# Patent

## Title
Methods and materials for conferring resistance to pests and pathogens of plants

## Abstract

Methods and materials for conferring pest resistance to plants are provided. Plants are transformed with a silencing construct homologous to a gene of a plant pest that is essential for the survival, development, or pathogenicity of the pest. This results in the plant producing RNAi to the selected gene, which, when ingested by the pest results in silencing of the gene and a subsequent reduction of the pest's ability to harm the plant. In other embodiments, the pest's reduced ability to harm the plant is passed on to pest progeny. Methods and materials for depathogenesis of pests is also provided.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This is a divisional of U.S. patent application Ser. No. 11/256,428 filed 21 Oct. 2005, which claims priority to U.S. Provisional Patent Application Ser. Nos. 60/657,821 filed 1 Mar. 2005 and 60/621,542 filed 21 Oct. 2004, each of which is incorporated by reference herein by its entirety.


BACKGROUND OF THE INVENTION
Plant pests (e.g. fungal pathogens, bacteria, nematodes, insects, viruses, etc.) cause major losses of food and fiber throughout the world, especially in developing countries. Losses include direct production or pre-harvest losses, postharvest and storage losses, and the diminution of food quality and safety (e.g. by production of mycotoxins). Other resultant losses from plant pests are observed in plants valued for aesthetic, olfactory, or ecologic properties.
Plant pests can sometimes be controlled by application of chemicals (e.g. fungicides, insecticides, nematicides, parasiticidals), manipulation or management of the microenvironment or by genes for resistance to the pathogen.
Discovery and introduction of a “new” gene for resistance frequently causes the development or selection of a new race of the pathogen able to infect plants containing that “new” gene. This has best been demonstrated by the rusts and smuts of cereal crops, but it also occurs with soil borne diseases such as black shank of tobacco and root and stem rot of soybean, caused by Phytophthora nicotianae and P. sojae, respectively. There are at least two races of P. nicotianae and more than 70 races of P. sojae, all requiring different genes or combinations of genes for disease resistance.
The fungal genus Phytophthora comprises many species of very destructive pathogens which cause serious diseases of plants. These include blights, damping-offs, cankers, fruit rots, root rots, wilts, and many other symptoms that affect a wide variety of food, fiber and oil crops including avocado, cacao, canola, citrus, pepper, potato, soybean, tobacco, tomato, pine, rubber, oak trees, etc.
In the past decade the phenomenon of gene silencing or RNA interference (RNAi) has been described and characterized in organisms as diverse as plants, fungi, nematodes, hydra and humans (Zamore and Haley, 2005). It is considered to be an ancient defense mechanism wherein the host organism recognizes as foreign a double-stranded RNA molecule and hydrolyzes it. The resulting hydrolysis products are small RNA fragments of 21-30 nucleotides in length, called small interfering RNAs (siRNAs). The siRNAs then diffuse or are carried throughout the host, where they hybridize to the mRNA for that specific gene and cause its hydrolysis to produce more siRNAs. This process is repeated each time the siRNA hybridizes to its homologous mRNA, effectively preventing that mRNA from being translated, and thus “silencing” the expression of that specific gene.
Fire et al. (2003) describe a method of administering RNA to a host wherein the RNA is composed of sense and antisense sequences homologous to a gene of that host cell to silence that host cell's gene. See U.S. Pat. No. 6,506,559.
Results by van West et al. (1999) demonstrated internuclear gene silencing in Phytophthora infestans. They transformed P. infestans with the infl elicitin gene in both the sense and antisense orientations. This resulted in silencing of both of the transgenes, as well as the endogenous gene. By somatic fusion of a silenced transgenic strain and a wild-type strain, they demonstrated two essential points; first, that the presence of the transgene itself is not necessary to maintain the silenced state; and second, the involvement of a trans-acting factor capable of transferring the silencing signal between nuclei, later found to be the siRNAs.
Waterhouse et al. (WO9953050A1) have described methods of conferring viral resistance to a plant by transforming the plant with a gene silencing construct directed towards a plant viral gene.
Wang et al. (US20030180945A1) describe a method of gene silencing in a fungus by providing the fungus with a gene silencing construct directed towards a fungal gene.
What is needed in the art is a plant resistance method that is readily adaptable to changes in the biological environment such as a new and serious plant disease. For example, there were no occurrences of Asian rust in soybean in the USA until November 2004. Asian rust is predicted to cause losses estimated at 1 to 7 billion dollars (USDA) in 2005-2006.
The art would also be substantially advanced if there existed a versatile genetic method of conferring resistance to a plant against a spectrum of fungi, insects, nematodes, bacteria, etc.
BRIEF SUMMARY OF THE INVENTION
The present invention relates to isolated TC polynucleotides, polypeptides, vectors and host cells expressing isolated TC polynucleotides capable of conferring increased drought and salt tolerance to plants. The related polynucleotides, polypeptides, vectors and cells of the present invention are also capable of imparting specific traits to plants, such as decreased chlorophyll loss in response to salt stress, reduced production of H2O2 and other reactive oxygen species (ROS), and increased production of γ-tocopherol.
There is now provided in the present invention a method of conferring pest resistance to a plant comprising the step of transforming a host plant cell with a heterologous polynucleotide, said heterologous polynucleotide comprising:

    
    
        (a) an antisense sequence having homology to a pest pathogenicity gene, and
        (b) a sense sequence substantially complementary to said antisense sequence;
    
    


wherein a transcript of the heterologous polynucleotide is capable of hybridizing to form a double-stranded region comprising nucleotides encoded by the antisense sequence and the sense sequence;
wherein said sense and antisense sequences are each at least about 19 nucleotides in length; and
wherein the antisense sequence is homologous to the pathogenicity gene of at least two pests from different species whereby the plant is resistant to said two pests as compared to substantially the same plant lacking the heterologous polynucleotide.
In another embodiment, the present invention provides a method for conferring pest resistance to a plant comprising a step of transforming a host plant cell with a heterologous polynucleotide, said heterologous polynucleotide comprising:

    
    
        (a) a first antisense sequence having homology to a first pest pathogenicity gene;
        (b) a second antisense sequence having homology to a second pest pathogenicity gene;
        (c) a first sense sequence substantially complementary to said first antisense sequence; and
        (d) a second sense sequence substantially complementary to said second antisense sequence,
    
    


wherein a transcript of the heterologous polynucleotide is capable of hybridizing to form a double-stranded region comprising nucleotides encoded by the first antisense sequence and the first sense sequence,
wherein a transcript of the heterologous polynucleotide is capable of hybridizing to form a double-stranded region comprising nucleotides encoded by the second antisense sequence and the second sense sequence, and
wherein said first and second sense sequences and first and second antisense sequences are each at least about 19 nucleotides in length.
Accordingly, a plant can now be made resistant to a plurality of pests belonging to the same or to different members selected from the group consisting of insects, bacteria, fungi, plants, and nematodes.
In another embodiment, the present invention provides a method for conferring pest resistance to a plant comprising a step of transforming a host plant cell with a heterologous polynucleotide, said heterologous polynucleotide comprising:

    
    
        (a) a first promoter operably linked to an antisense sequence wherein said antisense sequence is homologous to a pest pathogenicity gene and operably linked to a terminator;
        (b) a second promoter operably linked to a sense sequence wherein said sense sequence is substantially complementary to said antisense sequence and operably linked to a terminator;
    
    


wherein said second promoter is a strong promoter relative to the first promoter,
wherein a transcript of the heterologous polynucleotide is capable of hybridizing to form a double-stranded region between sequences encoded by the antisense sequence and the sense sequence,
wherein said sense and antisense sequences are each at least about 19 nucleotides in length.
In another embodiment, the present invention provides a method for conferring pest resistance to a plant comprising the step of transforming a host plant cell with a heterologous polynucleotide, said heterologous polynucleotide comprising:

    
    
        (a) an antisense sequence having homology to a pest pathogenicity gene, and
        (b) a sense sequence substantially complementary to said antisense sequence,
    
    


wherein a transcript of the heterologous polynucleotide is capable of hybridizing to form a double-stranded region comprising nucleotides encoded by the antisense sequence and the sense sequence,
wherein said sense and antisense sequences are each at least about 19 nucleotides in length,
wherein the pest is selected from the group consisting of insects, bacteria, fungi, and nematodes, and
wherein the pest pathogenicity gene and the pest are selected from those taught herein.



BRIEF SUMMARY OF THE SEVERAL VIEWS OF THE DRAWINGS
FIG. 1 is the genetic map of the pCAMBIA1201 plant transformation plasmid used as an example throughout this teaching and which is the backbone of pVZA100, pVZA200, pVZA300 and pVZA400.
FIG. 2 is a drawing of the insert in the intermediary plasmid pVZA1.
FIG. 3 is the pVZA100 silencing cassette cloned in plasmid pVZA3 and ready for transfer to pCAMBIA1201.
FIG. 4 is autoradiograms showing the siRNAs isolated from transgenic tobacco and from transgenic Phytophthora nicotianae. FIGS. 4A and 4B show the results of hybridization of the cutinase gene probe to siRNAs from wild type and transgenic tobacco plants and P. nicotianae, respectively.
FIG. 5 shows plant reactions to a natural epidemic of the blue mold disease of tobacco caused by Peronospora tabacina in the greenhouse. The numbers in the photographs refer to the different disease ratings as set forth in Table 11.
FIGS. 6A-6D show shoots emerging from transgenic soybean embryos.
FIG. 7 shows the results of soybean transformed with pVZA100. FIG. 7A shows transformed soybean calli. FIG. 7B show GUS and hygromycin phosphotransferase genes detected in the transformed soybean callus.
FIG. 8 shows roots and minitubers produced in the culture plates of potato cells transformed with pVZA100.
FIG. 9 shows a polycistronic gene silencing construct capable of silencing essential genes in three fungal species causing serious diseases of soybeans.
FIG. 10 shows gene silencing constructs based on rDNA and capable of silencing these essential genes in Phytophthora, in Phakopsora and in both species.
FIG. 11 shows a wild type tobacco plant (A) and a pVZA100 transgenic tobacco plant (B), susceptible and resistant to Phytophthora nicotianae, respectively.
FIG. 12 is an autoradiogram showing the siRNAs isolated from a transgenic tobacco plant (3) and from Phytophthora nicotianae growing on a silenced transgenic tobacco plant (4). Lane 1 shows the 35 nt oligonucleotides VZA 3F (SEQ ID NO: 3) and VZA 4R (SEQ ID NO: 4) serving as the molecular size control. Lane 2 shows the 620 nt messenger RNA from the wild type fungus. Lane 3 shows the siRNAs from a silenced transgenic tobacco plant. Lane 4 shows the siRNAs from the non-pathogenic fungus isolated from the resistant transgenic tobacco.
FIG. 13 shows transgenic soybean plants resistant to Phytophthora sojae. 
FIG. 14 shows the effects of pVZA300 and pVZA400 on the growth and development of transgenic Phytophthora nicotianae and Phytophthora sojae. Microscopic examination of cultures of P. nicotianae and P. sojae bombarded with pCAMBIA1201, pVZA100 and pVZA200 indicated that all grew normally like the wild type and with no visual malformations (FIG. 14A). Similar results were observed for cultures of P. nicotianae reisolated from infected wild type tobacco or tobacco transformed with pVZA100. However, those cultures of P. nicotianae bombarded with pVZA300 or pVZA400 (FIGS. 14B, C, D) and those of P. sojae also bombarded with pVZA300 or pVZA400 (FIGS. 14E, F) grew very slowly, their hyphae were severely malformed, and some cultures died.
FIGS. 15-19 show heterologous polynucleotides designed in accordance with the present invention.
FIGS. 20A-20G show the nucleotide sequences of the cutinase genes of 25 isolates comprising 9 species of Phytophthora. 



DETAILED DESCRIPTION OF THE INVENTION
Brief Description of the Sequences
SEQ ID NO: 1 is the PCR primer VZA 1F as used according to the subject invention.
SEQ ID NO: 2 is the PCR primer VZA 2R as used according to the subject invention.
SEQ ID NO: 3 is the PCR primer VZA 3F as used according to the subject invention.
SEQ ID NO: 4 is the PCR primer VZA 4R as used according to the subject invention.
SEQ ID NO: 5 is the silencing construct pVZA100 used according to the subject invention.
SEQ ID NO: 6 is the silencing construct pVZA200 used according to the subject invention.
SEQ ID NO: 7 is the silencing construct pVZA300 used according to the subject invention.
SEQ ID NO: 8 is the silencing construct pVZA400 used according to the subject invention.
SEQ ID NO: 9 is the forward PCR primer GUS as used according to the subject invention.
SEQ ID NO: 10 is the reverse PCR primer GUS as used according to the subject invention.
SEQ ID NO: 11 is the PCR primer pVZA100 and hygromycin phosphotrasferase as used according to the subject invention.
SEQ ID NO: 12 is the PCR primer pVZA100 and hygromycin phosphotrasferase as used according to the subject invention.
SEQ ID NO. 13 is the primer VZA 1 65R as used according to the subject invention.
SEQ ID NO. 14 is the primer VZA 2 69F as used according to the subject invention.
SEQ ID NO. 15 is the primer VZA 3 73F as used according to the subject invention.
SEQ ID NO. 16 is the primer VZA 3 73F as used according to the subject invention.
SEQ ID NO. 17 is the primer VZA 1 65 as used according to the subject invention.
SEQ ID NO. 18 is the primer VZA 6 70F as used according to the subject invention.
SEQ ID NO. 19 is the PCR primer VZA 7 72F as used according to the subject invention.
SEQ ID NO. 20 is the PCR primer VZA 8 79F as used according to the subject invention.
SEQ ID NO. 21 is the PCR VZA 9 84F primer as used according to the subject invention.
SEQ ID NO. 22 is the PCR VZA 10 87F primer as used according to the subject invention.
SEQ ID NO. 23 is the PCR VZA 11 89F primer as used according to the subject invention.
SEQ ID NO. 24 is the PCR VZA 12 90F primer as used according to the subject invention.
SEQ ID NO. 25 is the PCR VZA 13 91F primer as used according to the subject invention.
SEQ ID NO. 26 is the PCR VZA 14 81F primer as used according to the subject invention.
SEQ ID NO. 27 is the PCR VZA 15 82F primer as used according to the subject invention.
SEQ ID NO. 28 is the PCR primer VZA 6 83F as used according to the subject invention.
SEQ ID NO. 29 is the PCR primer VZA 17 85F as used according to the subject invention.
SEQ ID NO. 30 is the PCR VZA 18 86F primer as used according to the subject invention.
SEQ ID NO. 31 is the PCR VZA 19 64R primer as used according to the subject invention.
SEQ ID NO. 32 is the PCR VZA 20 77F primer as used according to the subject invention.
SEQ ID NO. 33 is the PCR VZA 21 63F primer as used according to the subject invention.
SEQ ID NO. 34 is the PCR VZA 22 66R primer as used according to the subject invention.
SEQ ID NO. 35 is the PCR VZA 23 80F primer as used according to the subject invention.
SEQ ID NO. 36 is the PCR VZA 24 92F primer as used according to the subject invention.
SEQ ID NO. 37 is the PCR primer VZA 25 93F as used according to the subject invention.
SEQ ID NO. 38 is the PCR primer VZA 2R as used according to the subject invention.
SEQ ID NO. 39 is the PCR primer cathepsin as used according to the subject invention.
SEQ ID NO. 40 is the PCR primer elicitin as used according to the subject invention.
SEQ ID NO. 41 is the PCR primer rDNA FP as used according to the subject invention.
SEQ ID NO. 42 is the PCR primer rDNA RP as used according to the subject invention.
SEQ ID NO. 43 is the marker Phialophora gregata ribosomal RNA gene (rDNA) as used according to the subject invention.
SEQ ID NO. 44 is the genotype B DNA marker Phialophora gregata ribosomal RNA gene (rDNA) as used according to the subject invention.
SEQ ID NO. 45 is the polymerase II subunit Sclerotinia sclerotiorum partial rpb2 gene for RNA as used according to the subject invention.
SEQ ID NO. 46 is the ribosomal RNA gene Puccinia hordei 18S as used according to the subject invention.
SEQ ID NO. 47 is the hexose transporter (hxt2) gene Sclerotinia sclerotiorum as used according to the subject invention.
SEQ ID NO. 48 is Fusarium solani pisi cutinase mRNA as used according to the subject invention.
SEQ ID NO. 49 is the genome or conserved protein motif of Phytophthora nicotianae as used according to the subject invention.
SEQ ID NO. 50 is a ribosomal DNA sequence found in the Phakopsora pachyrhizi genome as used according to the subject invention.
SEQ ID NO. 51 is a Fusarium sambucinum translation elongation factor 1 alpha as used according to the subject invention.
SEQ ID NO. 52 is an elicitin gene sequence found in the Phytophthora infestans genome as used according to the subject invention.
SEQ ID NO. 53 is a partial rpb2 gene for RNA polymerase II subunit Sclerotinia sclerotiorum as used according to the subject invention.
SEQ ID NO. 54 is rRNA Stenocarpella maydis as used according to the subject invention.
SEQ ID NO. 55 is the 18S ribosomal RNA from a Cercospora zeae-maydis Group II as used according to the subject invention.
SEQ ID NO. 56 is Cathepsin B protease from Myzus persicae as used according to the subject invention.
SEQ ID NO. 57 is cysteine protease from Leptinotarsa decemlineata as used according to the subject invention.
SEQ ID NO. 58 is Verticillium dahliae rDNA as used according to the subject invention.
SEQ ID NO. 59 is partial sequence of 5.8S ribosomal RNA found in the Peronospora berteroae genome as used according to the subject invention.
SEQ ID NO. 60 is a genome subunit of cytochrome oxidase Meloidogyne chitwoodi as used according to the subject invention.
SEQ ID NO. 61 is ribosomal RNA P. scribneri as used according to the subject invention.
SEQ ID NO. 62 is ribosomal RNA Heterodera glycines as used according to the subject invention.
SEQ ID NO. 63 is RNA polymerase II sequence Magnaporthe grisea as used according to the subject invention.
SEQ ID NO. 64 is rRNA Puccinia hordei 18S as used according to the subject invention.
SEQ ID NO. 65 is the partial sequence 5.8S ribosomal RNA Pseudoperonospora humuli as used according to the subject invention.
SEQ ID NO. 66 is Botrytis cinerea cytochrome P450 monoxygenase as used according to the subject invention.
SEQ ID NO. 67 is rRNA Pseudomonas syingae as used according to the subject invention.
SEQ ID NO. 68 is Clavibacter michiganensis michiganensis Cel A gene as used according to the subject invention.
SEQ ID NO. 69 is Clavibacter michiganensis endo B-glucosidase gene as used according to the subject invention.
DEFINITIONS
As used herein, the following definitions apply:
Cell (or host plant cell) means a cell or protoplast of a plant cell and includes isolated cells and cells in a whole plant, plant organ, or fragment of a plant. It also includes non-isolated cells.
Double stranded region means a region of a polynucleotide wherein the nucleotides are capable of hydrogen bonding to each other. Such hydrogen bonding can be intramolecular or intermolecular (e.g. single transcription unit forming a double stranded region with the so-called hairpin or two transcription units that align appropriately for complementary sequences to hydrogen bond). To be a double stranded region, according to the present invention, it is not necessary for 100% of the nucleotides to be complementary and hydrogen bonded within a region. It is merely necessary for sufficient base pairing to occur to give the RNA a substantial double stranded character (e.g. an indicative melting point).
Exogenous gene means a gene that is not normally present in a given host genome in the present form. In this respect, the gene itself may be native to the host genome, however the exogenous gene will comprise the native gene altered by the addition or deletion of one or more different regulatory elements or additional genes.
Gene or genes means nucleic acid sequences (including both RNA or DNA) that encode genetic information for the synthesis of a whole RNA, a whole protein, or any functional portion of such whole RNA or whole protein sufficient to possess a desired characteristic.
Heterologous polynucleotide means any polynucleotide that is not found in a non-transformed host plant. A polynucleotide is not excluded from being a heterologous polynucleotide by the presence of endogenous polynucleotide sequences.
Homologous, in the context of the relationship between the antisense sequence and a pest pathogenicity gene (i.e. the DNA strand that binds RNA polymerase and directs transcription of the pest pathogenicity gene mRNA which is the antisense strand), means having sequence similarity sufficient to allow hybridization in vivo, in vitro, and/or ex vivo under low stringency conditions between the antisense sequence and the pest pathogenicity gene mRNA.
Host plant cell resistance means that a plant is less susceptible to the deleterious effects of a plant pest when compared to a host plant cell not transformed with said construct.
Inhibition of gene expression means a decrease in the level of protein and/or mRNA product from a target gene. The consequences of inhibition can be confirmed by examination of the outward properties of the cell or organism (as presented below in the examples) or by biochemical techniques such as RNA solution hybridization, nuclease protection, Northern hybridization, polymerase chain reaction (PCR), reverse transcription (RT) reverse transcription PCR(RT/PCR), gene expression monitoring with a microarray, antibody binding, enzyme linked immunosorbent assay (ELISA), Western blotting, radioimmunoassay (RIA), other immunoassays, and fluorescence activated cell analysis (FACS).
Marker gene means a gene that imparts a distinct phenotype to a cell expressing the marker gene and thus allows such transformed cells to be distinguished from cells that do not contain the gene.
Operably linked means joined as part of the same nucleic acid molecule, suitably positioned and oriented for transcription to be initiated from the promoter.
Pest pathogenicity gene means any pest gene that serves a role in such a pest's deleterious effects on a host plant. Deleterious effects means, by way of example, a negative effect on plant growth (numbers and/or size of the plant), development, quality, and/or reproduction. By way of example only, a pest pathogenicity gene may be one that serves a role in any of pest growth, development, replication and/or reproduction, or pest invasion or infection of the host plant.
Pathogenic, in reference to a pest, means a pest's ability to cause deleterious effects on a plant. Pathogenicity is augmented by any pest pathogenicity genes.
Plant pest means any living organism that is deleterious to plant growth (numbers and/or size of the plant), development, quality, yield, and/or reproduction. Non-limiting examples of such pests are insects, fungi, nematodes, bacteria, and viruses.
Quality means any aspect of a plant that can be considered desirable. By way of non-limiting example, quality can refer to a plant's appearance, yield, flavor, nutritional value, aroma, or content.
Substantially complementary, with respect to the sense and antisense sequences means sufficiently complementary to allow for formation of a double stranded molecule.
Transformation means a process of introducing an exogenous DNA sequence (e.g., a vector, a recombinant DNA molecule) into a cell or protoplast in which that exogenous DNA is incorporated into a chromosome or is capable of autonomous replication in a manner such that a heterologous RNA is transcribed.
Transcript means RNA encoded by DNA. In context of sense and antisense transcripts of the present invention, such sense and antisense transcripts can be part of the same polynucleotide or they can be 2 separate polynucleotides (i.e., each having its own 5′ and 3′ end).
Treating a plant pest means a method to reduce, eliminate, reverse, or prevent the deleterious effects of a plant pest on the plant.
Vector means a DNA molecule capable of replication in a host cell and/or to which another DNA segment can be operatively linked so as to bring about replication of the attached segment. A plasmid is an exemplary vector.
Underlying the various embodiments of the present invention is treating a plant to confer pest resistance by transforming a host plant cell with a heterologous polynucleotide comprising:

    
    
        (a) an antisense sequence having homology to a pest pathogenicity gene, and
        (b) a sense sequence substantially complementary to said antisense sequence;
    
