# Patent

## Title
Methods and compositions for delaying senescence and improving disease tolerance and yield in plants

## Abstract

The present invention provides novel compositions for use to enhance crop performance. Specifically, the present invention provides for delayed senescence and/or improved yield enhancement in various crops. The present invention also provides for combinations of compositions and methods that provide for delayed senescence and/or improved yield.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This non-provisional U.S. patent application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 61/534,351, filed on Sep. 13, 2011 and incorporated herein by reference in its entirety.


INCORPORATION OF SEQUENCE LISTING
The sequence listing that is contained in the file named “40_77(58620).txt”, which is 259,094 bytes (measured in operating system MS-Windows), created on Sep. 11, 2012, is filed herewith by electronic submission and incorporated herein by reference in its entirety. The sequence listing contains SEQ ID NO: 1-659.
INCORPORATION BY REFERENCE OF TABLES OF PROVISIONAL APPLICATION
A “Table 1” was provided as a part of Provisional U.S. Patent Application No. 61/534,351 in a file named “40_77_58620_A_TAB1.txt” which was 57,572 bytes in size (measured in MS-Windows®) and that comprised SEQ ID NO:1-18. “Table 1” of Provisional U.S. Patent Application No. 61/534,351 is incorporated herein by reference in its entirety.
A “Table 2” was provided as a part of Provisional U.S. Patent Application No. 61/534,351, in file named “40_77_58620 A_TAB2.txt” which was 83,568 bytes in size (measured in MS-Windows®) and that comprised SEQ ID NO:19-331. “Table 2” of Provisional U.S. Patent Application No. 61/534,351 is incorporated herein by reference in its entirety.
A “Table 3” was provided as a part of Provisional U.S. Patent Application No. 61/534,351, in file named “40_77_58620_A_TAB3.txt” which was 12,078 bytes in size (measured in MS-Windows®) and that comprised SEQ ID NO:332-621. “Table 3” of Provisional U.S. Patent Application No. 61/534,351 is incorporated herein by reference in its entirety.
BACKGROUND OF THE INVENTION
Leaf senescence occurs at the end of the plant life cycle, and is controlled by a number of internal factors such as plant hormones and developmental stage, and can be accelerated by environmental stresses (Lim, Kim et al. 2007). Ethylene is a plant hormone with a key role in control of leaf senescence. Application of exogenous ethylene can induce leaf senescence, and suppression of ethylene biosynthesis or response can delay leaf senescence (Lim, Kim et al. 2007). Leaf senescence that has been delayed by reduced ethylene levels or response will eventually be initiated, and it will then proceed normally.
An example of the role of ethylene on crop productivity in field conditions was reported for soybean. Stressful high temperature conditions will reduce yield in soy and other crops. Ethylene production was shown to increase in soybean leaves in response to high temperature, and triggered premature leaf senescence (Djanaguiraman and Prasad 2010). However, prevention of ethylene response by use of the inhibitor 1-methyl cyclopropene (1-MCP) in plants grown at high temperature reduced leaf senescence and allowed increased yield compared to untreated controls also grown at high temperature. 1-MCP is a gas, and not appropriate for application in open fields. Therefore, other methods to delay leaf senescence are needed.
Ethylene also plays a major role in the ripening of fruits and vegetables. Ripening is an important phase of fruit development involving changes in fruit cellular metabolism leading to the development of a soft and edible ripe fruit with desirable quality. However, the softening that accompanies excessive ripening increases postharvest losses and reduces the shelf life of fruits and vegetables during handling, transportation, and storage. Ethylene, as a fruit ripening phytohormone, triggers many events of cell metabolism including initiation of ripening and senescence in fruits and vegetables, particularly in climacteric fruits (reviewed by (Lin, Zhong et al. 2009; Bapat, Trivedi et al. 2010)). Two systems, system 1 and 2 of ethylene production have been described in plants (McMurchie, McGlasson et al. 1972). System 1 operates during normal growth and development and during stress responses while system 2 functions during floral senescence and fruit ripening. System 2 is autocatalytic and it is stimulated by ethylene. Manipulation of ethylene biosynthesis, especially the system 2, in fruits and vegetables can be of strategic importance to enhance the shelf life and to reduce the postharvest losses of these crops.
The ethylene signal transduction pathway is relatively well understood (Lin, Zhong et al. 2009). Ethylene is perceived by a family of two-component histidine kinase-like receptors encoded by the ETHYLENE RESPONSE 1 (ETR1) and related genes. The CONSTITUTIVE TRIPLE RESPONSE 1 (CTR1) gene encodes a Raf-like serine/threonine kinase that has been shown to interact with ethylene receptors. ETHYLENE INSENSITIVE 2 (EIN2) is a positive regulator of ethylene signaling and has been placed downstream of the ethylene-receptor/CTR1 complex. The N-terminus of EIN2 has homology with NRAMP ion transporters, but its exact function in ethylene signaling is not understood. Further downstream of EIN2 are transcription factors, including EIN3-encoded proteins, that regulate genes in response to ethylene.
Mutants of EIN2 have delayed senescence in Arabidopsis, without an effect on flowering time or development (Aeong Oh, Park et al. 1997).
Ethylene insensitivity has been shown to provide improvements in tolerance to certain diseases in certain plants as well as reduced disease tolerance to certain diseases in certain plants (van Loon, L. C., et al. Trends in Plant Sci. 11(4): 184, 2006).
SUMMARY OF THE INVENTION
Provided herein are compositions and methods that provide for delayed leaf senescence, improved disease tolerance, and increased crop yield that results from suppression of EIN2 expression. In certain embodiments, delayed leaf senescence and increased crop yield that results from suppression of EIN2 expression is obtained by topical applications of compositions comprising polynucleotides and transfer agents to plants. For the purpose of extending functional stay-green, the ability to control the timing of application so that EIN2 genes are suppressed only during grain fill or under environmentally stressed conditions makes the compositions and methods provided herein particularly useful. Many crops will derive benefit from this method, including but are not limited to, corn, wheat, rice, soybean, cotton, Canola, tomato, alfalfa, melon, lettuce, cucumber, and broccoli.
Also provided herein are methods and compositions that provide for reductions in expression of EIN2 target polynucleotide and protein molecules in at least the cells of a plant root for improved resistance to nematodes. Nematodes that can be controlled by the methods and compositions provided herein include, but are not limited to, root knot nematodes (such as Meloidogyne sp.), cyst nematodes (such as Globodera sp. and Heterodera sp.), lesion nematodes (such as Pratylenchus sp.), and the like. In certain embodiments, EIN2 expression is reduced in plant root cells from which nematodes feed by providing topically to plant leaves, shoots, roots and/or seeds compositions comprising polynucleotides that comprise at least 18 contiguous nucleotides that are essentially identical or essentially complementary to an EIN2 gene or to a transcript of the EIN2 gene; and a transfer agent.
Also provided herein are compositions and methods for controlling plant fungal diseases. Plant fungal diseases that can be controlled with the methods and compositions provided herein include, but are not limited to, obligate biotrophic powdery mildew, downy mildew, and rust fungal infestations in plants. Plant fungal diseases that can be controlled with the methods and compositions provided herein also include, but are not limited to, fungal pathogens such as those causing anthracnose stalk rot, Diplodia Stalk or Ear Rot, Gibberella Stalk or Ear Rot, and Fusarium Stalk Rot in corn, or causing Take-all in wheat, Fusarium head blight in barley and wheat, or causing rice blast. In certain embodiments, methods and compositions for reducing expression of one or more host plant EIN2 polynucleotide and/or protein molecules in one or more cells or tissues of the plant such that the plant is rendered less susceptible to fungal infections from the order Erysiphales, the family Peronosporaceae or the order Pucciniales, are provided. In certain embodiments, nucleotide and amino acid sequences of plant EIN2 genes which can be downregulated by methods and compositions provided herein to increase plant resistance to powdery mildew, downy mildew, rust infection, or fungal pathogens such as those causing anthracnose stalk rot, Diplodia Stalk or Ear Rot, Gibberella Stalk or Ear Rot, and Fusarium Stalk Rot in corn, or causing Take-all in wheat, Fusarium head blight in barley and wheat, or causing rice blast are disclosed. Exemplary powdery mildew fungi of the order Erysiphales which are controlled by the compositions and methods provided herein include, but are not limited to, Blumeria graminis f. sp. hordei, Blumeria graminis forma specialis (f. sp.) tritici, Golovinomyces orontii, Golovinomyces cichoracearum, Oidium neolycopersici, Oidium lycopersici, Erysiphe pisi, Erisyphe necator and Sphaerotheca fuliginea among others. Exemplary downy mildew of the family Peronosporaceae include Pseudoperonospora humuli, Pseudoperonospora cubensis, Plasmopara viticola, Peronospora tabacina, Bremia lactucae, and Plasmopara halstedii. Exemplary rusts of the order Pucciniales which are controlled by the compositions and methods provided herein include, but are not limited to, Phakopsora meibomiae, Phakopsora pachyrhizi, Puccinia graminis, Puccinia recondita, Uromyces phaseoli and Uromyces appendeculatus. Other exemplary fungal pathogens which are controlled by the compositions and methods provided herein include, but are not limited to, Colletotrichum graminicola, Stenocarpella (or Diplodia) maydis, Gibberella zeae, Fusarium moniliforme, Gaeumannomyces graminis, Fusarium graminearum, Magnaporthe grisea (also known as Pyricularia grisea or Pyricularia oryzae), Septoria nodorum, and Septoria tritici. 
Also provided herein are compositions and methods for controlling plant bacterial diseases. Plant bacterial diseases that can be controlled with the methods and compositions provided herein include, but are not limited to, Disease Pathogen Bacterial leaf blight and stalk rot Pseudomonas avenae subsp. avenae, Bacterial leaf spot Xanthomonas campestris pv. Holcicola, Bacterial stalk rot Enterobacter dissolvens=Erwinia dissolvens, Bacterial stalk and top rot Erwinia carotovora subsp. carotovora, Erwinia chrysanthemi pv. zeae, Bacterial stripe Pseudomonas andropogonis, Chocolate spot Pseudomonas syringae pv. coronafaciens, Goss's bacterial wilt and blight Clavibacter michiganensis subsp. (leaf freckles and wilt) nebraskensis=Corynebacterium michiganense pv. andnebraskense, Holcus spot Pseudomonas syringae pv. syringae, Purple leaf sheath Hemiparasitic bacteria Seed rot-seedling blight Bacillus subtilis, Stewart's disease (bacterial wilt) Pantoea stewartii=Erwinia stewartii, Corn stunt (achapparramiento, Spiroplasma kunkelii maize stunt, Mesa Central or Rio Grande maize stunt). In certain embodiments, methods and compositions for reducing expression of one or more host plant EIN2 polynucleotide and/or protein molecules in one or more cells or tissues of the plant such that the plant is rendered less susceptible to any of the aforementioned bacterial diseases.
Also provided herein are compositions and methods that provide for delayed fruit senescence that results from suppression of EIN2 expression by topical applications of compositions comprising polynucleotides and transfer agents to plants. For the purpose of delaying fruit senescence, the ability to control the timing of application so that EIN2 genes are suppressed only during fruit ripening and/or only in detached fruit makes the compositions and methods provided herein particularly useful. Ethylene has many roles in growth and development and response to the environment that we do not want to otherwise disrupt. Many crops will derive benefit from this method, including, but not limited to, tomato, alfalfa, melon, lettuce, cucumber, and broccoli. Polynucleotides that can be used to suppress EIN2 include, but are not limited to, any of: i) polynucleotides comprising at least 18 contiguous nucleotides that are essentially identical or essentially complementary to an EIN2 gene or to a transcript of the gene of Table 2 (SEQ ID NO:1-18, or 622-623); ii) polynucleotides comprising at least 18 contiguous nucleotides that are essentially identical or essentially complementary to a polynucleotide of SEQ ID NO:19-331, or 624-658, or, iii) polynucleotides comprising at least 18 contiguous nucleotides that are essentially identical or essentially complementary to a polynucleotide of SEQ ID NO: 332-621.
In an aspect of the invention, the polynucleotide molecules are provided in compositions that can permeate or be absorbed into living plant tissue to initiate systemic gene inhibition or regulation. In certain embodiments of the invention, the polynucleotide molecules ultimately provide to a plant, or allow the in planta production of, RNA that is capable of hybridizing under physiological conditions in a plant cell to RNA transcribed from a target endogenous EIN2 gene or target EIN2 transgene in the plant cell, thereby effecting regulation of the target gene. In other embodiments of the invention, the polynucleotide molecules disclosed herein are useful for ultimately providing to a plant, or allowing the in planta production of, RNA that is capable of hybridizing under physiological conditions to RNA transcribed from a EIN2 target gene of the plant, thereby effecting regulation of the target gene. In certain embodiments, regulation of the target genes, such as by silencing or suppression of the target gene, leads to the upregulation of another gene that is itself affected or regulated by the target gene's expression.
In certain aspects or embodiments of the invention, the topical application of a composition comprising an exogenous polynucleotide and a transfer agent to a plant or plant part according to the methods described herein does not necessarily result in nor require the exogenous polynucleotide's integration into a chromosome of the plant. In certain aspects or embodiments of the invention, the topical application of a composition comprising an exogenous polynucleotide and a transfer agent to a plant or plant part according to the methods described herein does not necessarily result in nor require transcription of the exogenous polynucleotide from DNA integrated into a chromosome of the plant. In certain embodiments, topical application of a composition comprising an exogenous polynucleotide and a transfer agent to a plant according to the methods described herein also does not require that the exogenous polynucleotide be physically bound to a particle, such as in biolistic mediated introduction of polynucleotides associated with gold or tungsten particles into internal portions of a plant, plant part, or plant cell. An exogenous polynucleotide used in certain methods and compositions provided herein can optionally be associated with an operably linked promoter sequence in certain embodiments of the methods provided herein. However, in other embodiments, an exogenous polynucleotide used in certain methods and compositions provided herein is not associated with an operably linked promoter sequence. Also, in certain embodiments, an exogenous polynucleotide used in certain methods and compositions provided herein is not operably linked to a viral vector.
In certain embodiments, methods for delaying senescence, improving disease tolerance, and/or improving yield in a plant comprising topically applying compositions comprising a polynucleotide and a transfer agent that suppress the target EIN2 gene are provided. In certain embodiments, methods for selectively suppressing the target EIN2 gene by topically applying the polynucleotide composition to a plant surface at one or more selected seed, vegetative, or reproductive stage(s) of plant growth are provided. Such methods can provide for EIN2 gene suppression in a plant or plant part on an as needed or as desired basis. In certain embodiments, methods for selectively suppressing the target EIN2 gene by topically applying the polynucleotide composition to a plant surface at one or more pre-determined seed, vegetative, or reproductive stage(s) of plant growth are also provided. Such methods can provide for EIN2 gene suppression in a plant or plant part that obviates any undesired or unnecessary effects of suppressing the EIN2 genes expression at certain seed, vegetative, or reproductive stage(s) of plant development.
In certain embodiments, methods for selectively delaying senescence, improving disease tolerance, and/or improving yield in a plant by topically applying the polynucleotide composition to the plant surface at one or more selected seed, vegetative, or reproductive stage(s) are provided. Such methods can provide for a delayed senescence, improving disease tolerance, and/or improved yield in a plant or plant part on an as needed or as desired basis. In certain embodiments, methods for selectively delaying senescence, improving disease tolerance, and/or improving yield in a plant by topically applying the polynucleotide composition to the plant surface at one or more predetermined seed, vegetative, or reproductive stage(s) are provided. Such methods can provide for the delayed senescence, improving disease tolerance, and/or improved yield in a plant or plant part that obviates any undesired or unnecessary effects of providing the delayed senescence and/or improved yield at certain seed, vegetative, or reproductive stage(s) of plant development.
Polynucleotides that can be used to suppress an EIN2 gene include, but are not limited to, any of: i) polynucleotides comprising at least 18 contiguous nucleotides that are essentially identical or essentially complementary to EIN2 gene or to a transcript of the gene comprising or consisting of a sequence of SEQ ID NO:1-18, 622, or 623 in the sequence listing; ii) polynucleotides comprising at least 18 contiguous nucleotides that are essentially identical or essentially complementary to a polynucleotide comprising or consisting of a sequence of SEQ ID NO:19-331 or SEQ ID NO: 624-658 in the sequence listing; or, iii) polynucleotides comprising at least 18 contiguous nucleotides that are essentially identical or essentially complementary to a polynucleotide comprising or consisting of a sequence of SEQ ID NO:332-613 in the sequence listing
Provided herein are compositions and methods that provide for delayed leaf senescence, improving disease tolerance, and increased crop yield that results from suppression of EIN2 expression by topical applications of compositions comprising polynucleotides and transfer agents. For the purpose of extending functional stay-green, the ability to control the timing of application so that EIN2 genes are suppressed only during grain fill or under environmentally stressed conditions makes the compositions and methods provided herein especially effective. In certain embodiments, methods and compositions provided herein can delay leaf senescence during optimal growing conditions for a crop to improve yield. In certain embodiments, methods and compositions provided herein can delay premature leaf senescence caused various stresses including drought, heat and nutrient limitation can promote leaf senescence prematurely, and thus enhance crop yields under environmentally stressed conditions. Ethylene has certain roles in growth and development and response to the environment where suppression of EIN2 may be undesirable. The controlled suppression of EIN2 permitted by methods and compositions provided herein can avoid such undesirable suppression. Many crops will derive benefit from this method, including, but not limited to, corn, wheat, rice, soybean, cotton, Canola, tomato, alfalfa, melon, lettuce, cucumber and broccoli. Certain embodiments of the invention are directed to methods for producing a plant exhibiting delayed senescence, improving disease tolerance, and/or improved yield comprising topically applying to a plant surface a composition that comprises:
