CA2122785A1 - Composition and method of using plant lectins to control insects - Google Patents

Abstract

ABSTRACT OF THE INVENTION

The present invention provides a composition and method of using PNA lectin, amaranthin lectin or Con A lectin to protect plants otherwise susceptible to infestation by an insect selected from Western corn rootworm or Northern corn rootworm.

Description

2~227~

COMPOSITION AND METHOD OF USING PLANT LECTINS TO CONTROL
INSECTS
Field of the Invention The present invention relates to the fields of genetic engineering and plant husbandry. More specifically, the invention provides methods and ; compounds for co~trolling or combating insects in agriculture or horticulture.
Back~round of the Invention Maize crops are attacked by phytophagous insects including the corn rootworms. The western corn rootworm, Diabrotica virgifera virgifera9 and the northern corn I rootworm, D. barberi, are the most serious insect pests of i 15 dent corn, Zeamays, in the major corn producing state~
of the north central United States and in Canada. Both species are univoltine. In the corn belt, these beetles are present in cornfields from July through frost, feeding on corn pollen, silks, immature kernels and foliage as well a~ pollen of other plants. Fro~ late July through September, oviposition occurs primarily in cornfields; few eggs are laid in other crops. Eggs remain in the soil until the following spring with hatch ~-~
beginning in late May and early June. The larvae can , ~.
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survive only on the root~ of corn and on the root~ o~ a ~ ~-limited number of grasses. Larval feeding may reduce ;
the amount ol water and nutrients supplied to developing - -plants, and extensive root damage makes plantq more susceptible to lodging thereby resulting in diPficulty in harvesting the cropO Larval feeding may also facilitate infection by root and stalk rot fungi, resulting in further damage. Pupation occurs in the soil, and adult emergence begins in early July in the Midwest.
Control of such insects has traditionally been partially addressed by cultural and breeding methods.
An effective way to reduce these losses i~ to use crop cultivars having genes for pest resistance (see Painter (1951), Insect Resistance in Crop Plants, Macmillan: New York). Plant breeders have attempted to reduce losse cau~ed by insect attaek by incorporating in~ect resistance genes into their varieties via conventional breeding programs.
Classical approache to host plant re~istance9 though remarkably successful in some instances, are rather empirical. One limitation of the classical approach is that these types of resistance are likely to be under the control of many genes, and so are difficult for the plant breeder to fully exploit. Additionally, the movement of genes for resistance from one plant to another is restricted to species that can be interbred.
Moreover, often resistant varieties have shown a yield depression and so have not been economically viable.
Chemical insecticides have been heavily relied upon to control insects. These a~ents typically are applied on or banded into the soil, or to the plant :: .:
C-50,243 ~2-.~ ,~, .

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foliage or in bait ~tations. A major problem in the use of many pe~ticides i9 the ability of insects to become resistant to the applied agents. Thi~ phenomenon occur~
through selection of the most reqistant members of the insect population during repeated application of the agent. A need, therefore, exists for new insect control ; agents, particularly agents that have a mode of action different from conventional insecticides.
As alternatives to synthetic compounds, certain 0 naturally-occurring agents have been isolated and developed as pesticides. These include plant and microbial secondary metabolites and proteins9 and natural predators or pathogens of insects (including other insects, fungi, bacteria and viruses).
Furthermore, as recombinant DNA technology has advanced, genes from a donor organism may be transferred to a recipient organism resulting in a new phenotype in the recipient. In the case of transgenic plants, this phenotype may provide resistance to insect damage if the introduced gene encodes a polypeptide, the action of which results in a deleterious effect on the pest. A ~
very limited number of such polypeptides have been -deqcribed, e.g., polypeptides from Bacillus thuringiensis, various proteinaceous protease and amylase inhibitors and various plant lectins.
Plant tissues, including roots, leaves, fruits and seeds, often contain substantial concentrations of 3 lectins. Lectins are defined as proteinQ o~ non-immunoglobulin nature capable of specific recognition and reversible binding, with high affinity, to carbohydrate moieties of glycoproteins, glycolipids or polysaccharides without altering the covalent structure of any of the recognized glycosyl ligands (see Goldstein 50,243 -3-;

i^` _4_ 2~2278~

and Hayeq (1978), Adv. Carbohydr. Chem. Biochem., 35:127-3~0).
Lectins are alqo often quite abundant in vegetative plant organ~ such as rootq, leave~, rhizome~
and ~tems. Some o~ these are vacuolar while others ~uch as the chitin-binding Datur~ seed lectin are extracellular. Vacuolar lectins also occur in cereal seed~, but are much les~ abundant and occur only in specific cell layers of the embryo [e.g. wheat germ agglutinin in the coleorhiza and rootcap of the wheat embryo (see Etzler (1985), "Distribution and function of ?lant lectins", In. The Chemistry and Biology of Lectins, ed. I. E. Liener, N. Sharon, I~ J. Goldstein, New York- Academic)].
Lectins have been implicated in a variety of roles dealing with the defense mechanism~ o~ plants (see Etzler (1985), supra) . In 1976 Janzen etal implicated lectin~ in conferring insect resistance by suggesting that the inability of the cowpea weevil, Callosobruchus maculatus F, to attack seeds of P. vulgaris was partly due to the presence of lectinq. Recently Murdock etal -(1990), Phytochemi~try, 29(1):85-89 published a study showing the effects of 17 plant lectins against the cowpea weevil. Moreover, ~tudies with pea lectin (see Boulter et al. (1990), croP Protectiont 9:351-354) have shown it to enhance plant resistance to Heliothis uirescens :- --(tobacco budworm). -EP-A-0351924 (SheLl Internationale Research Maatschappi; B.V.) relates to a transgenic plant, preferably a plant from a member of the genus Solanaceae, comprising a foreign lectin gene, preferably a lectin from a member of the genus Leguminoseae. In particular, C-50,243 _4_ :

