance

Peioxidases (E C 1 1 1 1 7, donor hydrogen-peroxidase oxidoreductase) have been implicated in a numbei of physiological functions that may be important in plant-pathogen interactions 1 hese include ligmfication (Waltei , M H in "Genes Involved m Plant Defense" T Bollei and T Mtins, eds Spnngei-Verlag, Wien, New York pp 327-352 [1992]), cioss-lmking ot cell wall components (Bradley et al Cell 70 21-30 [1992]), wound healing (Sheif e/ al Plant Physiol 101 201 -208 [1993]) and auxm oxidation (Grambow and Langenbeck-Schwich, Planta 157 131 -137 [ 1983 ]) Some isoforms of peioxidases are also shown to be inducible by pathogens (Svalhein and Robci tson, Physiol Plant Physiol 78 261 -267 [1990], Kerby and Sommerville, Plant Physiol 100 397-402 [1992]) and bv wounding (Lagπmim and Rothstein, Plant Physiol 84 438-442 [1987]) In addition theie is substantial coπelative evidence suggesting that peroxidase has a lole in disease lesistance Association of some peioxidase isoforms with systemic acquired resistance and hypei sensitive responses have been demonstrated (Ye et al Physiol Mol Plant Pathol 36 523-531 [1 90] , li wing and Kuc, Physiol Mol Plant Pathol 37 355-366 [1990]) A high level of constitutive peioxidase expiession (as well as othei defence-related enzymes) in a hybrid between hicotiana glutmosu \ N debneyi was also found to be associated with resistance to a number of tobacco pathogens including Phytophthora parasitica var mcotiana (Goy et al Physiol Mol Plant Pathol 41 11 -21 [1992]) High levels of peroxidase activity has been used as a maiker for resistance to downy mildew in mus melon (Reuveni et al Phvtopthol 4 82 749-753 [1992]) Peroxidase enzymes can geneiate toxic ladicals which aie inhibitory to the growth of fungal pathogens in viti o (Peng and Kuc, Phytopathol 82 696-699 [1992]) In animal systems, peroxidases have also been implicated m defense against miciobial and protozoan pathogens (Smith et al Science 268 284-286 [ 1995] and Odell and Segal, Biochim Biophys Acta 971 266-274 [1988])

Seveial investigatois have cloned and studied the legulation and function of paiticulai peioxidase isogenes fiom vaπous species (Lagπmini et al , Proc Natl Acad Sci USA 84 7542 7546 [ 1983], Buffard et al Proc Natl Acad Sci USA 87 8874-8878 [1990], Robeits and

Kolattukudy, Mol Gen Genet 217 223-232 [1989]) Expression of particulai peroxidase isogenes during the infection piocess has also been demonstrated For instance, in tomato al least thi et diffeient peioxidase genes are induced by infection (Mohan and Kolattukudy, Plant Physiol 92 276 280 [1990], Sheif ef al [Supra], Vera et al Mol Plant Mici obe Interact 6 790-794 [ 1993]) Ei ysiph gi amims f sp hordei infection in barley differentially induces two distinct peioxidase isogenes (I hordal-Chi istensen et al , Physiol Mol Plant Pathol 40 395-409 [1992])

Pi evious woik at the Cooperative Research Centre foi 1 lopical Plant Pathology has show n that infection ot the tropical foiage legume Stylosanth s humilis by Colletoti ichum ioeospoi ionics also induces peioxidase activity Several peroxidase cDNA clones were isolated from S humilis and a peroxidase isogene corresponding to Sphxό was found to be strongly induced by the pathogen 4 houis after inoculation (Harrison et al Mol Plant Mic Intel 8 398-406 [ 1995]) This time point precedes the primary penetration event demonstrating that early lecognition and signalling pi cess are involved in peioxidase gene expression during fungal infection

It is evident fiom these studies that only some of the isoforms of peioxidases may be involved in plant pathogen interaction Constitutive expression of such peroxidase isogenes in tiansgenic plants may lead to a disease resistant phenotype

Seveial investigatois have constitutively expressed peioxidase isogenes in tiansgenic plants (Sheif and Kolattukudy, Plant J 3 829-833 [1993], Lagπmini et al J Amei Soc oi t Sci 117 1012-1016 [1992], Lagπmini, Plant Physiol 96 577-583 [1991]) Ilowevei , theie has been no lepoit legardmg disease resistant phenotypes of such plants expressing high levels of peioxidases

Austiaha Patent Application No AU-B-52183/90 discloses a cucumbei basic peioxidase cDNA clone and chimaeπc genes constructed using this clone for possible expression in transgenic plants for enhanced disease resistant phenotype. However, the peroxidase gene descnbed in this document does not have any close overall homology to the Shpx6 peroxidase gene Additionally, inoculation data is not given in AU-B-52183/90 so there is no evidence of the successful application of cucumbci basic peroxidase in genetically engineering disease resistance in transgenic plants

