Plant virus protein enabled nucleic acid delivery for RNA interference in plant pests

Abstract

Provided herein a spherical nanoparticle (SNP) comprised of a plant virus coat protein and dsRNA for controlling plant parasitic nematodes.

Description

PLANT VIRUS PROTEIN ENABLED NUCLEIC ACID DELIVERY FOR RNA INTERFERENCE IN PLANT PESTSCROSS-REFERENCE TO RELATED PATENT APPLICATIONS

[0001] This application claims priority under 35 U.S.C. § 119(e) of U.S. Provisional Application No.: 63 / 560,553, filed March 1, 2024, the content of which is incorporated herein by reference.STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under grant no. NIFA-2020- 67021-31255, awarded by the National Institute of Food and Agriculture (NIFA). The government has certain rights in the invention.BACKGROUND OF THE DISCLOSURE

[0003] Soil is the primary substrate for plant growth, offering the basic nutrients, water, and physical support required for agricultural production. Additionally, soil contains a significantly greater proportion of stored carbon than aboveground vegetation and is critically important for the regulation of carbon dioxide and other greenhouse gases. Soil also provides a habitat for many of the diverse organisms needed for ecosystem processes. Soil fertility is often considered the most important quality characteristic for crop production, but it is not indicative of soil health. Soil fertility considers the optimal characteristics needed to grow crops, but soil health encompasses the many complex abiotic and biotic interactions that are required for ecosystem services. In the short term, high fertility inputs may help to increase crop yields, but this does not address long-term ecosystem functions that sustainable soil management can provide. Embedded in the concept of soil health is the impact of soil-borne pests and pathogens, which account for 10-20% of annual crop losses. In a worldwide survey of growers, soil-borne pests and pathogens such as nematodes, arthropods, fungi, oomycetes, bacteria, and weeds were listed as one of the most important limitations to productivity.

[0004] Phytoparasitic nematodes are non-segmented roundworms that have adapted to feed and breed on the roots of plants. More than 4100 species have been identified and they can bedescribed as sedentary or migratory depending on the permanence of their feeding site in the root system. Sedentary parasites such as root-knot nematodes (e.g., Meloidogyne spp.) and cyst nematodes (e.g., Heterodera and Globodera spp.) have a major economic impact on agriculture, causing annual yield losses of US$157 billion. Root-knot nematodes can attack more than 2000 plant species and are responsible for 5% of all crop losses worldwide. The recent appearance of M. enterolobii in US states Florida, North Carolina, Louisiana, and South Carolina has prompted a range of internal and external quarantine measures. Like insects, nematodes molt between juvenile stages and in root-knot nematodes the first molt occurs within the egg. The hatchlings are therefore second-stage juveniles (J2) and this is a vermiform stage in the soil and rhizosphere of host plants, allowing the invasion of nearby roots. J2 nematodes typically do not travel more than ~1 m in their lifetime based on their intrinsic motility, but spreading is facilitated by agricultural practices such as the movement of contaminated soil between fields via farming equipment, as well as by events such as flooding. Symptoms caused by phytoparasitic nematodes include wilting, yellowing and stunting, but these are often misdiagnosed as the consequences of abiotic stress.

[0005] The design of nematode control strategies has been facilitated by the huge amount of data available for Caenorhabditis elegans, a widely used model organism and the first animal with a fully sequenced and annotated genome. Genome analysis has revealed a large number of conserved signaling pathways involved in animal development, immunity, behavior, and neurotoxicity responses. Although C. elegans feeds on bacteria, it shares many genes with phytoparasitic nematodes due to these conserved pathways. Any genes only found in phytoparasitic nematodes are therefore likely to reflect unique lifestyle attributes and can therefore help to identify suitable targets. These include genes involved in the development and function of sensory organs that allow the location of host plants, signaling pathways and metabolic pathways required for host-nematode interactions, and pathways required for the development and function of the stylet, which punctures plant cells and extracts nutrients.

[0006] Current measures to control pathogenic nematodes

[0007] The management of soil-borne phytoparasitic nematodes is challenging. In most countries, nematodes are controlled by crop rotation, but the choice of alternative crops is limited by the broad host range of root-knot nematodes. Cultural control methods offer themost environmentally sustainable approaches but they require careful planning and land available for rotation. Crop rotation with non-host plants can reduce the population of phytoparasitic nematodes; crop rotation also can increase soil fertility because different crops use and replenish different nutrients at different rates. On the other hand, complexity in management and increased labor (different crops may require different cultivation techniques) and initial costs for set up (seeds and equipment needs) as well as limited crop choices lower the economic returns. Nematode-resistant cultivars also offer a promising solution, but genetic resistance to nematodes is species-dependent, limited to a few crops, and takes years to engineer. Resistance to root-knot nematodes is provided by the dominant Mi resistance gene, which was introgressed from Solanum peruvianum to cultivated tomato in the 1940s. The Mi gene has been incorporated in tomato, but root-knot nematode resistance breaks down if the soil temperature is too high and the Mi gene does not confer resistance against one of the most aggressive species, M. enterolobii.

[0008] Other non-chemical approaches include flooding, solarization, and steaming. Flooding creates an anaerobic environment that inactivates and kills phytoparasitic nematodes. Solarization involves placing transparent plastic over the soil to trap solar radiation and increase the soil temperature enough to kill nematodes, but this is ineffective if the inoculum level is high or if the heat does not penetrate to a sufficient depth. Lastly, steaming soil (at 160°F for 30 minutes) can be used as a pasteurization process to control nematodes. Often a combination of methods is used, however these alternative nematode control strategies are less effective and less consistent than that of chemical nematicides and the economic benefits are therefore often lower.

[0009] The first generation of nematicides were highly toxic and volatile fumigants such as methyl bromide, but their use has been banned based on their detrimental environmental impact (e.g., thinning of the ozone layer and indiscriminate killing of beneficial insects) and health risks (e.g., sterility and cancer). Even so, many commercial growers still fumigate their fields with chemicals such as 1,3 -di chloropropene (1,3-D), methyl isothiocyanate (MITC) generators, chloropicrin, or dimethyl disulfide (DMDS) before planting, despite these risks. DMDS combined with chloropicrin has been effective for the management of nematodes and purple nutsedge (Cyperus rotundus), an invasive weed. However, the pungent odor of DMDS(decaying fish and overpowering garlic) has resulted in further restrictions. Other chemical nematicides that are highly effective include aryl formamide compounds, carbamates, organophosphates, and fenamiphos but their efficacy is limited by their slow diffusion through soil, which means that higher doses are required, increasing the risk of human exposure. Many of these chemicals target the nervous system, which is highly conserved between nematodes and humans. Only a few biochemical targets are therefore available in the field of nematicides.

[0010] Given the limitations of conventional nematicides, novel treatments are required to enhance nematicidal efficacy while minimizing effects on the environment, agricultural ecosystems, and human health. The latest generation of nematicides has a lower risk profile, and some are already available to growers. However, the most promising developments involve biochemical control strategies such as RNA interference (RNAi) combined with nanomaterials that facilitate targeted delivery and controlled release.SUMMARY OF THE DISCLOSURE

[0011] The foundation of most food production systems underpinning global food security is the careful management of soil resources. Embedded in the concept of soil health is the impact of diverse soil-borne pests and pathogens, but nematodes represent a particular challenge. Phytoparasitic root-knot nematodes such as Meloidogyne spp. and cyst nematodes such as Heterodera and Globodera spp. are severe threats to agriculture, accounting for annual yield losses of US$157 billion. The control of soil-borne phytoparasitic nematodes conventionally relies on the use of chemical nematicides, which can have adverse effects on the environment and human health due to their persistence in soil, plants, and water. Nematode-resistant plants offer a promising alternative, but genetic resistance is speciesdependent, limited to a few crops, and establishing resistant cultivars often takes years. Novel approaches for the control of phytoparasitic nematodes are therefore required, which specifically target these parasites in the ground while minimizing the impact on the environment, agricultural ecosystems, and human health.

[0012] In addition to the development of next-generation, environmentally safer nematicides, promising biochemical strategies include the combination of RNA interference (RNAi) withnanomaterials that ensure the targeted delivery and controlled release of double-stranded RNA. Genome sequencing has identified more than 75 genes in root knot and cyst nematodes that have been targeted with RNAi so far. But despite encouraging results, the delivery of dsRNA to nematodes in the soil remains inefficient. Applicant provides herein a state-of-the- art RNAi approaches targeting phytoparasitic nematodes and consider the potential benefits of nanotechnology to improve dsRNA delivery.

[0013] Applicant also provides herein a biological pest control strategy using proteins from plant viruses loaded with nucleic acids as the therapy. Applicant has developed a protein nanotechnology - called spherical nanoparticles (SNPs) - that packages dsRNA molecules and other nucleic acids, for use in crop protection against nematodes. Nucleic acid delivery - using coding or non-coding RNA - is a powerful tool in medicine (COVID19 vaccines), and also provided herein is a specialized tool for agronomy applications.

[0014] In one aspect, provided herein are SNPs comprising or consisting essentially of a plant virus coat protein and double stranded ribonucleic acid (dsRNA) that is homologous to a RNAi target nematode associated gene selected from the group of directed to or controlling nematode development or nematode reproduction, or a nematode housekeeping gene, or a parasitism effector gene. In one aspect, the SNP comprising a plant virus coat protein and dsRNA are used to control a nematode by contacting with or delivering to the nematode the SNP.

[0015] In another aspect, provided herein are SNPs comprising or consisting essentially of a plant virus coat protein and a nematicide. In one aspect, the SNP comprising a plant virus coat protein and nematicide are used to control nematode growth, development or reproduction by contacting with, or delivering to the nematode the SNP.

[0016] As described herein, the SNP as described herein is ingested or absorbed by a nematode. In one aspect, the nematode is a plant parasitic nematode.

[0017] In another aspect, provided herein are methods to make SNPs that control nematode growth, development and reproduction. According to one aspect, a plant virus is transformed into a spherical shape and dsRNA is loaded onto the viral coat protein. In another aspect, a plant virus is transformed into a spherical shape and nematicides (for example ivermectin orabamectin) are loaded onto the transformed virus. In a further aspect, both the nematicide and the dsRNA are loaded into the transformed viral coat protein.BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG. 1 : Activation of the core RNAi pathway. In the first step, dsRNA is cut by the ribonuclease Dicer into small dsRNA duplexes 21-27 nucleotides in length with short overhangs. In the second step, these dsRNA duplexes are loaded onto AGO proteins and incorporated into the RISC complex. The guide strand is retained whereas the passenger strand is degraded. In the third step, the RISC complex cuts mRNA that is complementary to the guide strand or suppresses protein synthesis (depending on the origin of the dsRNA and complementarity to the target). The silencing response can be amplified by RdRPs that convert cleaved mRNA into secondary dsRNAs.

[0019] FIGS. 2A - 2C: Current and novel methods for the RNAi -based control of phytoparasitic nematodes. (FIG. 2A) Delivery of dsRNA by HIGS, where plants are genetically modified to produce dsRNA that targets pathogens (transient expression or stable transgenic plants), and VIGS, where plant viruses act as vectors to produce dsRNA in plants. (FIG. 2B) Exogenous application of dsRNA for the control of nematodes. In a laboratory setting, dsRNA can be delivered to nematodes by directly soaking them in dsRNA, by feeding them on dsRNA (optionally encapsulated in polymers), or by feeding them on recombinant bacteria expressing dsRNA. In an agricultural setting, free dsRNA is rapidly degraded in the soil, the persistence of polymers used to encapsulate dsRNA can have implications for human and environmental health, and the release of GMOs (recombinant bacteria) into the environment raises biosafety concerns, which need to be addressed. (FIG. 2C) Use of protein-based nanomaterials for the delivery of dsRNA in agricultural settings. Plant virus nanoparticles and nanomaterials derived therefrom can be used to deliver nematicides or dsRNA for the control of phytoparasitic nematodes. Such novel nanomaterials are biodegradable and have excellent soil mobility.

[0020] FIGS. 3A - 3C: Spherical nanoparticles (SNPs) enable the control of plant-parasitic nematodes with RNA interference (RNAi). (FIG. 3A) Loading of double stranded RNA (dsRNA) into SNPs. To deliver dsRNA (the trigger for RNAi) to soil-living nematodes, SNPformulations loaded with dsRNA were developed. Loading of dsRNA into SNPs was achieved by heat-induced transformation of the rod-shaped plant virus TMGMV into spheres. The heat induced transformation can occur at a temperature of 98°C that is maintained over a period of 30 seconds. (FIG. 3B) Application of dsRNA-loaded SNPs. The functionality of dsRNA-loaded SNPs was demonstrated by silencing the model gene mCherry in transgenic (red fluorescence) Caenorhabditis elegans. (FIG. 3C) Evaluation of silencing efficiency. Transgenic nematodes were observed under a fluorescence microscope >60 h after ingesting dsRNA-loaded SNPs. This timepoint was chosen to ensure degradation of pre-existing mCherry (half-life time of ~50 h in eukaryotes).[0021 | FIGS. 4A - 4F: Proof of concept of SNP uptake and dsRNA functionality using the mCherry transgene as a model target. (FIG. 4A) Scanning electron microscopy (SEM) images of spherical nanoparticle (SNP) formulations prepared using purified tobacco mild green mosaic virus (TMGMV) coat protein. SNP formulations were prepared using 2.5 mg / mL TMGMV coat protein. (FIG. 4B) Overlay of bright field and fluorescence images of C. elegans 3 h after the ingestion of native and Cy5-labled SNPs. (FIG. 4C) Agarose gel electrophoresis of dsRNA. Templates for dsRNA synthesis were generated by amplifying the mCherry gene (861 bp) using primers that add T7 promoters. Single-stranded RNA (ssRNA) was produced from this template when only one primer contained a T7 promoter, and doublestranded RNA (dsRNA) was produced in a single reaction when both primers contained a T7 promoter (Table 2). Lane 1, size marker; lane 2, sense strand RNA; lane 3, dsRNA prepared by annealing sense and antisense ssRNA; lane 4, antisense strand RNA; lane 5, dsRNA produced in a single step. (FIG. 4D) Fluorescence microscopy of C. elegans after incubation in a solution of 5, 50 or 500 ng / pL free dsRNA for 16 h at 16 °C followed by ~60 h to allow the degradation of existing mCherry. (FIG. 4E) Efficacy of silencing in (n = 14-26) juvenile nematodes depending on the dsRNA concentration assessed after ~60 h. (FIG. 4F) Efficacy of silencing in adult nematodes as described for panel (FIG. 4E). Error bars represent the standard deviation. The increase in standard deviation at 50 and 500 ng / pL dsRNA results from only a fraction of the nematode population being affected by RNAi. High concentrations of free dsRNA are required to induce silencing.[0022| FIGS. 5A - 5D: Characterization and optimization of dsRNA-SNP formulations. (FIG. 5A) Agarose gel electrophoresis of dsRNA-loaded SNPs cast from TMGMV coat protein. SNPs were loaded with Alexa Fluor 555-labeled dsRNA as a model for standard siRNAs. Gel electrophoresis of SNPs was performed with 4% agarose gels in TAE buffer at 90 V for 50 min, using glycerol for loading samples; optionally 50-200 mM NaOH was used to disassemble SNPs before electrophoresis. After gel electrophoresis, nucleic acids were stained with GelRed and detected at 302 / 590 nm (excitation / emission), the fluorophore was detected at 534 / 607 nm, and protein was stained with Coomassie and detected at 302 / 590 nm. The TMGMV coat protein (17.5 kDa) and dsRNA (21 bp) were co-localized in intact SNPs (black arrows); dsRNA remained intact after loading into SNPs. (FIGS. 5B-5D) Scanning microscopy (SEM) images of SNP formulations prepared with long (861 bp) dsRNA. SNP formulations were prepared with 2.5 mg / mL TMGMV coat protein (constant) and 0, 50, 100 or 200 ng / pL dsRNA (FIG. 5A); the highest concentration of dsRNA (200 ng / pL) was optionally supplemented with 10, 100 or 250 mM monovalent (FIG. 5C) or bivalent (FIG. 5D) cations. For SEM, samples were diluted 10-fold in DI water and dried onto silicon wafers. Concentrations of dsRNA >50 ng / pL resulted in formation of aggregates (right two boxes in FIG. 5B); charge neutralization with cations restored SNP formation under these conditions. Bivalent cations (Mg2+) restored SNP formation more efficiently than monovalent cations (Na+). It was possible to influence the SNP size through the concentration of cations; small SNPs were deemed most suitable for feeding to nematodes (FIG. 5C right box and FIG. 5D middle box).

[0023] FIGS. 6A - 6C: Comparative efficacy of free and encapsulated dsRNA. (FIG.6A) Fluorescence images of C. elegans after incubation with free dsRNA or dsRNA-loaded SNPs. Transgenic nematodes were incubated with similar volumes of free dsRNA (500 ng / pL) or dsRNA-SNPs (cast with 2.5 mg / mL coat protein and 200 ng / pL dsRNA) at 20 °C for 16 h before transfer to NGM plates. Before treatment (0 h) and at 24-h intervals after ~60 h, nematodes were fixed for the detection of mCherry by confocal microcopy. (FIG. 6B) Efficacy of silencing in juvenile nematodes (N= 38-52 nematodes for each setting) assessed after ~60 h. (FIG. 6C) Efficacy of silencing in adult nematodes as described for panel (FIG. 6B) In box plots, vertical lines show the median, boxes show the first to third quartiles, andwhiskers show the minimum and maximum (n.a. - not assessed, n.s. - not significant, *p 0.05, **p < 0.01, ***p < 0.001).(0024] FIGS. 7A - 7D: Testing dsRNA-SNPs under near-field conditions. (FIG. 7A) Schematic representation of the soil column mobility assays with dsRNA-loaded SNPs. (FIG. 7B) SDS-PAGE analysis of dsRNA-loaded SNPs following their elution from soil columns. SNPs were cast with 2.5 mg / mL TMGMV coat protein, 200 ng / pL dsRNA and 100 mM MgCh. SNPs were sonicated at 30% amplitude for 30 s on ice to disperse aggregates. Soil mobility was tested with 12.5 mL Pro-Mix soil in 15-mL tubes (10 cm bed height) and 1 mg dsRNA-loaded SNPs. The column was infused with 100 mM MgCh at 150 mL / h. Black arrows indicate intact dsRNA and coat protein. (FIG. 7C) Fluorescence images of C. elegans after incubation with soil column elution fractions. Transgenic nematodes were incubated with the soil column elution fractions from panel (FIG. 7B) for 16 h at room temperature before transfer to NGM plates. After ~80 h, nematodes were fixed for confocal microscopy. (FIG. 7D) Silencing efficacy of different soil column elution fractions. In box plots, vertical lines are the median, boxes are the first to third quartiles, and whiskers show the minimum and maximum (*p < 0.05, **p < 0.01, ***p < 0.001).

[0025] FIGS. 8A - 8B: Compatibility of TMGMV and TMGMV coat protein with dsRNA. (FIG. 8A) White light images of SNPs after ultra-centrifugation. SNPs were cast with 2.5 mg / mL TMGMV or TMGMV coat protein, optionally supplemented with Alexa Fluor 555- labeled dsRNA. Black arrows indicate SNP pellets that were collected in 1.5 mL tubes by centrifugation at 52000 rpm. (FIG. 8B) Fluorescence images of SNPs after ultracentrifugation. The same samples as in (FIG. 8A) were observed at 534 / 607 nm (excitation / emission) to visualize labeled dsRNA. The fluorophore-labeled dsRNA remained in the supernatant when casting SNPs from TMGMV; native TMGMV already contains its own genomic RNA. In contrast, the fluorophore-labeled dsRNA was co-localized with the SNP pellet when using only the TMGMV coat protein for casting SNPs.