a. at least one polynucleotide that comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to a EIN2 gene or to a transcript of the gene; and
b. a transfer agent, wherein the plant exhibits an improvement in delayed senescence and/or improved yield that results from suppression of the EIN2 gene. In certain embodiments, the polynucleotide molecule comprises sense ssDNA, sense ssRNA, dsRNA, dsDNA, a double stranded DNA/RNA hybrid, anti-sense ssDNA, or anti-sense ssRNA. In certain embodiments, the polynucleotide is selected from the group consisting of SEQ ID NO:19-621, 624-657, and 658, or wherein the polynucleotide comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to SEQ ID NO:1-18, 622, or 623. In certain embodiments: (a) the plant is a corn plant, the gene or the transcript is a corn EIN2 gene or transcript, and the polynucleotide molecule is selected from the group consisting SEQ ID NO: 27-60, and 61 or the polynucleotide comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to SEQ ID NO:2 or SEQ ID NO:3; (b) the plant is a soy plant, the gene or the transcript is a soy EIN2 gene or transcript, and the polynucleotide molecule is selected from the group consisting of SEQ ID NO:191-295, and 296, or the polynucleotide comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to any one of SEQ ID NO:11-16; (c) the plant is a Canola plant, the gene or the transcript is a Canola EIN2 gene or transcript, and the polynucleotide molecule is selected from the group consisting of SEQ ID NO: 19-25, and 26, or the polynucleotide comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to SEQ ID NO:1; (d) the plant is a cucumber plant, the gene or the transcript is a cucumber EIN2 gene or transcript, and the polynucleotide molecule is selected from the group consisting of SEQ ID NO:62-106, 627-657, and 658, or the polynucleotide comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO: 622, or SEQ ID NO:623; (e) the plant is a lettuce plant, the gene or the transcript is a lettuce EIN2 gene or transcript, and the polynucleotide molecule is selected from the group consisting of SEQ ID NO:107-111, and 112, or the polynucleotide comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to SEQ ID NO:6; (f) the plant is a rice plant, the gene or the transcript is a rice EIN2 gene or transcript, and the polynucleotide molecule is selected from the group consisting of SEQ ID NO:113-189, and 190, or the polynucleotide comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to any one of SEQ ID NO:7-10; (g) the plant is a tomato plant the gene or the transcript is a tomato EIN2 gene or transcript, and the polynucleotide molecule is selected from the group consisting of SEQ ID NO:297-331, 624, 625, and 626, or the polynucleotide comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to any one of SEQ ID NO:17-18; (h) the plant is a corn or rice plant and the polynucleotide comprises a sequence selected from the group consisting of SEQ ID NO: 332-560, and 561; (i) the plant is a cucumber or soy plant and the polynucleotide comprises a sequence selected from the group consisting of SEQ ID NO: 562-588, and 589; (j) the plant is a cucumber or tomato plant of and the polynucleotide comprises a sequence selected from the group consisting of SEQ ID NO:590, and 591; or, (k) the plant is a rice or tomato plant and the polynucleotide comprises a sequence selected from the group consisting SEQ ID NO: 592-620, and 621. In certain embodiments, the composition comprises any combination of two or more polynucleotide molecules. In certain embodiments, the polynucleotide is at least 18 to about 24, about 25 to about 50, about 51 to about 100, about 101 to about 300, about 301 to about 500, or at least about 500 or more residues in length. In certain embodiments, the composition further comprises a non-polynucleotide herbicidal molecule, a polynucleotide herbicidal molecule, a polynucleotide that suppresses an herbicide target gene, an insecticide, a fungicide, a nematocide, or a combination thereof. In certain embodiments, the composition further comprises a non-polynucleotide herbicidal molecule and the plant is resistant to the herbicidal molecule. In certain embodiments, the transfer agent comprises an organosilicone preparation. In certain embodiments, the polynucleotide is not operably linked to a viral vector. In certain embodiments, the polynucleotide is not integrated into the plant chromosome.
Further embodiments of the invention are directed to: a plant made according to the above-described method; progeny of the plant that exhibits improvement in delayed senescence, improved disease tolerance, and/or improved yield; seed of the plant, wherein seed from the plant exhibits improvement in delayed senescence, improved disease tolerance, and/or improved yield; and a processed product of the plant, the progeny plant, or the seed, wherein the processed product exhibits improvement in delayed senescence, improved disease tolerance, and/or improved yield. In certain embodiments, the processed product exhibits an improved attribute relative to a processed product of an untreated control plant and wherein the improved attribute results from the delayed senescence, improved disease tolerance, and/or improved yield. An improved attribute of a processed product can include, but is not limited to, decreased mycotoxin content, improved nutritional content, improved storage characteristics, improved flavor, improved consistency, and the like when compared to a processed product obtained from an untreated plant or plant part.
An additional embodiment of the invention is directed to a composition comprising a polynucleotide molecule that comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to a EIN2 gene or transcript of the gene, wherein the polynucleotide is not operably linked to a promoter; and, a transfer agent. In certain embodiments, the polynucleotide is selected from the group consisting of wherein the polynucleotide is selected from the group consisting of SEQ ID NO:19-621, 624-657, and 658, or wherein the polynucleotide comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to SEQ ID NO:1-18, 622, or 623. In certain embodiments, the polynucleotide is at least 18 to about 24, about 25 to about 50, about 51 to about 100, about 101 to about 300, about 301 to about 500, or at least about 500 or more residues in length. In certain embodiments, the composition further comprises a non-polynucleotide herbicidal molecule, a polynucleotide herbicidal molecule, a polynucleotide that suppresses an herbicide target gene, an insecticide, a fungicide, a nematocide, or a combination thereof. In certain embodiments, the transfer agent is an organosilicone preparation. In certain embodiments, the polynucleotide is not physically bound to a biolistic particle. Another embodiment of the invention is directed to a method of making a composition comprising the step of combining at least: a) a polynucleotide molecule comprising at least 18 contiguous nucleotides that are essentially identical or essentially complementary to a EIN2 gene or a transcript of the gene, wherein the polynucleotide is not operably linked to a promoter or a viral vector; and, b) a transfer agent. In certain embodiments, the polynucleotide is obtained by in vivo biosynthesis, in vitro enzymatic synthesis, or chemical synthesis. In certain embodiments, the method further comprises combining with the polynucleotide and the transfer agent at least one of a non-polynucleotide herbicidal molecule, a polynucleotide herbicidal molecule, an insecticide, a fungicide, and/or a nematocide. In certain embodiments, the transfer agent is an organosilicone preparation. In certain embodiments, the polynucleotide is selected from the group consisting of wherein the polynucleotide is selected from the group consisting of SEQ ID NO:19-621, 624-657, and 658, or wherein the polynucleotide comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to SEQ ID NO:1-18, 622, or 623.
Yet another embodiment of the invention is directed to a method of identifying a polynucleotide for delaying senescence, improving disease tolerance, and/or improving yield in a plant comprising: a) selecting a population of polynucleotides that are essentially identical or essentially complementary to a EIN2 gene or transcript of the gene; b) topically applying to a surface of at least one of the plants a composition comprising at least one polynucleotide from the population and a transfer agent to obtain a treated plant; and, c) identifying a treated plant that exhibits suppression of the EIN2 gene or exhibits delayed senescence and/or improved yield, thereby identifying a polynucleotide that delays senescence and/or improves yield in a plant. In certain embodiments, the polynucleotide is selected from the group consisting of wherein the polynucleotide is selected from the group consisting of SEQ ID NO:19-621, 624-657, and 658, or the polynucleotide comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to SEQ ID NO:1-18, 622, or 623. In certain embodiments: (a) the plant is a corn plant, the gene or the transcript is a corn EIN2 gene or transcript, and the polynucleotide molecule is selected from the group consisting SEQ ID NO: 27-60, and 61 or the polynucleotide comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to SEQ ID NO:2 or SEQ ID NO:3; (b) the plant is a soy plant, the gene or the transcript is a soy EIN2 gene or transcript, and the polynucleotide molecule is selected from the group consisting of SEQ ID NO:191-295, and 296, or the polynucleotide comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to any one of SEQ ID NO:11-16; (c) the plant is a Canola plant, the gene or the transcript is a Canola EIN2 gene or transcript, and the polynucleotide molecule is selected from the group consisting of SEQ ID NO: 19-25, and 26, or the polynucleotide comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to SEQ ID NO:1; (d) the plant is a cucumber plant, the gene or the transcript is a cucumber EIN2 gene or transcript, and the polynucleotide molecule is selected from the group consisting of SEQ ID NO:62-106, 627-657, and 658, or the polynucleotide comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO: 622, or SEQ ID NO:623; (e) the plant is a lettuce plant, the gene or the transcript is a lettuce EIN2 gene or transcript, and the polynucleotide molecule is selected from the group consisting of SEQ ID NO:107-111, and 112, or the polynucleotide comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to SEQ ID NO:6; (f) the plant is a rice plant, the gene or the transcript is a rice EIN2 gene or transcript, and the polynucleotide molecule is selected from the group consisting of SEQ ID NO:113-189, and 190, or the polynucleotide comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to any one of SEQ ID NO:7-10; (g) the plant is a tomato plant the gene or the transcript is a tomato EIN2 gene or transcript, and the polynucleotide molecule is selected from the group consisting of SEQ ID NO:297-331, 624, 625, and 626, or the polynucleotide comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to any one of SEQ ID NO:17-18; (h) the plant is a corn or rice plant and the polynucleotide comprises a sequence selected from the group consisting of SEQ ID NO: 332-560, and 561; (i) the plant is a cucumber or soy plant and the polynucleotide comprises a sequence selected from the group consisting of SEQ ID NO: 562-588, and 589; (j) the plant is a cucumber or tomato plant of and the polynucleotide comprises a sequence selected from the group consisting of SEQ ID NO:590, and 591; or, (k) the plant is a rice or tomato plant and the polynucleotide comprises a sequence selected from the group consisting SEQ ID NO: 592-620, and 621.
A further embodiment of the invention is directed to a plant comprising an exogenous polynucleotide that comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to a EIN2 gene or transcript of the gene, wherein the exogenous polynucleotide is not operably linked to a promoter or to a viral vector, is not integrated into the chromosomal DNA of the plant, and is not found in a non-transgenic plant; and, wherein the plant exhibits delayed senescence and/or improved yield that results from suppression of the EIN2 gene. In certain embodiments, the plant further comprises an organosilicone compound, or a metabolite thereof, or a component thereof. In certain embodiments, the polynucleotide is selected from the group consisting of wherein the polynucleotide is selected from the group consisting of SEQ ID NO:19-621, 624-657, and 658, or the polynucleotide comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to SEQ ID NO:1-18, 622, or 623. In certain embodiments: (a) the plant is a corn plant, the gene or the transcript is a corn EIN2 gene or transcript, and the polynucleotide molecule is selected from the group consisting SEQ ID NO: 27-60, and 61 or the polynucleotide comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to SEQ ID NO:2 or SEQ ID NO:3; (b) the plant is a soy plant, the gene or the transcript is a soy EIN2 gene or transcript, and the polynucleotide molecule is selected from the group consisting of SEQ ID NO:191-295, and 296, or the polynucleotide comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to any one of SEQ ID NO:11-16; (c) the plant is a Canola plant, the gene or the transcript is a Canola EIN2 gene or transcript, and the polynucleotide molecule is selected from the group consisting of SEQ ID NO: 19-25, and 26, or the polynucleotide comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to SEQ ID NO:1; (d) the plant is a cucumber plant, the gene or the transcript is a cucumber EIN2 gene or transcript, and the polynucleotide molecule is selected from the group consisting of SEQ ID NO:62-106, 627-657, and 658, or the polynucleotide comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO: 622, or SEQ ID NO:623; (e) the plant is a lettuce plant, the gene or the transcript is a lettuce EIN2 gene or transcript, and the polynucleotide molecule is selected from the group consisting of SEQ ID NO:107-111, and 112, or the polynucleotide comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to SEQ ID NO:6; (f) the plant is a rice plant, the gene or the transcript is a rice EIN2 gene or transcript, and the polynucleotide molecule is selected from the group consisting of SEQ ID NO:113-189, and 190, or the polynucleotide comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to any one of SEQ ID NO:7-10; (g) the plant is a tomato plant the gene or the transcript is a tomato EIN2 gene or transcript, and the polynucleotide molecule is selected from the group consisting of SEQ ID NO:297-331, 624, 625, and 626, or the polynucleotide comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to any one of SEQ ID NO:17-18; (h) the plant is a corn or rice plant and the polynucleotide comprises a sequence selected from the group consisting of SEQ ID NO: 332-560, and 561; (i) the plant is a cucumber or soy plant and the polynucleotide comprises a sequence selected from the group consisting of SEQ ID NO: 562-588, and 589; (j) the plant is a cucumber or tomato plant of and the polynucleotide comprises a sequence selected from the group consisting of SEQ ID NO:590, and 591; or, (k) the plant is a rice or tomato plant and the polynucleotide comprises a sequence selected from the group consisting SEQ ID NO: 592-620, and 621.
An additional embodiment of the invention is directed to a plant part comprising an exogenous polynucleotide that comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to a EIN2 gene or transcript of the gene, wherein the exogenous polynucleotide is not operably linked to a promoter or to a viral vector and is not found in a non-transgenic plant; and, wherein the plant part exhibits delayed senescence, improved disease tolerance, and/or improved yield that results from suppression of the EIN2 gene. In certain embodiments, the plant part further comprises an organosilicone compound or a metabolite thereof. In certain embodiments, the polynucleotide is selected from the group consisting of wherein the polynucleotide is selected from the group consisting of SEQ ID NO:19-621, 624-657, and 658, or the polynucleotide comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to SEQ ID NO:1-18, 622, or 623. In certain embodiments: (a) the plant part is a corn plant part, the gene or the transcript is a corn EIN2 gene or transcript, and the polynucleotide molecule is selected from the group consisting SEQ ID NO: 27-60, and 61 or the polynucleotide comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to SEQ ID NO:2 or SEQ ID NO:3; (b) the plant part is a soy plant part, the gene or the transcript is a soy EIN2 gene or transcript, and the polynucleotide molecule is selected from the group consisting of SEQ ID NO:191-295, and 296, or the polynucleotide comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to any one of SEQ ID NO:11-16; (c) the plant part is a Canola plant part, the gene or the transcript is a Canola EIN2 gene or transcript, and the polynucleotide molecule is selected from the group consisting of SEQ ID NO: 19-25, and 26, or the polynucleotide comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to SEQ ID NO:1; (d) the plant part is a cucumber plant part, the gene or the transcript is a cucumber EIN2 gene or transcript, and the polynucleotide molecule is selected from the group consisting of SEQ ID NO:62-106, 627-657, and 658, or the polynucleotide comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO: 622, or SEQ ID NO:623; (e) the plant part is a lettuce plant part, the gene or the transcript is a lettuce EIN2 gene or transcript, and the polynucleotide molecule is selected from the group consisting of SEQ ID NO:107-111, and 112, or the polynucleotide comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to SEQ ID NO:6; (f) the plant part is a rice plant part, the gene or the transcript is a rice EIN2 gene or transcript, and the polynucleotide molecule is selected from the group consisting of SEQ ID NO:113-189, and 190, or the polynucleotide comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to any one of SEQ ID NO:7-10; (g) the plant part is a tomato plant part the gene or the transcript is a tomato EIN2 gene or transcript, and the polynucleotide molecule is selected from the group consisting of SEQ ID NO:297-331, 624, 625, and 626, or the polynucleotide comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to any one of SEQ ID NO:17-18; (h) the plant part is a corn or rice plant part and the polynucleotide comprises a sequence selected from the group consisting of SEQ ID NO: 332-560, and 561; (i) the plant part is a cucumber or soy plant part and the polynucleotide comprises a sequence selected from the group consisting of SEQ ID NO: 562-588, and 589; (j) the plant part is a cucumber or tomato plant part of and the polynucleotide comprises a sequence selected from the group consisting of SEQ ID NO:590, and 591; or, (k) the plant part is a rice or tomato plant part and the polynucleotide comprises a sequence selected from the group consisting SEQ ID NO: 592-620, and 621. Another embodiment of the invention is directed to a plant that exhibits delayed senescence, improved disease tolerance, and/or improved yield, wherein the plant was topically treated with a composition that comprises: (a) at least one polynucleotide that comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to a EIN2 gene or to a transcript of the gene; and, (b) a transfer agent; and, wherein the plant exhibits delayed senescence, improved disease tolerance, and/or improved yield that results from suppression of the EIN2 gene. Ion certain embodiments, the transfer agent comprises an organosilicone preparation.



