Patent Grant
8383887
The entire contents of a paper copy of the “Sequence Listing” and a computer readable form of the sequence listing on diskette, containing the file named 408204_SequenceListing_ST25.txt, which is 15 kilobytes in size and was created on May 6, 2010, are herein incorporated by reference.
The present disclosure relates generally to genetically modified plants, and more particularly to methods of using genetically modified crop plants to control the negative effects of certain disease-causing organisms in crop plants.
Members of the saprophytic fungus genus Aspergillus produce aflatoxin, a strictly regulated and highly carcinogenic metabolite in plants. Alfatoxin-producing members of Aspergillus include A. flavus, which commonly afflicts many important food crops including the cereal crops maize, sorghum, pearl millet, rice, wheat, and oilseeds including peanut, soybean, sunflower and cotton. A. flavus causes ear rot on corn that results in aflatoxin contamination and the presence of aflatoxin results in a large loss of marketable crop by farmers each year.
The native habitat of Aspergillus is in soil, decaying vegetation, hay, and grains undergoing organic decay. It commonly invades all types of organic substrates whenever favorable growth conditions exits. Favorable conditions include a relatively high (7% or higher) moisture content and higher ambient temperatures. Thus, A. flavus is widely present under common crop field and storage conditions, and can threaten significant contamination of a crop before harvest or in storage. Moreover, host crops are more susceptible to Aspergillus infection and resulting aflatoxin contamination under stressful growing conditions, including drought. At this time, few options are available for effective control of this pathogen. Breeding programs to generate aflatoxin-resistant cultivars of agricultural significance have not met much success. A need remains for methods to prevent and control aflatoxin contamination in the field.
In one aspect, the present disclosure provides a method for reducing aflatoxin accumulation in a crop plant, the method comprising: selecting a crop plant line susceptible to infection with Aspergillus flavus; and transforming a plant from the selected plant line with a DNA sequence encoding a bacterial NADP-specific glutamate dehydrogenase enzyme so that the plant expresses the bacterial NADP-specific glutamate dehydrogenase enzyme in an amount sufficient to reduce aflatoxin accumulation in comparison to an amount of aflatoxin accumulation in an untransformed plant from the A. flavus susceptible plant line. The method can further comprise growing the plant in conditions associated with A. flavus infection of the plant.
In another aspect, the present disclosure provides a method of using a transgenic gdhA+ plant line, the method comprising: controlling aflatoxin contamination of a food crop, wherein the transgenic gdhA+ plant line is a food crop plant line susceptible to A. flavus infection, by growing a plant of the transgenic gdhA+ plant line in conditions associated with A. flavus infection; measuring the amount of aflatoxin accumulation in the plant; and comparing the amount of aflatoxin accumulation in the plant to the amount of aflatoxin accumulation in a gdhA− plant from the food crop plant line susceptible to A. flavus infection.
In another aspect, the present disclosure provides a method of controlling aflatoxin contamination of a crop comprising: selecting an A. flavus susceptible crop plant line; transforming a plant from the selected plant line with a DNA sequence encoding a bacterial NADP-specific glutamate dehydrogenase enzyme to produce a transgenic gdhA+ plant line; and growing a plant of the transgenic gdhA+ plant line in conditions associated with A. flavus infection.
In another aspect, the present disclosure provides a method of controlling root rot in plants infected with Fusarium virguliforme, the method comprising: selecting a F. virguliforme susceptible plant line; and transforming a plant from the selected plant line with a DNA sequence encoding a bacterial NADP-specific glutamate dehydrogenase enzyme so that the plant expresses the bacterial NADP-specific glutamate dehydrogenase enzyme in an amount sufficient to reduce root rot in comparison to root rot in an untransformed plant from the .F. virguliforme susceptible plant line. The method may further comprise growing the plant in conditions associated with F. virguliforme infection of the plant.
In another aspect, the present disclosure provides method of using a transgenic gdhA+ plant line, the method comprising: controlling root rot in a crop plant, wherein the transgenic gdhA+ plant line is derived from a crop plant line susceptible to Fusarium virguliforme infection, by growing a plant of the transgenic gdhA+ plant line in conditions associated with F. virguliforme infection; measuring the amount of root rot in the plant; and comparing the amount of root rot in the plant to the amount of root rot in a gdhA− plant from the F. virguliforme susceptible crop plant line.
