TABLE 1.
Summary of RNA spray studies in plants.
| Target organism | Target gene (dsRNA length) | Plant species | Nanoformulation | Applied dsRNA (μ g) | Application method | Efficacies | Durability | References | |
| Viruses | Sugarcane Mosaic Virus (SCMV) | Coat protein (∼150 bp) | Zea mays | Escherichia coli HT115 (DE3) | 3 μg/L | Spray of bacteria-produced dsRNA | Total inhibition of virus infection | 30 days post inoculation (dpi) | Gan et al., 2010 |
| Pepper mild mottle virus (PMMoV) Cucumber mosaic virus (CMV) | PMMoV Replicase (977 bp) CMV Replicase (2b) (330 bp) | Vigna unguiculata Nicotiana tabacum | Layered double hydroxide (LDH) clay nanosheets | 1.25 μg of dsRNA and/or 3.75 μg of LDH | Sprayed with an atomizer | Total inhibition of virus infection (systemic protection) | Up to 20 days after a single spray | Mitter et al., 2017 | |
| Bean common mosaic virus (BCMV) | Nuclear inclusion b protein (480 bp) Coat protein (461 bp) | Nicotiana benthamiana Vigna unguiculata | 100 μg | Mechanical inoculation with carborundum as an abrasive and sprayed with an atomizer | Reduction in infection to 45% and to 8.3% | Analyzed for up to 10 days | Worrall et al., 2019 | ||
| Tomato yellow leaf curl virus (TYLCV) | Coat protein (CP) (680–700 bp) | Nicotiana benthamiana Solanum lycopersicum | 1.25 μg of pDNA, 3.75 μg of LDH | Sprayed using an atomizer at ∼125 μl/cm2 | Delivered systemically. Lower incidence and severity. Symptom expression rate reduced to 41.7% | Up to 35 days | Liu et al., 2020 | ||
| Insects | Sitobeon avenae | Salivary sheath protein (491bp) | Hordeum vulgare | Naked dsRNA | 20 ng/μl | Foliar spray on leaves | Reduced transcript level of 60% | Monitored up to 5 days | Biedenkopf et al., 2020 |
| Leptinotarsa decemlineata | Inhibitor of apoptosis, Actin; HSP70; Dynamin (300–600 bp) | Solanum tuberosum | Escherichia coli HT115 (DE3) | 15 μl of 0.85 μg/μl (larvae) and 2.5 μg/μl (adults) dsRNA spread on each disk surface. Expressed by bacteria heat−killed in 4ml sprayed | dsRNA spread on each disk surface and dried for 10 min | ∼100% larval mortality (dsIAP or dsActin); 80% adult mortality (dsActin) Feeding CPB with heat−killed bacteria induced significantly higher mortality (70–90%) | Mortality recorded until 11th day and up to 6th for the bacteria expressed assay | Máximo et al., 2020 | |
| Actin (297bp) | Solanum tuberosum | naked dsRNA | 12 μL (100, 30, 10, 3, 1, and 0.3 ng) | Purpose-built spraying device on potato leaf disks (Ø = 2 cm) | German strain and Spanish field strain E02 almost 100% mortality (30 ng dsActin). Spanish strain E01 showed only 30% mortality. By day four > 95% of the exposed larvae were dead | Monitored up 5 days | Mehlhorn et al., 2020 | ||
| Mesh gene (417 bp) | Solanum tuberosum | Naked dsRNA | 10 μg/ml. | 25 m2 plots of potato plants were sprayed under field conditions | Observed field mortality was slightly lower compared to laboratory trials | Monitored up 13 day | Petek et al., 2020 | ||
| Phaedon cochleariae | Cactus, srp54k, rop, α-SNAP Shibire, PP-α, hsc70–3, rpn7, rpt3; (317 and 599 bp) | Brassica oleracea | Naked dsRNA | 0.3 μg (9.6 g/ha), 1 μg (32 g/ha) to 3 μg (96 g/ha) | Custom-built spraying device, multi-well plate foliar RNAi screening procedure | Suppression of transcript level rpt3 (94.5%), srp54k (94.1%), rpn7 (93.9%), α-SNAP (84.9%), shibire (81.3%), PP-α (80.5%) hsc70–3 (75.9%) | Monitored over 10 days | Mehlhorn et al., 2021 | |
| Helicoverpa armigera | Juvenile hormone methyltransferase, Acetylcholine esterase; (21 nt) | Chickpea | Chitosan nanoparticles (CNPs) | One milliliter of CNPs-dsRNA (200 μg:1,000 ng wt/wt) | Sprayed over the plant canopy with a hand-held mist sprayer | 100% insect mortality | Monitored up to 5 days | Kolge et al., 2021 | |