SUMMARY OF THE INVENTION One of the obiects of the present invention is to provide a method of genetically engineeπng plants so as to provide plants having an enhanced disease resistance phenotype with respect to wild type plants Anothei object of the present invention is to provide transgenic plants capable of constitutive expression of a peioxidase activity thereby providing an enhanced disease lesistance phenotype with lespect to the wild type plants

Accoiding to a first embodiment of the invention, there is provided a method of engineei mg a plant to fungal lesistance, the method comprising intioducing into cells ot the plant a DNA construct comprising

(a) a piomotei constitutively operative in the plant cell, and

(b) a DNA sequence encoding a peioxidase isozyme operatively linked to said piomoter, whei ein said DNA sequence is selected fiom

(l) Shpx6 herein defined, (n) a sequence which hybridises to Shpx6 undei stringent conditions and which encodes a protein having peroxidase activity, (m) a fragment of a DNA sequence accoiding to (i) oi (n), which fi agment encodes a piotein having essentially the same activity as the peioxidase isozyme encoded by Shpx6 According to a second embodiment of the invention, theie is provided a plant cell haibo ing a

DNA construct compπsing

(a) a promoter constitutively operative in the plant cell, and

(b) a DNA sequence encoding a peroxidase isozyme operatively linked to said pi omotei , wherein said DNA sequence is selected from (I) Shpxό herein defined,

(n) a sequence which hybndises to Shpx6 under stringent conditions and which encodes a protein having peroxidase activity, (in) a fragment of a DNA sequence according to (l) oi (n), which fiagment encodes a protein having essentially the same activity as the peioxidase isozyme encoded by Shpxό

Accoiding to a thud embodiment of the invention, theie is provided a plant compnsing cells according to the second embodiment.

According to a fourth embodiment of the invention there is provided reproductive matenal, vegetative material or other regenerable tissue of the plant according to the third embodiment

Other aspects of the invention will become apparent from a reading of the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic representation of the relevant portion of the binaiy vectoi (pGA643) containing a promoter-Sphx6-terminator construct.

Figuie 2 shows the level of total leaf peroxidase activity in transgenic 'I , and 1 , tobacco families and in an untransformed control family

Figure 3 shows the level of total leaf peroxidase activity in transgenic T, and T-, canola families and in an untransformed control family.

Figuie 4 shows inoculation data of transgenic and control tobacco families with Phvihopthora parasitica cv nicotiana Figuie 5 shows inoculation data of transgenic and control canola families ( 1 , , I , and 'I ,) with

Lepto p/uieria maculans

Figure 6 shows glasshouse inoculations of adult plants of transgenic (T,) and contiol canola families with Leptosphaeria maculans.

Figure 7 shows inoculation data of transgenic (T, and T₂) and control canola families with Sclerotinia sclerotonum

BEST MODE AND OTHER MODES OF PERFORMING THE INVENTION The following abbreviations are used hereafter PCR polymerase chain reaction

BAP 6-benzylamιnopuπne MS Murashige-Skoog medium (Murashige and Skoog, Physiologea Plantarum

15:473-497 [1962], the entire contents of which are mcorpoiated heiein by cross-reference). NΛA napthalene acetic acid

^"I lie present invention describes a process for the production of transgenic plants which have enhanced disease resistance. In this piocess, a chimaeπc gene is constructed and tiansf erred to plants using any of the well established methods of plant transformation which include Agrohac ternini mediated tiansformation (Horsch et al , Science 227: 1229-1231 [1 85]), electioporation into protoplasts (Fromm et al , Nature 319:791-793 [1986]) and biolistic bombaidment with DNΛ coated tungsten oi gold particles (Klein et al , Proc. Natl Acad Sci. USA 85:8502-8505 [ 1988]) ^'I ransgenic plant cells including the DNA construct of the invention can be propagated using conditions appropriate to the particular plant. Similarly, whole plants, oi piopagatmg matenal of the plant, can be prepared from the initial transgenic cells using known methods and conditions.

Chimaeric genes according to the invention have as a basis the peroxidase isogene which can be isolated from the tropical forage legume Stylosanthes humilis. This isogene has been designated Sphxό and is described in Harrison et al. , Mol. Plant-Microbe Interact. 8:398-406 (1995), the entire contents of which is incorporated herein by cross-reference.