DESCRIPTION OF THE DRAWINGS
FIG. 1 A, B. Illustrates the hypocotyl lengths of tomato seedlings treated with EIN2 triggers or control treatments, and grown in the presence of 100 μm ACC in the dark. SEQ ID NO: 624, 625, and 626 are double-stranded RNAs containing sequences from the EIN2 transcript (Table 3). SEQ ID NO:659 is a control double-stranded RNA sequence from the jelly-fish green fluorescent protein (GFP). Two independent experiments are shown in FIGS. 1A and 1B. N=15-44 seedlings. Error bars represent 95% confidence intervals.
FIGS. 2A and B. Expression of the ethylene-responsive E4 gene in tomato seedlings treated with EIN2 triggers or control treatments, and grown in the presence of 100 μm ACC in the dark. Samples were taken from the experiment shown in FIGS. 1A and 1B. N=3 (FIG. 2A) or 9 (FIG. 2B). Error bars represent the 95% confidence interval.



DETAILED DESCRIPTION
I. Definitions
The following definitions and methods are provided to better define the present invention and to guide those of ordinary skill in the art in the practice of the present invention. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.
Where a term is provided in the singular, the inventors also contemplate aspects of the invention described by the plural of that term.
As used herein, the terms “DNA,” “DNA molecule,” and “DNA polynucleotide molecule” refer to a single-stranded DNA or double-stranded DNA molecule of genomic or synthetic origin, such as, a polymer of deoxyribonucleotide bases or a DNA polynucleotide molecule.
As used herein, the terms “DNA sequence,” “DNA nucleotide sequence,” and “DNA polynucleotide sequence” refer to the nucleotide sequence of a DNA molecule.
As used herein, the term “gene” refers to any portion of a nucleic acid that provides for expression of a transcript or encodes a transcript. A “gene” thus includes, but is not limited to, a promoter region, 5′ untranslated regions, transcript encoding regions that can include intronic regions, and 3′ untranslated regions.
As used herein, the terms “RNA,” “RNA molecule,” and “RNA polynucleotide molecule” refer to a single-stranded RNA or double-stranded RNA molecule of genomic or synthetic origin, such as, a polymer of ribonucleotide bases that comprise single or double stranded regions.
Unless otherwise stated, nucleotide sequences in the text of this specification are given, when read from left to right, in the 5′ to 3′ direction. The nomenclature used herein is that required by Title 37 of the United States Code of Federal Regulations §1.822 and set forth in the tables in WIPO Standard ST.25 (1998), Appendix 2, Tables 1 and 3.
As used herein, a “plant surface” refers to any exterior portion of a plant. Plant surfaces thus include, but are not limited to, the surfaces of flowers, stems, tubers, fruit, anthers, pollen, leaves, roots, or seeds. A plant surface can be on a portion of a plant that is attached to other portions of a plant or on a portion of a plant that is detached from the plant.
As used herein, the phrase “polynucleotide is not operably linked to a promoter” refers to a polynucleotide that is not covalently linked to a polynucleotide promoter sequence that is specifically recognized by either a DNA dependent RNA polymerase II protein or by a viral RNA dependent RNA polymerase in such a manner that the polynucleotide will be transcribed by the DNA dependent RNA polymerase II protein or viral RNA dependent RNA polymerase. A polynucleotide that is not operably linked to a promoter can be transcribed by a plant RNA dependent RNA polymerase.
As used herein, SEQ ID NO:19-331, though displayed in the Sequence Listing in the form of ssDNA, encompass dsDNA equivalents, dsRNA equivalents, ssRNA equivalents, ssRNA complements, ssDNA as shown, and ssDNA complements.
As used herein, SEQ ID NO: 332-621, and 624-659, though displayed in the Sequence Listing in the form of ssDNA, encompass dsDNA equivalents, dsRNA equivalents, ssRNA equivalents, a ssRNA complement, ssDNA as shown, and ssDNA complements.
As used herein, a first nucleic-acid sequence is “operably” connected or “linked” with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to an RNA and/or protein-coding sequence if the promoter provides for transcription or expression of the RNA or coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, are in the same reading frame.
As used herein, the phrase “organosilicone preparation” refers to a liquid comprising one or more organosilicone compounds, wherein the liquid or components contained therein, when combined with a polynucleotide in a composition that is topically applied to a target plant surface, enable the polynucleotide to enter a plant cell. Exemplary organosilicone preparations include, but are not limited to, preparations marketed under the trade names “Silwet®” or “BREAK-THRU®” and preparations provided in Table 1. In certain embodiments, an organosilicone preparation can enable a polynucleotide to enter a plant cell in a manner permitting a polynucleotide mediated suppression of target gene expression in the plant cell.
As used herein, the phrases “delayed senescence and/or improved yield” or “delaying senescence or improving yield” refer to any measurable delay in the onset or progress of a senescence process and/or any measurable improvement in yield. In certain embodiments, a delay in a senescence process and/or an improvement in yield in a plant or plant part can be determined in a comparison to a control plant or plant part that has not been treated with a composition comprising a polynucleotide and a transfer agent. When used in this context, a control plant is a plant that has not undergone treatment with polynucleotide and a transfer agent. Such control plants would include, but are not limited to, untreated plants or mock treated plants.
As used herein, the phrase “improved disease tolerance” refer to any measurable increase in a plants resistance to fungal-, bacterial-, and/or nematode-induced damage. In certain embodiments, an improvement in fungal-, bacterial-, and/or nematode-resistance in a plant or plant part can be determined in a comparison to a control plant or plant part that has not been treated with a composition comprising a polynucleotide and a transfer agent. When used in this context, a control plant is a plant that has not undergone treatment with polynucleotide and a transfer agent. Such control plants would include, but are not limited to, untreated plants or mock treated plants.
As used herein, a “senescence process” refers to any pre- or post-harvest process whereby any visual, physical, and/or biochemical property of a plant or plant part changes as a result of aging.
As used herein, the phrase “provides for a reduction”, when used in the context of a transcript or a protein in a plant or plant part, refers to any measurable decrease in the level of transcript or protein in a plant or plant part. In certain embodiments, a reduction of the level of a transcript or protein in a plant or plant part can be determined in a comparison to a control plant or plant part that has not been treated with a composition comprising a polynucleotide and a transfer agent. When used in this context, a control plant or plant part is a plant or plant part that has not undergone treatment with polynucleotide and a transfer agent. Such control plants or plant parts would include, but are not limited to, untreated or mock treated plants and plant parts.
As used herein, the phrase “wherein said plant does not comprise a transgene” refers to a plant that lacks either a DNA molecule comprising a promoter that is operably linked to a polynucleotide or a recombinant viral vector.
As used herein, the phrase “suppressing expression” or “suppression”, when used in the context of a gene, refers any measurable decrease in the amount and/or activity of a product encoded by the gene. Thus, expression of a gene can be suppressed when there is a reduction in levels of a transcript from the gene, a reduction in levels of a protein encoded by the gene, a reduction in the activity of the transcript from the gene, a reduction in the activity of a protein encoded by the gene, any one of the preceding conditions, or any combination of the preceding conditions. In this context, the activity of a transcript includes, but is not limited to, its ability to be translated into a protein and/or to exert any RNA-mediated biologic or biochemical effect. In this context, the activity of a protein includes, but is not limited to, its ability to exert any protein-mediated biologic or biochemical effect. In certain embodiments, a suppression of gene expression in a plant or plant part can be determined in a comparison of gene product levels or activities in a treated plant to a control plant or plant part that has not been treated with a composition comprising a polynucleotide and a transfer agent. When used in this context, a control plant or plant part is a plant or plant part that has not undergone treatment with polynucleotide and a transfer agent. Such control plants or plant parts would include, but are not limited to, untreated or mock treated plants and plant parts.
As used herein, the term “transcript” corresponds to any RNA that is produced from a gene by the process of transcription. A transcript of a gene can thus comprise a primary transcription product which can contain introns or can comprise a mature RNA that lacks introns.
As used herein, the term “liquid” refers to both homogeneous mixtures such as solutions and non-homogeneous mixtures such as suspensions, colloids, micelles, and emulsions.
II. Overview
Provided herein are certain methods and polynucleotide compositions that can be applied to living plant cells/tissues to suppress expression of target EIN2 genes and that provide delayed senescence, improved disease tolerance, and/or improved yield to a crop plant in need of the benefit. Also provided herein are plants and plant parts exhibiting delayed senescence and/or improved yield as well as processed products of such plants or plant parts. The compositions may be topically applied to the surface of a plant, such as to the surface of a leaf, and include a transfer agent. Aspects of the method can be applied to various crops, for example, including but not limited to: i) row crop plants including, but not limited to, corn, soybean, cotton, canola, sugar beet, alfalfa, sugarcane, rice, and wheat; ii) vegetable plants including, but not limited to, tomato, potato, sweet pepper, hot pepper, melon, watermelon, cucumber, eggplant, cauliflower, broccoli, lettuce, spinach, onion, peas, carrots, sweet corn, Chinese cabbage, leek, fennel, pumpkin, squash or gourd, radish, Brussels sprouts, tomatillo, garden beans, dry beans, or okra; iii) culinary plants including, but not limited to, basil, parsley, coffee, or tea; iv) fruit plants including but not limited to apple, pear, cherry, peach, plum, apricot, banana, plantain, table grape, wine grape, citrus, avocado, mango, or berry; v) a tree grown for ornamental or commercial use, including, but not limited to, a fruit or nut tree; or, vi) an ornamental plant (e. g., an ornamental flowering plant or shrub or turf grass). The methods and compositions provided herein can also be applied to plants produced by a cutting, cloning, or grafting process (i. e., a plant not grown from a seed) include fruit trees and plants. Fruit trees produced by such processes include, but are not limited to, citrus and apple trees. Plants produced by such processes include, but are not limited to, avocados, tomatoes, eggplant, cucumber, melons, watermelons, and grapes as well as various ornamental plants.
Examples of the fungal plant diseases controlled by materials and compositions provided herein include, but are not limited to, diseases caused by phytopathogenic fungi (in particular of the classes of Ascomycetes, Deuteromycetes, Oomycetes and Basidiomycetes) such as Magnaporthe grisea, Cochliobolus miyabeanus, Rhizoctonia solani and Gibberella fujikuroi on rice; Erysiphe graminis, Fusarium graminearum, F. avenacerum, F. culmorum, Microdochium nivale, Puccinia striiformis, P. graminis, P. recondita, P. hordei, Typhula sp., Micronectriella nivalis, Ustilago tritici, U. nuda, Tilletia caries, Pseudocercosporella herpotrichoides, Rhynchosporium secalis, Septoria tritici, Leptosphaeria nodorum and Pyrenophora teres on wheat and barley; Diaporthe citri, Elsinoe fawcetti, Penicillium digitatum, P. italicum, Phytophthora parasitica and Phytophthora citrophthora on citrus; Monilinia mali, Valsa ceratosperma, Podosphaera leucotricha, Alternaria alternata apple pathotype, Venturia inaequalis, Colletotrichum acutatum and Phytophtora cactorum on apple; Venturia nashicola, V. pirina, Alternaria alternata Japanese pear pathotype, Gymnosporangium haraeanum and Phytophthora cactorum on pear; Monilinia fructicola, Cladosporium carpophilum and Phomopsis sp. on peach; Elsinoe ampelina, Glomerella cingulata, Uncinula necator, Phakopsora ampelopsidis, Guignardia bidwellii and Plasmopara viticola on grape; Gloeosporium kaki, Cercospora kaki and Mycosphaerella nawae on persimmon; Colletotrichum lagenarium, Sphaerotheca fuliginea, Mycosphaerella melonis, Fusarium oxysporum, Pseudoperonospora cubensis and Phytophthora sp. on Cucurbitales vegetables; Alternaria solani, Cladosporium fulvum and Phytophthora infestans on tomato; Phomopsis vexans and Erysiphe cichoracearum on eggplant; Alternaria japonica, Cercosporella brassicae, Plasmodiophora brassicae and Peronospora parasitica on Brassicaceae vegetables; Puccinia allii and Peronospora destructor on leek; Cercospora kikuchii, Elsinoe glycines, Diaporthe phaseolorum var. sojae, Phakopsora pachyrhizi and Phytophthora sojae on soybean; Colletotrichum lindemuthianum of kidney bean; Cercospora personata, Cercospora arachidicola and Sclerotium rolfsii on peanut; Erysiphe pisi on pea; Alternaria solani, Phytophthora infestans, Phytophthora erythroseptica and Spongospora subterranean f sp. subterranean on potato; Sphaerotheca humuli and Glomerella cingulata on strawberry; Exobasidium reticulatum, Elsinoe leucospila, Pestalotiopsis sp. and Colletotrichum theae-sinensis on tea; Alternaria longipes, Erysiphe cichoracearum, Colletotrichum tabacum, Peronospora tabacina and Phytophthora nicotianae on tobacco; Cercospora beticola, Thanatephorus cucumeris, and Aphanidermatum cochlioides on sugar beet; Diplocarpon rosae, Sphaerotheca pannosa and Peronospora sparsa on rose; Bremia lactucae, Septoria chrysanthemi-indici and Puccinia horiana on chrysanthemum and Compositae vegetables; Alternaria brassicicola on radish; Sclerotinia homeocarpa and Rhizoctonia solani on turf; Mycosphaerella fijiensis and Mycosphaerella musicola on banana; Plasmopara halstedii on sunflower; and various diseases on crops caused by Pythium spp. (e.g., Pythium aphanidermatum, Pythium debaryanum, Pythium graminicola, Pythium irregulare, Pythium ultimum), Botrytis cinerea, Sclerotinia sclerotiorum, Aspergillus spp., Penicillium spp., Fusarium spp., Gibberella spp., Trichoderma spp., Thielaviopsis spp., Rhizopus spp., Mucor spp., Corticium spp., Phoma spp., Rhizoctonia spp., Diplodia spp., Polymyxa spp. and Olpidium spp.
Exemplary plants protected from plant-parasitic nematodes species associated with them by the methods and compositions provided herein include, but are not limited to: alfalfa: Pratylenchus spp., Meloidogyne hapla, Meloidogyne incognita, Meloidogyne javanica, Ditylenchus dipsaci, Paratylenchus spp., Xiphinema spp.; banana: Pratylenchus coffeae, Meloidogyne incognita, Meloidogyne arenaria, Meloidogyne javanica, Radopholus similis, Helicotylenchus multicinctus, Rotylenchulus reniformis; cereals (barley, wheat, rye): Pratylenchus spp., Meloidogyne naasi; chickpea: Pratylenchus spp., Meloidogyne spp., Heterodera cajani, Rotylenchulus reniformis, Hoplolaimus seinhorsti; citrus: Pratylenchus spp., Meloidogyne spp., Tylenchulus semipenetrans, Radopholus similis, Radopholus citrophilus, Hemicycliophora arenaria, Bolonolaimus longicaudatus, Trichodorus spp., Paratrichodorus spp., Xiphinema spp.; clover: Pratylenchus spp., Meloidogyne spp., Heterodera trifolii; corn: Pratylenchus brachyurus, Pratylenchus hexincisus, Pratylenchus penetrans, Pratylenchus scribneri, Pratylenchus zeae, Meloidogyne incognita, Paratrichodorus minor, Longidorus spp., Hoplolaimus columbus; cotton: Pratylenchus spp., Meloidogyne incognita, Belonolaimus longicaudatus, Rotylenchulus reniformis, Hoplolaimus galeatus, Tylenchorhynchus spp., Paratrichodorus minor; grapes: Pratylenchus vulnus, Meloidogyne spp., Xiphinema spp., Tylenchulus semipenetrans, Rotylenchulus reniformis; grasses: Pratylenchus spp., Longidorus spp., Paratrichodorus christiei, Xiphinema spp., Ditylenchus spp.; peanut: Pratylenchus spp., Meloidogyne hapla, Meloidogyne arenaria, Criconemella spp., Belonolaimus longicaudatus; pigeon pea: Pratylenchus spp., Meloidogyne spp., Heterodera cajani, Rotylenchulus reniformis, Hoplolaimus seinhorsti; potato: Pratylenchus spp., Meloidogyne spp., Globodera rostochiensis, Globodera pallida, Trichodorus primitivus, Ditylenchus spp., Paratrichodorus spp., Nacobbus aberrans; rice: Pratylenchus spp., Meloidogyne spp., Aphelenchiodes besseyi, Ditylenchus angustus, Hirchmanniella spp., Heterodera oryzae; small fruits: Pratylenchus spp., Meloidogyne spp.; Xiphinema spp., Longidorus spp., Paratrichodorus christiei, Aphelenchoides spp.; soybean: Pratylenchus spp., Meloidogyne incognita, Meloidogyne javanica, Heterodera glycines, Belonolaimus spp., Hoplolaimus columbus; sugar beet: Pratylenchus spp., Meloidogyne spp., Heterodera schachtii, Ditylenchus dipsaci, Nacobbus aberrans, Trichodorus spp., Longidorus spp., Paratrichodorus spp.; sugar cane: Pratylenchus spp., Meloidogyne spp., Radopholus spp., Heterodera spp., Hoplolaimus spp., Helicotylenchus spp., Scutellonema spp., Belonolaimus spp., Tylenchorhynchus spp., Xiphinema spp., Longidorus spp., Paratrichodorus spp.; tobacco: Pratylenchus spp., Meloidogyne spp., Tylenchorhynchus claytoni, Globodera tabacum, Trichodorus spp., Xiphinema americanum, Ditylenchus dipsaci, Paratrichodorus spp.; and tomato: Pratylenchus spp., Meloidogyne spp.
Materials and compositions provided herein also provide for improved tolerance to bacterial diseases including but not limited to: Disease Pathogen Bacterial leaf blight and stalk rot Pseudomonas avenae subsp. avenae, Bacterial leaf spot Xanthomonas campestris pv. Holcicola, Bacterial stalk rot Enterobacter dissolvens=Erwinia dissolvens, Bacterial stalk and top rot Erwinia carotovora subsp. carotovora, Erwinia chrysanthemi pv. zeae, Bacterial stripe Pseudomonas andropogonis, Chocolate spot Pseudomonas syringae pv. coronafaciens, Goss's bacterial wilt and blight Clavibacter michiganensis subsp. (leaf freckles and wilt) nebraskensis=Corynebacterium michiganense pv. andnebraskense, Holcus spot Pseudomonas syringae pv. syringae, Purple leaf sheath Hemiparasitic bacteria Seed rot-seedling blight Bacillus subtilis, Stewart's disease (bacterial wilt) Pantoea stewartii=Erwinia stewartii, and Corn stunt (achapparramiento, Spiroplasma kunkelii maize stunt, Mesa Central or Rio Grande maize stunt).
Without being bound by theory, the compositions and methods of the present invention are believed to operate through one or more of the several natural cellular pathways involved in RNA-mediated gene suppression as generally described in Brodersen and Voinnet (2006), Trends Genetics, 22:268-280; Tomari and Zamore (2005) Genes & Dev., 19:517-529; Vaucheret (2006) Genes Dev., 20:759-771; Meins et al. (2005) Annu. Rev. Cell Dev. Biol., 21:297-318; and Jones-Rhoades et al. (2006) Annu. Rev. Plant Biol., 57:19-53. RNA-mediated gene suppression generally involves a double-stranded RNA (dsRNA) intermediate that is formed intra-molecularly within a single RNA molecule or inter-molecularly between two RNA molecules. This longer dsRNA intermediate is processed by a ribonuclease of the RNAase III family (Dicer or Dicer-like ribonuclease) to one or more shorter double-stranded RNAs, one strand of which is incorporated into the RNA-induced silencing complex (“RISC”). For example, the siRNA pathway involves the cleavage of a longer double-stranded RNA intermediate to small interfering RNAs (“siRNAs”). The size of siRNAs is believed to range from about 19 to about 25 base pairs, but the most common classes of siRNAs in plants include those containing 21 to 24 base pairs (See, Hamilton et al. (2002) EMBO J., 21:4671-4679).
Polynucleotides
As used herein, “polynucleotide” refers to a DNA or RNA molecule containing multiple nucleotides and generally refers both to “oligonucleotides” (a polynucleotide molecule of 18-25 nucleotides in length) and longer polynucleotides of 26 or more nucleotides. Embodiments of this invention include compositions including oligonucleotides having a length of 18-25 nucleotides (18-mers, 19-mers, 20-mers, 21-mers, 22-mers, 23-mers, 24-mers, or 25-mers), or medium-length polynucleotides having a length of 26 or more nucleotides (polynucleotides of 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, or about 300 nucleotides), or long polynucleotides having a length greater than about 300 nucleotides (e. g., polynucleotides of between about 300 to about 400 nucleotides, between about 400 to about 500 nucleotides, between about 500 to about 600 nucleotides, between about 600 to about 700 nucleotides, between about 700 to about 800 nucleotides, between about 800 to about 900 nucleotides, between about 900 to about 1000 nucleotides, between about 300 to about 500 nucleotides, between about 300 to about 600 nucleotides, between about 300 to about 700 nucleotides, between about 300 to about 800 nucleotides, between about 300 to about 900 nucleotides, or about 1000 nucleotides in length, or even greater than about 1000 nucleotides in length, for example up to the entire length of a target EIN2 gene including coding or non-coding or both coding and non-coding portions of the target EIN2 gene). Where a polynucleotide is double-stranded, its length can be similarly described in terms of base pairs.
Polynucleotide compositions used in the various embodiments of this invention include compositions including oligonucleotides, polynucleotides, or a mixture of both, including: RNA or DNA or RNA/DNA hybrids or chemically modified oligonucleotides or polynucleotides or a mixture thereof. In certain embodiments, the polynucleotide may be a combination of ribonucleotides and deoxyribonucleotides, for example, synthetic polynucleotides consisting mainly of ribonucleotides but with one or more terminal deoxyribonucleotides or synthetic polynucleotides consisting mainly of deoxyribonucleotides but with one or more terminal dideoxyribonucleotides. In certain embodiments, the polynucleotide includes non-canonical nucleotides such as inosine, thiouridine, or pseudouridine. In certain embodiments, the polynucleotide includes chemically modified nucleotides. Examples of chemically modified oligonucleotides or polynucleotides are well known in the art; see, for example, U.S. Patent Publication 2011/0171287, U.S. Patent Publication 2011/0171176, U.S. Patent Publication 2011/0152353, U.S. Patent Publication 2011/0152346, and U.S. Patent Publication 2011/0160082, which are herein incorporated by reference. Illustrative examples include, but are not limited to, the naturally occurring phosphodiester backbone of an oligonucleotide or polynucleotide which can be partially or completely modified with phosphorothioate, phosphorodithioate, or methylphosphonate internucleotide linkage modifications, modified nucleoside bases or modified sugars can be used in oligonucleotide or polynucleotide synthesis, and oligonucleotides or polynucleotides can be labeled with a fluorescent moiety (e. g., fluorescein or rhodamine) or other label (e. g., biotin).
Polynucleotides can be single- or double-stranded RNA, single- or double-stranded DNA, double-stranded DNA/RNA hybrids, and modified analogues thereof. In certain embodiments of the invention, the polynucleotides that provide single-stranded RNA in the plant cell may be: (a) a single-stranded RNA molecule (ssRNA), (b) a single-stranded RNA molecule that self-hybridizes to form a double-stranded RNA molecule, (c) a double-stranded RNA molecule (dsRNA), (d) a single-stranded DNA molecule (ssDNA), (e) a single-stranded DNA molecule that self-hybridizes to form a double-stranded DNA molecule, (f) a single-stranded DNA molecule including a modified Pol III gene that is transcribed to an RNA molecule, (g) a double-stranded DNA molecule (dsDNA), (h) a double-stranded DNA molecule including a modified Pol III gene that is transcribed to an RNA molecule, and (i) a double-stranded, hybridized RNA/DNA molecule, or combinations thereof. In certain embodiments, these polynucleotides can comprise both ribonucleic acid residues and deoxyribonucleic acid residues. In certain embodiments, these polynucleotides include chemically modified nucleotides or non-canonical nucleotides. In certain embodiments of the methods, the polynucleotides include double-stranded DNA formed by intramolecular hybridization, double-stranded DNA formed by intermolecular hybridization, double-stranded RNA formed by intramolecular hybridization, or double-stranded RNA formed by intermolecular hybridization. In certain embodiments where the polynucleotide is a dsRNA, the anti-sense strand will comprise at least 18 nucleotides that are essentially complementary to the target EIN2 gene. In certain embodiments the polynucleotides include single-stranded DNA or single-stranded RNA that self-hybridizes to form a hairpin structure having an at least partially double-stranded structure including at least one segment that will hybridize to RNA transcribed from the gene targeted for suppression. Not intending to be bound by any mechanism, it is believed that such polynucleotides are or will produce single-stranded RNA with at least one segment that will hybridize to RNA transcribed from the gene targeted for suppression. In certain embodiments, the polynucleotides can be operably linked to a promoter—generally a promoter functional in a plant, for example, a pol II promoter, a pol III promoter, a pol IV promoter, or a pol V promoter.