| Henosepilachna vigintioctopunctata | Ecdysone receptor (EcR) | Solanum tuberosum | Escherichia coli HT115 (DE3) | 0.5 μg/mL | dsEcR-immersed foliage and dsEcR-E. coli directly sprayed to the foliage of greenhouse-growing potato plants | Only 40% of the treated larvae formed Pupae (6–8 days). Of them, 60% exhibited a defective phenotype | Monitored for up to 10 days | Wu et al., 2021 | |
| Fungi | Fusarium graminearum | Cytochrome P450 lanosterol C-14α-demethylases (CYP51A, CYP51B, CYP51C) (CYP3RNA) (791 nt; 21–24 nt) | Hordeum vulgare | Naked dsRNA | dsRNA was diluted in 500 μL water to a final concentration of 20 ng/μL | Detached barley leaves were sprayed using a spray flask | Reduction of transcript level: 72% (CYP51A), 90% (CYP51B), and 71% (CYP51C); inhibition of fungal growth | Analyzed for up to 8 days | Koch et al., 2016 |
| Botrytis cinerea Verticillium spp. | Dicer-like (DCL)1 DCL2 (315 bp) | Arabidopsis thaliana Nicotiana tabacum Solanum lycopersicum Fragaria rosales Vitis vinifera Lactuta sativa Allium cepa Rosaceae | naked dsRNA | 20 ng/μL | dsRNA were dropped onto the surface of fruits, vegetables and rose petals and B. cinerea Inoculum was applied on the same spot | Reduced fungal growth | Analyzed for up to 8–10 days after | Wang et al., 2016 | |
| Fusarium asiaticum | Myosin5 (Myo5) (∼500 bp) | Triticum aestivum | Naked dsRNA | 400 ng of fluorescein-Myo5-8 dsRNA | Sprayed using a spray flask | Inhibition of mycelial growth (31–70%), interference in life cycle and virulence, cell wall defects, life cycle disruption and virulence reduction | 9 h unless the dsRNA was continuously supplied | Song et al., 2018 | |
| Fusarium graminearum | 294 nt (FgCYP51A) 220 nt (FgCYP51B) 238 nt (FgCYP51C) 514 nt (FgCYP51A/CYP51B) 458 nt (FgCYP51B/CYP51C) 532 nt (FgCYP51A/CYP51C) | Hordeum vulgare | Naked dsRNA | dsRNA was diluted in 500 μL water to a final concentration of 20 ng/μL | Barley leaves were detached and sprayed using a spray flask | Reduced fungal growth and transcript level to less than 10% | Monitored up to 5 days | Koch et al., 2019 | |
| DICER-like1 and 2; ARGONAUTE1 and 2; AGO-interacting protein FgQIP; RecQ Helicase;RNA-dependent RNA polymerases (∼1,000 bp) | Central role in different steps of sexual and asexual reproduction, in fungal pathogenicity and DON production | Gaffar and Koch, 2019 | |||||||
| 500–, 800–, 1,518 nt (FgCYP51A) 400–, 800–, 1,575 nt (FgCYP51B) 400–, 800–, 1,548 nt (FgCYP51C) | Inhibition of fungal infection symptoms up to 82% | Höfle et al., 2020 | |||||||
| 365 nt, 1,529 nt (FgAGO1/AGO2) 355 nt, 1,570 nt (FgAGO1/DCL1) 366 nt, 1,528 nt (FgAGO1/DCL2) 374 nt, 1,783 nt (FgAGO2/DCL1) 1,741 nt (FgAGO2/DCL2) 1,782 nt (FgDCL1/DCL2) | Inhibition of fungal infection up to 60%; reduced transcript level up to 79% | Werner et al., 2020 | |||||||
| Phakopsora pachyrhizi | Acetyl-CoA acyltransferase 40S ribosomal protein S16, Glycine cleavage system H protein; (200–400 bp) | Glycine max | Diethyl-pyrocarbonate | 1 ml of diluted dsRNA (20 μg dsRNA) | Each box (six detached individual leaflets) was evenly sprayed | Average of over 73% reduction of pustule numbers 75% reduction in biomass accumulation | Monitored up to 2 weeks | Hu et al., 2020 | |
| Phytophthora infestans | Sorbitol dehydrogenase, Translation elongation factor 1-α, Phospholipase-D like 3, Glycosylphosphatidylinositol-anchored acidic serine-threonine rich HAM34-like protein, Heat shock protein-90; (402bp-536bp) | Solanum tuberosum | Escherichia coli HT115 (DE3) 0.5% Nanoclay solution (5, 10, and 20 ppm) | 100–, 150–, 250– and 500 ng and 1 μg dsRNA | Sprayed with dsRNA-nano clay formulation using automizer | Reduction in growth, sporulation and symptom expression, 15 days, control collapsed and wilted while dsRNA nano clay sprayed plants were erect and healthy | 15 days | Sundaresha et al., 2021 | |