~~ _5_ ~ ~ 22 ~8~

it discloses that Conaual~aensiformis lectin (Concanavalin A or Con A lectin), lentil lectin, and favin9 or pea lectin, may be used to control insects. The reference~
however, does not specify against which insects any of the lectins is efective 9 with the exception of pea lectin which was shown to provide some degree of insect resistance against the bean weevil, Callosobruchus maculatus ) .
EP-A-0427529 (Pioneer Hi-Bred International, Inc.) discloses that selected plant lectins have been found to be larvicidal against particular common insect pests of agricultural crops. Specifically, this reference only teaches that certain lectins have sufficient insecticidal (larvicidal) activity to be operative in a plant cell expression system~ More specifically9 the reference teaches the control of larvae selec~ed from European corn borer, corn rootworm and cutworm9 by administering enterally to the larvae a 20 larvicidal amount of a lec~in selected from Artocarpus ~
~ntegrifolia lectin (jacalin), Bauhiniap~rpureaalba (camel's --foot tree) lectin9 Cod~umfragile lectin, elderberry bark lectin, Gr~ffoniasimplicifolia lectin, Phytolaccaamericana lectin, Maclurapom~fera (osage orange) lectin, R~cinus communis (castor bean) agglutinin, Triticum uulgare lectin (Wheat germ agglutinin), and Vicia uillosa lectin. The reference further teaches that Arachishypogaea (peanut lectin) and Concanavalin A do not show significant insecticidal activity (i.e., less than a 25 percent mortality and/or 40 percent weight loss after a 7-day exposure to lectins topically applied to the diet) ' against Southern corn rootworm and European corn borer.

Notwithstanding the need to ;dentify new lectins and modes of delivery which may be useful in C-50,243 -5-~" -6- 212278~

crop protection against insects selected from Wester~
corn rootworm or Northern corn rootwor~, no publication to date has taught or suggested the use of peanut agglutinin (PNA) lectin from Arachis hypogaea, amaranthin lectin from Amaranthuscaudatus and/or Con A lectin from 5 Canaualiaensiformis to inhibit the growth of insects selected from Western corn rootworm or Northern corn rootworm. This is perhap~ due to the fact that it is generally assu~ed by skilled artisans that compounds having no significan~ activity against Southern corn rootworm would also not have significant activity against Western corn rootworm or Northern corn rootworm.
:-:
An object of the present invention is to provide a method for protecting a plant or a part thereof from an insect selected from Western corn rootworm or Northern corn rootworm.
~ .
A further object of the present invention i~ to provide novel topically applied insect controlling compositions which are capable of protecting from attack a plant or a part thereof otherwise susceptible to insect infestation by an insect selected from Western -~
corn rootworm or Northern corn rootwor~.
A ~till further object of the present invention is to provide a process for preparing genetically ~-transformed ho~t cells comprising the transformation of host cells with a gene encoding a protein capable of having a deleterious effect, upon ingestion, by an insect selected from Western corn rootworm or Northern corn rootwor~.
Yet another object of the present invention is to provide transgenic plants capable o~ expressing a C-50,243 -6-_7_ 21227~5 insect controlling protPin in a plant or a part thereof otherwise susceptible to insecS infestation by an insect selected from Western corn root~orm or Northern corn rootworm.
Other objects and advantages of the present invention will become apparent from the description of the invention provided hereunder.
Summary of the Invention Accordin~ly, one a~pect of the invention relate~ to a method of controlling an insect selected from Western corn rootworm or Northern corn root~orm.
It is more specifically concerned with providing a lectin selected from the PNA lectin9 amaranthin lectin and Con A lectin in, on or near a plant tissue otherwise susceptible to attack by an insect selected from Western CDrn rootworm or Northern corn roatworm, whereby the plant tissue has improved resistance to such insects.
In a second aspect, the present invention relates to an insecticidal composition of at least one lectin selected from peanut agglutinin lectin, amaranthin lectin and Con A lectin, wherein the composition is capable of improving the resistance of plants or parts thereof susceptible to insect infestation by disrupting the normal life processes of an insect selected from Western corn rootworm or Northern corn rootworm.
In other aspects, the invention is directed to expression vehicles capable of affecting the production of such aforementioned proteins in suitable host cells.
It includes the host cells and cell cultures which i C-50,243 ~7_ "

