Gregory R. Heck

Monsanto Company · Department of Chemistry

This invention relates to methods of controlling gene expression or gene suppression in eukaryotic cells. One aspect of this invention includes modifying the degree of silencing of a target gene by use of a modified suppression element. Another aspect includes providing a eukaryotic cell having a desired phenotype resulting from transcription in th...

Background: Glyphosate-resistant goosegrass has recently evolved and is homozygous for the double mutant of EPSPS (T102 I, P106 S or TIPS). These same mutations combined with EPSPS over-expression, have been used to create transgenic glyphosate-resistant crops. Arabidopsis thaliana (Wt EPSPS Ki ≈ 0.5 μM) was engineered to express a variant AtEPSPS...

Environmental RNAi (eRNAi) is a sequence-specific regulation of endogenous gene expression in a receptive organism by exogenous double-stranded RNA (dsRNA). Although demonstrated under artificial dietary conditions and via transgenic plant presentations in several herbivorous insects, the magnitude and consequence of exogenous dsRNA uptake and the...

Environmental RNAi (eRNAi) is a sequence-specific regulation of endogenous gene expression in a receptive organism by exogenous double-stranded RNA (dsRNA). Although demonstrated under artificial dietary conditions and via transgenic plant presentations in several herbivorous insects, the magnitude and consequence of exogenous dsRNA uptake and the...

The present invention relates to control of pest infestation by inhibiting one or more biological functions. The invention provides methods and compositions for such control, By feeding one or more recombinant double stranded RNA molecules provided by the invention to the pest, a reduction in pest infestation is obtained through suppression of gene...

The present invention provides a recombinant DNA molecule that confers herbicide tolerance to transgenic corn plants. Also provided by the present invention are methods for growing a corn plant that tolerate application of glyphosate comprising planting and growing seeds comprising the recombinant DNA molecule and applying glyphosate to the corn pl...

The expanding use of RNA interference (RNAi) in agricultural biotechnology necessitates tools for characterizing and quantifying double-stranded RNA (dsRNA)-containing transcripts that are expressed in transgenic plants. We sought to detect and quantify such transcripts in transgenic maize lines engineered to control western corn rootworm (Diabroti...

A novel Bacillus thuringiensis crystal protein exhibiting insect inhibitory activity is disclosed. Growth of Lygus insects is significantly inhibited by providing the novel crystal protein in Lygus insect diet. Polynucleotides encoding the crystal protein, transgenic plants and microorganisms that contain the polynucleotides, isolated peptides deri...

The compositions and methods disclosed herein provide novel DNA molecules that encode glyphosate resistant EPSPS proteins and plants containing these new proteins. The plants that express the new EPSPS proteins are themselves tolerant to the herbicidal effects of glyphosate.

This invention provides a non-natural transgenic plant cell expressing at least one invertebrate miRNA in planta for suppression of a target gene of an invertebrate pest or of a symbiont associated with the invertebrate pest, recombinant DNA constructs for expression of at least one invertebrate miRNA in planta and methods of use thereof, a non-nat...

Long double-stranded RNAs (long dsRNAs) are precursors for the effector molecules of sequence-specific RNA-based gene silencing in eukaryotes. Plant cells can contain numerous endogenous long dsRNAs. This study demonstrates that such endogenous long dsRNAs in plants have sequence complementarity to human genes. Many of these complementary long dsRN...

RNA interference (RNAi) has previously been shown to be effective in western corn rootworm (WCR, Diabrotica virgifera virgifera LeConte) larvae via oral delivery of synthetic double-stranded RNA (dsRNA) in an artificial diet bioassay, as well as by ingestion of transgenic corn plant tissues engineered to express dsRNA. Although the RNAi machinery c...

Primer sequences used in the study. (DOCX)

Details of gene sequences used in the study. (DOCX)

The role of non-coding RNAs (ncRNAs), both short and long ncRNAs, in the regulation of gene expression has become evident in recent years. Non-coding RNA-based regulation is achieved through a variety of mechanisms; some are relatively well-characterized, while others are much less understood. MicroRNAs (miRNAs), a class of endogenous small RNAs, f...

Plants contain significant quantities of small RNAs (sRNAs) derived from various sRNA biogenesis pathways. Many of these sRNAs play regulatory roles in plants. Previous analysis revealed that numerous sRNAs in corn, rice and soybean seeds have high sequence similarity to animal genes. However, exogenous RNA is considered to be unstable within the g...