| Botryotinia fuckeliana | Chitin synthase class III and DCL1 and DCL2 | Fragaria ananassa | Escherichia coli-derived anucleated minicells | 125–1,000 ng/ml | Topical spray application | Selectively knocked-down the target genes and led to significant fungal growth inhibition in vitro. Compensatory relationship between DCL1 and DCL2 gene transcripts | 12 days | Islam et al., 2021 | |
| Fusarium oxysporum | CYP51, chitin synthase 1, Elongation factor 2 (732bp) | Solanum lycopersicum | Layered double hydroxide nanosheets | 300 μg of dsRNAs in 3 mL of ddH2O per plant | Spraying on plant leaves avoiding stems | Reduced fungal growth dsCYP51showed 93% reduction in transcript abundance | Monitored up to 8 days | Mohamed and Youssef, 2021 | |
| Phytophthora infestans | Guanine-nucleotide binding protein β-subunit, haustorial membrane protein, cutinase, endo-1,3(4)-β-glucanase (436 bp) | Solanum tuberosum | Naked dsRNA | 20 ng/μL | dsRNA sprayed on potato leaves in a detached leaf assay | Decreased disease progression, smaller and aberrant Mycelial phenotype | 5 days | Kalyandurg et al., 2021 | |
| Plants | Nicotiana benthamiana | Green fluorescence protein (GFP), (21–, 22–, and 24 nt) | Nicotiana benthamiana | Naked dsRNA | High-pressure spraying procedure (HPSP) | Local and systemic silencing exception of siR24, delayed and weak local silencing (10 dpa), other GFP siRNAs induced local silencing 2 dpa | 20 days post application | Dalakouras et al., 2016 | |
| GFP (322, 139 bp) | 200 μl of dsRNA midGFP (10, 20, 200, and 240 ng/μl) 200 μl of dsRNA-5’GFP (24, 48, and 240 ng/μl) | None of the samples sprayed with dsRNA-midGFP (0/15), dsRNA-5’GFP (0/9) or water (0/9) showed silencing up to 3 weeks after spraying | – | Uslu et al., 2020 | |||||
| (CaMV) 35S promotor (333bp) | Naked dsRNA | 50 μg in 500 μl | High-pressure spraying (carborundum was added) | Methylation of the 35S promotor was observed 10 days after spraying | 10 days post spraying | Dalakouras and Ganopoulos, 2021 | |||
| GFP (124 bp) | Carbon dots (CD) surfactant BreakThru S279 was added to the CD-dsRNA complexes at a final concentration of 0.4% (v/v) | 12 ng/μL (3.8 μL/cm2, 45 ng/cm2) | Spray application was done with Iwata HP-M1 handheld airbrush sprayer with air pressure set to 82 kPA (∼12 PSI) | MgCheH transcript levels showed a 79% reduction in the phenotypic tissues at five days after treatment. Reduction of 88% in GFP protein levels was observed | Monitored up to 12 days | Schwartz et al., 2020 | |||
| GFP (22 nt) | 60 μl of solution/plant (3-4-leaf transgenic seedling) | No difference was observed in the extent or frequency of systemic silencing comparing the events containing or not the partial transposase. Expression was reduced to 48 and 72%. | Up to 14 days | Hendrix et al., 2021 | |||||
| GFP (siRNA loading) | DNA nanostructures | 100 nM for both the nanostructure and the siRNA duplex | Infiltrating nanostructures with a 1-ml needleless syringe and without using any surfactant. | 40–59% (varies with DNA structures) in both mRNA and protein level | Internalization into plant cells 12h post-infiltration; gene silencing disappears 7days post-infiltration | Zhang et al., 2020 | |||
| Gold nanoclusters (AuNCs) | 25 ng siRNA per 1 μg AuNCs | 32–35% reduction in GFP 3 days postinfiltration | Internalization into plant cells 0,5-1 h post-infiltration | Zhang et al., 2021 | |||||
| Single-walled carbon nanotubes | 100 nM siRNAs | 95–92% (mRNA level) ∼40% (protein level) | Internalization into plant cells 4 h post-infiltration | Demirer et al., 2020 |