WO2006101854A2

WO2006101854A2 - Compositions and methods for controlling fungal diseases

Info

Publication number: WO2006101854A2
Application number: PCT/US2006/009181
Authority: WO
Inventors: Harold N. Trick; Barbara Valent
Original assignee: Kansas State University Research Foundation
Priority date: 2005-03-16
Filing date: 2006-03-15
Publication date: 2006-09-28
Also published as: WO2006101854A3

Abstract

The present invention relates to compositions and methods for controlling fungal infestation of plants and crops. In particular, the present invention provides vectors comprising sequences designed to control fungal plant diseases by RNA interference (RNAi) and transgenic plants transformed with such vectors.

Description

COMPOSITIONS AND METHODS FOR CONTROLLING FUNGAL DISEASES

This invention was made with government support under The Kansas State University Plant Biotechnology Center Grant #KS693. The Government may have certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for controlling fungal infestation of plants and crops, m particular, the present invention provides vectors comprising sequences designed to control fungal plant diseases by RNA interference

(RNAi) and transgenic plants transformed with such vectors.

BACKGROUND OF THE INVENTION

Rice is the staple food for one half of the world's population and diseases of this crop are of special concern. One of the most widespread and devastating diseases is rice blast which causes significant crop losses in all rice-growing regions including those in Asia and Latin America. Major epidemics covering vast areas occur on a regular basis causing severe food shortages to entire nations.

Rice blast causes between 11% and 30% crop losses annually. This represents a loss of 157 million tons of rice. Rice blast is presently controlled using resistant cultivars or by application of fungicides, although problems are associated with both forms of management. Where blast is prevalent, resistant cultivars have an expected field life of only 2 -3 growing seasons due to the generation of newly virulent forms of the fungus. Fungicide resistance is also of concern and there is a considerable need for environmentally compatible, novel fungicides offering durable management of rice blast. This disease represents one of the world's largest fungicide markets and is one of the only agricultural markets where fungicide development costs can be justified for a single-disease stand-alone product.

Novel approaches to rice blast disease management are needed to complement current strategies, and prolong the effectiveness of available resistance genes.

SUMMARY OF THE INVENTION

Accordingly, in some embodiments, the present invention provides a transgenic plant comprising heterologous nucleic acid sequences encoding a double stranded fungal RNA sequence, wherein the double stranded RNA sequence inhibits the proliferation of fungi uptakiiig the double stranded fungal RNA sequence. In preferred embodiments, the fungi is M. grisea fungi or F. graminearum, and the plant is rice.

In preferred embodiments, the heterologous nucleic acid sequences are operably linked to the same promoter. In other preferred embodiments, the heterologous nucleic acid sequences are separated by a loop sequence. In yet other preferred embodiments, the promoter is a tissue specific promoter. In other preferred embodiments, the promoter is a constitutive promoter.

In preferred embodiments, the heterologous nucleic acid sequences are operably linked to separate promoters. In other preferred embodiments, one of the heterologous nucleic acid sequences is complementary to an RNA sequence selected from the group consisting of M. grisea fungi or F. graminearum farnesyl pyrophosphate synthetase, citrate synthetase, isocitrate dehydrogenase [NAD] subunit 1 — mitochondrial precursor, ABCl, and RNA Polymerase. In preferred embodiments, the heterologous nucleic acid sequences are at least 21 bases in length. In other preferred embodiments, the present invention provides seeds, leaves, roots, and stems from the transgenic plant.

In preferred embodiments, the double stranded RNA is complementary to a fungal gene required for pathogenecity.

In certain embodiments, the present invention provides a vector comprising heterologous nucleic acid sequences encoding a double stranded fungal RNA sequence, wherein the double stranded RNA sequence inhibits the proliferation of fungi ingesting the double stranded fungal RNA sequence. In preferred embodiments, the fungi is M. grisea fungi or F. graminearum fungi, and the plant is rice.

In preferred embodiments, the heterologous nucleic acid sequences are operably linked to the same promoter. In other preferred embodiments, the heterologous nucleic acid sequences are separated by a loop sequence. In other preferred embodiments, the promoter is a tissue specific promoter. In yet other preferred embodiments, the promoter is a constitutive promoter. In preferred embodiments, the heterologous nucleic acid sequences are operably linked to separate promoters. In preferred embodiments, one of the heterologous nucleic acid sequences is complementary to an RNA sequence selected from the group consisting of M. grisea fungi or F. graminearum farnesyl pyrophosphate synthetase, citrate synthetase, isocitrate dehydrogenase [NAD] subunit 1 - mitochondrial precursor, ABCl, and RNA

Polymerase. In preferred embodiments, the heterologous nucleic acid sequences are at least 21 bases in length.

In certain embodiments, the present invention provides a method for controlling fungi comprising a) providing transgenic plant tissue comprising heterologous DNA sequences encoding a double stranded fungal RNA; and b) growing the transgenic plant so that the double stranded fungal RNA is expressed in plant tissue; wherein the proliferation of fungi feeding on the plant tissue is reduced as compared to fungi feeding on non- transgenic plant tissue. In preferred embodiments, the fungi are M. grisea fungi or F. graminearum fungi, and the plant is rice. In preferred embodiments, the heterologous DNA sequences are located on a vector.

In other preferred embodiments, the heterologous DNA sequences are operably linked to a promoter. In yet other preferred embodiments, the heterologous DNA sequences are operably linked to the same promoter. In other preferred embodiments, the promoter is a tissue specific promoter. In preferred embodiments, the promoter is a constitutive promoter. In preferred embodiments, the heterologous DNA sequences are separated by a loop sequence, hi other preferred embodiments, one of the heterologous nucleic acid sequences is complementary to an RNA sequence selected from the group consisting of M. grisea fungi or F. graminearum farnesyl pyrophosphate synthetase, citrate synthetase, isocitrate dehydrogenase [NAD] subunit 1 - mitochondrial precursor, ABCl, and RNA Polymerase. hi preferred embodiments, the heterologous nucleic acid sequences are at least 21 bases in length. In preferred embodiments, the double stranded fungal RNA is complementary to a fungal gene required for pathogenecity.

DESCRIPTION OF THE FIGURES Figure 1 is the nucleic acid sequence for M. grisea farnesyl pyrophosphate synthetase (SEQ ID NO:1).

Figure 2 is a nucleic acid subsequence of M. grisea farnesyl pyrophosphate synthetase (SEQ ID NO:2). Figure 3 is an amino acid sequence for M. grisea farnesyl pyrophosphate synthetase (SEQ ID NO:3).

Figure 4 is the nucleic acid sequence for M. grisea citrate synthetase (SEQ ID NO:4). Figure 5 is a nucleic acid subsequence of M. grisea citrate synthetase (SEQ ID

NO:5).

Figure 6 is an amino acid sequence for M. grisea citrate synthetase (SEQ ID NO: 6). Figure 7 is the nucleic acid sequence for M. grisea isocitrate dehydrogenase [NAD] subunit 1, mitochondrial precursor (SEQ ID NO:7). Figure 8 is a nucleic acid subsequence of M. grisea isocitrate dehydrogenase [NAD] subunit 1, mitochondrial precursor (SEQ ID NO:8).

Figure 9 is an amino acid sequence for M. grisea isocitrate dehydrogenase [NAD] subunit 1, mitochondrial precursor (SEQ ID NO:9).

Figure 10 is the nucleic acid sequence for M. grisea ABCl (SEQ ID NO: 10). Figure 11 is a nucleic acid subsequence of M. grisea ABCl (SEQ ID NO:11).

Figure 12 is a nucleic acid sequence of/⁷, graminearum farnesyl pyrophosphate synthetase (SEQ ID NO: 12).

Figure 13 is a nucleic acid subsequence of F. graminearum farnesyl pyrophosphate synthetase (SEQ ID NO: 13). Figure 14 is an amino acid sequence of F. graminearum farnesyl pyrophosphate synthetase (SEQ ID NO: 14).

Figure 15 is a nucleic acid sequence of F. graminearum farnesyl citrate synthetase (SEQ ID NO: 15).

Figure 16 is a nucleic acid subsequence of F. graminearum farnesyl citrate synthetase (SEQ ID NO: 16).

Figure 17 is an amino acid sequence of F. graminearum farnesyl citrate synthetase (SEQ ID NO: 17).

Figure 18 is a nucleic acid sequence of F. graminearum isocitrate dehydrogenase (SEQ ID NO: 18). Figure 19 is an amino acid sequence for F. graminearum isocitrate dehydrogenase

(SEQ ID NO: 19).

DEFINITIONS

To facilitate an understanding of the present invention, a number of terms and phrases as used herein are defined below: The term "plant" is used in it broadest sense. It includes, but is not limited to, any species of woody, ornamental or decorative, crop (e.g., rice) or cereal, fruit or vegetable plant, and photosynthetic green algae. It also refers to a plurality of plant cells that are largely differentiated into a structure that is present at any stage of a plant's development. Such structures include, but are not limited to, a fruit, shoot, stem, leaf, flower petal, etc.

The term "plant tissue" includes differentiated and undifferentiated tissues of plants including those present in roots, shoots, leaves, pollen, seeds and tumors, as well as cells in culture (e.g., single cells, protoplasts, embryos, callus, etc.). Plant tissue maybe in planta, in organ culture, tissue culture, or cell culture. The term "plant part" as used herein refers to a plant structure, a plant organ, or a plant tissue.

The term "crop" or "crop plant" is used in its broadest sense. The term includes, but is not limited to, any species of plant or algae edible by humans or used as a feed for animals or used, or consumed by humans, or any plant or algae used in industry or commerce. The term plant cell "compartments or organelles" is used in its broadest sense. The term includes but is not limited to, the endoplasmic reticulum, Golgi apparatus, trans Golgi network, plastids including chloroplasts, proplastids, and leucoplasts, sarcoplasmic reticulum, glyoxysomes, mitochondrial, chloroplast, and nuclear membranes, and the like. As used herein, the term "rice blast disease," "blast" or other similar terms refer to a fungal disease of the rice crop caused by the Magnaporthe grisea fungus. The disease can strike all aerial parts of the plant. Infections occur on the leaves, causing diamond-shaped lesions with a gray or white center to appear, or on the panicles, which turn white and die before being filled with grain. Once on a rice plant, the fungus rapidly produces thousands of spores, which are carried readily through the air, by wind or rain, onto neighboring plants.

As used herein, the term "fungi" is used in its broadest sense. Generally, fungi are classified within the Fungal Kingdom of living things, and are characterized for absorbing food in solution directly through their cell walls and reproducing through spores. Examples of fungi include, but are not limited to, fungi from the ascomycota fungal phyla (e.g., Magnaporthe grisea), fungi from the basidiomycota fungal phyla, from the chytridiomycota fungal phyla, fungi from the glomeromycota fungal phyla and fungi from the zygomycota fungal phyla.

As used herein, the term "farnesyl pyrophosphate synthetase" when used in reference to a protein or nucleic acid refers to a protein or nucleic acid encoding a protein that shares greater than about 50% identity with any of SEQ ID NOs: 1, 2, 3, 12, 13 and 14. Thus, the term farnesyl pyrophosphate synthetase encompasses both proteins that are identical to wild-type farnesyl pyrophosphate synthetase and those that are derived from wild type farnesyl pyrophosphate synthetase protein (e.g., variants of farnesyl pyrophosphate synthetase).

As used herein, the terms "farnesyl pyrophosphate synthetase gene" and " farnesyl pyrophosphate synthetase nucleic acid sequence" refer to the full length M. grisea fungi or F. graminearum farnesyl pyrophosphate synthetase nucleic acid sequences, as well as sequences provided as SEQ ID NOs: 1, 2, 12 and 13, sequences which bind to SEQ ID NOs: 1, 2, 12 and 13 under conditions of high stringency, and M. grisea fungi or F. graminearum farnesyl pyrophosphate synthetase nucleic acid sequences available in public databases. These terms encompass fragments of the farnesyl pyrophosphate synthetase sequences and include DNA, cDNA, and RNA (e.g., mRNA) sequences.