In another aspect, the present disclosure provides method of controlling root rot in a crop comprising: selecting a Fusarium virguliforme susceptible crop plant line; transforming a plant from the selected plant line with a DNA sequence encoding a bacterial NADP-specific glutamate dehydrogenase enzyme to produce a transgenic gdhA+ plant line; and growing a plant of the transgenic gdhA+ plant line in conditions associated with F. virguliforme infection.
In another aspect, the present disclosure provides method of screening a crop for plants transformed with a gdhA gene, the method comprising: exposing a plurality of putatively transformed plants to Aspergillus flavus or to Fusarium. virguliforme; and selecting the plants that show resistance to the effects of the A. flavus or F. virguliforme. In the method, when the plurality of plants are exposed to A. flavus, selecting the plants that show resistance to the effects of the A. flavus can comprise selecting plants that show a decreased level of aflatoxin accumulation relative to a reference plant untransformed with the gdhA gene, or can comprise selecting plants that show a decreased level of ear rot relative to a reference plant untransformed with the gdhA gene. In the method, when the plurality of plants are exposed to F. virguliforme, selecting the plants that show resistance to the effects of the F. virguliforme can comprises selecting plants that show a decreased level of root rot relative to a reference plant untransformed with the gdhA gene.
In any of the above methods, a food crop plant line can be a cereal plant line, including for example a maize, sorghum, pearl millet, rice, or wheat plant line, or an oilseeds plant line, such as a peanut, soybean, sunflower, or cotton plant line. Alternatively, the plant line can be a tobacco plant line. In ay of the above methods, the DNA sequence may comprise the Kozac consensus sequences.
FIGS. 6A1, 6A2 and 6A3 show the DNA (SEQ ID NO:12) sequence of the mutagenized gdhA gene used for plant expression in corn.
FIGS. 6B1, 6B2 and 6B3 show the DNA sequence (SEQ ID NO:13) including the SphI site of the mutagenized gdhA gene used for plant expression in corn.
FIGS. 7A1, 7A2 and 7A3 shows the mutagenized gdhA gene (SEQ ID NO:14) with the added restriction sites for use in Zea mays.
Described herein are the results of successful experiments that show for the first time that corn plants transformed with the gdhA gene (gdhA+ corn) are resistant to aflatoxin accumulation. Additionally, results described herein show that corn and tobacco plants transformed with the gdhA gene (gdhA+ plant) are also resistant to root rot following infection with Fusarium virguliforme. These surprising findings provide the basis in part for various methods described herein. Additionally, the discovery provides the basis for the development of new markers for novel sources of resistance to ear rot, aflatoxin accumulation and root rot, and establishes the gdhA gene as an important tool for marker-assisted breeding programs.
More specifically, laboratory assays show that kernels from corn plants with the gdhA gene exhibit a reduction in the conidiation of A. flavus. The findings indicate that the fungus produces significantly less conidia on the embryos of gdhA+ corn kernels than on those of gdhA− kernels. As a further advantage, the aflatoxin resistance conferred on plants expressing the gdhA gene is coincident with other desirable characteristics of the plants, including higher tolerance to stressful environmental conditions, resistance to certain herbicides, and resistance to root rot, together with nutritional equivalence to unaltered corn.
Methods of Producing Transgenic Plants Containing the gdhA Gene
Plants containing a bacterial gdhA gene and their use in growing a transgenic crop that is resistant to herbicides of the phosphinothricin class have previously been described in U.S. Pat. Nos. 5,998,700 and 6,329,573 both under the title “Plants Containing a Bacterial gdhA Gene and Methods of Use Thereof.” Described herein are methods for controlling alfatoxin contamination in plants susceptible to Aspergillus. The findings disclosed herein also establish that the gdhA gene can be used as a specific marker in plant breeding programs seeking to reduce aflatoxin accumulation in plants infected by Aspergillus.
As used herein, the term transgenic plant refers to plants having exogenous genetic sequences that are introduced into the genome of a plant by a transformation method and the progeny thereof.