Table S1.Public sRNA datasets: organism and sample source distribution.

Table S3.Abundant miRNAs in rice, soy and corn seeds.

Small RNA probes for northern blot analysis.

Table S4.Reads from human datasets map to non-human animal miRNAs.

Figure S1.Comparison of plant miRNA abundance between insect and diet source plant tissues sRNA datasets (A, B and C), and between plant miRNAs from all plant libraries and all insect libraries (D) in the run.

Table S2.Public sRNA datasets and presence of plant miRNAs.

Table S5.Reads from insect libraries map to lettuce-specific sequence.

Table S6.sRNA sequences and raw counts from insect feeding experiment parsed against miRBase v17.

The plant bugs Lygus hesperus Knight (Hemiptera: Miridae) and L. lineolaris (Palisot de Beauvois) have emerged as economic pests of cotton in the United States. These hemipteran species are refractory to the insect control traits found in genetically modified commercial varieties of cotton. In this article, we report the isolation and characterizat...

Statistical analysis of qRT-PCR expression data of SCL6-III (At3g606030) in miR171_2B and miR171_3B plants versus wild type control. One-sized T-test Results from ANOVA. Results are from comparison of each event (homozygous (ho) or hemizygous (he)) versus the wild type control within each tissue, and adjusted p-values were computed from Dunnett's t...

Effect of miRNA decoy structure on efficacy. (A) Sequence of miRMON1 decoys with bulged (3B, 4B, 5B, 6B, 7B) structures between nucleotides 10 and 11 of the miRNA. (B) Expression of miRMON1-targeted GFP reporter co-expressed with 3B, 4B, 5B, 6B or 7B miRNA decoys in N. benthamiana leaves measured by fluorescence microscopy. Leaves were co-transform...

Structure of miRNA decoys and reporter cassette. (A) Diagram of the interaction between bulge and mismatch miRNA decoys and a corresponding miRNA. (B) Diagram of the reporter gene with miRNA target site incorporated into the 3′ UTR. (TIF)

Leaf inclination of wild-type Arabidopsis and miR171 decoy events. Rosette leaf inclination is decreased in plants expressing a miR171 decoy vs. wild-type, with the most dramatic decrease seen in miR171_3B events. (TIF)

Floral phenotypes of miR171_3B Arabidopsis. miR171_3B plants exhibit a closed flower bud phenotype. Sepals and petals were removed, revealing the altered carpel. (TIF)

Statistical analysis of qRT-PCR expression data of SCL6-III (At3g606030) in miR171_3B transgenic decoy events versus miR171_2M transgenic decoy events. Two-sized T-test results from ANOVA. Results are from comparison of two constructs within each tissue type. No adjustments were done to the raw p-values. (DOC)

Sequence information. (DOC)

Predicted endogenous miRNA decoy sites in Arabidopsis thaliana. For each unique miRNA sequence, the columns are miRNA ID (random if multiple loci encodes the same sequence), sequence (5′ – 3′), and all other miRNA IDs if they share the same sequence. For each predicted decoy, there are three rows listing (1) the query miRNA sequence (3′ – 5′), (2)...

Eukaryotic organisms possess a complex RNA-directed gene expression regulatory network allowing the production of unique gene expression patterns. A recent addition to the repertoire of RNA-based gene regulation is miRNA target decoys, endogenous RNA that can negatively regulate miRNA activity. miRNA decoys have been shown to be a valuable tool for...

Proteins that are synthesized on cytoplasmic ribosomes but function within plastids must be imported and then targeted to one of six plastid locations. Although multiple systems that target proteins to the thylakoid membranes or thylakoid lumen have been identified, a system that can direct the integration of inner envelope membrane proteins from t...

Introduction Target-Site Genomics Nontarget-Site Genomics The Biochemistry and Physiology Connection Screening Candidate Genes for Functionality Conclusions Acknowledgments References

Introduction Mechanisms of Engineering Glyphosate Resistance Development of First-Generation GR Crops Identification of at-Risk Tissues for Glyphosate Injury Development of Second-Generation GR Crops Use of Alternative Herbicides for Weed Resistance Management Engineering Crop Resistance to the Glufosinate Herbicide Engineering Crop Resistance to t...

Glyphosate is a broad spectrum herbicide that inhibits the activity of endogenous plant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Glyphosate resistance has been engineered in multiple crops by constitutive expression of an EPSPS variant from Agrobacterium sp. CP4 (CP4 EPSPS) that binds glyphosate less efficiently. The first glyphosate-re...

