WO2001021821A2

WO2001021821A2 - Insect-resistant rice plants

Info

Publication number: WO2001021821A2
Application number: PCT/EP2000/008877
Authority: WO
Inventors: Frank Michiels; Stefan Jansens; Harish Kumar; David Lobo; Jan Samson
Original assignee: Aventis Cropscience N.V.
Priority date: 1999-09-17
Filing date: 2000-09-07
Publication date: 2001-03-29
Also published as: WO2001021821A3; AU7518100A; CN1375009A

Abstract

Novel rice plants, as well as methods for controlling Lepidopteran rice insects, particularly rice leaffolders, rice stemborers, or rice skippers, rice cutworms, rice armyworms, rice caseworms, are provided by means of application of a Cry9C protein to such rice insects. In a preferred embodiment of the invention such application is by expression of a Cry9C protein in rice plants.

Description

INSECT-RESISTANT RICE PLANTS

INTRODUCTION

This invention relates to a method for controlling rice insect pests, particularly Lepidopteran rice stemborers, rice skippers, rice cutworms, rice armyworms, rice caseworms or rice leaffolders, preferably an insect selected from the group consisting of: Chilo suppressalis, Scirpophaga incertulas, Sesamia inferens, Cnaphalocrocis medinalis, Hereitogramma licarisalis, Naranga aenescens, Mycalesis gotama, Marasmia patnalis, Marasmia exigua, Marasmia ruralis, Nymphula depunctalis, Scirpophaga innotata, Spodoptera litura, Chilo polychrysus, Rupela albinella, Diatraea saccharalis, Spodoptera frugiperda, Mythimna unipuncta, Chilo zacconius and Parnara guttata, most particularly Chilo suppressalis, Scirpophaga incertulas, Marasmia patnalis and Cnaphalocrocis medinalis, which method comprises applying to an area or plant to be protected, a Cry9C protein as defined herein. In a preferred embodiment of this invention, such Cry9C protein is expressed throughout a rice plant by stably incorporating a DNA encoding a Cry9C protein in the rice genome.

This invention also relates to rice plants with resistance to rice pests by expression of a Cry9C protein and to an insecticide composition that is active against rice pests, particularly Lepidopteran rice stemborers, rice skippers, rice cutworms, rice caseworms or rice leaffolders, preferably an insect selected from the group consisting of: Chilo suppressalis, Scirpophaga incertulas, Sesamia inferens, Cnaphalocrocis medinalis, Hereitogramma licarisalis, Naranga aenescens, Mycalesis gotama, Marasmia patnalis, Marasmia exigua, Marasmia ruralis, Nymphula depunctalis, Scirpophaga innotata, Spodoptera litura, Chilo polychrysus, Rupela albinella, Diatraea saccharalis, Spodoptera frugiperda, Mythimna unipuncta, Chilo zacconius and Parnara guttata, most particularly Chilo suppressalis, Marasmia patnalis and Cnaphalocrocis medinalis and Scirpophaga incertulas, and that comprises the Cry9C crystals, crystal protein or active fragments thereof as an active ingredient.

This invention further relates to the use of a DNA sequence which encodes an insecticidal Cry9C protein as defined herein for obtaining insect resistance in rice plants.

BACKGROUND OF THE INVENTION

(i) Field of the Invention

Bacillus thuringiensis ("Bt") is a Gram-positive bacterium which produces crystals upon sporulation. The crystals are composed of proteins which have been shown to be toxic against insect larvae. These crystal proteins and their corresponding genes have been classified based on their amino acid sequence (Crickmore et al., 1998). The Cry9C protein is an insecticidal protein originally found in Bacillus thuringiensis (Lambert et al., 1996). Because of its interesting activity against corn insect pests, transgenic corn plants expressing a Cry9C protein have been successfully used to protect corn fields from insect infestation (Jansens et al., 1997). The Cry9C protein and variants thereof and their expression in several plants has been described in PCT patent publications WO 94/05771 , WO 94/24264, WO 99/00407 and in US Patents 5,885,571 and 5,861 ,543 (the contents of which are incorporated herein by reference). Insect pests are important constraints for rice production and occur in all rice growing environments. Insects reduce rice yields substantially and losses due to insects in Asia (excluding China) have been reported to be about 31 .5 % (Heinrichs, 1994). (ii) Description of Related Art

Insect resistance in several types of rice to some of the damaging Lepidopteran rice insect pests has been obtained by expressing genes encoding insecticidal Cryl Ab or Cryl Ac proteins of Bacillus thuringiensis (e.g., Fujimoto et al., 1993; Wunn et al., 1996; Wu et al. 1997; Ghareyazie et al., 1997; Nayak et al., 1997; and Cheng et al., 1998), a plant lectin (Rao et al., 1998) or protease inhibitors (Xu et al., 1996; Duan et al., 1996) in rice plants. Toxicity of isolated Bacillus thuringiensis crystal proteins to some Lepidopteran rice insect pests has been evaluated for Cryl Aa, Cryl Ab, Cryl Ac, Cryl B, Cryl C, Cryl D, Cryl E, Cryl F, Cryl G, and Cry2A proteins (Karim et al., 1997; Lee et al., 1997b).

To prevent or delay resistance development in rice insect pests constantly exposed to plants expressing a Bt protein, it is preferred to have different sources of resistance available in rice plants at high doses, and preferably to combine expression of resistance genes with a different mode of action in a single plant. This invention provides new rice plants expressing a Cry9C protein which provides a high dose effect on relevant Lepidopteran rice insect pest, allowing the development of new commercial rice plants which are better protected against insect attack and which at the same time prevent or delay the development of resistance in such insects.

At the General Meeting of The International Program on Rice Biotechnology, September 20-24 (1999) in Phuket, Thailand, Cohen et al. Presented an optimization of the high dose plus refuge resistance management strategy for Bt rice (see abstract p. 25).

SUMMARY OF THE INVENTION

In accordance with this invention, rice plants resistant to 1 ) Lepidopteran rice stemborers, rice skippers, rice cutworms, rice armyworms, rice caseworms and/or rice leaffolders, 2) an insect selected from the group consisting of: Chilo suppressalis, Scirpophaga incertulas, Sesamia inferens, Cnaphalocrocis medinalis, Hereitogramma licarisalis, Naranga aenescens, Mycalesis gotama, Marasmia patnalis, Marasmia exigua, Marasmia ruralis, Nymphula depunctalis, Scirpophaga innotata, Spodoptera litura, Chilo polychrysus, Rupela albinella, Diatraea saccharalis, Spodoptera frugiperda, Mythimna unipuncta, Chilo zacconius and Parnara guttata, or 3) an insect selected from the group consisting of: Chilo suppressalis, Scirpophaga incertulas, Marasmia patnalis and Cnaphalocrocis medinalis are provided. Such rice plants contain, stably integrated in their genome, a plant-expressible DNA sequence encoding a protein comprising the amino acid sequence from amino acid position 44 to amino acid position 658 of SEQ ID NO: 2, or a DNA sequence encoding a protein comprising the amino acid sequence of SEQ ID NO: 4. Further in accordance with this invention, seeds, grain and processed grain of these rice plants are provided, comprising a DNA sequence encoding a Cry9C protein.

In another embodiment of the invention, these rice plants also comprise in their genome a DNA encoding a protein selected from the group consisting of: Cryl B, Cryl C, Cryl D, Cryl E, Cryl Aa, Cryl Ab, Cryl Ac, Cryl I, Cryl J, Cry2A, Cry6B, Cry3A, a protease inhibitor, a cowpea trypsin inhibitor, protease inhibitor II, cystatin, GNA lectin, an insecticidal protein of Xhenorhabdus or Photorhabdus spp., and a protein conferring resistance to glufosinate ammonium.