The polynucleotide molecules of the present invention are designed to modulate expression by inducing regulation or suppression of an endogenous EIN2 gene in a plant and are designed to have a nucleotide sequence essentially identical or essentially complementary to the nucleotide sequence of an endogenous EIN2 gene of a plant or to the sequence of RNA transcribed from an endogenous EIN2 gene of a plant, which can be coding sequence or non-coding sequence. These effective polynucleotide molecules that modulate expression are referred to herein as “a trigger, or triggers”. By “essentially identical” or “essentially complementary” it is meant that the trigger polynucleotides (or at least one strand of a double-stranded polynucleotide) have sufficient identity or complementarity to the endogenous gene or to the RNA transcribed from the endogenous EIN2 gene (e.g. the transcript) to suppress expression of the endogenous EIN2 gene (e.g. to effect a reduction in levels or activity of the gene transcript and/or encoded protein). In certain embodiments, the trigger polynucleotides provided herein can be directed to an EIN2 transgene present in the plant. Polynucleotides of the methods and compositions provided herein need not have 100 percent identity to a complementarity to the endogenous EIN2 gene or to the RNA transcribed from the endogenous EIN2 gene (i.e. the transcript) to suppress expression of the endogenous EIN2 gene (i.e. to effect a reduction in levels or activity of the gene transcript or encoded protein). Thus, in certain embodiments, the polynucleotide or a portion thereof is designed to be essentially identical to, or essentially complementary to, a sequence of at least 18 or 19 contiguous nucleotides in either the target gene or messenger RNA transcribed from the target gene (e.g. the transcript). In certain embodiments, an “essentially identical” polynucleotide has 100 percent sequence identity or at least about 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence identity when compared to the sequence of 18 or more contiguous nucleotides in either the endogenous target gene or to an RNA transcribed from the target gene (e.g. the transcript). In certain embodiments, an “essentially complementary” polynucleotide has 100 percent sequence complementarity or at least about 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence complementarity when compared to the sequence of 18 or more contiguous nucleotides in either the target gene or RNA transcribed from the target gene.
In certain embodiments, polynucleotides used in the methods and compositions provided herein can be essentially identical or essentially complementary to any of: i) conserved regions of EIN2 genes of both monocot and dicot plants; ii) conserved regions of EIN2 genes of monocot plants; or iii) conserved regions of EIN2 genes of dicot plants. Such polynucleotides that are essentially identical or essentially complementary to such conserved regions can be used to improve delayed senescence and/or improved yield by suppressing expression of EIN2 genes in various dicot
Polynucleotides containing mismatches to the target gene or transcript can thus be used in certain embodiments of the compositions and methods provided herein. In certain embodiments, a polynucleotide can comprise at least 19 contiguous nucleotides that are essentially identical or essentially complementary to said gene or said transcript or comprises at least 19 contiguous nucleotides that are essentially identical or essentially complementary to the target gene or target gene transcript. In certain embodiments, a polynucleotide of 19 continuous nucleotides that is essentially identical or essentially complementary to the endogenous target gene or to an RNA transcribed from the target gene (e.g. the transcript) can have 1 or 2 mismatches to the target gene or transcript. In certain embodiments, a polynucleotide of 20 or more nucleotides that contains a contiguous 19 nucleotide span of identity or complementarity to the endogenous target gene or to an RNA transcribed from the target gene can have 1 or 2 mismatches to the target gene or transcript. In certain embodiments, a polynucleotide of 21 continuous nucleotides that is essentially identical or essentially complementary to the endogenous target gene or to an RNA transcribed from the target gene (e.g. the transcript) can have 1, 2, or 3 mismatches to the target gene or transcript. In certain embodiments, a polynucleotide of 22 or more nucleotides that contains a contiguous 21 nucleotide span of identity or complementarity to the endogenous target gene or to an RNA transcribed from the target gene can have 1, 2, or 3 mismatches to the target gene or transcript. In designing polynucleotides with mismatches to an endogenous target gene or to an RNA transcribed from the target gene, mismatches of certain types and at certain positions that are more likely to be tolerated can be used. In certain exemplary embodiments, mismatches formed between adenine and cytosine or guanosine and uracil residues are used as described by Du et al. Nucleic Acids Research, 2005, Vol. 33, No. 5 1671-1677. In certain exemplary embodiments, mismatches in 19 base pair overlap regions can be at the low tolerance positions 5, 7, 8 or 11 (from the 5′ end of a 19 nucleotide target) with well tolerated nucleotide mismatch residues, at medium tolerance positions 3, 4, and 12-17, and/or at the high tolerance nucleotide positions at either end of the region of complementarity (i.e. positions 1, 2, 18, and 19) as described by Du et al. Nucleic Acids Research, 2005, Vol. 33, No. 5 1671-1677. It is further anticipated that tolerated mismatches can be empirically determined in assays where the polynucleotide is applied to the plants via the methods provided herein and the treated plants assayed for suppression of EIN2 gene expression or appearance of delayed senescence and/or improved yield.
In certain embodiments, polynucleotide molecules are designed to have 100 percent sequence identity with or complementarity to one allele or one family member of a given target EIN2 gene coding or non-coding sequence. Target EIN2 genes include both the EIN2 genes of Table 2 (i.e. SEQ ID NO:1-18, 622, and 623) as well as orthologous EIN2 genes obtainable from other crops. In other embodiments, the polynucleotide molecules are designed to have 100 percent sequence identity with or complementarity to multiple alleles or family members of a given target gene.
In certain embodiments, polynucleotide compositions and methods provided herein typically effect regulation or modulation (e. g., suppression) of gene expression during a period during the life of the treated plant of at least 1 week or longer and typically in systemic fashion. For instance, within days of treating a plant leaf with a polynucleotide composition of this invention, primary and transitive siRNAs can be detected in other leaves lateral to and above the treated leaf and in apical tissue. In certain embodiments, methods of systemically suppressing expression of a gene in a plant, the methods comprising treating said plant with a composition comprising at least one polynucleotide and a transfer agent, wherein said polynucleotide comprises at least 18 or at least 19 contiguous nucleotides that are essentially identical or essentially complementary to a gene or a transcript encoding an EIN2 gene of the plant are provided, whereby expression of the gene in said plant or progeny thereof is systemically suppressed in comparison to a control plant that has not been treated with the composition.
Compositions used to suppress a target gene can comprise one or more polynucleotides that are essentially identical or essentially complementary to multiple genes, or to multiple segments of one or more genes. In certain embodiments, compositions used to suppress a target gene can comprise one or more polynucleotides that are essentially identical or essentially complementary to multiple consecutive segments of a target gene, multiple non-consecutive segments of a target gene, multiple alleles of a target gene, or multiple target genes from one or more species.
In certain embodiments, the polynucleotide includes two or more copies of a nucleotide sequence (of 18 or more nucleotides) where the copies are arranged in tandem fashion. In another embodiment, the polynucleotide includes two or more copies of a nucleotide sequence (of 18 or more nucleotides) where the copies are arranged in inverted repeat fashion (forming an at least partially self-complementary strand). The polynucleotide can include both tandem and inverted-repeat copies. Whether arranged in tandem or inverted repeat fashion, each copy can be directly contiguous to the next, or pairs of copies can be separated by an optional spacer of one or more nucleotides. The optional spacer can be unrelated sequence (i. e., not essentially identical to or essentially complementary to the copies, nor essentially identical to, or essentially complementary to, a sequence of 18 or more contiguous nucleotides of the endogenous target gene or RNA transcribed from the endogenous target gene). Alternatively the optional spacer can include sequence that is complementary to a segment of the endogenous target gene adjacent to the segment that is targeted by the copies. In certain embodiments, the polynucleotide includes two copies of a nucleotide sequence of between about 20 to about 30 nucleotides, where the two copies are separated by a spacer no longer than the length of the nucleotide sequence.
Tiling
Polynucleotide trigger molecules can be identified by “tiling” gene targets in random length fragments, e.g. 200-300 polynucleotides in length, with partially overlapping regions, e.g. 25 or so nucleotide overlapping regions along the length of the target gene. Multiple gene target sequences can be aligned and polynucleotide sequence regions with homology in common are identified as potential trigger molecules for multiple targets. Multiple target sequences can be aligned and sequence regions with poor homology are identified as potential trigger molecules for selectively distinguishing targets. To selectively suppress a single gene, trigger sequences may be chosen from regions that are unique to the target gene either from the transcribed region or the non-coding regions, e.g., promoter regions, 3′ untranslated regions, introns and the like.
Polynucleotides fragments are designed along the length of the full length coding and untranslated regions of a EIN2 gene or family member as contiguous overlapping fragments of 200-300 polynucleotides in length or fragment lengths representing a percentage of the target EIN2 gene. These fragments are applied topically (as sense or anti-sense ssDNA or ssRNA, dsRNA, or dsDNA) to determine the relative effectiveness in providing the delayed senescence and/or improved yield phenotype. Fragments providing the desired activity may be further subdivided into 50-60 polynucleotide fragments which are evaluated for providing the delayed senescence and/or improved yield phenotype. The 50-60 base fragments with the desired activity may then be further subdivided into 19-30 base fragments which are evaluated for providing the delayed senescence and/or improved yield phenotype. Once relative effectiveness is determined, the fragments are utilized singly, or in combination in one or more pools to determine effective trigger composition or mixture of trigger polynucleotides for providing the delayed senescence and/or improved yield phenotype.
Coding and/or non-coding sequences of EIN2 gene families in the crop of interest are aligned and 200-300 polynucleotide fragments from the least homologous regions amongst the aligned sequences are evaluated using topically applied polynucleotides (as sense or anti-sense ssDNA or ssRNA, dsRNA, or dsDNA) to determine their relative effectiveness in providing the delayed senescence and/or improved yield phenotype. The effective segments are further subdivided into 50-60 polynucleotide fragments, prioritized by least homology, and reevaluated using topically applied polynucleotides. The effective 50-60 polynucleotide fragments are subdivided into 19-30 polynucleotide fragments, prioritized by least homology, and again evaluated for induction of the delayed senescence and/or improved yield phenotype. Once relative effectiveness is determined, the fragments are utilized singly, or again evaluated in combination with one or more other fragments to determine the trigger composition or mixture of trigger polynucleotides for providing the yield/quality phenotype.
Coding and/or non-coding sequences of EIN2 gene families in the crop of interest are aligned and 200-300 polynucleotide fragments from the most homologous regions amongst the aligned sequences are evaluated using topically applied polynucleotides (as sense or anti-sense ssDNA or ssRNA, dsRNA, or dsDNA) to determine their relative effectiveness in inducing the delayed senescence and/or improved yield phenotype. The effective segments are subdivided into 50-60 polynucleotide fragments, prioritized by most homology, and reevaluated using topically applied polynucleotides. The effective 50-60 polynucleotide fragments are subdivided into 19-30 polynucleotide fragments, prioritized by most homology, and again evaluated for induction of the delayed senescence and/or improved yield phenotype. Once relative effectiveness is determined, the fragments may be utilized singly, or in combination with one or more other fragments to determine the trigger composition or mixture of trigger polynucleotides for providing the delayed senescence and/or improved yield phenotype.
Also, provided herein are methods for identifying a preferred polynucleotide for providing delayed senescence and/or improved yield in a plant. Populations of candidate polynucleotides that are essentially identical or essentially complementary to a EIN2 gene or transcript of the EIN2 gene can be generated by a variety of approaches, including but not limited to, any of the tiling, least homology, or most homology approaches provided herein. Such populations of polynucleotides can also be generated or obtained from any of the polynucleotides or genes provided herewith in Table 2. Such populations of polynucleotides can also be generated or obtained from any genes that are orthologous to the genes provided herewith in Table 2. Such polynucleotides can be topically applied to a surface of plants in a composition comprising at least one polynucleotide from said population and a transfer agent to obtain treated plants. Treated plants that exhibit suppression of the EIN2 gene and/or exhibit an improvement in delayed senescence and/or improved yield are identified, thus identifying a preferred polynucleotide that improves delayed senescence and/or improved yield in a plant. Suppression of the EIN2 gene can be determined by any assay for the levels and/or activity of a EIN2 gene product (i.e. transcript or protein). Suitable assays for transcripts include, but are not limited to, semi-quantitative or quantitative reverse transcriptase PCR® (qRT-PCR) assays. Suitable assays for proteins include, but are not limited to, semi-quantitative or quantitaive immunoassays, biochemical activity assays, or biological activity assays. In certain embodiments, the polynucleotides can be applied alone. In other embodiments, the polynucleotides can be applied in pools of multiple polynucleotides. When a pool of polynucleotides provides for suppression of the EIN2 gene and/or an improvement in delayed senescence and/or improved yield are identified, the pool can be de-replicated and retested as necessary or desired to identify one or more preferred polynucleotide(s) that improve delayed senescence and/or improved yield in a plant.
Methods of making polynucleotides are well known in the art. Such methods of making polynucleotides can include in vivo biosynthesis, in vitro enzymatic synthesis, or chemical synthesis. In certain embodiments, RNA molecules can be made by either in vivo or in vitro synthesis from DNA templates where a suitable promoter is operably linked to the polynucleotide and a suitable DNA-dependent RNA polymerase is provided. DNA-dependent RNA polymerases include, but are not limited to, E. coli or other bacterial RNA polymerases as well as the bacteriophage RNA polymerases such as the T7, T3, and SP6 RNA polymerases. Commercial preparation of oligonucleotides often provides two deoxyribonucleotides on the 3′ end of the sense strand. Long polynucleotide molecules can be synthesized from commercially available kits, for example, kits from Applied Biosystems/Ambion (Austin, Tex.) have DNA ligated on the 5′ end that encodes a bacteriophage T7 polymerase promoter that makes RNA strands that can be assembled into a dsRNA. Alternatively, dsRNA molecules can be produced from expression cassettes in bacterial cells that have regulated or deficient RNase III enzyme activity. Long polynucleotide molecules can also be assembled from multiple RNA or DNA fragments. In some embodiments design parameters such as Reynolds score (Reynolds et al. Nature Biotechnology 22, 326-330 (2004) and Tuschl rules (Pei and Tuschl, Nature Methods 3(9): 670-676, 2006) are known in the art and are used in selecting polynucleotide sequences effective in gene silencing. In some embodiments random design or empirical selection of polynucleotide sequences is used in selecting polynucleotide sequences effective in gene silencing. In some embodiments the sequence of a polynucleotide is screened against the genomic DNA of the intended plant to minimize unintentional silencing of other genes.
While there is no upper limit on the concentrations and dosages of polynucleotide molecules that can be useful in the methods and compositions provided herein, lower effective concentrations and dosages will generally be sought for efficiency. The concentrations can be adjusted in consideration of the volume of spray or treatment applied to plant leaves or other plant part surfaces, such as flower petals, stems, tubers, fruit, anthers, pollen, leaves, roots, or seeds. In one embodiment, a useful treatment for herbaceous plants using 25-mer polynucleotide molecules is about 1 nanomole (nmol) of polynucleotide molecules per plant, for example, from about 0.05 to 1 nmol polynucleotides per plant. Other embodiments for herbaceous plants include useful ranges of about 0.05 to about 100 nmol, or about 0.1 to about 20 nmol, or about 1 nmol to about 10 nmol of polynucleotides per plant. In certain embodiments, about 40 to about 50 nmol of a ssDNA polynucleotide is applied. In certain embodiments, about 0.5 nmol to about 2 nmol of a dsRNA is applied. In certain embodiments, a composition containing about 0.5 to about 2.0 mg/mL, or about 0.14 mg/mL of dsRNA or ssDNA (21-mer) is applied. In certain embodiments, a composition of about 0.5 to about 1.5 mg/mL of a long dsRNA polynucleotide (i.e. about 50 to about 200 or more nucleotides) is applied. In certain embodiments, about 1 nmol to about 5 nmol of a dsRNA is applied to a plant. In certain embodiments, the polynucleotide composition as topically applied to the plant contains the at least one polynucleotide at a concentration of about 0.01 to about 10 milligrams per milliliter, or about 0.05 to about 2 milligrams per milliliter, or about 0.1 to about 2 milligrams per milliliter. Very large plants, trees, or vines may require correspondingly larger amounts of polynucleotides. When using long dsRNA molecules that can be processed into multiple oligonucleotides, lower concentrations can be used. To illustrate embodiments of the invention, the factor 1×, when applied to oligonucleotide molecules is arbitrarily used to denote a treatment of 0.8 nmol of polynucleotide molecule per plant; 10×, 8 nmol of polynucleotide molecule per plant; and 100×, 80 nmol of polynucleotide molecule per plant.
The polynucleotide compositions of this invention are useful in compositions, such as liquids that comprise polynucleotide molecules, alone or in combination with other components either in the same liquid or in separately applied liquids that provide a transfer agent. As used herein, a transfer agent is an agent that, when combined with a polynucleotide in a composition that is topically applied to a target plant surface, enables the polynucleotide to enter a plant cell. In certain embodiments, a transfer agent is an agent that conditions the surface of plant tissue, e. g., seeds, leaves, stems, roots, flowers, or fruits, to permeation by the polynucleotide molecules into plant cells. The transfer of polynucleotides into plant cells can be facilitated by the prior or contemporaneous application of a polynucleotide-transferring agent to the plant tissue. In some embodiments the transferring agent is applied subsequent to the application of the polynucleotide composition. The polynucleotide transfer agent enables a pathway for polynucleotides through cuticle wax barriers, stomata and/or cell wall or membrane barriers into plant cells. Suitable transfer agents to facilitate transfer of the polynucleotide into a plant cell include agents that increase permeability of the exterior of the plant or that increase permeability of plant cells to oligonucleotides or polynucleotides. Such agents to facilitate transfer of the composition into a plant cell include a chemical agent, or a physical agent, or combinations thereof. Chemical agents for conditioning or transfer include (a) surfactants, (b) an organic solvent or an aqueous solution or aqueous mixtures of organic solvents, (c) oxidizing agents, (d) acids, (e) bases, (f) oils, (g) enzymes, or combinations thereof. Embodiments of the method can optionally include an incubation step, a neutralization step (e.g., to neutralize an acid, base, or oxidizing agent, or to inactivate an enzyme), a rinsing step, or combinations thereof. Embodiments of agents or treatments for conditioning of a plant to permeation by polynucleotides include emulsions, reverse emulsions, liposomes, and other micellar-like compositions. Embodiments of agents or treatments for conditioning of a plant to permeation by polynucleotides include counter-ions or other molecules that are known to associate with nucleic acid molecules, e. g., inorganic ammonium ions, alkyl ammonium ions, lithium ions, polyamines such as spermine, spermidine, or putrescine, and other cations. Organic solvents useful in conditioning a plant to permeation by polynucleotides include DMSO, DMF, pyridine, N-pyrrolidine, hexamethylphosphoramide, acetonitrile, dioxane, polypropylene glycol, other solvents miscible with water or that will dissolve phosphonucleotides in non-aqueous systems (such as is used in synthetic reactions). Naturally derived or synthetic oils with or without surfactants or emulsifiers can be used, e. g., plant-sourced oils, crop oils (such as those listed in the 9th Compendium of Herbicide Adjuvants, publicly available on the worldwide web (internet) at herbicide.adjuvants.com can be used, e. g., paraffinic oils, polyol fatty acid esters, or oils with short-chain molecules modified with amides or polyamines such as polyethyleneimine or N-pyrrolidine. Transfer agents include, but are not limited to, organosilicone preparations.
In certain embodiments, an organosilicone preparation that is commercially available as Silwet® L-77 surfactant having CAS Number 27306-78-1 and EPA Number: CAL.REG.NO. 5905-50073-AA, and currently available from Momentive Performance Materials, Albany, N.Y. can be used to prepare a polynucleotide composition. In certain embodiments where a Silwet L-77 organosilicone preparation is used as a pre-spray treatment of plant leaves or other plant surfaces, freshly made concentrations in the range of about 0.015 to about 2 percent by weight (wt percent) (e. g., about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.5 wt percent) are efficacious in preparing a leaf or other plant surface for transfer of polynucleotide molecules into plant cells from a topical application on the surface. In certain embodiments of the methods and compositions provided herein, a composition that comprises a polynucleotide molecule and an organosilicone preparation comprising Silwet L-77 in the range of about 0.015 to about 2 percent by weight (wt percent) (e. g., about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.5 wt percent) is used or provided. In certain embodiments of the methods and compositions provided herein, a composition that comprises a polynucleotide molecule and an organosilicone preparation comprising Silwet L-77 in the range of about 0.3 to about 1 percent by weight (wt percent) or about 0.5 to about 1% by weight (wt percent) is used or provided. In certain embodiments, any of the commercially available organosilicone preparations provided in the following Table 1 can be used as transfer agents in a polynucleotide composition. In certain embodiments where an organosilicone preparation of Table 1 is used as a pre-spray treatment of plant leaves or other surfaces, freshly made concentrations in the range of about 0.015 to about 2 percent by weight (wt percent) (e. g., about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.5 wt percent) are efficacious in preparing a leaf or other plant surface for transfer of polynucleotide molecules into plant cells from a topical application on the surface. In certain embodiments of the methods and compositions provided herein, a composition that comprises a polynucleotide molecule and an organosilicone preparation of Table 1 in the range of about 0.015 to about 2 percent by weight (wt percent) (e. g., about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.5 wt percent) is used or provided.