The evolution of glyphosate resistance in weedy species places an environmentally benign herbicide in peril. The first report of a dicot plant with evolved glyphosate resistance was horseweed, which occurred in 2001. Since then, several species have evolved glyphosate resistance and genomic information about nontarget resistance mechanisms in any o...

The mechanism of glyphosate resistance in horseweed was investigated. Eleven biotypes of putative sensitive (S) and resistant (R) horseweed were obtained from regions across the United States and examined for foliar retention, absorption, translocation, and metabolism of glyphosate. Initial studies used spray application of 14C-glyphosate to simula...

Commercial biotechnology solutions for controlling lepidopteran and coleopteran insect pests on crops depend on the expression of Bacillus thuringiensis insecticidal proteins, most of which permeabilize the membranes of gut epithelial cells of susceptible insects. However, insect control strategies involving a different mode of action would be valu...

Bioassay screening of Bacillus thuringiensis culture supernatants identified strain EG2158 as having larvicidal activity against Colorado potato beetle (Leptinotarsa decemlineata) larvae. Ion-exchange fractionation of the EG2158 culture supernatant resulted in the identification of a protein designated Sip1A (secreted insecticidal protein) of appro...

5-Enol-pyruvylshikimate-3-phosphate synthase from Agrobacterium sp. CP4 (CP4 EPSPS) confers tolerance to the nonselective herbicide glyphosate (marketed under the trade name Roundup1) when sufficiently expressed in transgenic plants. Dual CP4 EPSPS transgene cassettes were transformed into corn (Zea mays L.) under the transcriptionat regulatory con...

Crop resistance can be generated through a selection process or the introduction of a gene via transformation. We will refer to the latter as engineered herbicide resistance. The literature contains numerous studies on engineered plant resistance to various herbicide classes. There is a large distinction between demonstrating a resistance phenotype...

The herbicide Roundup® whose active ingredient is glyphosate (N-[phosphonomethy1]-glycine) was introduced as a broad spectrum, post emergent herbicide in 1974. Extensive research by a number of groups showed that glyphosate’s herbicidal activity resulted from inhibition of the enzyme 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPS) a key enzyme...

AGL15 (AGAMOUS-like 15), a member of the MADS domain family of regulatory factors, accumulates preferentially throughout the early stages of the plant life cycle. In this study, we investigated the expression pattern and possible roles of postembryonic accumulation of AGL15. Using a combination of reporter genes, RNA gel blot analysis, and immunoch...

Gibberellins are noted for their ability to induce expression of genes, such as alpha-amylase, in the aleurone layers of cereals. However, a number of mRNA species in the mature imbibed aleurone cell of barley, such as a storage globulin (Heck et al., Mol Gen Genet 239: 209-218 1993), are simultaneously and specifically repressed by gibberellin. In...

To extend our knowledge of genes expressed during early embryogenesis, the differential display technique was used to identify and isolate mRNA sequences that accumulate preferentially in young Brassica napus embryos. One of these genes encodes a new member of the MADS domain family of regulatory proteins; it has been designated AGL15 (for AGAMOUS-...

To extend our knowledge of genes expressed during early embryogenesis, the differential display technique was used to identify and isolate mRNA sequences that accumulate preferentially in young Brassica napus embryos. One of these genes encodes a new member of the MADS domain family of regulatory proteins; it has been designated AGL15 (for AGAMOUS-...

On Jul 15, 1996 this sequence version replaced gi:790638.

We report identification of a 2189 bp cDNA clone from barley corresponding to a single-copy gene, Beg1 (Barley embryo globulin), on chromosome 4, which encodes a storage globulin. In barley, the major protein reserve in the aleurone layer belongs to the 7S globulin class of proteins found in many seeds. Electrophoretically and antigenically similar...

We have isolated and sequenced a 1400-base-pair cDNA derived from gibberellic acid-treated aleurone cell mRNA. This sequence contains an open reading frame that would code for a protein of 361 amino acids. The carboxyl-terminal two-thirds of the predicted amino acid sequence is closely related to that of the rat lysosomal thiol protease cathepsin H...

In a 67-year old man, an intra-abdominal fusiform cell sarcoma developed ; twenty years after thorotrast application by arteriography of the right leg. ; Thorotrast deposition was detected by x rays in the liver and spleen. The ; sarcoma had affected the omentum, the mesentery, and the intestinal fluid, ; penetrated the pancreas, and formed a retro...