As used herein, the term "citrate synthase" when used in reference to a protein or nucleic acid refers to a protein or nucleic acid encoding a protein that shares greater than about 50% identity with any of SEQ ID NOs: 4, 5, 6, 15, 16 and 17. Thus, the term citrate synthase encompasses both proteins that are identical to wild-type citrate synthase and those that are derived from wild type citrate synthase protein (e.g., variants of citrate synthase). As used herein, the terms "citrate synthase gene" and "citrate synthase nucleic acid sequence" refer to the full length M. grisea fungi or F. graminearum citrate synthase nucleic acid sequence, as well as sequence provided as SEQ ID NOs: 4, 5, 15 and 16, sequences which bind to SEQ ID NOs: 4, 5, 15 and 16 under conditions of high stringency, and M. grisea fungi or F. graminearum citrate synthase nucleic acid sequences available in public databases. These terms encompass fragments of the citrate synthase sequences and include DNA, cDNA, and RNA (e.g., mRNA) sequences.

As used herein, the term "isocitrate dehydrogenase [NAD] subunit 1 - mitochondrial precursor" when used in reference to a protein or nucleic acid refers to a protein or nucleic acid encoding a protein that shares greater than about 50% identity with any of SEQ ID NOs: 7, 8 and 9. Thus, the term isocitrate dehydrogenase [NAD] subunit 1 - mitochondrial precursor encompasses both proteins that are identical to wild-type isocitrate dehydrogenase

[NAD] subunit 1 - mitochondrial precursor and those that are derived from wild type isocitrate dehydrogenase [NAD] subunit 1 - mitochondrial precursor protein (e.g., variants of isocitrate dehydrogenase [NAD] subunit 1 - mitochondrial precursor). As used herein, the terms "isocitrate dehydrogenase [NAD] subunit 1 - mitochondrial precursor gene" and "isocitrate dehydrogenase [NAD] subunit 1 - mitochondrial precursor nucleic acid sequence" refer to the full length M. grisea isocitrate dehydrogenase [NAD] subunit 1 - mitochondrial precursor nucleic acid sequence, as well as sequence provided as SEQ ID NOs: 7 and 8, sequences which bind to SEQ ID NOs: 7 and 8 under conditions of high stringency, and M. grisea isocitrate dehydrogenase [NAD] subunit 1 - mitochondrial precursor nucleic acid sequences available in public databases. These terms encompass fragments of the isocitrate dehydrogenase [NAD] subunit 1 - mitochondrial precursor sequences and include DNA, cDNA, and RNA (e.g., mRNA) sequences.

As used herein, the term "isocitrate dehydrogenase" when used in reference to a protein or nucleic acid refers to a protein or nucleic acid encoding a protein that shares greater than about 50% identity with any of SEQ ID NOs: 18 and 19. Thus, the term isocitrate dehydrogenase encompasses both proteins that are identical to wild-type isocitrate dehydrogenase and those that are derived from wild type isocitrate dehydrogenase {e.g., variants of isocitrate dehydrogenase).

As used herein, the terms "isocitrate dehydrogenase gene" and "isocitrate dehydrogenase nucleic acid sequence" refer to the full length F. graminearum isocitrate dehydrogenase nucleic acid sequence, as well as sequence provided as SEQ ID NO: 18, sequences which bind to SEQ ID NO: 18 under conditions of high stringency, and F. graminearum isocitrate dehydrogenase nucleic acid sequences available in public databases. These terms encompass fragments of the isocitrate dehydrogenase sequences and include DNA, cDNA, and RNA {e.g., mRNA) sequences.

As used herein, the term "ABCl" when used in reference to a protein or nucleic acid refers to a protein or nucleic acid encoding a protein that shares greater than about 50% identity with any of SEQ ID NOs: 10 and 11. Thus, the term ABC 1 encompasses both proteins that are identical to wild-type ABCl and those that are derived from wild type ABCl protein {e.g., variants of ABCl).

As used herein, the terms "ABCl gene" and "ABCl nucleic acid sequence" refer to the full length M. grisea ABCl nucleic acid sequence, as well as sequence provided as SEQ

ID NOs: 10 and 11, sequences which bind to SEQ ID NOs: 10 and 11 under conditions of high stringency, and M. grisea ABCl nucleic acid sequences available in public databases. These terms encompass fragments of the ABCl sequences and include DNA, cDNA, and RNA {e.g., mRNA) sequences. The term "host cell" refers to any cell capable of replicating and/or transcribing and/or translating a heterologous gene.

The term "heterologous," when used in reference to DNA sequences or genes, means a DNA sequence encoding a protein, polypeptide, RNA, or a portion of any thereof, whose exact amino acid sequence is not normally found in the host cell, but is introduced by standard gene transfer techniques.

The term "RNA interference" or "RNAi" refers to the silencing or decreasing of gene expression by iRNA (interfering RNAs) or siRNAs (small interfering RNAs). It is the process of sequence-specific, post-transcriptional gene silencing in animals and plants, initiated by iRNA that is homologous in its duplex region to the sequence of the silenced gene. The gene may be endogenous or exogenous to the organism, present integrated into a chromosome or present in a transfection vector that is not integrated into the genome. The expression of the gene is either completely or partially inhibited. RNAi may also be considered to inhibit the function of a target RNA; the function of the target RNA may be complete or partial.

The term "interfering RNA (iRNA)" refers to a double stranded RNA molecule that mediates RNA interference (RNAi). At least one strand of the duplex or double-stranded region of an iRNA is substantially homologous to or substantially complementary to a target RNA molecule. The strand complementary to a target RNA molecule is the "antisense strand;" the strand homologous to the target RNA molecule is the "sense strand," and is also complementary to the iRNA antisense strand. iRNAs may also contain additional sequences; non-limiting examples of such sequences include linking sequences, or loops, as well as stem and other folded structures.

The iRNA can serve as a source of siRNA. siRNAs generally comprise a duplex, or double-stranded region, of about 18-25 nucleotides long; often siRNAs contain from about two to four unpaired nucleotides at the 3' end of each strand. At least one strand of the duplex or double-stranded region of a siRNA is substantially homologous to or substantially complementary to a target RNA molecule. The strand complementary to a target RNA molecule is the "antisense strand;" the strand homologous to the target RNA molecule is the "sense strand," and is also complementary to the siRNA antisense strand. siRNAs may also contain additional sequences; non-limiting ex,amples of such sequences include linking sequences, or loops, as well as stem and other folded structures. siRNAs appear to function as key intermediaries in triggering RNA interference in invertebrates and in vertebrates, and in triggering sequence-specific RNA degradation during posttranscriptional gene silencing in plants.

The term "target RNA molecule" refers to an RNA molecule to which at least one strand of the short double-stranded region of an iRNA is homologous or complementary. Typically, when such homology or complementary is about 100%, the siRNA is able to silence or inhibit expression of the target RNA molecule. Although it is believed that processed mRNA is a target of siRNA, the present invention is not limited to any particular hypothesis, and such hypotheses are not necessary to practice the present invention. Thus, it is contemplated that other RNA molecules may also be targets of siRNA. Such targets include unprocessed mRNA, ribosomal RNA, and viral RNA genomes.

As used herein, the term "loop sequence" refers to a nucleic acid sequence that is placed between two nucleic sequences that are complementary to each other and wliich forms a loops when the complementary nucleic acid sequences hybridize to one another.

The term "fungal target RNA" as used herein refers to an RNA that is expressed in a fungus (e.g., M. grisea).

The term "double stranded fungal RNA sequence" refers to an iRNA that is specific for a fungal target RNA (e.g., farnesyl phyrophosphate synthetase).

The term "inhibits the proliferation of fungi" refers to a reduction in fungal parasitism of a host organism. The inhibition of proliferation can be mediated in a variety of ways, including, but not limited to, decreasing the fitness of the fungus by decreasing or inhibiting the expression of genes required for fungal metabolism (e.g., farnesyl phyrophosphate synthetase, citrate synthase).

The terms "protein" and "polypeptide" refer to compounds comprising amino acids joined via peptide bonds and are used interchangeably. As used herein, "amino acid sequence" refers to an amino acid sequence of a protein molecule. "Amino acid sequence" and like terms, such as "polypeptide" or "protein," are not meant to limit the amino acid sequence to the complete, native amino acid sequence associated with the recited protein molecule. Furthermore, an "amino acid sequence" can be deduced from the nucleic acid sequence encoding the protein. The term "portion" when used in reference to a protein (as in "a portion of a given protein") refers to fragments of that protein. The fragments may range in size from four amino acid residues to the entire amino sequence minus one amino acid.

The term "homology" when used in relation to amino acids refers to a degree of similarity or identity. There may be partial homology or complete homology {i.e., identity). "Sequence identity" refers to a measure of relatedness between two or more proteins, and is given as a percentage with reference to the total comparison length. The identity calculation takes into account those amino acid residues that are identical and in the same relative positions in their respective larger sequences. Calculations of identity may be performed by algorithms contained within computer programs.

The term "homolog" or "homologous" when used in reference to a polypeptide refers to a high degree of sequence identity between two polypeptides, or to a high degree of similarity between the three-dimensional structure or to a high degree of similarity between the active site and the mechanism of action, hi a preferred embodiment, a homolog has a greater than 60% sequence identity, and more preferable greater than 75% sequence identity, and still more preferably greater than 90% sequence identity, with a reference sequence.

The terms "variant" and "mutant" when used in reference to a polypeptide refer to an amino acid sequence that differs by one or more amino acids from another, usually related polypeptide. The variant may have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties (e.g., replacement of leucine with isoleucine). More rarely, a variant may have "non-conservative" changes (e.g., replacement of a glycine with a tryptophan). Similar minor variations may also include amino acid deletions or insertions (in other words, additions), or both. Guidance in determining which and how many amino acid residues may be substituted, inserted or deleted without abolishing biological activity may be found using computer programs well known in the art, for example, DNAStar software. Variants can be tested in functional assays. Preferred variants have less than 10%, and preferably less than 5%, and still more preferably less than 2% changes (whether substitutions, deletions, and so on). The term "gene" refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises coding sequences necessary for the production of an RNA, or a polypeptide or its precursor (e.g., proinsulin). A functional polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence as long as the desired activity or functional properties (e.g., enzymatic activity, ligand binding, signal transduction, etc.) of the polypeptide are retained. The term "portion" when used in reference to a gene refers to fragments of that gene. The fragments may range in size from a few nucleotides to the entire gene sequence minus one nucleotide. Thus, "a nucleotide comprising at least a portion of a gene" may comprise fragments of the gene or the entire gene. The term "gene" also encompasses the coding regions of a structural gene and includes sequences located adjacent to the coding region on both the 5' and 3' ends for a distance of about 1 kb on either end such that the gene corresponds to the length of the full- length mKNA. The sequences which are located 5' of the coding region and which are present on the mRNA are referred to as 5 ' non-translated sequences. The sequences which are located 3' or downstream of the coding region and which are present on the mRNA are referred to as 3' non-translated sequences. The term "gene" encompasses both cDNA and genomic forms of a gene. A genomic form or clone of a gene contains the coding region interrupted with non-coding sequences termed "introns" or "intervening regions" or "intervening sequences." Introns are segments of a gene which are transcribed into nuclear

RNA (hiiRNA); introns may contain regulatory elements such as enhancers. Introns are removed or "spliced out" from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript. The mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide. In addition to containing introns, genomic forms of a gene may also include sequences located on both the 5' and 3' end of the sequences that are present on the RNA transcript. These sequences are referred to as "flanking" sequences or regions (these flanking sequences are located 5' or 3' to the non-translated sequences present on the mRNA transcript). The 5' flanking region may contain regulatory sequences such as promoters and enhancers that control or influence the transcription of the gene. The 3' flanking region may contain sequences that direct the termination of transcription, posttranscriptional cleavage and polyadenylation.