-8- 21 ~ 2 ~ 8 ~ 73776-94 result from transformation with these expresqion -~
vehicles.
Brief Description of the Drawinqs Figures 1 to 5 show an amino acid sequence (SEQ. ID. -NO. 1) and synthetic DNA sequence (SEQ. ID. NO. 2) used . . .
to express the amino acid sequence of the subunit which makes up the peanut agglutinin tetramer. -~
.
Detailed DescriDtion of the Invention The entire teachings o~ all references cited herein are hereby incorporated by reference.
:,'' ~:
It has now been surprisingly found that lectins : ~ -~
selected from peanut agglutinin (PNA~ lectin from Arachis hypogaea, amaranthin lectin from Amaran~huscaudatus and Con A lectin from Canavalia ensiformis can control insect growth (including larvae) of an insect selected from 20 Western corn rootworm or Northern corn rootworm. ~-~
An "insect controlling amount" is an amount oP
a lectin selected from PNA, amaranthin lectin and Con A
lectin sufficient to deleteriously disrupt the normal life processes of an insect selected from Western corn rootworm or Northern corn rootworm ~i.e. 9 amounts which - -~
are lethal (toxic) or sublethal (injuring, growth or development inhibiting or repelling)]. The mechanisms for the observed toxicity of these lectins against --Wester~ corn rootworm or Northern corn rootworm are not known but, without intending to be bound by theory ?
probably involves the binding of the lectin to the midgut epithelial cells of the insect and disrupting cell functions.

C-50,243 -8-2~2278~

Con A iqi a manno~e/glucose binding lectin isolated ~rom jackbeanqi (Canaualia ensiformis) . Con A is a metalloprotein composed of four ~at pH 7) carbohydrate-free subunits, Mr = 26,500 (Ciee The Le_tin~, Leiner et al.
(eds.), page 51 (1986). The amino acid and cDNA
5 sequences of Con A are known (see Carrington et al.
(1985), Nature, 313:64-67; Cunningham etal. ( 1975) ~
J. Biol. Chem., 250: 1503-1512; and Wang etal. ( 1975), J Biol Chem., 250: 1490-1502) .

PNA is a legume seed lectin derived from Arach~s hypogaea. It is composed of four identical subunits of molecular mass 27 kDa, each with one binding site, that is specific for D-galactose residues at non-reducing 15 terminal po~itions of glycoconjugates (qee Young, etal.
(1991), Eur. J. Biochem., 196:631-637) . The lectin ; recognizes both a- and ~-galactosides, and the disaccharide of highest known affinity is the T-antigen structure Gal(1~-3)GalNAc[2] (GalNAc, N-acetyl-D-galactosamine).
The amino acid sequence of PNA has been published (see Young etal. (1991), Eur. J. Biochem, 196:631-637). Since the amino acid sequence of PNA is known, it is possible to reverse translate the sequence and construct a DNA
sequence capable of expressing the protein. However, it 25 is also possible to isolate the entire genomic sequence of the Arachis hypogaea lectin.
Amaranthin is a lectin present in the seed of Amaranthuscaudatus9 which specifically binds the T-3 disaccharide ~Gal~1, 3GalNAc~-0-) . The lectin is a homodimer composed of a single type of subunit with Mr = 33,000-36,000 (see Rinderle e~al. ( 1989), J. Biol.
Chem., 264:16123-16131). The amino acid sequence of the Amaranthuscaudatus lectin, or at least a portion thereof, ` may be determined by N-terminal sequencing or sequencing ,. :
C-50,243 -9-2~22785 --1 o--of oligopeptides derived by proteolyqis. In addition, antisera can be prepared that specifically recognize~
the Amaranthuscaudatus lectin. Thereafter it iq possible to construct or iqolate a suitable DNA ~equence.
It should be understood that one may syntheqize~ ~`
or isolate substantially pure functional derivatives of the naturally-occurring PNA lectin, amaranthin lectin or Con A lectin. As used herein, the term "substantially ` `~
pure" is meant to describe proteins which are homogeneous by one or more purity or homogeneity characteristics. For example1 a substantially pure PNA
lectin, amaranthin lectin or Con A lectin will show constant and reproducible characteristics within standard experimental deviations for parameters such as molecular weight, chromatographic behavior and the like.
The term, however, is not meant to exclude artificial or synthetic mixtures of the PNA lectin, amaranthin lectin or Con A lectin with other compounds or the presence of minor impurities which do not interfere with the biological activity of the lectin.
Substantially pure PNA lectin, amaranthin lectin or Con A lectin may be purified directly from plants in which they are naturally occurring by any appropriate protein purification technique~ Exemplary ~ `
techniqueq include chromatographic techniques, such as `
gel filtration liquid chromatography, ion exchange chromatography, high performance liquid chromatography, 3 reverse phase chromatography or by the use of immunological reagents employing antibodies specific for the PNA lectin, amaranthin lectin or Con A lectin. It `~
is also possible to synthe~ize in uitro and purify the lectins from the constituent amino acids (see Merrifield (1963), J. Amer~ Chem. Soc., 85:2149-2154; and Solid C-50,243 _10_ : `