    


wherein said sense and antisense sequences are capable of hybridizing to each other to form a double-stranded region and wherein said sense and antisense sequences are each at least about 19 nucleotides in length.
Without being bound by theory, it is believed that plants transformed according to the present invention transcribe an RNA molecule(s) with a region homologous to and a region complementary to the pest pathogenicity gene, and wherein the transcript(s) form a double stranded RNA (dsRNA) molecule. The plant recognizes the dsRNA as a potential foreign substance (e.g. a substance of viral origin). The dicer enzyme of the plant cuts the double stranded RNA into pieces of single-stranded RNA of about 23 nucleotides in length, called small interfering RNAs (siRNAs). These siRNAs are consumed by invading pests that have entered the plant via the digestion of plant cells (e.g. cutin). Once absorbed, the siRNAs can be incorporated into the pest's RNA-induced silencing complexes (RISC). The RISC complex can then digest the mRNA of the pest's homologous gene limiting the pest's ability to harm the plant.
In one embodiment, pest resistance is conferred to a plant comprising the step of transforming a host plant cell with a heterologous polynucleotide, said heterologous polynucleotide comprising:

    
    
        (a) an antisense sequence having homology to a pest pathogenicity gene, and
        (b) a sense sequence substantially complementary to said antisense sequence,
    
    


wherein a transcript of the heterologous polynucleotide is capable of hybridizing to form a double-stranded region comprising nucleotides encoded by the antisense sequence and the sense sequence,
wherein said sense and antisense sequences are each at least about 19 nucleotides in length,
wherein said pest pathogenicity gene is selected from the group consisting of cutinases, kinases, ribosomal RNAs, adhesins, elicitins, and G-proteins; and wherein said pest is a fungus.
In one embodiment, pest resistance is conferred to a plant comprising the step of transforming a host plant cell with a heterologous polynucleotide, said heterologous polynucleotide comprising:

    
    
        (a) an antisense sequence having homology to a pest pathogenicity gene; and
        (b) a sense sequence substantially complementary to said antisense sequence,
    
    


wherein a transcript of the heterologous polynucleotide is capable of hybridizing to form a double-stranded region comprising nucleotides encoded by the antisense sequence and the sense sequence,
wherein said sense and antisense sequences are each at least about 19 nucleotides in length,
wherein said pest pathogenicity gene is selected from the group consisting of kinases, ribosomal RNAs, G-proteins, moulting factors, serine proteases, cysteine proteases, and juvenile hormone esterases; and wherein said pest is an insect.
In one embodiment, pest resistance is conferred to a plant comprising the step of transforming a host plant cell with a heterologous polynucleotide, said heterologous polynucleotide comprising:

    
    
        (a) an antisense sequence having homology to a pest pathogenicity gene; and
        (b) a sense sequence substantially complementary to said antisense sequence,
    
    


wherein a transcript of the heterologous polynucleotide is capable of hybridizing to form a double-stranded region comprising nucleotides encoded by the antisense sequence and the sense sequence,
wherein said sense and antisense sequences are each at least about 19 nucleotides in length;
wherein said pest pathogenicity gene is selected from the group consisting of kinases, ribosomal RNAs, G-proteins, cuticle collagen proteins, and cathepsin proteases, and wherein said pest is a nematode.
In one embodiment, pest resistance is conferred to a plant comprising the step of transforming a host plant cell with a heterologous polynucleotide, said heterologous polynucleotide comprising:

    
    
        (a) an antisense sequence having homology to a pest pathogenicity gene; and
        (b) a sense sequence substantially complementary to said antisense sequence,
    
    


wherein a transcript of the heterologous polynucleotide is capable of hybridizing to form a double-stranded region comprising nucleotides encoded by the antisense sequence and the sense sequence,
wherein said sense and antisense sequences are each at least about 19 nucleotides in length,
wherein said pest pathogenicity gene is selected from the group consisting of cutinases, mascerating enzymes, kinases, ribosomal RNAs, adhesins, and G-proteins; and wherein said pest is a bacterium.
In one embodiment, the host plant is a soybean and the pest is one member selected from the group consisting of Fusarium solani (e.g., sudden death), Sclerotinia sclerotiorum (e.g., white mold), Phialophora gregata (e.g., brown stem rot), Phakospora pachyrhizi (e.g., Asian rust) and Phytophthora sojae (e.g., root and stem rot).
In one embodiment, the host plant is a cruciferous plant and the pest is Alubgo (e.g., white rust).
In one embodiment, the host plant is tobacco and the pest is Phytophthora (e.g., stem rot, root rot).
In one embodiment, the host plant is potato and the pest is Phytophthora (e.g., late blight).
In one embodiment, the host plant is broccoli and the is Pythium (e.g., damping-off).
In one embodiment, the host plant is pea and/or sugar beet and the pest is Aphanomyces (e.g., root rot).
In one embodiment, the host plant is tobacco and the pest is Peronospora (e.g., blue mold).
It has further been discovered that gene silencing in the pest according to the present invention, can be conferred upon the progeny of the pest—progeny that never had direct contact with the transformed host plant.
In one embodiment, the invention further comprises the step of cultivating a pest with the transformed host plant cell.
In one embodiment, a transformed host plant cell of the present invention is co-cultivated with a pest.
Optionally, progeny of the pest are less pathogenic to a plant cell other than the transformed host plant cell.
In one embodiment, a plant regenerated from a host plant cell transformed according to the present invention, is cultivated in soil contaminated with a pest.
In another embodiment, a plant regenerated from a host plant cell transformed according to the present invention, is cultivated in soil at risk of being contaminated with a pest
In another embodiment, a pest is co-cultivated with a plant regenerated from a host plant cell transformed according to the present invention and then cultivated in soil contaminated with a pest.
In another embodiment, a pest is co-cultivated with a plant regenerated from a host plant cell transformed according to the present invention and then cultivated in soil at risk of being contaminated with a pest.
In one embodiment, a pest is co-cultivated with the transformed plant and then cultivated in soil at risk of being contaminated with a pest.
Pest Pathogenicity Gene Homology
It has been further discovered that gene silencing according to the present invention can confer resistance to a surprisingly broad range of pests and that exact identity between a region of the sense sequence and the pest infectious gene is not required for the present invention to be effective.
In one embodiment of the present invention, the pest pathogenicity gene is a gene that provides for cross-resistance. Such a pest gene is conserved within the pest phylum such that when used with the present invention, a plant is made resistant to a plurality of members of the pest phylum (i.e. cross-resistance). Moreover, pest pathogenicity genes can be chosen according to the present invention by bioinformatic homology analysis as known to those skilled in the art to provide cross-resistance that is cross-species, cross-genus, cross-family, or cross-order resistance. For example, as will become clear from the examples herein, a cutinase gene is useful in plants to confer resistance to a plurality of fungi species. The gene for rDNA is useful in the pests of the present invention (i.e. insects, bacteria, fungi, and nematodes). Other non-limiting examples of such conserved genes are kinases, adhesions, G-proteins, elicitins, macerating enzymes, DNA and RNA polymerases, elongation factors, moulting factors, serine proteases, cysteine proteases, juvenile hormone esterase, cuticle collagen proteins, and cathepsin proteases and others set forth below. In another embodiment, a pest pathogenicity gene is selected to lack sufficient homology with a plant gene to cause harmful effects of the heterologous polynucleotide on the host plant (e.g. prevent silencing of a plant gene). For example, in one embodiment, the homology between the antisense sequence and the pest pathogenicity gene is less than about 70%.
In one embodiment, the antisense sequence is homologous to the pest pathogenicity gene by at least about 70%.
Optionally, the homology is at least about 75%.
Optionally, the homology is at least about 80%.
Optionally, the homology is at least about 85%.
Optionally, the homology is at least about 90%.
Optionally, the homology is at least about 95%.
In one embodiment, a transcript of the sense sequence is homologous to a transcript of the pest pathogenicity gene (e.g. pest pathogenicity gene mRNA) by at least about 70%.
Optionally, the homology is at least about 75%.
Optionally, the homology is at least about 80%.
Optionally, the homology is at least about 85%.
Optionally, the homology is at least about 90%.
Optionally, the homology is at least about 95%.
The above-mentioned homology can optionally extend over a stretch of at least about 20 nucleotides, or at least about 25 nucleotides, or at least about 50 nucleotides, or at least about 100 nucleotides.
Optionally, homology can be demonstrated between the antisense sequence and the pest pathogenicity gene (i.e. the DNA strand that binds RNA polymerase and directs transcription of the pest pathogenicity gene mRNA) in any of at least three ways.

    
    
        (1) hybridization between the antisense sequence and corresponding region of the pest gene sense strand.
        (2) hybridization between the antisense transcript and the corresponding region of the pest pathogenicity gene.
        (3) hybridization between a transcript of a DNA complentary to the antisense sequence (i.e. antisense cDNA) and a corresponding region of the pest pathogenicity gene mRNA.
    
    


Hybridization, as set forth above, can be demonstrated under conditions of low stringency. Optionally, the conditions are ones of moderate to high stringency by techniques well known in the art, as described, for example, in Keller, G. H., M. M. Manak (1987) DNA Probes, Stockton Press, New York, N.Y., pp. 169-170.
Examples of moderate to high stringency conditions are provided herein. Specifically, hybridization of immobilized DNA on Northern blots with 32P-labeled gene-specific probes is performed using standard methods (Maniatis et al.). In general, hybridization and subsequent washes are carried out under moderate to high stringency conditions that allowed for detection of target sequences with homology to sequences exemplified herein. For double-stranded DNA gene probes, hybridization was carried out overnight at 20-25° C. below the melting temperature (Tm) of the DNA hybrid in 6×SSPE, 5× Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA. The melting temperature is described by the following formula from Beltz et al. (1983): Tm=81.5° C.+16.6 Log [Na+]+0.41 (% G+C)−0.61 (% formamide)−600/length of duplex in base pairs.
Washes are typically carried out as follows:

    
    
        (1) Twice at room temperature for 15 minutes in 1×SSPE, 0.1% SDS (low stringency wash).
        (2) Once at Tm-20° C. for 15 minutes in 0.2×SSPE, 0.1% SDS (moderate stringency wash).
    
    


For oligonucleotide probes, hybridization was carried out overnight at 10-20° C. below the melting temperature (Tm) of the hybrid in 6×SSPE, 5× Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA. Tm for oligonucleotide probes was determined by the following formula from Suggs et al. (1981): Tm (° C.)=2(number T/A base pairs)+4 (number G/C base pairs).
Washes were typically carried out as follows:

    
    
        (1) Twice at room temperature for 15 minutes 1×SSPE, 0.1% SDS (low stringency wash).
        (2) Once at the hybridization temperature for 15 minutes in 1×SSPE, 0.1% SDS (moderate stringency wash).
    
    


In general, salt and/or temperature can be altered to change stringency. With a labeled DNA fragment of greater than about 70 or so bases in length, the following conditions can be used:
Low: 1 or 2×SSPE, room temperature
Low: 1 or 2×SSPE, 42° C.
Moderate: 0.2× or 1×SSPE, 65° C.
High: 0.1×SSPE, 65° C.
Duplex formation and stability depend on substantial complementarity between the two strands of a hybrid, and, as noted above, a certain degree of mismatch can be tolerated. Therefore, polynucleotide sequences useful in the subject invention include mutations (both single and multiple), deletions, and insertions in the described sequences, and combinations thereof, wherein said mutations, insertions, and deletions permit formation of stable hybrids with a target polynucleotide of interest. Mutations, insertions, and deletions can be produced in a given polynucleotide sequence using standard methods known in the art. Other methods may become known in the future.
The mutational, insertional, and deletional variants of the polynucleotide sequences of the invention can be used in the same manner as the exemplified polynucleotide sequences so long as the variants have substantial sequence similarity with the original sequence. As used herein, substantial sequence similarity refers to the extent of nucleotide similarity that is sufficient to enable the variant polynucleotide to function in the same capacity as the original sequence. Preferably, this similarity is greater than 75%; more preferably, this similarity is greater than 90%; and most preferably, this similarity is greater than 95%. The degree of similarity needed for the variant to function in its intended capacity will depend upon the intended use of the sequence. It is well within the skill of a person trained in this art to make mutational, insertional, and deletional mutations that are designed to improve the function of the sequence or otherwise provide a methodological advantage.
Vectors
Those skilled in the art are well able to construct vectors of the present invention (including those based on naked DNA) and design protocols for recombinant gene expression. For further details see, for example, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al, 1989, Cold Spring Harbor Laboratory Press. Such applicable techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are in detail in Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992.
Specific procedures and vectors previously used with wide success upon plants are described by Bevan, Nucl. Acids Res. (1984) 12, 8711-8721), and Guerineau and Mullineaux, (1993). Plant transformation and expression vectors. In: Plant Molecular Biology Labfax (Croy RRD ed). Oxford, BIOS Scientific Publishers, pp 121-148.
Vectors according to the present invention may be provided isolated and/or purified (i.e. from their natural environment), in substantially pure or homogeneous form, or free or substantially free of other nucleic acid. Nucleic acid according to the present invention may be wholly or partially synthetic.
Host Plants
The present invention may be used for transformation of any plant, including, but not limited to, corn (Zea mays), canola (Brassica napus, Brassica rapa ssp.), alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), sunflower (Helianthus annuus), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium hirsutum), sweet potato (Ipomoea batatus), cassaya (Manihot esculenta), coffee (Cofea ssp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidental), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), oats, barley, vegetables, ornamentals, and conifers.
Optionally, plants of the present invention are crop plants (for example, cereals and pulses, maize, wheat, potatoes, tapioca, rice, sorghum, millet, cassaya, barley, pea, and other root, tuber, or seed crops. Important seed crops for the present invention are oil-seed rape, sugar beet, maize, sunflower, soybean, and sorghum. Horticultural plants to which the present invention may be applied may include lettuce, endive, and vegetable brassicas including cabbage, broccoli, and cauliflower, and carnations, geraniums, petunias, and begonias. The present invention may be applied to tobacco, cucurbits, carrot, strawberry, sunflower, tomato, pepper, chrysanthemum, poplar, eucalyptus, and pine.
Optionally, plants of the present invention include grain seeds, such as corn, wheat, barley, rice, sorghum, rye, etc.
Optionally, plants of the present invention include oil-seed plants. Oil seed plants include canola, cotton, soybean, safflower, sunflower, Brassica, maize, alfalfa, palm, coconut, etc.
Optionally, plants of the present invention include leguminous plants. Leguminous plants include beans and peas. Beans include guar, locust bean, fenugreek, soybean, garden beans, cowpea, mung bean, lima bean, fava bean, lentils, chickpea, etc.
Host plants useful in the present invention are row crops and broadcast crops. Non limiting examples of useful row crops are corn, soybeans, cotton, amaranth, vegetables, rice, sorghum, wheat, milo, barley, sunflower, durum, and oats.
Non-limiting examples of useful broadcast crops are sunflower, millet, rice, sorghum, wheat, milo, barley, durum, and oats.
Host plants useful in the present invention are monocots and dicots.
Non-limiting examples of useful monocots are rice, corn, wheat, palm trees, turf grasses, barley, and oats.
Non-limiting examples of useful dicots are soybean, cotton, alfalfa, canola, flax, tomato, sugar beet, sunflower, potato, tobacco, corn, wheat, rice, lettuce, celery, cucumber, carrot, cauliflower, grape, and turf grasses.
Host plants useful in the present invention include plants cultivated for aesthetic or olfactory benefits. Non limiting examples include flowering plants, trees, grasses, shade plants, and flowering and non-flowering ornamental plants.
Host plants useful in the present invention include plants cultivated for nutritional value.
Host Cell Types
One skilled in the art will recognize the wide variety of host cells that can be contacted with the heterologous polynucleotides according to the present invention. Non-limiting examples of such cells are those in embryogenic tissue, callus tissue types I, II, and III, hypocotyl, meristem, root tissue, tissues for expression in phloem, and the like.
Host plant cells can optionally be enriched for cells with a higher potential to be transformed and/or a higher potential to regenerate mature plants. Manual selection of host plant cells, e.g., by selecting embryogenic cells from the surface of a Type II callus, is one means that may be used to enrich for host plant cells prior to culturing (whether cultured on solid media or in suspension). Optionally, cells can be selected from those located at the surface of a cell cluster, and may further be identifiable by their lack of differentiation, their size and dense cytoplasm. In one embodiment, host plant cells are less differentiated, or not yet committed to differentiation. Thus, in one embodiment, cells are identified and selected from those cells which are cytoplasmically dense, relatively unvacuolated with a high nucleus to cytoplasm ratio (e.g., determined by cytological observations), small in size (e.g., 10-20 μm), and capable of sustained divisions and somatic proembryo formation.
Optionally, host plant cells are identified through the use of dyes, such as Evan's blue, which are excluded by cells with relatively non-permeable membranes, such as embryogenic cells, and taken up by relatively differentiated cells such as root-like cells and snake cells (so-called due to their snake-like appearance).
Optionally, host plant cells are identified through the use of isozyme markers of embryogenic cells, such as glutamate dehydrogenase, which can be detected by cytochemical stains (Fransz et al., 1989). One skilled in the art will recognize that isozyme markers such as glutamate dehydrogenase may lead to some degree of false positives from non-embryogenic cells such as rooty cells which nonetheless have a relatively high metabolic activity.
Almost all plant tissues may be transformed during dedifferentiation using appropriate techniques described herein. Recipient cell targets include, but are not limited to, meristem cells, Type I, Type II, and Type III callus, immature embryos and gametic cells such as microspores, pollen, sperm, and egg cells. It is contemplated that any cell from which a fertile plant may be regenerated is useful as a recipient cell. Type I, Type II, and Type III callus may be initiated from tissue sources including, but not limited to, immature embryos, immature inflorescenses, seedling apical meristems, microspores, and the like.
Those cells that are capable of proliferating as callus also are recipient cells for genetic transformation. The present invention provides techniques for transforming immature embryos and subsequent regeneration of fertile transgenic plants. Direct transformation of immature embryos obviates the need for long term development of recipient cell cultures. Pollen, as well as its precursor cells, microspores, may be capable of functioning as recipient cells for genetic transformation, or as vectors to carry foreign DNA for incorporation during fertilization. Direct pollen transformation would obviate the need for cell culture.
Meristematic cells (i.e., plant cells capable of continual cell division and characterized by an undifferentiated cytological appearance, normally found at growing points or tissues in plants such as root tips, stem apices, lateral buds, etc.) may represent another type of recipient plant cell. Because of their undifferentiated growth and capacity for organ differentiation and totipotency, a single transformed meristematic cell could be recovered as a whole transformed plant. In fact, it is proposed that embryogenic suspension cultures may be an in vitro meristematic cell system, retaining an ability for continued cell division in an undifferentiated state, controlled by the media environment.
Pests
Plant pests useful in the present invention (i.e., can be rendered non-pathogenic according to the present invention), include fungi, nematodes, insects, bacteria, and parasitic plants such as striga, dodder and mistletoe.
Non-limiting examples of useful fungi include those set forth in Table 4.
Non-limiting examples of such useful nematodes include those set forth in Table 3.
Non-limiting examples of such useful insects include aphids, leafhoppers, planthoppers, mealy bugs, and Lepidoptera larvae.
Plant pests usefully treated by the present invention include bacteria. Non-limiting examples of such bacteria are shown in Table 1.
Plant pests usefully treated by the present invention includes rusts. Non-limiting examples of such rust are shown in Table 5.
Plant pests usefully treated by the present invention include the downy mildews. Non-limiting examples of such are shown in Table 2.







TABLE 1







Pests - Bacteria








Disease
Causative Agent





Bacterial leaf blight and
Pseudomonas avenae subsp. avenae


stalk rot



Bacterial leaf spot
Xanthomonas campestris pv. holcicola


Bacterial stalk rot
Enterobacter dissolvens = Erwinia dissolvens


Bacterial stalk and top
Erwinia carotovora subsp. carotovora, Erwinia


rot
chrysanthemi pv. Zeae


Bacterial stripe
Pseudomonas andropogonis


Chocolate spot
Pseudomonas syringae pv. Coronafaciens


Goss's bacterial wilt and
Clavibacter michiganensis subsp.


blight(leaf freckles and
nebraskensis = Corynebacterium


wilt)
michiganense pv. Nebraskense


Holcus spot
Pseudomonas syringae pv. Syringae


Purple leaf sheath
Hemiparasitic bacteria + (See under Fungi)


Seed rot-seedling blight
Bacillus subtilis


Stewart's disease
Pantoea stewartii = Erwinia stewartii


(bacterial wilt)



Corn stunt (Mesa Central
achapparramiento, stunt, Spiroplasma kunkelii


or Rio Grande stunt)
















TABLE 2







Pests - Downy Mildews








Disease
Causative Agent





Brown stripe downy
Sclerophthora rayssiae var. zeae


mildew



Crazy top downy mildew
Sclerophthora macrospora = S. macrospore


Green ear downy mildew
Sclerospora graminicola


Java downy mildew
Peronosclerospora maydis = Sclerospora maydis


Philippine downy mildew
Peronosclerospora philippinensis = Sclerospora philippinensis


Sorghum downy mildew
Peronosclerospora sorghi = Sclerospora sorghi


Spontaneum downy mildew
Peronosclerospora spontanea = Sclerospora spontanea


Sugarcane downy mildew
Peronosclerospora sacchari = Sclerospora sacchari


Dry ear rot (cob, kernel and
Nigrospora oryzae (teleomorph: Khuskia oryzae)


stalk rot)



Ear rots, minor
Aspergillus glaucus, A. niger, Aspergillus spp., Cunninghamella sp.,



Curvularia pallescens, Doratomyces stemonitis = Cephalotrichum



stemonitis, Fusarium culmorum, Gonatobotrys simplex, 



Pithomyces maydicus, Rhizopus microsporus, R. stolonifer = 



R. nigricans, Scopulariopsis brumptii


Ergot (horse's tooth, diente
Claviceps gigantea (anamorph: Sphacelia sp.)


del caballo)



Eyespot
Aureobasidium zeae = Kabatiella zeae


Fusarium ear and stalk rot
Fusarium subglutinans = F. moniliforme var. subglutinans


Fusarium kernel, root and
Fusarium moniliforme teleomorph: Gibberella fujikuroi)


stalk rot, seed rot and



seedling blight



Fusarium stalk rot, seedling
Fusarium avenaceum (teleomorph: Gibberella avenacea)


root rot



Gibberella ear and stalk rot
Gibberella zeae (anamorph: Fusarium graminearum)


Gray ear rot
Botryosphaeria zeae = Physalospora zeae (anamorph: 



Macrophoma zeae)


Gray leaf spot
Cercospora leaf spot) Cercospora sorghi = C. sorghi var. maydis, 



C. zeae-maydis


Helminthosporium root rot
Exserohilum pedicellatum = Helminthosporium pedicellatum 



(teleomorph: Setosphaeria


Hormodendrum ear rot
(Cladosporium Cladosporium cladosporioides = Hormodendrum 



cladosporioides, rot), C. herbarum (teleomorph: Mycosphaerella



tassiana)


Hyalothyridium leaf spot
Hyalothyridium maydis


Late wilt
Cephalosporium maydis


Leaf spots, minor
Alternaria alternata, Ascochyta maydis, A. tritici, A. zeicola,



Bipolaris victoriae = Helminthosporium victoriae (teleomorph:



Cochliobolus victoriae), C. sativus (anamorph: Bipolaris 



sorokiniana = H. sorokinianum = H. sativum), Epicoccum nigrum,



Exserohilum prolatum = Drechslera prolata (teleomorph: 



Setosphaeria prolata) Graphium penicillioides, Leptosphaeria 



maydis, Leptothyrium zeae, Ophiosphaerella herpotricha, 



(anamorph: Scolecosporiella sp.), Paraphaeosphaeria michotii,



Phoma sp., Septoria zeae, S. zeicola, S. zeina


Northern corn leaf blight
Exserohilum turcicum = Helminthosporium turcicum, Setosphaeria 



turcica


Northern corn leaf spot
Cochliobolus carbonum


Helminthosporium ear rot
Bipolaris zeicola = Helminthosporium carbonum


(race 1)



Penicillium ear rot (blue
Penicillium spp., P. chrysogenum, P. expansum, P. oxalicum


eye, blue mold)



Phaeocytostroma stalk rot
Phaeocytostroma ambiguum, Phaeocytosporella


and root rot
zeae


Phaeosphaeria leaf spot
Phaeosphaeria maydis, Sphaerulina maydis


Physalospora ear rot
Botryosphaeria Botryosphaeria festucae = Physalospora zeicola, 



(anamorph: Diplodia frumenti)


Purple leaf sheath
Hemiparasitic bacteria and fungi


Pyrenochaeta stalk rot and
Phoma terrestris, Pyrenochaeta terrestris


root rot



Pythium root rot
Pythium spp., P. arrhenomanes, P. graminicola


Pythium stalk rot
Pythium aphanidermatum = P. butleri L.