The term "heterologous gene" refers to a gene encoding a factor that is not in its natural environment (i.e., has been altered by the hand of man). For example, a heterologous gene includes a gene from one species introduced into another species. A heterologous gene also includes a gene native to an organism that has been altered in some way (e.g., mutated, added in multiple copies, linked to a non-native promoter or enhancer sequence, etc.). Heterologous genes may comprise plant gene sequences that comprise cDNA forms of a plant gene; the cDNA sequences may be expressed in either a sense (to produce mRNA) or anti-sense orientation (to produce an anti-sense RNA transcript that is complementary to the mRNA transcript). Heterologous genes are distinguished from endogenous plant genes in that the heterologous gene sequences are typically joined to nucleotide sequences comprising regulatory elements such as promoters that are not found naturally associated with the gene for the protein encoded by the heterologous gene or with plant gene sequences in the chromosome, or are associated with portions of the chromosome not found in nature (e.g., genes expressed in loci where the gene is not normally expressed).

The term "oligonucleotide" refers to a molecule comprised of two or more deoxyribonucleotides or ribonucleotides, preferably more than three, and usually more than ten. The exact size will depend on many factors, which in turn depends on the ultimate function or use of the oligonucleotide. The oligonucleotide may be generated in any manner, including chemical synthesis, DNA replication, reverse transcription, or a combination thereof. The term "an oligonucleotide having a nucleotide sequence encoding a gene" or "a nucleic acid sequence encoding" a specified polypeptide refers to a nucleic acid sequence comprising the coding region of a gene or in other words the nucleic acid sequence which encodes a gene product. The coding region may be present in either a cDNA, genomic DNA or RNA form. When present in a DNA form, the oligonucleotide may be single- stranded (i.e., the sense strand) or double-stranded. Suitable control elements such as enhancers/promoters, splice junctions, polyadenylation signals, etc. may be placed in close proximity to the coding region of the gene if needed to permit proper initiation of transcription and/or correct processing of the primary RNA transcript. Alternatively, the coding region utilized in the expression vectors of the present invention may contain endogenous enhancers/promoters, splice junctions, intervening sequences, polyadenylation signals, etc. or a combination of both endogenous and exogenous control elements.

The terms "complementary" and "complementarity" refer to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. For example, for the sequence "A-G-T," is complementary to the sequence "T-C-A." Complementarity may be "partial," in which only some of the nucleic acids' bases are matched according to the base pairing rules.

Or, there may be "complete" or "total" complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as detection methods which depend upon binding between nucleic acids.

The term "homology" when used in relation to nucleic acids refers to a degree of complementarity. There may be partial homology or complete homology (i.e., identity). "Sequence identity" refers to a measure of relatedness between two or more nucleic acids, and is given as a percentage with reference to the total comparison length. The identity calculation takes into account those nucleotide residues that are identical and in the same relative positions in their respective larger sequences. Calculations of identity may be performed by algorithms contained within computer programs such as "GAP" (Genetics Computer Group, Madison, Wis.) and "ALIGN" (DNAStar, Madison, Wis.). A partially complementary sequence is one that at least partially inhibits (or competes with) a completely complementary sequence from hybridizing to a target nucleic acid is referred to using the functional term "substantially homologous." The inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or Northern blot, solution hybridization and the like) under conditions of low stringency. A substantially homologous sequence or probe will compete for and inhibit the binding (i.e., the hybridization) of a sequence that is completely homologous to a target under conditions of low stringency. This is not to say that conditions of low stringency are such that non-specific binding is permitted; low stringency conditions require that the binding of two sequences to one another be a specific (i.e., selective) interaction. The absence of non-specific binding may be tested by the use of a second target which lacks even a partial degree of complementarity (e.g., less than about 30% identity); in the absence of non-specific binding the probe will not hybridize to the second non-complementary target.

When used in reference to a double-stranded nucleic acid sequence such as a cDNA or genomic clone, the term "substantially homologous" refers to any probe which can hybridize to either or both strands of the double-stranded nucleic acid sequence under conditions of low stringency as described infra.

The term "hybridization" refers to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is impacted by such factors as the degree of complementary between the nucleic acids, stringency of the conditions involved, the T_m of the formed hybrid, and the G:C ratio within the nucleic acids. A single molecule that contains pairing of complementary nucleic acids within its structure is said to be "self-hybridized."

The term "T_m" refers to the "melting temperature" of a nucleic acid. The melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands. The equation for calculating the T_m of nucleic acids is well known in the art. As indicated by standard references, a simple estimate of the T_m value may be calculated by the equation: T_m = 81.5 + 0.41(% G + C), when a nucleic acid is in aqueous solution at 1 M NaCl (See e.g., Anderson and Young, Quantitative Filter Hybridization (1985) in Nucleic Acid Hybridization). Other references include more sophisticated computations that take structural as well as sequence characteristics into account for the calculation of T_m.

As used herein the term "stringency" refers to the conditions of temperature, ionic strength, and the presence of other compounds such as organic solvents, under which nucleic acid hybridizations are conducted. With "high stringency" conditions, nucleic acid base pairing will occur only between nucleic acid fragments that have a high frequency of complementary base sequences. Thus, conditions of "low" stringency are often required with nucleic acids that are derived from organisms that are genetically diverse, as the frequency of complementary sequences is usually less.

"High stringency conditions" when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 42°C in a solution consisting of 5X SSPE (43.8 g/1 NaCl, 6.9 g/1 NaH₂PO₄-H₂O and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS, 5X Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followed by washing in a solution comprising 0. IX SSPE, 1.0% SDS at 42°C when a probe of about 500 nucleotides in length is employed.

"Medium stringency conditions" when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 42°C in a solution consisting of 5X SSPE (43.8 g/1 NaCl, 6.9 g/1 NaH₂PO₄-H₂O and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS, 5X Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followed by washing in a solution comprising LOX SSPE, 1.0% SDS at 42⁰C when a probe of about 500 nucleotides in length is employed.

"Low stringency conditions" comprise conditions equivalent to binding or hybridization at 42⁰C in a solution consisting of 5X SSPE (43.8 g/1 NaCl, 6.9 g/1 NaH₂PO₄-H₂O and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH), 0.1 % SDS, 5X

Denhardt's reagent [5OX Denhardt's contains per 500 ml: 5 g Ficoll (Type 400, Pharamcia), 5 g BSA (Fraction V; Sigma)] and 100 μg/ml denatured salmon sperm DNA followed by washing in a solution comprising 5X SSPE, 0.1% SDS at 42°C when a probe of about 500 nucleotides in length is employed. It is well known that numerous equivalent conditions may be employed to comprise desired stringency conditions; factors such as the length and nature (DNA, RNA, base composition) of the probe and nature of the target (DNA, RNA, base composition, present in solution or immobilized, etc.) and the concentration of the salts and other components (e.g., the presence or absence of formamide, dextran sulfate, polyethylene glycol) are considered and the hybridization solution may be varied to generate conditions of low stringency hybridization different from, but equivalent to, the above listed conditions, hi addition, the art knows conditions that promote hybridization under conditions of high stringency (e.g., increasing the temperature of the hybridization and/or wash steps, the use of formamide in the hybridization solution, etc.).

When used in reference to a double-stranded nucleic acid sequence such as a cDNA or genomic clone, the term "substantially homologous" refers to any probe that can hybridize to either or both strands of the double-stranded nucleic acid sequence under conditions of low to high stringency as described above. When used in reference to a single-stranded nucleic acid sequence, the term

"substantially homologous" refers to any probe that can hybridize (i.e., it is the complement of) the single-stranded nucleic acid sequence under conditions of low to high stringency as described above.

"Amplification" is a special case of nucleic acid replication involving template specificity. It is to be contrasted with non-specific template replication (i.e., replication that is template-dependent but not dependent on a specific template). Template specificity is here distinguished from fidelity of replication (i.e., synthesis of the proper polynucleotide sequence) and nucleotide (ribo- or deoxyribo-) specificity. Template specificity is frequently described in terms of "target" specificity. Target sequences are "targets" in the sense that they are sought to be sorted out from other nucleic acid. Amplification techniques have been designed primarily for this sorting out.

Template specificity is achieved in most amplification techniques by the choice of enzyme. Amplification enzymes are enzymes that, under conditions they are used, will process only specific sequences of nucleic acid in a heterogeneous mixture of nucleic acid. For example, in the case of Q øreplicase, MDV-I RNA is the specific template for the replicase (Kacian et al. (1972) Proc. Natl. Acad. Sci. USA, 69:3038). Other nucleic acid will not be replicated by this amplification enzyme. Similarly, in the case of T7 RNA polymerase, this amplification enzyme has a stringent specificity for its own promoters (Chamberlin et al (1970) Nature, 228:227). hi the case of T4 DNA ligase, the enzyme will not ligate the two oligonucleotides or polynucleotides, where there is a mismatch between the oligonucleotide or polynucleotide substrate and the template at the ligation junction (Wu and Wallace (1989) Genomics, 4:560). Finally, Taq and Pfu polymerases, by virtue of their ability to function at high temperature, are found to display high specificity for the sequences bounded and thus defined by the primers; the high temperature results in thermodynamic conditions that favor primer hybridization with the target sequences and not hybridization with non-target sequences (H.A. Erlich (ed.) (1989) PCR Technology, Stockton Press).

The term "amplifiable nucleic acid" refers to nucleic acids that may be amplified by any amplification method. It is contemplated that "amplifiable nucleic acid" will usually comprise "sample template."

The term "sample template" refers to nucleic acid originating from a sample that is analyzed for the presence of "target" (defined below). In contrast, "background template" is used in reference to nucleic acid other than sample template that may or may not be present in a sample. Background template is most often inadvertent. It may be the result of carryover, or it may be due to the presence of nucleic acid contaminants sought to be purified away from the sample. For example, nucleic acids from organisms other than those to be detected may be present as background in a test sample.

The term "primer" refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, (i.e., in the presence of nucleotides and an inducing agent such as DNA polymerase and at a suitable temperature and pH). The primer is preferably single stranded for maximum efficiency in amplification, but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent. The exact lengths of the primers will depend on many factors, including temperature, source of primer and the use of the method.

The term "polymerase chain reaction" ("PCR") refers to the method of K.B. Mullis U.S. Patent Nos. 4,683,195, 4,683,202, and 4,965,188, that describe a method for increasing the concentration of a segment of a target sequence in a mixture of genomic DNA without cloning or purification. This process for amplifying the target sequence consists of introducing a large excess of two oligonucleotide primers to the DNA mixture containing the desired target sequence, followed by a precise sequence of thermal cycling in the presence of a DNA polymerase. The two primers are complementary to their respective strands of the double stranded target sequence. To effect amplification, the mixture is denatured and the primers then annealed to their complementary sequences within the target molecule. Following annealing, the primers are extended with a polymerase so as to form a new pair of complementary strands. The steps of denaturation, primer annealing, and polymerase extension can be repeated many times (i.e., denaturation, annealing and extension constitute one "cycle"; there can be numerous "cycles") to obtain a high concentration of an amplified segment of the desired target sequence. The length of the amplified segment of the desired target sequence is determined by the relative positions of the primers with respect to each other, and therefore, this length is a controllable parameter. By virtue of the repeating aspect of the process, the method is referred to as the "polymerase chain reaction" (hereinafter "PCR"). Because the desired amplified segments of the target sequence become the predominant sequences (in terms of concentration) in the mixture, they are said to be "PCR amplified."

With PCR, it is possible to amplify a single copy of a specific target sequence in genomic DNA to a level detectable by several different methodologies (e.g., hybridization with a labeled probe; incorporation of biotinylated primers followed by avidin-enzyme conjugate detection; incorporation of ³²P-labeled deoxynucleotide triphosphates, such as dCTP or dATP, into the amplified segment). In addition to genomic DNA, any oligonucleotide or polynucleotide sequence can be amplified with the appropriate set of primer molecules. In particular, the amplified segments created by the PCR process itself are, themselves, efficient templates for subsequent PCR amplifications. The terms "PCR product," "PCR fragment," and "amplification product" refer to the resultant mixture of compounds after two or more cycles of the PCR steps of denaturation, annealing and extension are complete. These terms encompass the case where there has been amplification of one or more segments of one or more target sequences.

The term "amplification reagents" refers to those reagents (deoxyribonucleotide triphosphates, buffer, etc.), needed for amplification except for primers, nucleic acid template, and the amplification enzyme. Typically, amplification reagents along with other reaction components are placed and contained in a reaction vessel (test tube, microwell, etc.).