11 21~27~

Phase PeQtide Synthesi_ (1969), (eds.) Stewart and Young).
Preferably, DNA encoding the PNA lectin, amaranthin lectin or Con A lectin may be prepared from chromosomal DNA, cDNA or DNA oY ~ynthetic origin by using well-known techniques. Genomic DNA and cDNA may be isolated by standard techniques (see Sambrook et al.
(1989), Molecular Cloning: A Laboratory Manual, published by Cold Spring Harbor Laboratory (U.S.A.);
Mullis etal. ( 1987), Meth. Enz , 1~5:335-350; Horton etal.
(1989), Gene, 77:61; and PCR Technolo~: PrinciDles and Applications for DNA Amplification, (ed.) Erlich (1989)). Specifically comprehended as part of thi~
invention are genomic DNA sequences encoding allelic variant forms of the genes encoding PNA lectin, amaranthin lectin or Con A lectin, aq well aq it~ 5' and 3' flanking regions. It iq also pos ible to prepare synthetic sequences by oligonucleotide synthesis (see Caruthers (1983), In: Methodology of DNA and RNA, (ed.) Weissman; or Beaucage etal. ( 1981), Tetrahedron Letters, 22:1859~1962).
Of course, one may incorporate modifications into the isolated sequences to generate a functional derivative of the PNA lectin, amaranthin lectin or Con A
lectin. Exemplary techniques for modifying oligonucleotide sequences include using polynucleotide mediated, site-directed mutagenesis (see Zoller etal.
3 ~1984), DNA, 3:479-488); Higuchi etal. ( 1988), Nucl.
Acids Res., 16:7351 7367; Ho e~al. ( 1989), Gene, 77:51-59; and Horton etal. ( 1989), Gene, 77:61; and PC~
TechnologY: Principles and Ap~lications for DNA
Amplification, (ed.) Erlich (1989~). Techniques for C~50,243 .
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such manipulationi3 are well known in the art and diisclosed by Sambrook etal. (1989), supra. ~:
Such modifications include the addition, deletion, or nonconservative substitution of a limited number of variou3 nucleotidei3 or the conservative substitution of many nucleotides, provided that the proper reading frame is maintained. A "functional derivative" of the PNA lectin, amaranthin lectin or Con ~-A lectin is a compound which posi3esses a biological activity that is substantially similar to a biological activity of the PNA lectin, amaranthin lectin or Con A
lectin. The term "functional derivative" is intended to include fragments and effectively homologous variants thereof.
A "fragment" is meant to refer to any ~;
insecticidally-active polypeptide subiqet of a PNA
lectin, amaranthin lectin or Con A lectin. An "e~ectively homologous variant" of a molecule such ais the PNA lectin, amaranthin lectin or Con A lectin is meant to refer to a molecule substantially similar in sequence and function to either the entire molecule or to a fragment thereof. For purposeis of this invention, 25 one amino acid sequence is effectively homologous to a ~
second amino acid sequence if at least 70 percent, -preferably at least 80 percent, and most pre~erably at least 90 percent o~ the active portions of the amino acid sequence are identical or equivalent. General 3 categories of potentially-equivalent amino acids are set forth below, wherein9 amino acids within a group may be ~;
substituted for other amino acids in that group~
glutamic aaid and aspartic acid; (2) lysine, arginine and histidine; (3) alanine, valine, leucine and isoleucine; (4) asparagine and glutamine; (5) threonine C-50,243 -12-_13_ and serine; (6) phenylalanine, tyrosine and tryptophan;
and (7) glycine and alanine~
Upon completion of the appropriate manipulation~, the gene of interest may be incorporated into an expre~ion vehicle ~uitable for transformation of a suitable ho~t cell. In general, the expression vector should contain all the DNA control sequences necessary for both maintenance and expression of a heterologous DNA sequence in a given host. Such control sequences generally include a promoter sequence (including a transcriptional start site), a leader sequence and a DNA sequence coding for translation ~tart-signal codon (generally obtained from either the gene to be expressed by the promoter or from a leader from a second gene which is known to work well or enhance expression in the selected host cell), a translation terminator codon, and a DNA sequence coding for a 3' non-translated region containing signals controlling termination of RNA synthesis and/or messenger RNA modification. Preferably, the vector ~ -should contain a marker gene, appropriate enhancer elements capable of optimizing protein expression in any -particular species and an intron in the 5' untranslated region, e.g., intron 1 from the maize alcohol dehydrogenase gene that enhances the steady state levels of m~NA, all of which is a matter of ordinary skill in the art utilizing the teachings of this disclosure.
3 The host cell to be transformed may be either ;~
prokaryotic and eukaryotic. The specific host cell -employed will depend upon its intended uses, as discussed in more detail below.

C-50,243 -13- -~

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Conventional technologie_ for introducing biological material into host cells include electroporation [see Shigekawa and Dower (1988), Biotechnique~, 6:742; Miller, etal. ( 1988), Proc. Natl.
Acad. Sci.USA, 85:856-860; and Powell, etal ( 1988), Appl. Environ. Microblol., 54~655-660]; direct DNA
uptake mechanisms [see Mandel and Higa (1972), J. Mol.
Biol., 53:159-162; Dityatkin, etal. (1972), Biochimica et Biophysica Acta, 281:319-323; Wigler, etal. (1979), Cell, 1o 16:77; and Uchimiya, etal. ( 1982), In: Proc. 5th Intl.
Con~. Plant Tissue and Cell Culture, A. Fujiwara (ed.), Jap. Assoc. for Plant Tissue Culture, Tokyo, pp. 507--508~; fusion mechanisms [see Uchidaz, etal. ( 1980), In:
Introduction of Macromolecules Into Viable Mammalian Cells, C. Baserga, G~ Croqe, and G. Rovera (eds.) Wiqtar Symposium Series, Vol. 1, A. R. Liss Inc., NY, pp. 169-185]; infectious agents [see Fraley, etal. ( 1986), CRC Crit. Rev. Plant Sci., 4:1-46): and Anderson (1984), Science, 226:401-409]; microinjection mechanisms [see Crossway, etal. ( 1986), Mol. Gen. Genet., 202:179-185];
and high velocity projectile mechanisms (see EP0 0 405 696).
Transformants are then isolated in accordance with conventional methods, usually employing a selection technique which allows for selection of the desired '~ organism as against unmodified organismq. The selected cells can be screened for expression of the appropriate lectin by appropriate assay techniques such as immunoblot analysi~, enzyme-linked immunosorbent assay, radioimmunoassay, or fluorescence-activated cell sorter analysis, immunohistochemistry and the like. - -The present invention contemplates agricultural compo~ition~ containing a lectin selected from PNA