Red kernel disease (ear
Epicoccum nigrum


mold, leaf and seed rot)



Rhizoctonia ear rot
Rhizoctonia zeae (teleomorph: Waitea circinata)


Rhizoctonia root rot and
Rhizoctonia solani, Rhizoctonia zeae


stalk rot



Root rots, minor
Alternaria alternata, Cercospora sorghi, Dictochaeta fertilis, 



Fusarium acuminatum (teleomorph: Gibberella acuminata), 



F. equiseti (teleomorph: G. intricans), F. oxysporum, 



F. pallidoroseum, F. poae, F. roseum, F. cyanogena,



(anamorph: F. sulphureum), Microdochium bolleyi, Mucor sp., 



Periconia circinata, Phytophthora cactorum, P. drechsleri, 



P. nicotianae var. parasitica, Rhizopus arrhizus


Rostratum leaf spot
Setosphaeria rostrata, Helminthosporium (anamorph: 


(leaf disease, ear and stalk
Exserohilum rostratum = Helminthosporium rostratum)


rot)



Rust, common corn
Puccinia sorghi


Rust, southern corn
Puccinia polysora


Rust, tropical corn
Physopella pallescens, P. zeae = Angiopsora zeae


Sclerotium ear rot
Sclerotium rolfsii (teleomorph: Athelia rolfsii)


(southern blight)



Seed rot-seedling blight
Bipolaris sorokiniana, B. zeicola = Helminthosporium carbonum, 



Diplodia maydis, Exserohilum pedicillatum, Exserohilum 



turcicum = Helminthosporium turcicum, Fusarium avenaceum,



F. culmorum, F. moniliforme, Gibberella zeae (anamorph: 



F. graminearum), Macrophomina phaseolina, Penicillium spp., 



Phomopsis sp., Pythium spp., Rhizoctonia solani, R. zeae,



Sclerotium rolfsii, Spicaria sp.


Selenophoma leaf spot
Selenophoma sp.


Sheath rot
Gaeumannomyces graminis


Shuck rot
Myrothecium gramineum


Silage mold
Monascus purpureus, M. rubber


Smut, common
Ustilago zeae = U. maydis


Smut, false
Ustilaginoidea virens


Smut, head
Sphacelotheca reiliana = Sporisorium holci-sorghi


Southern corn leaf blight
Cochliobolus heterostrophus (anamorph: Bipolaris maydis = 


and stalk rot
Helminthosporium maydis)


Southern leaf spot
Stenocarpella macrospora = Diplodia macrospora


Stalk rots, minor
Cercospora sorghi, Fusarium episphaeria, F. merismoides,



F. oxysporum, F. poae, F. roseum, F. solani (teleomorph: 



Nectria haematococca), F. tricinctum, Mariannaea elegans, 



Mucor sp., Rhopographus zeae, Spicaria sp.


Storage rots
Aspergillus spp., Penicillium spp. and other fungi


Tar spot
Phyllachora maydis


Trichoderma ear rot and
Trichoderma viride = T. lignorum teleomorph: Hypocrea sp.


root rot



White ear rot, root and stalk
Stenocarpella maydis = Diplodia zeae


rot



Yellow leaf blight
Ascochyta ischaemi, Phyllosticta maydis (teleomorph: Mycosphaerella 



zeae-maydis)


Zonate leaf spot
Gloeccercospora sorghi
















TABLE 3







Pests - Parasitic Nematodes








Disease
Pathogen





Awl
Dolichodorus spp., D. heterocephalus


Bulb and stem (Europe)
Ditylenchus dipsaci


Burrowing
Radopholus similes


Cyst
Heterodera avenae, H. zeae, Punctodera



chalcoensis


Dagger
Xiphinema spp., X. americanum,



X. Mediterraneum


False root-knot
Nacobbus dorsalis


Lance, Columbia
Hoplolaimus Columbus


Lance
Hoplolaimus spp., H. galeatus


Lesion
Pratylenchus spp., P. brachyurus, P. crenatus,



P. hexincisus, P. neglectus, P. penetrans,



P. scribneri, P. thornei, P. zeae


Needle
Longidorus spp., L. breviannulatus


Ring
Criconemella spp., C. ornata


Root-knot
Meloidogyne spp., M. chitwoodi, M. incognita,



M. javanica


Spiral
Helicotylenchus spp.


Sting
Belonolaimus spp., B. longicaudatus


Stubby-root
Paratrichodorus spp., P. christiei, P. minor,



Quinisulcius acutus, Trichodorus spp.


Stunt
Tylenchorhynchus dubius
















TABLE 4







Pests - Fungal








Disease
Fungal Pest





Anthracnose leaf blight and
Colletotrichum graminicola


anthracnose stalk rot
(teleomorph: Glomerella



graminicola), Glomerella



tucumanensis (anamorph:



Glomerella falcatum)


Aspergillus ear and kernel rot
Aspergillus flavus


Banded leaf and sheath spot
Rhizoctonia solani = Rhizoctonia



microsclerotia (teleomorph:



Thanatephorus cucumeris)


Black bundle disease
Acremonium strictum = Cephalosporium



acremonium


Black kernel rot
Lasiodiplodia theobromae =



Botryodiplodia theobromae


Borde blanco
Marasmiellus sp.


Brown spot (black spot,
Physoderma maydis


stalk rot)



Cephalosporium kernel rot
Acremonium strictum = Cephalosporium



acremonium


Charcoal rot
Macrophomina phaseolina


Corticium ear rot
Thanatephorus cucumeris = Corticium



sasakii


Curvularia leaf spot
Curvularia clavata, C. eragrostidis, =



C. maculans (teleomorph:



Cochliobolus eragrostidis),



Curvularia inaequalis, C. intermedia



(teleomorph: Cochliobolus



intermedius), Curvularia lunata



(teleomorph: Cochliobolus lunatus),



Curvularia pallescens (teleomorph:



Cochliobolus pallescens),



Curvularia senegalensis, C. tuberculata



(teleomorph: Cochliobolus tuberculatus)


Didymella leaf spot
Didymella exitalis


Diplodia ear rot and stalk rot
Diplodia frumenti (teleomorph:



Botryosphaeria festucae)


Diplodia ear rot, stalk rot,
Diplodia maydis = Stenocarpella


seed rot and seedling blight
maydis


Diplodia leaf spot or leaf
Stenocarpella macrospora = Diplodia


streak
macrospore
















TABLE 5







Pests - Rusts









Host plant
Common disease name
Rust Species





Barley
Crown rust
Puccinia coronate



Leaf rust
Puccinia hordei



Stem rust
Puccinia graminis



Stripe (yellow) rust
Puccinia striiformis


Corn
Common rust
Puccinia sorghi



Southern rust
Puccinia polysora



Tropical rust
Physopella pallescens, P. zeae =




Angiopsora zeae


Oats
Crown rust
Puccinia coronata



Stem Rust
Puccinia graminis


Rye
Stem rust
Puccinia gramminis. = P. graminis f. sp. secalis



Leaf (brown) rust
Puccinia recondita (anamorph:




Aecidium clematidis)


Sorghum
Rust
Puccinia purpurea


Sugarcane
Common Rust
Puccinia melanocephala. = P. erianthi



Orange Rust
Puccinia kuehnii


Wheat
Leaf (brown) rust
Puccinia triticina. = P. Recondita f. Sp. tritici =




P. tritici-duri



Stem (black) rust
Puccinia gramminis = P. graminis f. sp. tritici



Stripe (yellow) rust
Puccinia striiformis (anamorph: P. uredoglumarum)


Apple
American hawthorne rust
Gymnosporangium globosum



Cedar apple rust
Gymnosporangium juniperi-virginianae



Japanese apple rust
Gymnosporangium yamadae miyabe ex




yamada



Pacific Coast pear rust
Gymnosporangium libocedri



Quince rust
Gymnosporangium clavipes


Asparagus
Rust
Puccinia asparagi


Banana
Leaf rust
Uredo musae, uromyces musae


Bean
Rust
Uromyces appendiculatus


Beet
Seedling rust.
Puccinia subnitens


Carrot
Rust
Aecidium foeniculiuromyces graminis,




Uromyces lineolatus subsp. nearcticus


Chickpea
Rust
Uromyces ciceris-arietini, Uromyces




striatus


Coffee
Rust (orange or leaf rust)
Hemileia vastatrix



Rust (powdery or grey
Hemileia coffeicola



rust)



Cotton
Cotton rust
Puccinia schedonnardi



Southwestern rust
Puccinia cacabata



Tropical rust
Phakopsora gossypii


Eucalyptus
Rust
Puccini apsidii


Flax
Rust
Melampsora lini


Crape
Rust
Physopella ampelopsidis


Lettuce
Rust
Puccinia dioicae = P. extensicola var.




hieraciata


Asparagus
Asparagus rust
Puccinia asparagi


Onion
Onion rust
Puccinia allii


Pea
Rust
Uromyces fabae


peanut
Rust
Puccinia arachidis


Pear
American hawthorne rust
Gymnosporangium globosum



Kern's pear rust
Gymnosporangium kernianum



Pacific Coast pear rust
Gymnosporangium libocedri



Pear trellis rust
Gymnosporangium fuscum



Rocky Mountain pear rust
Gymnosporangium nelsonii


Poplar
European Poplar rust
Melampsora larici-populinakle



American leaf rust
Melampsora medusae


Potato
Common rust
Puccinia pittierianap



Deforming rust
Aecidium cantensis


Red clover
Rust
Uromyces trifolii-repentis


Soybean
Rust
Phakopsora pachyrhizi


Strawberry
Leaf rust
Phragmidium potentillae = Frommea




obtusa


Sunflower
Rust
Puccinia helianthi, P. xanthii,




Uromyces junci


Sweet potato
Red rust
Coleosporium ipomoeae









Pest Pathogenicity Genes

The skilled artisan can readily identify pest genes to target in the present invention. Such a gene could be any pest gene that serves a direct or indirect role in such a pest's deleterious effects on a host plant. By way of example only, such a gene may be one that serves a role in pest growth, development, replication and reproduction, and invasion or infection.
In one embodiment, the host plant does not contain a gene that is more than about 70% homologous to the pest pathogenicity gene.
Optionally, the homology is not more than about 60%.
Optionally, the homology is not more than about 50%.
In one embodiment, the host plant does not contain a gene that contains a 25 nucleotide stretch that is more than about 70% homologous to the pest pathogenicity gene.
In one embodiment, the host plant does not contain a gene that contains a 25 nucleotide stretch that is more than about 60% homologous to the pest pathogenicity gene.
In one embodiment, the host plant does not contain a gene that contains a 25 nucleotide stretch that is more than about 50% homologous to the pest pathogenicity gene.
In one embodiment, the host plant does not contain more than one gene that contains a nucleotide stretch that is more than about 70% homologous to the pest pathogenicity gene.
In one embodiment, the host plant does not contain more than one gene that contains a 25 nucleotide stretch that is more than about 60% homologous to the pest pathogenicity gene.
In one embodiment, the host plant does not contain more than one gene that contains a 25 nucleotide stretch that is more than about 50% homologous to the pest pathogenicity gene.