The term "reverse-transcriptase" or "RT-PCR" refers to a type of PCR where the starting material is mRNA. The starting mRNA is enzymatically converted to complementary DNA or "cDNA" using a reverse transcriptase enzyme. The cDNA is then used as a "template" for a "PCR" reaction.

The term "gene expression" refers to the process of converting genetic information encoded in a gene into RNA (e.g., mRNA, rRNA, tRNA, or snRNA) through "transcription" of the gene (i.e., via the enzymatic action of an RNA polymerase), and into protein, through

"translation" of mRNA. Gene expression can be regulated at many stages in the process.

"Up-regulation" or "activation" refers to regulation that increases the production of gene expression products (i.e., RNA or protein), while "down-regulation" or "repression" refers to regulation that decrease production. Molecules (e.g., transcription factors) that are involved in up-regulation or down-regulation are often called "activators" and "repressors," respectively.

The terms "in operable combination", "in operable order" and "operably linked" refer to the linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced. The term also refers to the linkage of amino acid sequences in such a manner so that a functional protein is produced.

The term "regulatory element" refers to a genetic element that controls some aspect of the expression of nucleic acid sequences. For example, a promoter is a regulatory element that facilitates the initiation of transcription of an operably linked coding region.

Other regulatory elements are splicing signals, polyadenylation signals, termination signals, etc.

Transcriptional control signals in eukaryotes comprise "promoter" and "enhancer" elements. Promoters and enhancers consist of short arrays of DNA sequences that interact specifically with cellular proteins involved in transcription (Maniatis, et al. , Science

236:1237, 1987). Promoter and enhancer elements have been isolated from a variety of eukaryotic sources including genes in yeast, insect, mammalian and plant cells. Promoter and enhancer elements have also been isolated from viruses and analogous control elements, such as promoters, are also found in prokaryotes. The selection of a particular promoter and enhancer depends on the cell type used to express the protein of interest. Some eukaryotic promoters and enhancers have a broad host range while others are functional in a limited subset of cell types (for review, see Voss, et al, Trends Biochem. ScL, 11 :287, 1986; and

Maniatis, et al, supra 1987).

The terms "promoter element," "promoter," or "promoter sequence" as used herein, refer to a DNA sequence that is located at the 5' end (i.e. precedes) the protein coding region of a DNA polymer. The location of most promoters known in nature precedes the transcribed region. The promoter functions as a switch, activating the expression of a gene.

If the gene is activated, it is said to be transcribed, or participating in transcription.

Transcription involves the synthesis of mRNA from the gene. The promoter, therefore, serves as a transcriptional regulatory element and also provides a site for initiation of transcription of the gene into mRNA.

Promoters may be tissue specific or cell specific. The term "tissue specific" as it applies to a promoter refers to a promoter that is capable of directing selective expression of a nucleotide sequence of interest to a specific type of tissue in the relative absence of expression of the same nucleotide sequence of interest in a different type of tissue. Tissue specificity of a promoter may be evaluated by, for example, operably linking a reporter gene to the promoter sequence to generate a reporter construct, introducing the reporter construct into the genome of a plant such that the reporter construct is integrated into every tissue of the resulting transgenic plant, and detecting the expression of the reporter gene (e.g., detecting mRNA, protein, or the activity of a protein encoded by the reporter gene) in different tissues of the transgenic plant. The detection of a greater level of expression of the reporter gene in one or more tissues relative to the level of expression of the reporter gene in other tissues shows that the promoter is specific for the tissues in which greater levels of expression are detected. The term "cell type specific" as applied to a promoter refers to a promoter which is capable of directing selective expression of a nucleotide sequence of interest in a specific type of cell in the relative absence of expression of the same nucleotide sequence of interest in a different type of cell within the same tissue. The term "cell type specific" when applied to a promoter also means a promoter capable of promoting selective expression of a nucleotide sequence of interest in a region within a single tissue. Cell type specificity of a promoter may be assessed using methods well known in the art, e.g., immunohistochemical staining. Briefly, tissue sections are embedded in paraffin, and paraffin sections are reacted with a primary antibody that is specific for the polypeptide product encoded by the nucleotide sequence of interest whose expression is controlled by the promoter. A labeled (e.g., peroxidase conjugated) secondary antibody that is specific for the primary antibody is allowed to bind to the sectioned tissue and specific binding detected (e.g., with avidin/biotin) by microscopy.

Promoters may be constitutive or regulatable. The term "constitutive" when made in reference to a promoter means that the promoter is capable of directing transcription of an operably linked nucleic acid sequence in the absence of a stimulus (e.g., heat shock, chemicals, light, etc.). Typically, constitutive promoters are capable of directing expression of a transgene in substantially any cell and any tissue. Exemplary constitutive plant promoters include, but are not limited to SD Cauliflower Mosaic Virus (CaMV SD; see e.g., U.S. Pat. No. 5,352,605, incorporated herein by reference), mannopine synthase, octopine synthase (ocs), superpromoter (see e.g., WO 95/14098), and ubi3 (see e.g., Garbarino and Belknap (1994) Plant MoI. Biol. 24: 119-127) promoters. Such promoters have been used successfully to direct the expression of heterologous nucleic acid sequences in transformed plant tissue. In contrast, a "regulatable" promoter is one which is capable of directing a level of transcription of an operably linked nuclei acid sequence in the presence of a stimulus (e.g., heat shock, chemicals, light, etc.) which is different from the level of transcription of the operably linked nucleic acid sequence in the absence of the stimulus.

The enhancer and/or promoter may be "endogenous" or "exogenous" or "heterologous." An "endogenous" enhancer or promoter is one that is naturally linked with a given gene in the genome. An "exogenous" or "heterologous" enhancer or promoter is one that is placed in juxtaposition to a gene by means of genetic manipulation (i.e., molecular biological techniques) such that transcription of the gene is directed by the linked enhancer or promoter. For example, an endogenous promoter in operable combination with a first gene can be isolated, removed, and placed in operable combination with a second gene, thereby making it a "heterologous promoter" in operable combination with the second gene. A variety of such combinations are contemplated (e.g., the first and second genes can be from the same species, or from different species.

The presence of "splicing signals" on an expression vector often results in higher levels of expression of the recombinant transcript in eukaryotic host cells. Splicing signals mediate the removal of introns from the primary RNA transcript and consist of a splice donor and acceptor site (Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual 2nd ed., Cold Spring Harbor Laboratory Press, New York, pp. 16.7-16.8). A commonly used splice donor and acceptor site is the splice junction from the 16S RNA of SV40. Efficient expression of recombinant DNA sequences in eukaryotic cells requires expression of signals directing the efficient termination and polyadenylation of the resulting transcript. Transcription termination signals are generally found downstream of the polyadenylation signal and are a few hundred nucleotides in length. The term "poly(A) site" or "poly(A) sequence" as used herein denotes a DNA sequence which directs both the termination and polyadenylation of the nascent RNA transcript. Efficient polyadenylation of the recombinant transcript is desirable, as transcripts lacking a poly(A) tail are unstable and are rapidly degraded. The poly(A) signal utilized in an expression vector may be "heterologous" or "endogenous." An endogenous poly(A) signal is one that is found naturally at the 3' end of the coding region of a given gene in the genome. A heterologous poly(A) signal is one which has been isolated from one gene and positioned 3¹ to another gene. A commonly used heterologous poly(A) signal is the SV40 poly(A) signal. The SV40 poly(A) signal is contained on a 237 bp BamΗUBcll restriction fragment and directs both termination and polyadenylation (Sambrook, supra, at 16.6-16.7). The term "selectable marker" refers to a gene which encodes an enzyme having an activity that confers resistance to an antibiotic or drug upon the cell in which the selectable marker is expressed, or which confers expression of a trait which can be detected {e.g., luminescence or fluorescence). Selectable markers may be "positive" or "negative." Examples of positive selectable markers include the neomycin phosphotransferase (NPTII) gene which confers resistance to G418 and to kanamycin, and the bacterial hygromycin phosphotransferase gene (hyg), which confers resistance to the antibiotic hygromycin. Negative selectable markers encode an enzymatic activity whose expression is cytotoxic to the cell when grown in an appropriate selective medium. For example, the ΗSV-tk gene is commonly used as a negative selectable marker. Expression of the HSV-tk gene in cells grown in the presence of gancyclovir or acyclovir is cytotoxic; thus, growth of cells in selective medium containing gancyclovir or acyclovir selects against cells capable of expressing a functional HSV TK enzyme.

The term "vector refers to nucleic acid molecules that transfer DNA segment(s) from one cell to another. The term "vehicle" is sometimes used interchangeably with "vector." The terms "expression vector" or "expression cassette" refer to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism. Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), and a ribosome binding site, often along with other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.

The terms "transfection", "transformation", "transfected" and "transformed" are used interchangeably and refer to the introduction of foreign DNA into cells. Transfection may be accomplished by a variety of means known to the art including calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, glass beads, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, viral infection, biolistics (i.e., particle bombardment) and the like.

The terms "infecting" and "infection" when used with a bacterium refer to co- incubation of a target biological sample, (e.g., cell, tissue, etc.) with the bacterium under conditions such that nucleic acid sequences contained within the bacterium are introduced into one or more cells of the target biological sample.

The terms "bombarding, "bombardment," and "biolistic bombardment" refer to the process of accelerating particles towards a target biological sample (e.g., cell, tissue, etc.) to effect wounding of the cell membrane of a cell in the target biological sample and/or entry of the particles into the target biological sample. Methods for biolistic bombardment are known in the art (e.g., U.S. Patent No. 5,584,807, the contents of which are incorporated herein by reference), and are commercially available (e.g., the helium gas-driven microprojectile accelerator (PDS-1000/He, BioRad). The term "microwounding" when made in reference to plant tissue refers to the introduction of microscopic wounds in that tissue. Microwounding may be achieved by, for example, particle bombardment as described herein.

The term "transgenic" when used in reference to a plant or fruit or seed (i.e., a "transgenic plant" or "transgenic fruit" or a "transgenic seed") refers to a plant or fruit or seed that contains at least one heterologous gene in one or more of its cells. The term

"transgenic plant material" refers broadly to a plant, a plant structure, a plant tissue, a plant seed or a plant cell that contains at least one heterologous gene in one or more of its cells. The terms "transformants" or "transformed cells" include the primary transformed cell and cultures derived from that cell without regard to the number of transfers. All progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same functionality as screened for in the originally transformed cell are included in the definition of transformants.

The term "wild-type" when made in reference to a gene refers to a gene that has the characteristics of a gene isolated from a naturally occurring source. The term "wild-type" when made in reference to a gene product refers to a gene product that has the characteristics of a gene product isolated from a naturally occurring source. A wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designated the "normal" or "wild-type" form of the gene. In contrast, the term "modified" or "mutant" when made in reference to a gene or to a gene product refers, respectively, to a gene or to a gene product which displays modifications in sequence and/or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product. It is noted that naturally-occurring mutants can be isolated; these are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product. The term "antisense" refers to a deoxyribonucleotide sequence whose sequence of deoxyribonucleotide residues is in reverse 5' to 3' orientation in relation to the sequence of deoxyribonucleotide residues in a sense strand of a DNA duplex. A "sense strand" of a DNA duplex refers to a strand in a DNA duplex that is transcribed by a cell in its natural state into a "sense mRNA." Thus an "antisense" sequence is a sequence having the same sequence as the non-coding strand in a DNA duplex. The term "antisense RNA" refers to a RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target gene by interfering with the processing, transport and/or translation of its primary transcript or mRNA. The complementarity of an antisense RNA may be with any part of the specific gene transcript, i.e., at the 5' non-coding sequence, 3' non-coding sequence, introns, or the coding sequence. In addition, as used herein, antisense RNA may contain regions of ribozyme sequences that increase the efficacy of antisense RNA to block gene expression. "Ribozyme" refers to a catalytic RNA and includes sequence-specific endoribonucleases. "Antisense inhibition" refers to the production of antisense RNA transcripts capable of preventing the expression of the target protein.