.
~ C-50,2~3 _14_ 15_ 21~7~5 lectin, amaranthin lectin or Con A lectin. Exemplary recombinant ho~t cells from which signi~icant quantities of the lectin may be expres3ed and i~olated include unicellular prokaryotic and eukaryotic strains.
Prokaryotic microbes that may be used as hosts include 5 Escherichiacol~, and other Enterobacteriaceae, Bacilli7 and various Pseudomonas. Common eukaryotic microbes include Saccharomyces cerevisiae and Pichia pastoris . Common higher eukaryotic ho~t cells include Sp2/0 or CHO cells.
Another preferred host i3 insect cells, for example Drosophila larvae, in ~hich the vector contain~ the Drosophila alcohol dehydrogenase promoter.
Alternatively, baculovirus vectors, e.g., Autographa californica nuclear polyhedrosis virus (see Miller etal.
(1983), Science, 219:715-721) may be engineered to express large amounts of the lectin in cultured insects cells ~see Andrew~ etal. ( 1988), Biochem J., 252:199-206).
The isolated protein then may be incorporated into an agricultural composition for application to plants or parts thereof which are susceptible to infestation. Often the agricultural composition will contain an agriculturally acceptable carrier. By the term "agriculturally acceptable carrier" is meant a substance which may be used to dissolve9 di~perse or diffuse or enhance an active compound in the composition without impairing the effectiveness of the compound and which by itself ha~ no detrimental effect on the soil, equipment, crops or agronomic environment.
The agricultural compositions may be applied in a wide variety of forms including powders, crystals, suspensions, dustsl pellets, granules, encapsulations, microencapsulations, aerosol , solutions, gels or other C-50,243 _15_ i ` -16- 21~2785 dispersions. In addition to appropriate liquid or ~olid carrierY, compositions may include adjuvant~ quch as emulsifying and wetting agents, qpreading agent~, dispersing agents 9 adhesive~ or agents which ~timulate insect feeding according to conventional agricultural practice~. Adjuvants for the formulation of insecticides are well known to tho~e skilled in the art.
The concentration of lectin will vary widely depending upon the nature of the particular formulation, 0 particularly whether it i3 a concentrate or if it i~ to be used directly~ The lectin generally will be present in at least 1 percent by weight and may be up to 100 percent by weight.
The presentation of the agricultural composition may be achieved by external application either directly or in the vicinity o~ the plants or plant parts. The agricultural composition~ may be applied to the environment of the insect pest(s~, e.g., plants, soil or water9 by spraying, dusting, sprinkling, or the like. ~ -The present invention further contemplates applying microbes and insect viruses transformed with a gene encoding the PNA lectin, amaranthin lectin or Con A
lectin or, or near a selected plant or plant part J, "~
otherwise susceptible to attack by a target insect. The microbes are selected to be capable of colonizing a plant tissue susceptible to insect infestation or of being applied as dead or non-viable cells encapsulating the selected lectin.
Characteristics of preferred microbes include non-phytotoxicity; ease of introducing genetic C-50,243 -16-_17_ 21~78~

sequenceq, availability o~ expression systems, efficiency of expreqqion and stability of the insecticide in the host. Characteristics of microbes for encapsulating selected lectins include protective qualities for the protein/ such aY thick cell walls, and intracellular packaging or formation of inclusion bodies or desiccation re~istance; lack of mammalian toxicity, attractiveness to pests for ingestion; ease of killing and fixing without damage to the lectin; and the ability to be treated to prolong the activity of the lectin.
Many different types of environments exist on, ~ -around and within the plant. Plant colonizing ;
microorganisms exist in a wide variety of important -plantq. Because of the unique physiological makeup of these plants, and their surrounding soil environments, sometimes the~e colonizer3 are highly adapted and may be unique to an individual plant, or specific for particular location on the plant. In spite of the -20 harshness in these environments, a large number of -~-diverse microorganisms, bacterial, algal and fungal, are known to inhabit one, or many, of these domains. The sum total of these environments is known as the phytosphere.
Within the phytosphere, some environmental subdivi~ion~ include the phylloplane (the aerial surfaces of the plant)~ the rhizoplane (the surfaces of non-aerial structures, e.g. roots), the rhizosphere (the volume of soil surrounding the plant and that is under 3 its influence) as well as the endophytic domains (the areas interior to the plant, aerial and root strucutures). The great diversity of environmental conditions for microorganisms in the rhizoplane, phylloplane, etc., may introduce various niche specific consideration~ when selecting appropriate colonizers for C-50,243 -17--18~ 27~