TABLE 6







Pests, Pest Pathogenicity Genes, and Host Plants









Pest or pathogen group
Pest pathogenicity gene
Host Plants





Fungi
Cutinases
All



Kinases
All



Ribosomal RNAs
All



Adhesins
All



G-proteins
All



Elicitins
All


Bacteria
Cutinases
All



Mascerating enzymes
All



Kinases
All



Ribosomal RNAs
All



Adhesins
All



G-proteins
All


Insects
Kinases
All



Ribosomal RNAs
All



G-proteins
All



Moulting factors
All



Serine proteases
All



Cysteine proteases
All



Juvenile hormone esterase
All


Nematodes
Kinases
All



Ribosomal RNAs
All



G-proteins
All



Cuticle collagen proteins
All



Cathepsin proteases
All









By way of example, cutinase is a gene useful according to the present invention. When a fungal hypha invades a plant cell and absorbs nutrients, it absorbs the siRNAs from the plant and silences the fungus' essential and constitutive cutinase.
By way of example only, such a gene may be one that serves a role in pest growth, development, replication and reproduction, and invasion or infection. Other non limiting examples include those in Table 6.
Sense (and Antisense) Sequences
Sense and antisense sequences according to the present invention can be any sequence with homology to a sense and antisense strand (respectively) of pest pathogenicity gene.
The sense and antisense sequences can be on the same DNA strand or on different DNA strands. When the sense and antisense sequences are on the same DNA strand, transcription of these sequences can be driven off the same promoter or off of different promoters (e.g. different copies of the same promoter or different promoter). When the sense and antisense sequences are on different DNA strands, transcription of these sequences is driven off of different promoters.
The sense and antisense sequences can be arranged in DNA as complementary, duplex DNA or the sequences can be in different regions of the DNA (i.e. not forming duplex DNA with each other).
Each of the sense and antisense sequences comprise at least about 19 nucleotides. Optionally, each sequence comprises at least about 50 nucleotides, optionally at least about 100 nucleotides, optionally at least about 150 nucleotides, optionally at least about 250 polynucleotides, optionally at least about 500 nucleotides, optionally at least about 600 polynucleotides.
In one embodiment, the sequences are modified from the pest pathogenicity gene to create or increase homology to the pathogenicity gene of more than one pest.
In another embodiment the sequences are modified from the pest pathogenicity gene to shorten an open reading frame.
In another embodiment the sequences are modified from the pest pathogenicity gene to result in less homology with a plant, animal, or human gene.
In another embodiment, sense and antisense sequences comprise duplicative regions of a pest pathogenicity gene. Selection of such regions is made according to the teachings of the invention herein (e.g. highly conserved regions).
In other embodiments, regions of a pest pathogenicity gene or even the entire coding region of a pest pathogenicity gene may be duplicated to comprise sense and antisense sequences to increase the length of the double stranded region formed by the sense and antisense sequences. While not bound by theory, it is believed that in some embodiments of the present invention, a threshold level of siRNA's must be formed to elicit useful gene silencing in the pest and to confer pest resistance to the plant. Duplication of gene sequences is one useful way of increasing the length of the double stranded region and increasing the number of siRNA.
This (gene duplication) approach is demonstrated in examples herein using the heterologous polynucleotide pVZA300 (SEQ ID 7) comprising the elicitin INF1 of Phytophthora infestans (GenBank locus AY766228). This sequence was selected because of its high homology to many other elicitin genes expressed by the genus Phytophthora, suggesting that it could silence many other elicitin genes and thereby provide resistance to many species of Phytophthora. The sequence selected was 282 nt long, therefore the sequence was repeated (=564 nt) in both the sense and antisense orientations to approximate the lengths of the sense and antisense portions of pVZA100 (SEQ ID 5), pVZA200 (SEQ ID 6) and pVZA400 (SEQ ID 8) that have been remarkably successful.
The success of this approach was demonstrated by the dramatic effect of pVZA300 in reducing the growth and causing malformation of hyphae of transformed Phytophthora nicotianae and Phytophthora sojae (FIG. 14 D, F) as compared to the effects of transformations with pCAMBIA1201, pVZA100, or pVZA200. The plasmids pVZA200 and pVZA300 are partially identical with both containing a cathepsin gene, but pVZA300 also contains the repeated elicitin gene. Therefore, the deleterious impact of pVZA300 on the fungi must be due to the elecitin gene. This has very positive implications for developing Phytophthora resistant plants and application of this approach to depathogenization of Phytophthora contaminated soils.
Transforming Methods
A wide variety of methods are available for introducing heterologous polynucleotides of the present invention into the target host under conditions that allow for stable maintenance and expression of the polynucleotide. The particular choice of a transformation technology will be determined by its efficiency to transform certain plant species as well as the experience and preference of the person practicing the invention with a particular methodology of choice. It will be apparent to the skilled person that the particular choice of a transformation system to introduce nucleic acid into plant cells is not essential to or a limitation of the invention, nor is the choice of technique for plant regeneration.
While various transformation methods are taught herein as separate methods, the skilled artisan will readily recognize that certain methods can be used in combination to enhance the efficiency of the transformation process. Non-limiting examples of such methods include bombardment with Agrobacterium coated microparticles (EP-A-486234) or microprojectile bombardment to induce wounding followed by co-cultivation with Agrobacterium (EP-A-486233).
Direct delivery can be used to transform plant hosts according to the present invention. By way of non-limiting example, such direct delivery methods include polyethylene glycol treatment, electroporation, liposome mediated DNA uptake (e.g. Freeman et al. Plant Cell Physiol. 29: 1353 (1984)), or the vortexing method (e.g. Kindle, PNAS U.S.A. 87: 1228 (1990d). One form of direct DNA delivery is direct gene transfer into protoplasts from, for example, embryogenic cell suspension cultures (Lazzeri and Lorz (1988) Advances in Cell Culture, Vol. 6, Academic press, p. 291; OziasAkins and Lorz (1984) Trends in Biotechnology 2: 1 19).
Pollination Pathway
The skilled artisan is aware of certain challenges of genotype-dependant transformation arising from low regeneration potential of cereals. Accordingly, in one embodiment of the present invention, transformation is accomplished by a genotype-independent transformation approach based on the pollination pathway (Ohta Y., 1986). In maize, high efficiency genetic transformation can be achieved by a mixture of pollen and exogenous DNA (Luo Z. X. and Wu R., 1988, Proc. Natl. Acad. Sci. USA 83:715-719). Maize can be bred by both self-pollination and cross-pollination techniques. Maize has separate male and female flowers on the same plant, located on the tassel and the ear, respectively. Natural pollination occurs in maize when wind blows pollen from the tassels to the silks that protrude from the tops of the ears.
Transformation of rice according to the present invention can also be accomplished via the pollen-tube pathway (Plant Molecular Biology Reporter 6:165-174). The major potential advantages of the pollen-tube pathway approach include: (a) genotype independence; (b) lack of mosaicism; (c) no need for complicated cell and tissue culture techniques.
Transformation of tomato and melon with heterologous polynucleotides according to the present invention can be accomplished into intact plants via pollination pathway (Chesnokov, Yu. V., et al, 1992, USSR Patent No. 1708849; Bulletin of the USSR Patents, No. 4; Chesnokov Yu. V. & Korol A. B. 1993; Genetika USSR, 29:1345-1355). The procedures of genetic transformation based on the pollination-fecundation pathway include: (i) employment of a mixture (paste) of the pollen and transforming DNA; (ii) delivery of the alien DNA into the pollen tube, after pollination; and (iii) microparticle bombardment of microspores or pollen grains.
Agrobacterium Technology
In one embodiment of the present invention, host plants are transformed using Agrobacterium technology. Agrobacterium-mediated transfer is a widely applicable system for introducing genes into plant cells because the DNA can be introduced into whole plant tissues, thereby bypassing the need for regeneration of an intact plant from a protoplast. The use of Agrobacterium-mediated plant integrating vectors to introduce DNA into plant cells is well known in the art. See, for example, the methods described by Fraley et al., (1985), Rogers et al., (1987) and U.S. Pat. No. 5,563,055, specifically incorporated herein by reference in its entirety.
Agrobacterium-mediated transformation can efficiently be used with dicotyledonous host plants of the present invention including, by way of non-limiting example, Arabidopsis, corn, soybean, cotton, canola, tobacco, tomato, and potato.
Agrobacterium-mediated transformation is also applicable to nearly all monocotyledonous plants of the present invention. By non-limiting example, such monocotyledonous plant technologies are adaptable to rice (Hiei et al., 1997; Zhang et al., 1997; (Ishida et al., 1996); U.S. Pat. No. 5,591,616, specifically incorporated herein by reference in its entirety), wheat (McCormac et al., 1998), and barley (Tingay et al., 1997; McCormac et al., 1998).
Agrobacterium-mediated transformation, according to the present invention, can be accomplished with cultured isolated protoplasts and by transformation of intact cells or tissues.
Agrobacterium-mediated transformation in dicotyledons facilitates the delivery of larger pieces of heterologous nucleic acid as compared with other transformation methods such as particle bombardment, electroporation, polyethylene glycol-mediated transformation methods, and the like. In addition, Agrobacterium-mediated transformation appears to result in relatively few gene rearrangements and more typically results in the integration of low numbers of gene copies into the plant chromosome.
Modem Agrobacterium transformation vectors are capable of replication in E. coli as well as Agrobacterium, allowing for convenient manipulations as described (Klee et al., 1985). Moreover, recent technological advances in vectors for Agrobacterium-mediated gene transfer have improved the arrangement of genes and restriction sites in the vectors to facilitate the construction of vectors capable of expressing various polypeptide coding genes. The vectors described (Rogers et al., 1987) have convenient multi-linker regions flanked by a promoter and a polyadenylation site for direct expression of inserted polypeptide coding genes and are suitable for present purposes. In addition, Agrobacterium containing both armed and disarmed Ti genes can be used for the transformations. In those plant strains where Agrobacterium-mediated transformation is efficient, it is the method of choice because of the facile and defined nature of the gene transfer.
When Agrobacterium (e.g. A. tumefaciens and A. rhizogenes) is used to transform plant cells according to the present invention, the DNA to be inserted can be cloned into special plasmids, namely either into an intermediate vector or into a binary vector. The intermediate vectors can be integrated into the Ti or Ri plasmid by homologous recombination owing to sequences that are homologous to sequences in the T-DNA. The Ti or Ri plasmid also comprises the vir region necessary for the transfer of the T-DNA. Intermediate vectors cannot replicate themselves in Agrobacteria. The intermediate vector can be transferred into Agrobacterium tumefaciens by means of a helper plasmid (conjugation).
Binary vectors can replicate themselves both in E. coli and in Agrobacteria. Such vectors can comprise a selection marker gene and a linker or polylinker which are framed by the right and left T-DNA border regions. They can be transformed directly into Agrobacteria (Holsters et al. [1978] Mol. Gen. Genet. 163:181-187). The Agrobacterium used as host cell is to comprise a plasmid carrying a vir region. The vir region is necessary for the transfer of the T-DNA into the plant cell. Additional T-DNA may be contained. The bacterium so transformed is used for the transformation of plant cells. Plant explants can advantageously be cultivated with Agrobacterium tumefaciens or Agrobacterium rhizogenes for the transfer of the DNA into the plant cell. Whole plants can then be regenerated from the infected plant material (for example, pieces of leaf, segments of stalk, roots, but also protoplasts or suspension-cultivated cells) in a suitable medium, which may contain antibiotics or biocides for selection. The plants so obtained can then be tested for the presence of the inserted heterologous polynucleotides. No special demands are made of the plasmids in the case of injection and electroporation. It is possible to use ordinary plasmids, such as, for example, pUC derivatives.
Other transformation technology includes whiskers technology. See U.S. Pat. Nos. 5,302,523 and 5,464,765, both to Zeneca.
Microprojectile Bombardment
Optionally, host plant cells are transformed in accordance with the invention by microprojectile bombardment (U.S. Pat. No. 5,550,318; U.S. Pat. No. 5,538,880; U.S. Pat. No. 5,610,042; and U.S. Pat. No. 5,590,390; each of which is specifically incorporated herein by reference in its entirety). In this embodiment, particles are coated with nucleic acids and delivered into cells by a propelling force. Exemplary particles include those comprised of tungsten, platinum, and preferably, gold. It is contemplated that in some instances DNA precipitation onto metal particles would not be necessary for DNA delivery to a recipient cell using microprojectile bombardment. However, it is contemplated that particles may contain DNA rather than be coated with DNA. Hence, it is proposed that DNA-coated particles may increase the level of DNA delivery via particle bombardment but are not, in and of themselves, necessary.
For the bombardment, cells in suspension are concentrated on filters or solid culture medium. Alternatively, immature embryos or other target cells are arranged on solid culture medium. The cells to be bombarded are positioned at an appropriate distance below the microprojectile stopping plate.
An illustrative embodiment of a transforming method for delivering subject vectors into plant cells is by acceleration is the Biolistics Particle Delivery System (BioRad, Hercules, Calif.), which is used to propel particles coated with DNA or cells through a screen, such as a stainless steel or Nytex screen, onto a filter surface covered with plant cells cultured in suspension. The screen disperses the particles so that they are not delivered to the recipient cells in large aggregates. While not bound by theory, it is believed that a screen intervening between the projectile apparatus and the cells to be bombarded reduces the size of projectiles aggregate and may contribute to a higher frequency of transformation by reducing the damage inflicted on the recipient cells by projectiles that are too large.
For the bombardment, cells in suspension are preferably concentrated on filters or solid culture medium. Alternatively, immature embryos or other target cells may be arranged on solid culture medium. The cells to be bombarded are positioned at an appropriate distance below the microprojectile stopping plate. If desired, one or more screens may be positioned between the acceleration device and the cells to be bombarded. Through the use of techniques set forth herein one may obtain up to 1000 or more foci of cells transiently expressing a marker gene. The number of cells in a focus which express the exogenous gene product 48 hours post-bombardment often range from 1 to 10 and average 1 to 3.
One skilled in the art can optimize the prebombardment culturing conditions and the bombardment parameters to yield the maximum numbers of stable transformants. Both the physical and biological parameters for bombardment are important in this technology. Physical factors are those that involve manipulating the DNA/microprojectile precipitate or those that affect the flight and velocity of either the macro- or microprojectiles. Biological factors include all steps involved in manipulation of cells before and immediately after bombardment, the osmotic adjustment of target cells to help alleviate the trauma associated with bombardment, and also the nature of the transforming DNA, such as linearized DNA or intact supercoiled plasmids. It is believed that pre-bombardment manipulations are especially important for successful transformation of immature embryos.
Accordingly, it is contemplated that the skilled artisan can adjust the bombardment parameters in small scale studies to fully optimize the conditions. One may particularly wish to adjust physical parameters such as gap distance, flight distance, tissue distance, and helium pressure. One may also minimize the trauma reduction factors (TRFs) by modifying conditions which influence the physiological state of the recipient cells and which may therefore influence transformation and integration efficiencies. For example, the osmotic state, tissue hydration and the subculture stage or cell cycle of the recipient cells may be adjusted for optimum transformation. Results from such small scale optimization studies are disclosed herein and the execution of other routine adjustments will be known to those of skill in the art in light of the present disclosure.
Transformation by microprojectile bombardment is widely applicable, and can be used to transform virtually any plant species. Monocots are optionally transformed by bombardment according to the present invention, for example maize (U.S. Pat. No. 5,590,390), barley (Ritala et al., 1994; Hensgens et al., 1993), wheat (U.S. Pat. No. 5,563,055, specifically incorporated herein by reference in its entirety), rice (Hensgens et al., 1993), oat (Torbet et al., 1995; Torbet et al., 1998), rye (Hensgens et al., 1993), sugarcane (Bower et al., 1992), and sorghum (Casa et al., 1993; Hagio et al., 1991);
Dicots are optionally transformed by bombardment according to the present invention, for example, tobacco (Tomes et al., 1990; Buising and Benbow, 1994), soybean (U.S. Pat. No. 5,322,783, specifically incorporated herein by reference in its entirety), sunflower (Knittel et al. 1994), peanut (Singsit et al., 1997), cotton (McCabe and Martinell, 1993), tomato (Van Eck et al. 1995), and legumes in general (U.S. Pat. No. 5,563,055, specifically incorporated herein by reference in its entirety).
Electroporation
In one embodiment, plant cells are transformed using the method of electroporation, See, for example, WO 87/06614 to Boyce Thompson Institute; U.S. Pat. Nos. 5,472,869 and 5,384,253, both to Dekalb; and WO 92/09696 and WO 93/21335, both to Plant Genetic Systems.
The method of Krzyzek et al. (U.S. Pat. No. 5,384,253, incorporated herein by reference in its entirety), is well suited for the present invention. In this method, certain cell wall-degrading enzymes, such as pectin-degrading enzymes, are employed to render the target recipient cells more susceptible to transformation by electroporation than untreated cells. Alternatively, recipient cells are made more susceptible to transformation by mechanical wounding.
The method of Tada, Y., et al (Efficient gene introduction to rice by electroporation and analysis of transgenic plants: Use of electroporation buffer lacking chloride ions, Theor Appl Genet, Vol. 80: 475-480, 1990) is, for example, suitable for transforming rice.
In one embodiment, to effect transformation by electroporation, friable tissues, such as a suspension culture of cells or embryogenic callus are used.
Optionally, immature embryos or other organized tissue are transformed directly. In this technique, cell walls are partially degraded by exposing them to pectin-degrading enzymes (pectolyases) or mechanically wounding in a controlled manner.
Non-limiting examples plant cell hosts that can be transformed by electroporation of intact cells according to the present invention are wheat (according to, for example, Zhou et al., 1993), tomato (according to, for example Hou and Lin, 1996), soybean (according to, for example Christou et al., 1987) and tobacco (Lee et al., 1989). See also U.S. Pat. No. 5,384,253; Rhodes et al., 1995; and D'Halluin et al., 1992
In one embodiment, plants are transformed for electroporation of protoplasts (according to, for example, Bates, 1994; Lazzeri, 1995). Non-limiting examples of plant cell hosts that can be transformed by electroporation of protoplasts cells according to the present invention are soybean plants (according to, for example, Dhir and Widholm in PCT Publication No. WO 92/17598, specifically incorporated herein by reference), barley (e.g., Lazerri, 1995), sorghum (e.g., Battraw et al., 1991), (e.g., Bhattacharjee et al., 1997), wheat (e.g., He et al., 1994) and tomato (e.g. Tsukada, 1989).
Viral Vectors
Furthermore, viral vectors can also be used to transform plants according to the present invention. For example, monocotyledonous plants can be transformed with a viral vector using the methods described in U.S. Pat. No. 5,569,597 to Mycogen Plant Science and Ciba-Giegy, now Novartis.
U.S. Pat. No. 5,589,367 also describes plant transformation with a viral vector.
U.S. Pat. No. 5,316,931 describes plant transformation with a viral vector.
A large number of cloning vectors useful for higher plants (including monocots and divots) according to the present invention comprise a replication system in E. coli and a marker that permits selection of the transformed cells. These vectors comprise, for example, pBR322, pUC series, M13 mp series, pACYC184, etc., and pCAMBIA 1201. Accordingly, the sequence comprising a heterologous polynucleotide of the present invention can be inserted into the vector at a suitable restriction site. The resulting plasmid is used for transformation into E. coli. The E. coli cells are cultivated in a suitable nutrient medium, then harvested and lysed. The plasmid is recovered. Sequence analysis, restriction analysis, electrophoresis, and other biochemical-molecular biological methods are generally carried out as methods of analysis. After each manipulation, the DNA sequence used can be cleaved and joined to the next DNA sequence. Each plasmid sequence can be cloned in the same or other plasmids. Depending on the method of inserting desired genes into the plant, other DNA sequences may be necessary. If, for example, the Ti or Ri plasmid is used for the transformation of the plant cell, then at least the right border, but often the right and the left border of the Ti or Ri plasmid T-DNA, are joined as the flanking region of the genes to be inserted (FIG. 1).
The use of T-DNA for the transformation of plant cells is described in EP 120 516; Hoekema (1985) In: The Binary Plant Vector System, Offset-durkkerij Kanters B. V., Alblasserdam, Chapter 5; Fraley et al., Crit. Rev. Plant Sci. 4:1-46; An et al. (1985) EMBO J. 4:277-287; and many subsequent references widely known and available to those skilled in the art.
Marker Genes
In order to improve the ability to identify transformants, one may desire to employ one or more marker genes into a heterologous polynucleotide of the present invention.
Marker genes, according to the present invention, include those that encode a selectable marker and those that encode a screenable marker, depending on whether the marker confers a trait that one can “select” for by chemical means, i.e., through the use of a selective agent (e.g., a herbicide, antibiotic, or the like), or whether it is simply a “reporter” trait that one can identify through observation or testing, i.e., by “screening”. Of course many examples of suitable marker genes or reporter genes are known to the art and can be employed in the practice of the invention.
Included within the terms selectable or screenable marker genes are also genes that encode a “secretable marker” whose secretion can be detected as a means of identifying or selecting for transformed cells. Examples include markers that encode a secretable antigen that can be identified by antibody interaction, or even secretable enzymes that can be detected by their catalytic activity. Secretable proteins fall into a number of classes, including small, diffusible proteins detectable, e.g., by ELISA, and proteins that are inserted or trapped in the cell wall (e.g., proteins that include a leader sequence such as that found in the expression unit of extensin or tobacco PR-S).
With regard to selectable secretable markers, the use of a gene that encodes a protein that becomes sequestered in the cell wall, and which protein includes a unique epitope is considered to be particularly advantageous. Such a secreted antigen marker would ideally employ an epitope sequence that would provide low background in plant tissue, a promoter-leader sequence that would impart efficient expression and targeting across the plasma membrane, and would produce protein that is bound in the cell wall and yet accessible to antibodies. A normally secreted wall protein modified to include a unique epitope would satisfy all such requirements.
One example of a protein suitable for modification in this manner is extensin, or hydroxyproline rich glycoprotein (HPRG). The use of the HPRG (Stiefel et al., 1990) is well characterized in terms of molecular biology, expression, and protein structure. However, any one of a variety of extensins and/or glycine-rich wall proteins (Keller et al., 1989) could be modified by the addition of an antigenic site to create a screenable marker.
Elements of the present disclosure are exemplified in detail through the use of particular marker genes. However in light of this disclosure, numerous other possible selectable and/or screenable marker genes will be apparent to those of skill in the art in addition to the one set forth herein below. Therefore, it will be understood that the following discussion is exemplary rather than exhaustive.
Selectable Markers
Selectable markers for use in connection with the present invention include, but are not limited to, a neo gene (Potrykus et al., 1985) that codes for kanamycin resistance and can be selected for using kanamycin, G418, and the like; a bar gene that codes for bialaphos resistance; a gene that encodes an altered EPSP synthase protein (Hinchee et al., 1988) thus conferring glyphosate resistance; a nitrilase gene such as b×n from Klebsiella ozaenae that confers resistance to bromoxynil (Stalker et al., 1988); a mutant acetolactate synthase gene (ALS) or acetohydroxyacid synthase gene (AHAS) that confers resistance to imidazolinone, sulfonylurea, or other ALS-inhibiting chemicals (European Patent Application 154,204); a methotrexate-resistant DHFR gene (Thillet et al., 1988); a dalapon dehalogenase gene that confers resistance to the herbicide dalapon (U.S. Pat. No. 5,780,708); or a mutated anthranilate synthase gene that confers resistance to 5-methyl tryptophan (PCT Publication No. WO 97/26366). Where a mutant EPSP synthase gene is employed, additional benefit may be realized through the incorporation of a suitable chloroplast transit peptide, CTP (U.S. Pat. No. 4,940,835). See also, Lundquist et al., U.S. Pat. No. 5,508,468.
An illustrative embodiment of a selectable marker gene capable of being used in embodiments of the present invention to select transformants are those that encode the enzyme phosphinothricin acetyltransferase, such as the bar gene from Streptomyces hygroscopicus or the pat gene from Streptomyces viridochromogenes (U.S. Pat. No. 5,550,318, which is incorporated by reference herein). The enzyme phosphinothricin acetyl transferase (PAT) inactivates the active ingredient in the herbicide bialaphos, phosphinothricin (PPT). PPT inhibits glutamine synthetase, (Murakami et al., 1986; Twell et al., 1989) causing rapid accumulation of ammonia and cell death.
Suitable selectable marker genes contemplated herein include an aminoglycoside phosphotransferase gene, neomycin phosphotransferase gene, G418 resistance gene, glyphosate resistance gene, hygromycin resistance gene, methotrexate resistance gene, imidazolinones resistance gene, sulfonylureas resistance gene, triazolopyrimidine herbicide resistance gene, ampicillin resistance gene, tetracycline resistance gene, bacterial kanamycin resistance gene, phosphinothricin resistance gene, chloramphenicol acetyltransferase (CAT) gene and luciferase gene, amongst others.
Screenable Markers
Screenable markers that may be employed include, but are not limited to, a beta-glucuronidase or uidA gene (GUS) that encodes an enzyme for which various chromogenic substrates are known; an R-locus gene, which encodes a product that regulates the production of anthocyanin pigments (red color) in plant tissues (Dellaporta et al., 1988); a beta-lactamase gene (Sutcliffe, 1978), which encodes an enzyme for which various chromogenic substrates are known (e.g., PADAC, a chromogenic cephalosporin); a xyle gene (Zukowsky et al., 1983), which encodes a catechol dioxygenase that can convert chromogenic catechols; an alpha-amylase gene (Ikuta et al., 1990); a tyrosinase gene (Katz et al., 1983), which encodes an enzyme capable of oxidizing tyrosine to DOPA and dopaquinone which in turn condenses to form the easily detectable compound melanin; a beta-galactosidase gene, which encodes an enzyme for which there are chromogenic substrates; a luciferase (lux) gene (Ow et al., 1986), which allows for bioluminescence detection; or even an aequorin gene (Prasher et al., 1985), which may be employed in calcium-sensitive bioluminescence detection, or a green fluorescent protein gene (Niedz et al., 1995).
Genes from the R gene complex are contemplated as useful screenable markers. The R gene complex encodes a protein that acts to regulate the production of anthocyanin pigments in most seed and plant tissue. Maize lines can have one, or as many as four, R alleles that combine to regulate pigmentation in a developmental and tissue specific manner. Thus, an R gene introduced into such cells will cause the expression of a red pigment and, if stably incorporated, can be visually scored as a red sector. If a line carries dominant alleles for genes encoding the enzymatic intermediates in the anthocyanin biosynthetic pathway (C2, A1, A2, Bz1 and Bz2), but carries a recessive allele at the R locus, transformation of any cell from that line with R will result in red pigment formation. Exemplary lines include Wisconsin 22 that contains the rg-Stadler allele and TR112, a K55 derivative that is r-g, b, P1. Alternatively any genotype of maize can be utilized if the C1 and R alleles are introduced together.
It is further contemplated that R gene regulatory regions may be employed in heterologous polynucleotides of the present invention in order to provide mechanisms for controlling the expression of such heterologous polynucleotides. More diversity of phenotypic expression is known at the R locus than at any other locus (Coe et al., 1988). It is contemplated that regulatory regions obtained from regions 5′ to the structural R gene would be valuable in directing the expression of genes, e.g., insect resistance, drought resistance, herbicide tolerance or other protein coding regions. For the purposes of the present invention, it is believed that any of the various R gene family members may be successfully employed (e.g., P, S, Lc, etc.). However, the most preferred will generally be Sn (particularly Sn:bol3). Sn is a dominant member of the R gene complex and is functionally similar to the R and B loci in that Sn controls the tissue specific deposition of anthocyanin pigments in certain seedling and plant cells, therefore, its phenotype is similar to R.
A further screenable marker contemplated for use in the present invention is firefly luciferase, encoded by the lux gene. The presence of the lux gene in transformed cells may be detected using, for example, X-ray film, scintillation counting, fluorescent spectrophotometry, low-light video cameras, photon counting cameras or multiwell luminometry. It is also envisioned that this system may be developed for populational screening for bioluminescence, such as on tissue culture plates, or even for whole plant screening.
Marker Gene Strandedness
According to the present invention, marker genes can be designed by selection of sequence and position within the heteropolynucleotide to result in a positive or negative marker. When a heteropolynucleotide contains sequences of a marker gene but does not contain sufficient complementary sequences to the marker gene, a transcript of the heteropolynucleotide will contain the marker gene in a single stranded region and it can be expressed. Thus expression is correlated with integration of the heteropolynucleotide into the host plant. When a heteropolynucleotide contains sequences of a marker gene and sufficient complementary sequences to the marker gene, a transcript of the heteropolynucleotide will contain the marker gene in a double stranded region and it will be silenced. Thus, expression of such a marker gene will indicate that the heteropolynucleotide is not being silenced in the host plant (i.e. “negative marker”). When a plant cell is transformed with a heteropolynucleotide comprising both a positive marker and a negative marker gene expresses the positive marker gene but does not express the negative marker gene, this indicates that the plant is transcribing and silencing at least part of the heteropolynucleotide. Thus expression is correlated with integration of the heteropolynucleotide into the host plant (i.e. positive sequences can be selected).
Choice of Marker Gene Sequence
A skilled artisan will now readily recognize how combinations of markers at different positions within a heterologous polynucleotide of the present invention can be effected. Such differences in position effect whether the marker is silenced or not (e.g. a gene within the region that forms a double stranded structure by way of hydrogen bonding is recognized as foreign by the plant's internal defense mechanisms and hydrolyzed into siRNA [i.e. “silenced”]).
For example, in one embodiment, a heterologous polynucleotide of the present invention contains the hypothetical A gene (that is, any marker gene of the present invention) upstream of the double stranded region and a hypothetical B gene within the double stranded region. A non-transformed cell is deficient in A expression (“A−). It is also B− and C−A transformed cell that functionally inactivates B region (e.g. by gene silencing of the region wherein the B gene lies) is A+ and B−. A transformed cell that fails to inactivate the B region is A+ and B+.
A skilled artisan will now recognize that the presence of antisense sequences of a marker gene, appropriately placed downstream of the sense sequences of the marker gene, will further facilitate gene silencing (e.g. double strand formation) of such marker gene.
It should also now be readily recognized that a marker localized downstream of a double stranded region will also be silenced if no promoter lies between such double stranded region and such marker.
In one embodiment, heterologous polynucleotides of the present invention include those with the structures shown in FIGS. 15-19.
Promoter and Regulatory Elements
According to the present invention, a heterologous polynucleotide, or a portion thereof, is capable of being transcribed in a plant. In plants, such transcription requires a promoter that is operably linked to the heterologous polynucleotide. Such a promoter can be endogenous to the plant. By way of example, a heterologous polynucleotide can be inserted adjacent to a constitutive or inducible plant promoter.
The heterologous polypeptide of the present invention can optionally contain a promoter. Such a promoter can be a plant promoter or a non-plant promoter that functions in a plant.
Plant promoters include but are not limited to ribulose-1,6-bisphosphate (RUBP) carboxylase small subunit (ssu), beta-conglycinin promoter, beta-phaseolin promoter, ADH promoter, heat-shock promoters, and tissue specific promoters such as a constitutive promoterwhere it is desirable to directing continuous gene expression in all cell types and at all times (e.g., actin, ubiquitin, CaMV 35S, and the like).
Promoters from a variety of sources can be used efficiently in plant cells to express foreign genes. For example, promoter regulatory elements of bacterial origin, such as the octopine synthase promoter, the nopaline synthase promoter, the mannopine synthase promoter; promoters of viral origin, such as the cauliflower mosaic virus (35S and 19S), 35T (which is a re-engineered 35S promoter, see U.S. Pat. No. 6,166,302, especially Example 7E) and the like may be used.
It may be desirable to use an inducible promoter, which is responsible for expression of genes in response to a specific signal, such as: physical stimulus (heat shock genes), light (RUBP carboxylase), hormone (Em), metabolites, chemical, and stress. Other desirable transcription and translation elements that function in plants may be used. Numerous plant-specific gene transfer vectors are known in the art.
It may be desirable to use a promoter that is active during a certain stage of the plant's development as well as active in plant tissues and organs. Examples of such include but are not limited to pollen-specific, embryo-specific, corn-silk-specific, cotton-fiber-specific, root-specific, seed-endosperm-specific promoters and the like. Tissue specific promoter regulatory elements can be used where it is desirable to promote transcription in tissues such as leaves or seeds (e.g., zein, oleosin, napin, ACP, globulin and the like).
While any promoter that functions in a plant cell (i.e. supports transcription of a heterologous polynucleotide) is useful in the present invention, it can be desirable to select a relatively stronger or weaker promoter. One embodiment of the present invention uses a strong promoter to drive the sense sequence of a pest pathogenicity gene and a weaker promoter to drive the antisense sequence of the pest pathogenicity gene. One skilled in the art can readily determine promoter strength empirically within the embodiments of the present invention. When an embodiment contains two promoters (driving two different transcripts), the relative transcription rate can be determined by quantifying the transcripts (e.g. quantitative PCR).