The term "posttranscriptional gene silencing" or "PTGS" refers to silencing of gene expression in plants after transcription, and appears to involve the specific degradation of mRNAs synthesized from gene repeats. The term "overexpression" refers to the production of a gene product in transgenic organisms that exceeds levels of production in normal or non-transformed organisms. The term "cosuppression" refers to the expression of a foreign gene that has substantial homology to an endogenous gene resulting in the suppression of expression of both the foreign and the endogenous gene. The term "altered levels" refers to the production of gene product(s) in transgenic organisms in amounts or proportions that differ from that of normal or non-transformed organisms.

The term "recombinant" when made in reference to a nucleic acid molecule refers to a nucleic acid molecule that is comprised of segments of nucleic acid joined together by means of molecular biological techniques. The term "recombinant" when made in reference to a protein or a polypeptide refers to a protein molecule that is expressed using a recombinant nucleic acid molecule.

The term "isolated" when used in relation to a nucleic acid, as in "an isolated oligonucleotide" refers to a nucleic acid sequence that is identified and separated from at least one contaminant nucleic acid with which it is ordinarily associated in its natural source. Isolated nucleic acid is present in a form or setting that is different from that in which it is found in nature.

The term "purified" refers to molecules, either nucleic or amino acid sequences, that are removed from their natural environment, isolated or separated. An "isolated nucleic acid sequence" is therefore a purified nucleic acid sequence. "Substantially purified" molecules are at least 60% free, preferably at least 75% free, and more preferably at least 90% free from other components with which they are naturally associated. The term "purified" or "to purify" also refer to the removal of contaminants from a sample. The removal of contaminating proteins results in an increase in the percent of polypeptide of interest in the sample. In another example, recombinant polypeptides are expressed in plant, bacterial, yeast, or mammalian host cells and the polypeptides are purified by the removal of host cell proteins; the percent of recombinant polypeptides is thereby increased in the sample.

The term "sample" is used in its broadest sense. In one sense it can refer to a plant cell or tissue. In another sense, it is meant to include a specimen or culture obtained from any source, as well as biological and environmental samples. Biological samples may be obtained from plants or animals (including humans) and encompass fluids, solids, tissues, and gases. Environmental samples include environmental material such as surface matter, soil, water, and industrial samples. These examples are not to be construed as limiting the sample types applicable to the present invention.

DESCRIPTION OF THE INVENTION

The present invention relates to compositions and methods for controlling fungal infestation of plants or crops. In particular, the present invention provides vectors comprising sequences designed to control fungi by RNA interference (RNAi) and transgenic plants transformed with such vectors. The compositions and methods of the present invention can be used to inhibit the growth and reproduction of a number of fungal species, including, but not limited to fungi from the ascomycota fungal phyla (e.g., Magnaporthe grisea), fungi from the basidiomycota fungal phyla, fungi from the chytridiomycota fungal phyla, fungi from the glomeromycota fungal phyla and fungi from the zygomycota fungal phyla.

In preferred embodiments, the present invention provides compositions and methods for preventing rice blast disease. Rice blast disease is a serious disease affecting rice crop. Rice blast disease is a powerful model system for studying fungal plant diseases in general, due to the ease of molecular and genetic analyses and the availability of genome sequences for both Magnaporthe grisea and rice. Although rice lines resistant to rice blast disease have been developed, the fungus readily mutates to overcome these resistances. Novel methods are required to break this "boom and bust" cycle of resistance. The present invention utilizes genetically engineered plants expressing RNA interference (RNAi) directed toward M. grisea and F. graminearum genes that are essential for producing disease (e.g., citrate synthase, farnesyl pyrophosphate, isocitrate dehydrogenase [NAD] subunit 1 - mitochondrial precursor). Additionally, the compositions and methods of the present invention may be used to prevent other types of plant and crop fungal infestation diseases including, but not limited to, Fusarium Head Scab in cereal species such as wheat and Barley, Phytophora root rot in cereal plant species, and Rusts (e.g., Leaf Rusts, Stem Rusts, and Stripe Rusts) in both soybean and cereals.

I. RNAi RNAi refers to the introduction of homologous double stranded RNA (dsRNA) to target a specific gene product, resulting in post transcriptional silencing of that gene. This phenomena was first reported in Caenorhabditis elegans by Guo and Kemphues (Par-1, A gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed, 1995, Cell, 81 (4) 611-620) and subsequently Fire et al. (Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans , 1998, Nature 391: 806-811) discovered that it is the presence of dsRNA, formed from the annealing of sense and antisense strands present in the in vitro RNA preps, that is responsible for producing the interfering activity.

The present invention contemplates the use of RNA interference (RNAi) to downregulate the expression of genes needed for fungal infestation of plants (e.g., pathogenecity genes) and fungal viability (e.g., RNA Polymerase). In both plants and animals, RNAi is mediated by RNA-induced silencing complex (RISC), a sequence- specific, multicomponent nuclease that destroys messenger RNAs homologous to the silencing trigger. RISC is known to contain short RNAs (approximately 22 nucleotides) derived from the double-stranded RNA trigger, although the protein components of this activity are unknown. However, the 22-nucleotide RNA sequences are homologous to the target gene that is being suppressed. Thus, the 22-nucleotide sequences appear to serve as guide sequences to instruct a multicomponent nuclease, RISC, to destroy the specific mRNAs. Carthew has reported (Curr. Opin. Cell Biol. 13(2):244-248 (2001) that eukaryotes silence gene expression in the presence of dsRNA homologous to the silenced gene. Biochemical reactions that recapitulate this phenomenon generate RNA fragments of 21 to 23 nucleotides from the double-stranded RNA. These stably associate with an RNA endonuclease, and probably serve as a discriminator to select mRNAs. Once selected, mRNAs are cleaved at sites 21 to 23 nucleotides apart. hi preferred embodiments, the dsRNA used to initiate RNAi, may be isolated from native source or produced by known means, e.g., transcribed from DNA. The promoters and vectors described in more detail below are suitable for producing dsRNA. RNA is synthesized either in vivo or in vitro. In some embodiments, endogenous RNA polymerase of the cell may mediate transcription in vivo, or cloned RNA polymerase can be used for transcription in vivo or in vitro. In other embodiments, the RNA is provided by transcription from a transgene in vivo or an expression construct. In some embodiments, the RNA strands are polyadenylated; in other embodiments, the RNA strands are capable of being translated into a polypeptide by a cell's translational apparatus, hi still other embodiments, the RNA is chemically or enzymatically synthesized by manual or automated reactions. In further embodiments, the RNA is synthesized by a cellular RNA polymerase or a bacteriophage RNA polymerase (e.g., T3, T7, SP6). If synthesized chemically or by in vitro enzymatic synthesis, the RNA may be purified prior to introduction into the cell. For example, RNA can be purified from a mixture by extraction with a solvent or resin, precipitation, electrophoresis, chromatography, or a combination thereof. Alternatively, the RNA may be used with no or a minimum of purification to avoid losses due to sample processing, hi some embodiments, the RNA is dried for storage or dissolved in an aqueous solution, hi other embodiments, the solution contains buffers or salts to promote annealing, and/or stabilization of the duplex strands. hi some embodiments, the dsRNA is transcribed from the vectors as two separate stands, hi other embodiments, the two strands of DNA used to form the dsRNA may belong to the same or two different duplexes in which they each form with a DNA strand of at least partially complementary sequence. When the dsRNA is thus produced, the DNA sequence to be transcribed is flanked by two promoters, one controlling the transcription of one of the strands, and the other that of the complementary strand. These two promoters may be identical or different. In some embodiments, a DNA duplex provided at each end with a promoter sequence can directly generate RNAs of defined length, and which can join in pairs to form a dsRNA. See, e.g., U.S. Pat. No. 5,795,715, incorporated herein by reference. RNA duplex formation may be initiated either inside or outside the cell.

Inhibition is sequence-specific in that nucleotide sequences corresponding to the duplex region of the RNA are targeted for genetic inhibition. RNA molecules containing a nucleotide sequence identical to a portion of the target gene are preferred for inhibition.

RNA sequences with insertions, deletions, and single point mutations relative to the target sequence have also been found to be effective for inhibition. Thus, sequence identity may optimized by sequence comparison and alignment algorithms known in the art (see Gribskov and Devereux, Sequence Analysis Primer, Stockton Press, 1991, and references cited therein) and calculating the percent difference between the nucleotide sequences by, for example, the Smith-Waterman algorithm as implemented in the BESTFIT software program using default parameters (e.g., University of Wisconsin Genetic Computing Group). Greater than 90% sequence identity, or even 100% sequence identity, between the inhibitory RNA and the portion of the target gene is preferred. Alternatively, the duplex region of the RNA may be defined functionally as a nucleotide sequence that is capable of hybridizing with a portion of the target gene transcript. The length of the identical nucleotide sequences may be at least 25, 50, 100, 200, 300 or 400 bases.

There is no upper limit on the length of the dsRNA that can be used. For example, the dsRNA can range from about 21 base pairs (bp) of the gene to the full length of the gene or more. In one embodiment, the dsRNA used in the methods of the present invention is about 1000 bp in length. In another embodiment, the dsRNA is about 500 bp in length. In yet another embodiment, the dsRNA is about 22 bp in length.

In some preferred embodiments, the sequences that mediate RNAi are from about 21 to about 23 nucleotides. That is, the isolated RNAs of the present invention mediate degradation of the target RNA (e.g., farnesyl pyrophosphate synthetase, citrate synthase).

The double stranded RNA of the present invention need only be sufficiently similar to natural RNA that it has the ability to mediate RNAi for the target RNA. In one embodiment, the present invention relates to RNA molecules of varying lengths that direct cleavage of specific mRNA to which their sequence corresponds. It is not necessary that there be perfect correspondence of the sequences, but the correspondence must be sufficient to enable the RNA to direct RNAi cleavage of the target mRNA. In a particular embodiment, the RNA molecules of the present invention comprise a 3' hydroxyl group. In some embodiments, the amount of target RNA (mRNA) is reduced in the cells of the target organism (e.g., M. griseά) exposed to target specific double stranded RNA as compared to target organisms that have not been exposed to target specific double stranded RNA.

RNAi silencing mechanisms are operative in fungi. Fungal post-transcriptional silencing (e.g., quelling) was first described in Neurospora crassa, a saprophytic relative of the blast fungus (Catalanotto, C, et al ., 2002 Genes Dev. 16:790-795; herein incorporated by reference in its entirety). Genome sequence analysis of N. crassa has identified components of the eukaryotic RNA silencing machinery (Borkovich, K.A., et al., 2004 Microbiology and Molecular Biology Reviews 68: 1-108; herein incorporated by reference in its entirety). RNAi has not been widely studied or used for functional analysis in plant pathogenic fungi due to ease in performing directed gene knock-out analyses by fungal transformation which can result in gene replacement by homologous recombination.

A critical factor for plant produced dsRNA to silence genes in an invading fungus is delivery of the RNAi molecules from the plant into the cytoplasm of the pathogen. dsRNAs are mobile in nematodes, plants and animals. Filamentous fungi closely resemble these higher eukaryotes in most biological processes examined. There is growing evidence for horizontal gene transfer in fungi (see, e.g., Marinori, G., et al, 1999 J. Bacteriology 181:6488-6496; Walton, J.D., 2000 Fungal Genetics and Biology 30:167-171; each herein incorporated by reference in their entireties). The precise transfer of genes or gene clusters suggests direct DNA uptake rather than transfer by fusion between distantly related microorganisms. Active endocytosis in M. grisea conidia and germtubes on rice leaf surfaces indicates a potential mechanism for uptake of dsRNAs (see, e.g., Atkinson, H., et al., 2002 Fungal Genetics and Biology 37:233-244; herein incorporated by reference in its entirety).

Accordingly, in some embodiments, the present invention provides isolated RNA molecules (double-stranded or single-stranded) that are complementary to sequences required for fungus viability (e.g., RNA Polymerase) and/or pathogenecity (e.g., citrate synthase, farnesyl pyrophosphate, isocitrate dehydrogenase [NAD] subunit 1 — mitochondrial precursor). M. grisea has been analyzed in detail at the genetic, cellular, and molecular level, and many critical pathogenicity genes have been identified (see, e.g., Howard, RJ. & Valent, B. 1996 Annnual Review of Microbiology 50:491-512; Talbot, NJ.