Further provided, in accordance with this invention, is a process for obtaining a rice plant and progeny thereof resistant to 1 ) Lepidopteran rice stemborers, rice skippers, rice cutworms, rice armyworms, rice caseworms or rice leaffolders, 2) an insect selected from the group consisting of: Chilo suppressalis, Scirpophaga incertulas, Sesamia inferens, Cnaphalocrocis medinalis, Hereitogramma licarisalis, Naranga aenescens, Mycalesis gotama, Marasmia patnalis, Marasmia exigua, Marasmia ruralis, Nymphula depunctalis, Scirpophaga innotata, Spodoptera litura, Chilo polychrysus, Rupela albinella, Diatraea saccharalis, Spodoptera frugiperda, Mythimna unipuncta, Chilo zacconius, and Parnara guttata, or 3) an insect selected from the group consisting of: Chilo suppressalis, Scirpophaga incertulas, Marasmia patnalis and Cnaphalocrocis medinalis, comprising transforming a rice plant with a plant-expressible DNA sequence encoding a protein comprising the amino acid sequence from amino acid position 44 to amino acid position 658 of SEQ ID NO: 2, or a DNA sequence encoding a protein comprising the amino acid sequence of SEQ ID NO: 4.

Also provided in this invention is a process for producing rice plants and reproduction material thereof, such as seeds, which are resistant to 1 ) Lepidopteran rice stemborers, rice skippers, rice cutworms, rice armyworms, rice caseworms or rice leaffolders, 2) an insect selected from the group consisting of: Chilo suppressalis, Scirpophaga incertulas, Sesamia inferens, Cnaphalocrocis medinalis, Hereitogramma licarisalis, Naranga aenescens, Mycalesis gotama, Marasmia patnalis, Marasmia exigua, Marasmia ruralis, Nymphula depunctalis, Scirpophaga innotata, Spodoptera litura, Chilo polychrysus, Rupela albinella, Diatraea saccharalis, Spodoptera frugiperda, Mythimna unipuncta, Chilo zacconius, and Parnara guttata, or 3) an insect selected from the group consisting of: Chilo suppressalis, Scirpophaga incertulas, Marasmia patnalis and Cnaphalocrocis medinalis, comprising: transforming rice plants with a plant-expressible DNA sequence encoding a protein comprising the amino acid sequence from amino acid position 44 to amino acid position 658 of SEQ ID NO: 2, or a DNA sequence encoding a protein comprising the amino acid sequence of SEQ ID NO: 4.

In another embodiment, a process is provided for controlling 1 ) Lepidopteran rice stemborers, rice skippers, rice cutworms, rice armyworms, rice caseworms or rice leaffolders, 2) an insect selected from the group consisting of: Chilo suppressalis, Scirpophaga incertulas, Sesamia inferens, Cnaphalocrocis medinalis, Hereitogramma licarisalis, Naranga aenescens, Mycalesis gotama, Marasmia patnalis, Marasmia exigua, Marasmia ruralis, Nymphula depunctalis, Scirpophaga innotata, Spodoptera litura, Chilo polychrysus, Rupela albinella, Chilo zacconius and Parnara guttata, or 3) an insect selected from the group consisting of: Chilo suppressalis, Marasmia patnalis, Scirpophaga incertulas and Cnaphalocrocis medinalis, comprising contacting said insect with an insecticidal amount of a protein comprising the amino acid sequence from amino acid position 44 to amino acid position 658 of SEQ ID NO: 2, or a DNA sequence encoding a protein comprising the amino acid sequence of SEQ ID NO: 4, as well as such a process wherein the contacting comprises the steps of transforming rice plants with a DNA sequence encoding said protein and cultivating said plants or progeny thereof containing said DNA sequence in a field. Also, the invention provides plants, seeds and grain obtained by any one of the above processes.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

In this invention, new transgenic rice plants, particularly Oryza sp., preferably Oryza sativa, are provided which are shown to be suitable for preventing damage from several Lepidopteran rice insects, particularly Lepidopteran rice stemborers, rice skippers, rice cutworms, rice armyworms, rice caseworms or rice leaffolders, preferably Chilo suppressalis, Scirpophaga incertulas, Sesamia inferens, Cnaphalocrocis medinalis, Hereitogramma licarisalis, Naranga aenescens, Mycalesis gotama, Marasmia patnalis, Marasmia exigua, Marasmia ruralis, Nymphula depunctalis, Scirpophaga innotata, Spodoptera litura, Chilo polychrysus, Rupela albinella, Chilo zacconius and Parnara guttata, most particularly Chilo suppressalis, most preferably Chilo species, Marasmia species, Cnaphalocrocis species and Scirpophaga species insects. This has been accomplished by expressing a DNA encoding a Cry9C protein in rice plants. Excellent control of Lepidopteran rice insects, particularly Lepidopteran rice stemborers, rice skippers, rice cutworms, rice armyworms, rice caseworms or rice leaffolders, more particularly Chilo suppressalis, Scirpophaga incertulas, Spodoptera litura, Marasmia patnalis, Marasmia exigua, Marasmia ruralis, Nymphula depunctalis, Scirpophaga innotata, Spodoptera litura, Chilo polychrysus, Rupela albinella, Diatraea saccharalis, Spodoptera frugiperda, Mythimna unipuncta, Chilo zacconius, and Cnaphalocrosis medinalis, is obtained with the plants of the invention. The Cry9C protein of this invention can also be used to control other relevant insect pests, e.g. the rice moth Corcyra cepalonica and others Lepidopteran storage pests.

In a preferred embodiment of this invention, the Cry9C protein is used to control rice insect pests, particularly Chilo suppressalis, Scirpophaga incertulas, Sesamia inferens, Cnaphalocrocis medinalis, Marasmia patnalis, , Marasmia exigua, Marasmia ruralis, Nymphula depunctalis, Scirpophaga innotata, Spodoptera litura, Chilo polychrysus, Rupela albinella, Diatraea saccharalis, Spodoptera frugiperda, Mythimna unipuncta, Chilo zacconius, Hereitogramma licarisalis, Naranga aenescens, Mycalesis gotama, Parnara guttata, preferably Chilo suppressalis, Scirpophaga incertulas, Marasmia patnalis and Cnaphalocrocis medinalis. Preferred targets to control with the Cry9C protein are Lepidopteran rice insects belonging to the group of rice stemborers, rice skippers, rice cutworms, rice armyworms, rice caseworms and rice leaffolders.

The Cry9C protein can be isolated in a conventional manner from several Bt strains or can be recombinantly expressed, as described in PCT patent publications WO 94/05771 , WO 94/24264, WO 99/00407 and in US Patents 5,885,571 and 5,861 ,543.

The term "Cry9C protein", as used herein, refers to an insecticidal protein comprising the amino acid sequence of SEQ ID NO: 2 from amino acid position 44 to amino acid position 658 or an insecticidal protein comprising the amino acid sequence of SEQ ID NO: 4 from amino acid position 1 to amino acid position 625 and variants thereof retaining insecticidal activity such as the protease-resistant Alanine variant disclosed in PCT patent publication WO 94/24264. This term includes the crystal protein, protoxin or insecticidally effective forms thereof such as the toxin form obtained by protease-digestion from the protoxin or the protein with the amino acid sequence of SEQ ID NO: 2 from an amino acid position between amino acid position 1 and amino acid position 44 and an amino acid position between amino acid position 658 and amino acid position 666, as well as fusion proteins or protease-resistant variants thereof. It is known that some amino acids can be substituted by other equivalent amino acids in a Cry9C protein while still retaining all or some insecticidal activity, see, e.g., PCT patent application WO 99/00407, incorporated herein by reference. These proteins are included within the scope of the term "Cry9C protein". The most preferred form of a Cry9C protein, in accordance with this invention, is a protein comprising the amino acid sequence from amino acid position 44 to amino acid position 658 of SEQ ID NO: 2, or a protein comprising the amino acid sequence of SEQ ID NO: 4.