As used herein, the term transformation methods refers to means for integrating new genetic coding sequences by the incorporation of these sequences into a plant of new genetic sequences through man's assistance. Though there are a large number of known methods to transform plants, certain types of plants are more amenable to transformation than are others. For example, corn is a readily transformable monocot and tobacco is a readily transformable dicot. The basic steps of transforming plants are known in the art. These steps are concisely outlined for example in U.S. Pat. No. 5,484,956 “Fertile Transgenic Zea mays Plants Comprising Heterologous DNA Encoding Bacillus Thuringiensis Endotoxin” and U.S. Pat. No. 5,489,520 “Process of Producing Fertile Zea mays Plants and Progeny Comprising a Gene Encoding Phosphinothricin Acetyl Transferase”. A description of a method for transforming tobacco (Nicotiana tabacum var. Petite Havana) and Zea mays plants with the gdhA gene is provided in U.S. Pat. No. 6,329,573 “Plants Containing the gdhA Gene and Methods of Use Thereof”.
Tobacco and corn lines that express a bacterial NADP-dependent glutamate dehydogenase have been shown to have a high tolerance to glucosinate-type herbicides, and the altered corn lines provide increased grain biomass production in dry environments while retaining nutritional equivalence to unaltered corn. (See U.S. Pat Nos. 5,998,700 and 6,329,573).
Plant cells such as maize can be transformed by a number of different techniques. Some of these techniques have been described and are known in the art including maize pollen transformation (see University of Toledo 1993 U.S. Pat. No. 5,177,010); biolistic gun technology (see U.S. Pat. No. 5,484,956); Whiskers technology (see U.S. Pat. Nos. 5,464,765 and 5,302,523); electroporation; Agrobacterium (see 1996 article on transformation of maize cells in Nature Biotechnology, Volume 14, June 1996) along with numerous other methods which may have slightly lower efficiency rates then those listed. Some of these methods require specific types of cells and other methods can be practiced on any number of cell types.
The use of pollen, cotyledons, meristems and ovum as the target issue can eliminate the need for extensive tissue culture work. However, the present state of the technology does not provide very efficient use of this material.
Generally, cells derived from meristematic tissue are useful. Zygotic embryos can also be used. Additionally, the method of transformation of meristematic cells of cereal is also taught in PCT application WO96/04392. Any of the various cell lines, tissues, plants and plant parts can and have been transformed by those having knowledge in the art. Methods of preparing callus from various plants are well known in the art and specific methods are detailed in patents and references used by those skilled in the art.
Cultures can be initiated from most of the above identified tissue. The material used herein was zygotic embryos. The embryos are harvested and then either transformed or placed in media. Osmotic cell treatments may be given to enhance particle penetration, cell survival, etc.
The only true requirement of the transformed material is that it can form a fertile transformed plant. The gene can be used to transform plants including both monocots and dicots. Plants that are produced as field crops are of particular interest and particularly those crops susceptible to mycotoxin-producing fungi such as the aflatoxin-producing fungus Aspergillus. These crops include for example the cereal crops maize, sorghum, pearl millet, rice, wheat, and the oilseeds peanut, soybean, sunflower and cotton, among others. Also of interest are plants susceptible to or Fusarium virguliforme, including tobacco plants including but not limited to Nicotiana tabacum. The gdhA gene can come from various non-plant genes (such as bacteria, yeast, animals, and viruses). The gdhA gene can also come from plants. The gene insert used herein was either an E. coli glutamate dehydrogenase gene or a mutagenized version thereof. Another gdhA gene of particular interest is from Chlorella.
The DNA used for transformation of these plants clearly may be circular, linear, double or single stranded. Usually, the DNA is in the form of a plasmid. The plasmid usually contains regulatory and/or targeting sequences which assists the expression of the gene in the plant. The methods of forming plasmids for transformation are known in the art. Plasmid components can include such items as: leader sequences, transit polypeptides, promoters, terminators, genes, introns, marker genes, etc. The structures of the gene orientations can be sense, antisense, partial antisense, or partial sense: multiple gene copies can be used.
The regulatory promoters employed in the present disclosure can be constitutive such as CaMv35S for dicots and polyubiquitin for monocots or tissue specific promoters such as CAB promoters, etc. Promoters may include but are not limited to octopine synthase, nopaline synthase, CaMv19S, and mannopine synthase. These regulatory sequences can be combined with introns, terminators, enhancers, leader sequences and the like in the material used for transformation.