TABLE 1





Name
CAS number
Manufacturer1,2







BREAK-THRU ® S 321
na
Evonik Industries AG


BREAK-THRU ® S 200
67674-67-3
Evonik Industries AG


BREAK-THRU ® OE 441
68937-55-3
Evonik Industries AG


BREAK-THRU ® S 278
27306-78-1
Evonik Industries AG


BREAK-THRU ® S 243
na
Evonik Industries AG


Silwet ® L-77
27306-78-1
Momentive Performance




Materials


Silwet ® HS 429
na
Momentive Performance




Materials


Silwet ® HS 312
na
Momentive Performance




Materials


BREAK-THRU ® S 233
134180-76-0
Evonik Industries AG


Silwet ® HS 508

Momentive Performance




Materials


Silwet ® HS 604

Momentive Performance




Materials





1Evonik Industries AG, Essen, Germany


2Momentive Performance Materials, Albany, New York






Organosilicone preparations used in the methods and compositions provided herein can comprise one or more effective organosilicone compounds. As used herein, the phrase “effective organosilicone compound” is used to describe any organosilicone compound that is found in an organosilicone preparation that enables a polynucleotide to enter a plant cell. In certain embodiments, an effective organosilicone compound can enable a polynucleotide to enter a plant cell in a manner permitting a polynucleotide mediated suppression of target gene expression in the plant cell. In general, effective organosilicone compounds include, but are not limited to, compounds that can comprise: i) a trisiloxane head group that is covalently linked to, ii) an alkyl linker including, but not limited to, an n-propyl linker, that is covalently linked to, iii) a poly glycol chain, that is covalently linked to, iv) a terminal group. Trisiloxane head groups of such effective organosilicone compounds include, but are not limited to, heptamethyltrisiloxane. Alkyl linkers can include, but are not limited to, an n-propyl linker. Poly glycol chains include, but are not limited to, polyethylene glycol or polypropylene glycol. Poly glycol chains can comprise a mixture that provides an average chain length “n” of about “7.5”. In certain embodiments, the average chain length “n” can vary from about 5 to about 14. Terminal groups can include, but are not limited to, alkyl groups such as a methyl group. Effective organosilicone compounds are believed to include, but are not limited to, trisiloxane ethoxylate surfactants or polyalkylene oxide modified heptamethyl trisiloxane.




One organosilicone compound believed to be ineffective comprises the formula:




In certain embodiments, an organosilicone preparation that comprises an organosilicone compound comprising a trisiloxane head group is used in the methods and compositions provided herein. In certain embodiments, an organosilicone preparation that comprises an organosilicone compound comprising a heptamethyltrisiloxane head group is used in the methods and compositions provided herein. In certain embodiments, an organosilicone composition that comprises Compound I is used in the methods and compositions provided herein. In certain embodiments, an organosilicone composition that comprises Compound I is used in the methods and compositions provided herein. In certain embodiments of the methods and compositions provided herein, a composition that comprises a polynucleotide molecule and one or more effective organosilicone compound in the range of about 0.015 to about 2 percent by weight (wt percent) (e. g., about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.5 wt percent) is used or provided.
In certain embodiments, the polynucleotide compositions that comprise an organosilicone preparation can comprise a salt such as ammonium chloride, tetrabutylphosphonium bromide, and/or ammonium sulfate. Ammonium chloride, tetrabutylphosphonium bromide, and/or ammonium sulfate can be provided in the polynucleotide composition at a concentration of about 0.5% to about 5% (w/v). An ammonium chloride, tetrabutylphosphonium bromide, and/or ammonium sulfate concentration of about 1% to about 3%, or about 2% (w/v) can also be used in the polynucleotide compositions that comprise an organosilicone preparation. In certain embodiments, the polynucleotide compositions can comprise an ammonium salt at a concentration greater or equal to 300 millimolar. In certain embodiments, the polynucleotide compositions that comprise an organosilicone preparation can comprise ammonium sulfate at concentrations from about 80 to about 1200 mM or about 150 mM to about 600 mM.
In certain embodiments, the polynucleotide compositions can also comprise a phosphate salt. Phosphate salts used in the compositions include, but are not limited to, calcium, magnesium, potassium, or sodium phosphate salts. In certain embodiments, the polynucleotide compositions can comprise a phosphate salt at a concentration of at least about 5 millimolar, at least about 10 millimolar, or at least about 20 millimolar. In certain embodiments, the polynucleotide compositions will comprise a phosphate salt in a range of about 1 mM to about 25 mM or in a range of about 5 mM to about 25 mM. In certain embodiments, the polynucleotide compositions can comprise sodium phosphate at a concentration of at least about 5 millimolar, at least about 10 millimolar, or at least about 20 millimolar. In certain embodiments, the polynucleotide compositions can comprise sodium phosphate at a concentration of about 5 millimolar, about 10 millimolar, or about 20 millimolar. In certain embodiments, the polynucleotide compositions will comprise a sodium phosphate salt in a range of about 1 mM to about 25 mM or in a range of about 5 mM to about 25 mM. In certain embodiments, the polynucleotide compositions will comprise a sodium phosphate salt in a range of about 10 mM to about 160 mM or in a range of about 20 mM to about 40 mM. In certain embodiments, the polynucleotide compositions can comprise a sodium phosphate buffer at a pH of about 6.8.
In certain embodiments, other useful transfer agents or adjuvants to transfer agents that can be used in polynucleotide compositions provided herein include surfactants and/or effective molecules contained therein. Surfactants and/or effective molecules contained therein include, but are not limited to, sodium or lithium salts of fatty acids (such as tallow or tallowamines or phospholipids) and organosilicone surfactants. In certain embodiments, the polynucleotide compositions that comprise a transfer agent are formulated with counter-ions or other molecules that are known to associate with nucleic acid molecules. Illustrative examples include, tetraalkyl ammonium ions, trialkyl ammonium ions, sulfonium ions, lithium ions, and polyamines such as spermine, spermidine, or putrescine. In certain embodiments, the polynucleotide compositions are formulated with a non-polynucleotide herbicide. Non-polynucleotide herbicidal molecules include, but are not limited to, glyphosate, auxin-like benzoic acid herbicides including dicamba, chloramben, and TBA, glufosinate, auxin-like herbicides including phenoxy carboxylic acid herbicide, pyridine carboxylic acid herbicide, quinoline carboxylic acid herbicide, pyrimidine carboxylic acid herbicide, and benazolin-ethyl herbicide, sulfonylureas, imidazolinones, bromoxynil, delapon, cyclohezanedione, protoporphyrinogen oxidase inhibitors, and 4-hydroxyphenyl-pyruvate-dioxygenase inhibiting herbicides.
In certain embodiments, the polynucleotides used in the compositions that are essentially identical or essentially complementary to the target gene or transcript will comprise the predominant nucleic acid in the composition. Thus in certain embodiments, the polynucleotides that are essentially identical or essentially complementary to the target gene or transcript will comprise at least about 50%, 75%, 95%, 98%, or 100% of the nucleic acids provided in the composition by either mass or molar concentration. However, in certain embodiments, the polynucleotides that are essentially identical or essentially complementary to the target gene or transcript can comprise at least about 1% to about 50%, about 10% to about 50%, about 20% to about 50%, or about 30% to about 50% of the nucleic acids provided in the composition by either mass or molar concentration. Also provided are compositions where the polynucleotides that are essentially identical or essentially complementary to the target gene or transcript can comprise at least about 1% to 100%, about 10% to 100%, about 20% to about 100%, about 30% to about 50%, or about 50% to a 100% of the nucleic acids provided in the composition by either mass or molar concentration.
Polynucleotides comprising ssDNA, dsDNA, ssRNA, dsRNA, or RNA/DNA hybrids that are essentially identical or complementary to certain plant target genes or transcripts and that can be used in compositions containing transfer agents that include, but are not limited to, organosilicone preparations, to suppress those target genes when topically applied to plants are disclosed in co-assigned U.S. patent application Ser. No. 13/042,856. Various polynucleotide herbicidal molecules, compositions comprising those polynucleotide herbicidal molecules and transfer agents that include, but are not limited to, organosilicone preparations, and methods whereby herbicidal effects are obtained by the topical application of such compositions to plants are also disclosed in co-assigned U.S. patent application Ser. No. 13/042,856, and those polynucleotide herbicidal molecules, compositions, and methods are incorporated herein by reference in their entireties. Genes encoding proteins that can provide tolerance to an herbicide and/or that are targets of a herbicide are collectively referred to herein as “herbicide target genes”. Herbicide target genes include, but are not limited to, a 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), a glyphosate oxidoreductase (GOX), a glyphosate decarboxylase, a glyphosate-N-acetyl transferase (GAT), a dicamba monooxygenase, a phosphinothricin acetyltransferase, a 2,2-dichloropropionic acid dehalogenase, an acetohydroxyacid synthase, an acetolactate synthase, a haloarylnitrilase, an acetyl-coenzyme A carboxylase (ACCase), a dihydropteroate synthase, a phytoene desaturase (PDS), a protoporphyrin IX oxygenase (PPO), a hydroxyphenylpyruvate dioxygenase (HPPD), a para-aminobenzoate synthase, a glutamine synthase, a cellulose synthase, a beta tubulin, and a serine hydroxymethyltransferase gene. The effects of applying certain compositions comprising polynucleotides that are essentially identical or complementary to certain herbicide target genes and transfer agents on plants containing the herbicide target genes was shown to be potentiated or enhanced by subsequent application of an herbicide that targets the same gene as the polynucleotide in co-assigned U.S. patent application Ser. No. 13/042,856. For example, compositions comprising polynucleotides targeting the EPSPS herbicide target gene were potentiated by glyphosate in experiments disclosed in co-assigned U.S. patent application Ser. No. 13/042,856.
In certain embodiments of the compositions and methods disclosed herein, the composition comprising a polynucleotide and a transfer agent can thus further comprise a second polynucleotide comprising at least 19 contiguous nucleotides that are essentially identical or essentially complementary to a transcript to a protein that confers resistance to a herbicide. In certain embodiments, the second polynucleotide does not comprise a polynucleotide that is essentially identical or essentially complementary to a transcript encoding a protein of a target plant that confers resistance to said herbicidal molecule. Thus, in an exemplary and non-limiting embodiment, the second polynucleotide could be essentially identical or essentially complementary to a transcript encoding a protein that confers resistance to a herbicide in a weed (such as an EPSPS encoding transcript) but would not be essentially identical or essentially complementary to a transcript encoding a protein that confers resistance to that same herbicide in a crop plant.
In certain embodiments, the polynucleotide compositions that comprise a transfer agent can comprise glycerin. Glycerin can be provided in the composition at a concentration of about 0.1% to about 1% (w/v or v/v). A glycerin concentration of about 0.4% to about 0.6%, or about 0.5% (w/v or v/v) can also be used in the polynucleotide compositions that comprise a transfer agent.
In certain embodiments, the polynucleotide compositions that comprise a transfer agent can further comprise organic solvents. Such organic solvents include, but are not limited to, DMSO, DMF, pyridine, N-pyrrolidine, hexamethylphosphoramide, acetonitrile, dioxane, polypropylene glycol, other solvents miscible with water or that will dissolve phosphonucleotides in non-aqueous systems (such as is used in synthetic reactions).
In certain embodiments, the polynucleotide compositions that comprise a transfer agent can further comprise naturally derived or synthetic oils with or without surfactants or emulsifiers. Such oils include, but are not limited to, plant-sourced oils, crop oils (such as those listed in the 9th Compendium of Herbicide Adjuvants, publicly available on the world wide web at herbicide.adjuvants.com, paraffinic oils, polyol fatty acid esters, or oils with short-chain molecules modified with amides or polyamines such as polyethyleneimine or N-pyrrolidine.
In aspects of the invention, methods include one or more applications of the composition comprising a polynucleotide and a transfer agent or one or more effective components contained therein. In certain embodiments of the methods, one or more applications of a transfer agent or one or more effective components contained therein can precede one or more applications of the composition comprising a polynucleotide and a transfer agent. In embodiments where a transfer agent and/or one or more effective molecules contained therein is used either by itself as a pre-treatment or as part of a composition that includes a polynucleotide, embodiments of the polynucleotide molecules are double-stranded RNA oligonucleotides, single-stranded RNA oligonucleotides, double-stranded RNA polynucleotides, single-stranded RNA polynucleotides, double-stranded DNA oligonucleotides, single-stranded DNA oligonucleotides, double-stranded DNA polynucleotides, single-stranded DNA polynucleotides, chemically modified RNA or DNA oligonucleotides or polynucleotides or mixtures thereof.
Compositions and methods of the invention are useful for modulating or suppressing the expression of an endogenous target gene or transgenic target gene in a plant cell or plant. In certain embodiments of the methods and compositions provided herein, expression of EIN2 target genes can be suppressed completely, partially and/or transiently to result in delayed senescence and/or improved yield. In various embodiments, a target gene includes coding (protein-coding or translatable) sequence, non-coding (non-translatable) sequence, or both coding and non-coding sequence. Compositions of the invention can include polynucleotides and oligonucleotides designed to target multiple genes, or multiple segments of one or more genes. The target gene can include multiple consecutive segments of a target gene, multiple non-consecutive segments of a target gene, multiple alleles of a target gene, or multiple target genes from one or more species. Examples of target genes of the present invention include endogenous EIN2 genes and EIN2 transgenes.
Target EIN2 genes and plants containing those target EIN2 genes can be obtained from: i) row crop plants including, but are not limited to, corn, soybean, cotton, canola, sugar beet, alfalfa, sugarcane, rice, and wheat; ii) vegetable plants including, but not limited to, tomato, potato, sweet pepper, hot pepper, melon, watermelon, cucumber, eggplant, cauliflower, broccoli, lettuce, spinach, onion, peas, carrots, sweet corn, Chinese cabbage, leek, fennel, pumpkin, squash or gourd, radish, Brussels sprouts, tomatillo, garden beans, dry beans, or okra; iii) culinary plants including, but not limited to, basil, parsley, coffee, or tea; iv) fruit plants including but not limited to apple, pear, cherry, peach, plum, apricot, banana, plantain, table grape, wine grape, citrus, avocado, mango, or berry; v) a tree grown for ornamental or commercial use, including, but not limited to, a fruit or nut tree; or, vi) an ornamental plant (e. g., an ornamental flowering plant or shrub or turf grass). The methods and compositions provided herein can also be applied to plants produced by a cutting, cloning, or grafting process (i. e., a plant not grown from a seed) include fruit trees and plants that include, but are not limited to, citrus, apples, avocados, tomatoes, eggplant, cucumber, melons, watermelons, and grapes as well as various ornamental plants. Such row crop, vegetable, culinary, fruit, tree, or ornamental plants exhibiting improvements in that result from suppressing expression of EIN2 are provided herein. Such row crop, vegetable, culinary, fruit, tree, or ornamental plant parts or processed plant products exhibiting improvements in Delayed senescence and/or improved yield that result from suppressing expression of EIN2 are also provided herein. Such plant parts can include, but are not limited to, flowers, stems, tubers, fruit, anthers, meristems, ovules, pollen, leaves, or seeds. Such processed plant products obtained from the plant parts can include, but are not limited to, a meal, a pulp, a feed, or a food product.
Without seeking to be limited by theory, it is believed that in certain embodiments that delays in leaf senescence provided by suppression of EIN2 can provide for improved yield in plants. It is believed that in certain embodiments, delays in senescence provided by suppression of EIN2 can enhance source capacity by delaying leaf senescence and extending the period during grain fill that the source leaves are actively photosynthesizing and exporting sugar. Even a small delay in senescence can significantly improve yield. It was calculated that a 2-day delay in senescence of Lolium temulentum would increase the amount of carbon fixed by 11% over the life of the plant (Thomas and Howarth 2000). In fact, in corn increased leaf longevity was associated with improvements of hybrid corn through breeding (Rajcan and Tollenaar 1999). Genetic analysis using a cross of a stay-green inbred corn with a normal inbred showed that there were 14 quantitative trait loci (QTL) for stay-green traits and that these traits were correlated with grain yield (Zheng, Wu et al. 2009). Correlation of stay-green phenotype with yield has also been demonstrated for rice (Fu, Yan et al. 2011).
An aspect of the invention provides a method for modulating expression of an EIN2 gene in a plant including (a) conditioning of a plant to permeation by polynucleotides and (b) treatment of the plant with the polynucleotide molecules, wherein the polynucleotide molecules include at least one segment of 18 or more contiguous nucleotides cloned from or otherwise identified from the target EIN2 gene in either anti-sense or sense orientation, whereby the polynucleotide molecules permeate the interior of the plant and induce modulation of the target EIN2 gene. The conditioning and polynucleotide application can be performed separately or in a single step. When the conditioning and polynucleotide application are performed in separate steps, the conditioning can precede or can follow the polynucleotide application within minutes, hours, or days. In some embodiments more than one conditioning step or more than one polynucleotide molecule application can be performed on the same plant. In embodiments of the method, the segment can be cloned or identified from (a) coding (protein-encoding), (b) non-coding (promoter and other gene related molecules), or (c) both coding and non-coding parts of the target EIN2 gene. Non-coding parts include DNA, such as promoter regions or the RNA transcribed by the DNA that provide RNA regulatory molecules, including but not limited to: introns, 5′ or 3′ untranslated regions, and microRNAs (miRNA), trans-acting siRNAs, natural anti-sense siRNAs, and other small RNAs with regulatory function or RNAs having structural or enzymatic function including but not limited to: ribozymes, ribosomal RNAs, t-RNAs, aptamers, and riboswitches. In certain embodiments where the polynucleotide used in the composition comprises a promoter sequence essentially identical to, or essentially complementary to at least 18 contiguous nucleotides of the promoter of the endogenous target EIN2 gene, the promoter sequence of the polynucleotide is not operably linked to another sequence that is transcribed from the promoter sequence.
Compositions comprising a polynucleotide and a transfer agent provided herein can be topically applied to a plant or plant part by any convenient method, e.g., spraying or coating with a powder, or with a liquid composition comprising any of an emulsion, suspension, or solution. Such topically applied sprays or coatings can be of either all or of any a portion of the surface of the plant or plant part. Similarly, the compositions comprising a transfer agent or other pre-treatment can in certain embodiments be applied to the plant or plant part by any convenient method, e. g., spraying or wiping a solution, emulsion, or suspension. Compositions comprising a polynucleotide and a transfer agent provided herein can be topically applied to plant parts that include, but are not limited to, flowers, stems, tubers, meristems, ovules, fruit, anthers, pollen, leaves, or seeds.
Application of compositions comprising a polynucleotide and a transfer agent to seeds is specifically provided herein. Seeds can be contacted with such compositions by spraying, misting, immersion, and the like.
In certain embodiments, application of compositions comprising a polynucleotide and a transfer agent to plants, plant parts, or seeds in particular can provide for the delayed senescence and/or improved yield in progeny plants, plant parts, or seeds derived from those treated plants, plant parts, or seeds. In certain embodiments, progeny plants, plant parts, or seeds derived from those treated plants, plant parts, or seeds will exhibit an improvement in delayed senescence and/or improved yield that result from suppressing expression of EIN2. In certain embodiments, the methods and compositions provided herein can provide for an improvement in delayed senescence and/or improved yield in progeny plants or seeds as a result of epigenetically inherited suppression of EIN2 gene expression. In certain embodiments, such progeny plants exhibit an improvement in delayed senescence and/or improved yield from epigenetically inherited suppression of EIN2 gene expression that is not caused by a transgene where the polynucleotide is operably linked to a promoter, a viral vector, or a copy of the polynucleotide that is integrated into a non-native location in the chromosomal DNA of the plant. Without seeking to be limited by theory, progeny plants or seeds derived from those treated plants, plant parts, or seeds can exhibit an improvement in delayed senescence and/or improved yield through an epigenetic mechanism that provides for propagation of an epigenetic condition where suppression of EIN2 gene expression occurs in the progeny plants, plant parts, or plant seeds. In certain embodiments, progeny plants or seeds exhibiting an improvement in delayed senescence and/or improved yield as a result of epigenetically inherited suppression of EIN2 gene expression can also exhibit increased methylation, and in particular, increased methylation of cytosine residues, in the endogenous EIN2 gene of the plant. Plant parts, including seeds, of the progeny plants that exhibit an improvement in the delayed senescence and/or improved yield as a result of epigenetically inherited suppression of EIN2 gene expression, can also in certain embodiments exhibit increased methylation, and in particular, increased methylation of cytosine residues, in the endogenous EIN2 gene. In certain embodiments, DNA methylation levels in DNA encoding the endogenous EIN2 gene can be compared in plants that exhibit the delayed senescence and/or improved yield and control plants that do not exhibit the delayed senescence and/or improved yield to correlate the presence of the delayed senescence and/or improved yield to epigenetically inherited suppression of EIN2 gene expression and to identify plants that comprise the epigenetically inherited delayed senescence and/or improved yield.
Various methods of spraying compositions on plants or plant parts can be used to topically apply to a plant surface a composition comprising a polynucleotide that comprises a transfer agent. In the field, a composition can be applied with a boom that extends over the crops and delivers the composition to the surface of the plants or with a boomless sprayer that distributes a composition across a wide area. Agricultural sprayers adapted for directional, broadcast, or banded spraying can also be used in certain embodiments. Sprayers adapted for spraying particular parts of plants including, but not limited to, leaves, the undersides of leaves, flowers, stems, male reproductive organs such as tassels, meristems, pollen, ovules, and the like can also be used. Compositions can also be delivered aerially, such as by a crop dusting airplane. In certain embodiments, the spray can be delivered with a pressurized backpack sprayer calibrated to deliver the appropriate rate of the composition. In certain embodiments, such a backpack sprayer is a carbon dioxide pressurized sprayer with a 11015 flat fan or equivalent spray nozzle with a customized single nozzle assembly (to minimize waste) at a spray pressure of about 0.25 MPa and/or any single nozzle sprayer providing an effective spray swath of 60 cm above the canopy of 3 to 12 inch tall growing plants can be used. Plants in a greenhouse or growth chamber can be treated using a track sprayer or laboratory sprayer with a 11001XR or equivalent spray nozzle to deliver the sample solution at a determined rate. An exemplary and non-limiting rate is about 140 L/ha at about 0.25 MPa pressure.
In certain embodiments, it is also contemplated that a plant part can be sprayed with the composition comprising a polynucleotide that comprises a transfer agent. Such plant parts can be sprayed either pre- or post-harvest to provide delayed senescence and/or improved yield in the plant part that results from suppression of EIN2 gene expression. Compositions can be topically applied to plant parts attached to a plant by a spray as previously described. Compositions can be topically applied to plant parts that are detached from a plant by a spray as previously described or by an alternative method. Alternative methods for applying compositions to detached parts include, but are not limited to, passing the plant parts through a spray by a conveyor belt or trough, or immersing the plant parts in the composition.
Compositions comprising polynucleotides and transfer agents can be applied to plants or plant parts at one or more developmental stages as desired and/or as needed. Application of compositions to pre-germination seeds and/or to post-germination seedlings is provided in certain embodiments. Seeds can be treated with polynucleotide compositions provided herein by methods including, but not limited to, spraying, immersion, or any process that provides for coating, imbibition, and/or uptake of the polynucleotide composition by the seed. Seeds can be treated with polynucleotide compositions using seed batch treatment systems or continuous flow treatment systems. Seed coating systems are at least described in U.S. Pat. Nos. 6,582,516, 5,891,246, 4,079,696, and 4,023,525. Seed treatment can also be effected in laboratory or commercial scale treatment equipment such as a tumbler, a mixer, or a pan granulator. A polynucleotide composition used to treat seeds can contain one or more other desirable components including, but not limited to liquid diluents, binders to serve as a matrix for the polynucleotide, fillers for protecting the seeds during stress conditions, and plasticizers to improve flexibility, adhesion and/or spreadability of the coating. In addition, for oily polynucleotide compositions containing little or no filler, drying agents such as calcium carbonate, kaolin or bentonite clay, perlite, diatomaceous earth or any other adsorbent material can be added. Use of such components in seed treatments is described in U.S. Pat. No. 5,876,739. Additional ingredients can be incorporated into the polynucleotide compositions used in seed treatments. Such ingredients include but are not limited to: conventional sticking agents, dispersing agents such as methylcellulose (Methocel A15LV or Methocel A15C, for example, serve as combined dispersant/sticking agents for use in seed treatments), polyvinyl alcohol (e.g., Elvanol 51-05), lecithin (e.g., Yelkinol P), polymeric dispersants (e.g., polyvinylpyrrolidone/vinyl acetate PVPNA S-630), thickeners (e.g., clay thickeners such as Van Gel B to improve viscosity and reduce settling of particle suspensions), emulsion stabilizers, surfactants, antifreeze compounds (e.g., urea), dyes, colorants, and the like that can be combined with compositions comprising a polynucleotide and a transfer agent. Further ingredients used in compositions that can be applied to seeds can be found in McCutcheon's, vol. 1, “Emulsifiers and Detergents,” MC Publishing Company, Glen Rock, N.J., U.S.A., 1996 and in McCutcheon's, vol. 2, “Functional Materials,” MC Publishing Company, Glen Rock, N.J., U.S.A., 1996. Methods of applying compositions to seeds and pesticidal compositions that can be used to treat seeds are described in US Patent Application publication 20080092256, which is incorporated herein by reference in its entirety.
Application of the compositions in early, mid-, and late vegetative stages of plant development is provided in certain embodiments. Application of the compositions in early, mid-, and late reproductive stages is also provided in certain embodiments. Application of the compositions to plant parts at different stages of maturation is also provided.
LITERATURE CITED