The "cry9C DNA", as used herein, refers to any DNA sequence encoding a Cry9C protein and includes naturally occurring, recombinant, semi-synthetic or synthetic DNA sequences and can include regulatory or other regions not coding for an amino acid sequence such as introns, leader or trailer sequences, promoters and 3' transcription termination and polyadenylation sequences. The "cry9C coding region" refers to any nucleotide sequence which starts with a start codon and ends with a stop codon and encodes a Cry9C protein. Because of the degeneracy of the genetic code, some amino acid codons can be replaced with others without changing the amino acid sequence of the protein. An example of a cry9C DNA in accordance with the invention is the DNA of Sequence ID No. 3. The "chimeric cry9C gene", as used herein, refers to a cry9C DNA consisting of a cry9C coding region flanked by regulatory elements, such as a promoter and a transcription termination and polyadenylation region which allow expression of a Cry9C protein in the cells of a plant, particularly a rice plant. For any other Bacillus thuringiensis crystal protein or a toxic fragment thereof used herein, the "Cry" nomenclature is used, as described by Crickmore et al. (1998). Whenever only one number and one character is used after "Cry", e.g. "Cryl B", this refers to all related sequences starting with the listed number and character, the protoxin form as well as any insecticidally effective fragments thereof such as the toxin form obtained after protease treatment (e.g., "Cryl B" includes all Cryl B forms such as "Cryl Ba1 " and "Cryl Bb1 ").

Plants "resistant to" or with "resistance against" insects, or "resistant to" or with "resistance against" insect infestation or attack, as used herein, refers to plants which, by action of man, particularly by transformation with a DNA in accordance with this invention, or by spraying with a composition in accordance with this invention, exhibit a significantly increased control of insects which otherwise damage plants of that species. This includes significantly increased feeding inhibition, growth inhibition or mortality of insects feeding on the plants. In a preferred embodiment of the invention, resistance against insects of the transgenic plants of the invention refers to complete protection to the insects which can be determined by the minor damage caused by the insects to such plants (e.g., for stemborers, no tunneling in the rice stem) or refers to the achievement of substantially the same yields as would have been obtained under the same conditions but in the absence of attack of that insect pest. In the most preferred embodiment, resistance against insects of a rice plant in accordance with the invention refers to the killing of at least 90 % of the insects, preferably at least 95 % of the insects, which attempt to feed on the Cry9C rice plants of the invention, this preferably within 10 days, most preferably within 5 days. A "plant-expressible DNA sequence", in accordance with this invention, is a DNA sequence capable of expressing in a plant a gene product, preferably a protein. In the most preferred embodiment, this is a DNA sequence comprising regulatory elements allowing proper transcription and translation of a desired protein from a coding sequence introduced in the plant's genome. Typically, a plant-expressible DNA sequence in accordance with this invention comprises a promoter region capable of transcription in plant cells, particularly rice. A plant-expressible DNA sequence can also contain other elements such as a 3' transcription termination and polyadenylation sequence or introns. The term "genome" of a plant or plant cell, as used herein, refers to the totality of genetic material present in a plant or plant cell, and includes but is not limited to mitochondrial, chloroplast and nuclear DNA. DNA "stably integrated" in the genome, as used herein, refers to a DNA which is integrated in such a manner that it can be passed on to the progeny of a plant, preferably by integration of such DNA in the genome, particularly nuclear DNA, of a plant. "Progeny" of a plant, as used herein, refers to further generations of a plant. This includes seeds, offspring plants and cells, tissues or whole plants obtainable from a plant or taken from a plant to obtain more plants or further generations with the same or different genetic material, including vegetative propagation. "Processed grain", as used herein, refers to grain which has been treated using one or several processes, particularly to grain processed to be used as feed or food. Processing of grain includes but is not limited to polishing, milling, parboiling, dehusking and the like. Processed and unprocessed grain derived from the Cry9C rice plants of the invention, and comprising the cry9C chimeric gene of the invention, is included within the scope of the invention.

In one embodiment of this invention, the Cry9C protein is combined with other insecticidal proteins for providing better protection against relevant rice insect pests and for preventing or delaying insect resistance development. Preferred proteins are selected from the group consisting of: Cryl B, Cryl C, Cryl D, Cryl E, Cryl Aa, Cryl Ab, Cryl Ac, Cryl I, Cryl J, Cry2A, Cry6B, Cry3A, protease inhibitors such as cowpea trypsin inhibitor (CpTI, Xu et al., 1996), protease inhibitor II (pinll, Duan et al., 1996) or cystatin (Irie et al., 1996), the GNA lectin (Galanthus nivalis agglutinin, Rao et al., 1998), an insecticidal protein of Xhenorhabdus or Photorhabdus spp. as described in Bowen et al. (1998) and ffrench-Constant and Bowen. (1999), PCT patent publications WO 98/08388, WO 99/03328, WO 95/00647, WO 97/1 7432, WO 98/05212, or WO 98/08932, or any Bacillus thuringiensis or Bacillus cereus-derived protein with insecticidal activity to rice insect pests, preferably rice stemborers, rice skippers, rice cutworms, rice armyworms, rice caseworms or rice leaffolders, particularly to Chilo spp.,

Chaphalocrocis spp., Marasmia spp. and Scirpophaga spp., more particularly Chilo suppressalis, Marasmia patnalis, Chaphalocrocis medinalis, and Scirpophaga incertulas. In a preferred embodiment, the Cry9C protein is combined with at least one of the proteins selected from the Cryl Ab, Cryl B, Cry2A and Cryl C (preferably Cryl Ca4) proteins or active fragments thereof, particularly by combined expression in a transgenic rice plant.

Also, for selection of transgenic plant cells and/or for providing herbicide resistance in plants in the field, the use of a DNA sequence encoding a protein conferring resistance to glufosinate ammonium is preferred so that the plant cell or plant containing it can grow normally in otherwise toxic concentrations of this molecule, particularly a phosphinothricin acetyl transferase from Streptomyces hygroscopicus or viridochromogenes, as described in PCT patent publication WO 87/05629, US patent 5,561 ,236, and US patent 5,276,268. In order to express the cry9C DNA in E. coli, in other bacteria or in plants, suitable restriction sites can be introduced, flanking the DNA. This can be done by site- directed mutagenesis, using well-known procedures (Stanssens et al., 1989; White et al., 1989).