[0026] FIG. 9: Investigation of dsRNA-loading capacity of SNPs without charge neutralization and dsRNA condensation. Agarose gel electrophoresis of SNP formulations prepared with increasing concentrations of dsRNA. SNPs were prepared with 2.5 mg / mL TMGMV coat protein (constant concentration) and 5-500 ng / pL dsRNA (861 bp). Samplesmixed with glycerol for loading were analyzed by 1% (w / v) agarose gel electrophoresis in TAE buffer at 100 V for 40 min. The nucleic acids were stained with GelRed and detected at 590 nm. Concentrations of dsRNA > 300 ng / pL inhibited SNP formation, as indicated by the presence of free dsRNA on the agarose gels (black arrow).

[0027] FIGS. 10A - 10B: Coating of SNPs with metal-organic polymers. (FIG. 10A) SEM images of dsRNA-loaded SNPs before and after coating with tannic acid. SNPs were prepared with 2.5 mg / mL TMGMV coat protein, 400 ng / pL dsRNA and 100 mM MgCh. To disperse potential aggregates, SNPs were sonicated at 30% amplitude for 30 s on ice then dialyzed against deionized water to remove free MgCh and coated by incubation in 30 or 150 mM tannic acid [Z. Wu, et al., ACS Applied Materials & Interfaces 14(11) (2022)]. Mg2+ions for tannic acid polymerization are already present in SNP formulations. Tannic acid concentrations of 150 mM gave rise to novel shapes, which are interesting for the delivery of cargos in the soil [U.P. Venkateswaran et al., ACS Agricultural Science & Technology (2023)]. (FIG. 10B) Analysis of coated dsRNA-SNPs by SDS-PAGE after elution from soil columns. SNPs were prepared as described in panel (FIG. 10A) and coated with 30 mM tannic acid. Soil mobility was tested with 12.5 mL Pro-Mix soil in 15-mL tubes (10 cm bed height) and 1 mg coated dsRNA-SNPs. The eluent was 100 mM MgCh in deionized water (infusion rate 150 mL / h). Soil column elution fractions were mixed in a 1 : 1 ratio with NEB Purple Loading Dye and separated on 4-12% Bis-Tris gels in MOPS buffer at 200 V for 45 min. Nucleic acids were stained with GelRed and detected at 590 nm. Protein was stained with Gel Code Blue and detected at 590 nm. Black arrows indicate intact dsRNA or TMGMV coat protein. Coating with tannic acid appeared to stabilize dsRNA-loaded SNPs, abolishing fractions containing only dsRNA.

[0028] FIGS. 11A - 11G: Labeling TMGMV with Cy-5 and disassembly into free Cy5- labeled coat protein. (FIGS. 11A - 11C) Size exclusion chromatography elution profiles of native TMGMV (FIG. 11 A), diazonium-coupled TMGMV (FIG. 11B) and Cy5-labeled TMGMV (FIG. 11C). Applicant used a Superpose 6 Increase 10 / 300 GL column on an Akta Pure system (Cytiva) with 100 mM KPO4 (pH 7.0) as the running buffer at a flow rate of 0.5 mL / min. Cy5 absorbance was detected at 647 nm. The Cy5 signal co-eluted with TMGMV. (FIG. HD) Free TMGMV coat protein before (left) and after (right) precipitation at theisoelectric point. (FIG. HE) Separation of precipitated TMGMV coat protein by centrifugation. (FIG. HF, FIG. 11G) Analysis of Cy5-labeled TMGMV coat protein by SDS-PAGE and detection at 710 nm. The Cy5 signal co-migrated with TMGMV coat protein.

[0029] FIG. 12: Quantification of silencing efficiency. Silencing efficiency in different settings (n=38-52 nematodes for each setting) was assessed after 60 h with the software ImageJ. Error bars represent the standard deviation. Silencing was more efficient with dsRNA-SNPs compared to free dsRNA. The results shown in FIG. 12 are the data from one time point in FIGS. 6B and 6C.DETAILED DESCRIPTION OF THE DISCLOSURE

[0030] Definitions

[0031] As it would be understood, the section or subsection headings as used herein is for organizational purposes only and are not to be construed as limiting or separating or both limiting and separating the subject matter described.

[0032] Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. The disclosures of these publications, patents and published patent specifications are hereby incorporated by reference into the present disclosure in their entireties to more fully describe the state of the art to which this invention pertains.

[0033] The practice of the present technology will employ, unless otherwise indicated, conventional techniques of organic chemistry, pharmacology, immunology, molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2ndedition (1989); Current Protocols In Molecular Biology (F. M. Ausubel, et al. eds., (1987)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, a Laboratory Manual, and Animal Cell Culture (R.I. Freshney, ed. (1987)).

[0034] As used in the specification and claims, the singular form “a,” “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes a plurality of cells, including mixtures thereof.

[0035] As used herein, the term “comprising” is intended to mean that the compounds, compositions and methods include the recited elements, but not exclude others. “Consisting essentially of’ when used to define compounds, compositions and methods, shall mean excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants, e.g., from the isolation and purification method and pharmaceutically acceptable carriers, preservatives, and the like. “Consisting of’ shall mean excluding more than trace elements of other ingredients. Embodiments defined by each of these transition terms are within the scope of this technology.

[0036] “Optional” or “optionally” means that the subsequently described circumstance may or may not occur, so that the description includes instances where the circumstance occurs and instances where it does not.

[0037] As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0038] All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied (+) or (-) by increments of 1, 5, or 10%. It is to be understood, although not always explicitly stated that all numerical designations are preceded by the term “about.” It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0039] As used herein, the term “about” is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value. The term “about” when used before a numerical designation, e.g., temperature, time, amount, and concentration, including range, indicates approximations which may vary by (+) or (-) 15%, 10%, 5%, 3%, 2%, or 1 %.

[0040] “Substantially” or “essentially” means nearly totally or completely, for instance, 95% or greater of some given quantity. In some embodiments, “substantially” or “essentially” means 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.

[0041] A “composition” as used herein, refers to an active agent, such as a compound as disclosed herein and a carrier, inert or active. The carrier can be, without limitation, solid such as a bead or resin, or liquid, such as phosphate buffered saline.

[0042] Carriers also include pharmaceutical excipients and additives proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-, tri, tetraoligosaccharides, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars and the like; and polysaccharides or sugar polymers), which can be present singly or in combination, comprising alone or in combination 1-99.99% by weight or volume. Exemplary protein excipients include serum albumin such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acid / antibody components, which can also function in a buffering capacity, include alanine, arginine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like. Carbohydrate excipients are also intended within the scope of this technology, examples of which include but are not limited to monosaccharides such as fructose, maltose, galactose, glucose, D- mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol) and myoinositol.

[0043] The term “contacting” means direct or indirect binding or interaction between two or more. A particular example of direct interaction is binding. A particular example of an indirect interaction is where one entity acts upon an intermediary molecule, which in turn acts upon the second referenced entity. Contacting as used herein includes in solution, in solid phase, in vitro, ex vivo, in a cell and in vivo. Contacting in vivo can be referred to as administering, or administration.[00441 An “agricultural product” intends vegetation in whole or in part and includes plants, trees, roots, flowers, limbs, shoots, stems, leaves or any other part thereof.[0045| As used herein, the terms "treating," "treatment" and the like mean obtaining a desired agricultural effect such as an amelioration. The effect may be prophylactic in terms of completely or partially preventing an infection of a plant, vegetation, or other agricultural product by a pest or insect. In one aspect, the term “treatment” excludes prophylaxis.

[0046] The term “ameliorate” means a detectable improvement in an agricultural product or vegetation, such as a plant, tree, flower, crop, root, stem, or leaf. A detectable improvement includes a subjective or objective decrease, reduction, inhibition, suppression, limit or control in the occurrence, frequency, severity, progression, or duration of an infection or presence of a pest or microorganism, such as for example C. elegans.

[0047] As used herein, the term “plant virus” includes viruses that infect plants or plant systems, e.g., leaves, root and / or stems. Plant viruses can be stably stored (and are stable without cold chain requirements). Plant viruses do not infect or replicate in mammalian cells, thus adding another layer of safety compared to oncolytic viral therapies. Non-limiting examples include tobacco mosaic virus (TMV), cytoplasmic type citrus leprosis virus, tobacco mild green mottle virus (TMGMV), physalis mottle virus like particle (PhMV), cowpea chlorotic mottle virus (CCMV), and cowpea mosaic virus (CPMV). Methods of replicating and producing virus for therapeutic application are known in the art and described in WO 2022 / 221692, published October 20, 2022, and incorporated herein by reference.

[0048] Virus-like Particles (VLPs) are generally composed of one or more viral proteins, such as, but not limited to, those proteins referred to as capsid, coat, shell, surface and / or envelope proteins, or particle-forming polypeptides derived from these proteins. VLPs can form spontaneously upon recombinant expression of the protein in an appropriate expression system. VLPs can also be engineered, e.g., comprising, or consisting essentially of, or yet further consisting of, one or more viral proteins that comprise, or consists essentially of, or yet further consists of, a modification. Methods for producing VLPs are known in the art. The presence of VLPs following recombinant expression of viral proteins can be detected using conventional techniques known in the art, such as by electron microscopy, biophysicalcharacterization, and the like. Further, VLPs can be isolated by known techniques, e.g., density gradient centrifugation and identified by characteristic density banding. See, for example, Baker et al. (1991) Biophys. J. 60: 1445-1456; and Hagensee et al. (1994) J. Viral. 68:4503-4505; Vincente, J Invertebr Pathol., 2011; Schneider Ohrum and Ross, Curr. Top. Microbial. Immunol., 354: 53073, 2012).

[0049] As used herein, the term “spherical nanoparticle (SNP)” intends a non-replicating, viral shell, derived from one or more plant viruses e.g., one or more plant viruses described herein). SNPs are generally composed of one or more viral proteins, such as, but not limited to, those proteins referred to as capsid, coat, shell, surface and / or envelope proteins, or particle-forming polypeptides derived from these proteins. SNPs can form spontaneously upon recombinant expression of the protein in an appropriate expression system. SNPs can also be engineered, e.g., comprising, or consisting essentially of, or yet further consisting of, one or more viral proteins that comprise, or consists essentially of, or yet further consists of, a modification. Methods for producing SNPs are known in the art. The presence of SNPs following recombinant expression of viral proteins can be detected using conventional techniques known in the art, such as by electron microscopy, biophysical characterization, and the like. Further, SNPs can be isolated by known techniques, e.g., density gradient centrifugation and identified by characteristic density banding. See, for example, Baker et al. (1991) Biophys. J. 60: 1445-1456; and Hagensee et al. (1994) J. Viral. 68:4503-4505;Vincente, J Invertebr Pathol., 2011; Schneider Ohrum and Ross, Curr. Top. Microbial. Immunol., 354: 53073, 2012).

[0050] As used herein, the terms “SNP” is used synonymously with “Virus-like particle” or “VLP” refers to a non-replicating, viral shell, derived from one or more viruses (e.g., one or more plant viruses described herein).

[0051] In one aspect, a VLP or SNP is a Tobacco mild green mosaic virus (TMGMV) derived VLP that comprises, or consists essentially of, or yet further consists of, one or more viral particles, e.g., a capsid, derived from Tobacco mild green mosaic virus (TMGMV) or a derivative thereof.

[0052] As used herein, the term “an equivalent thereof’ in reference to a polynucleotide or a protein (e.g., a capsid or coat protein) include a polynucleotide or a protein that comprise, or consists essentially of, or yet further consists of, at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identify to the respective polynucleotide or protein of which it is compared to, while still retaining a functional activity. In the instances with reference to a capsid or coat protein, a functional activity refers to the formation of a VLP, e.g., a rodshaped or spherical plant virus nanoparticles derived from Tobacco mild green mosaic virus (TMGMV) or derivative thereof.

[0053] As used herein, the term “an equivalent thereof’ in reference to a polynucleotide or a protein (e.g., a capsid or coat protein) include a polynucleotide or a protein that comprise, or consists essentially of, or yet further consists of, at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identify to the respective polynucleotide or protein of which it is compared to, while still retaining a functional activity. In the instances with reference to a capsid or coat protein, a functional activity refers to the formation of a VLP, e.g., a rodshaped or spherical plant virus nanoparticles derived from Tobacco mild green mosaic virus (TMGMV) or derivative thereof.

[0054] As used herein, the term “modification” includes, for example, substitutions, additions, insertions and deletions to the amino acid sequences, which can be referred to as “variants.” Exemplary sequence substitutions, additions, and insertions include a full length or a portion of a sequence with one or more amino acids substituted (or mutated), added, or inserted, for example of a capsid derived from the plant virus. In some instances, a capsid described herein includes, e.g., a modified capsid comprising, or consisting essentially of, or yet further consisting of, at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to its respective wild-type version. These or other modifications are intended in the scope “or derivative thereof.”

[0055] The term “sequence identity” refers to the percentage of bases or amino acids between two polynucleotide or polypeptide sequences that are the same, and in the same relative position. As such one polynucleotide or polypeptide sequence has a certain percentage of sequence identity compared to another polynucleotide or polypeptide sequence. For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences arecompared. The term “reference sequence” refers to a molecule to which a test sequence is compared. A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) having a certain percentage (for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of “sequence identity” to a reference sequence means that, when aligned, that percentage of bases (or amino acids) at each position in the test sequence are identical to the base (or amino acid) at the same position in the reference sequence. This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology. Preferably, default parameters are used for alignment. One alignment program is BLAST, using default parameters. In particular, programs are BLASTN and BLASTP, using the following default parameters: Genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non-redundant, GenBank + EMBL + DDB J + PDB + GenBank CDS translations + SwissProtein + SPupdate + PIR. Details of these programs can be found at the following Internet address: ncbi.nlm.nih.gov / blast / Blast.cgi.

[0056] Modified capsid polypeptides include, for example, non-conservative and conservative substitutions of the capsid amino acid sequences. In one aspect, the modified capsids are within the scope of the term “derivative thereof.”

[0057] As used herein, the term “conservative substitution” denotes the replacement of an amino acid residue by another, chemically or biologically similar residue. Biologically similar means that the substitution does not destroy a biological activity or function, e.g., assembly of a viral capsid.

[0058] Structurally similar means that the amino acids have side chains with similar length, such as alanine, glycine and serine, or a similar size. Chemical similarity means that the residues have the same charge or are both hydrophilic or hydrophobic. Particular examples of conservative substitutions include the substitution of a hydrophobic residue such as isoleucine, valine, leucine or methionine for another, the substitution of a polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, and the like. The term "conservative substitution" also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid. Such proteinsthat include amino acid substitutions can be encoded by a nucleic acid. Consequently, nucleic acid sequences encoding proteins that include amino acid substitutions are also provided.

[0059] Modified proteins also include one or more D-amino acids substituted for L-amino acids (and mixtures thereof), structural and functional analogues, for example, peptidomimetics having synthetic or non-natural amino acids or amino acid analogues and derivatized forms. Modifications include cyclic structures such as an end-to-end amide bond between the amino and carboxy -terminus of the molecule or intra- or inter-molecular disulfide bond.

[0060] Modified forms further include “chemical derivatives,” in which one or more amino acids has a side chain chemically altered or derivatized. Such derivatized polypeptides include, for example, amino acids in which free amino groups form amine hydrochlorides, p- toluene sulfonyl groups, carobenzoxy groups; the free carboxy groups form salts, methyl and ethyl esters; free hydroxl groups that form O-acyl or O-alkyl derivatives as well as naturally occurring amino acid derivatives, for example, 4-hydroxyproline, for proline, 5- hydroxylysine for lysine, homoserine for serine, ornithine for lysine etc. Also included are amino acid derivatives that can alter covalent bonding, for example, the disulfide linkage that forms between two cysteine residues that produces a cyclized polypeptide.

[0061] In one aspect, provided herein is a virus-like particle (VLP) or SNP derived from Tobacco mild green mosaic virus (TMGMV) and a dsRNA as described herein.

[0062] In one aspect, the dsRNA is entrapped in the VLP or covalently attached to the VLP. In another aspect, the diameter of VLP nanoparticle can range from about 1 nm to about 2 pm. In certain embodiments, the nanoparticle is less than about 2 pm, or less than about 1.5 pm, or less than about 1.25 pm or less than about 1 pm, or less than about .9 pm, or less than about 0.8 pm, or less than about 0.7 pm, or about less than about .5 pm in diameter. In other embodiments, the diameter of VLP nanoparticle is less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 200 nm, less than about 100 nm, or less than about 50 nm in diameter. In one aspect, the diameter of the VLP is from about 100 nm to about 200 nm, and ranges in between.

[0063] In some embodiments, the engineered SNP has a diameter of from about 15 nm to about 60 nm, or from about 15 nm to about 50 nm, or from about 10 nm to about 40 nm, or from about 20 or 25 nm to about 50 nm, or from about 20 or 25 nm to about 40 nm, or from about 15 nm to about 35 nm, or from about 15 nm to about 40 nm, or from about 15 nm to about 45 nm, or alternatively about 15 nm, or about 20 nm, or about 25 nm, or about 30 nm, or about 35 nm, or about 40 nm, or about 45nm, or about 50 nm, or about 55 nm, or about 60 nm.

[0064] In some embodiments, SNP is derived from Cowpea mosaic virus (CPMV). CPMV is a non-enveloped plant virus that belongs to the Comovirus genus. CPMV strains include, but are not limited to, SB (Agrawal, H.O. (1964). Meded. Landb. Hoogesch. Wagen. 64: 1) and Vu (Agrawal, H.O. (1964). Meded. Landb. Hoogesch. Wagen. 64: 1). Cowpea mosaic virus (CPMV) is a SNP and a plant-infecting member of the order Picornavirales, with a relatively simple, non-enveloped capsid that has been extensively studied and a positivesense, single-stranded RNA genome. For CPMV, the genome is bipartite, with RNA-1 (6 kb) and RNA-2 (3.5 kb) being separately encapsi dated. CPMV has an icosahedral capsid structure, which is ~30 nm in diameter and is formed from 60 copies each of a Large (L) and Small (S) coat protein. These two coat proteins are processed from a single RNA-2-encoded precursor polyprotein (VP60) by the action of the 24 K viral proteinase which is encoded by RNA-1. Thus capsid assembly, as well as viral infection, is dependent on the presence of both genomic segments in an infected plant cell. In some embodiments, the SNP particles have been treated, prepared and / or inactivated by methods known in the art. In some instances, CPMV produces a large capsid protein and a small capsid protein precursor (which generates a mature small capsid protein). In some cases, CPMV capsid is formed from a plurality of large capsid proteins and mature small capsid proteins. In some cases, the large capsid protein is a wild-type large capsid protein, optionally expressed by SB or Vu strain. In other instances, the large capsid protein is a modified large capsid protein, e.g., comprising, or consisting essentially of, or yet further consisting of, one or more substitutions, insertions, and / or deletions. In some cases, the large capsid protein comprises, or consists essentially of, or yet further consists of, the sequence as set forth in the UniProtKB ID P03599 (residues 460-833):[0065| MEQNLFALSLDDTSSVRGSLLDTKFAQTRVLLSKAMAGGDVLLDEYLYDV VNGQDFRATVAFLRTHVITGKIKVTATTNISDNSGCCLMLAINSGVRGKYSTDVYTIC SQDSMTWNPGCKKNFSFTFNPNPCGDSWSAEMISRSRVRMTVICVSGWTLSPTTDVI AKLDWSIVNEKCEPTIYHLADCQNWLPLNRWMGKLTFPQGVTSEVRRMPLSIGGGA GATQAFLANMPNSWISMWRYFRGELHFEVTKMSSPYIKATVTFLIAFGNLSDAFGFY ESFPHRIVQFAEVEEKCTLVFSQQEFVTAWSTQVNPRTTLEADGCPYLYAIIHDSTTG TISGDFNLGVKLVGIKDFCGIGSNPGIDGSRLLGAIAQ (SEQ ID NO: ), or an equivalent thereof.10066] In some cases, the mature small capsid protein is a wild-type mature small capsid protein, optionally expressed by SB or Vu strain. In other instances, the mature small capsid protein is a modified mature small capsid protein, e.g., comprising, or consisting essentially of, or yet further consisting of, one or more substitutions, insertions, and / or deletions. In some cases, the mature small capsid protein comprises, or consists essentially of, or yet further consists of, the sequence as set forth in the UniProtKB ID P03599 (residues 834- 1022):

[0067] GPVC AEASD VYSPCMIASTPPAPF SD VT AVTFDLINGKITP VGDDNWNTHIY NPPIMNVLRTAAWKSGTIHVQLNVRGAGVKRADWDGQVFVYLRQSMNPESYDART FVISQPGSAMLNFSFDIIGPNSGFEFAESPWANQTTWYLECVATNPRQIQQFEVNMRF DPNFRVAGNILMPPFPLSTETPPL (SEQ ID NO: ), or an equivalent thereof.