Examples of promoters that are relatively strong are the figwort mosaic virus promoter and the enhanced CaMV 35S promoter. An example of a promoter that is relatively weaker is the CaMV 35S promoter. One skilled in the art will readily appreciate that “weaker” and “stronger” promoters is a relative term, and they can be empirically identified by, for example, quantitative PCR with the appropriate primers against the transcript that is driven by the promoters.
Other elements such as matrix attachment regions, scaffold attachment regions, introns, enhancers, polyadenylation sequences and the like may be present and thus may improve the transcription efficiency or DNA integration. Such elements may or may not be necessary for DNA function, although they can provide better expression or functioning of the DNA. Such elements may be included in the DNA as desired to obtain optimal performance of the transformed DNA in the plant. Typical elements include but are not limited to Adh-intron 1, Adh-intron 6, the alfalfa mosaic virus coat protein leader sequence, the maize streak virus coat protein leader sequence, as well as others available to a skilled artisan.
Terminator
Heterologous polynucleotides of the present invention can optionally contain a terminator. The term “terminator” refers to a DNA sequence at the end of a transcriptional unit which signals termination of transcription. Eukaryotic terminators are 3′-non-translated DNA sequences containing a polyadenylation signal, which facilitates the addition of poly (A) sequences to the 3′-end of a primary transcript.
Terminators active in cells derived from viruses, yeast, molds, bacteria, insects, birds, mammals and plants are known and described in the literature and useful in the present invention. They may be isolated from bacteria, fungi, viruses, animals and/or plants.
Examples of terminators suitable for use in the present invention include the nopaline synthase (NOS) gene terminator of Agrobacterium tumefaciens, the terminator of the Cauliflower mosaic virus (CaMV) 35S gene, the zein gene terminator from Zea mays, the Rubisco small subunit (SSU) gene terminator sequences, Subclover stunt virus (SCSV) gene sequence terminators (International Patent Application No. PCT/AU95/00552), and the terminator of the Flaveria bidents malic enzyme gene NSEA3 (PCT/AU95/00552).
Those skilled in the art will be aware of additional promoter sequences and terminator sequences suitable for use in performing the invention. Such sequences may readily be used without any undue experimentation.
Embodiments of the present invention are taught herein where it is desirable to have more than one terminator. Examples of such are embodiments are where the sense and antisense sequences are to be contained on separate transcripts (i.e. each having its own 3′ and 5′ end).
Plant Regeneration and Propagation
Following transformation, a plant may be regenerated, e.g., from single cells, callus tissue, or leaf discs, as is standard in the art. Almost any plant can be entirely regenerated from cells, tissues, and organs of the plant. Available techniques are reviewed in Vasil et al. (1984) in Cell Culture and Somatic Cell Genetics of Plants, Vols. I, II, and III, Laboratory Procedures and Their Applications (Academic press); and Weissbach et al. (1989) Methods for Plant Molecular Biology, Academic Press, 1989.
During suspension culture development, small cell aggregates (10-100 cells) are formed, apparently from larger cell clusters, giving the culture a dispersed appearance. Upon plating these cells to solid media, somatic embryo development can be induced, and these embryos can be matured, germinated and grown into fertile seed-bearing plants. Alternatively, callus cells growing on solid culture medium can be induced to form somatic embryos from which fertile seed bearing plants may develop. The characteristics of embryogenicity, regenerability, and plant fertility are gradually lost as a function of time in suspension culture. Cryopreservation of suspension cells arrests development of the culture and prevents loss of these characteristics during the cryopreservation period.
The transformed plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting line having expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited, and then seeds harvested to ensure expression of the desired phenotypic characteristic has been achieved.
In addition to a plant, the present invention provides any clone of such a plant, seed, selfed or hybrid descendants, and any part of any of these, such as cuttings, pollen, or seed. The invention provides any plant propagule, that is any part which may be used in reproduction or propagation, sexual or asexual, including cuttings, seed, and so on. Also encompassed by the invention is a plant which is a sexually or asexually propagated off-spring, clone, or descendant of such a plant; or any part or propagule of said plant, off-spring, clone, or descendant. Plant extracts and derivatives are also provided.
Gene Stacking
A heterologous polynucleotide according to the present invention can comprise a plurality of sense sequences and a plurality of antisense sequences (i.e. a “polycistronic heterologous polynucleotide”). Thus, a heterologous polynucleotide can protect a plant against multiple pests.
One skilled in the art will now recognize that, by using gene stacking, a plant can be made resistant to a plurality of pests. The plurality of pests can be from the same phylum or from different phyla. For example, a plant can be made resistant to a plurality of fungal pests. As one non-limiting example of gene stacking according to the present invention, soybeans can be made resistant to brown stem rot (Phialophora gregata), stem and root rot (Phytophthora sojae), white mold disease (Sclerotinia sclerotiorum), sudden death syndrome (Fusarium solani pisi), and Asian rust (Phakopsora pachyrhizi).
Optionally, a plant can be made resistant to a plurality of insect pests. As one non-limiting example of gene stacking according to the present invention, potatoes can be made resistant to potato leafhopper (Empoasca filament), Colorado potato beetle (Leptinotarsa decemlineata), and green peach aphid (Myzus persicae).
Optionally, a plant can be made resistant to a plurality of fungal and non-fungal pests. As one non-limiting example of gene stacking according to the present invention, potatoes can be made resistant to potato golden nematode (Globodera rostochiensis), Colorado potato beetle (Leptinotarsa decemlineata), and potato late blight (Phytophthora infestans).
In one embodiment of the present invention, the heterologous polynucleotide comprises two different sense and two different antisense sequences.
In one embodiment of the present invention, the heterologous polynucleotide comprises at least three different sense and at least three different antisense sequences.
Plant pests often cause the infected plants to become yellow or brown, to be reduced in size, to produce less root exudates and to mature more rapidly. Therefore, controlling a single major pest of a plant may in turn make that plant more susceptible to other pests. If a fungal disease of a plant is controlled, then the healthy plant will remain large and green for a longer period, and therefore be attractive to and susceptible to insects and airborne fungal diseases. The green plant also may produce more root exudates which will attract more nematodes and root infecting fungi. It has been discovered herein that it can be surprisingly advantageous to transform a plant with a heterologous polynucleotide containing sequences homologous to a plurality of pests genes, wherein such genes are from pests of different taxons (e.g. subspecies, species, genera, families, orders, class).
Depathogenesis
It has been discovered that when a pest infects a host plant cell transformed according to the present invention, the pest becomes less pathogenic to the transformed host plant cell and non-transformed host plant cells. Moreover, in one embodiment, a pest infects a host plant cell transformed according to the present invention and then the gene silencing molecules derived from the plant by feeding are subsequently transmitted through mating (e.g. by cytoplasmic transmission, conjugation, hyphal anastomosis, etc.) to populations of the pest and produce a less pathogenic pest. In this way, populations of pests can be made non-pathogenic (i.e. less pathogenic) by, for example, a pest that is unable to multiply, and the macroenvironment (e.g. soil, plant foliage, plant debris, buildings) contaminated with a pest can be protected or decontaminated therefrom.
Optionally, progeny of the pest are less pathogenic to the transformed host plant cell and non-transformed host plant cells.
Accordingly, one embodiment of this invention is a mechanism for biological control, and it has great economic value in that it can be used to deplete important fields of pathogenic pest isolates of fungi and reduce the application of pesticides. In this embodiment the transgenic plant transmits the siRNAs to the pest by way of pest feeding behavior, and the pest loses its pathogenicity due to the subsequent silencing of its own pathogenic gene. Moreover, the pest progeny will in turn be nonpathogenic (e.g. by cytoplasmic transmission, mating, conjugation, hyphal anastomosis), thereby effectively cleaning up the local environment of that pest and preventing or significantly reducing its plant destroying behavior.
Depathogenesis, in one embodiment, is accomplished by co-cultivating nonpathogenic (either transformed directly or having obtained siRNAs), pests with pathogenic pests. Such co-cultivation is accomplished, by way of nonlimiting example, by broadcast of the transgenic pest to grow and infest the soil wherein the nonpathogenic pests then mate with like species. Such broadcast can be accomplished for fungi, by way of nonlimiting example, by cultivating transformed fungi on barley or wheat seeds and then distributing such seeds to soil that is in need of being depathogenized or protection from pathogenic pests.
This embodiment is suitable for protecting established valuable plants such as trees (e.g. redwoods, oaks, palms, maples, etc.).
Although the foregoing discussion uses fungal plant pathogens as an example, the strategy for depathogenesis can be adapted to any other plant pests that would transmit the “silenced” trait to their progeny, as will be readily apparent to the skilled artisan.
First, it is possible to “depathogenize” a pest by silencing a pest pathogenicity gene. The pest then could survive in the environment (e.g. soil) and spread the siRNAs throughout the population and no longer attack plants. Second, by silencing major structural genes or essential nutrition genes such as ribosomal RNA or elicitin genes, respectively, it is possible to debilitate or kill a pest thereby eliminating it from crop fields or lowering its presence below economic thresholds.
EXAMPLES
Example 1
Cloning Strategy
The plasmids pVZA100, pVZA200, pVZA300, and pVZA400 are embodiments that were designed for transformation of various plant species, including, but not limited to, tobacco (Nicotiana tobacum, cv. Xanthi), soybean (Glycine max cv. Williams 82), potato (Solanum tuberosm cv. Alpha) and corn (Zea mays cv. Hi II×B73). pVZA100 contains the full-length cutinase gene from Phytophthora nicotianae in both the sense (S) and antisense (AS) orientations, separated by a spacer region containing a portion (817 bp) of the E. coli β-glucuronidase gene (GUS) referred to as dGUS. pVZA100 expression in plants is under the control of the cauliflower mosaic virus (CaMV) 35S promoter and the cucumber mosaic virus (CMV) capsid protein gene 5′ untranslated region (5′ UTR). The termination signal also is from the CaMV 35S gene. The backbone of the pVZA100 plasmid is the plasmid pCAMBIA1201 (FIG. 1) (Roberts et al., 2000). Its plant selectable marker is the antibiotic hygromycin. The expression of hygromycin phosphotransferase in pCAMBIA 1201 also is controlled by the CaMV 35S promoter and terminator. The reporter gene in pCAMBIA 1201 is GUS with a catalase intron, also with the CaMV 35S promoter, but with the nopaline synthase (NOS) terminator.
Example 2
Construction of the Intermediary Plasmid pVZA1
Because of its versatility, the gene expression plasmid pUC18 cpexp (Slightom, 1991) was selected to contain the gene silencing cassette. This plasmid is pUC18, containing the CaMV 35S promoter, followed by the CMV capsid protein gene 5′ UTR and the CaMV 35S gene termination signal. A Bgl II site was engineered into the polylinker between the CMV 5′ UTR and the CaMV 35S terminator. To obtain dGUS, the plasmid pCAMBIA1201 (FIG. 1) was double digested with Nco I and Hinc II. The GUS fragment 821 bp in length, corresponding to nucleotides 9540 to 10361 of pCAMBIA1201, was isolated and purified by agarose gel electrophoresis. That fragment was then digested (blunted) at the 3′ Nco I site using mung bean nuclease to reduce it to 817 bp. This blunt-ended dGUS was then re-purified by agarose gel electrophoresis and ligated into the Bgl II site of pUC18 cpexp to yield the intermediary plasmid pVZA1 (FIG. 2).
Example 3
Construction of the pVZA2, pVZA3, pVZA100, pVZA200, pVZA300 and pVZA400 Plasmids
The full length cutinase gene from P. nicotianae was amplified in the antisense orientation by PCR from a cutinase clone in the plasmid pZErO-2.1 (Munoz and Bailey, 1998) using primers VZA 1F (SEQ ID NO: 1) and VZA 2R (SEQ ID NO: 2) containing the Not I and Nco I restriction sites, respectively. It was then purified by agarose gel electrophoresis, and ligated into the Not I and Nco I sites of the plasmid pVZA1. The resulting plasmid, pVZA2, was cloned in E. coli and purified. A copy of the cutinase gene in the sense orientation was amplified by PCR from the same cutinase clone in the plasmid pZErO-2.1 using primers VZA 3F and VZA 4R (SEQ ID NO: 3 and SEQ ID NO: 4, respectively) containing the Apa I and Xho I restriction sites, respectively. It was purified by agarose gel electrophoresis, and ligated into the Apa I and Xho I restriction sites in the plasmid pVZA2 to produce the final intermediary plasmid pVZA3 (FIG. 3). The portion containing dGUS and separating the cutinase gene in sense and antisense orientation is referred to as the silencing construct. The portion containing the silencing construct plus the regulatory elements for the construct (35S promoter, the 5′ UTR and the 35S terminator) is referred to as the silencing cassette (FIG. 3).
The silencing cassette was excised from plasmid pVZA3 by double restriction digestion with EcoR I and Hind III and purified by agarose gel electrophoresis. The plasmid pCAMBIA1201 was double digested with EcoR I and Hind III, and the silencing cassette was ligated into those sites to produce the final plant transformation plasmid pVZA100. The presence of the entire silencing cassette was confirmed by restriction digestion of pVZA100 with PstI (which releases the whole insert) and by digestions with the various restriction enzymes used in its assembly. The digestion products were separated by agarose gel electrophoresis and compared with known molecular standards. The sequence, 2201 nt, of the silencing construct was confirmed by sequencing of the DNA, and it is SEQ ID NO: 5. The following oligonucleotide pairs were used as PCR primers to detect the presence of the respective genes in pVZA100 and in transgenic plants: cutinase (SEQ ID NOs: 1 and 2, or SEQ ID NOs: 3 and 4); GUS (SEQ ID NOs: 9 and 10) and hygromycin phosphotransferase (SEQ ID NOs: 11 and 12).
Plant transformation plasmids pVZA200, pVZA300 and pVZA400 were based on the same principle as pVZA100. The silencing construct was an inverted repeat of the selected pest pathogenicity gene in both the sense and antisense orientations, separated by the dGUS fragment. However, the silencing constructs for pVZA200, pVZA300, and pVZA400 were synthesized commercially by GenScript Corporation (Piscataway, N.J.) with ApaI and Not I restriction sites at the 5′ and 3′ ends respectively, and cloned in the pUC57 vector.
The silencing construct of pVZA200 contained nucleotides 221 to 820 of the cathepsin B gene (GenBank locus AY702822) of the aphid Myzus persicae. This aphid is a major pest and the most efficient vector of many plant viruses. Cathepsin B is a cysteine protease, a digestive enzyme required for the survival and development of Myzus persicae. The 2031 nt sequence of the silencing construct for pVZA200 is shown as SEQ ID NO: 6.
The regulatory elements were then added to the pVZA200 silencing construct to yield the pVZA200 silencing cassette. For this, the entire pVZA100 silencing cassette was excised from pVZA100 by digestion with EcoR I and Hind III and purified by agarose gel electrophoresis. It was cloned into a similarly digested pUC19. This plasmid was then digested with ApaI and Not I. The pUC19 portion containing the regulatory elements from pVZA100 was purified and ligated to the pVZA200 silencing construct released from pUC57 by digestion with ApaI and Not I. The pVZA200 silencing cassette, now flanked by the regulatory elements from pVZA100, was increased in pUC19. The entire pVZA200 silencing cassette was excised from pUC19 by digestion with EcoR I and Hind III and then ligated into the EcoR I and Hind III sites of pCAMBIA1201 to complete the pVZA200 plant transformation plasmid. As with pVZA100, the presence of the entire silencing cassette was confirmed by restriction digestion of pVZA200 with PstI and by digestions with the various restriction enzymes used in its assembly. The digestion products were separated by agarose gel electrophoresis and compared with known molecular standards.
The following oligonucleotide pairs were used as PCR primers to detect the presence of the respective genes in pVZA200 and in transgenic plants: cathepsin (SEQ ID NOs: 38 and 39); GUS (SEQ ID NOs: 9 and 10) and hygromycin phosphotrasferase (SEQ ID NOs: 11 and 12).
The plant transformation plasmids pVZA300 and pVZA400 were planned similarly and synthesized. The silencing construct of pVZA300 was a double gene construct, containing gene sequences from two different plant pests. It was designed to contain the same cathepsin B gene inverted repeat around the dGUS fragment as the silencing construct of pVZA200. But in addition, at each end it contained the partial coding sequence for the elicitin INF1 of Phytophthora infestans (GenBank locus AY766228). This elicitin gene is required by Phytophthora infestans for pathogenicity and as a sterol receptor because Phytophthora spp. do not synthesize the sterols essential for their growth and development. The elicitin sequence was 282 nt long. Therefore, to obtain a sequence approaching 600 nt, the sequence was repeated to obtain 564 nt.
An ApaI restriction site was included at the 5′ end of the elicitin sense sequence, and a NotI restriction site was included at the 3′ end of the elicitin antisense sequence. The elicitin sense sequence was added to the 5′ end of the pVZA200 sequence, and the elicitin antisense sequence was added to the 5′ end of the pVZA200 sequence to complete the silencing construct of pVZA300. It is 3159 nt long and composed of the elicitin gene and cathepsin gene both in the sense orientation, followed by dGUS, followed by the cathepsin gene and elicitin gene, respectively, both in the antisense orientation.
The sequence of the silencing construct for pVZA300 is shown as SEQ ID NO: 7. This sequence was synthesized by GenScript Corporation and cloned in the pUC57 vector. The regulatory elements were then added as described for pVZA200 to complete the silencing cassette for pVZA300, and it was cloned into the EcoR I and Hind III sites of pCAMBIA1201. As previously, the presence of the entire silencing cassette was confirmed by restriction digestion of pVZA300 with PstI and by digestions with the various restriction enzymes used in its assembly. The digestion products were separated by agarose gel electrophoresis and compared with known molecular standards. The following oligonucleotide pairs were used as PCR primers to detect the presence of the respective genes in pVZA300 and in transgenic plants: elicitin and cathepsin overlap (SEQ ID NOs: 40 and 39) cathepsin (SEQ ID NOs: 38 and 39); GUS (SEQ ID NOs: 9 and 10) and hygromycin phosphotrasferase (SEQ ID NOs: 11 and 12).
The silencing construct of the plant transformation plasmid pVZA400 contained nt 1-600 of the ribosomal RNA genes (rDNA) of Phytophthora infestans (GenBank locus AJ854293) in the sense and antisense orientations, respectively, separated by dGUS. An ApaI and NotI restriction site were included on the 5′ and 3′ ends, respectively. The sequence of the silencing construct (2031 nt) for pVZA400 is shown as SEQ ID NO: 8. It was synthesized by GenScript Corporation and cloned in the pUC57 vector. The regulatory elements were then added as described for pVZA200 to complete the silencing cassette for pVZA400, and it was cloned into the EcoR I and Hind III sites of pCAMBIA1201. As previously, the presence of the entire silencing cassette was confirmed by restriction digestion of pVZA400 with PstI and by digestions with the various restriction enzymes used in its assembly. The digestion products were separated by agarose gel electrophoresis and compared with known molecular standards.
The following oligonucleotide pairs were used as PCR primers to detect the presence of the respective genes in pVZA400 and in transgenic plants: ribosomal DNA (SEQ ID NOs: 41 and 42); GUS (SEQ ID NOs: 9 and 10) and hygromycin phosphotrasferase (SEQ ID NOs: 11 and 12).
Example 4
Transformation Methods
Plant cells (including tobacco, soybean, potato and corn) were transformed with transformation plasmids pCAMBIA1201, pVZA100, pVZA200, pVZA300, and pVZA400 using biolistics or with pVZA100 using strain PGV 2260 of Agrobacterium tumefaciens. Fungal cultures were transformed only by biolistics with transformation plasmids pCAMBIA1201, pVZA100, pVZA200, pVZA300, and pVZA400.
Preparation of tungsten or gold microprojectiles for bombardment with the helium-driven biolistic device was according to the protocol of Shark et al. 1991. The microprojectiles were coated with either pCAMBIA1201, pVZA100, pVZA200, pVZA300, pVZA400, or with no DNA. For plant tissue and fungal culture transformation, the Dupont PDS-1000 apparatus was used under vacuum (23 mm Hg) and at 1200 PSI with tissues at 8 and 12 cm from the stopping plate. For seedling and plantlet transformation, a hand held homemade particle gun was used at 200 PSI, and held as close as possible to the developing meristem.
The transformations with Agrobacterium tumefaciens were performed essentially as described by Rogers et al. (1987). Although biolistics and Agrobacterium tumefaciens transformation are used to exemplify this portion of the subject invention, any method of introducing heterologous DNA into target cells for purposes of transformation that is known in the art could be used, as would readily be appreciated by the ordinarily skilled artisan, and as would be optimized depending on the target species.
Example 5
Tobacco Transformation with pVZA100 and Regeneration
Organogenic callus cultures of Nicotiana tabacum cv Xanthi were maintained in shake cultures in the laboratory and transferred monthly to fresh MS medium (Murashige and Skoog, 1962). Calli were removed from flasks aseptically, filtered free of liquid and spread on sterile filter paper in a sterile plastic Petri dish. They were bombarded with pVZA100 according to the methods described above, and the filter paper was transferred to phytohormone-free MS medium overnight. The following day the filter paper was transferred to MS medium containing 1 mg/l 6-benzyladenine, 0.1 mg/l napthaline acetic acid, 1 mg/l thiamine-HCl, 100 mg/l inositol, 30 g/l sucrose, 6 g/l phytagar and 30 ug/ml hygromycin for selection of the transgenic cells. For regeneration, the expanding calli were transferred to fresh MS medium every 2 weeks. Emerging shoots were then transferred to MS medium without phytohormones to form roots. Rooted plantlets were transferred to soil or tested directly for resistance to P. nicotianae. Plants were grown to maturity in soil and seed collected for subsequent analysis.
Example 6
Fungal Transformation and Selection Protocols
Five mm mycelial discs of Phytophthora nicotianae, P. sojae and P. infestans were bombarded according to the methods and with the constructs described in Examples 3 and 5. Bombarded cultures were placed on plates of V-8 agar and incubated at 27C overnight. The following day they were transferred to V-8 agar plates containing 400 ug/ml hygromycin and incubated at 27° C. for 3-4 days. Discs (5 mm) were cut from the new growth and transferred to Petri dishes containing 15 ml of V-8 broth and incubated at 27° C. for 2 days. The mycelial mats were washed with sterile water, pressed dry and incubated for 2 days with 10 ml of soil extract to induce sporangia formation. To induce zoospore release, the mats were placed in sterile demineralized water and chilled at 4° C. for 30-60 min. Zoospores were collected by centrifugation for 5 min. at 5,000 g and 4° C. For selection, the zoospore concentration was adjusted to approximately 1,000/ml and plated on V-8 agar containing 400 ug/ml hygromycin and X-gluc (Jefferson et al., 1986) and incubated for several days at 27° C.
Those isolates stably transformed with the GUS gene in pCAMBIA1201 appeared on the X-gluc agar as small blue colonies originating from single zoospores. They were transferred to individual plates of V-8 agar containing 400 ug/ml hygromycin, and the cultures consistently stained blue, positive for GUS, after many transfers. Those isolates transformed with the pVZA100, pVZA200, pVZA300, and pVZA400 silencing plasmids also initially appeared as small blue colonies originating from single zoospores. They were transferred to plates of V-8 agar containing 400 ug/ml hygromycin and X-gluc. After 2-3 transfers on this medium, those colonies which survived became white, indicating that the GUS gene had been silenced. All cultures were maintained at 27° C. and transferred monthly to fresh medium.
Example 7
Transformation of Corn, Soybeans and Potato
Corn was transformed by bombarding Type II callus from Hi II germplasm with gold particles coated with two plasmids, pVZA100 and pBAR184 (Frame et al., 2000). The pBAR184 was included so that the co-transformants could be selected and regenerated on media containing the herbicide bialaphos rather than hygromycin. Calli were grown and selected on N6-based media containing 2 mg/l each of 2,4-dichlorophenoxyacetic acid and bialaphos. For regeneration rooting, embryos were transferred to MS media containing 2 mg/l of bialaphos, and no phytohormones. Rooted plantlets were assayed by PCR.
Soybean was transformed both by Agrobacterium tumefaciens and biolistics. Mature embryos were excised aseptically from hydrated seeds of Williams soybean. The embryos were then soaked for 15 min. in a few milliters of a culture of Agrobacterium tumefaciens that had been grown on a shaker overnight at 28 C and contained either pCAMBIA1201 or pVZA10. The treated embryos were placed on MS medium containing 15 mg/L of hygromycin and phytohormones (BAP and NAA) and then on MS medium free of phytohormones for rooting. Embryo-like structures formed and germinated, and leaves were produced (FIGS. 6A-6D), but no plants were regenerated and rooted, despite testing of many media. Biolistic transformation of soybean was performed using the hand held homemade particle gun. Seedlings 8-10 days old and 6-10 cm tall were bombarded with gold particles coated with either pCAMBIA1201, pVZA100, pVZA200, pVZA300 or pVZA400 or with no DNA. The plants were grown in the greenhouse and assayed by PCR. Mature seed was collected and tested for resistance to P. sojae. 
Potato was transformed both by Agrobacterium tumefaciens and biolistics. Russett Burbank and Alpha potatoes were transformed by Agrobacterium tumefaciens. Tubers were surface disinfested, and the potato “eyes” were excised and plated on solid MS medium without phytohormones. The eyes germinated, and plantlets were produced by cutting a node section containing one leaf and placing the stem in fresh MS medium. Rooted plantlets 5-7 cm tall were produced in 10-15 days. The leaves were removed from the plantlets, and the stems were cut into pieces 1 cm long. The pieces were then soaked as above for 15 min. in a few milliters of a culture of Agrobacterium tumefaciens that had been grown on a shaker overnight at 28C and contained either pCAMBIA1201 or pVZA100. The pieces were blotted on sterile filter paper and the tissue culture procedure of Cearley and Bolyard (1997) was followed. Bulbous tuber-like and root-like structures were produced (FIG. 8), but no plants were regenerated and rooted, despite testing of many media. For biolistic transformation, potato plantlets 3-5 cm tall were transplanted to soil. After 2-3 days they were bombarded using the hand held homemade particle gun and gold particles coated with either pCAMBIA1201, pVZA100, pVZA200, pVZA300 or pVZA400 or with no DNA. The plants were grown in the greenhouse and assayed by PCR and for their reactions to aphids and P. infestans. 
Example 8
DNA and RNA Extraction from Plant and Fungal Tissues and PCR and RT/PCR Protocols
Total DNA was extracted from tobacco leaf tissue and mycelium of Phytophthora nicotianae by the method of Dellaporta et al., 1983. Usually a 100 mg sample of frozen tissue was ground to a fine powder in a microfuge tube containing liquid nitrogen, and then homogenized in 500 ul of extraction buffer with a motor-driven stainless steel pestle.
Total RNA for RT/PCR reactions was extracted from leaf tissue and fungal mycelium by the TRIzol method of Invitrogen. Usually 100 mg of tissue was frozen in liquid nitrogen in a 1.8 ml microfuge tube. It was ground to fine powder and then homogenized in 1.0 ml of TRIzol with a motor-driven pestle. The mixture was allowed to incubate for 5 min. at room temperature. Then 200 ul of chloroform were added and mixed by vortexing. The suspension was incubated 5 min. at room temperature. The tubes were centrifuged at 14,000 rpm for 15 min. at 4° C. The aqueous phase was transferred to a clean tube, 500 ul of isopropanol were added and the samples mixed and incubated 10 min. at room temperature. The tubes were centrifuged again for 10 min. at 4° C., and the supernatant was discarded. The pellet was rinsed once with 250 ul of 75% ethanol by vortexing and centrifuging 5 min. at 4° C. The RNA pellet was air dried briefly, dissolved in 20 ul of RNase-free water and stored at −70° C. until used.
A similar method was used for large preparations of total RNA for siRNA detection. Usually 1 gram of frozen leaf or fungal mycelium was ground to fine powder in liquid nitrogen and homogenized with 15 ml TRIzol reagent. Then 3 ml of chloroform were added and incubated as above. The supernatant was recovered by centrifugation and the RNA precipitated with 1⁄2 volume of isopropanol. The pellet was washed with 75% ethanol, air dried and resuspended in 80 ul of RNase-free water.
The PCR reactions for both plant and fungal tissues were performed essentially as described by Munoz and Bailey, 1998, except the reaction volumes totaled 50 ul. The reactions contained 1×PCR buffer (10× solution: 500 mM KCl, 100 mM Tris-HCl pH 9.0, and 1% Triton X-100), 2.5 mM MgCl2, 200 μM of each deoxyribonucleotide triphosphate (dNTP), 2.5 units of Taq DNA polymerase (Invitrogen or Promega), 100 pmol of the appropriate forward and reverse oligonucleotide primer, 1-10 ul of template and sufficient deionized water to equal 50 ul. DNA preparations used as templates were usually diluted to 50 ng per microliter, and 1 microliter was used as the template.
The PCR reaction conditions were standardized except for the annealing temperature, which was varied depending on the Tm of the oligonucleotide primers for the gene being amplified. The standardized conditions were 94° C. for 2 min. for melting, followed by 40 cycles of 1 min. at 94° C., 1 min. at the annealing temperature, and 2 min. at 72° C. for elongation. After 40 cycles there was 10 min. elongation at 72° C., and the reactions were held at 4° C. until assayed.
The RT/PCR reactions were carried out in 3 separate steps, DNase treatment, reverse transcription and then PCR, as recommended by Invitrogen. The DNAse treatment was performed to eliminate any DNA carried over in the RNA purification to ensure that only messenger RNA was being transcribed, and not the genomic DNA. The DNase mix contained per reaction: 3 ug of RNA, 1 ul 10×DNAse I buffer, 2 ul DNAse I, 0.5 ul RNase OUT and water (RNase-free) to 10 ul final volume. This was incubated at 26° C. for 30 min. Then 6.5 ul of 25 mM EDTA were added per tube and incubated for 15 min. at 65° C.
For the reverse transcriptase (RT) reactions a Mix I and Mix H were prepared. Mix I contained per reaction: 1 ul dNTPs, 4 ul 5×RT buffer, and 2 ul dithiothreitol. Mix II contained per reaction: 0.5 ul RNase OUT, 1 ul reverse transcriptase and 1 ul water. The appropriate oligonucleotide primers (1 ul each or water) were placed in the appropriate tubes and Mix I and II added appropriately. Mix I was placed in every tube, Mix II in the odd numbered tubes and 2.5 ul water in the even numbered tubes; then 10 ul of DNase treated RNA was added to each tube. The RT reactions were incubated at 42° C. for 60 min., and then at 70° C. for 15 min.
Example 9
Detection of siRNAs in Plant and Fungal Tissues
The procedure was essentially as described by Hutvagner et al., 2000. Total RNA was extracted from tobacco leaves or fungal mycelium as above. It was quantitated in a spectrophotometer at 260 nm and 80 ug were fractionated by denaturing polyacrylamide gel electrophoresis on 15% gels for 2.5 hrs at 0.025 Amp. The gels were stained with ethidium bromide, photographed, and the RNA transferred to hybond N+ (Amersham) for 1 hr. at 10 volts at 4° C. Hybridization was performed as described by Sambrook et al. (1989) for Northern blotting.
The probe was transcribed from a 500 bp PCR product of the cutinase gene and labeled with 32P by random priming. Membranes were prehybridized 1 hr at 50° C. with HYBAID buffer solution from Amersham and hybridized for at least 16 hrs at 50° C. Washes were performed twice with 5×SSC at room temperature. The hybridization signals were detected by phosphorimaging, using a Storm 860 phosphorimager scanner from Molecular Dynamics.
Example 10
GUS Staining of Plant and Fungal Tissues
Preparation of the X-GLUC substrate: For 200 ml, set up a beaker with 150 ml distilled water and add the following components: Sodium phosphate buffer pH 7.0 (1 M), 20 ml, EDTA pH 8.0 (500 mM), 4 ml, Potassium ferrocyanide K4Fe(CN)6.3H2O, 0.042 g, Triton X-100 0.2 ml. Stir for 10 min. Adjust pH 7.0 with NaOH. Dissolve 100 mg of X-GLUC in 2 ml DMSO or dimethyl-formamide. Add the X-GLUC in DMSO to the beaker. Filter sterilize the solution, dispense in 10 ml aliquots, and store at −20° C. For incorporation into growth media, add 100 mg of X-GLUC powder dissolved in DMSO to 250 ml V8 agar cooled and ready to dispense for fungal isolates or to MS media for plant materials.
Staining procedure: Add enough X-GLUC solution to cover the small explant of plant tissue and incubate at 37° C. for 3-24 hours. For fungal isolates or plant materials in X-gluc agar plates, incubate at 24-27° C. for 3-24 hours.
This procedure is essentially as described by Jefferson, et al., 1986.
Example 11
Plant Inoculation Tests with Fungi
Three methods were tested to inoculate tobacco with P. nicotianae. All gave satisfactory results, but the toothpick method was more rigorous and more dependable.
1. Toothpick method: This method was provided by Dr. A. Csinos (personal communication). Round wooden toothpicks were broken in half and autoclaved in V-8 broth. The sterile toothpicks were laid flat in plates of V-8 agar, and 6-8 mycelial discs (5 mm in diameter) of P. nicotianae were spaced equidistant on the plate. The plates were incubated at 27C for at least 2 weeks, when chlamydospores become obvious by microscopic examination. Plant inoculation is accomplished by standing 1 or 2 toothpicks in the soil next to the crown of the plant. The plants were covered with a plastic tent to maintain relative humidity at 100% and incubated at 27° C. Symptoms were usually apparent after 2-4 days, and susceptible plants were usually dead after 5-8 days. The reactions of resistant transgenic plants vary from being symptomless to producing restricted stem lesions 1-4 cm long, which do not interfere with plant growth and subsequent flower and seed development.
2. The mycelial soak method: Mycelia of P. nicotianae were grown overnight at 27° C. in V-8 broth. Transgenic seedlings or plants in soil were washed free of the medium and placed in 100 ml of sterile deionized water. Mycelial mats were shredded with forceps and added to the water. The plants were covered with a plastic tent to maintain relative humidity at 100% and incubated at 27° C. Symptoms were usually apparent after 2-4 days, and susceptible plants were usually dead after 5-8 days. The roots and crown of the susceptible plants were completely macerated (FIG. 11A). The roots of the resistant transgenic plants appear to be “burned” at the tips (FIG. 11B), or there were defined lesions at the crown, but this does not interfere with plant growth and subsequent flower and seed development after the plants were potted in soil.
3. Zoospore inoculation: Abundant sporangia were produced by placing strips of surface-sterilized tobacco leaves in growing cultures of P. nicotianae at 27° C. in V-8 broth overnight. Millions of zoospores were released, by placing the mats in sterile demineralized water and chilled at 4° C. for 30-60 min. Zoospores were collected by centrifugation for 5 min. at 5,000 g and 4° C.
Soybean plants were inoculated with P. sojae by two different methods. Seedlings were inoculated by cutting a 3 mm cylinder from an actively growing culture on V8 agar with a cork borer and placing the cylinder in the axil of one hypocotyl. The inoculum was held in place by Parafilm, and the plants enclosed in a plastic bag to give 100% humidity. Leaves of soybean plants were inoculated with P. sojae by placing a leaf in a Petri dish with 10 ml of sterile water and adding 1 or 2 plugs of inoculum as above.
Potato leaves were inoculated with P. infestans by placing a leaf in a Petri dish with 10 ml of sterile water and adding 1 or 2 plugs of inoculum as above, or by placing 10 ul of a zoospore suspension per leaflet.
In all inoculation tests, the plant or leaf reactions were read and recorded daily until the inoculated plants or leaves stopped dying or showed no evidence of infection.
Example 12
Molecular Characterization of P. Nicotianae Resistant Tobacco Plants
DNA and RNA were extracted from T0 and T2 generation transformed plants of Example 27. Polymerase chain reaction (PCR) and reverse transcription/PCR(RT/PCR) tests were performed on the DNA and RNA samples, respectively. The PCR reactions were performed to determine the presence of the transgenes in the plants. The RT/PCR reactions were performed to determine if the transgenes were being expressed or had been silenced. The results were the same for both the T0 and T2 generations, and are summarized in Table 7.