Furthermore, since the Cry9C protein can bind to a receptor different from the Cryl A type Bt proteins in insect gut membranes, it is useful to cross transgenic rice plants expressing the Cry9C protein with transgenic rice plants expressing a protein with a different binding site in the target insect compared to the Cry9C protein, i.e., a Cryl A or Cry2A protein. Similarly, rice can be transformed with the cry9C DNA and a DNA encoding a protein with a different binding site in the target insect compared to the Cry9C protein, i.e., a cryl A DNA such as a DNA encoding a truncated insecticidally-effective Cryl Ab5 protein, so as to obtain a transgenic rice plant or progeny thereof expressing these two insect control proteins, or a DNA encoding a Cry2Aa or Cry2Ab protein. A DNA encoding a cryl A protein can be selected from any of the DNA sequences which have already been successfully expressed in plants, particularly in rice or corn, and encoding any of the proteins listed as Cryl A by Crickmore et al. (1998 (see cited references or database accession numbers for full sequences)), including but not limited to insecticidal fragments of such proteins, preferably such DNA sequences encode a Cryl Ab or Cryl Ac protein, preferably the Cryl Ab5 protein of Hόfte et al. (1986) or an insecticidal protein fragment thereof. Several strategies for achieving combined expression of at least two Bt toxins in a plant have been reported in US Patent 5,866,784, and are included in the scope of this invention. The cry9C DNA, preferably the cry9C chimeric gene, can be stably inserted in a conventional manner into the nuclear genome of a rice cell, and the so-transformed cell can be regenerated in a conventional manner to produce a transformed rice plant and progeny (seed and further generations) thereof that is insect-resistant. In this regard, a disarmed Ti-plasmid, containing the insecticidally effective cry9C gene part, in Agrobacterium tumefaciens can be used to transform the rice cell, and thereafter, a transformed rice plant can be regenerated from the transformed plant cell using the procedures described, for example, in PCT patent publication WO 92/09696. Preferred tissues for transformation with Agrobacterium include but are not limited to mature seed-derived callus, immature embryo-derived callus and immature embryos. These tissues can be wounded prior to co-cultivation with Agrobacterium, and can be pre-induced with acetosyringone or other plant phenolic compounds as described in PCT patent publication WO 98/3721 2. Preferred Ti-plasmid vectors each contain the cry9C chimeric gene between the border sequences, or at least located to the left of the right border sequence, of the T-DNA of the Ti-plasmid. Of course, other types of vectors can be used to transform plant cells or plants, using procedures such as direct gene transfer (as described, for example in EP 0,233,247), pollen mediated transformation (as described, for example in EP 0,270,356, PCT publication WO 85/01 856, and US Patent 4,684,61 1 ), plant RNA virus-mediated transformation (as described, for example in EP 0,067,553 and US Patent 4,407,956), liposome- mediated transformation (as described, for example in US Patent 4,536,475), and other methods such as the rice transformation method of Shimamoto et al. (1989) and Datta et al. (1990) and the method for transforming monocots generally, as described in PCT publications WO 93/21 335 and WO 92/09696. Any known procedures for transforming rice plants are suitable for obtaining the transgenic rice plants in accordance with this invention.

The resulting transformed rice plant can be used in a conventional plant breeding scheme to produce more transformed rice plants with the same characteristics or to introduce the cry9C DNA in other rice varieties or in related plant species. Preferred rice plants include plants from the Oryza species, particularly Oryza sativa, preferably japonica, indica or javanica rice, whether soil, water, upland, rainfed shallow, deep water, floating or irrigated rice. Seeds, which are obtained from the transformed rice plants, contain the cry9C DNA as a stable genomic insert, either in the nuclear, mitochondrial or chloroplast genome. Cells of the transformed rice plant can be cultured in a conventional manner to produce the insecticidally effective Cry9C protein.

The cry9C DNA is inserted in the rice cell genome so that the inserted DNA is downstream (i.e., 3¹) of, and under the control of, a promoter which can direct the expression of the DNA in the plant cell. This is preferably accomplished by inserting the cry9C chimeric gene in the plant cell genome. Preferred promoters include: the strong constitutive 35S promoters (the "35S promoters") of the cauliflower mosaic virus of isolates CM 1841 (Gardner et al., 1981 ), CabbB-S (Franck et al., 1980) and CabbB-JI (Hull and Howell, 1987); promoters from the ubiquitin family (e.g., the maize ubiquitin promoter of Christensen et al., 1992, see also Cornejo et al., 1993), the gos2 promoter (de Pater et al., 1992), the emu promoter (Last et al., 1990), rice actin promoters such as the promoter described by Zhang et al. (1991 ), the TR1 ' promoter and the TR2¹ promoter (the "TR1 ' promoter" and "TR2¹ promoter", respectively) which drive the expression of the 1 ' and 2' genes, respectively, of the T-DNA (Velten et al., 1984). Alternatively, a promoter can be utilized which is not constitutive but rather is specific for one or more tissues or organs of the plant (e.g., leaves, stem, pith and/or root tissue) whereby the inserted cry9C gene part is expressed only in cells of the specific tissue(s) or organ(s). For example, the insecticidally effective cry9C gene part could be selectively expressed in rice leaves and stem or could be expressed by a promoter not active in pollen. Other promoters include light-inducible promoters such as the promoter of the rice ribulose-1 ,5-bisphosphate carboxylase small subunit gene or of another plant such as pea as disclosed in US Patent 5,254,799. Another alternative is to use a promoter whose expression is inducible (e.g., by wounding caused by feeding insects, by temperature or by chemical factors). Of course, any promoter known to be highly expressed in rice plants, particularly in leaves or stem, can be used in accordance with this invention.

The cry9C DNA is inserted in the rice genome so that the inserted gene part is upstream (i.e., 5¹) of suitable 3' end transcription regulation signals (i.e., transcript formation and polyadenylation signals). This is preferably accomplished by inserting the cry9C chimeric gene in the rice genome. Preferred polyadenylation and transcript formation signals include those of the octopine synthase gene (Gielen et al., 1984) and the T-DNA gene 7 (Velten and Schell, 1985), which act as 3 '-untranslated DNA sequences in transformed plant cells. The cry9C DNA can optionally be inserted in the plant genome as a hybrid gene (Vaeck et al., 1987) under the control of the same promoter as a selectable marker gene, so that the plant expresses a fusion protein retaining the activity of both proteins. As such, high expression levels of Cry9C protein can be selected by increasing the concentration of selectable marker agent in the culture medium. Further in accordance with this invention, a process is provided for making a rice plant resistant to Lepidopteran insects, particularly Chilo suppressalis, Scirpophaga incertulas, Sesamia inferens, Cnaphalocrocis medinalis, Marasmia patnalis, Marasmia exigua, Marasmia ruralis, Nymphula depunctalis, Scirpophaga innotata, Spodoptera litura, Chilo polychrysus, Rupela albinella, Diatraea saccharalis, Spodoptera frugiperda, Mythimna unipuncta, Chilo zacconius, Hereitogramma licarisalis, Naranga aenescens, Mycalesis gotama, Parnara guttata, preferably Chilo spp., Cnaphalocrocis spp., Marasmia spp., and Scirpophaga spp., most preferably Chilo suppressalis, Marasmia patnalis, Cnaphalocrocis medinalis and Scirpophaga incertulas. This process is characterized by transforming rice plants with a DNA sequence encoding a Cry 9C protein using known procedures. In a preferred process, in accordance with this invention, rice plants are rendered resistant to Lepidopteran rice stemborers, rice skippers, rice cutworms, rice armyworms, rice caseworms or rice leaffolders, by transforming rice plants with a DNA sequence encoding a Cry 9C protein.

Another embodiment in accordance with the invention is a process for producing rice plants and reproduction material thereof, such as seeds, which are resistant to infestation with an insect selected from the group consisting of: Chilo suppressalis, Scirpophaga incertulas, Sesamia inferens, Cnaphalocrocis medinalis, Marasmia patnalis, Marasmia exigua, Marasmia ruralis, Nymphula depunctalis, Scirpophaga innotata, Spodoptera litura, Chilo polychrysus, Rupela albinella, Diatraea saccharalis, Spodoptera frugiperda, Mythimna unipuncta, Chilo zacconius, Hereitogramma licarisalis, Naranga aenescens, Mycalesis gotama, Parnara guttata, preferably Chilo spp., Cnaphalocrocis spp., Marasmia spp., and Scirpophaga spp., most preferably Chilo suppressalis, Marasmia patnalis, Cnaphalocrocis medinalis and Scirpophaga incertulas, comprising: transforming rice cells with a DNA sequence encoding a Cry9C protein and regenerating plants and reproduction material thereof comprising said DNA sequence. In a preferred embodiment, a process is provided for producing rice plants and reproduction material thereof, such as seeds, which are resistant to infestation with a Lepidopteran rice leaffolder, rice skipper, rice cutworm, rice armyworm, rice caseworm or rice stemborer, which process comprises transforming rice cells with a DNA sequence encoding a Cry9C protein and regenerating plants and reproduction material thereof comprising said DNA sequence.