The chimaeric gene constructs of the invention comprise:

1 ) a DNA sequence encoding the Shpxό peroxidase (Genbank Accession # L361 10;

Harrison et al., supra; SEQ ID NO: l herein) or a sequence encoding a peroxidase having essentially the same characteristics as the Shpxό peroxidase; 2) a suitable promoter with or without other regulatory elements for constitutive or inducible expression in plants of the peroxidase encoded by (1 ); and optionally, 3) a suitable sequence for termination of transcription in plants.

As indicated above and in the description of the first and second embodiments, chimaeπc genes accoiding to the invention comprise not only the Shpxό peroxidase but also allelic vaπants and homologues of Shpxό. The homologue can be an alternative S. humilis gene or a gene of another plant species. The chimaeric genes can further include DNA sequences which hybridise with the Shpxό peroxidase sequence under stringent conditions. Such stringent conditions can be defined as follows:

Wash solution 0. lxSSPE/0.1 % SDS Wash temperature 65°C

Number of washes two

( l xSSPE is a solution consisting of 180 mM NaCI, 10 mM NaH O„ and 1 mM EDTA, and which has a pH of 7.4).

DNA sequences for inclusion in constructs according to the invention can be prepared or isolated using any of the methods known to those of skill in the art. Such methods are described in Sambrook et al. , Molecular Cloning: a Laboratory Manual, 2nd Ed. , Cold Spring Harbour Laboratory Press, Cold Spring Harbour NY (1989) and Λusebel et al. , Current Protocols in Molecular Biology, John Wiley & Sons, Inc., USA (1987-1995), the entire contents of which are incorporated herein by cross-reference. For example, an Shpxό homologue or allelic variant can be isolated from a genomic or cDNA library using hybridisation probes derived from the Sphxό sequence. The Sphxό sequence can also be used to derive oligonucleotide primers which can be used to amplify desired gene sequences by PCR. Harrison et al. (supra) describe a method of isolating Shpxό from S. humilis genomic DNA.

With reference to item (ii) above, the promoter can be selected to ensure strong constitutive expression of the peroxidase protein in most or all plant cells, it can be a promoter which ensures expression in specific tissues or cells that are susceptible to fungal infestation, and it can also be a promoter which ensures strong induction of expression during the infection process. Examples of other regulatory sequences which can be included in constructs are enhancers, untranslated regions of some transcripts and intron sequences from eukaryotic genes which can be used in combination with the suitable promoter. It will be appreciated by those of skill in the art that a promoter is not essential and the peroxidase encoded by the DNA sequence can be stably expressed in plant cells without any promoter present in the construct provided that insertion of the DNA sequence into the genome is in such a position that the sequence is operatively linked lo a native plant promoter or similar regulatory sequences.

Regarding item (iii) above, transcription terminators operative in plant cells are well known in the art and are descπbed, for example, in Ingelbrecht et at., The Plant Cell 1 :671 -680 (1989), the entire contents of which is incoφorated herein by cross-reference. A preferred terminator is the Sphxό terminator or the terminator of a homologue or allelic variant. However, depending on the site of insertion of the construct into a plant genome, a terminator may not be required and terminators naturally present in the genome of the transformed plant may be utilised. The DNA constructs of the invention can be introduced into both monocotyledonous and dicotyledonous plants. The plant is typically from a family of plants of agricultural importance such as cereals, legumes, oilseed plants, sugar and fibre plants. However, plants that are not of agricultural importance can be transformed with the subject DNA constructs so that they exhibit a greater degree of resistance to fungal infestation. Specific examples of plants which can be genetically modified with DNA constructs according to the invention are maize, banana, peanut, field peas, sunflower, tomato, canola, tobacco, wheat, barley, oats, potato, soybeans, cotton, carnations, and sorghum.

Plant cells can be transformed with DNA constructs of the invention according to a vaπety of known methods (Agrobacterium, Ti plasmids, electroporation, micro-injections, micro-projectile gun, and the like) as has been briefly discussed above. Two such suitable methods will now be described. Firstly, the DNA construct can be ligated into a binary vector carrying: i) left and right border sequences that flank the T-DNA of the Agrobacterium tumefaciens Ti plasmid; ii) a suitable selectable marker gene for the selection of antibiotic resistant plant cells; iii) origins of replication that function in either A. tumefaciens or Esche chia coli; and, iv) an antibiotic resistance gene that allows selection of plasmid-carrying cells of Y tumefaciens and E. coli. This binary vector carrying the chimaeric DNA construct can be introduced by either electroporation or triparental mating into A. tumefaciens strains carrying disarmed Ti plasmids such as strains LBA4404. GV3101 , and AGL1 or into A. rhizogenes strains such as R4 or NCCP1885. These Agrobacterium strains can then be co- cultivated with suitable plant explants or intact plant tissue and the transformed plant cells and/or regenerants selected by using antibiotic resistance.

