Double stranded RNA compositions for reducing asian citrus psyllid infestation and methods of use
Inventors
Hunter, Wayne B. • Gonzalez, Maria T. • Andrade, Eduardo C.
Assignees
Empresa Brasileira de Pesquisa Agropecuaria EMBRAPA • US Department of Agriculture USDA
Publication Number
US-10344291-B2
Publication Date
2019-07-09
Expiration Date
2037-01-25
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Abstract
dsRNA generated from D. citri trehalase gene is effective in reducing fitness and/or survival of D. citri. Thus genetically altered plants expressing the dsRNA and plants to which dsRNA solutions are applied increase D. citri mortality and reduce D. citri infestation. With reduced D. citri population, the spread of microorganisms for which D. citri is a vector is reduced. Such microorganisms include, but are not limited to, C. Liberibacter species, including: CLas, CLam, and CLaf. Thus, applying of the D. citri trehalase dsRNA to a plant reduces disease and/or microorganism transmission by killing D. citri that feed on the treated plant.
Core Innovation
The invention relates to the use of double-stranded RNA (dsRNA) generated from the Diaphorina citri trehalase gene to reduce the fitness and/or increase mortality of the Asian citrus psyllid (D. citri). This dsRNA, when ingested by D. citri, causes a sequence-specific RNA interference effect leading to gene silencing of the trehalase gene, which is important for insect energy metabolism. Consequently, genetically altered plants expressing such dsRNA or plants treated with dsRNA solutions show increased mortality of D. citri and reduced infestation levels.
The main problem addressed by the invention is the devastating impact of Huanglongbing (HLB) disease in citrus crops, especially caused by Candidatus Liberibacter species (such as CLas, CLam, and CLaf), which are transmitted by D. citri as a vector. There is currently no effective treatment for HLB, and controlling the D. citri vector is critical to limiting disease spread. Existing methods insufficiently control this pest while sparing beneficial insects. The invention solves this by providing sequence-specific dsRNA compositions that reduce D. citri populations without harming beneficial insects like pollinators.
The invention includes compositions containing D. citri trehalase dsRNA and methods of using these compositions by topical application (e.g., spraying or soil application) on plants to be fed upon by D. citri. It also encompasses genetically altered plants and microorganisms engineered to express the dsRNA, which, when fed upon by D. citri, reduce their fitness or survival. The dsRNA solutions can contain agriculturally acceptable carriers and optional additives to enhance stability or delivery. The invention utilizes the specificity of RNAi technology, targeting sequences unique to D. citri trehalase to minimize off-target effects.
Claims Coverage
The patent includes multiple independent claims covering compositions of dsRNA, compositions containing dsRNA solutions, methods for reducing D. citri infestation or fitness via dsRNA application, genetically altered plants expressing dsRNA, and methods employing recombinant plant viruses expressing the dsRNA sequences.
dsRNA composition targeting D. citri trehalase
A dsRNA comprising a sense sequence of at least 21 contiguous nucleotides equivalent to D. citri trehalase cDNA (SEQ ID NO: 26) and an anti-sense region complementary to the sense region with at least 95% identity, wherein ingestion or absorption of this dsRNA kills D. citri.
dsRNA solution with agriculturally acceptable carriers
A dsRNA solution comprising the D. citri trehalase dsRNA and an agriculturally acceptable carrier such as water, surfactant, liposome, lipid, protein, peptide, nanotube, chitin, or inactivated microorganisms, optionally including compounds that prevent dsRNA degradation or other additives for improved delivery or stability.
Methods of reducing D. citri infestation by topical or soil application
Methods for reducing D. citri infestation by administering a dsRNA solution with insecticidal activity to untreated plants in an amount effective to kill D. citri that ingest or absorb the dsRNA, including applications by spraying onto plants, applying to soil for root absorption, or applying directly to roots.
Methods of reducing D. citri fitness or survival using genetically altered plants
Methods for reducing D. citri fitness or survival by introducing dsRNA into wild-type plants to produce altered plants expressing the dsRNA, allowing D. citri to feed on them and ingest the dsRNA, with the dsRNA encoding sequences selected from defined regions of SEQ ID NOs: 4, 9, 14, or 26 (or their complements), delivered via transformation or topical/soil application.
Genetically altered plants expressing D. citri trehalase dsRNA
Genetically altered plants and parts thereof comprising expression vectors with promoters operably linked to D. citri trehalase sense and anti-sense polynucleotides forming dsRNA sequences derived from specified segments of SEQ ID NOs: 1, 6, 11, or 26, with promoters that may be the same or different, wherein D. citri are killed upon ingestion or absorption of the dsRNA.
Methods of making genetically altered plants producing dsRNA
Methods for producing genetically altered plants by transforming wild-type plant cells with expression vectors encoding the dsRNA sequences, selecting transformed cells, and growing these into genetically altered plants producing dsRNA that kills D. citri upon ingestion or absorption.
Methods employing recombinant plant viruses expressing dsRNA
Methods for reducing D. citri fitness or survival by infecting wild-type plants with recombinant plant viruses (DNA or RNA viruses) containing heterologous polynucleotides encoding D. citri trehalase dsRNA or fragments, whereby the virus produces dsRNA during replication in the plant, and feeding of D. citri on the infected plants causes ingestion of the insecticidal dsRNA.
The claims encompass compositions of sequence-specific dsRNA targeting D. citri trehalase, dsRNA-containing solutions with delivery carriers, methods of topical and genetic application to plants to control D. citri populations, genetically engineered plants producing dsRNA, and virus-induced gene silencing approaches to confer insecticidal effects on D. citri feeding on treated or altered plants.
Stated Advantages
Increased mortality and reduced infestation of D. citri by ingestion of dsRNA targeting trehalase.
Reduction in transmission of HLB disease-causing microorganisms by killing or reducing fitness of the D. citri vector.
Specificity to D. citri without harming beneficial insects such as honey bees and other pollinators.
Multiple delivery methods including topical spray, soil application, genetic transformation, or virus-induced gene silencing provide versatility.
Documented Applications
Reducing fitness and survival of D. citri on citrus and other host plants to reduce psyllid infestation.
Applying dsRNA solutions or genetically altering plants to increase D. citri mortality and thereby reduce spread of Huanglongbing (HLB) disease caused by Candidatus Liberibacter species such as CLas, CLam, and CLaf.
Using genetically engineered plants or recombinant viruses to produce dsRNA that controls D. citri populations in citrus groves or other affected citrus-producing regions.
Reducing pesticide impact by providing sequence-specific RNAi compositions targeting D. citri trehalase gene.
Interested in licensing this patent?