[0068] In some embodiments, the plant virus nanoparticle or SNP is derived from Cowpea chlorotic mottle virus (CCMV). CCMV is a spherical plant virus that belongs to the Bromovirus genus. Several strains have been identified and include, but not limited to, Carl (Ali, et al., 2007. J. Virological Methods 141 :84-86), Car2 (Ali, et al., 2007. J. Virological Methods 141 :84-86, 2007), type T (Kuhn, 1964. Phytopathology 54: 1441-1442), soybean (S) (Kuhn, 1968. Phytopathology 58: 1441-1442), mild (M) (Kuhn, 1979. Phytopathology 69:621-624), Arkansas (A) (Fulton, et al., 1975. Phytopathology 65: 741-742), bean yellow stipple (BYS) (Fulton, et al., 1975. Phytopathology 65: 741-742), R (Sinclair, ed. 1982. Compendium of Soybean Diseases. 2nded. The American Phytopathological Society, St. Paul. 104 pp.), and PSM (Paguio, et al., 1988. Plant Diseases 72(9): 768-770).

[0069] In some instances, the plant virus nanoparticle or SNP from CCMV comprise, or consists essentially of, or yet further consists of, a plurality of capsid proteins. In some instances, the capsid protein is a wild-type CCMV capsid, optionally expressed by Carl, Car2, type T, soybean (S), mild (M), Arkansas (A), bean yellow stipple (BYS), R, or PSM strain. In other instances, the capsid protein is a modified capsid protein, e.g., comprising, or consisting essentially of, or yet further consisting of, one or more substitutions, insertions, and / or deletions. In some cases, the CCMV capsid comprises, or consists essentially of, or yet further consists of, the sequence as set forth in the UniProtKB ID P03601 :|0070] MSTVGTGKLTRAQRRAAARKNKRNTRVVQPVIVEPIASGQGKAIKAWTGY SVSKWTASCAAAEAKVTSAITISLPNELSSERNKQLKVGRVLLWLGLLPSVSGTVKS CVTETQTTAAASFQVALAVADNSKDVVAAMYPEAFKGITLEQLTADLTIYLYSSAAL TEGDVIVHLEVEHVRPTFDDSFTPVY (SEQ ID NO: ), or an equivalent thereof.

[0071] In some cases, the virus or SNP from CCMV is prepared by the method as described in Ali et al., “Rapid and efficient purification of Cowpea chlorotic mottle virus by sucrose cushion ultracentrifugation,” Journal of Virological Methods 141 : 84-86 (2007).

[0072] In some embodiments, the plant virus nanoparticle or SNP is derived from Physalis mottle virus (PhMV). Physalis mottle virus (PhMV) is a +ssRNA virus from the family Tymoviridae that forms a ~30 nm-sized icosahedral capsid from 180 identical capsid proteins CPs, and can be recombinantly expressed and purified as a homogenous and stable SNP. In some instances, the plant virus nanoparticle or SNP from PhMV comprises, or consists essentially of, or yet further consists of, a plurality of coat proteins. In some instances, the coat protein is a wild-type PhMV coat protein. In other instances, the coat protein is a modified coat protein, e.g., comprising, or consisting essentially of, or yet further consisting of, one or more substitutions, insertions, and / or deletions. In some cases, the PhMV coat comprise, or consists essentially of, or yet further consists of, the sequence as set forth in the UniProtKB ID P36351 :

[0073] MDSSEVVKVKQASIPAPGSILSQPNTEQSPAIVLPFQFEATTFGTAETAAQVSL QTADPITKLTAPYRHAQIVECKAILTPTDLAVSNPLTVYLAWVPANSPATPTQILRVY GGQSFVLGGAISAAKTIEVPLNLDSVNRMLKDSVTYTDTPKLLAYSRAPTNPSKIPTA SIQISGRIRLSKPMLIAN (SEQ ID NO: ), or an equivalent thereof.

[0074] In some embodiments, the plant virus nanoparticle or SNP is derived from Sesbania mosaic virus (SeMV). SeMV is a positive stranded RNA virus that belongs to the genus Sobemovirus. In some instances, the virus or SNP from SeMV comprise, or consists essentially of, or yet further consists of, a plurality of capsid proteins. In some instances, the capsid protein is a wild-type SeMV capsid protein. In other instances, the capsid protein is a modified capsid protein, e.g., comprising, or consisting essentially of, or yet further consisting of, one or more substitutions, insertions, and / or deletions. In some cases, the SeMV capsid comprises, or consists essentially of, or yet further consists of, the sequence as set forth in the UniProtKB ID Q9EB06:

[0075] MAKRLSKQQLAKAIANTLETPPQPKAGRRRNRRRQRSAVQQLQPTQAGISM APSAQGAMVRIRNPAVSSSRGGITVLTHSELSAEIGVTDSIVVSSELVMPYTVGTWLR GVAANWSKYSWLSVRYTYIPSCPSSTAGSIHMGFQYDMADTVPVSVNQLSNLRGYV SGQVWSGSAGLCFINGTRCSDTSTAISTTLDVSKLGKKWYPYKTSADYATAVGVDV NIATPLVPARL VIALLDGS S ST AVAAGRIYCTYTIQMIEPT AS ALNN (SEQ ID NO : ), or an equivalent thereof. The virus can be obtained according to various methods known to those skilled in the art.

[0076] In some embodiments, the plant virus nanoparticle or SNP is derived from Tobacco mild green mosaic virus (TMGMV). TMGMV is a single-stranded RNA virus that belongs to the genus Tobamovirus . In some instances, the virus or SNP from TMGMV comprise, or consists essentially of, or yet further consists of, a plurality of capsid proteins. In some instances, the capsid protein is a wild-type TMGMV capsid protein. In other instances, the capsid protein is a modified capsid protein, e.g., comprising, or consisting essentially of, or yet further consisting of, one or more substitutions, insertions, and / or deletions. In some cases, the TMGMV capsid comprises, or consists essentially of, or yet further consists of, the sequence as set forth in the UniProtKB ID P03579:

[0077] MPYTINSPSQFVYLSSAYADPVQLINLCTNALGNQFQTQQARTTVQQQFADA WKPVPSMTVRFPASDFYVYRYNSTLDPLITALLNSFDTRNRIIEVDNQPAPNTTEIVN ATQRVDDATVAIRASINNLANELVRGTGMFNQAGFETASGLVWTTTPAT (SEQ ID NO: 1), or an equivalent thereof. 1

[0078] In some embodiments, the plant virus nanoparticle or SNP is derived from Tobacco mosaic virus (TMV). TMV is a positive-sense single-stranded RNA virus that belongs to the genus Tobamovirus. In some instances, the virus or SNP from TMV comprise, or consists essentially of, or yet further consists of, a plurality of capsid proteins. In some instances, the capsid protein is a wild-type TMV capsid protein. In other instances, the capsid protein is a modified capsid protein, e.g., comprising, or consisting essentially of, or yet further consisting of, one or more substitutions, insertions, and / or deletions. In some cases, the TMV capsid comprises, or consists essentially of, or yet further consists of, the sequence as set forth in the UniProtKB ID P69687:|0079| MSYSITTPSQFVFLSSAWADPIELINLCTNALGNQFQTQQARTVVQRQFSEVW KPSPQVTVRFPDSDFKVYRYNAVLDPLVTALLGAFDTRNRIIEVENQANPTTAETLD ATRRVDDATVAIRSAINNLIVELIRGTGSYNRSSFESSSGLVWTSGPAT (SEQ ID NO: ), or an equivalent thereof.(0080] In some embodiments, the plant virus nanoparticle or SNP is derived from a Citrus leprosis disease causing virus. In one embodiment, the Citrus leprosis disease causing virus is Citrus leprosis virus C (CiLV-C). CiLV-C is a bipartite, positive-sense, single stranded RNA virus. In some instances, the virus or SNP from CiLV-C comprise, or consists essentially of, or yet further consists of, a plurality of capsid proteins. In some instances, the capsid protein is a wild-type CiLV-C capsid protein. In other instances, the capsid protein is a modified capsid protein, e.g., comprising, or consisting essentially of, or yet further consisting of, one or more substitutions, insertions, and / or deletions. In some cases, the CiLV-C capsid comprises, or consists essentially of, or yet further consists of, the sequence as set forth in the UniProtKB ID Q1KZ58:

[0081] MSIVTFTLTDPSSALIAEIMQAIERHNVSVPEGLRDISKPTKKKQQSQPQQLSR ASARPQQLQPGPSGYQAKKPAKQKAEVVKPKQKQLAPPINKKAAKAKLYGLEQHC PKYAEAKGLQKQIGMTYYKISEPYALPDFKVMEASEDLVAVSEKDPMGSFEKRLYS MGFPKRPIKNVVPVFEFSDHYIVVFFPGSNAEIVKNVPKDSVSDYAEAQLAALLAAR QQINQIHELGDILPTNYLNVLDSGTQDVVVSDEEDDSDSAQ (SEQ ID NO: ), or an equivalent thereof.

[0082] In some embodiments, the plant virus nanoparticle or SNP is derived from Potato Virus X (P X) TMV is a positive-sense single-stranded RNA virus that belongs to the genus Potexvirus. In some instances, the virus or SNP from PVX comprise, or consists essentially of, or yet further consists of, a plurality of capsid proteins. In some instances, the capsid protein is a wild-type PVX capsid protein. In other instances, the capsid protein is a modified capsid protein, e.g., comprising, or consisting essentially of, or yet further consisting of, one or more substitutions, insertions, and / or deletions. PVX strain X3 is representative of PVX. In some cases, the PVX strain X3 capsid comprises, or consists essentially of, or yet further consists of, the sequence as set forth in the UniProtKB ID Pl 7782:10083] MSAPASTTQATGSTTSTTTKTAGATPATASGLFTIPDGDFFSTARAIVASNAV ATNEDLSKIEAIWKDMKVPTDTMAQAAWDLVRHCADVGSSAQTEMIDTGPYSNGIS RARLAAAIKEVCTLRQFCMKYAPVVWNWMLTNNSPPANWQAQGFKPEHKFAAFD FFNGVTNPAAIMPKEGLIRPPSEAEMNAAQTAAFVKITKARAQSNDFASLDAAVTRG RITGTTTAEAVVTLPPP (SEQ ID NO: ), or an equivalent thereof.

[0084] In embodiments where plant virus particles are used, the virus particles can be obtained from the extract of a plant infected by the plant virus or using the method disclosed herein. For example, cowpea mosaic virus can be grown in black eyed pea plants, which can be infected within 10 days of sowing seeds. Plants can be infected by, for example, coating the leaves with a liquid containing the virus, and then rubbing the leaves, preferably in the presence of an abrasive powder which wounds the leaf surface to allow penetration of the leaf and infection of the plant. Within a week or two after infection, leaves are harvested and viral nanoparticles are extracted. In the case of cowpea mosaic virus, 100 mg of virus can be obtained from as few as 50 plants. Procedures for obtaining plant picornavirus particles using extraction of an infected plant are known to those skilled in the art. See Wellink J., Meth Mol Biol, 8, 205- 209 (1998). Procedures are also available for obtaining virus-like particles. Saunders et al., Virology, 393(2):329-37 (2009). The disclosures of both of these references are incorporated herein by reference.]0085] Modes For Carrying Out the Disclosure[0086| In an aspect, provided herein are SNPs comprising or consisting essentially of a plant virus coat protein and double stranded ribonucleic acid (dsRNA) that encodes or is homologous to a RNAi target nematode associated gene selected from the group of a gene associated with nematode development, a gene associated with nematode reproduction, a nematode housekeeping gene, or a nematode parasitism effector gene. A dsRNA that is homologous to a RNAi target nematode associated gene is also referred herein to as a dsRNA that encodes a RNAi target nematode associated gene.

[0087] According to one embodiment, the SNP comprising or consisting essentially of a plant virus coat protein and dsRNA is used to control the growth, development and / reproduction of a nematode. The terms “coat protein” and “viral coat protein” are used interchangeably, and refer to a protein, at least a portion of which is present on the surface of a viral particle. In some embodiments, a coat protein refers to a protein that creates the tightly assembled structure of the protective shell (also referred to as a capsid) for a virus and prevents degradation of the viral genome, such as by environmental factors. Non-limiting examples of plant virus coat proteins in the SNP is selected from a coat protein from a plant virus selected from a tobacco mosaic virus (TMV), a cytoplasmic type citrus leprosis virus, a tobacco mild green mottle virus (TMGMV), a physalis mottle virus like particle (PhMV), a cowpea chlorotic mottle virus (CCMV), and a cowpea mosaic virus (CPMV). In a further aspect, the plant virus coat protein is a tobacco mild green mottle virus (TMGMV).100881 In some instances, an VLP described herein further comprise, or consists essentially of, or yet further consists of, a label or a tag, e.g., such as a detectable label. A detectable label can be attached to, e.g., to the surface of a VLP. In one aspect, a rod-shaped or spherical plant virus nanoparticles derived from Tobacco mild green mosaic virus (TMGMV) or derivative thereof further comprises, consists essentially thereof, or consists of the label or tag.100891 Non-limiting exemplary detectable labels also include a radioactive material, such as a radioisotope, a metal or a metal oxide. Radioisotopes include radionuclides emitting alpha, beta or gamma radiation. In particular embodiments, a radioisotope can be one or more of:3H,10B,18F,nC,14C,13N,18O,150,32P, P33,35S,35C1,45Ti,46Sc,47Sc,51Cr,52Fe,59Fe,57Co,60Cu,61Cu,62Cu,64Cu,67Cu,67Ga,68Ga,72As76Br,77Br,81mKr,82Rb,85Sr,89Sr,86Y,90Y,95Nb,94mTc, "mTc,97RU,103RU,105Rh,109Cd,mIn,113Sn,113mIn,114In, I125, 1131,140La,141Ce,149Pm,153Gd,157Gd,153Sm,161Tb,166Dy,166Ho,169Er,169Y,175Yb,177Lu,186Re,188Re,2O1T1,203Pb,211At,212Bi or225Ac.

[0090] Additional non-limiting exemplary detectable labels include a metal or a metal oxide. In particular embodiments, a metal or metal oxide is one or more of: gold, silver, copper, boron, manganese, gadolinium, iron, chromium, barium, europium, erbium, praseodynium, indium, or technetium. In additional embodiments, a metal oxide includes one or more of: Gd(III), Mn(II), Mn(III), Cr(II), Cr(III), Cu(II), Fe (III), Pr(III), Nd(III) Sm(III), Tb(III), Yb(III) Dy(III), Ho(III), Eu(II), Eu(III), or Er(III).

[0091] Further non-limiting exemplary detectable labels include contrast agents (e.g., gadolinium; manganese; barium sulfate; an iodinated or noniodinated agent; an ionic agent or nonionic agent); magnetic and paramagnetic agents (e.g., iron-oxide chelate); nanoparticles; an enzyme (horseradish peroxidase, alkaline phosphatase, P-galactosidase, or acetylcholinesterase); a prosthetic group (e.g., streptavidin / biotin and avidin / biotin); a fluorescent material (e.g., umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin); a luminescent material (e.g., luminol); or a bioluminescent material (e.g., luciferase, luciferin, aequorin).

[0092] Additional non-limiting examples of tags and / or detectable labels include enzymes (horseradish peroxidase, urease, catalase, alkaline phosphatase, beta-galactosidase, chloramphenicol transferase); enzyme substrates; ligands (e.g., biotin); receptors (avidin); GST-, T7-, His-, myc-, HA- and FLAG®-tags; electron-dense reagents; energy transfer molecules; paramagnetic labels; fluorophores (fluorescein, fluorscamine, rhodamine, phycoerthrin, phycocyanin, allophycocyanin); chromophores; chemi-luminescent (imidazole, luciferase, acridinium, oxalate); and bio-luminescent agents.

[0093] As set forth herein, a detectable label or tag can be linked or conjugated (e.g., covalently) to the SNP. In various embodiments a detectable label, such as a radionuclide or metal or metal oxide can be bound or conjugated to the agent, either directly or indirectly. A linker or an intermediary functional group can be used to link the molecule to a detectable label or tag. Linkers include amino acid or peptidomimetic sequences inserted between themolecule and a label or tag so that the two entities maintain, at least in part, a distinct function or activity. Linkers may have one or more properties that include a flexible conformation, an inability to form an ordered secondary structure or a hydrophobic or charged character which could promote or interact with either domain. Amino acids typically found in flexible protein regions include Gly, Asn and Ser. The length of the linker sequence may vary without significantly affecting a function or activity.

[0094] Linkers further include chemical moieties, conjugating agents, and intermediary functional groups. Examples include moieties that react with free or semi-free amines, oxygen, sulfur, hydroxy or carboxy groups. Such functional groups therefore include mono and bifunctional crosslinkers, such as sulfo-succinimidyl derivatives (sulfo-SMCC, sulfo- SMPB), in particular, disuccinimidyl suberate (DSS), BS3 (Sulfo-DSS), disuccinimidyl glutarate (DSG) and disuccinimidyl tartrate (DST). Non-limiting examples include diethylenetriaminepentaacetic acid (DTP A) and ethylene diaminetetracetic acid.(0095] Also provided herein is the VLP as described herein further comprising, or consisting essentially of, or yet further consisting of an additional agent. Non-limiting examples of such include otherwise insoluble compounds and pesticides for drug delivery in the soil, demonstrating desirable drug release and soil mobility characteristics. These are covalently linked to the SNP and / or entrapped within the SNP. The agents also can be covalently attached to the SNP by use of a linker.(0096] In some embodiments, the size of the SNP nanoparticle can range from about 1 nm to about 2 pm in diameter. In certain embodiments, the nanoparticle is less than about 2 pm, or less than about 1.5 pm, or less than about 1.25 pm or less than about 1 pm, or less than about .9 pm, or less than about 0.8 pm, or less than about 0.7 pm, or about less than about .5 pm in diameter. In other embodiments, the SNP nanoparticle is less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 200 nm, less than about 100 nm, or less than about 50 nm in diameter. In further embodiments, the nanoparticle is from about 75 nm to about 300 nm, or from about 75 nm to about 275 nm, or about 75 nm to about 225 nm, or from about 100 nm to about 300 nm, or from about 100 nm to about 250 nm, or from about 100 nm to about 200 nm, and ranges in between. In some embodiments the SNP can range from about lOOnm to about 200nm.