A second method of gene transfer to plants can be achieved by direct insertion of the gene in target plant cells Foi example, the DNA construct can be co-precipitated onto gold 01 tungsten particles along with a plasmid encoding a chimaeric gene for antibiotic resistance in plants The tungsten particles can be accelerated using a fast flow of helium gas and the paiticles allowed to bombaid a suitable plant tissue This can be an embryogenic cell culture, a plant explant, a callus tissue oi cell suspension or an intact meristem Plants can be recovered using the antibiotic lesistance gene foi selection and antibodies used to detect plant cells expressing the pei oxidase protein

As described above in the fourth embodiment, the invention provides repioductive matenal, vegetative material, oi other regenerable tissue of a plant which includes a DNA constiuct accoiding to the invention Seeds and pollen are included within the ambit of reproductive matenal and stem segments oi cuttings within the ambit of vegetative material

The invention will now be illustrated by the following non-limit g examples Geneial Methods

Manipulation of DNA and RNA was caiπed out using known methods such as those descnbed by Sambiook et al (Molecular Cloning a Laboratory Manual 2nd Ed , Cold Spring I laiboui Laboiatoiy Press, Cold Spring Harbour NY [1989])

Reagents and other material were obtained from commeicial souices oi as othei wise indicated

EXAMPLE 1 Construction of a chimaeric gene In this example, we describe the construction of a chimaeric gene compπsing a constitutive promotei and the coding region of Shpxό The coding legion of the Shpxό cDNA (nucleotides 42 to 1001 of the SEQ ID NO 1 sequence - see SEQ ID NO 2 for the amino acid sequence) was amplified fiom plasmid pBluescnpt II SK⁺ (Strategene) by the polymerase chain leaction (PCR) using the following oligonucleotide primers Pπmei 1 5' GGCTCTAGAAGTCGACATGGTrCG 3'

Pnmei 2 5' AACAGCTATGACCATG 3'

The Pnmei 1 and 2 sequences weie selected either wholly or at least paitially fiom plasmid sequence either side of the Shpxό inseit PCR pπmeis were designed to incoipoiate l estπction endonuclease sites to facilitate manipulation of the construct in geneial puipose cloning (pBluescnpt) and binaiy vectors for Agrobacterium based plant transformation

PCR products weie digested wit Xbal and ligated into pBluescnpt cut with the same enzyme Inseition of the Shpxό cDNA was verified by DNA sequencing of the insei t DNA sequencing was perfoimed on denatuied double stranded DNA templates using automated methods on an Applied Biosystems (ABI) 373A instrument with the ABI PRISM Dye Deoxy Terminatoi Cycle Sequencing Kit The sequence was verified on both strands with overlap Oligonucleotide pπmeis used foi DNA sequencing weie synthesised on a Beckman Ohgo 1000 DNA synthesisei

insei t was latei sepaiatcd from the pBluescnpt DNA with Xbal and cloned into a binary vectoi - pGA643 (An et al EMBO J 4 227-288 [1985]) This created a transcriptional fusion between the constitutive expiession piomotei and the Shpxό cDNA Figure 1 schematically lllustiates this fusion constiuct The binary vectoi carrying the chimaeric gene construct was then used to transfoim tobacco and canola using an Agrobacterium tumefaciens mediated transformation system

