Produces Higher Levels of RNAi to Silence Specific Genes, Protecting Plants Against Bacterial and Fungal Pathogens
These self-amplifying RNAs will silence specific plant host genes and thereby protect against multiple microbial pathogens such as CLas, the bacterium that causes citrus greening disease. However, this platform technology, which is in development, will work with any plant, not just citrus, using a key component nanoemulsion to deliver double-stranded RNA (dsRNA) into plant cells using foliar sprays efficiently and economically.
Applying double-stranded RNA by foliar spray efficiently silences specific host target genes and protects plants against both intracellular and intercellular bacterial and fungal pathogen attack. All animal and plant cells have highly regulated cell defense responses and programmed cell death mechanisms designed to limit the damage caused by invading pathogens. These defense responses and mechanisms serve as genetic brakes to prevent runaway damage spreading from the initial site of infection to the entire organism. Initiation of host programmed cell death requires sensors to recognize pathogen invasion, typically by a combination of factors that trigger or potentiate the response. Most pathogens have evolved to avoid triggering these responses, and even to actively suppress them to the point where the initially infected cells fail to react.
For example, CLas is a stealthy bacterial pathogen that causes citrus greening disease and systemically invades living host phloem cells and then multiplies, initially without any disease symptoms. It also multiples in living host insect vector cells. In Florida, CLas now has infected nearly every citrus grove; loss of production caused by this disease has cost the state’s economy in excess of $3.5 billion. One available strategy to manage citrus greening involves spray applications of dsRNA to suppress the genetic brakes on host defense responses, including programmed cell death. However, the cost of large-scale field application of sprayed dsRNA is exorbitant, and effects are typically short-lived, requiring multiple applications per growing season. University of Florida researchers are developing self-amplifying RNA that when applied by foliar spray both significantly amplifies and extends the duration of the effect of dsRNA in suppressing specific genetic brakes on host defense responses.
Application
Self-amplifying RNA to target and silence one or more genes of any plant species to reduce or eliminate damage induced by microbial pathogens
Advantages
- Self-amplifying RNA reduces the amount of dsRNA required for spray treatment by several orders of magnitude, increasing the effectiveness of treatment and enhancing its longevity
- Multiple plant and/or insect vector target genes may be included in the self-amplifying to simultaneously silence multiple genes
- Self-amplifying RNA does not create a GMO, is not infectious, and its effects are not permanent
- The siRNAs created from the dsRNA are phloem mobile thus will reach all parts of the plant
- Self-amplifying RNAs are readily modified target genes in many different plant species, increasing the number of potential applications
Technology
This platform technology will enable application of self-amplifying RNAs to produce much higher levels of RNA interference (RNAi) in plants than if dsRNA were applied alone. The self-amplifying RNA uses an RNA-dependent RNA polymerase (RdRp) based on a grapevine alphavirus to amplify both itself and a subgenomic region encoding a synthetic chimera within plant cells. The chimera encodes host RNA sequences from one or more plant or insect vector target genes that forms a dsRNA. dsRNA is recognized by the plant enzyme Dicer (RNAse III) and cleaved, creating small interfering RNAs (siRNAs) of about 21 nucleotides in length. In plants, these siRNAs are phloem mobile and become systemic. These siRNAs can also be acquired by insects feeding on these cells. Each 21-mer fragment is unwound by helicases (in any host cell) to form single-stranded 21-mers, including the antisense strand that causes RNAi by its incorporation into the RNA-induced silencing complex (RISC). The siRNA/RISC complex then specifically cleaves or blocks expression of the targeted messenger RNA (mRNA), thereby causing RNAi, including in insect vectors that feed upon the plant cells containing the siRNAs. In the case of target genes that encode genetic brakes on defenses including programmed cell death, such suppression can provide protection from intracellular and intercellular infections by bacterial and fungal infections.
In order for an aqueous spray to get past the waxy leaf cuticle, various processes must be used to overcome RNA’s highly anionic and hydrophilic nature, which typically inhibits cellular uptake. In order to penetrate into living phloem cells, aqueous solutions of dsRNA or self-amplifying RNA are encapsulated in negatively charged nanoemulsions consisting of lecithin, gelatin. The dsRNA nanoparticles become positively charged when in contact with acidic citrus phloem.
Brochure