2003 Ann. Rev. Microbiol. 57:177-202; each herein incorporated by reference in their entireties). The M. grisea genome is completely sequenced and publicly available. Identification of essential pathogenecity genes have been discovered through a variety of techniques including, but not limited to, high-throughput strategies. In experiments conducted during the course of the present invention, fungal genes specifically expressed by invasive hyphae (e.g., the specialized bulbous hyphae that grow within living rice cells and establish successful infection) have been identified through laser-capture microdissection of invaded rice cells. In other embodiments, genes specific to fungi are utilized for RNAi so as to minimize interactions against endogenous proteins of plants, livestock and humans. .

In some embodiments, probes that are specific for a fungal gene of interest are amplified from a DNA sample prepared from a fungus by using primers designed from fungus genomic DNA or cDNA. Genes amplified from fungus DNA are then used as probes for homologous genes from a genomic or cDNA libraries prepared from the fungus of interest (e.g., M. grisea). In other embodiments, degenerate primers based on the fungus sequences are utilized to amplify the gene of interest from a library derived from the fungus of interest (e.g., M. grisea). These genes are then inserted into an expression vector so that a fungal double stranded RNA corresponding to the gene of interest is produced when the vector is used to transfect a plant. Accordingly, in some embodiments, the present invention utilizes RNAi genes encoding dsRNA sequences that target fungal genes identified as having embryonic lethal or sterile RNAi phenotypes. The coding sequences for the target RNAs are available in public databases, including http://www.broad.mit.edu/annotation/fungi/magnaporthe/index.html. In still further embodiments, the genes utilized for RNAi are critical pathogenecity genes, including, but not limited to farnesyl pyrophosphate synthetase, citrate synthase, isocitrate dehydrogenase [NAD] subunit 1 - mitochondrial precursor, and ABCl (see, e.g., Howard, RJ. & Valent, B. 1996 Annual Review of Microbiology 50:491-512; Talbot, NJ. 2003 Annual Review of Microbiology 57:177-202; each herein incorporated by reference in their entireties). The methods and compositions of the present invention have been exemplified for the control of

M. grisea. However, it will be recognized that these materials and methods can be used in the control of other fungi. Accordingly the present invention provides the sequences for M. grisea farnesyl pyrophosphate synthetase (e.g., SEQ ID NOs: 1, 2 and 3), citrate synthase (e.g., SEQ ID NOS:4, 5 and 6), isocitrate dehydrogenase [NAD] subunit 1 - mitochondrial precursor (e.g., SEQ ID NOs 7, 8 and 9), and ABCl (SEQ ID NOs: 10 and 11).

Additionally, the present invention provides the sequences for F. graminearum farnesyl pyrophosphate synthetase (e.g., SEQ ID NOs: 12, 13 and 14), citrate synthase (e.g., SEQ ID NOs: 15, 16 and 17), and isocitrate dehydrogenase (e.g., SEQ ID NOs 18 and 19), As described above, the entire coding sequence of the genes can be used to make dsRNA for RNAi, or, alternatively, subsequences can be utilized. Additionally, homologous sequences from other fungi may be utilized for targeting the corresponding species of M. grisea. Such sequences can be conveniently identified by conducting BLASTN of BLASTP searches of GenBank as appropriate.

II. Transgenic Plants

In some embodiments, the present invention provides transgenic plants that express dsRNA molecules that correspond to target molecules in desired fungal species (e.g. M. grisea or Fusarium graminearium). It is contemplated that fungi feeding on the transgenic plants ingest the dsRNA molecules, which in turn decrease the abundance of target RNA within the fungal species. By targeting genes that are required for fertility or fitness of the fungus, fungal growth and reproduction is reduced thus reducing fungal induced plant damage (e.g., Rice Blast disease or Head Scab).

A heterologous gene encoding a RNAi gene of the present invention, which includes variants of the RNAi gene, includes any suitable sequence that encodes an double stranded molecule specific for a fungal target RNA. Preferably, the heterologous gene is provided within an expression vector such that transformation with the vector results in expression of the double stranded RNA molecule; suitable vectors are described below.

In yet other embodiments of the present invention, a transgenic plant comprises a heterologous gene encoding a RNAi gene of the present invention operably linked to an inducible promoter, and is grown either in the presence of the an inducing agent, or is grown and then exposed to an inducing agent. Li still other embodiments of the present invention, a transgenic plant comprises a heterologous gene encoding a RNAi gene of the present invention operably linked to a promoter which is either tissue specific or developmentally specific, and is grown to the point at which the tissue is developed or the developmental stage at which the developmentally-specific promoter is activated. Such promoters include seed and root specific promoters. In still other embodiments of the present invention, the transgenic plant comprises a RNAi gene of the present invention operably linked to constitutive promoter, hi further embodiments, the transgenic plants of the present invention express at least one double stranded RNA molecule at a level sufficient to reduce the proliferation of fungi as compared to the proliferation of fungi observed in a nontransgenic plant. 1. Plants

The methods of the present invention are not limited to any particular plant. Indeed, a variety of plants are contemplated, including but not limited to soybean, wheat, oats, milo, sorghum, cotton, tomato, potato, tobacco, pepper, rice, corn, barley, Brassica, Arabidopsis, sunflower, poplar, pineapple, banana, turf grass, and pine. Many commercial cultivars can be transformed with heterologous genes. In cases where that is not possible, noncommercial cultivars of plants can be transformed, and the trait for expression of the RNAi gene of the present invention moved to commercial cultivars by breeding techniques well- known in the art.

2. Vectors

The methods of the present invention contemplate the use of at least one heterologous gene encoding a RNAi gene of the present invention. Heterologous genes intended for expression in plants are first assembled in expression cassettes comprising a promoter. Methods which are well known to those skilled in the art may be used to construct expression vectors containing a heterologous gene and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are widely described in the art (See e.g., Sambrook. et at. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, N. Y., and Ausubel, F. M. et at

(1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N. Y).

In general, these vectors comprise a nucleic acid sequence of the invention encoding a RNAi gene of the present invention (as described above) operably linked to a promoter and other regulatory sequences (e.g., enhancers, polyadenylation signals, etc.) required for expression in a plant.

Promoters include but are not limited to constitutive promoters, tissue-, organ-, and developmentally-specific promoters, and inducible promoters. Examples of promoters include but are not limited to: constitutive promoter 35S of cauliflower mosaic virus; a wound-inducible promoter from tomato, leucine amino peptidase ("LAP," Chao et al. (1999) Plant Physiol 120: 979-992); a chemically-inducible promoter from tobacco,

Pathogenesis-Related 1 (PRl) (induced by salicylic acid and BTH (benzothiadiazole-7- carbothioic acid S-methyl ester)); a tomato proteinase inhibitor II promoter (PIN2) or LAP promoter (both inducible with methyl jasmonate); a heat shock promoter (US Pat 5,187,267); a tetracycline-inducible promoter (US Pat 5,057,422); and seed-specific promoters, such as those for seed storage proteins (e.g., phaseolin, napin, oleosin, and a promoter for soybean beta conglycin (Beachy et al. (1985) EMBO J. 4: 3047-3053)). In some preferred embodiments, the promoter is a phaseolin promoter. All references cited herein are incorporated in their entirety. The expression cassettes may further comprise any sequences required for expression of mRNA. Such sequences include, but are not limited to transcription terminators, enhancers such as introns, viral sequences, and sequences intended for the targeting of the gene product to specific organelles and cell compartments.

A variety of transcriptional terminators are available for use in expression of sequences using the promoters of the present invention. Transcriptional terminators are responsible for the termination of transcription beyond the transcript and its correct polyadenylation. Appropriate transcriptional terminators and those which are known to function in plants include, but are not limited to, the CaMV 35S terminator, the tail terminator, the pea rbcS E9 terminator, and the nopaline and octopine synthase terminator (See e.g., Odell et al. (1985) Nature 313:810; Rosenberg et al. (1987) Gene, 56:125;

Guerineau et al. (1991) MoI. Gen. Genet, 262:141; Proudfoot (1991) Cell, 64:671; Sanfacon et al. Genes Dev., 5:141 ; Mogen et al. (1990) Plant Cell, 2:1261; Munroe et al. (1990) Gene, 91:151; Ballad et al. (1989) Nucleic Acids Res. 17:7891; Joshi et al. (1987) Nucleic Acid Res., 15:9627). In addition, in some embodiments, constructs for expression of the gene of interest include one or more of sequences found to enhance gene expression from within the transcriptional unit. These sequences can be used in conjunction with the nucleic acid sequence of interest to increase expression in plants. Various intron sequences have been shown to enhance expression, particularly in monocotyledonous cells. For example, the introns of the maize Adhl gene have been found to significantly enhance the expression of the wild-type gene under its cognate promoter when introduced into maize cells (Calais et al. (1987) Genes Develop. 1: 1183). Intron sequences have been routinely incorporated into plant transformation vectors, typically within the non-translated leader.

In some embodiments of the present invention, the construct for expression of the nucleic acid sequence of interest also includes a regulator such as a nuclear localization signal (Calderone et al. (1984) Cell 39:499; Lassoer et al. (1991) Plant Molecular Biology 17:229), a plant translational consensus sequence (Joshi (1987) Nucleic Acids Research 15:6643), an intron (Luehrsen and Walbot (1991) MoI. Gen. Genet. 225:81), and the like, operably linked to the nucleic acid sequence encoding a polypeptide that inhibits the proliferation of fungi (e.g., farnesyl pyrophosphate synthetase, citrate synthase, isocitrate dehydrogenase [NAD] subunit 1 - mitochondrial precursor, ABCl).

In preparing a construct comprising a nucleic acid sequence encoding a RNAi gene of the present invention, various DNA fragments can be manipulated, so as to provide for the DNA sequences in the desired orientation (e.g., sense or antisense) orientation. For example, adapters or linkers can be employed to join the DNA fragments or other manipulations can be used to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resection, ligation, or the like is preferably employed, where insertions, deletions or substitutions (e.g., transitions and transversions) are involved.

Numerous transformation vectors are available for plant transformation. The selection of a vector for use will depend upon the preferred transformation technique and the target species for transformation. For certain target species, different antibiotic or herbicide selection markers are preferred. Selection markers used routinely in transformation include the nptll gene which confers resistance to kanamycin and related antibiotics (Messing and Vierra (1982) Gene 19: 259; Bevan et al. (1983) Nature 304:184), the bar gene which confers resistance to the herbicide phosphinothricin (White et al. (1990) Nucl Acids Res. 18:1062; Spencer et al. (1990) Theor. Appl. Genet. 79:625), the hph gene which confers resistance to the antibiotic hygromycin (Blochlinger and Diggelmann (1984)

MoI. Cell. Biol. 4:2929), and the dhfr gene, which confers resistance to methotrexate (Bourouis et al. (1983) EMBO J., 2:1099). hi some preferred embodiments, the vector is adapted for use in an Agrobacterium mediated transfection process (See e.g., U.S. Pat. Nos. 5,981,839; 6,051,757; 5,981,840; 5,824,877; and 4,940,838; all of which are incorporated herein by reference). Construction of recombinant Ti and Ri plasmids in general follows methods typically used with the more common bacterial vectors, such as pBR322. Additional use can be made of accessory genetic elements sometimes found with the native plasmids and sometimes constructed from foreign sequences. These may include but are not limited to structural genes for antibiotic resistance as selection genes.

There are two systems of recombinant Ti and Ri plasmid vector systems now in use. The first system is called the "cointegrate" system. In this system, the shuttle vector containing the gene of interest is inserted by genetic recombination into a non-oncogenic Ti plasmid that contains both the cis-acting and trans-acting elements required for plant transformation as, for example, in the pMLJl shuttle vector and the non-oncogenic Ti plasmid pGV3850. The second system is called the "binary" system in which two plasmids are used; the gene of interest is inserted into a shuttle vector containing the cis-acting elements required for plant transformation. The other necessary functions are provided in trans by the non-oncogenic Ti plasmid as exemplified by the pBINl 9 shuttle vector and the non-oncogenic Ti plasmid PAL4404. Some of these vectors are commercially available.