EXAMPLE 2 Preparation of transgenic plant cells Agi obacterium tumefaciens was transformed with the vector carrying the chimaeπc constiuct using electroporation (Nagel et al FEMS Microbiol Let 67 325-328 [1990]) Both tobacco and canola wei e tiansformed using A tumefaciens strain LBA4404 (GibcoBRL) l obacco (N tahacum) transfoimation was caπied out essentially according to Horsch et al (Science 227 1229-1231 [ 1985]) using leal discs and 100 mg/L kanamycin as selective agent For canola tiansf ormation seeds of a double haploid canola line (141-227) derived fiom cv Wcstar pioduced in the Crop Science Depaitmcnt of the Univeisity of Guelph and Ontaiio Ministiy of Agπcultuie weie obtained fiom Di W D Bewersdoif (Ci op Science Department of the University of Guelph, Ontaiio, Canada) Seeds from this line were suiface sterilised and germinated on MS salts (Murashige and Skoog, siipia) complemented with 3 % sucrose and 0 8 % agar under a iegime of 16 h light and 8 h dai at 24 °C Hypocotyl segments (5-10 mm in length) were taken from 5 to 6 day-old steπlc seedlings and preincubated for a day on callus-inducing medium including MS salts and vitamins, Λ ' suciosc, 1 mg/L 2,4-D and 0 8% Difco Bacto-agar Agrobacterium tumefaciens haibouπng peioxidase gene constructs was grown overnight in YEP medium (An et al Plant Physiology 81 301 -305 [ 1988]) with selective antibiotics Before cocultivation, the absorbance at A_W(1 of the bacteπal solution was detei mined and the number of bacteria was adjusted to lxl0^x per mL (A,_π =0 03) in liquid callus inducing medium Hypocotyl segments weie incubated in bacterial solution with gentle shaking for 5 min, blotted on steπle filter papers placed on callus inducing medium foi 2 3 days Attei cocultivation, the segments weie washed twice in liquid MS medium, blotted briefly on filtei paper and placed on MS medium solidified with Phytagai (Difco) or Phytagel (Sigma) and containing 150 mg/I timentin (l icaicillin) After 5-7 days incubation on this medium, segments wei e tiansteπ ed on shoot l egeneiation medium containing 3 mg/I BAP, 1 mg/L zeatin, 5 mg/L AgNO,, 25 mg/L kana vcin and 1 0 mg/L timentin Plates weie sealed with Micropoie tape (3M Health Caie MN, USA) 1 he initial plating densities were 40-50 explants pei plate This was I educed to 20-2^ pei plate m subsequent subcultures Hypocotyl segments weie subcultuied onto fiesh medium without AgNO, eveiy two weeks Differentiated shoots were transferred to jais Elongation and loot formation were established in a hormone-free medium containing half stiength MS and suciosc 25 mg/L kanamycin and 100 mg/L timentin The transgenic status of the shoots was assessed bv placing leaf discs on a medium containing 4 mg/L BAP, 0 5 mg/L NAA and 25-50 nm L kanamvcin for foui weeks Rooted transgenic shoots were tiansferred to soil and kept under a dome foi a tew weeks in conti oiled environment rooms before exposing the shoots to normal conditions

EXAMPLE 3 Peroxidase assays for the analysis of transgenic plant tissue expressing Shpxό

Fieshly harvested leaves from transgenic (T₍„ T,, T₂ and T-,) N tabaccum cv Xanthi and B napus cv Westar (141 -227) were frozen in liquid Ν₂ for storage and subsequently homogenised at 4°C in a microcentrifuge tube with a custom made tight-fitting stainless steel gπndei attached to an electric drill using 3 volumes per unit fresh weight of buffer (10 mM sodium phosphate, 1 "A sodium metabisulphite, pH 6 0) Homogenates were centrifuged at 14,000 rpm in a refrigerated miciofuge at 4°C foi 30 minutes and aliquots of supernatant frozen at -70°C Peroxidase assays wei e earned out accoiding to Rathmell and Sequeira (Plant Physiol 53 317-318 [1974]) Reactions contained 0 28 % guaiacol and 0 3 % H₂0₂ in 50 mM sodium phosphate buffei (pH 6 0) The l eaction late was momtoi ed at 470 nm Reaction rates were linear and propoitional to the enzyme concentiation added Total protein was determined using the Bio-Rad protein assay adapted for mici otitie plates

Figures 2 and 3 show the total leaf peroxidase activities of the T, and T-, liansgemc tobacco and canola families, respectively Depending on the transgenic family, constitutive ovei -expi ession of Sphxό lesulted with 2-3 fold inci eases in the total leaf peroxidase activity over untiansfoπned contiol plants In these figures, peroxidase activity in 10-20 plants from each transgenic and control family was measured and values for t were calculated in pairwise comparison of the tiansgenic families with the contiol family Standard deviations aie indicated as aπows Families with different denoted letteis show significant differences at P < 0 05

EXAMPLE 4 Development of transgenic Tl seed lines Genotype designations for transgenic plants used herein are in accordance with the following convention the initial plant resulting from a transformation event and having giown fiom tissue culture is designated a T„ plant Plants resulting from self pollination of the natural floweis oi the T„ plant ai e designated T,

Transgenic plants (T„) were giown to maturity. Flowers were allowed to self-pollinate and seed pods collected aftei normal desiccation Seeds fiom each individual plant weie collected and stoied separately. Each seed lot was tested by genetic segregation analysis to determine the number of Mende an loci beaπng the kanamycin resistant trait Seeds collected from each I plant weie germinated on MS medium containing 400 mg/L kanamycin The ratio of normal gieen (kan-i ) versus bleached (kan-s) cotyledons was deteimined Seedlings with green coloui weie tiansplanted to soil foi fuithei analyses

To pioduce a fuithei geneiation, seeds weie collected from T, plants and the above pi ocess repeated to produce T₂ plants. T, plants were similarly produced.

EXAMPLE 5 Evaluation of transgenic plant tissue expressing Shpxό for disease resistance In this example, we describe the response of transgenic plants capable of expressing Shpxό peroxidase to challenge from a variety of fungal pathogens.