TABLE 7





Gene Detection and Expression in Transgenic and Wild


Type Tobacco Plants


















Hygromycin




phosphotransferase
GUS











Tissue Assayed
PCR
RT-PCR
PCR
RT-PCR





Tobacco wild type
−
−
−
−


Tobacco + pCAMBIA1201
+
+
+
+


pVZA100 Transgenic line 4
+
+
+
−


pVZA100 Transgenic line 23
+
+
+
−


pVZA100 Transgenic line 26
+
+
+
−


pVZA100 Transgenic line 27
+
+
+
−


pVZA100 plasmid DNA
+
na
+
na














16S



Cutinase
Ribosomal RNA











Tissue Assayed
PCR
RT-PCR
PCR
RT-PCR





Tobacco wild type
−
−
+
+


Tobacco + pCAMBIA1201
−
−
+
+


pVZA100 Transgenic line 4
+
−
+
+


pVZA100 Transgenic line 23
+
−
+
+


pVZA100 Transgenic line 26
+
−
+
+


pVZA100 Transgenic line 27
+
−
+
+


pVZA100 plasmid DNA
+
na
−
na





+ = Positive (gel band)


− = Negative (no gel band)


na = Not applicable






In the upper panel of Table 7, the data in the first column demonstrate that the hygromycin resistance gene (hygromycin phosphotransferase) was not present in the wild type tobacco plants, but it was present in all the plants transformed with either the pCAMBIA 1201 plasmid alone or with pVZA100. The data in the second column demonstrate that the hygromycin phosphotransferase gene was expressed in all plants in which it was present. The data in the third column demonstrate that the GUS gene was not present in the wild type tobacco plants, but it was present in all the plants transformed with either the pCAMBIA 1201 plasmid alone or with pVZA100. The data in the fourth column demonstrate that the GUS gene was expressed only in the pCAMBIA 1201 transformed plants, where no GUS silencing construct was present. However, GUS was silenced in all transgenic plants containing the pVZA100 silencing construct.
In the lower panel of Table 7, the data in the first column demonstrate that the fungal cutinase gene was not present in the wild type tobacco plants, nor in those plants transformed with the pCAMBIA 1201 plasmid alone. However, it was present in all the plants transformed with pVZA100. The data in the second column demonstrate that the fungal cutinase gene was not expressed in either the wild type plants or in those plants transformed with either the pCAMBIA 1201 plasmid alone or with pVZA100. The data in the third column demonstrate that the 16S ribosomal RNA gene was present in the wild type and all of the transgenic tobacco plants, but not in the silencing construct. The data in the fourth column demonstrate that the 16S ribosomal RNA gene was expressed in all of the tobacco plants, whether wild type or transgenic.
When the wild type tobacco plants or those transformed with pCAMBIA1201 were inoculated with P. nicotianae, they were always susceptible, whereas those transformed with pVZA100, such as transgenic lines 4, 23, 26 and 27 were resistant (FIGS. 11A and B, respectively).
Example 13
Fungal Transformation and Characterization
Different cultures of either P. nicotianae or P. sojae are bombarded with either pCAMBIA1201, pVZA100, pVZA200, pVZA300, or pVZA400 as above, and single zoospore cultures are produced from the viable cultures.
Those P. nicotianae cultures bombarded with pCAMBIA1201 and pVZA100 were tested for pathogenicity on the normally susceptible wild type Xanthi tobacco. Those cultures transformed with pCAMBIA1201 remained pathogenic, whereas those cultures transformed with pVZA100 were nonpathogenic. Wild type and transgenic cultures of P. nicotianae were grown in liquid V8 medium and DNA and RNA were extracted as described above. Those samples were analyzed by PCR and RT/PCR as above. The results are summarized in Table 8.







TABLE 8





Gene Detection and Expression in Transgenic and Wild


Type Phytophthora nicotianae


















Hygromycin




phosphotransferase
GUS











Tissue Assayed
PCR
RT-PCR
PCR
RT-PCR





P. nicotianae wild type
−
−
−
−


P. nicotianae + pCAMBIA
+
+
+
+


1201






P. nicotianae + pVZA100
+
+
+
−


pVZA100 plasmid DNA
+
na
+
na













Cutinase
16S Ribosomal RNA











Tissue Assayed
PCR
RT-PCR
PCR
RT-PCR





P. nicotianae wild type
+
+
+
+


P. nicotianae + pCAMBIA
+
+
+
+


1201






P. nicotianae + pVZA100
+
−
+
+


pVZA100 plasmid DNA
+
na
−
na





+ = Positive (gel band)


− = Negative (no gel band)


na = Not applicable






In the upper panel of Table 8, the data in the first column demonstrate that the hygromycin phosphotransferase gene was not present in the wild type P. nicotianae, but it was present in those cultures of P. nicotianae transformed with either the pCAMBIA 1201 plasmid alone or with pVZA100. The data in the second column demonstrate that the hygromycin phosphotransferase gene was expressed in all cultures in which it was present.
The data in the third column demonstrate that the GUS gene was not present in the wild type P. nicotianae, but it was present in those cultures of P. nicotianae transformed with either the pCAMBIA 1201 plasmid alone or with pVZA100. The data in the fourth column demonstrate that the GUS gene was being expressed only in the pCAMBIA 1201 transformed P. nicotianae, where no GUS silencing construct was present. However, GUS was silenced in all transgenic P. nicotianae cultures containing the pVZA100 silencing construct.
In the lower panel of Table 8, the data in the first column demonstrate that the fungal cutinase gene was present in the wild type P. nicotianae, as well as those cultures transformed with either the pCAMBIA 1201 plasmid alone or with the pVZA100 silencing construct, because cutinase is a constituitive gene of P. nicotianae. The data in the second column demonstrate that the fungal cutinase gene was expressed in both the wild type P. nicotianae as well as in the cultures transformed with the pCAMBIA 1201 plasmid. However, the cutinase expression was silenced in those P. nicotianae cultures transformed with the pVZA100 silencing construct.
The data in the third column demonstrate that the 16S ribosomal RNA gene was present in the wild type and all of the transgenic P. nicotianae, but not in the silencing construct. The data in the fourth column demonstrate that the 16S ribosomal RNA gene was being expressed in both the wild type and transgenic P. nicotianae cultures.
Microscopic examination of cultures of P. nicotianae and P. sojae bombarded with pCAMBIA1201, pVZA100 and pVZA200 indicated that all grew normally like the wild type and with no visual malformations (FIG. 14 A). Similar results were observed for cultures of P. nicotianae reisolated from infected wild type tobacco or tobacco transformed with pVZA100. However, those cultures of P. nicotianae bombarded with pVZA300 or pVZA400 (FIG. 14 B, C, D) and those of P. sojae also bombarded with pVZA300 or pVZA400 (FIG. 14 E, F) grew very slowly, their hyphae were severely malformed, and some cultures died.
Transformation of P. nicotianae and P. sojae with pCAMBIA1201 and pVZA200 had no deleterious effects on the fungi because those plasmids do not contain fungal genes and cannot induce silencing in those fungi. Furthermore, transformation of P. nicotianae and P. sojae with pVZA100, which contains the P. nicotianae cutinase gene, allowed them to grow normally, but caused both species to become nonpathogenic, because the cutinase gene, which is essential for pathogenicity, was silenced by pVZA100.
Transformation of P. nicotianae and P. sojae with pVZA300 or pVZA400, which contain the P. infestans elicitin and ribosomal RNA genes, respectively, both had marked deleterious effects on the growth and development of both P. nicotianae and P. sojae. There was insufficient growth even to provide enough inoculum for pathogenicity tests. This suggests that the elicitin and ribosomal RNA genes were silenced in the transformed fungi, and that silencing either of these important genes is very detrimental or lethal to the fungi. This is further supported by the fact that both pVZA 200 and pVZA300 contain the Myzus persicae cathepsin gene. But in addition, pVZA300 contains the P. infestans elicitin gene. Since pVZA200 caused no visible effects, and pVZA300 caused detrimental effects, the elicitin gene appears to be responsible for the malformation caused by pVZA300.
As above, where P. nicotianae cutinase induced plant resistance to other species of Phytophthora, these results demonstrate that essential genes from one species of Phytophthora (P. infestans), when introduced in silencing constructs, can cause very detrimental effects on other species of Phytophthora (P. nicotianae and P. sojae), which should be useful in controlling Phytophthora spp. directly in plants or by depathogenesis of infested soil and fields.
Example 14
Demonstration of Gene Silencing in Tobacco by siRNA Detection
Further proof that gene silencing is occurring is the detection of the small interfering RNAs (siRNAs) in the purportedly silenced transformed plants (Examples 5 and 13) and transformed fungus (Example 13). The siRNAs are 21 to 25 nucleotides (nt) in length, with homology to the mRNA that is being “silenced”.
Total RNA was extracted from wild type and purportedly silenced transgenic T0 and T1 generation tobacco plants and wild type and purportedly silenced P. nicotianae. The RNA was separated by polyacrylamide gel electrophoresis, blotted to a membrane and hybridized with a probe transcribed from a 500 bp PCR product of the cutinase gene and labeled with 32P by random priming. The hybridization and detection conditions were as in Example 9 above.
FIGS. 4a and 4b show the results of hybridization of the cutinase gene probe to RNAs from wild type and transgenic tobacco plants and transgenic P. nicotianae. In FIG. 4a, lane 1 shows hybridization to a mixture of the 35 nt oligonucleotides VZA 3F (SEQ ID NO: 3) and VZA 4R (SEQ ID NO: 4). These were used to prime the PCR reaction to synthesize the template for production of the probe. Therefore, they are completely homologous to the template, and also of a known molecular size. Lanes 2 and 3 contain RNA from wild type tobacco plants. There is no hybridization because neither the cutinase gene nor the silencing construct are present in wild type plants, hence no siRNAs are produced. Lanes 4 to 11 contain the RNA from T0 and T1 generation plants of transgenic lines 4, 23, 26 and 27. The hybridization demonstrates the presence of the siRNAs in each of the transgenic plants, indicating the degradation of the cutinase mRNA, and the concomitant silencing of the cutinase gene in the transgenic plants.
In FIG. 4b, lane 1 again shows hybridization to a mixture of the 35 nt oligonucleotides VZA 3F and VZA 4R. Lane 2 shows hybridization to the RNA from the wild type P. nicotianae. The intense spot 620 nt in length corresponds to the full length transcript of the cutinase gene in wild type P. nicotianae. The cutinase gene is constituitive in this culture, and no silencing construct is present. Therefore, the full length mRNA transcript survives and is not digested to siRNAs. Lanes 3 to 6 contain the RNA from four cultures of P. nicotianae transformed with the silencing construct. The hybridization demonstrates the presence of siRNAs in each of the transgenic cultures of P. nicotianae, indicating the degradation of the cutinase mRNA, and the concomitant silencing of the cutinase gene in the transgenic P. nicotianae. 
Biological evidence for the silencing of the essential cutinase gene in transgenic P. nicotianae comes from the fact that those cultures transformed with the cutinase gene silencing construct, pVZA100, are rendered non-pathogenic, whereas those cultures transformed with the pCAMBIA 1201 plasmid alone remain equally pathogenic to the wild type cultures from which they were derived. Further evidence of gene silencing in the presence of the pVZA100 silencing construct is provided by the GUS gene. The GUS reaction was always positive in those cultures that were transformed with pCAMBIA1201 and were pathogenic. Whereas it was always negative in those cultures that were transformed with pVZA100 and were nonpathogenic. This correlates directly with the activity or silencing of the cutinase gene in the same culture.
Example 15
Highly Homologous Phytophthora Cutinase Sequences Useful Herein
Analysis of Phytophthora gene sequences provides a basis for designing heterologous polynucleotides that confer cross-species resistance. As an illustration of the use of bioinformatic analysis to select pest pathogenicity gene sequences useful according to the present invention, 25 different isolates of Phytophthora were cloned and sequenced. Isolates included the nine species P. capsici, P. cinnamomi, P. citricola, P. citrophthora, P. hevea, P. megakatya, P. megasperma (P. sojae), P. nicotianae and P. palmivora. Each of these sequences (See SEQ ID NOs: 13 to 37) are useful for polynucleotides of the present invention.
For example, nine of the sequences are identical and 11 are >99% identical to the cutinase gene sequence of P. nicotianae. See FIGS. 20A-20G. A shaded letter   indicates a difference in sequence.
Without being bound by theory, these cutinase sequences provide the molecular basis for the cross resistance demonstrated above and support the general application of gene sequencing and bioinformatic analysis to select other pest pathogenicity genes with similar outcomes.
Example 16
Intergeneric Resistance in Tobacco was Conferred by the P. Nicotianae Cutinase Gene-Silencing to a Disease Caused by a Peronospora Tabacina 
A natural epidemic of the blue mold disease of tobacco caused by Peronospora tabacina occurred in the greenhouse containing the tobacco plants. Clear differences in blue mold severity were observed. Three genotypes of plants were available for evaluation: Transgenic plants containing the cutinase gene and which had already been selected for resistance to P. nicotianae, cutinase transgenic plants which had not yet been selected for resistance to P. nicotianae, and wild type or non-transgenic Xanthi tobacco plants. The numbers in the photographs in FIG. 5 refer to the different disease ratings, not the different plant lines or genotypes.







TABLE 9







Reactions of Different Tobacco Genotypes to a Natural


Epidemic of Blue Mold of Tobacco Caused by


Peronospora tabacina in the Greenhouse










Disease Rating




and Analysis














Tobacco Genotype
1*
2
3
4
#Plants 1 + 2
#Plants 3 + 4
















Phytophthora resistant
34**
11
9
12
45 (68)***
21 (32)


Unselected transgenic
62
20
67
26
82 (47)
93 (53)


Wild Type Xanthi
 0
3
12
30
 3 (7)
42 (93)





*= Disease ratings.