In other embodiments of the invention, the nucleic acid sequence of interest is targeted to a particular locus on the plant genome. Site-directed integration of the nucleic acid sequence of interest into the plant cell genome may be achieved by, for example, homologous recombination using Agrobacterium-deήvQά sequences. Generally, plant cells are incubated with a strain of Agrobacterium which contains a targeting vector in which sequences that are homologous to a DNA sequence inside the target locus are flanked by Agrobacterium transfer-DNA (T-DNA) sequences, as previously described (U.S. Pat. No. 5,501,967). One of skill in the art knows that homologous recombination may be achieved using targeting vectors which contain sequences that are homologous to any part of the targeted plant gene, whether belonging to the regulatory elements of the gene, or the coding regions of the gene. Homologous recombination may be achieved at any region of a plant gene so long as the nucleic acid sequence of regions flanking the site to be targeted is known. In yet other embodiments, the nucleic acids of the present invention are utilized to construct vectors derived from plant (+) RNA viruses {e.g., brome mosaic virus, tobacco mosaic vims, alfalfa mosaic virus, cucumber mosaic virus, tomato mosaic virus, and combinations and hybrids thereof). Generally, the inserted polypeptide that inhibits the proliferation of fungi (e.g., farnesyl pyrophosphate synthetase, citrate synthase, isocitrate dehydrogenase [NAD] subunit 1 - mitochondrial precursor, ABCl) can be expressed from these vectors as a fusion protein (e.g., coat protein fusion protein) or from its own subgenomic promoter or other promoter. Methods for the construction and use of such viruses are described in U.S. Pat. Nos. 5,846,795; 5,500,360; 5,173,410; 5,965,794; 5,977,438; and 5,866,785, all of which are incorporated herein by reference. In some embodiments of the present invention the nucleic acid sequence of interest is introduced directly into a plant. One vector useful for direct gene transfer techniques in combination with selection by the herbicide Basta (or phosphinothricin) is a modified version of the plasmid pCIB246, with a CaMV 35S promoter in operational fusion to the E. coli GUS gene and the CaMV 35S transcriptional terminator (WO 93/07278). 3. Transformation Techniques

Once a nucleic acid sequence encoding a polypeptide that inhibits the proliferation of fungi (e.g., farnesyl pyrophosphate synthetase, citrate synthase, isocitrate dehydrogenase [NAD] subunit 1 - mitochondrial precursor, ABCl) is operatively linked to an appropriate promoter and inserted into a suitable vector for the particular transformation technique utilized (e.g., one of the vectors described above), the recombinant DNA described above can be introduced into the plant cell in a number of art-recognized ways. Those skilled in the art will appreciate that the choice of method might depend on the type of plant targeted for transformation. In some embodiments, the vector is maintained episomally. In other embodiments, the vector is integrated into the genome.

In some embodiments, the vector is introduced through ballistic particle acceleration using devices (e.g., available from Agracetus, hie, Madison, Wis. and Dupont, Inc., Wilmington, Del). (See e.g., U.S. Pat. No. 4,945,050; and McCabe et al. (1988) Biotechnology 6:923). See also, Weissinger et al. (1988) Annual Rev. Genet. 22:421; Sanford et al. (1987) Particulate Science and Technology, 5:27 (onion); Svab et al. (1990)

Proc. Natl. Acad. Sci. USA, 87:8526 (tobacco chloroplast); Christou et al. (1988) Plant Physiol., 87:671 (soybean); McCabe et al. (1988) Bio/Technology 6:923 (soybean); Klein et al. (1988) Proc. Natl. Acad. Sci. USA, 85:4305 (maize); Klein et al. (1988) Bio/Technology, 6:559 (maize); Klein et al. (1988) Plant Physiol., 91:4404 (maize); Fromm et al. (1990) Bio/Technology, 8:833; and Gordon-Kamm et al. (1990) Plant Cell, 2:603

(maize); Koziel et al. (1993) Biotechnology, 11 : 194 (maize); Hill et al. (1995) Euphytica, 85:119 and Koziel et al. (1996) Annals of the New York Academy of Sciences 792:164; Shimamoto et al. (1989) Nature 338: 274 (rice); Christou et al. (1991) Biotechnology, 9:957 (rice); Datta et al. (1990) Bio/Technology 8:736 (rice); European Patent Application EP 0 332 581 (orchardgrass and other Pooideae); Vasil et al. (1993) Biotechnology, 11: 1553

(wheat); Weeks et al. (1993) Plant Physiol., 102: 1077 (wheat); Wan et al. (1994) Plant Physiol. 104: 37 (barley); Jahne et al. (1994) Theor. Appl. Genet. 89:525 (barley); Knudsen and Muller (1991) Planta, 185:330 (barley); Umbeck et al. (1987) Bio/Technology 5: 263 (cotton); Casas et al. (1993) Proc. Natl. Acad. Sci. USA 90:11212 (sorghum); Somers et al. (1992) Bio/Technology 10:1589 (oat); Torbert et al. (1995) Plant Cell Reports, 14:635

(oat); Weeks et al. (1993) Plant Physiol., 102:1077 (wheat); Chang et al, WO 94/13822 (wheat) and Nehra et al. (1994) The Plant Journal, 5:285 (wheat).

In other embodiments, direct transformation in the plastid genome is used to introduce the vector into the plant cell (See e.g., U.S. Patent Nos 5,451,513; 5,545,817; 5,545,818; PCT application WO 95/16783). The basic technique for chloroplast transformation involves introducing regions of cloned plastid DNA flanking a selectable marker together with the nucleic acid encoding the RNA sequences of interest into a suitable target tissue (e.g. , using biolistics or protoplast transformation with calcium chloride or PEG). The 1 to 1.5 kb flanking regions, termed targeting sequences, facilitate homologous recombination with the plastid genome and thus allow the replacement or modification of specific regions of the plastome. Initially, point mutations in the chloroplast 16S rRNA and rpsl2 genes conferring resistance to spectinomycin and/or streptomycin are utilized as selectable markers for transformation (Svab et al. (1990) PNAS₅ 87:8526; Staub and Maliga, (1992) Plant Cell, 4:39). The presence of cloning sites between these markers allowed creation of a plastid targeting vector introduction of foreign DNA molecules (Staub and Maliga (1993) EMBO J., 12:601). Substantial increases in transformation frequency are obtained by replacement of the recessive rRNA or r-protein antibiotic resistance genes with a dominant selectable marker, the bacterial aadA gene encoding the spectinomycin- detoxifying enzyme aminoglycoside-3'-adenyltransferase (Svab and Maliga (1993) PNAS,

90:913). Other selectable markers useful for plastid transformation are known in the art and encompassed within the scope of the present invention. Plants homoplasmic for plastid genomes containing the two nucleic acid sequences separated by a promoter of the present invention are obtained, and are preferentially capable of high expression of the RNAs encoded by the DNA molecule.

In other embodiments, vectors useful in the practice of the present invention are microinjected directly into plant cells by use of micropipettes to mechanically transfer the recombinant DNA (Crossway (1985) MoI. Gen. Genet, 202:179). In still other embodiments, the vector is transferred into the plant cell by using polyethylene glycol (Krens et al. (1982) Nature, 296:72; Crossway et al. (1986) BioTechniques, 4:320); fusion of protoplasts with other entities, either minicells, cells, lysosomes or other fusible lipid- surfaced bodies (Fraley et al. (1982) Proc. Natl. Acad. Sci., USA, 79:1859); protoplast transformation (EP 0 292 435); direct gene transfer (Paszkowski et al. (1984) EMBO J., 3:2717; Hayashimoto et al (1990) Plant Physiol. 93:857). Li still further embodiments, the vector may also be introduced into the plant cells by electroporation (Fromm, et al. (1985) Proc. Natl Acad. Sci. USA 82:5824; Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83:5602). In this technique, plant protoplasts are electroporated in the presence of plasmids containing the gene construct. Electrical impulses of high field strength reversibly permeabilize biomembranes allowing the introduction of the plasmids. Electroporated plant protoplasts reform the cell wall, divide, and form plant callus.

In addition to direct transformation, in some embodiments, the vectors comprising a nucleic acid sequence encoding a RNAi gene of the present invention are transferred using Agrobacterium-mediated transformation (Hinchee et al. (1988) Biotechnology, 6:915;

Ishida et al. (1996) Nature Biotechnology 14:745). Agrobacterium is a representative genus of the gram-negative family Rhizobiaceae. Its species are responsible for plant tumors such as crown gall and hairy root disease, hi the dedifferentiated tissue characteristic of the tumors, amino acid derivatives known as opines are produced and catabolized. The bacterial genes responsible for expression of opines are a convenient source of control elements for chimeric expression cassettes. Heterologous genetic sequences {e.g., nucleic acid sequences operatively linked to a promoter of the present invention), can be introduced into appropriate plant cells, by means of the Ti plasmid of Agrobacterium tumefaciens. The Ti plasmid is transmitted to plant cells on infection by Agrobacterium tumefaciens, and is stably integrated into the plant genome (Schell (1987) Science, 237: 1176). Species which are susceptible infection by Agrobacterium may be transformed in vitro. Alternatively, plants may be transformed in vivo, such as by transformation of a whole plant by Agrobacteria infiltration of adult plants, as in a "floral dip" method (Bechtold N, Ellis J, Pelletier G (1993) Cr. Acad. Sci. m - Vie 316: 1194-1199).

4. Regeneration

After selecting for transformed plant material that can express the heterologous gene encoding a RNAi gene of the present invention, whole plants are regenerated. Plant regeneration from cultured protoplasts is described in Evans et al. (1983) Handbook of Plant Cell Cultures, Vol. 1 : (MacMillan Publishing Co. New York); and Vasil I. R. (ed.),

Cell Culture and Somatic Cell Genetics of Plants, Acad. Press, Orlando, Vol. I (1984), and Vol. Ill (1986). It is known that many plants can be regenerated from cultured cells or tissues, including but not limited to all major species of sugarcane, sugar beet, cotton, fruit and other trees, legumes and vegetables, and monocots {e.g., the plants described above). Means for regeneration vary from species to species of plants, but generally a suspension of transformed protoplasts containing copies of the heterologous gene is first provided. Callus tissue is formed and shoots may be induced from callus and subsequently rooted.

Alternatively, embryo formation can be induced from the protoplast suspension. These embryos germinate and form mature plants. The culture media will generally contain various amino acids and hormones, such as auxin and cytokinins. Shoots and roots normally develop simultaneously. Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. The reproducibility of regeneration depends on the control of these variables.

5. Generation of Transgenic lines

Transgenic lines are established from transgenic plants by tissue culture propagation. The presence of nucleic acid sequences encoding a RNAi gene of the present invention (including mutants or variants thereof) may be transferred to related varieties by traditional plant breeding techniques.

These transgenic lines are then utilized for evaluation of oil production and other agronomic traits.

EXPERIMENTAL The following examples are provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.

In the experimental disclosures which follow, the following abbreviations apply: N (normal); M (molar); mM (millimolar); μM (micromolar); mol (moles); mmol (millimoles); μmol (micromoles); nmol (nanomoles); pmol (picomoles); g (grams); mg (milligrams); μg

(micrograms); ng (nanograms); 1 or L (liters); ml (milliliters); μl (microliters); cm (centimeters); mm (millimeters); μm (micrometers); nm (nanometers); ⁰C (degrees Centigrade).

Example 1. Inhibition of Exogenously Applied EGFP Gene Expression in Fungus

Grown In Axenic Culture

Fungal strains with high levels of constitutive expression of an enhanced version of the jellyfish green fluorescent protein (EGFP, Clontech) have been constructed for use in live cell microscopy as it invades rice sheath epidermal cells. Loss of fluorescence due to silencing of EGFP in the fungus will be tested. hi experiments conducted during the course of the present invention, a rapid assay for live-cell microscopy of rice blast infection was developed. Spore suspensions were inoculated on the inner surface of rice leaf sheaths for various time periods up to 72 hours. To visualize the progress of infection, the inner surface of the sheath was trimmed off for microscopic investigation. Such selections, approximately 3 cell layers deep, were optically clear and were visualized without additional treatment.