Eight-week old transgenic tobacco plants from T, and T₂ transgenic families were inoculated with the fungal pathogen Phytophthora parasitica var. nicotiana (black shank disease of tobacco) using the methods described ty Robin and Guest (NZ J. Crop Port. Sci. 22: 159-166 [1994]). Ten plants were used for each family (transgenes and controls). Lesion lengths on the decapitated stems were measured daily for 8 days postinoculation. Values for t were calculated in pairwise comparison of the transgenic families with control untransformed plants.

The results of this experiment are presented in Figure 4 in which the filled bars represent T, families and the vertically hatched bars represent T₂ families. The error bars represent the standard deviation for each family. Families showing significant differences at P < 0.05 with respect to the controls are denoted by different letters above the error bars. Analysis of inoculation data from this experiment showed that the transgenic families with higher peroxidase activity had significantly better protection with respect to wild type plants.

For Leptosphaeria maculans (blackleg disease of canola) inoculations, cotyledons from T, , T₂ and T, canola seedlings were punctured and inoculated with the pycnidiospore suspension of 10" spores. Disease reaction, or index, was scored visually using a scale where "0" corresponded to complete resistance and "9" corresponded to complete susceptiblity to infection. Based on this scale, plants with an index of 0-3 were considered resistant, plants with an index of 4-6 moderately resistant, and plants with an index of 7-9 susceptible. Thirty plants or more were used for eacli transgenic family and untransformed control families. Duncan's Multiple Range Test were used to statistically compare transgenic families with the untransformed controls.

Data from these inoculation experiments are presented in Figure 5. In Figure 5, the horizontally hatched bars represent T, families, the vertically hatched bars represent T₂ families, and the filled bars represent T, families. Analysis of the data showed that some of the transgenic lines had significantly better protection against Leptosphaeria (P< 0.05). To measure the response ol^' adult plants to this pathogen, an inoculation experiment using 5-6 weeks old plants from T, families were also done in the glasshouse. In this experiment, plant survival rate for each family was calculated as the percentage of plants that reached maturity and set seed. Data from this inoculation experiment are presented in Figure 6. Analysis of data showed that transgenic lines which performed better in cotyledon inoculation tests displayed better survival rates.

For Sclerotinia sclerotorium inoculations, stems of 10 adult canola plants from each of T, and T-, families weie inoculated by securing a barley grain colonised by the fungus on the stem Lesion extension was measured daily Duncan's Multiple Range Test were used to statistically compai c transgenic families with untransformed contiol plants. Data fiom the inoculation expeπment aie presented in Figure 7 in which the filled bars represent T, families and the vertically hatched bais represent 1 ₂ families Experiments with T, families showed that some transgenic lines expi essing Shpxό had better piotection against the fungus due to lower rates of lesion extension on then stems. Howevei , next round inoculations done on T₂ families did not show any significant pi otection Peioxidase assays done on a subset of plants sampled before the inoculations also showed some reduction in the total peroxidase activity of these plants This suggested that stable expicssion of transgene (Shpxό) is necessaiy foi consistent disease lesistance l esponce of canola against S sclerotorium

SEQUENCE LISTING

(1) GENERAL INFORMATION:

( ) APPLICANT:

(A) NAME: COOPERATIVE RESEARCH CENTRE FOR TROPICAL PLANT PATHOLOGY

B) STREET: The University of Queensland

C) CITY: St Lucxa

D) STATE: Queensland

E) COUNTRY: Australia POSTAL CODE (ZIP) : 4067

NAME: GRAINS RESEARCH & DEVELOPMENT CORPORATION

STREET: National Circuit

CITY: Barton

STATE: ACT

COUNTRY: Australia

POSTAL CODE (ZIP) : 2600

NAME: KAZAN, Kemal (US only) STREET: 1/24 Durham Street CITY: St Lucia STATE: Queensland COUNTRY. Australia POSTAL CODE (ZIP) : 4067

NAME: GOULTER, Kenneth C. (US only)

STREET: 26 Emblem Street

CITY Jamboree Heights

STATE: Queensland

COUNTRY: Australia

POSTAL CODE (ZIP) : 4074

NAME: MANNERS, John M. (US only) STREET: 28 armington Street CITY: Paddmgton STATE: Queensland COUNTRY: Australia POSTAL CODE (ZIP) . 4064

(ii) TITLE OF INVENTION: Fungus Resistant Transgenic Plants

( ll) NUMBER OF SEQUENCES: 2

(iv) COMPUTER READABLE FORM- (A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.30 (EPO)

(2) INFORMATION FOR SEQ ID NO: 1:

(l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1144 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: cDNA to mRNA