**= Number of plants in that class.


***= Percentage of plants in that class.


1 = Symptomless


2 = Obvious symptoms


3 = Severe symptoms


4 = Dead or dying






Table 9 shows that among the P. nicotianae resistant plants, 68% of them were moderately affected by Peronospora tabacina (disease ratings 1+2), and 32% were severely affected (disease ratings 3+4); about a 2:1 ratio in favor of resistance to Peronospora tabacina. For the transgenic unselected plants, 47% were moderately affected and 53% severely affected; about a 1:1 ratio in favor of resistance to Peronospora tabacina. Of the wild type plants, 7% were moderately affected and 93% were severely affected; about a 13:1 ratio in favor of susceptibility. Therefore, FIG. 5 and Table 9 clearly demonstrate different levels of resistance in the different tobacco genotypes, and that the cutinase gene from P. nicotianae induces broad based resistance in tobacco to Peronospora tabacina, a fungus distantly related to P. nicotianae.
Table 10 demonstrates that Phytophthora nicotianae and Peronospora tabacina are not closely related taxonomically. Yet, as shown above, the Phytophthora nicotianae cutinase gene confers resistance to both Phytophthora nicotianae and Peronospora tabacina. Therefore, the Phytophthora nicotianae cutinase gene provides broad resistance against at least three different fungal species and three fungal diseases. It protects against two different species of Phytophthora, P. nicotianae in tobacco and P. sojae in soybeans, and it also protects tobacco against both Phytophthora nicotianae and Peronospora tabacina. These two fungal species are classified in the same order, but in distinctly different families and genera (Table 10).







TABLE 10







Taxonomic relationship between Phytophthora nicotianae


and Peronospora tabacina










Phytophthora nicotianae
Peronospora tabacina














Class
Oomycetes
Oomycetes



Order
Peronosporales
Peronosporales



Family
Pythiaceae
Peronosporaceae



Genus
Phytophthora
Peronospora



Species
Phytophthora nicotianae
Peronospora tabacina









Example 17
Soybeans Transformed and Made Resistant with Phytophthora Gene Silencing Constructs
Soybean tissue culture: Shoots emerged from many of the soybean embryos transformed with pVZA100 (Example 7) by A. tumefaciens and by biolistics (FIG. 6). Other calli on the plate remained green and became very friable (FIG. 7A). DNA was extracted from three samples each of the shoots and friable calli, and PCR was performed using the oligonucleotide primers designed to amplify the GUS (SEQ ID NOs: 9 and 10) and hygromycin phosphotransferase (SEQ ID NOs: 11 and 12) genes.
Both the GUS and hygromycin phosphotransferase genes (HPT) were detected in the soybean callus, as shown in FIG. 7B. The upper panel of FIG. 7B shows that all 6 of the samples (lanes 1-6) were transgenic for HPT (lane 7 is the plasmid control). The lower panel shows that at least 4 (lanes 1, 2, 4, 5) of the 6 samples were transgenic for the GUS gene (lane 7 is the plasmid control). These data demonstrate that the host plant cells were transgenic with the pVZA100 heterologous polynucleotide.
The continued growth of the plantlets and shoots on the medium containing 15 mg/L of hygromycin demonstrate that the calli expressed the heterologous polynucleotide.
The fact that the soybean calli were GUS negative in their staining reaction demonstrated that GUS expression in the host plant cell was silenced by the heterologous polynucleotide.
Next, the calli are grown, rooted, transplanted to soil, and grown to maturity to obtain pollen and seed. Non transformed plants are pollinated with pollen from a mature transformed plant, and the resultant fertilized seed is grown to a mature plant. Mature plants (F0 plants), plants grown from seeds of an F0 plant, and non-transformed plants regenerated from seeds from transformed plant pollen and non-transformed plant flowers all show resistance to Phytophthora sojae. 
Soybean plants: The example above with soybean tissue culture was clearly demonstrated in Example 27 wherein soybean plants transformed by bombardment with pVZA100 were resistant to P. sojae (FIG. 13). This result also demonstrates that broad resistance was conferred by the cutinase gene of P. nicotianae against P. sojae. 
Example 18
Potatoes Transformed and Made Resistant to Phytophthora Infestans 
From Example 7, both roots and minitubers were produced in the culture plates (FIG. 8). The plantlets, roots, and minitubers grew on the medium containing 15 mg/L of hygromycin demonstrating that the calli were transformed and expressing the hygromycin phosphotransferase transgene. Potato calli transformed with pCAMBIA1201 were GUS positive staining, whereas potato calli transformed with pVZA100 were GUS negative staining. This demonstrates that GUS was silenced in those tissues transformed with pVZA100.
PCR analysis demonstrates the presence of the transgenes. Progeny plants from cuttings and tubers test positive for siRNAs.
The potatoes transformed with the silencing construct are resistant to Phytophthora infestans. 
Potato plants: This example (18) with potato tissue culture was clearly demonstrated in Example 27 wherein potato plants transformed by bombardment with pVZA300 and pVZA400 were resistant to P. infestans. 
Example 19
Soybeans are Made Resistant to Brown Stem Rot (Phialophora Gregata rDNA)
Soybean host cells are transformed with a heterologous polynucleotide similar to pVZA100 (Example 3) except that the sense and antisense sequences correspond to Phialophora gregata ribosomal RNA gene (rDNA) (GenBank locus U66728, SEQ ID NO: 43). Soybean plants are generated from transformed cells and are successfully selected for conferred resistance to brown stem rot following inoculation with Phialophora gregata. Plants resistant to Phialophora gregata are also resistant to Phakopsora pachyrhizi and Aspergillus nidulans. 
Example 20
Soybeans are Made Resistant to Brown Stem Rot (Phialophora Gregata Genotype B DNA Marker)
Soybean host cells are transformed with a heterologous polynucleotide similar to pVZA100 (Example 3) except that the sense and antisense sequences correspond to Phialophora gregata ribosomal RNA gene (rDNA) (GenBank locus U66728, SEQ ID NO: 44). Soybean plants are generated from transformed cells and are successfully selected for conferred resistance to brown stem rot following inoculation with Phialophora gregata. Plants resistant to Phialophora gregata are also resistant to Phakopsora pachyrhizi and Aspergillus nidulans. 
Example 21
Soybeans are Made Resistant to Stem and Root Rot (Phytophthora Sojae Cutinase)
Soybean host cells are transformed with a heterologous polynucleotide similar to pVZA 100 (Example 3) except that the sense and antisense sequences correspond to Phytophthora sojae cutinase (i.e. SEQ ID NO: 36). Soybean plants are generated from transformed cells and are successfully selected for conferred resistance to stem and root rot following inoculation with Phytophthora sojae. 
Example 22
Soybeans are Made Resistant to White Mold Disease (Sclerotinia Sclerotiorum RNA Polymerase Subunit)
Soybean host cells are transformed with a heterologous polynucleotide similar to pVZA100 (Example 3) except that the sense and antisense sequences correspond to Sclerotinia sclerotiorum partial rpb2 gene for RNA polymerase II second largest subunit, exon 1, strain 484 (GenBank locus AJ745716, SEQ ID NO: 45). Soybean plants are generated from transformed cells and are successfully selected for conferred resistance to white mold disease following inoculation with Sclerotinia sclerotiorum. Plants resistant to Sclerotinia sclerotiorum are also resistant to Phakopsora, Aspergillus nidulans, Magnaporthae orizae, Candida sojae, Gibberella zeae, and Puccinia graminis. Transformed plants show normal growth, lacking a gene with high homology to the S. sclerotiorum rpb2 gene. Similar results are obtained using sense and antisense sequences corresponding to Puccinia hordei 18S ribosomal RNA gene, partial sequence of GenBank locus AY125412 (SEQ ID NO: 46).
Example 23
Soybeans are Made Resistant to White Mold Disease (Sclerotinia Sclerotiorum Hexose Transporter)
Soybean host cells are transformed with a heterologous polynucleotide similar to pVZA100 (Example 3) except that the sense and antisense sequences correspond to Sclerotinia sclerotiorum hexose transporter (hxt2) gene (GenBank locus AY647268.1, SEQ ID NO: 47). Soybean plants are regenerated from transformed cells and successfully selected for conferred resistance to white mold disease following inoculation with Sclerotinia sclerotiorum. Plants resistant to Sclerotinia sclerotiorum also demonstrate some resistance to Botrytis cinerea. 
Example 24
Soybeans are Made Resistant to Sudden Death Syndrome (Fusarium Solani Pisi Cutinase)
Soybean host cells are transformed with a heterologous polynucleotide similar to pVZA100 (Example 3) except that the sense and antisense sequences correspond to F. solani pisi cutinase mRNA (GenBank locus K02640.1, SEQ ID NO: 48) Soybean plants are regenerated from transformed cells and successfully selected for conferred resistance to sudden death syndrome following inoculation with Fusarium solani glycines. Plants resistant to Fusarium solani glycines show some increased resistance to A. nidulans. 
Example 25
Soybeans are Made Resistant to Asian Rust (Phakopsora Pachyrhizi Cutinase Homolog)
Soybean host cells are transformed with a heterologous polynucleotide similar to pVZA100 (Example 3) except that the sense and antisense sequences correspond to Phakopsora pachyrhizi conserved protein motif of Phytophthora nicotianae and found in the Phakopsora pachyrhizi genome (GenBank locus AC149367.2, SEQ ID NO: 49). Based on homology with Phytophthora nicotianae cutinase, this conserved protein motif is likely a cutinase homologue and involved in penetrance of the soybean cutin. Soybean plants are regenerated from transformed cells and successfully selected for conferred resistance to Asian rust following inoculation with Phakopsora pachyrhizi. 
Example 26
Soybeans are Made Resistant to Asian Rust (Phakopsora Pachyrhizi rDNA)
Soybean host cells are transformed with a heterologous polynucleotide similar to pVZA100 (Example 3) except that the sense and antisense sequences correspond to a ribosomal DNA sequence found in the Phakopsora pachyrhizi genome (GenBank locus AF333491, SEQ ID NO: 50). Soybean plants are generated from transformed cells and successfully selected for conferred resistance to Asian rust following inoculation with Phakopsora pachyrhizi. 
Example 27
Testing of Tobacco, Soybean, Potato and Corn for Transgenesis, Resistance to Phytophthora spp. and Resistance to Aphids
pVZA100 in Tobacco: Mature tobacco plants produced in Example 5 by transformation with pVZA100 were tested for resistance against P. nicotianae. Three methods were tested for inoculation with P. nicotianae. All gave satisfactory results, but as explained above, the toothpick method was more rigorous and more dependable. Generally about 20% of the T0 plants transformed with pVZA100 (initial transgenic generation) survived infection with P. nicotianae, whereas 0% of the wild type plants and those plants transformed with pCAMBIA 1201 survived.
Surviving plants transformed with pVZA100 were grown to maturity in soil, and seed was collected for subsequent analysis. The seed progeny (=T1 generation) were grown on MS medium containing 30 ug/ml hygromycin, and the surviving plants again tested for resistance to P. nicotianae. 
Surviving transformed plants also were grown to maturity in soil and seed collected for subsequent analysis. A group of seed progeny (=T2 generation) of four different lines (lines #4, #23, #26 and #27; =different transformation events) of these plants were inoculated twice successively to ensure that any survivors were resistant to P. nicotianae. In the first inoculation 192 of 442 plants were resistant (=43%). Upon reinoculation: 150 of 192 plants were resistant (=78%). In summary, 150 of 442 plants were resistant (=34%). All of these 150 resistant plants were also assayed for GUS activity and found to be negative.
Reactions by tobacco plants are shown in FIG. 11. Plant A shows reactions typical of wild type tobacco plants or those transformed with pCAMBIA1201. The plants are susceptible, there are major stem lesions, and the root system is essentially destroyed. Such plants usually die 5-8 days after inoculation. Plant B is from Transgenic line 26 (see below). It is typical of those plants transformed with pVZA100. It was highly resistant to P. nicotianae and showed no symptoms other than small lesions on the tips of the upper roots. Such plants grow to maturity and set fertile seed.
pVZA100 in Soybean: Soybean plants were bombarded with pVZA100. PCR with the appropriate primers demonstrated that 15 of 32 bombarded plants were transgenic. Transgenic plants were cultivated to produce seeds and seed plants were tested for resistance to P. sojae. As shown in FIG. 13, 43 of 495 seed plants tested were resistant to P. sojae (FIG. 13) whereas 100% of a similar number of wild-type and pCAMBIA1201 transformed (i.e. “control-transformed”) plants died.
pVZA100 in Potato: Potato plants were bomarded with pVZA100. PCR with the appropriate primers demonstrated that 15 of 18 bombarded plants were transgenic. Transgenic plants are tested for resistance to P. infestans. 100% of these plants are resistant as compared to 0% resistance in wild-type or control-transformed plants.
pVZA100 in Corn: Corn plants were bombarded with pVZA100. PCR with the appropriate primers demonstrated that 33 of 44 bombarded plants were transgenic. Transgenic plants are cultivated to produce seeds and seed plant are tested for resistance to Pythium graminicola. 25% of the F1 plants tested are resistant as compared to 0% resistance in wild-type or control-transformed plants.
The above results demonstrate that plants useful in the present invention include monocots and dicots. Similarly, corn plants are transformed with pVZA100 and tested and illustrate cross resistance to Phytophthora and Peronospora. 
pVZA200 in Potato: Potato plants were bomarded with pVZA200 (Myzus persicae Cathepsin B). PCR with the appropriate primers demonstrated that 7 of 12 bombarded plants were transgenic. Transgenic plants are tested for resistance to aphids (Myzus persicae). 80% of such plants, compared to 0% of control or wildtype plants, are resistant.
Aphids after feeding on transformed plants are examined. It is determined that silencing cathespin B in aphids according to the present invention not only protects the plant (i.e. causes aphid resistance in plants) but also reduces the aphid population.
According to the present invention and this example, insect resistance is conferred to plants through transformation with a silencing construct containing an insect gene such as Cathepsin B.
pVZA300 in Potato: Potato plants were bomarded with pVZA300, a construct containing sense and antisense sequence homologous and complementary to Cathepsin B from Myzus persicae and elicitin INF1 of Phytophthora infestans. PCR with the appropriate primers demonstrated that 15 of 18 bombarded plants were transgenic. Such transgenic plants are tested for resistance to aphids (Myzus persicae) and to Phytophthora infestans. 
In one set of experiments, one group of transformed potato plants are challenged with aphids only and one group is challenged with Phytophthora infestans only.
For the aphid challenged plants, 80% of such plants tested are resistant as compared to 0% resistance in wild-type or control-transformed plants.
For the Phytophthora infestans challenged plants, 75% of such plants tested were resistant as compared to 0% resistance in non-transformed or control plants.
In another set of experiments, similarly transformed and non-transformed potato plants are challenged with aphids and Phytophthora infestans at the same time. 60% of the transgenic plants tested are resistant to both pests as compared to 0% resistance in wild-type or control-transformed plants.
After feeding on transformed plants, Phytophthora infestans is isolated and cultured. Such cultures either grow very slowly or die. Microscopic examination reveals that the hyphae are severely malformed.
Moreover, silencing rRNA according to the present invention further illustrates an embodiment whereby the pathogen population in the soil is dramatically reduced.
pVZA300 in Soybeans: Soybean plants were bomarded with pVZA300 (a construct containing sense and antisense sequence homologous and complementary to Cathepsin B from Myzus persicae and elicitin INF1 of Phytophthora infestans). PCR with the appropriate primers demonstrated that 60% of bombarded plants are transgenic. Transgenic plants are cultivated to produce seeds and seed plants are grown. Such F1 plants are tested for resistance to aphids (Myzus persicae) and to Phytophthora sojae.
In one set of experiments, one group of transformed soybean plants are challenged with aphids only and one group is challenged with Myzus persicae only.
For the aphid challenged plants, 70% of the F1 plants tested are resistant as compared to 0% resistance in wild-type or control-transformed plants.
For the Phytophthora sojae challenged plants, 15% of the F1 plants tested are resistant as compared to 0% resistance in wildtype and in control transformed plants.
In another set of experiments, transformed and non-transformed soybeans plants are challenged with aphids and Phytophthora sojae at the same time. 10% of the F1 plants tested are resistant to both pests as compared to 0% resistance in wild-type or control-transformed plants.
According to the present invention and these examples, insect resistance and fungus resistance is conferred in a plant (both for moncot and dicot species) by a single heterologous polynucleotide comprising a plurality of genes (i.e. by gene stacking). Moreover, a surprising level of pest resistance is conferred in plants by silencing genes from more than one pest.
After feeding on transformed plants, Phytophthora sojae is isolated and cultured. Such cultures either grow very slowly or die. Microscopic examination reveals that the hyphae are severely malformed.
pVZA400 in Soybeans: Soybean plants were bomarded with pVZA400 comprising antisense and sense sequences homologous and complementary to the rRNA gene of Phytophthora infestans. PCR with the appropriate primers demonstrated that 12 of 18 bombarded plants were transgenic. Transgenic plants are cultivated to produce seeds and seed plants are tested for resistance to Phytophthora sojae. 15% of the F1 plants that are tested are resistant as compared to 0% resistance in non-transformed or control transgenic plants.
After feeding on transformed plants, Phytophthora sojae is isolated and cultured. Such cultures either grow very slowly or die. Microscopic examination reveals that the hyphae are severely malformed.
Next, wild-type soybeans are challenged with Phytophthora sojae cultures derived from the pVZA400-transformed plants. Such P. sojae cultures are non pathogenic in comparison to sojae cultures which are derived from wild-type or control-transformed soybeans.
The above results illustrate the usefulness of silencing of rRNA in a pest (e.g. fungi) and further illustrates cross resistance to a pest species other than the pest from which the pest pathogenicity gene was constructed.
After feeding on transformed plants, Phytophthora sojae is isolated and cultured. Such cultures either grow very slowly or die. Microscopic examination reveals that the hyphae are severely malformed.
Moreover, silencing rRNA according to the present invention further illustrates an embodiment whereby the pathogen population in the soil is dramatically reduced.
pVZA400 in Potato: Potato plants were bomarded with pVZA400 comprising antisense and sense sequences mologous and complementary to the rRNA gene of Phytophthora infestans. PCR with the appropriate primers demonstrated that 6 of 12 bombarded plants were transgenic. Transgenic plants were cultivated and tested for resistance to Phytophthora infestans. These plants were tested and showed resistance in 100% of these plants as compared to 0% resistance in wild-type or control-transformed plants.
After feeding on transformed plants, Phytophthora infestans is isolated and cultured. Such cultures either grow very slowly or die. Microscopic examination reveals that the hyphae are severely malformed.
The above results the usefulness of a single silencing construct of rRNA in conferring pest resistance in a plurality of plants to a pest (e.g. fungi).
Example 28
Potato is Made Resistant to Tuber Rot (Fusarium Sambucinum Translation Elongation Factor 1 Alpha)
Potato host cells are transformed with a heterologous polynucleotide similar to pVZA100 (Example 3) except that the sense and antisense sequences correspond to a Fusarium sambucinum translation elongation factor 1 alpha DNA sequence (GenBank locus AJ543605, SEQ ID NO: 51). Potato plants are generated from transformed cells and successfully selected for conferred resistance to Fusarium sambucinum tuber rot.
Example 29
Tomato is Made Resistant to Late Blight (Phytophthora Infestans Elicitin Genet)
Tomato host cells are transformed with a heterologous polynucleotide similar to pVZA100 (Example 3) except that the sense and antisense sequences correspond to an elicitin gene sequence found in the Phytophthora infestans genome (GenBank locus AY766228, SEQ ID NO: 52). Tomato plants are generated from transformed cells and successfully selected for conferred resistance to late blight of tomato following inoculation with Phytophthora infestans. 
Example 30
Canola is Made Resistant to Stem Rot (Sclerotinia Sclerotiorum RNA Polymerase Subunit)
Canola host cells are transformed with a heterologous polynucleotide similar to pVZA100 (Example 3) except that the sense and antisense sequences correspond to a Sclerotinia sclerotiorum partial rpb2 gene for RNA polymerase II second largest subunit, exon 1, strain 484 1096 bp (GenBank locus AJ745716, SEQ ID NO: 53). Canola plants are generated from transformed cells and successfully selected for conferred resistance to stem rot following inoculation with Sclerotinia sclerotiorum. 
Example 31
Cotton is Made Resistant to Cotton Wilt (Fusarium Solani Pisi Cutinase Homolog)
Cotton host cells are transformed with a heterologous polynucleotide similar to pVZA100 (Example 3) except that the sense and antisense sequences correspond to a cutinase gene from F. solani pisi (GenBank locus K02640.1, SEQ ID NO: 48). This sequence is picked herein because of the homology of genes within the genus Fusarium. Cotton plants are generated from transformed cells and successfully selected for conferred resistance to cotton wilt following inoculation with Fusarium oxysporum. 
Example 32
Corn is Made Resistant to Mycotoxins (F. Solani Pisi Cutinase)
Corn host cells are transformed with a heterologous polynucleotide similar to pVZA100 (Example 3) except that the sense and antisense sequences correspond to a cutinase gene from an F. solani pisi (GenBank locus K02640.1, SEQ ID NO: 48). This sequence is picked herein because of the homology of genes within the genus Fusarium. Corn plants are generated from transformed cells and selected for increased resistance to mycotoxins following inoculation with Fusarium moniliforme. 
Example 33
Corn is Made Resistant to Stalk Rot (Stenocarpella Maydis rDNA)
Corn host cells are transformed with a heterologous polynucleotide similar to pVZA100 (Example 3) except that the sense and antisense sequences correspond to Stenocarpella maydis rRNA (GenBank locus AY332489, SEQ ID NO: 54). This sequence is picked herein because of the homology of genes within the genera Stenocarpella and Fusarium. Corn plants are generated from transformed cells and selected for increased resistance to stalk rot following inoculation with Stenocarpella maydis and several Fusarium spp.
Example 34
Corn is Made Resistant to Leaf Blight (Cercospora Zeae-Maydis 18s rDNA)
Corn host cells are transformed with a heterologous polynucleotide similar to pVZA100 (Example 3) except that the sense and antisense sequences correspond to 18S ribosomal RNA from a Cercospora zeae-maydis Group II (GenBank locus AF291710, SEQ ID NO: 55). Corn plants are generated from transformed cells and successfully selected for conferred resistance to leaf blight following inoculation with Cercospora zeae-maydis. 
Example 35
Potato is Made Resistant to Green Peach Aphid (Myzus Persicae Cathepsin B Protease)
Potato host cells are transformed with a heterologous polynucleotide similar to pVZA100 (Example 3) except that the sense and antisense sequences correspond to Cathepsin B protease from Myzus persicae (GenBank locus AY702822, SEQ ID NO: 56). Potato plants are generated from transformed cells and successfully selected for conferred resistance to green peach aphids following inoculation with Myzus persicae. 
Example 36
Potato is made resistant to Colorado potato beetle (Leptinotarsa decemlineata cysteine protease)
Potato host cells are transformed with a heterologous polynucleotide similar to pVZA100 (Example 3) except that the sense and antisense sequences correspond to cysteine protease from Leptinotarsa decemlineata (GenBank locus AY159377, SEQ ID NO: 57). Potato plants are generated from transformed cells and successfully selected for conferred resistance to Colorado potato beetle following inoculation with Leptinotarsa decemlineata. Such regenerated potatoes demonstrate increased resistance to each of the following: Callosobruchus maculates, Diabrotica virgifera virgifera, Acanthoscelides obtectus, Helicoverpa armigera, Leptinotarsa decemlineata, Anthonomus grandis, Triatoma infestans, Tenebrio molitor, Toxoptera citricida, Aphis gossypii, Pandalus borealis, Myzus persicae, Delia radicum, Sarcophaga peregrine, Sitophilus zeamais, Boophilus microplus, Hypera postica, Frankliniella occidentalis, and H. americanus. 
Example 37
Potato is Made Resistant to Verticillium Wilt (V. Dahliae rDNA)
Potato host cells are transformed with a heterologous polynucleotide similar to pVZA100 (Example 3) except that the sense and antisense sequences correspond to Verticillium dahliae rDNA (GenBank locus AF108478, SEQ ID NO: 58). Potato plants are generated from transformed cells and successfully selected for conferred resistance to Verticillium wilt following inoculation with Verticillium dahliae. 
Example 38
Soybeans are Made Resistant to Downy Mildew (Peronospora Berteroae rDNA)
Soybean host cells are transformed with a heterologous polynucleotide similar to pVZA100 (Example 3) except that the sense and antisense sequences correspond to internal transcribed spacer 1 partial sequence of 5.8S ribosomal RNA found in the Peronospora berteroae genome (GenBank locus AY531450, SEQ ID NO: 59). Soybean plants are generated from transformed cells and successfully selected for conferred resistance to downy mildew following inoculation with Peronospora manchurica. Such selected soybeans also have increased resistance to Hyaloperonospora species (e.g. H. camelinae, H. niessleana, H. parasitica, and H. thlaspeos-perfoliati) and Peronospora species (e.g. P. arabidopsidis, P. arabis-alpinae, P. buniadis, P. cardaminopsidis, P. cochleariae, P. dentariae, P. galligena, P. hesperidis, P. iberidis, P. isatidis, P. nesleae, P. sisymbrii-loeselii, P. sisymbrii-officinalis, P. sisymbrii-sophiae, P. thlaspeos-alpestris, and P. thlaspeos-arvensis).
Example 39
Potato is Made Resistant to Potato Root Knot Nematode (Meloidogyne Chitwoodi Cytochrome Oxidase II)
Potato host cells are transformed with a heterologous polynucleotide similar to pVZA100 (Example 3) except that the sense and antisense sequences correspond to a sequence found in the Meloidogyne chitwoodi genome (i.e. cytochrome oxidase subunit II gene, (GenBank locus AY757882, SEQ ID NO: 60). Potato plants are generated from transformed cells and successfully selected for conferred resistance to potato root knot following inoculation with Meloidogyne chitwoodi. Such selected potato plants are also found to be resistant to Bombyx mandarina, Bombyx mori, Chilo tumidicostalis, Hemileuca, Hydrotaea aenescens, Meloidogyne hapla, Meloidogyne partityla, Oecetis avara, Papilio aegeus, Papilio demoleus, Papilio erithonioides, Papilio hipponous, Papilio oribazus, Papilio protenor, Papilio troilus, Papilio xuthus, Sabethes cyaneus, and Sarnia cynthia ricini. 
Example 40
Soybeans are Made Resistant to Nematode Infestation (Pratylenchus Scribneri and Heterodera Glycines Ribosomal RNA Genes)
The present invention can be used to produce host plants with increased resistance to additional nematodes. Nematode polynucleotide sequences that are known and are searchable in public databases such as the NCBI/NIH GenBank demonstrate conserved nucleotide motifs among different nematode genera. Conserved nucleotide motifs strongly suggest that these sequences are associated with viability and/or parasitism and are functionally conserved and expressed in both Meloidogyne incognita (root-knot nematode) and Globodera rostochiensis and Globdera pallids (potato cyst nematodes), and also in the well studied Heterodera glycines and Caenorhabditis elegans. Thus, the use of these sequences and variants thereof, is advantageous because such RNAi can be designed to have broad RNAi specificity and are thus useful for controlling a large number of plant parasitic nematodes in planta. Because genes identified herein are associated with nematode survival and/or parasitism, RNAi inhibition of these genes (arising from contacting nematodes with compositions comprising RNAi molecules such as, for example, by allowing nematodes to feed on plants transformed as taught herein) prevents and/or reduces parasitic nematode growth, development, and/or parasitism.
Nematode feeding is a useful indicator of the effectiveness of the methods of the present invention. The Pratylenchus scribneri in vitro feeding assay uses a corn root exudate as a feeding stimulus and both the red dye Amaranth, or potassium arsenate, as feeding indicators. Feeding is confirmed after seven days by the presence of red stained intestinal cells in live worms exposed to the Amaranth or death of worms exposed to arsenate. P. scribneri can be cultured on wild type roots of corn, rice and Arabidopsis, and on Agrobacterium rhizogenes-induced hairy roots of sugar beet and tomato. P. scribneri is very valuable in evaluating transgenic hairy roots (ideally developed by use of Agrobacterium rhizogenes strains known in the art as transformation vectors) because of the non-specific feeding of these worms.
Corn, rice, tomato, and sugar beets are transformed with a heterologous polynucleotide containing complementary sense and antisense sequences of P. scribneri ribosomal RNA (GenBank locus SEQ ID NO:61). P. scribneri is tested on a feeding assay using transformed and wild type corn, rice, tomato, and sugar beets. Transformed plants demonstrate increased resistance to P. scribneri. 
Soybeans are transformed with a heterologous polynucleotide containing complementary sense and antisense sequences of Heterodera glycines ribosomal RNA (GenBank locus AY667456, SEQ ID NO: 62). Heterodera glycines is tested on a feeding assay using transformed and wild type soybeans. Transformed soybeans demonstrate increased resistance to Heterodera glycines. 
Example 41
Various Plants are Made Resistant to Infection by Various Phytophthora spp. (Phytophthora Nicotianae Cutinase)
In one embodiment, the pVZA100 heterologous polynucleotide (Example 3) is used to transform a variety of plants to confer resistance to various Phytophthora species. For example, resistance is conferred against Phytophthora spp.-induced leaf and stem infections when any of Chrysalidocarpus palm, Catharanthus, Dendrobium, poinsettia, impatiens, sage, spathiphyllum, or onion is transformed with the pVZA100 (SEQ ID NO: 5). Resistance is conferred against Phytophthora spp.-induced root rot when any of poinsettia, tomato, pineapple, or anthurium is transformed with the pVZA100.
Resistance is conferred against Phytophthora spp.-induced crown and collar rots when any of watermelon, protea, or African violet is transformed with the pVZA100.
Resistance is conferred against Phytophthora spp.-induced fruit rots when any of tomato, papaya, or eggplant is transformed with the pVZA100.
Example 42
Agave is Made Resistant to Agave Root Rot (Fusarium Solani Pisi Cutinase)
Agave host cells are transformed with a heterologous polynucleotide similar to pVZA100 (Example 3) except that the sense and antisense sequences correspond to a cutinase gene from Fusarium solani pisi (GenBank locus K02640.1, SEQ ID NO: 48) This sequence is picked herein because of the homology of genes within the genus Fusarium. Agave plants are generated from transformed cells and successfully selected for conferred resistance to Agave rot following inoculation with Fusarium oxysporum. 
Example 43
Rice is Made Resistant to Blast Disease (Magnaporthe Grisea RNA Polymerase II)
Rice host cells are transformed with a heterologous polynucleotide similar to pVZA100 (Example 3) except that the sense and antisense sequences correspond to a Magnaporthe grisea RNA polymerase II sequence of (GenBank locus XM 362268, SEQ ID NO: 63). Transformation was accomplished by electroporation or Agrobacterium tumefaciens. Rice plants are generated from transformed cells and successfully selected for conferred resistance to rice blast disease following inoculation with Magnaporthe grisea. 
Example 44
Various Plants are Made Resistant to Phytophthora-Induced Root Disease (Phytophthora Citricola cutinase)
Various plants cells including those from Rhododendron (e.g. Rh. catawbiense, Rh. simsii), Erica sp., Calluna sp., avocado trees, citrus trees, and hops are transformed with the heterologous polynucleotide pVZA100 (Example 3). This construct was chosen because of the homology between Phytophthora nicotianae SEQ ID NO: 14 and Phytophthora citricola SEQ ID NO: 34. Host plants are generated from transformed cells and successfully selected for conferred resistance to disease following inoculation with Phytophthora citricola. 
Example 45
Barley is Made Resistant to Barley Leaf Rust (Puccinia Hordei 18S Ribosomal RNA)
Barley cells are transformed with the heterologous polynucleotide similar to pVZA100 (Example 3) except that the sense and antisense sequences correspond to Puccinia hordei 18S rRNA (GenBank locus AY125412, SEQ ID NO:64). Host plants are generated from transformed cells and successfully selected for conferred resistance to barley leaf rust following inoculation with Puccinia hordei. 
Example 46
Barley is Made Resistant to Downy Mildew (Pseudoperonospora Humuli rDNA)
Barley cells are transformed with the heterologous polynucleotide similar to pVZA100 (Example 3) except that the sense and antisense sequences correspond to sequences from Pseudoperonospora humuli isolate HV 148 internal transcribed spacer 1, partial sequence; 5.8S ribosomal RNA (GenBank locus AY198305, SEQ ID NO: 65). Host plants are generated from transformed cells and successfully selected for conferred resistance to downy mildew following inoculation with Pseudoperonospora humuli. 
Example 47
Barley is Made Resistant to Gray Mold Disease (Botrytis Cinerea Cytochrome P450 Monoxygenase)
Barley cells are transformed with the heterologous polynucleotide similar to pVZA100 (Example 3) except that the sense and antisense sequences correspond to sequences from Botrytis cinerea for aba2 gene for cytochrome P450 monoxygenase, exons 1-5 (GenBank locus AJ851088, SEQ ID NO: 66). Host plants are generated from transformed cells and successfully selected for conferred resistance to gray mold disease following inoculation with Botrytis cinerea. 
Example 48
Tomato is Made Resistant to Tomato Speck Disease (Pseudomonas Syringae rDNA)
Tomato cells are transformed with the heterologous polynucleotide similar to pVZA100 (Example 3) except that the sense and antisense sequences correspond to Pseudomonas syringae ribosomal RNA (GenBank locus AY342210, SEQ ID NO: 67). Host plants are generated from transformed cells and are successfully selected for conferred resistance to tomato speck disease following inoculation with Pseudomonas syringae. 
Example 49
Tomato is Made Resistant to Bacterial Wilt (Clavibacter Michiganensis Michiganensis Cel A Gene)
Tomato cells are transformed with the heterologous polynucleotide similar to pVZA100 (Example 3) except that the sense and antisense sequences correspond to Clavibacter michiganensis michiganensis Cel A gene (GenBank locus AY007311, SEQ ID NO: 68). Host plants are generated from transformed cells and are successfully selected for conferred resistance to bacterial wilt following inoculation with Clavibacter michiganensis michiganensis. 
Example 50
Corn is Made Resistant to Goss's Bacterial Wilt and Blight (Clavibacter michiganensis Endo β-Glucosidase Gene
Corn cells are transformed with the heterologous polynucleotide similar to pVZA100 (Example 3) except that the sense and antisense sequences correspond to Clavibacter michiganensis gene for endo β-glucosidase (GenBank locus X62582, SEQ ID NO: 69). Host plants are generated from transformed cells and are successfully selected for conferred resistance to Goss's bacterial wilt and blight (leaf freckles and wilt) following inoculation with Clavibacter michiganensis. 
Example 51
A Silencing Construct Method to Protect Soybeans Against One or Two Fungal Diseases
A soybean host plant cell is transformed with either of the gene silencing constructs of FIG. 10 and mature plants are generated therefrom. Mature plants are screened for resistance to two major fungal diseases, namely soybean root and stem rot caused by Phytophthora sojae and Asian rust of soybean caused by Phakopsora pachyrhizi. Plants are identified that are resistant to each and to both of these two diseases. Those plants transformed with the upper construct are resistant to soybean root and stem rot. Those plants transformed with the middle construct are resistant to Asian rust of soybean. Those plants transformed with the lower construct are resistant to both soybean root and stem rot and Asian rust of soybean.
Example 52
A Polycistronic Silencing Construct Method to Protect Soybeans Against Three Fungal Diseases
A soybean host plant cell is transformed with a polycistronic gene silencing construct of FIG. 9 and mature plants are generated therefrom. Mature plants are screened for resistance to three major fungal diseases, namely soybean root and stem rot caused by Phytophthora sojae, soybean sudden death syndrome caused by Fusarium solani f. sp. glycines, and Asian rust of soybean caused by Phakopsora pachyrhizi. Plants are identified that are resistant to each and all of these three diseases.
Example 53
Depathogenesis of Phytophthora Species by Feeding on Transgenic Plants or by Direct Transformation with pVZA 100, pVZA300 or pVZA400
Tobacco host cells were transformed with the silencing construct pVZA 100 (SEQ ID NO: 5) and plants were generated therefrom. Such transformed plants were highly resistant to P. nicotianae. Typical reactions by tobacco plants are shown in FIG. 11. Plant A shows reactions typical of wild type tobacco plants or those transformed with pCAMBIA1201. The plants are susceptible, there are major stem lesions, and the root system is essentially destroyed. Such plants usually die 5-8 days after inoculation. Plant B is from Transgenic line 26 (see below). It is typical of those plants transformed with pVZA100. It is highly resistant to P. nicotianae and shows no symptoms other than small lesions on the tips of the upper roots. Such plants grow to maturity and set fertile seed.
When P. nicotianae was re-isolated from the restricted lesions on the resistant plant and re-tested on wild type plants, it was no longer pathogenic. Isolation of RNA from this fungus and hybridization with the radioactive cutinase probe as above in Example 14 indicates the presence of siRNAs for the cutinase gene in P. nicotianae, thereby explaining the loss of pathogenicity. In FIG. 12, lane 1 again shows the 35 nt oligonucleotides VZA 3F (SEQ ID NO: 3) and VZA 4R (SEQ ID NO: 4) serving as the molecular size control, lane 2 shows the 620 nt messenger RNA from the wild type fungus, lane 3 shows the siRNAs from a silenced transgenic tobacco plant, and lane 4 shows the siRNAs from the nonpathogenic fungus isolated from the resistant transgenic tobacco.
As shown in Example 13, transforming species of a pest directly with silencing constructs or by feeding pest species on plants transformed with silencing constructs had major effects on the pathogenicity, growth and survival of the pest (in this example, the fungi). These results support directly or indirectly the utility of this embodiment of the present invention to minimize crop losses to pests and to enhance agricultural productivity.
Example 54
Depathogenesis: Watermelon Transformed with a Cutinase Silencing Heterologous Polynucleotide (F. Oxysporum Cutinase)
In Florida and Georgia watermelons can be planted in a field for only one year. The farmer must then wait 6 or 7 years to use that field again because a single year of use results in the high build up of pathogenic isolates of Fusarium oxysporum. To use a field in successive years, the farmer must resort to expensive and severe annual treatments with methyl bromide or other potent chemicals.
Watermelons are regenerated from a host plant cell transformed with a heterologous polynucleotide similar to pVZA 100 (Example 3) except that the sense and antisense sequences correspond to the cutinase gene in F. oxysporum. F. oxysporum infects the watermelon and the F. oxysporum cutinase gene is silenced. Other watermelon species (not transformed according to the present invention) are planted within six feet of the regenerated (transgenic) watermelon. Such other watermelon species are infected by F. oxysporum to a lesser extent than would be predicted were it not for the cultivation of the regenerated (transgenic) watermelons adjacent to them.
Example 55
Broad Based Plant Resistance and Depathogenesis with Ribosomal RNA Silencing Heterologous Polynucleotides
Optionally, to effectively implement depathogenesis, a broad-based or cross generic protection against several species of pathogenic fungi can be obtained. This can be accomplished by utilizing silencing constructs containing ribosomal DNA (rDNA) genes. The rDNA sequences of fungal pathogens are similar enough to function in the control of closely related fungal species, but are sufficiently dissimilar to permit differentiation among groups of fungi, thereby enabling the selective targeting of specific groups of fungal pathogens with single or multiple or polycistronic gene silencing constructs.
This approach is illustrated in Table 12. The 18S rDNA sequences of Phytophthora sojae and Phakopsora pachyrhizi were “blasted” against the data in the NCBI/NIH GenBank. The Phytophthora sojae sequence showed strong homology (100 to 93%) to the sequences of 16 additional closely related fungal species, but it did not show significant homology to that of Phakopsora pachyrhizi. Conversely, the 18S rDNA sequence of Phakopsora pachyrhizi showed strong homology (100 to 93%) to the sequences of 19 additional closely related fungal species, but it did not show significant homology to that of Phytophthora sojae. Therefore, individual gene silencing constructs can be developed in accord with the teachings herein to control the group of fungi related to Phytophthora sojae or to Phakopsora pachyrhizi, or alternatively a polycistronic gene silencing construct can be developed to control both groups simultaneously.