Further in accordance with this invention, a method is provided for controlling Lepidopteran insect pests, particularly Lepidopteran rice leaffolders, rice skippers, rice cutworms, rice armyworms, rice caseworms or rice stemborers, more particularly Chilo suppressalis, Scirpophaga incertulas, Sesamia inferens, Cnaphalocrocis medinalis, Hereitogramma licarisalis, Naranga aenescens, Mycalesis gotama, Marasmia patnalis, Marasmia exigua, Marasmia ruralis, Nymphula depunctalis, Scirpophaga innotata, Spodoptera litura, Chilo polychrysus, Rupela albinella, Diatraea saccharalis, Spodoptera frugiperda, Mythimna unipuncta, Chilo zacconius, or Parnara guttata, preferably Chilo spp., Cnaphalocrocis spp., Marasmia spp., and Scirpophaga spp., most preferably Chilo suppressalis, Marasmia patnalis, Cnaphalocrocis medinalis and Scirpophaga incertulas, which comprises contacting said insects with a Cry9C protein in an insecticidally effective amount. In a most preferred embodiment, such contacting is by sowing, planting or cultivating rice transformed to express a Cry9C protein in a field. In this case, only rice insect pests attempting to feed on the rice plants will be affected by the Cry9C protein.

In another embodiment of the current invention, such insects are contacted with the Cry9C protein by spraying compositions comprising insecticidally effective amounts of Cry9C protein to the rice plants. This composition can be formulated in a conventional manner using the Cry9C protein as an active ingredient, together with suitable carriers, diluents, emulsifiers and/or dispersants (e.g., as described by Bernhard and Utz, 1 993). This insecticide composition can be formulated as a wettable powder, pellets, granules or dust or as a liquid formulation with aqueous or non-aqueous solvents as a foam, gel, suspension, concentrate, etc. The concentration of the Cry9C protein in such a composition will depend upon the nature of the formulation and its intended mode of use. Generally, an insecticide composition of this invention can be used to protect a rice field for 1 or 2 weeks against Lepidoptera with each application of the composition. For more extended protection (e.g., for a whole growing season), additional amounts of the composition should be applied periodically in case a sprayable composition is used. Bt cells or crystals, crystal proteins, protoxins, toxins, and insecticidally effective protoxin portions and other insecticides, as well as fungicides, biocides, herbicides and fertilizers, can be employed along with the Cry9C protein to provide additional advantages or benefits. Generally, the concentration of the Cry9C protein in a composition will be at least about 0.1 % by weight of the formulation to about 100% by weight of the formulation, more often from about 0.1 5% to about 0.8% by weight of the formulation. Also, in accordance with this invention is provided a method for producing processed or unprocessed rice grain, comprising planting Cry9C rice of the invention in the field, harvesting and treating seeds from such rice to obtain processed or unprocessed grain. An improved yield is obtainable in rice plants transformed or treated in accordance with the invention. Toxicity of a protein to a Lepidopteran rice insect, such as a Lepidopteran rice leaffolder, rice armyworm, rice cutworm, rice caseworm, rice skipper, rice stemborer, Chilo suppressalis, Scirpophaga incertulas, Sesamia inferens, Cnaphalocrocis medinalis, Hereitogramma licarisalis, Naranga aenescens, Mycalesis gotama, Marasmia patnalis, Marasmia exigua, Marasmia ruralis, Nymphula depunctalis, Scirpophaga innotata, Spodoptera litura, Chilo polychrysus, Rupela albinella, Diatraea saccharalis, Spodoptera frugiperda,

Mythimna unipuncta, Chilo zacconius, or Parnara guttata, can be measured using established procedures. A suitable procedure in accordance with this invention is drenching a solution containing the protein (at determined concentration) on a layer of rice seeds or leaves. After addition of larvae to these seeds or leaves, the number of dead larvae are counted and the larvae still alive are weighed after determined time periods. Appropriate control samples are run in the same test so as to determine the background mortality and standard larval growth under the same conditions. Similar assays using artificial diet with protein applied thereon or incorporated therein or rice seeds or rice leaf material with protein applied thereon can be used as described, e.g., in Lee et al. (1997a) or Theunis et al. (1998). Toxicity to insects of transgenic rice expressing a protein in accordance with the invention can be measured following routine protocols with appropriate controls such as by using a cut stem in vitro test or a whole plant assay as described, e.g., by Nayak et al. (1997) or Ghareyazie et al. (1997).

Rice pests can be caught in a rice field and can be cultivated following known procedures (see, e.g., Kamano and Sato, 1985, for Chilo suppressalis cultivation). For Scirpophaga incertulas, e.g., the method as described by Yannian et al. (1988) can be used. The taxonomy of a Lepidopteran rice insect pest can be determined by procedures known to a person of ordinary skill in the art, see, e.g. Barrion and Litsinger (1994).

A method for controlling Lepidopteran rice insect pests, particularly Lepidopteran rice stem borers, rice caseworms, rice cutworms, rice armyworms or rice leaffolders, more particularly an insect selected from the group consisting of: Chilo suppressalis, Scirpophaga incertulas, Sesamia inferens, Cnaphalocrocis medinalis, Hereitogramma licarisalis, Naranga aenescens, Mycalesis gotama, Marasmia patnalis, Marasmia exigua, Marasmia ruralis, Nymphula depunctalis, Scirpophaga innotata, Spodoptera litura, Chilo polychrysus, Rupela albinella, Diatraea saccharalis, Spodoptera frugiperda, Mythimna unipuncta, Chilo zacconius, and Parnara guttata, preferably Chilo spp., Cnaphalocrocis spp., Marasmia spp., and Scirpophaga spp., most preferably Chilo suppressalis, Marasmia patnalis, Cnaphalocrocis medinalis and Scirpophaga incertulas, in accordance with this invention preferably comprises applying (e.g., spraying or expressing in a transgenic plant), to a field or area to be protected, an insecticidal amount of the Cry9C protein. The field or area to be protected can include, for example, the habitat of the insect pests, rice growing in the field, or an area where rice is to be grown.

The following Examples illustrate the invention but are not meant to limit the scope of the invention. The sequence listing referred to in the description and the examples is as follows:

Sequence Listing:

SEQ ID NO: 1 - naturally occurring Cry9C protein and cry9C DNA sequence.

SEQ ID NO: 2 - amino acid sequence of the Cry9C protein of SEQ ID NO: 1

SEQ ID NO: 3 - cry9C DNA and Cry9C amino acid sequence for expression in rice.

SEQ ID NO: 4 - amino acid sequence of the Cry9C protein of SEQ ID NO: 3.

Unless otherwise stated in the Examples, all procedures for making and manipulating recombinant DNA are carried out by the standard procedures described in Sambrook et al., Molecular Cloning - A Laboratory Manual, Second Ed., Cold Spring Harbor Laboratory Press, NY (1989), and in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA. Standard materials and methods for plant molecular biology work are described in Plant Molecular Biology Labfax (1993) by R.R.D. Croy, jointly published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications (UK).

EXAMPLES

Example 1 : Insecticidal activity of Cry9C protein to rice insect pests. Using insect feeding assays, the toxicity of the Cry9Ca protein to selected rice insects was evaluated.