[0097] Also provided is a plurality of SNPs as described herein, wherein the SNPs are the same or different from each other and / or the agents are the same or different from each other and / or the TMGMV or a derivative thereof are the same or different.

[0098] According to some embodiments the target nematode associated gene is from a plant parasitic nematode. According to other embodiments the target nematode associated gene is from a human or animal parasitic nematode. According to other embodiments the target nematode associated gene is from a free-living nematode. According to one embodiment, the target nematode associated gene is listed in Table 1. According to some embodiments, the target nematode associated gene is a nematode development gene, a nematode reproductive gene, a nematode housekeeping gene, or a nematode parasitism gene.

[0099] According to one embodiment, the nematode development gene may encode a protein selected from chitin synthase, CPN-1, a dual oxidase protein, a RNA-binding protein, and troponin C. The nematode development gene may be selected from chs, cpn-1, duoxI, pos-1, and tnc. According to other embodiments, the nematode development gene may be selected from other genes related to nematode development According to other embodiments, the nematode development gene may encode other proteins related to nematode development.10.1.00 [ According to one embodiment, the nematode reproductive gene may encode a protein selected from an aminopeptidase and a major sperm protein. The nematode reproductive gene may be selected from amp-1, and msp. According to other embodiments, the nematode reproductive gene may be selected from other genes related to nematode reproduction.According to other embodiments, the nematode reproductive gene may encode other proteins related to nematode reproduction.[0101 | According to one embodiment, the nematode housekeeping gene may encode a protein selected from a phosphatase, a G-protein-coupled receptor, a cathepsin B cysteine proteinase, a cysteine proteinase, a dicer, cytochrome C, a FMRF amide-like neuropeptide, a glyceraldehyde-3 -phosphate dehydrogenase, heat shock protein 70, heat shock protein 90, a ribosomal protein, small ribosomal protein 3a, small ribosomal protein 4, a synaptobrevin, a spliceosomal SR protein, integrase, isocitrate lyase, 26S proteasome, a putative transcription factor, myosin heavy chain, a spliceosome subunit, a splicing factor, a pre-mRNA splicingfactor, a tropomyosin, paramyosin, and a coatomer complex subunit. The nematode housekeeping gene may be selected from ppm-1, srh-1, cb-1, cp-I, cpl-1, dcr-1.1, cyt-2.1 ,flp- 1 ,flp-6,flp-12,flp-14, flp-18. gpd-1, hsp-1. hsp90, r ps-23. rps-3a. rps-4. snb-1. spk-1. ICL, rpn7, Tisll, myo3, PV010,prp-17, tmy-1, unc-15, and Y25. According to other embodiments, the nematode housekeeping gene may be selected from other nematode housekeeping genes. According to other embodiments, the nematode housekeeping gene may encode other proteins related to housekeeping genes.

[0102] According to one embodiment, the parasitism genes (i.e. parasitism effector genes) may encode a protein selected from a protein similar to a plant ring H2 zinc finger protein, a cellulose-binding protein, a predicted protein with similarity to plant ubiquitins, a protein with similarity to the SKP1 protein of plants, an aspartic protease, P-l,4-endoglucanase, calreticulin, chorismite mutase, a C-type lectin, a glutathione-S transferase, a secreted peptide, a putative effector gene (ortholog of secreted peptide Mil6D10), a translationally controlled tumor protein, a body wall troponin C, a movement related protein, a venom allergen-like protein, Meloidogyne secreted protein 1, a putative esophageal gland cell secretory protein 2 (Meloidogyne secreted protein 2), a putative esophageal gland cell secretory protein 3 (Meloidogyne secreted protein 3), a putative esophageal gland cell secretory protein 5 (Meloidogyne secreted protein 5), esophageal gland cell secretory protein8D05, a putative esophageal gland cell secretory protein 9 (Meloidogyne secreted protein 9), a putative esophageal gland cell secretory protein 10 (Meloidogyne secreted protein 10), an effector protein, parasitism protein 16D10, a putative esophageal gland cell secretory protein ^ Meloidogyne secreted protein 18), a putative esophageal gland cell secretory protein 20 (Meloidogyne secreted protein 20), a putative esophageal gland cell secretory protein 23 (Meloidogyne secreted protein 23), a putative esophageal gland cell secretory protein 24 (Meloidogyne secreted protein 24), a putative esophageal gland cell secretory protein 40 (Meloidogyne secreted protein 40), a pectate lyase, pectate lyase 2, pectate lyase 3, polygalacturonase, a lipid binding protein, a secreted amphid protein, serine protease, and xylanase. The parasitism effector gene may be selected from 10A06, 3B05, 4G06, 8H07, asp- 7, eng-1, crl. cm, ctl, gsts-1, syv46, 16D10L, TCTP, pat-10, unc-87, msp-1, vap-1, msp2, msp3, msp5, msp-9, 8D05, msp-10, spl2, msp-16, mspl8, msp20, msp23, msp24, msp40,pel, pell, pel2,pel3, pg, far-1, pat-10, pat-10, ams-1, ser-1, spl, aw3 xyl2. According to otherembodiments, the parasitism effector gene may be selected from other genes related to nematode parasitism. According to other embodiments, the parasitism effector gene may encode other proteins related to nematode parasitism.

[0103] According to one embodiment, the plant virus coat protein is present in the SNP at a concentration of about Img / mL to about 5mg / mL. More preferably, the plant virus coat protein is at a concentration of about 2mg / mL to about 3mg / mL. More preferably, the plant virus coat protein is at a concentration of about 2.5mg / mL. According to one embodiment, the dsRNA is present in the SNP at a concentration of about 3ng / pL to about 600ng / pL. More preferably, the dsRNA is at a concentration of about 5ng / pL to about 500ng / pL. More preferably, the dsRNA is at a concentration of about 5ng / pL to about 400ng / pL. In one embodiment, the dsRNA is at a concentration of about 400ng / pL. Preferably, the quantity of dsRNA does not inhibit SNP formation.

[0104] In an aspect, a SNP comprising dsRNA can be used to control a nematode. In another aspect, a SNP comprising a plant virus coat protein and a nematicide. The nematode can be controlled by contacting the nematode with a SNP. According to one embodiment, the nematode is directly contacted with the SNP. According to one embodiment, the SNP can be ingested orally or absorbed through the epidermis. According to one embodiment, the nematode controlled is an egg, juvenile, or adult. According to one embodiment, the nematode is a plant parasitic nematode. The nematode may be a root-associated plant parasitic nematode. The nematode may be a plant parasitic nematode selected from Meloidogyne arliella. Meloidogyne incognita, Meloidogyne chilwoodi, Meloidogyne enlerolobii, Meloidogyne graminicola, Meloidogyne hapla, Meloidogyne arenaria, Meloidogyne javanica, Heterodera glycines, Heterodera schactii, Bursaphelenchus xylophilus, Radopholus similis, Globodera pallida, Globodera rostochiensis, Pratylenchus vulnus, Pratylenchus coffeae, Pratylenchus penetrans and Ditylenchus destructor. According to other embodiments, the nematode is a different plant parasitic nematode. According to other embodiments the nematode is a human or animal parasitic nematode. According to another embodiment, the nematode is a free living nematode.

[0105] According to the embodiment of FIG. 2C, the SNP may be added to a medium such as soil or water for the purposes of nematode control. The SNPs described herein are mobilethrough soil and can come into contact with a nematode in the soil. For example, a SNP can travel at least 10 centimeters through soil and remain intact.

[0106] In some embodiments, the SNP is coated with tannic acid. In some embodiments the tannic acid is at a concentration of about lOmM to about 200mM. More preferably, the tannic acid is at a concentration of about 30mM to about 150mM. In some aspects, the tannic acid is at a concentration about 30mM, about 40mM, about 50mM, about 60mM, about 70mM, about 80mM, about 90mM, about lOOmM, about HOmM, about 120mM, about 120mM, about 130mM, about 140mM, or about 150mM. In one aspect, the tannic acid is at a concentration of about 30mM. In some embodiments, the tannic acid changes the shape of the SNP from spherical to a different shape, such as more oblong. The tannic acid can stabilize the SNP and prevent leakage of SNP material within the SNP, such as dsRNA or nematicide.

[0107] In some embodiments, the SNP comprises bivalent cations. In some embodiments, the SNP comprises Mg2+as the bivalent cations. In some aspects, the SNP comprises MgCh at a concentration of about 5mM to about 500mM. In some aspects, the MgCh is at a concentration of about 50mM to about 150mM. In some aspects, the MgCh is at a concentration of about 50mM, about 60mM, about 70mM, about 80mM, about 90mM, about lOOmM, about HOmM, about 120mM, about 130mM, about 140mM, or about 150mM. In one aspect, the MgCh is about lOOmM. In some embodiments, Mg+ions from the MgCh are used in the polymerization of tannic acid in the SNPs.

[0108] In some embodiments, the SNP comprises monovalent cations. In some embodiments, the SNP comprises Na+as the monovalent cations. In some aspects, the SNP comprises NaCl at a concentration of about 5mM to about 500mM. In some aspects, the NaCl is at a concentration of about 50mM, about 75mM, about lOOmM, about 125mM, about 150mM, about 150mM, about 175mM, about 200mM, about 225mM, about 250mM, about 275mM, or about 300mM.

[0109] According to some embodiments, in the SNPs comprising dsRNA, the dsRNA in the SNP triggers RNAi. Once in contact with the nematode in the soil, the dsRNA triggers RNAi. The SNPs described herein do not degrade rapidly compared to free dsRNA.

[0110] In some respects, the SNPs comes in contact with the nematode in the rhizosphere of a plant. In some respects, the SNPs come in contact with the nematode on a plant root in the soil. The dsRNA triggers RNAi in the nematode.

[0111] According to some embodiments, for the SNPs comprising nematicide, the nematicide will target the nematode directly or indirectly, once in the soil.101.12] In another aspect, provided herein are methods to make SNPs which can be used to control nematodes. According to one aspect, a plant virus is transformed into a spherical shape and dsRNA is loaded onto the transformed virus. In another aspect, a plant virus is transformed into a spherical shape and nematicides are loaded onto the transformed virus. In one embodiment, the SNPs may be formed using heat transformation according to the steps of FIG. 3A

[0113] Compositions

[0114] In another aspect, provided herein is a composition comprising, consisting essentially of, or consisting of a SNP as provided herein, and at least one carrier, suitable for its intended use, e.g. in soils or other agricultural environments. In one aspect, the SNP is rod-shaped or spherical plant virus nanoparticles derived from Tobacco mild green mosaic virus (TMGMV) or derivative thereof.

[0115] Compositions comprising, or consisting essentially of, or consisting of the SNP composition alone or in combination of other agents can be manufactured by means of conventional mixing, dissolving, granulating, dragee-making levigating, emulsifying, encapsulating, entrapping, or lyophilization processes. These can be formulated in conventional manner using one or more carriers, diluents, excipients, or auxiliaries which facilitate processing of the combinations of compounds provided herein into preparations which can be used in agriculture.

[0116] In some embodiments, the agricultural formulations include, but are not limited to, lyophilized formulations, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast melt formulations, tablets, capsules, pills,delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations (e.g., nanoparticle formulations), and mixed immediate and controlled release formulations. 0317] In one aspect, the nanoparticle is combined with a carrier, such as an organic solvent (e.g., water) or mineral clay, adjuvants such as stickers or spreaders, stabilizers, safeners, or other chemicals that improve or enhance pesticidal activity. See, for example, http : / / npic. orst.edu / factsheets / formuladons.hunl, and references cited therein.

[0118] In some embodiments, the composition includes a carrier or carrier materials selected on the basis of compatibility with the composition disclosed herein, and the release profile properties of the desired dosage form. Exemplary carrier materials include, e.g., binders, suspending agents, disintegration agents, filling agents, surfactants, solubilizers, stabilizers, lubricants, wetting agents, diluents, and the like. Agriculturally compatible carrier materials include, but are not limited to, acacia, gelatin, colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, maltodextrin, glycerine, magnesium silicate, polyvinylpyrrollidone (PVP), cholesterol, cholesterol esters, sodium caseinate, soy lecithin, taurocholic acid, phosphotidylcholine, sodium chloride, tricalcium phosphate, dipotassium phosphate, cellulose and cellulose conjugates, sugars sodium stearoyl lactylate, carrageenan, monoglyceride, diglyceride, pregelatinized starch, and the like.

[0119] In some instances, the compositions further include pH adjusting agents or buffering agents which include acids such as acetic, boric, citric, lactic, phosphoric and hydrochloric acids, bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate and tris-hydroxymethylaminomethane, and buffers such as citrate / dextrose, sodium bicarbonate and ammonium chloride. Such acids, bases and buffers are included in an amount required to maintain pH of the composition in an acceptable range.

[0120] In some instances, the composition includes one or more salts in an amount required to bring osmolality of the composition into an acceptable range. Such salts include those having sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions, suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate.[01211 In some embodiments, the compositions include, but are not limited to, sugars like trehalose, sucrose, mannitol, maltose, glucose, or salts like potassium phosphate, sodium citrate, ammonium sulfate and / or other agents such as heparin to increase the solubility and stability.10.1221 In some instances, the compositions further include diluent which are used to stabilize compounds because they can provide a more stable environment. Salts dissolved in buffered solutions (which also can provide pH control or maintenance) are utilized as diluents in the art, including, but not limited to a phosphate buffered saline solution. In certain instances, diluents increase bulk of the composition to facilitate compression or create sufficient bulk for homogenous blend for capsule filling. Such compounds can include e.g., lactose, starch, mannitol, sorbitol, dextrose, microcrystalline cellulose such as AVICEL®, dibasic calcium phosphate, dicalcium phosphate dihydrate, tricalcium phosphate, calcium phosphate, anhydrous lactose, spray-dried lactose, pregelatinized starch, compressible sugar, such as Di- PAC® (Amstar), mannitol, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose acetate stearate, sucrose-based diluents, confectioner's sugar, monobasic calcium sulfate monohydrate, calcium sulfate dihydrate, calcium lactate trihydrate, dextrates, hydrolyzed cereal solids, amylose, powdered cellulose, calcium carbonate, glycine, kaolin, mannitol, sodium chloride, inositol, bentonite, and the like.

[0123] In some cases, the compositions include disintegration agents or disintegrants to facilitate the breakup or disintegration of a substance. The term “disintegrate” includes both the dissolution and dispersion of the dosage form when contacted with gastrointestinal fluid. Examples of disintegration agents include a starch, e.g., a natural starch such as com starch or potato starch, a pregelatinized starch such as National 1551 or AMIJEL®, or sodium starch glycolate such as PROMOGEL® or EXPLOTAB®, a cellulose such as a wood product, methylcrystalline cellulose, e.g., AVICEL®, AVICEL® PH101, AVICEL®PH102, AVICEL® PHI 05, ELCEMA® Pl 00, EMCOCEL®, VIVACEL®, MING TIA®, and SOLKA- FLOC®, methylcellulose, croscarmellose, or a cross-linked cellulose, such as cross-linked sodium carboxymethylcellulose (AC-DLSOL®), cross-linked carboxymethylcellulose, or cross-linked croscarmellose, a cross- linked starch such as sodium starch glycolate, a crosslinked polymer such as crospovidone, a cross-linked polyvinylpyrrolidone, alginate such asalginic acid or a salt of alginic acid such as sodium alginate, a clay such as VEEGUM® HV (magnesium aluminum silicate), a gum such as agar, guar, locust bean, Karaya, pectin, or tragacanth, sodium starch glycolate, bentonite, a natural sponge, a surfactant, a resin such as a cation-exchange resin, citrus pulp, sodium lauryl sulfate, sodium lauryl sulfate in combination starch, and the like.[01241 In some instances, the compositions include filling agents such as lactose, calcium carbonate, calcium phosphate, dibasic calcium phosphate, calcium sulfate, microcrystalline cellulose, cellulose powder, dextrose, dextrates, dextran, starches, pregelatinized starch, sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethylene glycol, and the like.101251 Lubricants and glidants are also optionally included in the compositions described herein for preventing, reducing or inhibiting adhesion or friction of materials.

[0126] Exemplary lubricants include, e.g., stearic acid, calcium hydroxide, talc, sodium stearyl fumerate, a hydrocarbon such as mineral oil, or hydrogenated vegetable oil such as hydrogenated soybean oil (STEROTEX®), higher fatty acids and their alkali-metal and alkaline earth metal salts, such as aluminum, calcium, magnesium, zinc, stearic acid, sodium stearates, glycerol, talc, waxes, STEAROWET®, boric acid, sodium benzoate, sodium acetate, sodium chloride, leucine, a polyethylene glycol (e.g., PEG-4000) or a methoxypolyethylene glycol such as CARBOWAX™, sodium oleate, sodium benzoate, glyceryl behenate, polyethylene glycol, magnesium or sodium lauryl sulfate, colloidal silica such as SYLOID™, CAB-O-SIL®, a starch such as corn starch, silicone oil, a surfactant, and the like.

[0127] Plasticizers include compounds used to soften the microencapsulation material or film coatings to make them less brittle. Suitable plasticizers include, e.g., polyethylene glycols such as PEG 300, PEG 400, PEG 600, PEG 1450, PEG 3350, and PEG 800, stearic acid, propylene glycol, oleic acid, triethyl cellulose and triacetin. Plasticizers can also function as dispersing agents or wetting agents.10128 ] Solubilizers include compounds such as triacetin, triethyl citrate, ethyl oleate, ethyl caprylate, sodium lauryl sulfate, sodium doccusate, vitamin E TPGS, dimethylacetamide, N-methylpyrrolidone, N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropylmethyl cellulose, hydroxypropyl cyclodextrins, ethanol, n-butanol, isopropyl alcohol, cholesterol, bile salts, polyethylene glycol 200-600, glycofurol, transcutol, propylene glycol, and dimethyl isosorbide and the like.101291 Stabilizers include compounds such as any antioxidation agents, buffers, acids, preservatives and the like. Exemplary stabilizers include L-arginine hydrochloride, tromethamine, albumin (human), citric acid, benzyl alcohol, phenol, disodium biphosphate dehydrate, propylene glycol, metacresol or m-cresol, zinc acetate, poly sorb ate-20 or TWEEN® 20, or trometamol.

[0130] Suspending agents include compounds such as polyvinylpyrrolidone, e.g., polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30, vinyl pyrrolidone / vinyl acetate copolymer (S630), polyethylene glycol, e.g., the polyethylene glycol can have a molecular weight of about 300 to about 6000, or about 3350 to about 4000, or about 7000 to about 5400, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, hydroxymethylcellulose acetate stearate, polysorbate-80, hydroxy ethylcellulose, sodium alginate, gums, such as, e.g., gum tragacanth and gum acacia, guar gum, xanthans, including xanthan gum, sugars, cellulosics, such as, e.g., sodium carboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, polysorbate-80, sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan monolaurate, povidone and the like.