TABLE 11







GenBank sequences ribosomal DNA producing significant


alignments with target species








Phytophthora sojae 18s ribosomal
Phakopsora pachyrhizi 18s


DNA The identities of the
ribosomal DNA The identities of


sequences ranges from 100
the sequences ranges from


to 93% over 129 nucleotides.
100 to 93% over 89 nucleotides.










Fungus Species = (17)
(bits)
Fungus Species = (18)
(bits)





Phytophthora sojae
278
Phakopsora pachyrhizi
278


Phytophthora vignae
194
Phakopsora meibomiae
153


Phytophthora drechsleri
174
Uromyces aemulus
131


Phytophthora cinnamomi
174
Puccinia striiformis
123


Plasmopara viticola
168
Pandora neoaphidis
121


Phytophthora melonis
167
Puccinia allii
119


Phytophthora cryptogea
165
Piromyces sp.
119


Phytophthora pistaciae
161
Dioszegia sp.
117


Hyaloperonospora parasitica
161
Bullera sp. T
117


Phytophthora niederhauserii
161
Dioszegia crocea
117


Phytophthora sinensis
153
Cadophora sp.
117


Phytophthora cajani
153
Dioszegia hungarica
117


Pythium insidiosum
153
Phialophora sp.
117


Phytophthora palmivora
151
Anaeromyces sp.
117


Phytophthora ramorum
149
Phialophora melinii
117


Peronospora corydalis
149
Puccinia triticina
115


Pythium rostratum
141
Neocudoniella radicella
115




Pinctada nigra
115




Peziza vesiculosa
115




Phillipsia domingensis
115









Example 56
Control of Multiple Plant Pests Using Multiple Silencing Heterologous Polynucleotides
Plants are often attacked by a multiplicity of pests. Optionally, it may be useful to control several pests on the same plant. Methods for gene stacking to control multiple pests and the useful gene sequences (for example, genes encoding ribosomal RNAs, cutinases, cathepsins and other essential enzymes and proteins) are taught herein. A nonlimiting example is soybeans and the simultaneous control of Phytophthora root and stem rot, Asian rust, the soybean cyst nematode, sudden death syndrome and aphids. Exemplary genes to stacked for this purpose include SEQ ID NO: 14 for Phytophthora root and stem rot, SEQ ID NO: 50 for Asian rust, SEQ ID NO: 62 for the soybean cyst nematode, SEQ ID NO: 48 for sudden death syndrome, and SEQ ID NO: 56 for aphids.
Similarly, corn can be made resistant to root and stalk rots, mycotoxins, leaf blights, stalk and ear borers, aphids and nematodes. Similarly, cotton can be made resistant to boll rot, Verticillium and Fusarium wilts, seedling blights, damping off, boll weevil, bollworms, aphids, and loopers. Similarly, canola can be made resistant to black leg, stem rot, black spot, damping off and seedling blights, flea beetles, weevils, and aphids.
Similarly, wheat can be made resistant to leaf and stem rust, head blight, Septoria blotches, take-all, smuts, powdery and downy mildew, aphids and nematodes. Similarly, tomato can be made resistant to late blight, anthracnose, gray mold, Verticillium wilt, nematodes, and aphids. Similarly, potato can be made resistant to late blight, early blight, Verticillium wilt, nematodes, Colorado potato beetle, and aphids.
All references cited herein are incorporated by reference herein for all that they teach and for all purposes, to the extent they are not inconsistent with the explicit teachings herein. It is to be understood that, while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
The Sequence Listings are incorporated herein by reference.

## Claims

1. A method of conferring fungal resistance to a plant comprising expressing in the plant:
(a) an antisense sequence having at least 20 contiguous nucleotides of at least one sequence selected from the group consisting of SEQ ID NOs: 8, 43, 44, 46, 50, 54, 55, 58, 59 and 65;
(b) a sense sequence substantially complementary to the antisense sequence, wherein a transcript of the antisense sequence and a transcript of the sense sequence are capable of hybridizing to form a double-stranded RNA; and whereby the plant expressing said double stranded RNA is resistant to a fungus expressing at least one ribosomal RNA gene having substantial homology to the antisense sequence.

2. The method of claim 1, wherein the antisense sequence is homologous to at least 20 contiguous nucleotides of at least one additional sequence selected from the group consisting of SEQ ID NOs: 8, 43, 44, 46, 50, 54, 55, 58, 59 and 65, whereby the plant is resistant to a fungus expressing at least one ribosomal RNA gene having substantial homology to the at least one additional sequence.

3. The method of any one of claims 1 or 2, wherein the transcript of the antisense sequence and the transcript of the sense sequence form a double stranded region.

4. The method of claim 1, further comprising expressing in the plant:
(c) a second antisense sequence having at least 20 contiguous nucleotides of a second sequence selected from the group consisting of SEQ ID NOs: 8, 43, 44, 46, 50, 54, 55 58, 59 and 65; and
(d) a second sense sequence substantially complementary to the second antisense sequence; wherein a transcript of the second antisense sequence and a transcript of the second sense sequence are capable of hybridizing to form a double-stranded region; and whereby the plant is resistant to a fungus expressing at least one ribosomal RNA gene having substantial homology to the second antisense sequence.

5. The method of claim 4, wherein the transcript of the second antisense sequence and the transcript of the second sense sequence form a double stranded region.

6. A transgenic plant that is resistant to fungus, the transgenic plant comprising:
(a) an antisense sequence having at least 20 contiguous nucleotides of at least one sequence selected from the group consisting of SEQ ID NOs: 8, 43, 44, 46, 50, 54, 55, 58, 59 and 65;
(b) a sense sequence substantially complementary to the antisense sequence, wherein a transcript of the antisense sequence and a transcript of the sense sequence are capable of hybridizing to form a double-stranded RNA; and
whereby the plant expressing said double-stranded RNA is resistant to a fungus expressing at least one ribosomal RNA gene having substantial homology to the antisense sequence.

7. The transgenic plant of claim of claim 6, wherein the antisense sequence is homologous to at least 20 contiguous nucleotides of at least one additional sequence selected from the group consisting of
SEQ ID NOs: 8, 43, 44, 46, 50, 54, 55, 58 59 and 65, whereby the plant is resistant to a fungus expressing at least one ribosomal RNA gene having substantial homology to the at least one additional sequence.

8. The transgenic plant of any one of claims 6 or 7, wherein the transcript the antisense sequence and the transcript of the sense sequence form a double stranded region.

9. The transgenic plant of claim 6, further comprising:
(c) a second antisense sequence having at least 20 contiguous nucleotides of a second sequence selected from the group consisting of SEQ ID NOs: 8, 43, 44, 46, 50, 54, 55, 58, 59 and 65; and
(d) a second sense sequence substantially complementary to the second antisense sequence; wherein a transcript of the second antisense sequence and a transcript of the second sense sequence are capable of hybridizing to form a double-stranded region; and whereby the plant is resistant to a fungus expressing at least one ribosomal RNA gene having homology to the second antisense sequence.

10. The transgenic plant of claim 9, wherein the transcript the second antisense sequence and the transcript of the second sense sequence form a double stranded region.