The isolated DNA is then transformed into the plant. Many dicots can easily be transformed with Agrobacterium. Some monocots are more difficult to transform. As previously noted, there are a number of useful transformation processes. The improvements in transformation technology are beginning to eliminate the need to regenerate plants from cells. Since 1986, the transformation of pollen has been published and recently the transformation of plant meristems has been published. The transformation of ovum, pollen, and seedlings meristem greatly reduce the difficulties associated with cell regeneration of different plants or genotypes within a plant can be present.
The most common method of transformation is referred to as gunning or microprojectile bombardment. This biolistic process has small gold-coated particles coated with DNA shot into the transformable material. Techniques for gunning DNA into cells, tissue, callus, embryos, and the like are well known in the prior art.
After the transformation of the plant material is complete, the next step is identifying the cells or material that has been transformed. In some cases, a screenable marker can be employed, such as the beta-glucuronidase gene of the uidA locus of E. coli. Thus, the cells expressing the colored protein are selected for either regeneration or further use. In many cases, the transformed material is identified by a selectable marker. The putatively transformed material is exposed to a toxic agent, such as A. flavus or Fusarium virguliforme, in varying amounts. The cells that are not transformed with the selectable marker that provides resistance to the toxic agent die. Cells or tissues containing the resistant selectable marker generally proliferate. It has been noted that although selectable markers protect the cells from some of the toxic affects of the herbicide or antibiotic, the cells may still be slightly affected by the toxic agent by having slower growth rates. The present disclosure thus provides a selectable marker for identifying transformed plant materials in the presence of Aspergillus flavus or Fusarium virguliforme. In fact, when combined with the PAT or bar gene which is known to give resistance to phosphinothricin, the cells or plants after exposure to the herbicide often evidence increased growth by weight, and appear more vigorous and healthy.
If the transformed material consists of cell lines then these lines are regenerated into plants. The cell lines are treated to induce tissue differentiation. Methods of regeneration of cellular material have been well known in the art since early 1982. The plants resulting from either the transformation process or the regeneration process are transgenic plants.
To evaluate various plant lines for susceptibility to A. flavus or Fusarium virguliforme, and the effect of gdhA expression in transformed lines, various lines and hybrids can be tested as provided in the Examples. For example, various gdhA− corn lines can be tested as described in Example 4 to select lines that demonstrate at least a moderate level of ear rot following exposure to Aspergillus. Such lines can then be used to evaluate, as described in the Examples, the effect of transformation with gdhA on aflatoxin resistance in each line. Exemplary corn lines are listed in Table 1, which is an exemplary seed inventory at Southern Illinois University at Carbondale (Carbondale, Ill.).
Similarly, various gdhA− plant lines, such as a tobacco plant line, can be tested as described in Example 8 to select lines that demonstrate at least a moderate level of root rot following exposure to Fusarium virguliforme. Untransformed and transformed plants from such lines can be used to evaluate, as described in the Examples, the effect of transformation with gdhA on root rot resistance in each line. Any plant from the known wide host range in which F. virguliforme causes root rot can be used, including for example legumes such as soybean, pea, snap bean, alfalfa, and green bean.
Expression of gdhA in transformed plants impacts certain metabolic pathways. The levels of plant metabolites in gdhA+ and gdhA− plants can be compared to help establish the impact of gdhA expression on plant function. Tissue from gdhA+ plants is extracted, analyzed for the levels of selected metabolites and those levels compared to those in unaltered plants (gdhA−).
According to the methods disclosed herein, expression of the bacterial NADP-specific glutamate dehydrogenase in transformed plants is in an amount sufficient to reduce or eliminate the effects of Aspergillus flavus or Fusarium virguliforme in comparison to the effects of these infectious agents observed in an untransformed plant from the susceptible plant line. That a sufficient amount of expression has been achieved is readily determined, for example, by methods described herein. For example, a sufficient amount of bacterial NADP-specific glutamate dehydrogenase expression in transformed plants is achieved when plants demonstrate a reduced severity of ear rot following exposure to A. flavus, in comparison to untransformed plants also exposed to A. flavus. Alternatively or in addition, a sufficient amount of bacterial NADP-specific glutamate dehydrogenase expression in transformed plants is achieved when plants demonstrate a reduction in aflatoxin accumulation following exposure to A. flavus, in comparison to untransformed plants also exposed to A. flavus. Measures of ear rot severity and aflatoxin accumulation are readily obtained using methods described herein or as otherwise well-known in the art. For example, positive or negative reference or cut-off values for ear rot severity or may be established using the prior established effects of A. flavus on transformed or untransformed plants. Similarly, a sufficient amount of bacterial NADP-specific glutamate dehydrogenase expression in transformed plants is achieved when plants demonstrate a reduction in severity of root rot following exposure to F. virguliforme, in comparison to untransformed plants also exposed to F. virguliforme. Measures of root rot severity are readily obtained using methods described herein or as otherwise well-known in the art.
