RNA-based control of powdery mildew
Inventors
PURALEWSKI, Adriana • Lawrence, Christopher • DEVISETTY, Upendra Kumar • MISHRA, Sambit Kumar • Sridharan, Krishnakumar • CADLE DAVIDSON, Lance E. • Underhill, Anna N. • FANG, Yufeng • KOKULAPALAN, Wimalanathan
Assignees
US Department of Agriculture USDA • Greenlight Biosciences Inc
Publication Number
US-12077763-B2
Publication Date
2024-09-03
Expiration Date
2042-03-08
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Abstract
Disclosed herein are compositions and methods that involve recombinant polynucleotide molecules, such as single or double-stranded DNA or RNA molecules, also referred to “triggers”, that are useful for controlling or preventing E. necator infection, or recombinant DNA constructs for making such RNA molecules or for making transgenic grape plants resistant to E. necator infection. In some embodiments, polynucleotide triggers are provided as topically applied agents for controlling or preventing infection of a plant by E. necator.
Core Innovation
The invention relates to compositions and methods involving recombinant polynucleotide molecules, such as single or double-stranded DNA or RNA molecules referred to as "triggers", useful for controlling or preventing infection by Erysiphe necator (E. necator), the fungal pathogen causing grape powdery mildew. The compositions include recombinant DNA constructs for producing such RNA molecules or for generating transgenic grape plants resistant to E. necator infection. Polynucleotide triggers are provided as topically applied agents, such as sprays, for controlling or preventing infection of plants by E. necator.
The problem being solved is the significant economic impact of grape powdery mildew worldwide, specifically by E. necator, which requires extensive pesticide applications and causes losses in quality and yield. Current management relies heavily on chemical fungicides, to which E. necator rapidly develops resistance, creating a high-risk situation. The heavy use of chemical treatments also raises environmental and human health concerns. Therefore, there is an urgent need for alternative, sustainable, and environmentally friendly methods to control E. necator. The invention aims to provide such alternative methods by applying RNA interference (RNAi) technology through exogenous double-stranded RNA applications or transgenic expression in grape plants to decrease or eliminate E. necator growth.
Claims Coverage
The patent contains one independent composition claim concerning a double-stranded RNA molecule composition for controlling E. necator. The inventive features focus on the specific RNA sequences, their structure, length, and application form, and on recombinant DNA constructs encoding these RNA molecules.
Double-stranded RNA molecule composition targeting E. necator
A composition comprising a double-stranded RNA molecule having two strands each about 600 nucleotides or less in length, where the first strand comprises the nucleotide sequence of SEQ ID NO: 215 and the second strand is complementary thereto, causing mortality or suppression of E. necator when contacted or transfected.
Formulation and additional components of the RNA composition
The composition can be formulated as solids, liquids, powders, suspensions, sprays, or carrier particulates and may include carrier agents, surfactants, organosilicones, polynucleotide or non-polynucleotide pesticidal molecules, safeners, or pathogen growth regulators.
Recombinant DNA construct encoding the double-stranded RNA molecule
A recombinant DNA construct encoding one or more strands of the double-stranded RNA molecule comprising SEQ ID NO: 215 and its complement for producing the RNAi agents against E. necator.
Inclusion of conserved target regions from multiple E. necator strains
The first RNA strand can comprise a conserved region of a target gene present in two or more strains of E. necator, specifically strains such as LNYM, NY90, G14, Pumocnh, SHNC1, BLMT2, Dresden2, NCAES6, CH36, and CH19, improving broad-spectrum efficacy.
RNA strand length range
The strands of the double-stranded RNA molecule are between about 400 and about 600 nucleotides in length, preferably between about 500 and 600 nucleotides.
The independent claim covers compositions of double-stranded RNA molecules with specific sequence and length designed to control E. necator by RNAi, their formulation and additional components, recombinant DNA constructs encoding these RNAs, and the targeting of conserved sequences across various E. necator strains, providing a broad and specific approach to grape powdery mildew control.
Stated Advantages
Allows selective control of E. necator by silencing essential fungal genes via RNA interference.
Provides an environmentally friendly alternative to chemical fungicides, reducing health and environmental risks.
Targets E. necator specifically, minimizing adverse effects on non-target species.
Mitigates the risk of fungicide resistance development by using sequence-specific RNA molecules.
Can be applied topically as sprays, enabling flexible and practical use in vineyards.
Enables production of resistant transgenic grape plants expressing RNA triggers for durable protection.
Documented Applications
Use of recombinant polynucleotide molecules, including double-stranded RNA triggers, for controlling or preventing infection of grape plants by E. necator through topical application such as spraying on grapevine leaves.
Producing transgenic grape plants that express RNA triggers conferring resistance to E. necator infection.
Formulation of compositions comprising double-stranded RNA molecules for application to plants or environments to suppress growth, pathogenicity, or propagation of E. necator.
Use of recombinant DNA constructs encoding RNA molecules targeting E. necator genes for development of resistant grape plants.
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