utilization as recombinant technology delivery agent~.
Conver~ely, some genera (or sp~cies and ~trains) have evolutionally adapted for particular niche~ but can be found acro~3 a wide variety of plants.
These phytosphere organisms of interest may include many microorganisms, some microorganisms are member~ of the prokaryotes (bacteria), or members of the taxon of Protista (algae7 fungi or protozoa).
Microorganisms of interest are epiphytic or endophytic bacteria, e.g., the genera of Pseudomonas, Erwinia, Enterobacter, Serratia, Klebsiella, Xanthomonas, Xanthobacter, Methylobacterium, Clavibacter, Streptomyces ( or other Actinomycetales ), Azospir~llium , Bacillus , Beijerinckia , Rhizobium, Bradyrhizobium, Derxia, Flauobacterium, Gluconobacter, Rhodopseudomonas, Rhodobacter, Acetobacter, :
Arthrobacter, Azotobacter, Azomonas, Acaligenes; Acinetobacter, Agrobacterium, Leuconostoc, Methylophilus and Lactobacillus or apathogenic forms and ~trains of phytopathogenic bacteria; epiphytic or endophytic fungi, particulary yeast, e.g. Saccharomyces, Cryptococcus, Kluyvermyces, Sporobolomyces, Rhodotorula and Aureobasidium; versicular-arbuscular mycorrhial (VAM) fungi such as Glomus9 Gipaspora, Acaulospora and Scutellospora; other fungi such as 25 Trichoderma, Alternaria, Cladosporium, Eptcoccum, Ascochyta, Cephalosprium, Mycosphaerella, Stemphyllium, Ulocladi~m, Phialophora or Fusarium; or apathogenic strains of phytopathogenic fungi.
3 Of particular interest are bacterial species, e.g. Pseudomonas syringae, Pseudomonas fluorescens, Pseudomonas cepacia, Pse~domonas marginalis, Pseudomonas p~tida, Serratia marcescens, Enterobacter agglomerans, Acetobacter xylinum~ Agrobacterium tumefaciens, Rhodopseudomonas spheroides, Xanthomonas campestris, Xanthomonas maltophilia, ' ~
C-50,243 -18-'. ~

~1227~5 _19_ Clavibacter xyli, RhizobiL~m meliloti, Rhizob~um loti, Rhizo70ium legum~nosarum, Bradyrhizobium japonicum, Alcaligenes entrophus~ :
Alcaligenes denitrificans, Azosp~rillum brasilense, Azobacter uinlandii and Azo~octer chroococcum; plant colonizing species ~
such as Rhodotorula rubra, Rhodotorula marina, Rhodotorula ~ ;
aurantiaca, Cryptococcus aldibus~ Cryptococcus diffluens, :
Cryptococcus laurentii, Saccharomyces rosei, Saccharomyces ~:
pretoriensis, Saccharomyces cervisiae, Sporobolomyces roseus, Sporobolomyces odorus, Kluyueromyces ueronae and Aureobasidium :-: -0 pollulans; and other fungi such as Cladosporium ~ ~-sladosporioides, Glomus globiferum, Glomus derserticola, Glomus aggregatum and Trichoderma har2ianum.
Exemplary insect viruses include baculo~iruses that infect Heliothisvirescens (cotton bollworm), Orgyla pse~dotsugata (Douglas fir tussock moth), Lyman~riadispar (gyp~y moth), Autographicacalifornica (alPalfa looper), Neodiprionserti~er (European pine fly) and Laspeyresia pomonella (coddling moth), all of which have been registered and used as pesticides (see US 4,745,051 and EP 175 852).
The recombinant microbes and insect viruses may be formulated in a variety of ways. They may be employed in wettable powders, granules or dust~, or by mixing with various inert materials, such as inorganic mineralq (phyllosilicates, carbonate~, sulfates, phosphates, and the like) or botanical materials (powdered corncobs, rice hulls, walnut shells, and the 3 like). The formulations may include spreader-sticker adjuvants, stabilizing agents, other insecticidal additives, surfactants, and bacterial nutrients or other agents to enhance growth or stabilize bacterial cells.
Liquid formulations may be aqueous-ba~ed or non-aqueous and employed as foams, gels, suspensions, emulsifiable :~, C-50,243 -19-. :
':

~ 20 ~ 7 8 ~
.

concentrates, or the like. The ingredientq may include rheological agent~, surfactants, emulsi~iers, dispersants, or polymers.
Alternatively, t~e PNA lectin, amaranthin lectin or Con A lectin can be incorporated into the tissues of a ~usceptible plant so that in the course of infesting the plant the insect consumes in~ect-controlling amountq of the incorporated lectin. One method of doing this is to incorporate the lectin in a 0 non-phytotoxic vehicle which is adapted for systemic adminiqtration to the susceptible plants.
The present invention further contemplates (a) culturing cells or tissues from at least one plant susceptible to infestation by Western corn rootworm or Northern corn rootworm; (b) introducing into the cells of the cell or ti~sue culture a vector containing a structural gene encoding a PNA lectin, amaranthin lectin or Con A lectin, operably linked to plant regulatory sequences which cause expression of the gene in the cells, and (c) regenerating insect-resistant whole plants from the cell or tissues culture.