[0131] Surfactants include compounds such as sodium lauryl sulfate, sodium docusate, Tween 60 or 80, triacetin, vitamin E TPGS, sorbitan monooleate, polyoxyethylene sorbitan monooleate, polysorbates, polaxomers, bile salts, glyceryl monostearate, copolymers of ethylene oxide and propylene oxide, e.g., PLURONIC® (BASF), and the like. Additional surfactants include polyoxyethylene fatty acid glycerides and vegetable oils, e.g., polyoxyethylene (60) hydrogenated castor oil, and polyoxyethylene alkyl ethers and alkylphenyl ethers, e.g., octoxynol 10, octoxynol 40. Sometimes, surfactants are included to enhance physical stability or for other purposes.

[0132] Viscosity enhancing agents include, e.g., methyl cellulose, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, hydroxypropylmethyl cellulose acetate stearate, hydroxypropylmethyl cellulose phthalate, carbomer, polyvinyl alcohol, alginates, acacia, chitosans and combinations thereof.[01331 Wetting agents include compounds such as oleic acid, glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, sodium docusate, sodium oleate, sodium lauryl sulfate, sodium doccusate, triacetin, Tween 80, vitamin E TPGS, ammonium salts and the like.

[0134] In a further aspect, the composition is frozen or lyophilized and can contain agents to preserve stability and activity.10135] In some embodiments, one or more compositions disclosed herein are contained in a kit. Accordingly, in some embodiments, provided herein is a kit comprising, consisting essentially of, or consisting of one or more compositions disclosed herein and instructions for their use.

[0136] Dosage and Dosage Formulations

[0017] In some embodiments, the compositions may be administered or delivered to an environment, either alone or as part of a formulation, once a week, once a day, twice a day, three times a day, or four times a day, or even more frequently.10138] Administration of the SNPs formulation alone or in combination with the additional agent and compositions containing same can be affected by any method that enables delivery to the site of action.{0139] Thus, the skilled artisan would appreciate, based upon the disclosure provided herein, that the dose and dosing regimen is adjusted in accordance with methods well-known in the art.

[0140] Uses of the SNPs and Compositions Containing Same

[0141] Provided herein is a method of treating an agricultural environment such as example soil, feed or plants (leaf, stalk or roots), comprising, or consisting essentially of, or yet further consisting of delivering or contacting the environment a SNP formulation or a composition as described herein. They also are useful to deliver pesticides and other insoluble compounds to inhibit pathogenic infestations of plants, roots, and soil. In one aspect the method is performed with a plurality of SNPs as described herein, wherein the SNPs are the same or different from each other and / or the agents are the same or different from each other and / or the TMGMV or a derivative thereof are the same or different.

[0142] In one aspect, the method is practiced with a SNP derived from Tobacco mild green mosaic virus (TMGMV) or a derivative thereof optionally conjugated to an agent. In a further aspect, it is conjugated to the agent. In one aspect, the agent or derivative thereof is conjugated by being entrapped in the SNP or covalently attached to the SNP. In another aspect, the method of the disclosure is practiced with a SNP having a diameter from about 1 nm to about 2 pm. In certain embodiments, the nanoparticle is less than about 2 pm, or less than about 1.5 pm, or less than about 1.25 pm or less than about 1 pm, or less than about .9 pm, or less than about 0.8 pm, or less than about 0.7 pm, or about less than about .5 pm in diameter. In other embodiments, the diameter of SNP nanoparticle in the method is less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 200 nm, less than about 100 nm, or less than about 50 nm in diameter. In one aspect, the diameter of the SNP in the method is from about 100 nm to about 200 nm, and ranges in between.

[0143] In some instances, an SNP used in the methods described herein further comprise, or consists essentially of, or yet further consists of, a label or a tag, e.g., such as a detectable label. A detectable label can be attached to, e.g., to the surface of a SNP. In one aspect, a rod-shaped and / or spherical plant virus nanoparticles derived from Tobacco mild green mosaic virus (TMGMV) or derivative thereof further comprises, consists essentially thereof, or consists of the label or tag.

[0144] Kits10145] As used herein, a kit or article of manufacture described herein include a carrier, package, or container that is compartmentalized to receive one or more containers such asvials, tubes, and the like, each of the contained s) comprising, or consisting essentially of, or yet further consisting of, one of the separate elements to be used in a method described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. In one embodiment, the containers are formed from a variety of materials such as glass or plastic.10146] The articles of manufacture provided herein contain packaging materials. Examples of agricultural packaging materials include, but are not limited to, blister packs, bottles, tubes, bags, containers, bottles, and any packaging material suitable for a selected formulation and intended mode of administration and treatment.

[0147] A kit typically includes labels listing contents and / or instructions for use, and package inserts with instructions for use. A set of instructions will also typically be included.

[0148] While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the disclosure. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0149] Experimental

[0150] Experiment No. 1:

[0151] Biochemical control of nematodes by RNAi

[0152] RNAi explained

[0153] RNA interference (RNAi) is a highly conserved defense mechanism triggered by double stranded RNA (dsRNA), which inhibits gene expression by the specific cleavage or translational repression of the corresponding mRNA

[0047] , The mechanism is widely conserved in eukaryotes, including nematodes [48-50], RNAi was first observed in tobacco plants recovering from a viral infection in 1928 [51, 52], but it was 70 years before themolecular mechanism was discovered in the nematode C. elegans, leading to the 2006 Nobel Prize in Physiology or Medicine

[0053] ,

[0154] The highly conserved core mechanism (FIG. 1) is triggered when dsRNA is detected in a eukaryotic cell

[0054] , This can be invading viral dsRNA

[0055] , synthetic RNA duplexes

[0049] or aberrant transcripts

[0056] typically produced by transposons or transgenes

[0057] , In the first step, the dsRNA is cleaved by a ribonuclease (RNase III) called Dicer, yielding small RNA duplexes 21-27 nucleotides in length with short overhangs

[0058] , These duplexes are termed small interfering RNAs (siRNAs) if they are perfectly complementary and processed from long dsRNA, or microRNAs (miRNAs) if there are mismatches and they originate from hairpin-containing primary transcripts

[0059] , The siRNA / miRNA molecule is then loaded onto an Argonaute (AGO) protein and incorporated into the RNA-induced silencing complex (RISC). AGO proteins are the directed binding partners of small RNAs and can be loaded with siRNAs and miRNAs

[0060] , They also serve as platform for additional proteins in gene silencing such as glycine-tryptophan (GW) proteins

[0060] , During RISC maturation, the passenger RNA strand is ejected while the other strand remains bound to the RISC complex and guides it to a complementary mRNA

[0061] , The RISC-siRNA / RISC-miRNA then cleaves the mRNA or recruit repressing and / or destabilizing proteins that block translation (both types of small RNAs can share similar mechanisms) [61, 62], Optionally, the silencing response can be amplified by an RNA-dependent RNA polymerase (RdRP), which uses the target mRNA as a template to synthesize new dsRNA [63, 64], This dsRNA is then cut by Dicer, giving rise to secondary siRNAs that match not only the initiating sequence in the primary siRNA but also upstream and downstream sequences

[0065] , This mode of amplification has been described in many but not all eukaryotes [65, 66],

[0155] RNAi is well documented in nematodes [67, 68], The dsRNA can be taken up orally or absorbed directly from the environment by soaking in a dsRNA solution. Systemic RNAi following oral uptake appears possible because nematodes have a mildly alkaline gut lumen, which prevents acid-induced nucleic acid degradation

[0069] , The systemic trafficking of dsRNA in nematodes is facilitated by the expression of the systemic RNA interferencedeficient gene (SID-l), which encodes a transmembrane channel involved in dsRNA uptake from the environment and cell-to-cell translocation

[0070] , More dsRNA is produced in vivo byRdRP, thus increasing the dose and longevity of the active principle [71, 72], This allows the RNAi effects to be inherited for at least two generations

[0073] , RNAi should therefore be a feasible biochemical strategy for nematode control in the field.

[0156] RNAi technology in agriculture

[0157] RNAi has been widely used as a research tool for the functional analysis of genes

[0074] , but has also been investigated as a medical intervention

[0075] and as an agricultural pest and disease control strategy, mainly targeting fungi

[0076] , viruses [77, 78], arthropods

[0079] , and nematodes (see below). RNAi-based control has been used for the protection of crops

[0079] and forests

[0080] , In addition to pest control, RNAi has also been investigated as a strategy to increase the tolerance of plants to salinity, drought, extreme temperatures, heavy metals, and other forms of abiotic stress

[0081] ,

[0158] RNAi target genes

[0159] RNAi target genes in nematodes can be assigned to four broad categories: genes associated with nematode (1) development and (2) reproduction, as well as (3) housekeeping genes, and (4) parasitism effector genes

[0082] , Bioinformatics is needed to mine gene targets effectively. The ideal target should be highly specific, avoiding potential cross-kingdom adverse effects caused by exposure to dsRNA (e.g., contact with beneficial insects such as honey bees, or the human consumption of crops containing dsRNA). The target gene should also be strongly transcribed, and protein synthesis should occur during the early stages of development so that the effects are lethal, thus preventing the emergence of mature feeding adults

[0083] , The target genes explored thus far are listed in Table 1. Promising examples include the FMRF-like peptide (FLP) genes flp-14 and flp-18, which encode neuropeptides that are core components of many nematode biological processes including feeding, locomotion, parasitism, and the sensory system

[0084] , Similarly, 16D10 encodes a secreted parasitism peptide that is conserved across root-knot nematodes and mediates early signaling events in host interactions. Host-induced gene silencing (HIGS) targeting these genes by expressing dsRNA in transgenic tobacco or Arabidopsis plants was shown to confer resistance against M. incognita [84, 85], Other examples include the developmental genes mispc3, encoding a signal peptidase required to process secreted proteins, and miduox,encoding an oxidase that facilitates tyrosine cross-linking in the developing cuticle. HIGS targeting these genes conferred resistance against M. incognita, and the efficacy increased when targeting both genes simultaneously

[0086] , Other promising targets include rpn7, which is essential for the integrity of the 26S proteasome. This eliminates misfolded proteins and short-lived proteins involved in cell cycle regulation, DNA repair, signal transduction, apoptosis, and metabolic regulation [87, 88], HIGS targeting this gene affected reproduction, motility and infectivity in AT. incognita

[0089] ,

[0160] Table 1 : Overview of RNAi target genes in phytoparasitic nematodes. Targets have been broadly classified as housekeeping genes, parasitism effector genes, developmental genes, and reproduction genes.

[0161] The table is updated from previous literature reviews [83, 136, 137], Genes are listed in alphabetical order within the different classes.

[0012] The nematode may be selected from Meloidogyne arliella. Meloidogyne incognita, Meloidogyne chitwoodi, Meloidogyne enlerolobii, Meloidogyne graminicola, Meloidogyne hapla, Meloidogyne arenaria, Meloidogyne javanica, Heterodera glycines, Heterodera schactii, Bursaphelenchus xylophilus, Radopholus similis, Globodera pallida, Globodera rostochiensis, Pratylenchus vulnus, Pratylenchus coffeae, Pratylenchus penetrans and Ditylenchus destructor. In Table 1: Ma means Meloidogyne artiella, Mi means Meloidogyne incognita, Me means Meloidogyne chitwoodi, Me means Meloidogyne enterolobii, Mg means Meloidogyne graminicola, Mj means Meloidogyne javanica, Hg means Heterodera glycines, Hs means Heterodera schactii, Bx means Bursaphelenchus xylophilus, Rs means Radopholus similis, Gp means Globodera pallida, Gr means Globodera rostochiensis, Pv means Pratylenchus vulnus, Pc means Pratylenchus coffeae, Pp means Pratylenchus penetrans, and Dd means Ditylenchus destructor. A gene name with a nematode abbreviation included means that the gene sequence is from a nematode genome of that species.

[0163] As seen in Table 1, the nematode development gene may be selected from chs, cpn-1 duoxI, pos-1, and tnc. chs encodes chitin synthase, cpn-1 encodes CPN-1, duoxl encodes dual oxidase 1, pos-1 encodes RNA-binding protein pos-1, and tnc encodes troponin C.[0164| As seen in Table 1, the nematode reproductive gene may be selected from amp-1, and msp. amp-1 encodes aminopeptidase and msp encodes major sperm protein.

[0165] As seen in Table 1, the nematode housekeeping gene may be selected from According to one embodiment, the nematode housekeeping gene be selected from ppm-1, srh-1, cb-1, cp-I, cpl-1, dcr-1.1, cyt-2.1,flp-l,flp-6,flp-12,flp-14, flp-18, gpd-1, hsp-1, hsp90, rps-23, rps-3a, rps-4, snb-1, spk-1, EST, ICL, rpn7, Tisll, myo3, 010,prp-17, tmy-1, unc-15, and Y25. ppm-1 encodes a phosphatase, srh-1 encodes a G-protein coupled receptor, cb-1 encodes cathepsin B cysteine proteinase, cp-I encodes a cysteine proteinase, cpl-1 encodes a cysteine proteinase, dcr-1.1 encodes a dicer, cyt-2.1 encodes cytochrome C,flp-1 encodes a FMRF amide-like neuropeptide, flp-6 encodes a FMRF amide-like neuropeptide, flp-12 encodes a FMRF amide-like neuropeptide, flp-14 encodes a FMRF amide-like neuropeptide, flp-18 encodes a FMRF amide-like neuropeptide, gpd-1 encodes glyceraldehyde-3 -phosphate dehydrogenase, hsp-1 encodes heat shock protein 70, hsp90 encodes heat shock protein 90, rps-23 encodes a ribosomal protein, rps-3a encodes small ribosomal protein 3a, rps-4 encodes small ribosomal protein 4, snb-1 encodes synaptobrevin, spk-1 encodes splicesomal SR protein, ICL encodes isocitrate lyase, rpn7 encodes a 26S proteasome, Tisll encodes a putative transcription factor, myo3 encodes a myosin heavy chain, PV010 encodes a splicesome subunit, prp-17 encodes a pre-mRNA splicing factor, tmy-1 encodes tropomyosin, unc-15 encodes paramyosin, and Y25 encodes a coatom er complex subunit. EST are expressed sequence tags that can encode proteins including integrase and splicing factors.

[0016] As seen in Table 1, the parasitism effector parasitism effector gene may be selected from 10A06, 3B05, 4G06, 8H07, asp-1, eng-1, crt, cm, ctl, gsts-1, syv46, 16D10L, TCTP, pat- 10, unc-87, msp-1 / vap-1 , msp-1 / vap-1 msp2, msp3, msp5, msp-9 / 8D05, msp-9 / 8D05, msp-10, spl2, msp-16, mspl8, msp20, msp23, msp24, msp40 , pel, pell , pel2 , pel3 , pg, far-1 , pat-10, pat-10, ams-1, ser-1, spl, and xyl2. 10A06 encodes a secretory protein with similarity to plant ring H2 zinc finger proteins, 3B05 encodes a cellulose binding protein, 4G06 encodes a predicted protein with similarity to plant ubiquitins, 8H07 encodes a protein with similarity to the SKP1 protein of plants, asp-1 encodes an aspartic protease, eng-1 encodes P-1,4- endoglucanase, crt encodes calreticulin , cm encodes chorismite mutase, ctl encodes a C-typelectin, gsts-1 encodes a glutathione-S transferase, syv46 encodes a secreted peptide, 16D10L encodes a putative effector gene, TCTP encodes a translationally controlled tumor protein, pat-10 encodes a body wall troponin C, unc-87 encodes two movement related proteins, vap- 1 encodes a venom allergen-like protein, mspl encodes a Meloidogyne secreted protein 1, msp2 encodes a putative esophageal gland cell secretory protein 2 (Meloidogyne secreted protein 2), msp3 encodes a putative esophageal gland cell secretory protein 3 (Meloidogyne secreted protein 3), msp5 encodes a putative esophageal gland cell secretory protein 5 (Meloidogyne secreted protein 5), msp-9 encodes a putative esophageal gland cell secretory protein 9 (Meloidogyne secreted protein 9), 8D05 encodes esophageal gland cell secretory protein 8D05 (also known as Meloidogyne secreted protein 9), msp-10 encodes a putative esophageal gland cell secretory protein 10 (Meloidogyne secreted protein 10), spl2 encodes an effector protein, msp-16 encodes parasitism protein 16D10 (Meloidogyne secreted protein 16D10), mspl8 encodes a putative esophageal gland cell secretory protein 18 (Meloidogyne- secreted protein 18), msp20 encodes a putative esophageal gland cell secretory protein 20 (Meloidogyne-SQVCQ protein 20), msp23 encodes a putative esophageal gland cell secretory protein 23 (Meloidogyne-secreted protein 23), msp24 encodes a putative esophageal gland cell secretory protein 24 (Afe / oztfogyzze-secreted protein 24), msp40 encodes a putative esophageal gland cell secretory protein 40 (Afe / oztfogyzze-secreted protein 40), pel encodes a pectate lyase, pell encodes pectate lyase, pel2 encodes pectate lyase 2, pel3 encodes pectate lyase 3, pg encodes a polygalacturonase far- encodes a lipid binding protein, pat-10 encodes body wall tropoin C, ams-1 encodes a secreted amphid protein, ser-1 encodes a serine protease, spl encodes a serine protease, and xyl2 encodes a xylanase.

[0167] Nanotechnology

[0168] Strategies for the delivery of dsRNA

[0169] The four major strategies for the delivery of dsRNA are host-induced gene silencing (HIGS), virus-induced gene silencing (VIGS)

[0138] , bacterium-induced gene silencing (BIGS)

[0139] , and the exogenous application of dsRNA using foliar sprays or other methods (FIG. 2A). HIGS is typically achieved using transgenic plants, as demonstrated in Arabidopsis plants engineered to induce RNAi against M. incognita by targeting the pathogenicity factor 16D10

[0140] , However, HIGS may not be feasible in many agriculturalsettings, because it requires the deliberate release of genetically modified organisms (GMOs). VIGS involves the production of dsRNA by plant viruses. Tobacco rattle virus (TRV) has been chosen as a vector for nematode control because it propagates in the syncytia induced by root-knot nematodes. A TRV vector producing dsRNA targeting the housekeeping gene glyceraldehyde 3 -phosphate dehydrogenase (Hs-GAPDH) was able to control the beet cyst nematode Heterodera schachlii. although it did not prevent the initial infestation

[0141] , In fact, impediment to the broad application of VIGS is that only incomplete silencing is achieved - more effective delivery techniques are urgently needed. To the best of Applicant’s knowledge BIGS has not been applied for nematode control. Lastly, while exogenous application of dsRNA for the control of nematodes has been demonstrated in vitro it is not applicable for field conditions (FIG. 2B). This reflects the need to deliver the rather fragile dsRNA molecules into the root zone, deep in the soil. Naked dsRNA is labile in the soil environment, resulting in complete degradation within 48 h

[0142] , Although this provides an inbuilt safety mechanism to prevent the accumulation of dsRNA in the environment, it highlights the need for a delivery system that preserves dsRNA long enough for it to take effect. Lessons can be learned from the field of nanomedicine, where nanomaterials are used to control the delivery, targeting and release of active pharmaceutical ingredients, including RNA-based drugs FIG. 2C).101701 Nanotechnology for the delivery of dsRNA10.1711 The foliar delivery of dsRNA has been achieved using several nanomaterials, including clay nanosheets [145, 146], chitosan nanoparticles

[0147] , polymer nanoparticles

[0148] , and peptide assemblies

[0149] , In each case, the nanoparticles enhanced the uptake of dsRNA into the plant, increasing the efficacy of RNAi against viruses and insects. Nevertheless, in all these examples, the dsRNA was delivered to the host rather than directly to the target pest. Given that nematodes can take up dsRNA orally or through their epidermis, there is an opportunity to use nanotechnology for the direct targeting of nematode pests.