Several methods are available for producing milligram quantities of dsRNA molecules for in vitro assays. dsRNA corresponding to EGFP will be produced in vitro from PCR fragments designed to be flanked by T7 promoter sequences using a T7 RNA

Polymerase and the Megascript™ (Ambion) system. dsRNA is formed from the annealing of sense and antisense strands present in the in vitro RNA preps.

EGFP-labeled fungus growing in liquid media in wells of microtiter plates will be treated with purified dsRNA. Spores of the fungus will be germinated in the presence of different concentrations of dsRNA in different liquid media, and the fluorescence will be checked at

12, 24 and 48 and 72 hours (using a fluorescence microscope and fluorescence plate reader for quantitation). The extent of which RNAi decreases fungal EGFP expression will be determined. The destabilized EGFP ( d2EGFP) version available from Clontech for EGFP may be substituted in these experiments. d2EGFP is engineered with a C-terminal PEST domain that targets the protein for degradation, and has a half-life of approximately 2 hours in vivo. hi the presence of 1, 16-hexadecanediol, a monomer component of plant cuticles, the fungus produces germ tubes and appressoria as if it were on the plant surface. Considering that the fungus undergoes extensive endocytosis throughout the development process (see, e.g., Atkinson, H., et al., 2002 Fungal Genetics and Biology 37:233-244; herein incorporated by reference in its entirety), the uptake of dsRNA and silencing will be enhanced under these conditions. Conditions that enhance uptake of DNA during transformation will be utilized (e.g., treating the fungus with Li Acetate in PEG). Conditions that enhance uptake of DNA in intact fungal cells also enhance uptake of dsRNAs. Controls are needed to assure that LiOAc and other treatments do not block gene expression. Finally infected leaf sheath pieces will be incubated with dsRNA to test for silencing inplanta. This experiment, if successful, will lead to an ideal bioassay of dsRNA efficacy.

Example 2. Test Efficacy of dsRNAs Produced in Transgenic Rice Interferes with Fungal EGFP Expression during Infection.

A transgenic strategy will directly test if the specialized invasive hyphae produced by the fungus inside rice cells are subject to RNAi silencing by dsRNA produced by the plant. Intracellular invasive hyphae are morphologically distinct from typical hyphae produced on agar medium, and are specialized for nutrient uptake from living plant cells, hi addition, the developing invasive hypha within a plant cell changes that cell's structure and metabolism. Therefore, the transgenic approach will be successful even if the in vitro assay fails to show RNA interference.

Constructs will be made for stable rice transformation experiments using Agrobacterium tumifaciens. At least 200 bp of the candidate genes will be PCR-amplified using two sets of primers. The vector will be constructed so that the transcribed RNA will produce dsRNA with a stem-loop structure. The plant promoter on this expression vector is the maize ubiquitin promoter, which has strong expression in monocots. This plant expression cassette will be subcloned into pZP200 or pCambia-type plasmids as binary vectors for Agrobacterium-medi&ted transformation.

Rice transformation will be performed with slight modifications (see, e.g., Cheng, X., et al., 1997 Methods in Biotechnology, Vol. 3: Recombination protein from plants: Production and Isolation of Clinically useful compounds; herein incorporated by reference in its entirety). Immature seeds will be disinfected and placed on callus induction medium in 24h light @ 30° C for 10 to 14 days. Callus will be isolated and maintained on callus induction medium and transferred every 3 weeks. To transform rice calli, tissue will be co- cultivated with Agrobacterium harboring the binary plasmid on callus induction medium containing 200 μm acetosyringone for 2 to 3 days. Selection medium will contain 50 mg/L Hygromycin and 200 mg/L Timentin to prevent Agrobacterium growth. After 2 to 6 weeks resistant microcalli will be isolated and placed on regeneration medium. After another 6 weeks regenerated plantlets will be transferred to rooting medium and then to soil. Total time to recover transgenic plants will be 4 to 6 months. Transgenic seeds should be recovered within 6 to 12 months after transformation depending on the cultivar. Transformants will be analyzed by standard molecular techniques. Leaf sheath assays will be performed on TO transformants without sacrificing the plants, as well as on progeny derived from these plants. When transgenic progeny are obtained, standard infection assays as described (see, e.g., Valent, B., et al., 1991 Genetics 127:87-101; herein incorporated by reference in its entirety) will be performed. EGFP expression by fungus growing in rice leaves will be assessed by confocal microscopy (see, e.g., Czyrnmek, K. J., et al., 2002 Mycologia 94(2):280-289; herein incorporated by reference in its entirety).

Example 3. Targeting Essential Fungal Pathogenicity Genes for RNAi Interference and Demonstrating Disease Control. Pathogenicity genes critical for colonizing rice will be targeted. Example of targeted pathogenicity genes include, but are not limited to, farnesyl pyrophosphate synthetase and other key enzymes in the ergosterol pathway. Ergosterol is a key sterol specific for fungal plasma membranes, and it is important for membrane fluidity. Physiological effects of mutants in the erg pathway include infertility, slow growth and reduced conidiation in

Neurospora sp. Genes critical for fungal growth within plant tissue will also be targeted, including, but not limited to the critical efflux pump ABCl (see e.g., Talbot, NJ., et al., 2003 Ann. Rev. Microbiol. 57:177-202; herein incorporated by reference in its entirety) and genes such as RNA polymerase that are generally essential for fungal viability but unlike their counterparts in plants and animals.

Primers for each gene were designed using the gene sequences from Genbank. Polymerase chain reactions (PCR) were performed using cDNA template from Magnaporthe grisea to obtain a 652 base pair (bp) fragment of FPS, a 795bp fragment of ID, a 243bp fragment of CS, and a 264bρ fragment of ABCl . The PCR products were purified using the Qiaquick PCR purification kit. The purified DNA of each gene were then ligated into the pGEM-T Easy vector. PCR fragments were then subcloned in sense orientation to pENTR 4 (Invitrogen) and finally into the pANDA vector system (Miki, D and Shimamoto, K. (2004) Plant and Cell Physiology 45: 490; herein incorporated by reference in its entirety) for Agrobacterium-mediated transformation with RNAi expression in plants.

Rice transformation was accomplished as described in Example 2. To test the efficacy of the transgenic plants to rice blast either a leaf sheath assay or a detached leaf assay will be performed For the detached leaf assay, three different fungal spore concentrations 10⁵, 10⁴, and 10³ will be used. A gelatin will be used as a negative control and three leaves per plate will be inoculated. Plated will be scored at seven and ten days for infection.

All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology, plant biology, biochemistry, or related fields are intended to be within the scope of the following claims.

Claims

CLAIMSWe claim:

1. A transgenic plant comprising heterologous nucleic acid sequences encoding a double stranded fungal RNA sequence, wherein said double stranded RNA sequence inhibits the proliferation of fungi ingesting said double stranded fungal RNA sequence.

2. The transgenic plant of Claim 1, wherein said fungus is M. grisea.

3. The transgenic plant of Claim 1, wherein said fungus is F. graminearum.

4. The transgenic plant of Claim I₅ wherein said plant is rice.

5. The transgenic plant of Claim 1 , wherein said heterologous nucleic acid sequences are operably linked to the same promoter.

6. The transgenic plant of Claim I₅ wherein said heterologous nucleic acid sequences are separated by a loop sequence.

7. The transgenic plant of Claim 5, wherein said promoter is a tissue specific promoter.

8. The transgenic plant of Claim 5, wherein said promoter is a constitutive promoter.

9. The transgenic plant of Claim 1, wherein said heterologous nucleic acid sequences are operably linked to separate promoters.

10. The transgenic plant of Claim 1, wherein said transgenic plant comprises at least two heterologous nucleic acid sequences each encoding a double stranded fungal RNA sequence, wherein each double stranded fungal RNA sequence inhibits the proliferation of said fungi ingesting said double stranded fungal RNA sequences.

11. The transgenic plant of Claim 1 , wherein one of said heterologous nucleic acid sequences is complementary to an RNA sequence selected from the group consisting of farnesyl pyrophosphate synthetase, citrate synthetase, isocitrate dehydrogenase [NAD] subunit 1 - mitochondrial precursor, ABCl, and RNA Polymerase.

12. The transgenic plant of Claim 11 , wherein said heterologous nucleic acid sequences are at least 21 bases in length.

13. Seeds from the transgenic plant of Claim 1.

14. Leaves from the transgenic plant of Claim 1.

15. Roots from the transgenic plant of Claim 1.

16. Stems from the transgenic plant of Claim 1.

17. Floral tissues from the transgenic plant of Claim 1.

18. Panicles from the transgenic plant of Claim 1.

19. The transgenic plant of Claim 1, wherein double stranded RNA is complementary to a fungal gene required for pathogenecity.

20. A vector comprising heterologous nucleic acid sequences encoding a double stranded fungal RNA sequence, wherein said double stranded RNA sequence inhibits the proliferation of fungi ingesting said double stranded fungal RNA sequence.

21. The vector of Claim 20, wherein said fungi is M. grisea fungi.

22. The vector of Claim 20, wherein said fungi is F. graminearum.

23. The vector of Claim 20, wherein said vector comprises at least two heterologous nucleic acid sequences each encoding a double stranded fungal RNA sequence, wherein each double stranded fungal RNA sequence inhibits the proliferation of said fungi ingesting said double stranded fungal RNA sequences.

24. The vector of Claim 20, wherein said heterologous nucleic acid sequences are operably linked to the same promoter.

25. The vector of Claim 20, wherein said heterologous nucleic acid sequences are separated by a loop sequence.

26. The vector of Claim 24, wherein said promoter is a tissue specific promoter.

27. The vector of Claim 24, wherein said promoter is a constitutive promoter.

28. The vector of Claim 20, wherein said heterologous nucleic acid sequences are operably linked to separate promoters.

29. The vector of Claim 20, wherein one of said heterologous nucleic acid sequences is complementary to an RNA sequence selected from the group consisting of M. grisea farnesyl pyrophosphate synthetase, citrate synthetase, isocitrate dehydrogenase [NAD] subunit 1 - mitochondrial precursor, ABCl, and RNA Polymerase.

30. The vector of Claim 29, wherein said heterologous nucleic acid sequences are at least 21 bases in length.

31. A transgenic plant comprising the vector of Claim 20.

32. A method for controlling fungi comprising: a) providing transgenic plant tissue comprising heterologous DNA sequences encoding a double stranded fungal RNA; and b) growing said transgenic plant so that said double stranded fungal RNA is expressed in plant tissue; wherein the proliferation of fungi feeding on said plant tissue is reduced as compared to fungi feeding on non-transgenic plant tissue.

33. The method of Claim 32, wherein said fungi is M. grisea fungi.

34. The method of Claim 32, wherein said fungi is F. graminearum.

35. The method of Claim 32, wherein said plant is rice.

36. The method of Claim 32, wherein said heterologous DNA sequences are located on a vector.

37. The method of Claim 36, wherein said heterologous DNA sequences are operably linked to a promoter.

38. The method of Claim 36, wherein said heterologous DNA sequences are operably linked to the same promoter.

39. The method of Claim 36, wherein said promoter is a tissue specific promoter.

40. The method of Claim 36, wherein said promoter is a constitutive promoter.

41. The method of Claim 32, wherein said heterologous DNA sequences are separated by a loop sequence.

42. The method of Claim 32, wherein one of said heterologous nucleic acid sequences is complementary to an RNA sequence selected from the group consisting of M. grisea faπiesyl pyrophosphate synthetase, citrate synthetase, isocitrate dehydrogenase [NAD] subunit 1 - mitochondrial precursor, ABCl, and RNA Polymerase.

43. The method of Claim 32, wherein said heterologous nucleic acid sequences are at least 21 bases in length.

44. The method of Claim 32, wherein double stranded fungal RNA is complementary to a fungal gene required for pathogenecity.