The following examples are thus included to demonstrate various aspects and iterations of the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to function well in the practice of the invention. Those of skill in the art should, however, 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, therefore all matter set forth or shown in the accompanying drawings is to be interpreted as illustrative and not in a limiting sense.
In the examples set forth herein below, the gdhA gene refers to the DNA sequence of the gdhA gene of Escherichia coli, which encodes a 447 amino acid polypeptide subunit of NADP-specific glutamate dehydrogenase as presented in 1982 in Nucleic Acids Research, Volume II, Number 15, 1983. The present examples will illustrate the gdhA gene transformed into a monocot (corn) plant, and a dicot (tobacco) plant. A complete description of a method for transforming a dicot plant (the tobacco Nicotiana tabacum var. Petite Havana) with the gdhA gene is also provided for example in U.S. Pat. No. 6,329,573, “Plants Containing the gdhA Gene and Methods of Use Thereof”.
A. Sequences and Formation of Plasmid
A bacterial glutamate dehydrogenase (gdhA) gene, shown in
The uidA gene from pBI121.1 (pBI121 plasmid is commercially available from Clontech Laboratories, Palo Alto, Calif.), (Jefferson, 1987) was removed by restriction digest with XbaI and SacI and the gel eluted PCR products were ligated into the resulting 9.7 kb fragment of pBI121.1. The amino acid sequence of the GDH enzyme produced by the gdhA gene is shown in (SEQ ID NO:10)
B. Construction of Plasmids to Transfer E. Coli gdhA to Zea mays
The pBI121::GDH plasmid (shown in
The modified E. coli gdhA gene (shown in
The 3′ EcoRI SphI adapter is between nosT and the plasmid for corn transformation. This gives pUBGDH1 (shown in
The plasmid pUBGDH1 (shown in
Because the pBI121::GDH plasmid was not suitable for Zea mays transformation or gene expression, another plasmid vector was used to achieve gdhA gene transfer and expression. The 1.8 kbp SmaI to EcoRI fragment of pBI121::SSU::GDH1 was isolated and ligated with an EcoRI/SmaI adapter and SmaI digested pUC18. This produced the plasmid pUCSSUGDH1 which was amplified in E. coli DH5. Digestion of pUCSSUGDH1 with SmaI allowed recovery of the SSU::gdhA::nosT as a 1.8 kbp fragment (
Corn kernels from the gdhA− and gdhA+ corn lines were analyzed for levels of a variety metabolites and those levels compared. Table 2 lists the names and molecular formulae (in comparable form) of seven metabolites that were present at different levels in gdhA+ corn compared to gdhA− corn. The third column lists the factor for relative level of the metabolite in gdhA+ corn as compared to the level observed in gdhA-− corn.
Corn kernels from the corn lines DAL (gdhA−) and LL3 (gdhA+) were inoculated with conidia from the aflatoxin B1 (AFB1) producing A. flavus isolate NRLL3357. Endosperms and embryos of corn kernels differ substantially in their chemical composition; endosperms are composed mainly of starch and other carbohydrates, whereas embryos are richer in lipids and proteins. Either the endosperms or the embryos of kernels were wounded by inserting a 26 G hypodermic needle to a depth of 1 mi. The endosperm-wounded kernels were dissected, separating the embryo and endosperm tissues, prior to inoculation. Wounded kernels were surface sterilized with 5% sodium hypochlorite, inoculated with 103 conidia of A. flavus to the wound site, and incubated at 28° C. in a moist chamber to enhance fungal growth. Innoculation of corn kernels with conidia was performed on either embryos or endosperms of corn kernels. Each experimental unit consisted of three kernels placed in a Petri-plate. Three-kernel samples were collected 14 days after inoculation, and fungal growth was assessed by counting the produced conidia using a hemacytometer.