[0172] Nanomaterials derived from plant viruses offer a promising alternative to synthetic nanoparticles. Plant viruses have evolved to remain stable in soil, some are soil-borne, and many are transmitted by nematode vectors [150, 151], hence the natural vector interactions can be harnessed and plant viruses repurposed for vector control strategies. Indeed, platformssuch as tobacco mild green mosaic virus (TMGMV)

[0152] and red clover necrotic mottle virus (RCNMV)

[0153] have been already developed for the encapsulation of nematicides to reduce exposure risks while enhancing their efficacy at the root level (Fig 2C). Unlike synthetic nanoparticles, these biological platforms offer a higher degree of environmental biocompatibility, and Applicant has demonstrated that rod-shaped and spherical TMGMV nanoparticles have superior mobility in the soil [143, 152], Notably, BioProdex Inc. has commercialized TMGMV as a bioherbicide under the US EP A Pesticide Product Label SOLVINIX LC. Thus, methods for the mass-production of TMGMV to meet commercial needs have been established

[0154] , On the lab scale, plant viruses can be produced at 500 mg per 100 g leaves, using rapid and cost-effective molecular farming approaches

[0155] , Plant virus nanoparticles and their genome-free counterparts (virus-like particles) are produced using inexpensive media (soil and water) and do not require toxic solvents or reagents. The nucleoprotein assemblies provide an impeccable design and engineering space and are amenable to both chemical and genetic modifications to introduce new functionalities

[0155] , Given that viruses have evolved to protect their payload (i.e., their genome) and deliver it efficiently, they can be regarded as naturally occurring nanocarriers of nucleic acids. Plant viruses are biodegradable, yet they are exceptionally robust in the harsh open-field environment. Finally, and most importantly, from a human health perspective, plant viruses are non-infectious to mammals, making them safe to use on food and feed crops. From an agronomical point, SNPs are non-infectious toward plants either.

[0173] The control of nematodes using the HIGS approach has been experimentally demonstrated using transgenic plants, but these are classed as GMOs

[0140] , Transient dsRNA delivery is therefore likely to be favored because this encompasses various GMO-free approaches that are more cost-effective and address the safety concerns of transgenic plants, making them more likely to achieve public acceptance

[0079] , The transition from GMOs to transient delivery is already underway in the agrifood industry more generally. For example, the transgenic maize variety SmartStax Pro (Mon87411) was the first RNAi -based genetically engineered product from Bayer, and was approved by the US government in 2017 and by China in 2021

[0156] , However, given its GMO background, interest in Smart-Stax Pro maize is decreasing

[0079] and companies such as Bayer, Syngenta and Greenlight Biosciences are developing spray-induced gene silencing products to replace it

[0156] ,

[0174] Although HIGS based on transient dsRNA delivery overcomes challenges associated with the GMO approach, environmental safety must be considered when dsRNA is transiently produced by or exogenously applied to crops. This is necessary to protect beneficial insects (e.g., honey bees), livestock and humans from exposure to or the consumption of dsRNA. RNAi approaches are rationally designed for target specificity, and no sequence-dependent adverse effects against non-target organisms have been observed in laboratory studies

[0157] , Notably, many plant viruses have dsRNA replication intermediates that are present in plant material consumed by humans, but no adverse effects have been reported

[0158] , This probably reflects the multiple biological barriers that any dsRNA in the human diet needs to overcome in order to avoid degradation and clearance, including stomach acid, nucleases, the reticuloendothelial system, renal filtration, and cell membranes. Nevertheless, dsRNA is a potent immunomodulator because it is a pathogen-associated molecular pattern (PAMP) that triggers the innate immune response by signaling through pattern recognition receptors (PRRs). Although there is no precedent for RNAi-induced adverse effects in humans following the consumption of dsRNA, the increasing use of RNAi in agriculture means that the risks should not be ignored

[0079] ,

[0175] In conclusion, nanotechnology platforms based on plant viruses and other nanomaterials adapted for the delivery of chemical or biochemical reagents to soil may provide an eco-friendly and publicly acceptable strategy for the protection of crops against nematodes and other pests. The COVID-19 pandemic has highlighted the value of nanotechnology platforms for the delivery of nucleic acids (here mRNA vaccines)

[0159] , The adoption of similar approaches in agriculture could lead to long-term sustainable solutions for the management of nematode pests.

[0176] Experiment No. 2:

[0177] Materials and methods

[0178] Synthesis and purification of dsRNA

[0179] Short (21 bp) dsRNA pre-labeled with Alexa Fluor 555 (BLOCK-iT Alexa Fluor Red Fluorescent Control) was obtained from Thermo Fisher Scientific. Long (861 bp) dsRNA was synthesized by amplifying the mCherry gene directly from C. elegans RBW2642 genomicDNA using Q5 polymerase and the corresponding buffer (New England Biolabs). Primer overhangs (Table 2) were used to introduce T7 promoters at both ends of the PCR product, which was then used as the template for dsRNA synthesis with the MEGAscript T7 Transcription Kit (Thermo Fisher Scientific). The dsRNA was purified using the LiCl precipitation method (Thermo Fisher Scientific) and resuspended in nuclease-free water. The dsRNA concentration was quantified with a NanoDrop 2000 spectrophotometer, using a dsRNA-specific conversion factor of 46.18 pg / rnL / A260nm

[0037] , The correct size of the dsRNA was confirmed by agarose gel electrophoresis.

[0180] Table 2: Primers used for dsRNA synthesis

[0181] T7 promoter sequences introduced as primer overhangs are shown in capital letters. All primers were obtained from Integrated DNA Technologies.

[0182] Preparation of TMGMV

[0183] Bulk TMGMV obtained from BioProdex was purified by dialysis against 10 mM KPO4 (pH 7.0) for 48 h at 4 °C using 12-14 kDa dialysis membranes (Thermo Fisher Scientific), followed by ultracentrifugation over a sucrose cushion (30% sucrose in 10 mM KPO4, pH 7.0) at 42,000 rpm for 2.5 h at 4 °C with an Optima L-90k centrifuge and a 50.2 Ti rotor (Beckman Coulter). The supernatant was discarded and the TMGMV pellet was resuspended in 10 mM KPO4 (pH 7.0) on a rotary shaker at 4 °C overnight

[0036] , Purified TMGMV was quantified with a NanoDrop 2000 spectrophotometer, using an extinction coefficient of e = 3.0 mL / mg / cm. After purification, TMGMV was stored in 10 mM KPO4 (pH 7.0) at 4 °C.

[0184] Free TMGMV coat protein was prepared in 50-mL tubes by mixing one volume of purified TMGMV (20 mg / mL) with two volumes of acetic acid (final concentration 67%), followed by incubation on ice for 20 min [38, 39], Precipitated virus RNA was removed by centrifugation (20,000 x g, 20 min, 4 °C) and the supernatant was processed by ultracentrifugation as above for 2 h. The ultracentrifugation supernatant was discarded and the pellet was resuspended in deionized water on a rotary shaker at 4 °C overnight. Purified coat protein was stored at 4 °C, with the pH at 3.35 due to residual acetic acid. The coat protein concentration was determined using a bicinchoninic acid (BCA) assay.

[0185] Preparation of Cy5-labeled TMGMV coat protein

[0186] TMGMV was labeled with Cy5 using copper-catalyzed azide-alkyne cycloaddition

[0040] as described in the Supporting Information. Free Cy5-labeled TMGMV coat protein was obtained by disassembling the TMGMV particles in 67% acetic acid as described above for native TMGMV, with modifications. Briefly, 300 pL of Cy5-labeled TMGMV (10 mg / mL) was mixed with 600 pL acetic acid (final concentration 67%) and incubated for 20 min on ice in the dark. The viral RNA was removed by centrifugation as described above. Next, 200 pL of the supernatant was mixed with 25 pL 3.0 M sodium acetate (pH 4.75) and 175 pL deionized water on ice to precipitate coat proteins at their isoelectric point

[0041] , The pellet was recovered by centrifugation as above and dissolved in deionized water by adjusting the pH to ~3.3 using 0.5 M HC1 (free coat protein was prone to aggregation at neutral or alkaline pH but remained soluble under acidic conditions). Cy5-labeled TMGMV coat protein was used immediately to produce Cy5-labeled SNPs.

[0187] Preparation of SNPs

[0188] For the preparation of empty SNPs, 5.0 pL of the TMGMV or TMGMV coat protein stock was mixed with 5.0 pL deionized water resulting in a final concentration of 2.5 mg / mL. The samples were heated to 98 °C for 30 s in an MJ Research PTC-200 thermal cycler (Table 3). For the preparation of dsRNA-loaded SNPs, Applicant added < 500 ng / pL (final concentration) of dsRNA in deionized water to the reaction mixture (optionally with Na+or Mg2+at final concentrations of 10-250 mM). To disperse aggregates, SNPs were pooled insterile 1.5-mL reaction tubes and sonicated for 30 s with a Q500 Ultra Sonicator (Q Sonica) on ice, using an amplitude of 30% (pause on 3, pause off 1).

[0189] Table 3: Thermal cycler protocol for the casting of TMGMV coat protein into SNPs.101901 All SNP formulations were cast in a total volume of 10 pL (in PCR tubes) in a MJ Research PTC-200 thermal cycler. To keep the temperature profile consistent, larger batches of SNPs were prepared by running multiple reactions in parallel.[0191 J Coating of SNPs with tannic acid

[0012] SNPs were prepared as described above using 2.5 mg / mL TMGMV coat protein, 400 ng / pL dsRNA and 100 mM MgCh (final concentrations). To disperse aggregates, SNPs were sonicated on ice as described above. SNPs were then dialyzed against deionized water at 4 °C overnight to remove free MgCh (because Mg2+ions induce the polymerization of tannic acid) and coated by incubation in 30 or 150 mM tannic acid (Sigma- Aldrich) at 20 °C for 15 min

[0042] , Finally, SNPs were dialyzed against deionized water overnight at 4 °C to remove free tannic acid.

[0193] Agarose gel electrophoresis

[0014] The dsRNA was analyzed by agarose gel electrophoresis in TAE buffer (Thermo Fisher Scientific) at 90 V for 50 min, using 4% (w / v) agarose for the short dsRNA and 1% (w / v) agarose for the long dsRNA. Applicant used 5% (w / v) glycerol in the loading buffer, and optionally 50-200 mM NaOH to disassemble SNPs before electrophoresis. Unlabeled nucleic acids in the gel were stained with GelRed (Thermo Fisher Scientific) and detected at 302 / 590 nm (excitation / emission) using the FluorChem R system (Bio-Techne). Alexa Fluor555-labeled dsRNA was detected at 534 / 607 nm, and protein was stained with Gel Code Blue reagent (Thermo Fisher Scientific) and detected at 302 / 590 nm.

[0015] Scanning electron microscopy (SEM)

[0196] SNP preparations were diluted 10-fold in deionized water and dried onto silicon wafers bound onto aluminum mounting studs with carbon tape. Samples were dried for > 24 h at 20 °C before coating with iridium using a CTC sputter coater with a 95% sputter setpoint and a coating time of 30 s. Images were acquired using a Zeiss Sigma 500 scanning electron microscope at 3-5 kV under vacuum.[0197.1 Soil mobility assays

[0198] Applicant packed 12.5 mL Pro-Mix BX soil (Greenhouse Megastore) into 15-mL tubes (10 cm bed height) fitted with a double layer of cheesecloth at the bottom. The soil was pre-wetted with deionized water and left until dripping had stopped. An aliquot of 1 mg dsRNA-loaded SNPs was then added to the column, which was irrigated with 100 mM MgCh in deionized water at a constant rate of 150 mL / h using a syringe pump. The effluent was collected in 1-mL fractions for analysis by SDS-PAGE. Optionally, C. elegans RBW2642 was incubated in the effluent to assess the efficacy of silencing with SNPs.

[0199] SDS-PAGE

[0200] Soil column elution fractions were mixed in a 1 : 1 ratio with NEB Purple Loading Dye (New England Biolabs) and separated on 4-12% Bis-Tris gels in MOPS buffer (both from Thermo Fisher Scientific) at 200 V for 45 min. Nucleic acids were then stained with GelRed and detected at 302 / 590 nm (excitation / emission), whereas protein was stained with Gel Code Blue reagent (Thermo Fisher Scientific) and detected at 302 / 590 nm.

[0201] Cultivation of C. elegans

[0202] Escherichia coli OP-50, wild-type C. elegans (N2 laboratory strain) and transgenic C. elegans expressing mCherry (RBW2642) were obtained from the Caenorhabditis Genetics Center (University of Minnesota). All nematodes were cultivated on 100 * 15 mm sterile polystyrene Petri dishes (Thermo Fischer Scientific) with solid nematode growth medium(NGM; 3.0 g / L NaCl, 17.5 g / L agar, 2.5 g / L peptone) supplemented with 0.001 M MgSCh, 0.001 M CaCh, 0.005 g / L cholesterol (Sigma-Aldrich) and 0.025 M KPO4(pH 6.0) at 20 °C in the dark. Plates were seeded with E. coll OP-50 as a primary food source.

[0203] Feeding studies with labeled SNPs

[0204] Wild-type C. elegans (N2 laboratory strain) were washed off 7-day-old NGM plates using S-medium

[0043] and were washed twice in the same medium by centrifugation (2000 x g, 2 min, room temperature). The nematodes were then incubated with SNPs or Cy5-labled SNPs for 3 h at 20 °C in 100 pL S-medium, before washing as described above and fixation with 4% (v / v) formaldehyde.

[0205] Silencing experiments and confocal imaging

[0206] Transgenic C. elegans (RBW2642) nematodes were washed off 7-day-old NGM plates as described above for the feeding studies and were incubated with 5, 50 or 500 ng / pL long dsRNA (or without dsRNA as a control) in soaking buffer (3 g / L KH2PO4, 6 g / L Na2HPC>4, 5 g / L NaCl) in 96 well plates (-100 nematodes / well) for 16 h at 16 °C. The nematodes were then transferred to NGM plates for -60 h before fixing with 4% (v / v) formaldehyde. Free dsRNA and dsRNA-loaded SNPs were compared by incubating C. elegans RBW2642 with similar volumes of free dsRNA (500 ng / pL) or dsRNA-SNPs (cast with 200 ng / pL dsRNA) in S-medium at 20 °C for 16 h before transfer to NGM plates. After -60 h, the nematodes were collected from the plates at 24-h intervals and fixed with 4% (v / v) formaldehyde. Fixed nematodes were visualized on a BZ-X710 all-in one microscope (Keyence Corporation) using a BZ-X Cy5 or TxRed filter set and the accompanying imaging analysis software. Silencing efficacy was quantified using ImageJ

[0044] ,

[0207] Statistical methods

[0208] Data from silencing experiments were tested for normality using a Shapiro-Wilk test and equal variance was confirmed using an F-test

[0045] , Unpaired two-sample, two-sided t- tests were used to compare normally distributed data, with a significance level of a = 0.05. Welch’s t-test was used if variances differed significantly between datasets. A Mann-Whitney U-test (Wilcoxon test) was used to compare non-normally distributed data

[0046] , In allstatistical tests, N refers to the total number of samples from both groups, whereas n refers to the number of samples from a single group. OriginPro 2024 (OriginLab) was used for all statistical tests.

[0209] Results and discussion

[0210] SNPs are taken up by nematodes

[0211] To establish the feasibility of the approach, Applicant first determined whether nematodes would interact with and ingest the dsRNA-containing SNPs. In previous study, SNPs were produced from wild-type TMGMV

[0036] , whereas here Applicant produced them from purified TMGMV coat protein in an attempt to avoid the incorporation of residual genomic RNA. The A26o:28Onm ratio of the coat protein preparation was 0.77 (Table 4) compared to 1.20 for intact TMGMV

[0047] , thus confirming that much of the virus RNA (absorbance maximum at 260 nm) had been removed during purification. However, an A26o:28Onm ratio of 0.65 would be expected for completely pure coat protein formulations

[0039] , indicating the presence of some residual virus RNA in the coat protein preparations.

[0212] Using a coat protein concentration of 2.5 mg / mL, SNPs readily formed at an incubation temperature of 98 °C. Applicant analyzed the size distribution based on SEM images and found that 77% of SNPs had a diameter of 100-200 nm, whereas 23% were larger than 200 nm, and 3% were ~1 pm in diameter (FIG. 4A). Applicant deemed this size range suitable for the strategy because nanoparticles and microparticles up to 1 pm in diameter are taken up by C. elegans

[0048] , To confirm the ingestion of SNPs by C. elegans. Applicant produced SNPs from native and Cy5-labeled TMGMV coat protein

[0049] followed by feeding assays. Cy5-labeled TMGMV was prepared by coupling diazonium to tyrosine side chains exposed on the virion surface, followed by the copper-catalyzed azide-alkyne cycloaddition of Cy5 (FIGS. 11A-11C). Size exclusion chromatography co-elution profiles confirmed that Cy-5 was conjugated to TMGMV (FIG. 11C). The conjugation efficiency was ~7% (or 150 Cy5 molecules per TMGMV particle), as previously reported

[0050] ,

[0213] Cy5-labeled coat protein was obtained by disassembling Cy5-labeled TMGMV in acetic acid as previously described for native TMGMV [38, 39], This approach is suitable because Cy5 is stable in acid but not alkaline solutions. Following disassembly and theprecipitation of virus RNA, free TMGMV coat protein was precipitated at its isoelectric point by adding sodium acetate (FIG. 11D). Precipitated coat protein was separated by centrifugation (FIG. HE) and dissolved in deionized water by adjusting the pH to ~3.3. SDS-PAGE confirmed the colocalization of coat protein and Cy-5 (FIG. HF, FIG. 11G). Cy5-labeled coat protein (2.5 mg / mL) was converted into Cy5-labeled SNPs at 98 °C, because these conditions have previously yielded SNP sizes suitable for nematode feeding, as discussed above

[0048] ,

[0214] Native and Cy5-labeled SNPs were fed to ~50 nematodes in 100 pL S-medium in 96- well plates at a concentration of 1 mg / mL. Dosing was based on previous feeding studies with Cy5-labeled TMGMV

[0050] , After incubation for 3 h and two washes in S-medium, Applicant observed the nematodes by fluorescence microscopy, revealing fluorescent digestive tracts in those fed on the Cy5-labeled SNPs and the absence of autofluorescence in those fed on unlabeled SNPs (FIG. 4B).

[0215] The ingestion of dsRNA by mammals hinders RNAi because the RNA undergoes extensive degradation and cannot surmount biological barriers to reach the systemic circulation

[0051] , In contrast, ingested dsRNA triggers RNAi in many invertebrates, including C. elegans

[0052] , The uptake of ingested dsRNA into C. elegans intestinal cells is enabled by the intestinal transmembrane protein SID-2

[0053] , followed by release from the internalized vesicles by the dsRNA channel SID-1 [52, 54], The ingestion of dsRNA has also been shown to induce RNAi in phytoparasitic cyst and root knot nematodes, typically using octopamine or resorcinol treatment to induce feeding [55-58], These observations indicate that pathways for dsRNA uptake and transport also exist in phytoparasitic nematodes, thus enabling RNAi triggered by the oral uptake of dsRNA. SID-1 and SID-2 homologs, however, are absent in phytoparasitic nematodes such as Meloidogyne and Globodera spp., therefore the uptake of dsRNA in these species probably involves endocytosis, as reported in insects [54, 59],

[0216] Table 4: UV-Vis spectroscopy data for purified TMGMV coat protein.[0217| The A260:280nm absorbance ratio was used to assess the purity of free TMGMV coat protein [S.K. Chan et al., ChemBioChem 23(11); (2022) ]. The A26o:28Onm ratio of intact TMGMV particles is 1.20 [ I. Gonzalez-Gamboa et al., Scientific Reports 14(1) (2024)].