    Aeong Oh, S., J.-H. Park, et al. (1997). “Identification of three gerietic loci controlling leaf senescence in Arabidopsis thaliana.” The Plant Journal 12(3): 527-535.
    Bapat, V. A., P. K. Trivedi, et al. (2010). “Ripening of fleshy fruit: Molecular insight and the role of ethylene.” Biotechnology Advances 28(1): 94-107.
    Djanaguiraman, M. and P. V. V. Prasad (2010). “Ethylene production under high temperature stress causes premature leaf senescence in soybean.” Functional Plant Biology 37(11): 1071-1084.
    Fu, J.-D., Y.-F. Yan, et al. (2011). “Population-specific quantitative trait loci mapping for functional stay-green trait in rice (Oryza sativa L.).” Genome 54(3): 235-243.
    Ghosh, S., V. S. Meli, et al. (2011). “The N-glycan processing enzymes α-mannosidase and β-D-N-acetylhexosaminidase are involved in ripening-associated softening in the non-climacteric fruits of capsicum.” Journal of Experimental Botany 62(2): 571-582.
    Hu, Z., L. Deng, et al. (2011). Biologia Plantarum 55(1): 27-34. Lim, P. O., H. J. Kim, et al. (2007). “Leaf Senescence.” Annual Review of Plant Biology 58(1): 115-136.
    McMurchie, E. J., W. B. McGlasson, et al. (1972). “Treatment of Fruit with Propylene gives Information about the Biogenesis of Ethylene.” Nature 237(5352): 235-236.
    Meli, V. S., S. Ghosh, et al. (2010). “Enhancement of fruit shelf life by suppressing N-glycan processing enzymes.” Proceedings of the National Academy of Sciences.
    Rajcan, I. and M. Tollenaar (1999). “Source:sink ratio and leaf senescence in maize:—I. Dry matter accumulation and partitioning during grain filling.” Field Crops Research 60(3): 245-253.
    Thomas, H. and C. J. Howarth (2000). “Five ways to stay green.” Journal of Experimental Botany 51: 329-337.
    Wellburn, A. R. (1994). “The spectral determination of chlorophylls a and b, as well as total carotenoids, using various solvents with spectrophotometers of different resolution.” Journal of Plant Physiology 144: 307-313.
    Zheng, H. J., A. Z. Wu, et al. (2009). “QTL mapping of maize (Zea mays) stay-green traits and their relationship to yield.” Plant Breeding 128(1): 54-62.


The following examples are included to demonstrate examples of certain preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches the inventors have found function well in the practice of the invention, and thus can be considered to constitute examples of preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
EXAMPLES
Example 1. EIN2 Target Genes Target Gene Sequences
The EIN2 genes provided in Table 4 and the sequence listing, or their corresponding transcripts, can be used as targets of polynucleotide compositions comprising a polynucleotide that of at least 18 contiguous nucleotides that are essentially identical or essentially complementary to those genes or transcripts. The genes provided in Table 2 and the sequence listing, protein sequences encoded by those genes, or sequences contained within those genes can also be used to obtain orthologous EIN2 genes from plants not listed in Table 2 and the sequence listing. Such orthologous genes and their transcripts can then serve as targets of polynucleotides provided herein or as a source of polynucleotides that are specifically designed to target the orthologous genes or transcripts.