The vector chosen to transform the plant cells should be selected to cause sufficient expression to -~
result in the production of an insect controlling amount of protein. Exemplary vectors include pDAB219~-No~ and -~
pDAB303-Not (see U.S. Application Serial Number 07/936,164. filed on August 27, 1992) and pDAB219i and pDAB303 (see U.S. Application Serial Number 07/936,163, filed on August 27, 1992).
At the 5' end of the structural gene will be provided a constitutive promoter active in plant cells, ~-~

:' ' -C-50,243 -20-~~~ -21- 2122 l85 ~ ~
. -,. ~ ,~
e.g. 9 nopaline syntha~e, octopine synthase and ~annopine synthase promoters from the tumor-inducing plasmids of Agrobac~rium tumefaciens, or the CaMV 19S and 35S promoters (JP 63287485) or the ubiquitin promoter or the rice actin promoter (W0 9109948). It may also be pre~erable to provide localized expre~sion of or over production of the PNA lectin~ amaranthin lectin or Con A lectin.
Examples of tissue specific promoters, for the control of an insect selected from Western corn rootworm or Northern corn rootworm, include the root specific promoters such as maize metallothionein (EP 452269 to De Framond), the rape (Brassica napus L.) extensin gene promoter (W0/9113992 to Croy etal.) and the maize root-preferential cationic peroxidase promoter (U.S.
Application Serial Number 07/956,704 to Folkerts etal.;
DowElanco). At the 3' terminus of the structural gene will be provided a termination sequence which is ~unctional in plants, e.g., bacterial, opine, viral, and plant genes. Suitable transcript termination regions include termination regions known to those skilled in the art, such as the nos 3', tmL 3', or acp 3'.
In order to optimize the transcriptional and translational ePficiency of such systems, it is possible to examine the frequency o~ codon usage and determine which codons are, in essence, preferred within the transcriptional and translational systems normally present in that plant. Using such preferred usage codons, it is possible to construct a protein coding sequence which may result in a significantly enhanced level of transcriptional and translational efficiency of the genes encoding PNA lectin, amaranthin lectin or Con A lectin compared to what would be achieved by taking a genomic coding sequence (~or a discussion of making a ~' ''' ~

C-50,243 -21~

-22- 2l22 78 5 gene codon-biased for expression in plants see generally, EP O 359 472 to Lubrizol Genetic3 Inc;
EP O 385 962 to Monsanto Company; and WO 91/16432 to Plant Geneticc~ Systems N.V.). An exemplary DNA sequence codon-biased for expression in maize is de3cribed in Example 2, below, and set forth in Figure 1.
The appropriate procedure to produce mature transgenic plants may be chosen in accordance with well-known techniques. Once whole plant3 have been obtained, they can be sexually or clonally reproduced in such a manner that at least one copy of the sequence is pre~ent in the cells of the progeny of the reproduction. Such procedures may be chosen in accordance with the plant species used.
Mature plant~, grown from the transformed plant cells, may be selfed to produce an inbred plant. In hybrid plant~, typically one parent may be transformed and the other parent may be the wild type. The parent will be crossed to form first generation hybrids (F1), which are selfed to produced second generation hybrids ~F2). F2 hybrids with the genetic makeup of ~ -lectin/lectin are chosen and selfed to produce inbred plants.
Conventional plant breeding method~ can be used to transfer a~,ong different plants the structural gene of PNA lectin, amaranthin lectin or Con A lectin via -crossing and backcrossing. Such methods comprise the further steps of (a) sexually crossing the insect-' resistant plant with a plant from the insect-susceptible variety; (b) recovering reproductive material from the progeny of the cross; and (c) growing insect-resistant plants from the reproductive material. Where desirable I

, C-50,243 -22-: :. . .

-23- 2~ 2~7~

or necesqary, the agronomic characteristic of the susceptible variety can be substantially preserved by expanding thi~ method to include the further ~teps of repetitively (d) backcrosqing the in~ect-resistant progeny with insect-susceptible plants from the susceptible variety; and (e) selecting for expres~ion of insect resistance (or an as~ociated marker gene) among the progeny?? of the backcross, until the desired percentage of the characteri~tics of the su~ceptible variety are present in the progeny along with the gene imparting insect resistance. Subsequently, the inbreds according to this invention may be crossed with another inbred line to produce the hybrid.
The present invention further contemplates using, with the PNA lectin, amaranthin lectin or Con A
lectin, adjuvants, chemical or biological additives in an effort to expand the spectrum of target pests, to extend the duration of effectiveness or to help stabilize the agricultural composition of the selected lectin. Exemplary of such potentiators include other lectins, amphipathic proteins or proteinase inhibitors.
While clearly directed to protecting maize plants from infestation by insects selected from Western corn rootworm or Northern corn rootworm, the present invention contemplates protecting any plant of a taxon, if it is found to be susceptible to infestation and damage by insects selected from Western corn rootworm or 3 Northern corn rootworm. -~
Examples The present invention is illustrated in further detail by the following examples. The examples are for C-50,243 -23~

-24- 2l~27 8~

the purposss oP illu~tration only, and are not to be construed as limiting the scope of the present invention, All part~ and percentages are by weight unles3 otherwise specifically noted.
In carrying out the following example~, all DNA
manipulations were done according to standard procedures, unless otherwise indicated. See Sambrook et al. (1989), Molecular Cloning: A LaboratorY Manual, published by Cold Spring Harbor Laboratory (U.S.A.) ExamPle 1 Second instar western corn rootworm (WCR) larvae were used to screen lectins for growth inhibitory effects. It was necessary to use second inYtar WCR
because an artificial diet which will support neonate growth of this species is not available. In these bioassays 0.25 ml of diet was placed in each well of a 24-well microtitre plate and the lectin (dissolved in water) was added to each well a~ a 30 ~l aliquot. This aliquot was allowed to dry and individual larvae were placed in each well. The WCR larvae were weighed at the beginning of the assay (2,5-3.0 mg) and allowed to feed for 3.5 days prior to a second determination of weight gainO The screening level of lectin was 2.0 mg/g diet and significant growth inhibition was arbitrarily set at 40 percent.