Given the well-established link between conidation and toxin biosynthesis, the reduction of A. flavus conidiation in transgenic (gdhA+) corn plants was expected to produce a reduction in accumulation of the mycotoxin (aflatoxin B1).
Several corn lines were assessed for A. flavus colonization and AFB1 accumulation during the 2006 and 2007 growing seasons (Table 3). Corn lines used in field studies were the inbreds LL3-272, LL3-775 and DL1-005, all of which are transgenic lines developed at Southern Illinois University at Carbondale. LL3-272 and LL3-775 express gdhA whereas DL1-005 does not. The hybrid line B73xLL3-272 was developed at SIU by crossing B73 with LL3-272 (gdhA+). The corn line M182 was known to be resistant to A. flavus whereas the inbred B73 was known to be susceptible to the fungus.
Hybrids were planted in single-row plots with 12 plants per row. Plots were arranged in a randomized complete block design with 3 replications. Primary ears of each plant were inoculated 20 days following the mid-silk growth stage with a conidial suspension of A. flavus isolate NRLL3357 using the pinbar method (see Zummo and Scott, Plant Disease, 1992, 76:771-773). Ears were harvested 60 days after midsilk and visually rated for rotting. Ears were harvested 60 days after mid-silk and visually rated for rotting on a scale of 1 to 10, where 1 corresponded to 10% of inoculated area rotted, and 10 indicated 100% of inoculated area rotted. All ears tested were at least minimally susceptible, i.e. showed at least some sign of infection. However, gdhA+ lines showed significantly less rot.
A total of fourteen corn lines were assessed for A. flavus colonization and AFB1 accumulation during the 2006 and 2007 growing seasons. All lines were previously shown to be susceptible to A. flavus. Six of these lines, four hybrids and two isolines, were transgenic expressing the gdhA gene (gdhA+). The plants were planted in single-row plots with twelve plants per row. Plots were arranged in a randomized complete block design with two replications. Primary ears of each plant were inoculated twenty days following the midsilk growth stage with a conidial suspension of A. flavus isolate NRLL3357 using the pinbar method (Zummo and Scott 1992). Ears were harvested 60 days after midsilk and visually rated for rotting on a scale of 1 to 10, where 1 corresponded to 10% of inoculated area rotted, and 10 indicated 100% of inoculated area rotted. Wounded kernels from the inoculated area of the ear and the surrounding two rows were manually collected and analyzed for aflatoxin B1 using High Pressure Liquid Chromatography (HPLC).
Results are reported in Table 4. Inbred line LL3-272 is a transgenic line developed at Southern Illinois University at Carbondale. LL-3, LL3-2, LL3-7, LL200, LL3-272 are lines from different event transformations (selfed at least three times). DL5 is a line from corn transformed with an empty vector. Controls included Mp420, Pioneer, B73, H99 and DL5. H99 is the parent line used in the original transformation.
Results revealed up to 56% reduction in fungal colonization in transgenic, gdhA+ expressing lines. HPLC analysis showed that gdhA+ expressing lines had up to 70% less accumulated aflatoxin compared to lines not expressing the gdhA gene.
aMeans followed by different letters are significantly different at the 0.05 probability level by the least significant ratio test.
bRatings on 1 to 10 scale: 1 = 10%, TO 10 = 100% of inoculated area rotted.
Real-time PCR was used to assess expression levels of a corn lipoxygenase gene in transgenic corn plants. No significant differences were found in expression levels of the gene in transgenic plants (gdhA+) as compared to unaltered plants.