The rice leaffolders Cnaphalocrocis medinalis and Marasmia patnalis were tested using a rice leaf dip assay. Whole rice leaves of a leaffolder-susceptible rice variety (Taichung native 1 variety (TN1 )) were used to determine the LC50 concentration of purified Cry9Ca(R1 64K) protein (a Cry9Ca protein wherein the amino acid at position 1 64 (arginine) has been replaced by lysine to obtain improved stability to proteases). The leaves were dipped in a serial solution of 0, 0.025, 0.1 , 0.39, 1 .56, 6.25, 25 and 100 microgram/ml Cry9C(R1 64K) trypsin- treated active toxin. All dilutions were made with sterile distilled water with 0.01 % Triton X-100. After air-drying for 30 minutes, each leaf was placed in a Petri dish and ten neonate larvae were added per concentration. Five leaves were used for every concentration, each leaf representing a replicate. Mortality was scored 96 hours after infestation. The LC50 values were calculated by the Probit-Least Square Method employing the Field Trial/Agricultural Research Manager Software (ARM version 6.0.2; Gylling Data Management (GDM), 1 999). For Cnaphalocrocis medinalis, an LC50 value of 0.956 microgram/ml was obtained (fiducial limits at 95 % confidence level: 0.64-1 .52 microgram/ml), for Marasmia patnalis, an LC50 value of 1 .230 microgram/ml was obtained (fiducial limits at 95 % confidence level: 0.82-1 .92 microgram/ml). Thus, the Cry9C protein is highly toxic to the rice leaffolders Cnaphalocrocis medinalis and Marasmia patnalis. The striped stem borer, Chilo suppressalis, was tested using a rice leaf sheath dip assay with leaf sheaths of a stem borer-susceptible rice variety. Leaf sheaths were dipped in a serial dilution of Cry9C(R163K) tryps in-treated active toxin. Five concentrations were tested, the negative control was: 0 microgram/ml Cry9Ca protein, all dilutions were made with sterile distilled water with 0.01 % Triton X- 100. After drying the leaf sheath was placed in a Petri dish and five neonate larvae were used per concentration. Mortality was scored after 96 hours. The bioassay was replicated ten times. The LC50 values were calculated by the Probit- Least Square Method employing the Field Trial System/Agricultural Research Manager Software (ARM version 6.0.2; Gylling Data Management (GDM), 1999). An LC50 value of 6.44 microgram/ml was obtained (95 % confidence level fiducial limits: 5.74-7.23). Thus, the Cry9C protein was found to be highly toxic to the striped stem borer.

Similar results are also found for the above insects when using the native Cry9Ca protein (SEQ ID NO: 2).

In a similar insect leaf dip feeding assay using neonate larvae, high toxicity was also found for the Cry1 Ca4 protein to the rice cutworm, Spodoptera litura (LC50 value : 0.19 microgram/ml (fiducial limits at 95% confidence level: 0.02-0.62). Serial dilutions of Cryl Ca4 solutions (0.1 , 0.39, 1 .56, 6.25, 25, and 100 microgram/ml in sterile distilled water, plus 0.01 % Triton X-100) were screened for activity to Spodoptera litura neonates. Punched (6 cm diameter) Ricinus leaves were dipped in a Cryl C solution and air-dried. The treated leaves were placed in Petri-dishes fitted with a moist filter paper mat, and were infested with 10 neonates in each replicate. After incubation for 96 hours, the mortality was determined. Neonates of S. litura were obtained using egg batches laid within one day, incubated at 25 °C (+/- 1 °C) to ensure uniform hatching. Other Cryl Ca proteins give similar results in this assay. The same assay with Spodoptera litura but using the Cry9Ca(R1 64K) toxic protein fragment in several replicated leaf-dip assays resulted in an LC50 value of 19.63 microgram/ml (95% confidence level fiducial limits: 12.61 -33.55). Thus, both Cryl C and Cry9C are highly toxic proteins for the rice cutworm Spodoptera litura.

Example 2: Transformation of rice with a cry9C chimeric gene.

For transformation of rice plants, the vector system as described by Deblaere et al. (1985, 1987) was used. The vector system consists of an Agrobacterium strain and two plasmid components: 1 ) an intermediate cloning vector, and 2) a non- oncogenic Ti-plasmid.

The intermediate cloning vector was essentially derived from pGSC1 700 (Cornelissen and Vandewiele, 1 989). It contained an artificial T-region consisting of the left and right border sequences of the TL-DNA from pTiB6S3 and multilinker cloning sites allowing the insertion of chimeric genes between the T-DNA border repeats. The DNA inserted between the T-DNA border repeats encodes two genes, a chimeric bar gene and a chimeric cry9C gene. The chimeric bar gene which confers resistance to glufosinate ammonium or phosphinothricin comprises the coding region from the phosphinothricin acetyl transferase gene of Streptomyces hygroscopicus (PCT patent publication WO 87/05629), under the control of the CaMV 35S promoter (Odell et al., 1985), and the CaMV 35S 3' transcript termination and polyadenylation region (Mogen et al., 1990). The chimeric bar gene is useful as selectable marker and also confers herbicide resistance in the field to commercial formulations containing as active ingredient glufosinate ammonium. The chimeric cry9C gene comprises the coding region of SEQ ID NO: 3, under the control of the CaMV 35S promoter (Odell et al., 1985), and flanked by the CaMV 35S 3' transcript termination and polyadenylation region (Mogen et al., 1990). The acceptor Agrobacterium strain carries a non-oncogenic (disarmed) Ti plasmid from which the internal T-DNA has been deleted but the normal vir transfer genes have been retained. The plasmid also has a region of homology that allows cointegrate formation with the intermediate cloning vector. The intermediate vector is constructed in E. coli. It was transferred to the acceptor Agrobacterium tumefaciens strain via a triparental mating involving an E. coli strain that carries a mobilization helper plasmid (Van Haute et al., 1 983, Deblaere et al., 1987). The structure of the T-DNA in the resulting Agrobacterium strain was confirmed by Southern blot hybridization (Deblaere et al., 1985).

Agrobacterium-mediated gene transfer of the intermediate cloning vector resulted in transfer to the rice genome of the DNA fragment between the T-DNA border repeats. As target tissue for transformation, immature embryo or callus derived thereof from japonica and indica rice cultivars were used which had been cut into small pieces, using the technique described in PCT patent publication WO 92/09696. After co-cultivation of this tissue with Agrobacterium, the transformed rice cells were selected by addition of glufosinate ammonium to the rice tissue culture medium.

Calli grown on glufosinate ammonium were transferred to regeneration medium. When plantlets with roots and shoots developed, they were transferred to soil, and placed in the greenhouse. The transformation was confirmed by phosphinothricin acetyl transferase assay, by glufosinate ammonium application to leaves, and by quantitative ELISA, PCR and Southern blot analysis. When the plants flowered, they were self-pollinated, and seeds were harvested when mature. Cry9C transformants are also obtained using electroporation of wounded compact embryogenic callus as described in US Patent 5,679,558. Also, besides the above chimeric gene, other chimeric cry9C genes were constructed for expression in rice, including different plant-expressible promoters such as the ubiquitin and gos2 promoter (Christensen et al., 1992, de Pater et al., 1992). These chimeric genes were also used to transform rice plants with the above Agrobacterium transformation protocol. Rice plants having a single copy of the cry9C chimeric gene integrated into their genome were selected by means of Southern blot. Expression levels of the Cry9C protein in leaf and stem material were analyzed using ELISA assays and Western blot. Seeds of successfully transformed plants were grown into plants which were tested for expression of the Cry9C protein and insect resistance. Following the above protocol, also indica rice plants were transformed with the chimeric Cry9C gene. Upon transfer to the greenhouse these plants were evaluated for transformation and insect resistance.