TABLE 2







Target EIN2 genes












SEQ







ID







NO
Source
Name|Reference
Gene
Type
Length















1
Canola
BRANA-03JUN08-CLUS4975_1
EIN2
cDNA
1473


2
Corn
Zm_B73_CR03.G24460.1.cdna
EIN2
cDNA
4315


3
Corn
Zm_B73_CR03.G24460.1.2kbPromoter
EIN2
Promoter
2003


4
Cucumber
CumMe_WSH_CR08.G13373580.1.cdna
EIN2
cDNA
4372


5
Cucumber
CumSa_CL_CR06.G4545310.1.cdna
EIN2
cDNA
3768


6
lettuce
TC23690
EIN2
cDNA
1189


7
Rice
LOC_Os03g49400.1_CDNA
EIN2
cDNA
5154


8
Rice
LOC_Os07g06130.3_cDNA
EIN2
cDNA
4791


9
Rice
LOC_Os03g49400.1_2KB_Promoter
EIN2
Promoter
2000


10
Rice
LOC_Os07g06130.3_2Kb_Promoter
EIN2
Promoter
2000


11
Soy
Gm_W82_CR03.G375100.1.cdna
EIN2
cDNA
4629


12
Soy
Gm_W82_CR10.G25060.1.cdna
EIN2
cDNA
4525


13
Soy
Gm_W82_CR13.G158730.1.cdna
EIN2
cDNA
4005


14
Soy
Gm_W82_CR13.G158730.1.2kbPromoter
EIN2
Promoter
2003


15
Soy
Gm_W82_CR10.G25060.1.2kbPromoter
EIN2
Promoter
2003


16
Soy
Gm_W82_CR03.G375100.1.2kbPromoter
EIN2
Promoter
2003


17
Tomato
Sl_H1706_CR09.G245610.1.cdna
EIN2
cDNA
4294


18
Tomato
Sl_H1706_CR09.G245610.1.2KbPromoter
EIN2
Promoter
2000


622
Cucumber
Cucumis sativus EIN2 coding sequence
EIN2
coding
3872


623
Cucumber
Cucumis sativus EIN2 5′UTR
EIN2
5′UTR
2001









The sequence listing contains the target DNA sequences from plant species for EIN2 genes listed in Table 2. For each gene having a DNA sequence provided in the sequence listing and listed in listed in Table 2 single stranded or double stranded DNA or RNA fragments in sense or antisense orientation or both are mixed with an organosilicone preparation that comprises the compositions of the topical application method. This composition is topically applied to plants to effect expression of the target genes in the specified plant to obtain the desired delays in senescence and/or improvements in yield.
Example 2. Polynucleotides that can be Used to Suppress EIN2 Expression in Various Plants
An exemplary set of polynucleotides that can be used to suppress expression of EIN2 genes in various plants is provided herewith in the sequence listing as SEQ ID NOS: 19-331. The SEQ ID NOS: 19-331 describe polynucleotide sequences from a variety of dicot and monocot plants as indicated that are useful for downregulating EIN2 expression using methods described here. The SEQ ID NOS: 19-331 describe polynucleotide sequences that can be applied to plants as ssDNA, ssRNA, dsDNA, dsRNA, and/or as DNA/RNA hybrids to provide for delayed senescence and/or improved yield. Subfragments of at least 18 contiguous nucleotides of the SEQ ID NOS: 19-331 polynucleotide sequences can also be applied to plants as ssDNA, ssRNA, dsDNA, dsRNA, and/or as DNA/RNA hybrids to provide for delayed senescence and/or improved yield. Other regions of EIN2 genes can also be targeted to modify expression including the use of antisense DNA oligonucleotides against coding regions and/or targeting promoter regions using sense/antisense dsRNA, sense or antisense ssDNA as well as sense/antisense double stranded DNA. For example, a polynucleotide that comprises at least 18 contiguous nucleotides that are essentially identical or essentially complementary to SEQ ID NO: 1-18 can be used to downregulate expression of those EIN2 genes.
Example 3. Method for Identifying Preferred Polynucleotides
A method for testing the entire sequence of each gene for selecting effective trigger molecules is described. Polynucleotides fragments are designed to cover the full length coding and untranslated regions of the gene in Table 2 and the sequence listing as full-length sequences or as contiguous overlapping fragments of 200-300 bases length. These fragments are applied topically as sense or anti-sense ssDNA or ssRNA, dsRNA, or dsDNA to determine the relative effectiveness in providing the trait phenotype. Fragments providing the desired activity are further subdivided into 50-60 polynucleotide fragments which are evaluated for providing the trait phenotype. The 50-60 base fragments with the desired activity are subdivided into 19-30 base fragments which are evaluated for providing the trait phenotype. Fragments are tested singly, or in combination in one or more pools to determine effective trigger formulations for generation of the trait phenotype. Exemplary triggers developed in this manner are provided herewith in the sequence listing as SEQ ID NO:19-331 and as SEQ ID NO: 614-659.
Example 4. Method for Identifying Conserved Preferred Polynucleotides
Triggers can also be developed to simultaneously suppress multiple EIN2 gene family members by alignment of coding and/or non-coding sequences of gene families in the crop of interest (Table 2), and choosing 200-300 base fragments from the most similar regions of the aligned sequences for evaluation in the topical application method (as sense or anti-sense ssDNA or ssRNA, dsRNA, or dsDNA) to determine their relative effectiveness in inducing the trait phenotype. The effective segments are subdivided into 50-60 base fragments, prioritized by greatest similarity, and re-evaluated in a topical application method. The effective 50-60 base fragments are subdivided into 19-30 base fragments, prioritized by greatest similarity, and again evaluated for induction of the trait/benefit phenotype. Once relative effectiveness is determined, the fragments may be utilized singly, or in combination with one or more other fragments to determine the trigger formulation for providing the trait phenotype. Exemplary triggers developed in this manner are provided herewith in the sequence listing as SEQ ID NO:332-613.
Example 5 Methods for Topical Application of Polynucleotide Molecules that Suppress EIN2
Tomato plants at the 2-leaf stage grown in a peat moss, composted bark and perlite soil mix are spotted with polynucleotides, either ssDNA and/or dsRNA oligos or long dsRNAs directed to the promoter and/or targeting the coding region of the EIN2 gene or gene family. The nucleotide solution applied consists of 40-50 nmoles of each ssDNA oligonucleotide or 0.5-2 nmoles dsRNA, 0.3% Silwet L77, 5 mM Na2HPO4 and 2% ammonium sulfate in a final volume of 40 μL. Two mature leaves are spotted with 20 μL of the nucleotide solution for a total of 40 μL per plant.
Corn plants are germinated in potting medium and grown in the greenhouse for approximately 10 days. ssDNA and/or dsRNA polynucleotides directed to the promoter and/or targeting the coding region of the EIN2 gene or gene family are spotted onto the first and second leaves. The nucleotide solution applied consists of 40-50 nmoles of each ssDNA oligonucleotide or 0.5-2 nmoles dsRNA, 0.5% Silwet L77, 20 mM Na2HPO4 and 2% ammonium sulfate in a final volume of 50 μL. Two mature leaves are spotted with 25 μL each of the nucleotide solution for a total of 50 μL per plant.
Alternatively, corn plants grown in the greenhouse are treated 20 days after pollination by spraying leaves with a solution containing 0.14 mg/mL of dsRNA or ssDNA (21-mer) or 0.5 to 1.5 mg/mL long dsRNA polynucleotides targeted to the EIN2 gene or gene family with 0.5% Silwet L77, 20 mM Na2HPO4 and 2% ammonium sulfate.
To delay fruit ripening, tomato and melon plants grown in the greenhouse are treated at the stage before fruit production by spraying leaves with a solution containing 0.14 mg/L ssDNA oligonucleotide or dsRNA polynucleotides targeted to the EIN2 gene or gene family with 0.5% Silwet L77, 20 mM Na2HPO4 and 2% (NH4)2SO4.
Example 6. Assay for Delayed Leaf Senescence
Various methods are used to demonstrate efficacy of polynucleotide triggers that target EIN2 genes as follows:
Method 1: After treatment with topical polynucleotides targeting the EIN2 gene or with a non-efficacious control polynucleotide, corn plants are kept in the greenhouse under optimal growing conditions or with limiting water and grown to maturity. Yellowing of leaves is assessed at approximately 40 days after pollination, and the percentage of each leaf that has turned yellow is recorded. Leaves are then sampled, and chlorophyll is extracted and quantified (Wellburn 1994). Plants treated with efficacious polynucleotides have leaves that have a higher percentage of green leaf area and more chlorophyll per leaf weight compared with plants treated with non-efficacious control polynucleotides. Photosynthesis of comparable leaves of each of these plants is then measured using a LiCor or PAM fluorescence monitor. Leaves of plants treated with efficacious polynucleotides have a higher rate of photosynthesis compared to controls.
Method 2: One week after topical application of the EIN2 polynucleotides, a hole punch is used to sample treated leaves or leaves from above the treatment site. These leaf discs are placed on 3 layers of wet Whatman No. 1 filter paper and placed in the dark at 25 C (Hu, Deng et al. 2011). One week after sampling, chlorophyll is extracted from the leaf pieces and measured spectrophotometrically using standard methods (Wellburn 1994). Leaf pieces from plants treated with efficacious polynucleotides have a higher concentration of chlorophyll compared with plants treated with non-efficacious control polynucleotides.
To examine the effect of suppression of the EIN2 gene on leaf senescence that occurs in response to drought stress, water is with-held from plants after they have been sprayed with the solution described above containing the EIN2 trigger sequences or with a control solution containing a non-efficacious control polynucleotide. Samples are taken from leaves after drought symptoms are visible to compare chlorophyll content using methods described above. In addition, photosynthesis of comparable leaves of each of these plants is measured using a LiCor or PAM fluorescence monitor. Leaves of plants treated with efficacious polynucleotides contain a higher concentration of chlorophyll and have a higher rate of photosynthesis compared to controls.
Example 7. Assays for Delayed Fruit Ripening that Results from EIN2 Suppression Fruit Shelf Life
To assay for delays in fruit ripening that are mediated by application of EIN2 polynucleotides, fruit deterioration will be analyzed by time-lapse photography as described in (Meli, Ghosh et al. 2010) and (Ghosh, Meli et al. 2011). Fruits with topically induced suppression of EIN2 will be stored in various temperatures with control fruits for the analyses.
Textural Analysis:
The impact of topically induced suppression of EIN2 in fruit would be increased firmness. Fruit firmness will be determined using a Texture Analyzer (e.g. TA-XT plus) as described in (Meli, Ghosh et al. 2010) and (Ghosh, Meli et al. 2011).
Cell Wall Structure Analysis:
Topically induced suppression of EIN2 in fruit would delay changes in cell wall structure that are observed in ripening fruit. Cell wall structure in EIN2 polynucleotide treated and control plants will be analyzed by using microscopy. Analysis of cell wall structure by microscopy is as described in (Meli, Ghosh et al. 2010) and (Ghosh, Meli et al. 2011).
Example 8. Additional Methods for Measuring Delayed Leaf Senescence in EIN2 Polynucleotide Treated Plants and Controls
The following procedure can be used to measure leaf senescence. Zea mays plants that have been topically treated will be monitored for yellowing of leaves will be visually assessed at approximately 40 days after pollination, and the percentage of each leaf that has turned yellow is recorded. Leaves will be sampled using a hole punch, and chlorophyll will be extracted and quantified using the following method. Each leaf disc is placed in a microcentrifuge tube and 1 ml of ice-cold 80% acetone is added. The tissue is then ground with a blue pestle with sand, until it is completely disintegrated. The tube is centrifuged in a microcentrifuge for 5 minutes at maximum speed at 4° C. The supernatant is removed and absorbance is measured in a spectrophotometer in a 1 ml glass cuvette at 663.2 nm, 646.8 nm and 710 nm. Chlorophyll a and b concentrations (μg/ml) are calculated using the following equations:

Chla=12.25*(A663.2−A710)−2.79*(A646.8−A710)

Chlb=21.5*(A646.8−A710)−5.1*(A663.2−A710 

Leaves of plants treated with efficacious polynucleotides of SEQ ID NO:19-633, or with efficacious polynucleotides that are essentially complementary or identical to Zea mays sequence provided in Table 2 and the sequence listing will have a higher percentage of green leaf area and more chlorophyll per leaf weight compared with plants treated with non-efficacious control polynucleotides. Photosynthesis of comparable leaves of each of these plants will be measured using a LiCor photosynthesis system (LiCor Biosciences) or PAM fluorescence monitor (Heinz Walz GmbH) following the manufacturer's recommended procedures. Leaves of plants treated with efficacious polynucleotides have a higher rate of photosynthesis compared to leaves of plants treated with non-efficacious control polynucleotides.
Chlorophyll will be extracted from leaves one week after topical treatment using a hole punch on treated leaves or leaves from above the treatment site. Leaf discs are placed on 3 layers of wet Whatman No. 1 filter paper and placed in the dark at 25° C. One week after sampling, chlorophyll is extracted from the leaf pieces and measured spectrophotometrically using the method described above. Leaf pieces from plants treated with efficacious polynucleotides have a higher concentration of chlorophyll compared with leaf pieces from plants treated with non-efficacious control polynucleotides.
Example 9. Additional Methods for Measuring Delayed Leaf Senescence in EIN2 Polynucleotide Treated Plants and Controls
The following procedure is used for determining the effect of gene regulation on leaf senescence that occurs in response to drought stress. Water is with-held from plants after topical spray application. Samples are taken from leaves after drought symptoms are visible to compare chlorophyll content using methods described above. Photosynthesis of comparable leaves of each of these plants is measured using a LiCor or PAM fluorescence monitor, as described above. Leaves of plants treated with efficacious polynucleotides contain a higher concentration of chlorophyll and will have a higher rate of photosynthesis compared to leaves of plants treated with non-efficacious control polynucleotides.
Example 10. Additional Assays for Delays in Fruit Ripening Mediated by Application of EIN2 Polynucleotides
Fruit deterioration will be analyzed by time-lapse photography. Pink tomato fruits with topically induced suppression of EIN2 will be stored at 25° C. with control fruits for the analyses. Analysis will entail taking photographs every 5 days during the storage to record deterioration. Additionally, textural analysis will be performed at 10, 20, 30 and 40 days of storage using texture analyzer (TA-XT) and a 75-mm-wide P75 compression plate that compresses the fruit to a vertical displacement of 5 mm with a test speed of 1 mm/second. The firmness will be defined as the response force to a 5×g applied force. The impact of topically induced suppression of EIN2 will be increased firmness.
Assay for Cell Wall Structure
Cell wall structure in pink fruit and fruit harvested every 2 subsequent days will be analyzed using microscopy. The fruit will be cut into sections, immersed in 0.05% calcofluor and rinsed with distilled water. The stained fruit sections will be mounted in water under a coverslip and photographed using a microscope.
Example 11. Topical Oligonucleotide Application and Fungal Testing Methods
Application of oligonucleotides to leaves for powdery mildew control. Barley seeds are planted in 2 inch pots in the greenhouse. Five days later, barley seedlings are sprayed with nucleotides, either ssDNA and/or dsRNA oligos directed to the promoter and/or targeting the coding region of a target gene of interest such as from Table 2 or Table 3. The nucleotide solution applied consists of 6-20 nm of each ssDNA oligonucleotide or 0.5-4 nm dsRNA, 0.1 to 0.3% L77 silwet, 50 mM NaPO4 in a final volume of 40 uL water. Two to 4 days post spraying seedlings are infected with dry spores of barley powdery mildew (Blumeria graminis. 1 sp. hordei) and 7 days post infection, disease development is scored for the percentage of leaf area covered with powdery mildew.
Cucumber seeds are planted in a 3-inch square pot and thinned to one plant per pot after emergence. When the first true leaf is fully expanded and the second leaf is opening a nucleotide solution of either ssDNA and/or dsRNA oligos directed to the promoter and/or targeting the coding region of a target gene of interest such as from Table 2 or Table 3 is applied to the first true leaf or the cotyledons. The nucleotide solution applied consists of 6-20 nm of each ssDNA oligonucleotide or 0.5-4 nm dsRNA, 0.1 to 0.3% L77 silwet, 50 mM NaPO4 in a final volume of 40 uL water. Two days later the entire cucumber plant is inoculated with a shower of dry spores of cucumber powdery mildew (Podosphaera xanthii) shaken off diseased plants. Disease severity will be evaluated on the treated leaf and succeeding leaves 10 days later and at subsequent intervals.
Tomato seeds are planted in a 3-inch square pot and thinned to one plant per pot after emergence. Two weeks old tomato seedlings are treated with 6-20 nm of each ssDNA oligonucleotide or 0.5-4 nm dsRNA (from one of the polynucleotides from Table 2 or Table 3, 0.2-0.5% L77 silwet, 50 mM NaPO4, 1% ammonium sulfate in a final volume of 30 uL water. Two to 4 days post spraying plants are innoculated with dry spores of tomato powdery mildew (Oidium neolycopersici) and 13 days post infection, disease development is scored for the percentage of leaf area covered with powdery mildew.
Application of Oligonucleotides to Leaves for Phytophthora Control
Pepper seeds and cucumber seeds are planted in vermiculite in 2 inch pots in the greenhouse. For topical nucleotide treatment, three weeks old pepper seedlings are used and 2 weeks old cucumber seedlings are used.
Seedlings are sprayed with nucleotides, either ssDNA and/or dsRNA oligos directed to the promoter and/or targeting the coding region of a target gene of interest such as from Table 2 or Table 3. The nucleotide solution applied consists of 6-20 nm of each ssDNA oligonucleotide or 0.5-4 nm dsRNA, 0.1 to 0.3% L77 silwet, 50 mM NaPO4 in a final volume of 40 uL water. Two to 4 days post spraying seedlings are infected with zoospores of Phytophthora capsici. Phytophthora capsici infection will be done either by spraying leaf tissue to test shoot dieback or by flooding soil to check root rot. Ten days post infection and at subsequent intervals, disease development will be scored with disease index ranging from 0 (no disease) to 4 (severe disease).
Zoospore inoculums are prepared with the following procedure. Grow mycelium on V8 agar medium for a week at 25° C., cut medium with mycelium into 4 mm squares, rinse with sterilized water trice at 20 min interval, soak in water in the hood with light on at room temperature for 24 h, transfer plate into 4° C. and keep for 1 h, take out and keep at room temperature for 1 h, check zoospores quantity and quality under dissection microscope. Adjust zoospore concentration to 500/ml with water for inoculation.
Example 12: Topical Oligonucleotide Application and Bacterial Testing Methods
Application of Oligonucleotides to Leaves for Pseudomonas syringae Control.
Tomato seeds are planted in 2 inch pots in the greenhouse. Two weeks later, tomato seedlings are sprayed with nucleotides, either ssDNA and/or dsRNA oligos directed to the promoter and/or targeting the coding region of a target gene of interest such as from Table 2 or Table 3. The nucleotide solution applied consists of 6-20 nm of each ssDNA oligonucleotide or 0.5-4 nm dsRNA, 0.1 to 0.3% L77 silwet, 50 mM NaPO4, 1% ammonium sulfate in a final volume of 40 uL water. Two to 4 days post nucleotide treatment; plants are inoculated by spraying Pseudomonas. syringae pv. tomato suspension until run-off. P. syringae suspension is prepared as described below. P. syringae pv. tomato DC3000 are grown at 28° C. in liquid LB medium with half strength of salt for overnight. Prior to inoculation, bacterium culture is diluted 50 times with 0.025% silwet 77. Inoculated plants are incubated under mist conditions in the greenhouse with a temperature regime of 28/22° C. (day/night) and natural illumination.
Example 13: Topical Application of Polynucleotide Molecules that Suppress EIN2
Tomato Seed Soak Method
Another trigger application method involved soaking seed in solutions containing the EIN2 trigger molecules, which are then taken up by the germinating seedlings. Approximately 45 tomato seeds (Oregon Spring variety) were soaked in a 1-mL solution containing 1 nmol double-stranded RNA, 5 mM Na2HPO4 (pH 6.8), and 0.01% Silwet L77 overnight at room temperature. Seeds (approximately 15 per box) were then transferred to seed germination boxes containing 12 mL 100 μM 1-aminocyclopropane-1-carboxylic-acid (ACC), and incubated in the dark for 1 week at 25° C.
Ethylene sensitivity can be tested by measuring the length of hypocotyls of seedlings grown in the dark in the presence of the ethylene precursor, ACC, which is converted by the seedling to ethylene (Lanahan et al., 1994). Seedlings grown in the dark in the absence of ACC or ethylene have long hypocotyls. However, seedlings grown in the presence of ethylene in the dark normally have short hypocotyls. Seedlings that are insensitive to ethylene due to a genetic mutation of the ethylene receptor have long hypocotyls even in the presence of ethylene when grown in the dark.
The treatment with dsRNA triggers that target the EIN2 gene also results in longer hypocotyls for seedlings grown in the dark with 100 μM ACC, suggesting these seedlings have become insensitive to ethylene (FIG. 1). The long double-stranded RNA triggers used in these experiments are fragments of the tomato EIN2 cDNA, Sl_H1706_CR09.G245610.1.cDNA (Sequence ID NO 17 in Table 2). The coordinates and sizes of the EIN2 triggers are show in Table 3. A control double-stranded RNA sequence (SEQ ID NO 659) was taken from the green fluorescent protein (GFP). Hypocotyls of the seedlings treated with water, buffer or SEQ ID NO 659 have short hypocotyls, as expected (FIGS. 1A and 1B). However, treatment with either the SEQ ID 624, or SEQ ID 625, or SEQ ID 626 dsRNA molecules that target EIN2 tomato sequence results in longer hypocotyls. The error bars represent 95% confidence intervals. The regulation of gene expression by ethylene in plant tissues has been studied, and this research identified a gene called E4 that is transcriptionally activated by ethylene in both tomato fruit and in leaves (Montgomery et al., 1993). The E4 gene encodes a methionine sulfoxide reductase, an enzyme which likely functions to repair proteins damaged by oxidative stress (Dai and Wang, 2012), but the E4 gene can be used as a marker for ethylene response. RNA was isolated from shoots of seedlings treated as described above, and qPCR was conducted to quantify E4 mRNA. As shown in FIGS. 2A and 2B, E4 gene expression is relatively high in samples from seedlings treated with buffer or the GFP control trigger but is significantly reduced in samples from seedlings treated with EIN2 triggers. There is a fairly good correlation between treatments that produce long hypocotyls (FIG. 1A,B) and those that have reduced E4 expression (FIG. 2A,B). Thus, we observed 2 independent phenotypes that suggest a loss of ethylene sensitivity from treatment with dsRNA triggers targeting the EIN2 gene.
Table 3. Description of long dsRNA triggers tested with seed soak method in tomato. Coordinates of EIN2 dsRNAs are with respect to Sl_H1706_CR09.G245610.1.cDNA (Sequence ID NO:17 in Table 2 and the sequence listing).







TABLE 3







Description of long dsRNA triggers tested with seed soak method


in tomato. Coordinates of EIN2 dsRNAs are with respect to 


Sl_H1706_CR09.G245610.1.cDNA 


(Sequence ID NO: 17 in Table 2 and the sequence listing).