(m) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Stylosanthes humilis

(B) STRAIN: Paterson (F) TISSUE TYPE: Stem

(vii) IMMEDIATE SOURCE: (B) CLONE: Shpxδ

(ix) FEATURE: (A) NAME/KEY: sιg_peptιde

(B) LOCATION:42..113

(ix) FEATURE:

(A) NAME/KEY: mat_peptιde (B) LOCATION:114..1001

(x) PUBLICATION INFORMATION:

(A) AUTHORS: Harrison, S J

Curtis, M D Mclntyre, C L

Maclean, D J Manners, J M

(B) TITLE: Differential expression of peroxidase isogenes during the early stages of infection of the tropical forage legume Stylosanthes humilis by

Colletotrichum gloeosporioides

(C) JOURNAL: Mol. Plant Microb. Interact. (D) VOLUME: 8

(E) ISSUE: 3

(F) PAGES: 398-406

(G) DATE: 1995

(K) RELEVANT RESIDUES IN SEQ ID NO: 1: FROM 1 TO 1144

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

TTTCGAATAA TTTCATTACT TTTATATATT ATTGCATTGC CATGGCAATT CTTGCAATTA 60

GCAAAGTTTG TTTGATAATA TTGGTGATGA GCCTTATAGG ATTAGGATCA GGTCAATTGT 120

CATCAAATTT TTATGCAACA ACATGTCCGA ATGCACTTTC AACGATTAGG TCAGGAGTGA 180

ACTCTGCTGT GAGCAAAGAA GCTCGCATGG GAGCTTCCCT TCTTCGCCTT CATTTCCATG 240

ATTGCTTTGT TCAAGGATGT GATGCATCAG TGTTATTAGA TGATACATCA AATTTCACAG 300

GAGAAAAAAC AGCACGTCCT AATGCTAATT CAATTAGAGG TTTTGAAGTC ATAGACACCA 360

TAAAATCTCA AGTAGAGAGC TTGTGTCCTG GTGTTGTTTC TTGTGCTGAT ATTCTTGCTG 420

TTGCTGCTAG AGATTCTGTT GTTGCTCTTG GTGGACCCAG TTGGACAGTG CAACTGGGAA 480

GAAGAGACTC AACTACAGCA AGTTTAAGCT TAGCTAACTC AGATTTGGCT GCTCCCACTT 540

TGGATCTCAG TGGTCTAATC TCTGCTTTCT CTAAGAAAGG TTTATCAACT AGTGAAATGG 600

TTGCCCTATC AGGAGGGCAT ACAATTGGGC AAGCAAGATG CACAAGCTTT AGAACAAGGA 660

TATACACTGA GAGCAACATA GATCCCAATT TTGCCAAATC ATTGCAAGGA AATTGCCCTA 720

ATACCACAGG CAATGGTGAC AACAACTTGG CCCCAATTGA CACAACTAGT CCAACAAGGT 780

TTGACAATGG TTACTATAAG AACTTGCTAG TGAAAAAGGG TCTCTTCCAC TCTGATCAAC 840

AACTCTTCAA TGGAGGATCC ACAGATTCTC AAGTGAATGG TTATGCCTCC AACCCTTCAA 900

GTTTCTGCTC TGATTTTGGC AATGCTATGA TTAAGATGGG TAACATTAGT CCACTCACTG 960

GATCCAGTGG CCAGATTAGG ACCAATTGCA GGAAGACCAA TTAGGATCAT ATGATAAAAT 1020

AAT AATAAT ATAGATAAAA AATATATATA TATATATAAT AATAATAATA ATTAAATAAA 1080

CCGAATATAG TTTCTAGCTT ATAACTTTTG TTTTATTTTT TAATGTTGAA GAAATTAAAA 1140 GGGT 1144

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 320 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Stylosanthes humilis (B) STRAIN: Paterson

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met Ala He Leu Ala He Ser Lys Val Cys Leu He He Leu Val Met 1 5 10 15

Ser Leu He Gly Leu Gly Ser Gly Gin Leu Ser Ser Asn Phe Tyr Ala 20 25 30

Thr Thr Cys Pro Asn Ala Leu Ser Thr He Arg Ser Gly Val Asn Ser 35 40 45

Ala Val Ser Lys Glu Ala Arg Met Gly Ala Ser Leu Leu Arg Leu His 50 55 60

Phe His Asp Cys Phe Val Gin Gly Cys Asp Ala Ser Val Leu Leu Asp 65 70 75 80

Asp Thr Ser Asn Phe Thr Gly Glu Lys Thr Ala Arg Pro Asn Ala Asn 85 90 95

Ser He Arg Gly Phe Glu Val He Asp Thr He Lys Ser Gin Val Glu 100 105 110

Ser Leu Cys Pro Gly Val Val Ser Cys Ala Asp He Leu Ala Val Ala 115 120 125

Ala Arg Asp Ser Val Val Ala Leu Gly Gly Pro Ser Trp Thr Val Gin 130 135 140 Leu Gly Arg Arg Asp Ser Thr Thr Ala Ser Leu Ser Leu Ala Asn Ser 145 150 155 160