Also, crosses between the above Cry9C indica rice lines and indica rice plants transformed with a chimeric gene encoding a truncated Cryl Ab5 protein (with the amino acid sequence of amino acid position 2 to 61 6 of Hδfte et al., 1 986, preceded by a methionine and alanine amino acid) are made to improve the resistance of the plants to the insects and prevent or delay the development of insect resistance. Chimeric genes encoding the truncated Cryl Ab5 protein comprising the ubiquitin or gos2 promoter as described above have been used to obtain the Cryl Ab5-expressing rice plants. Similarly, plant-expressible chimeric genes encoding an active fragment of the Cryl Ca4 protein are made to transform rice plants in accordance with the above description.

Example 3: Insect resistance of transformed Cry9C-expressing japonica type rice plants.

The insecticidal activity against Lepidopteran rice pests of the ELISA-positive and herbicide-resistant T1 progeny plants obtained form the japonica rice plants of Example 2 transformed with the 35S-cry9C chimeric gene, was initially evaluated by recording the growth rate and mortality of Chilo suppressalis larvae fed on these plants in greenhouse trials. These results were compared with the growth rate and mortality of larvae fed leaves from untransformed rice plants of variety Chiyonishiki. Four rice transformation events (ROS-01 2-2805, -2401 , -2702, -3703, having been confirmed as transformed plants expressing Cry9C protein) and one untransformed negative control, Chiyonishiki rice, were evaluated for Chilo suppressalis resistance in two greenhouse experiments. The events were sown in the greenhouse and when reaching the fourth leaf stage were sprayed with glufosinate ammonium to segregate non-transgenic and transgenic plants.

When plants started tillering, they were infested on two consecutive days with one eggmass per plant. Plant damage was scored using a 0, no damage, to 3, heavy damage, scale; 20-35 days and 70 days after infestation. The infestations were repeated just prior to booting of the plants. External damage to the stem was scored 44-52 days after the infestations. 70 days after the infestations the plants were dissected and the number of stems with tunnels was counted per plant. The plant damage scoring of 2.5-3.0 of Chiyonishiki proves that the vegetative stage infestations were successful and very high damage was caused to the non- transformed plants. The same can be said for the pre-boot infestations where most of the Chiyonishiki control plants showed tunnels of Chilo suppressalis. Three of the transformed rice events, ROS1 2-2805, -2401 , -2702 showed a very low plant damage after the vegetative stage infestation with a maximum plant damage score of 0.25. All transgenic Cry9C rice plants showed a lower number of stems with tunnels than Chiyonishiki control plants. One of the 4 events evaluated, ROS1 2-3703, was completely protected and showed no tunnels at all.

Thus, a significant and high mortality rate is obtained among Chilo larvae fed on transformed rice plants expressing the Cry9C protein compared to larvae fed the leaves of untransformed plants. Also, plants comprising higher concentrations of the Cry9C protein have been found to be better protected against insects than plants with lower concentrations. In separate insect toxicity assays with the transgenic Cry9C plants expressing high amounts of the Cry9C protein, good control was obtained for assays with other Lepidopteran rice stemborers and rice leaffolders, particularly Scirpophaga incertulas, Marasmia patnalis, and Cnaphalocrocis medinalis.

The transformed plants can also be useful to obtain resistance against other Lepidopteran rice pests such as rice skippers, Sesamia inferens, Spodoptera litura, Hereitogramma licarisalis, Naranga aenescens, Mycalesis gotama, or Parnara guttata.

Example 4: Insect resistance of transformed Cry9C-expressing indica type rice plants. The insecticidal activity against Lepidopteran rice pests of the ELISA-positive and herbicide-resistant T1 progeny plants obtained from the indica rice plants of Example 2 transformed with the Pgos2-cry9C or Pubi-cry9C chimeric genes, was initially evaluated by recording the growth rate and mortality of Scirpophaga incertulas and Cnaphalocrocis medinalis larvae fed on these plants in greenhouse trials. These results were compared with the growth rate and mortality of larvae fed leaves from untransformed rice plants of the same indica variety. Seven rice transformation events (having been confirmed as transformed plants expressing Cry9C protein, see enclosed Table 1 ) and one untransformed negative control, indica type rice, were evaluated for Scirpophaga incertulas and

Cnaphalocrocis medinalis resistance in a greenhouse experiment (results see in Table 1 ). The events were sown in the greenhouse, transplanted and when reaching the fourth leaf stage were sprayed with glufosinate ammonium to segregate non-transgenic and transgenic plants. Scirpophaga incertulas larvae were collected in the field. They developed to adults in the lab, and eggmasses were collected.

Infestation with five eggmasses or larvae of Scirpophaga incertulas per plant tiller was done 4-5 weeks after transplanting. Plant damage by Scirpophaga incertulas was scored by counting the number of tillers showing deadheart, the phenotype typical for plant damage by stemborers, 20-35 days after infestation and comparison to the total number of tillers of the plant. Following scoring of the damage rate, the plants were dissected and the number of recovered larvae was determined on plants showing insect damage (results see Table 1 below). The high plant damage score (50-100% of all tillers showing deadheart) of the non- transformed indica type control plants proves that the vegetative stage infestations were successful and very high damage was caused to the non-transformed plants. Of the damaged plants 2-4 larvae could be recovered per plant. Three of the transformed rice events, ROS045-01001 , ROS046-00401 , ROS048- 00207 showed a very low plant damage after the vegetative stage infestation with each event containing 1 -2 tillers showing deadheart among the total number tillers of all plants together. The other events were completely protected from insect damage by Scirpophaga incertulas.

Infestation with 5 eggmasses or larvae of Cnaphalocrocis medinalis per plant tiller was done 6-8 weeks after transplanting. Plant damage by Cnaphalocrocis medinalis was scored using a damage scoring scale from 0, no damage, to 9, whole plant dried. Following the damage rate scoring, the plants were dissected and the number of recovered larvae was determined on plants showing insect damage (results see Table 1 ). The high plant damage score of 8 of the non-transformed indica variety proves that the vegetative stage infestations were succesful and very high damage was caused to the non-transformed plants. Of the damaged plants 9-1 1 larvae could be recovered.

All transformed rice events showed a very low plant damage rate of 1 -2 after the vegetative stage infestation showing complete protection from insect damage by

Cnaphalocrocis medinalis. Some minor damage is normal, since the insects need to ingest rice plant material in order to experience the toxicity of the Cry9C protein.

Thus, these greenhouse plant infestations confirm that a significant and high mortality rate is obtained among Lepidoptera larvae fed on transformed rice plants expressing the Cry9C protein compared to larvae fed the leaves of untransformed plants.

Needless to say, this invention is not limited to the above rice plants and the particular Cry9C protein used. Rather, the invention also includes any mutant or variant of the Cry9C protein retaining insecticidal activity or having improved insecticidal activity to Lepidopteran rice insect pests, such as a protein having substantially the amino acid sequence of the Cry9C protein and having substantially the insecticidal activity of the Cry9C protein (e.g., an insecticidal protein with an amino acid sequence identity of at least 90 %, particularly at least 95 %, to the protein of SEQ ID NO: 2 or 4 or insecticidally-effective fragments thereof, and which is insecticidal to Chilo suppressalis and Scirpophaga incertulas). Also, the invention is not limited to the above exemplified rice plants but includes any rice variety, hybrid, inbred or elite line. Also, any plant which is susceptible to damage by Lepidopteran rice stemborers, rice skippers, rice cutworms, rice armyworms, rice caseworms or rice leaffolders, preferably an insect selected from the group consisting of: Chilo suppressalis, Scirpophaga incertulas, Sesamia inferens, Cnaphalocrocis medinalis, Hereitogramma licarisalis, Naranga aenescens, Mycalesis gotama, Marasmia patnalis, Marasmia exigua, Marasmia ruralis, Nymphula depunctalis, Scirpophaga innotata, Spodoptera litura, Chilo polychrysus, Rupela albinella, Diatraea saccharalis, Spodoptera frugiperda, Mythimna unipuncta, Chilo zacconius, and Parnara guttata, most particularly Chilo suppressalis, is included in the invention as a preferred target for transformation with a DNA encoding a Cry9C protein. These plants which can also be used in accordance with the invention in a method for controlling such insects include but are not limited to other Graminea plants, including water oat, sugarcane, sorghum, wheat, millet, monophagous rice and Indian corn. Also in accordance with the invention a process is provided for protecting a plant other than rice from any of the above listed insects, comprising transforming the plants with a DNA encoding an Cry9C protein or applying the Cry9C protein to such plants. This invention is not limited to the use of Agrobacterium tumefaciens Ti-plasmids for transforming rice cells with a DNA encoding an insecticidally effective Cry9C protein. Other known techniques for rice transformation, such as by means of direct gene transfer using liposomes, electroporation or particle gun bombardement or by means of vector systems based on plant viruses or pollen, can be used for transforming rice with a cry9C chimeric gene. Expression can be in stably transformed rice plant cells with cry9C integrated in the genome, particularly the nuclear genome, or can be in transiently transformed rice plant cells.