[0218] RNAi can be assessed rapidly in transgenic nematodes expressing fluorescent reporter genes

[0219] Applicant chose C. elegans RBW2642 as a model to study the efficacy of RNAi because the fluorescent reporter protein mCherry is constitutively and ubiquitously expressed in this strain (driven by the hsp90!daf21 promoter

[0060] ). The knockdown of the mCherry transgene can be assessed quantitatively by measuring the fluorescence intensity and does not affect nematode health or survival. To generate the dsRNA probe, Applicant amplified the mCherry gene directly from the genome of C. elegans RBW2642 by PCR using extended primers that introduced T7 promoters at both ends, allowing the production of dsRNA in a single reaction (FIG. 4C). The rationale for the use of long dsRNA covering the entire mCherry gene (861 bp) was to increase the melting temperature of dsRNA, and thus prevent denaturation during the thermal shape transition process that produces the SNPs. Progress made in genome sequencing means the same approach can be used to generate dsRNA targeting essential genes in phytoparasitic nematodes [61-63], A review of RNAi applications in phytoparasitic nematodes provides a summary of suitable targets

[0020] ,

[0220] Applicant investigated the functionality of the dsRNA by soaking C. elegans RBW2642 in free dsRNA solutions of 0, 5, 50 and 500 ng / pL (FIGS. 4D-4F). Applicant acquired images after ~60 h to ensure that all pre-existing mCherry (half-life ~30 h in eukaryotes

[0064] ) had broken down. High concentrations of free dsRNA (500 ng / pL) reducedmCherry fluorescence by up to -50%, thus confirming its ability to induce RNAi (FIGS. 4D- 4F). Interestingly, RNAi was observed in the body of the transgenic nematodes but not in the pharynx (FIG. 4D). This has previously been linked to the developmental stage where RNAi is induced

[0065] , Specifically, pharyngeal muscle cells require nuclear rather than cytoplasmic RNAi

[0065] , Because the development stage can affect silencing efficacy, the sustained delivery of dsRNA that spans multiple nematode generations is desirable.

[0221] SNPs can be used to encapsulate dsRNA

[0222] Building on previous studies with SNPs as nanocarriers for nematicides

[0036] , Applicant tested the ability of SNPs to encapsulate dsRNA during thermal shape transition. Applicant tested the encapsulation of dsRNA into SNPs prepared from TMGMV coat protein (as well as those prepared from assembled TMGMV particles) using Alexa Fluor 555-labeled dsRNA as a model for standard short interfering RNAs (siRNAs) of -21 bp. Applicant collected SNPs by ultracentrifugation and monitored the fluorophore using a gel documentation system (FIG. 8). Interestingly, the majority of the labeled dsRNA remained in the supernatant when using assembled TMGMV to cast the SNPs, whereas the labeled dsRNA colocalized with SNPs when using free coat protein (FIG. 8). This may reflect charge repulsion caused by the genomic RNA in the wild-type TMGMV particles, whereas most viral RNA is removed during the preparation of free coat protein. The analysis of dsRNA- loaded SNPs by agarose gel electrophoresis confirmed that the labeled dsRNA remained colocalized with SNPs, indicating encapsulation (FIG. 5A). Whereas free dsRNA moves toward the anode, the SNPs do not move into the gel, probably due to their high molecular weight and large size. Interestingly, empty (no dsRNA) SNPs formed from purified coat protein showed a weak signal for nucleic acids, indicating the presence of residual viral RNA, consistent with the UV-Vis spectroscopy data (Table 4). To optimize the process, such residual RNA can be removed by treatment with RNase during the preparation of free coat protein from TMGMV.

[0223] To determine whether the dsRNA cargo remained intact following its encapsulation into SNPs, Applicant treated the dsRNA-laden SNPs with NaOH to release the encapsulated dsRNA (FIG. 5A). This is important because the dsRNA was exposed to a temperature of 98 °C for 30 s and siRNAs typically have a melting temperature in the range 20-60 °C [66, 67],Analysis of the disassembled SNPs by agarose gel electrophoresis confirmed that the encapsulated dsRNA remained intact after thermal transitioning, as shown by the difference in mobility compared to ssRNA, indicating that the brief, high-temperature exposure was not sufficient for denaturation (FIG. 4C, FIG. 5A).10224] Having shown that dsRNA is loaded preferentially into SNPs prepared from free coat protein, Applicant continued to use this method in subsequent experiments. Next, Applicant tested the encapsulation of long dsRNA (861 bp) because this preferentially interacts with the dsRNA-binding protein RDE-4 and is efficiently processed by Dicer into multiple siRNAs, thus increasing the efficacy of silencing

[0068] , Additionally, the transmembrane protein SID-2 only transfers dsRNA molecules in the size range 50-1500 bp

[0052] , Applicant therefore switched from the short dsRNA (sufficient for preliminary experiments) to the longer molecule for experiments involving efficacy testing. To investigate the dsRNA loading capacity of SNPs, Applicant produced SNPs with a constant concentration of 2.5 mg / mL coat protein but different amounts of dsRNA (0-500 ng / pL). The characterization of SNP morphology by SEM revealed that dsRNA concentrations exceeding 50 ng / pL resulted in the formation of aggregates during thermal transitioning (FIG. 5B, right two images). When the concentration of dsRNA was increased to 300-500 ng / pL, encapsulation was completely abolished and the dsRNA remained in the supernatant (FIG. 9). The encapsulation of long dsRNA into SNPs thus required further optimization.

[0225] As a first approach, Applicant neutralized the negative charge of the RNA backbone with monovalent (FIG. 5C) and bivalent cations (FIG. 5D), both of which restored SNP formation in the presence of 200 ng / pL dsRNA (FIG. 5C, FIG. 5D). The bivalent cation Mg2+was more efficient than the monovalent cation Na+in that only 10 mM MgCh was required, compared to 250 mM NaCl (FIG. 5C, FIG. 5D). The acidic pH of the coat protein formulations (pH < 5.0) also facilitated the efficient loading of dsRNA into SNPs. Specifically, the combination of multivalent cations with an acidic buffer induces long, stretched DNA molecules to form compact spheres

[0069] ,

[0226] In the absence of bivalent cations, it was only possible to load up to 0.02 mg dsRNA per 1.0 mg SNPs without inducing aggregation (FIG. 5B, Table 5). With the optimized protocol, using Mg2+ions for charge neutralization (2.5 mg / mL coat protein, 400 ng / pLdsRNA, 100 mM MgCh), it was possible to load up to up to 0.20 mg dsRNA per 1.0 mg of SNPs 100-200 nm in diameter, thus increasing the dsRNA loading capacity of SNPs by an order of magnitude. It was also possible to influence the size of SNPs using different concentrations of cations, allowing the production of SNPs with sizes up to 1.0 pm (FIGS. 5C and 5D,), which are suitable for feeding to nematodes as discussed above

[0048] ,Table 5: Investigation of the dsRNA-loading capacity of SNPs without charge neutralization and condensation of dsRNA as determined by densitometry. ImageJ was used to quantify free and encapsulated dsRNA when gradually increasing the concentration of dsRNA during SNP casting (FIG. 9).10227]

[0228] dsRNA-loaded SNPs are more efficient for RNAi than free dsRNA

[0229] Having loaded SNPs with high concentrations of dsRNA (0.02 mg dsRNA per 1.0 mg SNPs), Applicant investigated their silencing efficacy compared to free dsRNA. Applicant fed groups of nematodes on native SNPs (control), dsRNA-loaded SNPs, or free dsRNA, waited ~60 h for the mCherry present before feeding to be broken down, then imaged the nematodes at ~24-h intervals and quantified the fluorescence (FIG. 6A). Applicant normalized the fluorescence intensity to different-sized regions of interest (ROIs) to ensure that the results were consistent regardless of the size of each nematode specimen

[0070] ,

[0230] Although free dsRNA was present at a higher concentration, silencing was more potent when the dsRNA was encapsulated in SNPs (FIG. 6B, FIG. 6C). This may reflect the more efficient uptake of encapsulated dsRNA and / or its greater stability compared to free dsRNA, which should be investigated in future experiments. The minimal fluorescence in juvenile nematodes ~60 h post-feeding was 51±36% with free dsRNA and 25±12% with encapsulated dsRNA. The difference was statistically significant (Mann-Whitney U-test, a = 0.05, p < 0.001, N= 71). In adult nematodes, the minimal fluorescence was 60±17% with free dsRNA and 25±4% with encapsulated dsRNA under the same conditions, and again the difference was statistically significant (two-sided, two-sample t-test, a = 0.05, p < 0.001, N = 15). The silencing efficacy Applicant observed with free dsRNA was similar to that reported in earlier studies

[0071] ,

[0231] SNPs loaded with dsRNA retain silencing activity following elution from a soil column(0232] Applicant tested the soil mobility of dsRNA-loaded SNPs by replicating a previous soil-column setup (FIG. 7A

[0049] ) using Pro-Mix soil (10 cm bed height) infused with deionized water (150 mL / h) to mimic real-life irrigation. Applicant also added 100 mM MgCh to model the cations present in plant fertilizer, making the experiment close to typical application conditions in the field.

[0233] The SNPs were sonicated briefly to disperse aggregates, and the soil columns were pre-wetted before Applicant loaded them with 1.0 mg of the SNPs. Elution fractions (1 mL) were analyzed by SDS-PAGE for the presence of dsRNA and TMGMV coat protein (FIG. 7B). Both intact dsRNA and coat protein were detected in the elution fractions (FIG. 7B, black arrow), indicating that dsRNA-loaded SNPs can move through the soil. Interestingly, early elution fractions (3 mL) contained dsRNA but not coat protein (FIG. 7B), suggesting that a fraction, namely ~ 30%, of SNPs were disassembled on the soil column leading to premature release of their dsRNA cargo. Thus, some optimization to increase the dsRNA- laden SNP formulation in soil could be explored (see also below). Collectively this data suggests SNPs are not only suitable for the feeding of nematodes with encapsulated dsRNA, but could also be used for the sustained release of dsRNA in the soil.

[0234] Importantly, SNPs induced silencing even after passing through soil columns that mimic the intended field application (FIG. 7C, FIG. 7D). Silencing was less effective with elution fractions lacking the TMGMV coat protein (free dsRNA, 3 mL elution fraction), compared to elution fractions containing both dsRNA and coat protein (7 mL elution fraction). These results agreed with experiments comparing free dsRNA and encapsulated dsRNA, indicating that encapsulated dsRNA was more efficacious than free dsRNA. As stated earlier, this may reflect the more efficient update of encapsulated dsRNA and / or the greater stability of the encapsulated dsRNA compared to free dsRNA. Overall, these experiments confirmed that dsRNA-loaded SNPs can deliver dsRNA in the soil, thus enabling the control of phytoparasitic nematodes by RNAi.

[0235] Potential for the further optimization of dsRNA-loaded SNPs

[0236] As stated above, some dsRNA was released prematurely from the SNPs when passing them over soil columns prior to nematode feeding. In an effort to stabilize the formulation and prevent premature disassembly and dsRNA release, Applicant coated the dsRNA-laden SNPs with a metal-organic coating based on tannic acid, because this polymer is easy to synthesize, biodegradable, and can be used to functionalize surfaces [42, 72], Moreover, coating with tannic acid is synergistic with the addition of metal ions required to load the dsRNA into SNPs because metal ions are also required for the polymerization of tannic acid.

[0237] For coating experiments, Applicant prepared SNPs by mixing 2.5 mg / mL TMGMV coat protein, 400 ng / pL dsRNA and 100 mM MgCh. SEM images revealed that SNPs readily formed after heating to 98 °C, and that the size range was comparable to that of SNPs produced with free TMGMV coat protein (FIG. 10A). Applicant dialyzed the SNPs overnight against deionized water to remove free Mg2+ions, followed by incubation in 30 or 150 mM tannic acid as previously described

[0042] , After another round of dialysis to remove free tannic acid, the nanoparticle morphology was again characterized by SEM, revealing that the SNPs were coated with a polymer network (FIG. 10A). However, the SNPs formed clusters before and after coating with tannic acid, indicating that the dispersion of SNPs before coating requires further optimization; for example by adjusting the sonication settings or by adding low concentrations of organic solvents. Of note, at high concentrations of tannic acid (150 mM), the structural integrity of the SNPs may be compromised - clusters of smaller structures were apparent as well as the formation of stick-like shapes (FIG. 10A).

[0238] Applicant investigated the mobility and stability of the coated SNPs in soil column experiments as described above (30 mM tannic acid conditions were used). Applicant found that the tannic acid coating stabilized the dsRNA-loaded SNPs and prevented the premature release of dsRNA (FIG. 10B) Overall, these exploratory experiments indicated that coating dsRNA-loaded SNPs with metal-organic polymers can optimize their properties, and future experiments should systematically investigate the effect of different types of metal ions as well as tannic acid concentrations on the shape and stability of SNPs.

[0239] Conclusions[02401 RNAi offers tremendous potential for targeted pest control but faces several delivery barriers in field settings, including the inefficient uptake and rapid environmental degradation of dsRNA

[0077] , Applicant overcame these issues by encapsulating dsRNA into SNPs prepared from coat proteins of a plant virus (TMGMV). Applicant increased the dsRNA loading capacity of these SNPs by 10-fold (from 0.02 to 0.20 mg dsRNA per mg of 100-200 nm SNPs) by charge neutralization and the compaction of dsRNA using the bivalent cation Mg2+under acidic conditions (pH < 5.0). This effect was originally described for DNA

[0069] and can therefore be used to encapsulate DNA into SNPs as well. The ability to encapsulate charged cargos within SNPs by charge neutralization will be useful beyond nucleic acids, for example for the encapsulation of proteins, and Applicant will explore this possibility in the future. The concentration and type of cation influences the size of SNPs, with higher concentrations of cations forming smaller SNPs. Applicant used 100 mM Mg2+to produce SNPs in the range 100-200 nm, which is ideal for feeding to nematodes

[0048] , The same approach can be used to tailor the size of SNPs for other applications.[02411 As proof of concept, Applicant used dsRNA-loaded SNPs to silence the reporter gene mCherry in transgenic C. elegans. The dsRNA-loaded SNPs were readily ingested by nematodes and induced efficient silencing, reducing mCherry fluorescence by 76.2±13.6%. Applicant confirmed that dsRNA-loaded SNPs retain their silencing activity following elution from a soil column, therefore enabling the delivery of dsRNA to soil-dwelling nematodes. The logical next step is to transfer the same concept to lethal genes in phytoparasitic nematodes, more than 75 of which have been identified thus far

[0020] ,

[0242] The dsRNA-loaded SNPs used in this Experiment are derived from the plant virus TMGMV, which can be produced rapidly and in a scalable manner by plant molecular farming. Nanomaterials derived from this plant virus are inexpensive, easy to produce, and therefore well suited for applications in agriculture, which is characterized by narrow profit margins and requires cost efficient pest control strategies

[0078] , Although TMGMV has been approved by the EPA as a bioherbicide [32, 33], the nanomaterials add an additional layer of safety because Applicant removed the virus genome during the preparation of free coat proteins. Furthermore, the new dsRNA cargo is target specific, so these nanomaterials offermajor advantages over commonly used chemical pesticides, which have adverse effects on human health and the environment. The data also indicated that there is room for further optimization, for example by coating dsRNA-loaded SNPs with metal-organic polymers to improve their stability and mobility in soil. Overall, these properties make dsRNA-loaded SNPs a promising tool for targeted pest control strategies in agriculture.

[0243] Experiment No.: 3:

[0244] Labeling of TMGMV with Cy5

[0245] Diazonium salt was prepared as previously described [I. Gonzalez-Gamboa et al., ChemBioChem 23(18) (2022)]. Briefly, 1.27 M 4-ethynylaniline (in methanol), 0.32 M p- toluenesulfonic acid (in deionized water) and 3.0 M sodium nitrite (in deionized water) were prepared and precooled at -20 °C. Next, the 4-ethynylaniline solution was added to the precooled acid while mixing on ice. Gradually adding the nitrile solution to the mixture resulted in a color change from beige to red after 30-60 min, indicating the formation of diazonium salts. The diazonium slurry was then centrifuged (10,000xg, 2 min, 4 °C), the supernatant was discarded, and the diazonium salt was resuspended in 70% ice-cold ethanol. Aliquots of 1 mL were stored at -20 °C or used immediately for tyrosine modification.

[0246] Diazonium was coupled to tyrosine residues exposed on the TMGMV as previously described [I. Gonzalez-Gamboa et al., ChemBioChem 23(18) (2022)]. TMGMV (20 mg / mL in 100 mM borate buffer, pH 8.5) was pre-cooled on ice and diazonium was added at a mass ratio of 40: 1 (diazoniunrTMGMV). The reaction was incubated on ice for 30 min before ultracentrifugation over a 30% (w / v) sucrose cushion (50,000 rpm at 4 °C using a Beckman Optima MAX-XP centrifuge and a TLA-55 rotor). The pellet (diazonium-TMGMV) was resuspended in 10 mM KPO4 buffer (pH 7.0) on a rotary shaker at 4 °C overnight.

[0247] Applicant mixed diazonium-TMGMV (final concentration 5.0 mg / mL) with 10 mM KPO4 buffer, Cy5 (final concentration 1.2 mg / mL; five-fold molar excess per coat protein), aminoguanidine (final concentration 2.0 mM), CuSO4 (final concentration 1.0 mM) premixed with tris-hydroxypropyltriazolylmethylamine (final concentration 0.5 mM), and L- ascorbic acid (final concentration 2.0 mM) in this order. The reaction was incubated on ice in the dark for 1 h before centrifugation on a 40% (w / v) sucrose cushion (42,000 rpm, 70 min,4°C; Beckman Optima MAX-XP, TLA-55 rotor). The pellet was gently washed with 10 mM KPO4 buffer and resuspended in the same buffer on a rotary shaker at 4 °C overnight. Free Cy5 dye was removed with Amicon Ultra 100 kDa spin filters (4000 x g, 4°C). The samples (Cy5-labeled TMGMV) were stored in 10 mM KPO4 buffer at -80 °C in the dark.Equivalents

[0248] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs.

[0249] The present technology illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising,” “including,” “containing,” etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the present technology claimed.

[0250] Thus, it should be understood that the materials, methods, and examples provided here are representative of preferred aspects, are exemplary, and are not intended as limitations on the scope of the present technology.

[0251] It should be understood that although the present invention has been specifically disclosed by certain aspects, embodiments, and optional features, modification, improvement and variation of such aspects, embodiments, and optional features can be resorted to by those skilled in the art, and that such modifications, improvements and variations are considered to be within the scope of this disclosure.

[0252] The present technology has been described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the present technology. This includes the generic description of the present technology with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.[02531 In addition, where features or aspects of the present technology are described in terms of Markush groups, those skilled in the art will recognize that the present technology is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0254] Embodiments

[0255] 1. A spherical nanoparticle (SNP) comprising a plant virus coat protein and double stranded ribonucleic acid (dsRNA) that is homologous to a RNAi target nematode associated gene selected from the group of nematode development, nematode reproduction, nematode housekeeping gene, and parasitism effector gene, optionally wherein the dsRNA is 50-1500bp in length.