Coordinates 
Size of 
Trigger 


Trigger
of EIN2 cDNA in 
trigger
SEQ ID


name
SEQ ID NO: 17
(bp)
NO













PCR1
2484..2676
192
624


PCR2
87..473
356
625


PCR3
474..935
461
626


GFP

25
659









The regulation of gene expression by ethylene in plant tissues has been studied, and this research identified a gene called E4 that is transcriptionally activated by ethylene in both tomato fruit and in leaves (Montgomery et al., 1993). The E4 gene encodes a methionine sulfoxide reductase A enzyme, which likely functions to repair proteins damaged by oxidative stress (Dai and Wang, 2012), but the E4 gene can be used as a marker for ethylene response. RNA was isolated from shoots of seedlings treated as described above, and qPCR was conducted to quantify E4 mRNA. As shown in FIGS. 2A and 2B, E4 gene expression is relatively high in samples from seedlings treated with buffer or the GFP control trigger but is significantly reduced in samples from seedlings treated with EIN2 triggers. There is a fairly good correlation between treatments that produce long hypocotyls (FIGS. 1A and 1B) and those that have reduced E4 expression (FIGS. 2A and 2B).
Thus, we have observed 2 independent phenotypes that suggest a loss of ethylene sensitivity from treatment with dsRNA triggers targeting the EIN2 gene.
Lettuce Seed Soak Method
Absence of the ethylene-induced triple response phenotype has been used to select lettuce lines that were insensitive to ethylene, and these lines then showed reduced ethylene-induced chlorophyll loss in mature heads (Saltveit et al., 2003). We have used the seed soak method to demonstrate suppression of ethylene response by treatment of seeds with dsRNAs containing lettuce EIN2 transcribed sequences.
Dai C., Wang M.-H. (2012) Characterization and functional analysis of methionine sulfoxide reductase A gene family in tomato. Molecular Biology Reports 39:6297-6308. DOI: 10.1007/s11033-012-1451-0. Lanahan M. B., Yen H. C., Giovannoni J. J., Klee H. J. (1994) The never ripe mutation blocks ethylene perception in tomato. The Plant Cell Online 6:521-30. DOI: 10.1105/tpc.6.4.521. Montgomery J., Goldman S., Deikman J., Margossian L., Fischer R. L. (1993) Identification of an ethylene-responsive region in the promoter of a fruit ripening gene. Proceedings of the National Academy of Sciences 90:5939-5943. Saltveit M. E., Ochoa O., Campos-Vargas R., Michelmore R. (2003) Lines of lettuce selected for ethylene insensitivity at the seedling stage displayed variable responses to ethylene or wounding as mature heads. Postharvest Biology and Technology 27:277-283. DOI: 10.1016/s0925-5214(02)00119-9.
Example 14. Topical Oligonucleotide Application and Nematode Testing Methods
Application of Oligonucleotides to Leaves for Nematode Control.
Ten day old cucumber plants grown in sand are spotted with nucleotides, either ssDNA and/or dsRNA oligos directed to the promoter and/or targeting the coding region of a target EIN2 gene of interest such as from Table 2 and the sequence listing. The nucleotide solution applied consists of 6-20 nm of each ssDNA oligonucleotide or 0.5-1 nm dsRNA, 0.1% L77 silwet, 50 mM NaPO4 in a final volume of 40 uL water. Two cotyledon or leaves are spotted with 20 uL of the nucleotide solution for a total of 40 uL per plant. After 6-24 hours, 1000 vermiform eggs or 1000 J2 Meloidogyne incognita (RKN) are inoculated into each pot. Watering of the test plants is then restricted to only water as needed to prevent wilt for a period of 24 hours. After the 24 hour restricted watering, normal sub-irrigation watering is done for the duration of the test. Cucumber plants are harvested approximately 14 days after inoculation by washing sand off the roots. A root gall rating and visual phytotoxicity rating is assigned using the following scales: Gall rating scale (Gall: % root mass galled): 0=0-5%; 1=6-20%; 2=21-50%; and 3=51-100%. Visual phytotoxicity scale is also assigned (Vis. tox; visual reduction in root mass compared to the control): rs1=mild stunting; rs2=moderate stunting; rs3=severe stunting
Experiments in soybeans using soy cyst nematodes (SCN) are similar to the cucumber RKN assay except for the following changes. Soybean seeds are planted in 100% sand in two inch square plastic pots. The oligonucleotide solution is applied when the soybeans show the first trifoliate beginning to emerge, about 10 to 12 days after planting. At least six hours after application of the oligonucleotide solution, the nematode soybean cyst nematode (SCN) innoculum (1000 vermiform eggs or 1000 J2s) is applied to the pots. Watering of the test plants is then restricted to only water as needed to prevent wilt for a period of 24 hours. After the 24 hour restricted watering, normal sub-irrigation watering is done for the duration of the test. Twenty eight days after inoculation the plants are harvested and cysts counted. Experiments in corn using lesion nematodes are similar to above except for the following changes. Corn plants growin in a sand:turface mix 2:1 in 4 inch deep pots. Treatment with oligonucleotide solution is done when the plants are approximately 8-10 old. At least six hours after inoculation of the oligonucleotide solution, plants are inoculated with 2 μm of P. scribneri infested corn roots which are then removed from the pot after 7 days. Watering of the test plants is then restricted to only water as needed to prevent wilt for a period of 24 hours after inoculation. After the 24 hour restricted watering, normal sub-irrigation watering as needed is done for the duration of the test. 12-14 days post inoculation, plants are harvested and nematodes extracted for 6 days from the cut up roots in a mist tent.
Application of Oligonucleotides to Seeds for Nematode Control
Cucumber seeds are soaked approximately 5-72 hours in nucleotides, either ssDNA and/or dsRNA oligos directed to the promoter and/or targeting the coding region of a target of interest such as from Table 2 or Table 3. Seeds can also be soaked in water for a few hours prior to soaking in oligonucleotide solution. Soaking solution consists of 20 nm of each ssDNA nucleotide or 0.03-1 nm dsRNA, 0.1% silwet L77, 50 mM NaPO4 in a final volume 200 uL in water. The radicals of the cucumber seeds emerge within 72 hours, after which the seeds are placed on germination paper until root length is approximately 2 inches. Seedlings are transplanted to sand vials for RKN inoculation 24 hours later. Ten mL dry sand is added to each vial and seedlings are planted by tilting the vial and laying the seedling in the correct orientation so that the cotyledons are just above the sand and then tilting back to cover the radicles with sand. 3.3 ml water is added to each vial and the vials placed in racks under fluorescent light banks. 500 vermiform eggs or 300 J2 RKN are inoculated in each tube in 50 uL of deionized or spring water. Harvest of the cucumber plants is performed 10 to 12 days after inoculation by washing sand off the roots. A root gall rating and visual phytotoxicity rating is assigned using the following scales: Gall rating scale (Gall: % root mass galled): 0=0-5%; 1=6-20%; 2=21-50%; and 3=51-100%. The average of the triplicate gall rating is then calculated: green=0.00-0.33 (no galls); yellow=0.67-1.33 (mild galling); orange=1.67-2.33 (moderate galling); red=2.67-3.00 (severe galling). Visual phytotoxicity scale is also assigned (Vis. tox; visual reduction in root mass compared to the control): rs1=mild stunting; rs2=moderate stunting; rs3=severe stunting.
Experiments in soybeans using soy cyst nematodes (SCN) are similar to RKN assays except for the following changes. After 5-72 hours of soaking soybean seeds are planted in 100% sand in two inch square plastic pots. Seeds can also be soaked in water for a few hours prior to soaking in oligonucleotide solution. Seven days after planting the soybean seed, the nematode soybean cyst nematode (SCN) inoculum (1000 vermiform eggs or 1000 J2s) are applied to the pot. Watering of the test plants is then restricted to only water as needed to prevent wilt for a period of 24 hours. After the 24 hour restricted watering, normal sub-irrigation watering is done for the duration of the test. Twenty eight days after inoculation the test is harvested and cysts counted
Experiments in corn using lesion nematodes are similar to above except for the following changes. After 5-72 hours of soaking corn seeds are planted in a sand:turface mix 2:1 in 4 inch deep pots. Seeds can also be soaked in water for a few hours prior to soaking in oligonucleotide solution. Inoculum of 2 μm of roots P. scribneri infested corn roots are applied at seeding and removed from the pot after 7 days. Watering of the test plants is then restricted to only water as needed to prevent wilt for a period of 24 hours after inoculation. After the 24 hour restricted watering, normal sub-irrigation watering as needed is done for the duration of the test. 12-14 days post inoculation, plants are harvested and nematodes extracted for 6 days from the cut up roots in a mist tent.
RKN and SCN J2s are prepared from hatchbowls using the following solutions: RKN solution: 1 L aerated tap water, 1 ml of 50 mg/ml kanamycin, 0.5 ml of 20 mg/ml imazalil sulfate; SCN solution: 1 L aerated tap water, 1 ml of 50 mg/ml kanamycin, 0.5 ml of 20 mg/ml imazalil sulfate, 1430 mg zinc sulfate Hatchbowls are autoclaved 6 oz bowls, lined with screen mesh and paper filter. Approximately 20 ml of appropriate hatch solution is poured into each bowl. Eggs are then place in the bowls and covered with foil. The bowls are then placed in a 25° C. incubator overnight. The next day the hatched J2's are extracted, additional solution added as needed and replaced in the incubator. Each bowl is used for 2 weeks and then disposed.
Example 15. Nematode Control in Cucumber Via Oligonucleotide Application by Seed Soak
Cucumber seeds were soaked approximately 72 hours in dsDNA oligos directed to the promoter and/or targeting the coding region of a cucumber EIN2 gene. Soaking solution consists of 20 nm of each ssDNA nucleotide or 0.03-1 nm dsRNA, 0.1% silwet L77, 50 mM NaPO4 in a final volume 200 uL in water. The radicals of the cucumber seeds emerge within 72 hours, after which the seeds were placed on germination paper until root length is approximately 2 inches. Seedlings are transplanted to sand vials for Root Knot Nematode (RKN) inoculation 24 hours later. Ten mL dry sand is added to each vial and seedlings are planted by tilting the vial and laying the seedling in the correct orientation so that the cotyledons are just above the sand and then tilting back to cover the radicles with sand. 3.3 ml water is added to each vial and the vials were placed in racks under fluorescent light banks. 500 vermiform eggs or 300 J2 RKN are inoculated in each tube in 50 uL of deionized or spring water. Harvest of the cucumber plants is performed 10 to 12 days after inoculation by washing sand off the roots. A root gall rating and visual phytotoxicity rating was assigned using the following scales: Gall rating scale (Gall: % root mass galled): 0=0-5%; 1=6-20%; 2=21-50%; and 3=51-100%. The average of the triplicate gall rating was then calculated: green=0.00-0.33 (no galls); yellow=0.67-1.33 (mild galling); orange=1.67-2.33 (moderate galling); red=2.67-3.00 (severe galling). Visual phytotoxicity scale was also assigned (Vis. tox; visual reduction in root mass compared to the control): rs1=mild stunting; rs2=moderate stunting; rs3=severe stunting.
The results of this experiment shown in Table 6 indicate that certain EIN2 oligonucleotide pools exhibited RKN control.







TABLE 4







Treatment regimens.













Treat-








ment

Nucleo-






Num-

tide






ber #
Description:
ID
Oligos
NaPO4
water
total
















1
Ein2 Pool 1 asDNA
T8140-43
4 × 10
40
120
200



CDS 80 nmol







2
Ein2 Pool 2 asDNA
T8144-47
4 × 10
40
120
200



CDS 80 nmol







3
Ein2 Pool 3 asDNA
T8148-51
4 × 10
40
120
200



CDS 80 nmol







4
Ein2 Pool 4 asDNA
T8152-55
4 × 10
40
120
200



CDS 80 nmol







5
Ein2 Pool 1 asDNA
T8156-59
4 × 10
40
120
200



prom 80 nmol







6
Ein2 Pool 2 asDNA
T8160-63
4 × 10
40
120
200



prom 80 nmol







7
Ein2 Pool 3 asDNA
T8164-67
4 × 10
40
120
200



prom 80 nmol







8
Ein2 Pool 4 asDNA
T8168-71
4 × 10
40
120
200



prom 80 nmol







9
GFP asDNA

40
40
120
200


10
blank

 0
40
160
200
















TABLE 5







Nucleotide Sequences









Nucleotide
SEQ



ID
ID NO:
Sequence





T8140
627
CTGATAGCCATGGACTGTGGCAGGC


 


T8141
628
AGCCTGATGAATCCAACTGACCGTT


 


T8142
629
CTGCACCACCACCACCCAAGGTATG


 


T8143
630
CAAGCACCCAAGCCATTTTGCAACT


 


T8144
631
ACACTCCACGAGCTGTTATTCTACC


 


T8145
632
TAAAGAAATTTCTCCCTGGCAGCTA


 


T8146
633
CCCGACCCATCTCCCTTGCCTCAGC


 


T8147
634
AATGAAGGAGATTCTTTCATGCGGA


 


T8148
635
TTCATTCCAGAACCTGGTCTCCTAT


 


T8149
636
CCCCATAGTTCAGGCCGACTTTCCA


 


T8150
637
ATACGAGGCTTCGAAAATGCAGGAT


 


T8151
638
TTTGGAGGCAGAAGCATGGTGGCAT


 


T8152
639
TTGACCTCTGCTGGAATGCTTGGGG


 


T8153
640
CTTGCCAGGTTTTGCAGCAGGAGGC


 


T8154
641
TCCAGAAGCATTGCAGCAGTGGTGC


 


T8155
642
GGCAAGAGATGGCTATCTCCACATC


 


T8156
643
gattccataagaagtgcttcaataa


 


T8157
644
caattctaatttcaccctcataaat


 


T8158
645
tctctctctttttttcatccccttt


 


T8159
646
ctgcttttgcttccacactcccaat


 


T8160
647
agatgcttcagtaacgccagagagt


 


T8161
648
tgaatccatgaattcagtgtggaca


 


T8162
650
tattcacctgggttttccgtactgg


 


T8163
651
agaaaaaaaaaggagatagggttgt


 


T8164
652
aaaaccacggatccgatgcaagcta


 


T8165
653
agaaattggccacgagaaatgaaga


 


T8166
654
cccaacccagaacccagaacccaaa


 


T8167
655
aaaaaataccggggactttcatcga


 


T8168
656
tcaaaattaatagagccccattaca


 


T8169
657
ctggttcggccttaccttaccgcct


 


T8170
658
aagaaaactcgatttagggaggtac


 


T8171
659
gcagctaattccccacgaaatcagt
















TABLE 6







Results of Oligonucleotide Treatment on Nematode Resistance











Trt.

Rep Score (% 




No.
Treatment
root mass galled)
AVG
stdev

















1
Ein2 asDNA CDS T8140-43
45
45
45
45
45.00
0.00


2
Ein2 asDNA CDS T8144-47
30
30
45
45
37.50
8.66


3
Ein2 asDNA CDS T81448-51
25
35
30
35
31.25
4.79


4
Ein2 asDNA CDS T8152-55
35
25
40

33.33
7.64


5
Ein2 asDNA prom T8156-59
35
35
45
30
36.25
6.29


6
Ein2 asDNA prom T8160-63
45
30
40
25
35.00
9.13


7
Ein2 asDNA prom T8164-67
40
40
35
30
36.25
4.79


8
Ein2 asDNA prom T8168-71
45
40
60
40
46.25
9.46



GFP asDNA
50
55
40
50
48.75
6.29



blank
45
50
50
55
50.00
4.08

















Anova: Single







Factor









Similar protocols were also used to test single oligonucleotides for inhibition of RKN in cucumbers. Data shown in Table 7 indicates that certain single oligonucleotides could provide RKN control.







TABLE 7







Single oligonucleotide mediated control of RKN











Rep Score




Treatment
(% root mass galled)
AVG
stdev
















Ein2 CDS T8148
55
45
45
45
47.50
5.00


Ein2 CDS T8149
50
40
60
45
48.75
8.54


Ein2 CDS T8151
40
45
40
40
41.25
2.50


Ein2 CDS T8152
40
35
40
45
40.00
4.08


Ein2 CDS T8153
55
50
55
60
55.00
4.08


Ein2 CDS T8154
60



60.00



Ein2 CDS T8155
40
45
35
50
42.50
6.45


Ein2 CDS T8160
60
60
60

60.00
0.00


Ein2 CDS T8161
50
50
60
45
51.25
6.29


Ein2 CDS T8162
50
40
45
40
43.75
4.79


Ein2 CDS T8163
50
50
65
75
60.00
12.25


Ein2 CDS T8160-63
40
40
60
50
47.50
9.57


Ein2 CDS T8148-51
50
45
40
40
43.75
4.79


Ein2 CDS T8152-55
50
50
40
50
47.50
5.00


GFP asDNA
45
60
50
65
55.00
9.13


blank
60
60


60.00
0.00

















Anova: Single Factor









Example 16. Barley Powdery Mildew Assay Protocol
Barley seeds (Perry variety) are planted about 1⁄4′′ into soil in 2 inch pots in the growth chamber and grown at 25° C. with a 16 hr light cycle in 50% humidity. Before polynucleotide application the plants are randomized. Application of polynucleotides (either ssDNA oligos and/or dsRNA) is performed by pipet application where 54 of solution containing nucleotides is applied to both sides of the first leaf. The nucleotide solution applied consists of  ̃3-15 nm of each ssDNA oligonucleotide or  ̃0.5-1 nm dsRNA, 0.1-0.3% Silwet L-77, 5 mM NaPO4, and 1% AMS in Gibco ultra pure water. Two days post treatment seedlings are infected with barley powdery mildew (Blumeria graminis f sp. hordei). The growth chamber settings for the infection are as follows: 23° C., with a 12 hr light cycle in 70% humidity. At seven days post infection disease severity is scored for the percentage of leaf area covered with powdery mildew. Data is analyzed using Anova Single Factor Analysis (α=0.1). The 1⁄2 LSD is calculated and custom error bars created for the bar graphs. Percent disease reduction is compared to formulation blank and nucleic acid control
Example 17 Application of Topical Polynucleotides to Leaves or Seed for Phytophthora Control Growth Chamber Whole Plant Assay
Soybean seeds are planted in vermiculite in 3 inch pots and allowed to grow for 8 to 11 days. Unifoliate leaves are topically treated with 5 L total volume of polynucleotides (either ssDNA oligos and/or dsRNA) in a solution containing  ̃3-15 nm of each ssDNA oligonucleotide or  ̃0.5-1 nm dsRNA, 0.1-0.3% Silwet L-77, 5 mM NaPO4, and 1% AMS in Gibco ultra pure water. One day after topical polynucleotide application, pots are inoculated with 3 to 5 ml of ground up inoculum. P. sojae inoculum is grown on V8 agar, ground in a Cuisinart blender and pushed through a syringe. Plants are harvested 21 to 26 days after inoculation. Roots are weighed and checked for disease symptoms.
Petri Plate Seedling Assay
Soybean seeds are sterilized with 4 ml of 37% HCL into 100 ml of 100% bleach over a 3 day period. Bleach and HCL is replaced each day. (Sterile soybeans are critical for the assay to be useful.) Sterile seeds are soaked overnight in a solution comprising polynucleotides (either ssDNA oligos and/or dsRNA) in a solution containing  ̃3-15 nm of each ssDNA oligonucleotide or  ̃0.5-1 nm dsRNA, 0.1-0.3% Silwet L-77, 5 mM NaPO4, and 1% AMS in Gibco ultra pure water and transferred to a square petri plate with 8 layers of moistened Whatman filter paper. Seeds are germinated in the dark at 25 C for 5 days, then hypocotyls are wounded and inoculated with a small plug of P. sojae. Seedlings are kept under lights at room temperature and hypocotyls are checked for disease symptoms at 3 and 6 days.

## Claims

1. A method for producing a tomato plant exhibiting delayed senescence and/or improved disease tolerance, the method comprising topically applying to a tomato plant surface a composition that comprises:
(a) at least one double-stranded RNA (dsRNA) polynucleotide that comprises at least 18 contiguous nucleotides that are identical or complementary to a tomato EIN2 gene transcript, wherein the said dsRNA polynucleotide is not operably linked to a promoter or to a viral vector; and
(b) a transfer agent comprising a surfactant comprising an organosilicone preparation that conditions the tomato plant surface to permeation by the dsRNA polynucleotide into cells of the tomato plant;

wherein said tomato plant exhibits delayed senescence and/or improved disease tolerance in comparison to a control plant that has not been treated with a composition comprising a polynucleotide and a transfer agent and wherein the delayed senescence and/or improved disease tolerance results from suppression of said tomato EIN2 gene.

2. The method of claim 1, wherein said dsRNA polynucleotide comprises at least 18 contiguous nucleotides that are identical or complementary to SEQ ID NO:17.

3. The method of claim 1, wherein one strand of said dsRNA polynucleotide molecule is complementary to a sequence selected from the group consisting of SEQ ID NO: 624, 625, and 626.

4. The method of claim 1, wherein said composition further comprises one or more additional dsRNA polynucleotides that are essentially identical or complementary to a different target gene or transcript thereof.

5. The method of claim 1, wherein said dsRNA polynucleotide is at least 18 to about 24, about 25 to about 50, about 51 to about 100, about 101 to about 300, about 301 to about 500, or at least about 500 or more residues in length.

6. The method of claim 1, wherein said composition further comprises a non-polynucleotide herbicidal molecule, a polynucleotide herbicidal molecule, a polynucleotide that suppresses an herbicide target gene, an insecticide, a fungicide, a nematocide, or a combination thereof.

7. The method of claim 1, wherein said composition further comprises a non-polynucleotide herbicidal molecule and said plant is resistant to said herbicidal molecule.

8. The method of claim 1, wherein the improved disease tolerance is tolerance to Alternaria solani, Cladosporium fulvum, Oidium neolycopersici, Oidium lycopersici, Phytophthora infestans, Pratylenchus spp., Meloidogyne spp., or Pseudomonas syringae.