It is believed that any method for transforming rice so that it expresses the Cry9C protein at sufficient levels can be used to develop in sect- resistant rice plants. In this regard, it may be preferred to express at least 2 non-competitively binding Bt proteins, such as a Cryl Aa, Cryl Ab, Cryl C or Cryl B protein together with the Cry9C protein in one plant to prevent or delay the development of insect resistance.

Table 1

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Claims

CLAIMS:

1 . A rice plant resistant to Lepidopteran rice stemborers, rice skippers, rice cutworms, rice armyworms, rice caseworms or rice leaffolders, comprising stably integrated in its genome, a plant-expressible DNA sequence encoding a protein comprising the amino acid sequence from amino acid position 44 to amino acid position 658 of SEQ ID NO: 2, or a DNA sequence encoding a protein comprising the amino acid sequence of SEQ ID NO: 4.

2. A rice plant resistant to an insect selected from the group consisting of: Chilo suppressalis, Scirpophaga incertulas, Sesamia inferens, Cnaphalocrocis medinalis, Hereitogramma licarisalis, Naranga aenescens, Mycalesis gotama, Marasmia patnalis, Marasmia exigua, Marasmia ruralis, Nymphula depunctalis, Scirpophaga innotata, Spodoptera litura, Chilo polychrysus, Rupela albinella, Diatraea saccharalis, Spodoptera frugiperda, Mythimna unipuncta, Chilo zacconius, and Parnara guttata, comprising stably integrated in its genome, a plant- expressible DNA sequence encoding a protein comprising the amino acid sequence from amino acid position 44 to amino acid position 658 of SEQ ID NO:

2. or a DNA sequence encoding a protein comprising the amino acid sequence of SEQ ID NO: 4.

3. A rice plant which is resistant to Chilo suppressalis, Marasmia patnalis, Scirpophaga incertulas or Cnaphalocrocis medinalis, comprising stably integrated in its genome, a plant-expressible DNA sequence encoding a protein comprising the amino acid sequence of SEQ ID NO: 2 from amino acid position 44 to amino acid position 658, or a DNA sequence encoding a protein comprising the amino acid sequence of SEQ ID NO: 4.

4. A seed, grain, processed grain of the plant of any one of claims 1 to 3, comprising a DNA sequence encoding said protein.

5. The processed grain of claim 4 which is milled, polished, dehusked, and/or parboiled grain.

6. The plant of any one of claims 1 to 3 which also comprises in its genome a DNA encoding a protein selected from the group consisting of: Cryl B, Cryl C, Cryl D, Cryl E, Cryl Aa, Cryl Ab, Cryl Ac, Cryl I, Cryl J, Cry2A, Cry6B, Cry3A, a protease inhibitor, cowpea trypsin inhibitor, protease inhibitor II, cystatin, the GNA lectin, an insecticidal proteins of Xhenorhabdus or Photorhabdus spp., and a protein conferring resistance to glufosinate ammonium.

7. A process for obtaining a rice plant and progeny thereof resistant to against Lepidopteran rice stemborers, rice skippers, rice cutworms, rice armyworms, rice caseworms or rice leaffolders, comprising transforming a rice plant with a plant- expressible DNA sequence encoding a protein comprising the amino acid sequence of SEQ ID NO: 2 from amino acid position 44 to amino acid position 658, or a DNA sequence encoding a protein comprising the amino acid sequence of SEQ ID NO: 4.

8. A process for obtaining a rice plant and progeny thereof resistant to an insect selected from the group consisting of: Chilo suppressalis, Scirpophaga incertulas, Sesamia inferens, Cnaphalocrocis medinalis, Hereitogramma licarisalis, Naranga aenescens, Mycalesis gotama, Marasmia patnalis, Marasmia exigua,

Marasmia ruralis, Nymphula depunctalis, Scirpophaga innotata, Spodoptera litura, Chilo polychrysus, Rupela albinella, Diatraea saccharalis, Spodoptera frugiperda, Mythimna unipuncta, Chilo zacconius, and Parnara guttata, comprising transforming a rice plant with a plant-expressible DNA sequence encoding a protein comprising the amino acid sequence of SEQ ID NO: 2 from amino acid position 44 to amino acid position 658, or a DNA sequence encoding a protein comprising the amino acid sequence of SEQ ID NO: 4.

9. A process for obtaining a rice plant and progeny thereof resistant to Chilo suppressalis, Marasmia patnalis, Cnaphalocrocis medinalis, or Scirpophaga incertulas, comprising transforming a rice plant with a plant-expressible DNA sequence encoding a protein comprising the amino acid sequence from amino acid position 44 to amino acid position 658 of SEQ ID NO: 2, or a DNA sequence encoding a protein comprising the amino acid sequence of SEQ ID NO: 4.

10. A process for controlling a Lepidopteran rice stemborer, rice skipper or rice leaffolder, comprising contacting said insect with an insecticidal amount of a protein comprising the amino acid sequence from amino acid position 44 to amino acid position 658 of SEQ ID NO: 2, or a DNA sequence encoding a protein comprising the amino acid sequence of SEQ ID NO: 4.

1 1 . A process for controlling an insect selected from the group consisting of: Chilo suppressalis, Scirpophaga incertulas, Sesamia inferens, Cnaphalocrocis medinalis, Hereitogramma licarisalis, Naranga aenescens, Mycalesis gotama, Marasmia patnalis, Marasmia exigua, Marasmia ruralis, Nymphula depunctalis, Scirpophaga innotata, Spodoptera litura, Chilo polychrysus, Rupela albinella, Diatraea saccharalis, Spodoptera frugiperda, Mythimna unipuncta, Chilo zacconius, and Parnara guttata, comprising contacting said insect with an insecticidal amount of a protein comprising the amino acid sequence from amino acid position 44 to amino acid position 658 of SEQ ID NO: 2, or a DNA sequence encoding a protein comprising the amino acid sequence of SEQ ID NO: 4.

12. A process for controlling Chilo suppressalis, Cnaphalocrocis medinalis, Scirpophaga incertulas or Marasmia patnalis, comprising contacting said insect with an insecticidal amount of a protein comprising the amino acid sequence from amino acid position 44 to amino acid position 658 of SEQ ID NO: 2, or a DNA sequence encoding a protein comprising the amino acid sequence of SEQ ID NO: 4.

1 3. The process of any one of claims 10 to 12, wherein said contacting comprises the step of transforming rice plants with a DNA sequence encoding said protein and the step of cultivating said plants or progeny thereof containing said DNA sequence in a field.

14. A plant and progeny thereof obtained by the process of any one of claims 7 to 9 comprising said plant-expressible DNA sequence.