C-50,243 -24--25- 212278 ~

Table 1: Effect of Lectins from a Variety of Plant Source on Growth of Western Corn Rootworm Larvae :
Growth ~ :
Inhibition* -Plant Source WCR
Amaranthuscaudatus 42, (n= 29) ~ ~ ~c~- -Arach~shypogaea 75, ~n= 121) Canaualia ensiform~s 40, (n= 157) J
*Growth Inhibition was defined as percent inhibition at 0.2~ dietary lectin -': .. ~: :

Example 2 :
~:
The amino acid sequence for the subunit which makes up the PNA tetramer was obtained from the :::
literature (see Young etal. (1991), supra). This sequence was then reverse translated using preferred maize codon .
usage (see ~ada etal. (1990) Nucl. Acids Res., 18:2367) : . ;
and is set forth in Figure 1.

C-50,243 -25- :

.h'~ ~
'.
~: .

-26- 212~5 The strategy employed to construct the gene is substantially as set forth in United States Patent 5,023,171 to Ho and ~orton (Mayo Foundation ~or Medical Education and Research). Polymerase chain reaction (PCR) techniques are used to build the gene in two parts and then attach the t~o portions. The S' and 3' portions of the gene are synthesized in sequential PCR
reactions from overlappinq oligonucleotide primers.
These oligonucleotides are designed to produce 20 base pair overlaps during each PCR reaction. The two halves of the gene have at least a 30 base pair overlap to .
facilitate the attachment of the two parts using PCR.
The gene is constructed so as to permit a maize expressible ER signal peptide sequence to be added, in addition to stop codons at the 3' terminus and appropriate restriction enzyme site~ at the 3' (Sac 1 and 5' (Nco 1) termini to facilitate insertion into pDAB303-Not (for a description o~ pDAB303-Not, see United States Application 07/936,164 to Walshetal., filed August 27, 1992; DowElanco).
Although the invention has been described in considerable detail, with reference to certain preferred embodiments thereof, it will be understood that variations and modi~ications can be affected within the spirit and scope of the invention as described above and as defined in the appended claims.
3o , C-50,243 -26-

Claims (16)

1. A method of protecting a plant or a part thereof against insect infestation by insects selected from Western corn rootworm (Diabrotica virgifera virgifera)or Northern corn rootworm (D. barberi), said method comprising presenting to a loci wherein said insect(s) is to be controlled or combated with an insect controlling amount of PNA lectin, amaranthin lectin, Con A lectin, or an effectively homologous derivative thereof.

2. The method of Claim 1 wherein the Western corn rootworm or Northern corn rootworm is controlled by administering enterally an insect controlling amount of the lectin.

3. The method according to Claim 2 wherein the lectin is administered enterally by incorporating the lectin or combination thereof in the diet of the insect.

4. The method according to Claim 3 wherein the diet of the larvae is an insect controlling composition comprising the lectin.

5. The method of Claim 1, wherein the lectin is applied to maize plants.

6. The method of Claim 1, wherein the corn rootworm is a Western corn rootworm.

7. A transgenic maize plant and its sexual progeny resistant to attack by an insect selected from Western corn rootworm or Northern corn rootworm, said transgenic maize plant expressing an insect controlling amount of PNA lectin, Con A lectin, or an effectively homologous derivative thereof.

8. The transgenic maize plant and its sexual progeny of Claim 7, wherein the plant comprises a DNA
sequence stably incorporated into its genome downstream of a promoter sequence active in the plant to cause expression of the lectin sequence at levels which provide an insect controlling amount of the lectin, said DNA sequence encoding PNA lectin, Con A lectin, or an effectively homologous derivative thereof.

9. The transgenic maize plant and its sexual progeny of Claim 8, wherein the DNA sequence encodes PNA
lectin, Con A lectin, or an effectively homologous derivative thereof.

10. The transgenic maize plant of Claim 7, wherein the DNA sequence has a nucleotide sequence as set forth in Figure 1.

11. An agricultural composition containing a carrier and an insect controlling or combating amount of PNA lectin, amaranthin lectin or Con A lectin, or effectively homologous derivatives thereof.

12. The agricultural composition of Claim 11, wherein the lectin is a protein having the amino acid sequence set forth in Figure 1, or an effectively homologous derivative thereof.

13. A biologically functional expression vehicle containing a promoter effective to promote expression of a downstream coding sequence in plant cells, a DNA coding region encoding PNA lectin, amaranthin lectin, Con A lectin, or an effectively homologous derivative thereof and a termination sequence effective to terminate transcription or translation of the genetic construction product in plant cells, the genetic construction being effective to express in the cells of the plant an insect controlling amount of PNA
lectin or Con A lectin.

14. The biologically functional expression vehicle of Claim 13, wherein the DNA sequence encodes a lectin selected from PNA lectin, Con A lectin, or an effectively homologous derivative thereof.

15. The biologically functional expression vehicle of Claim 13, wherein the DNA sequence has a nucleotide sequence as set forth in Figure 1.

16. The biologically functional expression vehicle of Claim 13, wherein the expression vehicle is pDAB303-Not.