[0256] 2. The SNP of embodiment 1, wherein the plant virus coat protein a coat protein from a plant virus selected from a tobacco mosaic virus (TMV), a cytoplasmic type citrus leprosis virus, a tobacco mild green mottle virus (TMGMV), a physalis mottle virus like particle (PhMV), a cowpea chlorotic mottle virus (CCMV), and a cowpea mosaic virus (CPMV).

[0257] 3. The SNP of embodiment 2, wherein the plant virus coat protein is a coat protein from TMGMV.

[0258] 4. The SNP of embodiment 1, wherein the nematode development gene encodes a protein selected from chitin synthase, CPN-1 a dual oxidase, a RNA-binding protein, and troponin C.

[0259] 5. The SNP of embodiment 1, wherein the nematode reproductive gene encodes a protein selected from an aminopeptidase and a major sperm protein.

[0260] 6. The SNP of embodiment 1, wherein the housekeeping gene encodes a protein selected from a phosphatase, a G-protein-coupled receptor, cathepsin B cysteine proteinase, a cysteine proteinase, a dicer, cytochrome C, a FMRF amide-like neuropeptide, glyceraldehyde-3 -phosphate dehydrogenase, heat shock protein 70, heat shock protein 90, a ribosomal protein, small ribosomal protein 3a, small ribosomal protein 4, a synaptobrevin, aspliceosomal SR protein, integrase, isocitrate lyase, 26S proteasome, a putative transcription factor, myosin heavy chain, a spliceosome subunit, a splicing factor, a pre-mRNA splicing factor, a tropomyosin, paramyosin, and a coatomer complex subunit.

[0261] 7. The SNP of embodiment 1, wherein the parasitism effector gene encodes a protein selected from a protein similar to plant ring H2 zinc finger proteins, a cellulose- binding protein, a predicted protein with similarity to plant ubiquitins, a protein with similarity to the SKP1 protein of plants, an aspartic protease, P-l,4-endoglucanase, calreticulin, chorismite mutase, a C-type lectin, a glutathione-S transferase, a secreted peptide, a putative effector gene (ortholog of secreted peptide Mil6D10), a translationally controlled tumor protein, a body wall troponin C, a movement related protein, a venom allergen-like protein, Meloidogyne secreted protein 1, a putative esophageal gland cell secretory protein 2 (Meloidogyne secreted protein 2), a putative esophageal gland cell secretory protein 3 (Meloidogyne secreted protein 3), a putative esophageal gland cell secretory protein 5 (Meloidogyne secreted protein 5), esophageal gland cell secretory protein 8D05, a putative esophageal gland cell secretory protein 9 (Meloidogyne secreted protein 9), a putative esophageal gland cell secretory protein 10 (Meloidogyne secreted protein 10), effector protein, parasitism protein 16D10, a putative esophageal gland cell secretory protein 18 (Meloidogyne secreted protein 18), a putative esophageal gland cell secretory protein 20 (Meloidogyne secreted protein 20), a putative esophageal gland cell secretory protein 23 (Meloidogyne secreted protein 23), a putative esophageal gland cell secretory protein 24 (Meloidogyne secreted protein 24), a putative esophageal gland cell secretory protein 40 (Meloidogyne secreted protein 40), a pectate lyase, pectate lyase 2, pectate lyase 3, polygalacturonase, a lipid binding protein, movement related proteins, a secreted amphid protein, serine protease, and xylanase.

[0262] 8. The SNP of embodiment 1, where in the nematode development gene is selected from, selected from chs, cpn-1 , duoxl. pos-1. and inc

[0263] 9. The SNP of embodiment 7, wherein the nematode development gene is selected from Ma-chs, Hg-chs. Hg-cpn-1. Mi-duoxl , Mi-pos-1. mA Mi-lnc.

[0264] 10. The SNP of embodiment 1, wherein the nematode reproduction gene is selected from amp-1, and msp.(0265] 11. The SNP of embodiment 10, wherein the nematode reproduction gene is selected from Hg-amp-1, and Hg-msp.(0266] 12. The SNP of embodiment 1, wherein the nematode housekeeping gene is selected from ppm-1, srh-1, cb-1, cp-1, cpl-1, dcr-1.1, cyt-2.1,flp-l,flp-6,flp-12,flp-14, flp- 18, gpd-1, hsp-1, hsp90, rps-23, rps-3a, rps-4, snb-1, spk-1, ICL, rpn7, Tisll, myo3, PV010, prp-17, tmy-1, unc-15, and Y25

[0267] 13. The SNP of embodiment 12, wherein the nematode housekeeping gene is selected from Bx-ppm-1, Bx-srh-1, Rs-cb-1, Gp-cp-I, Hg-cp-I, Mi-cpl-1, Mi-dcr-1.1, Bx-cyt- 2.1, Gp-flp-1, Gp-flp-6, Gp-flp-12, Gp-flp-14, Gp-flp-18, Mi-flp-14, Mi-flp-18, Hs-gpd-1, Bx-hsp-1, Mi-hsp90, Hg-rps-23, Hg-rps-3a, Hg-rps-4, Hg-snb-1, Hg-spk-1, Mi-ICL, Mi- rpn7, Mj-Tisl l, Bx-myo3, Pv-010, Hg-prp-17, Bx-tmy-1, Dd-unc-15, and Hg-Y25.

[0268] 14. The SNP of embodiment 1, wherein the parasitism effector gene is selected from 10A06, 3B05, 4G06, 8H07, asp-1, eng-1, crt, cm, ctl, gsts-1, syv46, 16D10L, TCTP, pat-10, msp-1, vap-1, msp2, msp3, msp5, msp-9, 8D05, msp-10, spl2, msp-16, mspl8, msp20, msp23, msp24, msp40, pel, pell, pel2, pel3, pg, far-1, pat- 10, unc-87, ams-1, ser-1, spl, and xyl2.

[0269] 15. The SNP of embodiment 14, wherein the parasitism effector gene is selected from Hs-10A06, Hs-3B05, Hs-4G06, Hs-8H07, Mi-asp-1, Gr-eng-1, Hg-eng-1, Mi-crt, Hgcm, Hg-ctl, Mi-gsts-1, Hg-syv46, Mc-16D10L, Me-TCTP, Mg-pat-10, Mg-unc-87, Mi- msp-1 / Mi-vap-1, Mi-msp-1 / Mi-vap-1, Mi-msp2, Mi-msp3, Mi-msp5, Mi-msp-9 / Mi8D05, Mi-msp-9 / Mi8D05, Mi-msp-10, Mi-spl2, Mi-msp-16, Mi-mspl8, Mi-msp20, Mi-msp23, Mi-msp24, Mi-msp40, Hg-pel, Mi-pell, Mi-pel2, Mi-pel3, Mi-pg, Mj -far-1, Pc-pat- 10, Pp- pat-10, Pc-unc-87, Pp-unc-87, Gr-ams-1, Mi-ser-1, Mi-spl, and Mi-xyl2.

[0270] 16. The SNP of embodiment 1, wherein the dsRNA is present in the SNP at a concentration from about 3ng / pL to about 600ng / pL.

[0271] 17. The SNP of embodiment 1, wherein the dsRNA is present in the SNP at a concentration up to about 400 ng / pL.(0272] 18. The SNP of embodiment 1, wherein the dsRNA is present at a concentration of about 400ng / pL.(0273] 19. The SNP of embodiment 1, wherein the plant virus coat protein is present at a concentration of about 2.0 mg / ml to about 3.0 mg / mL, optionally about 2.5 mg / mL.

[0274] 20. The SNP of embodiment 1, wherein the SNP is coated with tannic acid.

[0275] 21. The SNP of embodiments 20, wherein the tannic acid is at a concentration of about 10 mM to about 200 mM.10276] 22. The SNP of embodiment 20, wherein the tannic acid is at a concentration of about 30mM.

[0277] 23. The SNP of any one of embodiments 1-22, having a diameter of up to IpM.

[0278] 24. The SNP of embodiment 1-22, having a diameter of about lOOnm to about 200nm.

[0279] 25. The SNP of any one of embodiments 1-24, further comprising MgCh.

[0280] 26. The SNP of embodiment 25, wherein the MgCh is at a concentration of 50mM to 150mM.

[0281] 27. A SNP comprising: a. a plant virus coat protein; b. double stranded ribonucleic acid (dsRNA) that is homologous to a RNAi target nematode associated gene selected from the group of nematode development, nematode reproduction, nematode housekeeping gene, and parasitism effector gene, c. tannic acid, andd. MgCh.

[0282] 28. A composition comprising the SNP of any one of embodiments 1-27 and a pharmaceutically acceptable carrier.

[0283] 29. A method for controlling a nematode comprising contacting a nematode with the SNP of any one of embodiments 1 to 27 or the composition of embodiment 28.

[0284] 30. The method of embodiment 29, wherein the nematode is an egg, juvenile, or adult.

[0285] 31. The method of embodiment 29, wherein the nematode is a plant parasitic nematode.10286] 32. The method of embodiment 31, wherein the nematode is a root-associated plant parasitic nematode.

[0287] 33. The method of embodiment 31 or 32, wherein the plant parasitic nematode is at least one of Meloidogyne arliella. Meloidogyne incognita, Meloidogyne chitwoodi, Meloidogyne enlerolobii, Meloidogyne graminicola, Meloidogyne hapla, Meloidogyne arenaria, Meloidogyne javanica, Heterodera glycines, Heterodera schactii, Bursaphelenchus xylophilus, Radopholus similis, Globodera pallida, Globodera rostochiensis, Pratylenchus vulnus, Pratylenchus coffeae, Pratylenchus penetrans and / or Ditylenchus destructor.

[0288] 34. The method of embodiment 30, wherein the nematode is a free-living nematode, optionally wherein the free-living nematode is Caenorhabditis elegans.

[0289] 35. The method of any one of embodiments 29 to 34, wherein the contacting is to the nematode orally or through the epidermis of the nematode.(0290] 36. The method of any one of embodiments 29 to 35, wherein the contacting occurs according to the steps of FIG. 2C.

[0291] 37. The method of any one of embodiments 29 to 36, wherein the contacting occurs in soil.

[0292] 38. The method of embodiment 37, wherein the soil is a rhizosphere of a plant.

[0293] 39. The method of any one of embodiments 29 to 36, wherein the contacting occurs on a plant root.

[0294] 40. A method to make SNPs of any one of embodiments 1 to 15 comprising transforming a plant virus into a spherical shape and loading dsRNA onto the transformed plant virus.|0295] 41. The method of embodiment 40, wherein the plant virus is transformed using heat transformation.10296] 42. The method of embodiment 40, wherein the heat transformation comprises the steps according to FIG. 3 A.REFERENCES FOR EXPERIMENT NO: 1[1] D. Pennock, N. McKenzie, and L. Montanarella, Status of the world’s soil resources - Main report, Food and Agriculture Organization of the United Nations and Intergovernmental Technical Panel on Soils 650 (2015).[2] T. Ontl, L. Schulte, Soil carbon storage, Nature Education Knowledge, 3 (10), 35, 2012.[3] C.N. Friedrichsen, S. Hagen-Zakarison, M.L. Friesen, C.R. McFarland, H. Tao, J. Wulfhorst, Soil health and well-being: redefining soil health based upon a plurality of values, Soil Security 2 (2021) 100004.[4] G.S. Toor, Y.-Y. Yang, S. Das, S. Dorsey, G. Felton, Soil health in agricultural ecosystems: current status and future perspectives, Advances in Agronomy 168 (2021) 157- 201.[5] P. Trivedi, M. Delgado-Baquerizo, I.C. Anderson, B.K. Singh, Response of soil properties and microbial communities to agriculture: implications for primary productivity and soil health indicators, Frontiers in Plant Science 7 (2016) 990.[6] Y.A. Nion, K. Toyota, Recent trends in control methods for bacterial wilt diseases caused by Ralstonia solanacearum, Microbes and Environments 30(1) (2015) 1-11.[7] A.K. Jaiswal, Y. Elad, I. Paudel, E.R. Graber, E. Cytryn, O. Frenkel, Linking the belowground microbial composition, diversity and activity to soilbome disease suppression and growth promotion of tomato amended with biochar, Scientific reports 7(1) (2017) 44382.[8] K. Lambert, S. Bekal, Introduction to plant-parasitic nematodes, The plant Health instructor (2002).[9] W. Decraemer, E. Geraert, Ectoparasitic nematodes, Plant Nematology, CABI Wallingford UK2006, pp. 153-184.

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Claims

WHAT IS CLAIMED IS:

1. A spherical nanoparticle (SNP) comprising a plant virus coat protein and double stranded ribonucleic acid (dsRNA) that is homologous to a RNAi target nematode associated gene selected from the group of nematode development, nematode reproduction, nematode housekeeping gene, and parasitism effector gene, optionally where in the dsRNA is 50-1500 base pairs (bp) in length.

2. The SNP of claim 1, wherein the plant virus coat protein is a coat protein from a plant virus selected from a tobacco mosaic virus (TMV), a cytoplasmic type citrus leprosis virus, a tobacco mild green mottle virus (TMGMV), a physalis mottle virus like particle (PhMV), a cowpea chlorotic mottle virus (CCMV), and a cowpea mosaic virus (CPMV).

3. The SNP of claim 1, wherein the nematode development gene encodes a protein selected from chitin synthase, CPN-1, a dual oxidase, a RNA-binding protein, and troponin C.

4. The SNP of claim 1, wherein the nematode reproductive gene encodes a protein selected from an aminopeptidase and a major sperm protein.

5. The SNP of claim 1, wherein the housekeeping gene encodes a protein selected from a phosphatase, a G-protein-coupled receptor, cathepsin B cysteine proteinase, a cysteine proteinase, a dicer, cytochrome C, a FMRF amide-like neuropeptide, glyceraldehyde-3- phosphate dehydrogenase, heat shock protein 70, heat shock protein 90, a ribosomal protein, small ribosomal protein 3a, small ribosomal protein 4, a synaptobrevin, a spliceosomal SR protein, integrase, isocitrate lyase, 26S proteasome, a putative transcription factor, myosin heavy chain, a spliceosome subunit, a splicing factor, a pre-mRNA splicing factor, a tropomyosin, paramyosin, and a coatomer complex subunit.

6. The SNP of claim 1, wherein the parasitism effector gene encodes a protein selected from a protein similar to plant ring H2 zinc finger proteins, a cellulose-binding protein, a predicted protein with similarity to plant ubiquitins, a protein with similarity to the SKP1 protein of plants, an aspartic protease, P-l,4-endoglucanase, calreticulin, chorismite mutase, a C-type lectin, a glutathione-S transferase, a secreted peptide, a putative effector gene (ortholog of secreted peptide Mil6D10), a translationally controlled tumor protein, a body wall troponin C, a movement related protein, a venom allergen-like protein, Meloidogynesecreted protein 1, a putative esophageal gland cell secretory protein 2 (Meloidogyne secreted protein 2), a putative esophageal gland cell secretory protein 3 (Meloidogyne secreted protein 3), a putative esophageal gland cell secretory protein 5 (Meloidogyne secreted protein 5), esophageal gland cell secretory protein 8D05, a putative esophageal gland cell secretory protein 9 (Meloidogyne secreted protein 9), a putative esophageal gland cell secretory protein 10 (Meloidogyne secreted protein 10), effector protein, parasitism protein 16D10, a putative esophageal gland cell secretory protein 18 (Meloidogyne secreted protein 18), a putative esophageal gland cell secretory protein 20 (Meloidogyne secreted protein 20), a putative esophageal gland cell secretory protein 23 (Meloidogyne secreted protein 23), a putative esophageal gland cell secretory protein 24 (Meloidogyne secreted protein 24), a putative esophageal gland cell secretory protein 40 (Meloidogyne secreted protein 40), a pectate lyase, pectate lyase 2, pectate lyase 3, polygalacturonase, a lipid binding protein, movement related proteins, a secreted amphid protein, serine protease, and xylanase.

7. The SNP of claim 1, where in the RNAi target nematode associated gene is selected from: a) the nematode development gene selected from chs, cpn-1, duoxl. pox-1, and inc b) the nematode reproduction gene selected from amp-1, and msp,- c) the nematode housekeeping gene selected from ppm-1, srh-1, cb-1, cp-1, cpl-1, dcr-1.1, cyt-2.1,flp-l,flp-6,flp-12,flp-14, flp-18, gpd-1, hsp-1, hsp90, rps-23, rps-3a, rps-4, snb-1, spk-1, ICL, rpn7, Tisll, myo3, PV010,prp-17, tmy-1, unc-15, and Y25 and d) the parasitism effector gene selected from 10A06, 3B05, 4G06, 8H07, asp-1, eng-1, crt, cm, ctl, gsts-1, syv46, 16D10L, TCTP, pat-10, msp-1, vap-1, msp2, msp3, msp5, msp-9, 8D05, msp-10, spl2, msp-16, mspl8, msp20, msp23, msp24, msp40 , pel, pell , pel2 , pel3 , pg, far-1, pat-10, unc-87, ams-1, ser-1, spl, aw3 xyl28. The SNP of claim 1, wherein the dsRNA is present in the SNP at a concentration from about 3ng / pL to about 600ng / pL.

9. The SNP of claim 1, wherein the dsRNA is present at a concentration of about 400ng / pL.

10. The SNP of claim 1, wherein the plant virus coat protein is present at a concentration of about 2.0 mg / ml to about 3.0 mg / mL, optionally about 2.5 mg / mL.

11. The SNP of claim 1, wherein the SNP is coated with tannic acid.

12. The SNP of claim 11, wherein the tannic acid is at a concentration of about 10 mM to about 200 mM, optionally at a concentration of about 30mM.

13. The SNP of any one of claims 1-12, having a diameter of up to IpM.

14. The SNP of any one of claims 1-13, further comprising MgCh, optionally wherein theMgCh is at a concentration of about 50mM to about 150mM.

15. A SNP comprising: a) a plant virus coat protein; b) double stranded ribonucleic acid (dsRNA) that is homologous to a RNAi target nematode associated gene selected from the group of nematode development, nematode reproduction, nematode housekeeping gene, and parasitism effector gene, c) tannic acid, and d) MgCh.

16. A composition comprising the SNP of any one of claims 1-15 and a pharmaceutically acceptable carrier.

17. A method for controlling a nematode comprising contacting a nematode with the SNP of any one of claims 1 to 15 or the composition of claim 16, wherein the nematode is an egg, juvenile, or adult, optionally wherein the contacting is to the nematode orally or through the epidermis of the nematode.

18. The method of claim 17, wherein the nematode is a plant parasitic nematode, optionally wherein the plant parasitic nematode is at least one of: Meloidogyne artiella, Meloidogyne incognita, Meloidogyne chitwoodi, Meloidogyne enlerolobii, Meloidogyne graminicola, Meloidogyne hap la, Meloidogyne arenaria, Meloidogyne javanica, Heterodera glycines, Heterodera schactii, Bursaphelenchus xylophilus, Radopholus similis, Globodera pallida, Globodera rostochiensis, Pratylenchus vulnus, Pratylenchus coffeae, Pratylenchus penetrans or Ditylenchus destructor.

19. The method of claim 17, wherein the nematode is a free-living nematode, optionally wherein the free-living nematode is Caenorhabditis elegans.

20. A method to make SNPs of any one of claims 1 to 15 comprising transforming a plant virus into a spherical shape and loading dsRNA onto the transformed plant virus, optionally wherein the plant virus is transformed using head transformation.

Images