Asp Leu Ala Ala Pro Thr Leu Asp Leu Ser Gly Leu He Ser Ala Phe 165 170 175

Ser Lys Lys Gly Leu Ser Thr Ser Glu Met Val Ala Leu Ser Gly Gly 180 185 190

His Thr He Gly Gin Ala Arg Cys Thr Ser Phe Arg Thr Arg He Tyr 195 200 205

Thr Glu Ser Asn He Asp Pro Asn Phe Ala Lys Ser Leu Gin Gly Asn 210 215 220

Cys Pro Asn Thr Thr Gly Asn Gly Asp Asn Asn Leu Ala Pro He Asp 225 230 235 240

Thr Thr Ser Pro Thr Arg Phe Asp Asn Gly Tyr Tyr Lys Asn Leu Leu 245 250 255

Val Lys Lys Gly Leu Phe His Ser Asp Gin Gin Leu Phe Asn Gly Gly 260 265 270

Ser Thr Asp Ser Gin Val Asn Gly Tyr Ala Ser Asn Pro Ser Ser Phe 275 280 285

Cys Ser Asp Phe Gly Asn Ala Met He Lys Met Gly Asn He Ser Pro 290 295 300

Leu Thr Gly Ser Ser Gly Gin He Arg Thr Asn Cys Arg Lys Thr Asn 305 310 315 320

Claims

1 A method of engineering a plant to fungal resistance, the method comprising introducing into cells of the plant a DNA construct comprising:

(a) a promoter constitutively operative in the plant cell; and (b) a DNA sequence encoding a peroxidase isozyme operatively linked to said piomoter, wherein said DNA sequence is selected from: (I) Shpxό herein defined;

(n) a sequence which hybridises to Shpxό under stringent conditions and which encodes a protein having peroxidase activity; (in) a fragment of a DNA sequence according to (l) or (ii), which fragment encodes a protein having essentially the same activity as the peroxidase isozyme encoded by Shpxό

2 The method according to claim 1 , wherein said promoter is the 35S promoter of Cauliflower Mosaic Virus.

3 The method according to claim 1 , wherein said DNA sequence compπses SEQ ID NO 1

4. The method according to claim 1 , wherein said peroxidase isozyme has an amino acid sequence substantially corresponding to SEQ ID NO: 2.

5 The method according to claim 1 , wherein said fungal resistance is to Phytophthora parasitica,

Leptosphaeria maculans, or Sclerottnia sclerotorium.

6. A plant cell harbouring a DNA construct comprising: (a) a promoter constitutively operative in the plant cell; and

(b) a DNA sequence encoding a peroxidase isozyme operatively linked to said piomotei , wherein said DNA sequence is selected from'

(I) Shpxό herein defined;

(n) a sequence which hybridises to Shpxό under stringent conditions and which encodes a protein having peroxidase activity,

(in) a fragment of a DNA sequence according to (I) or (ii), which fragment encodes a protein having essentially the same activity as the peroxidase isozyme encoded by Shpxό.

7 The plant cell according to claim 6, wherein said promotei is the 35S promoter of Cauliflower Mosaic Virus.

8 The plant cell according to claim 6, wherein said DNA sequence comprises SEQ ID NO 1 .

9 The plant cell according to claim 6, wherein said peroxidase isozyme has an amino acid sequence substantially corresponding to SEQ ID NO: 2

10. The plant cell according to claim 6, wherein said fungal resistance is to Phytophthora parasitica, Leptosphaeria maculans, or Sclerotima sclerotorium.

1 1 The plant cell according to claim 6, wherein said DNA construct is incoφorated into the genome of said plant cell.

12. A plant comprising cells according to claim 6.

13. The plant according to claim 12 which is a monocot or a dicot.

14. The plant according to claim 12 which is from a family of plants selected from cereals, legumes, oilseed plants, sugar or fibre plants.

15. The plant according to claim 12 which is selected from maize, banana, peanut, field peas, sunflower, tomato, canola, tobacco, wheat, barley, oats, potato, soybeans, cotton, carnations, or sorghu .

16. Material of the plant according to claim 12, which material is selected from reproductive material, vegetative material, or other regenerable material.

17. Material according to claim 16, wherein said reproductive material is seed or pollen.

18. Material according to claim 16, wherein said vegetative material is a stem segment or a cutting.