To assess expression of the corn lipoxygenase gene, embryos of corn kernels from corn lines of interest can be inoculated with A. flavus as described in previous examples. Ten days after inoculation, total RNA is extracted from the embryos of the corn kernels. The RNA from each experimental unit (three kernels in a Petri-plate) is pooled. Real-Time PCR is used to assess the expression levels of corn lipoxygenase genes. Primers used in the Real-Time PCR experiment are designed based on the published sequences of lipoxygenase genes identified in corn (GenBank accession numbers: AF465643, and AF329371). Primers specific to the constitutively expressed α-tubulin genes are used as an internal standard (Giedt and Weil, 2000, Plant Journal, 24:815-823). The previously extracted RNA will contain both plant and fungal RNA if the corn kernels harbor any fungal growth. Real-Time PCR is also used to quantify the extent of colonization of the kernels by A. flavus by assessing the expression level of a constitutively expressed actin gene from A. flavus. Accumulation of AFB1 is assessed by quantifying the expression levels of the gene verA, one of the genes involed in AFB1 biosynthesis. Expression data is analyzed as described by (Tsitsigiannis et al., 2005, Molecular Plant-Microbe Interactions, 18:1081-1089). The expression level of the genes is determined by first normalizing the target RNA to the internal RNA (actin) using the 2(Ct actin)-(Ct target) formula (Livak and Schmittgen, 2001, Methods, 25:402-408; Pfaffl, 2001, Nucleic Acids Research, 29:2002-2007). The same approach is also used to validate expression data in the field. The results reveal differences in the expression patterns of the lipoxygenase genes amongst the different tested corn lines and whether challenging the different corn lines with A. flavus affects the expression level of these genes.
In this example the gdhA gene was shown to provide resistance to root rot and protected root growth in corn and tobacco plants when Fusarium virguliforme was present. Root weight was higher in gdhA+ plants than in gdhA− plants (Table 5). Root rot was much less in gdhA+ plants than in gdhA− plants (Table 5). Additionally, the root health depended on the amount of gdhA expression. Metabolites found altered in the roots of gdhA+ plants are believed to cause the observed resistance (Tables 6A-6J).
Methods were as follows: seed of Zea mays (Zm) and Nicotiana tabacum (Nt), either with or without the gdhA gene were germinated in a sand soil mix for 14 days. The plants were transferred to F. virguliforme infested media prepared as follows: Fusarium virguliforme strain Monticello 1, maintained on 5× Bilays medium at 19° C., was transferred onto PDA plates at 28° C. for inoculum preparation.
Cornmeal Sand Assay: The cornmeal method of inoculation was used with a few modifications. Two 1 cm×1 cm square pieces of the infested agar was transferred into a 50 ml volume of cornmeal and silica mix (1:1) and moistened with sterile water. This was kept in an incubator for 14 days. At the same time, seeds were sown on steamed sand. After 14 days, the inoculum was mixed with steamed sand/soil (1:1 v/v) in a 1:40 proportion. Pots with 0.5 L of media were filled and 14 day old seedlings were transferred into the filled pots. These were set in water-filled basins with enough water to keep the lower 1-2 inches flooded and the rest of the root zone moist. An equal number of non-inoculated plants were kept under identical conditions. Root infection was rated at day at 28 days after inoculation, using a 1 to 5 scale for describing root rot, with 1 healthy and 5 completely rotted.
Tables 6A through 6J list the various metabolites, by class of compound, that were found to be altered in gdhA+ Nicotiana tabacum plants, when compared to unaltered (gdhA−) plants.
amass is ±1 ppm, or 0.0002-0.00001 d
b% changes are ±2%
a: mass is ±1 ppm, or 0.0002-0.00001 d
b: % changes are ±2%
amass is ±1 ppm, or 0.0002-0.00001 d
b% changes are ±2%
amass is ±1 ppm, or 0.0002-0.00001 d
b% changes are ±2%
a: mass is ±1 ppm, or 0.0002-0.00001 d
b: % changes are ±2%
a: mass is ±1 ppm, or 0.0002-0.00001 d
b: % changes are ±2%
a: mass is ±1 ppm, or 0.0002-0.00001 d
b: % changes are ±2%
†Pesticide or Herbicide
‡Drug
amass is ±1 ppm, or 0.0002-0.00001 d
b% changes are ±2%
†Pesticide or Herbicide
‡Drug
amass is ±1 ppm, or 0.0002-0.00001 d
b% changes are ±2%
Fusarium virguliforme.
This application claims priority from U.S. Provisional Application Ser. No. 61/153,576 filed on Feb. 18, 2009, the disclosure of which is hereby incorporated by reference in its entirety.
| Number | Name | Date | Kind |
|---|---|---|---|
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| 5489520 | Adams et al. | Feb 1996 | A |
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| Number | Date | Country | |
|---|---|---|---|
| 20100223693 A1 | Sep 2010 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61153576 | Feb 2009 | US |
