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Project Number19-74-10023

Project titleExogenous application of RNAs for regulation of plant properties

Project LeadDubrovina Alexandra

AffiliationFederal Scientific Center of the East Asia Terrestrial Biodiversity, Far Eastern Branch of the Russian Academy of Sciences,

Implementation period 07.2019 - 06.2022 

Research area 04 - BIOLOGY AND LIFE SCIENCES, 04-206 - Physiology and biochemistry of plants

KeywordsExogenous RNAs, gene silencing, RNA interference, dsRNA, siRNA, miRNA, plant gene expression regulation, endogenous plant genes, transgenes, Arabidopsis thaliana, Vitis amurensis, stilbenes, anthocyanes



Currently, researchers are actively working on establishing transgenic plants to improve plant stress resistance, crop yield, or qualitative crop properties. Multiple investigations show considerable success in this field, since it is possible not only transfer various genes into a plant’s genome but also regulate expression level of target genes in plants. However, there are legislative limitations on sowing transgenic plants in our country and in the majority of the European countries. Indeed, there is not sufficient information on the consequences of genetic modifications, and this raises question of the safety of genetically-modified organisms. Therefore, development of new approaches to improve plant properties without genome modifications is an urgent task. It is known that in the course of RNA interference (or gene silencing process), which was discovered more than 15 years ago, double-straned RNAs (dsRNAs) are processed by specialized enzymes (DICER LIKE or DCL) into so-called small interfering RNA (siRNA or miRNA). These small interfering RNAs which then are included in the RNA-induced protein complex that provides degradation of any RNAs similar in the nucleotide sequence to the inducing RNAs. As a result, the homologous mRNAs are degraded. It is known that RNA interference serves for the regulation of various processes important for vital activity of plants, such as plant growth and development, adaptation to abiotic and biotic stresses, or synthesis biologically active compounds. Numerous investigations show that it is possible to switch off or decrease expression of particular genes for the regulation of stress tolerance, growth processes and other plant properties of plants via induction of RNA interference. However, application of this approach requires plant genome modifications or application of the weakened plant viruses. There are new intriguing messages in the literature that exogenously applied dsRNAs and siRNAs (by direct spraying, spraying under high pressure, spraying using materials promoting RNA adhesion, using protein carriers) are capable of entering into plant vascular system and plant cells and inducing the process of RNA interference. Recent investigations report on the substantial increase of plant fungal and viral resistance after direct spraying of plants with RNA solutions designed to inhibit important genes of the fungi or viruses. It has been shown that the RNA molecules are capable of entering plant cells and vascular system and then spreading across plant tissues and inducing local and system resistance against the viral or fungal infection by inhibiting the homologous RNAs of the pathogens. Besides, it has been shown that spraying plants by RNA solution under high pressure can lead to silencing of a transgene (shown using GFP and YFP transgenes). Spraying with chalcone synthase (CHS)-encoding siRNAs in a complex with protein carriers inhibited production of anthocyanes in Arabidopsis but CHS expression was no analyzed. Active studies in this direction are extremely urgent not for confirmation and detailed investigation of these facts but also for investigation of the possibility to regulate transcription of plant endogenous genes via this approach. Now little is known about the mechanisms and speed of RNA penetration into plant vascular system and plant cells and their distribution. It is also important to investigate the possibility of regulating expression of plant endogenous genes via RNA spraying and plant improvements in a desirable direction.Thus, the present project has a considerable scientific novelty and is important for getting new knowledge important for the development of plant agriculture. Approachability of the project aims is defined by the available literature data, our scientific and methodological base, the availability of the model system for carrying out necessary experiments, and our experience in the field of plant molecular biology and biotechnology. The proposed project is a continuation of the completing projects (initiative research of young scientists) of the Russian Science Foundation No. 17-74-10083 (Dubrovina AS) and No. 17-74-10082 (A. Tyunin AP).

Expected results
In this project, we are planning to analyze the effect of various conditions on the effectiveness of external plant treatments with RNA solutions on the expression of transgenes and endogenous plant genes in a model plant Arabidopsis thaliana L. and a valuable source of stilbenes Amur grapevine Vitis amurensis Rupr. We are going to obtain information on the effectiveness of different treatment conditions and the impact of some abiotic stresses on the silencing of target genes. In addition, we are going to investigate the mechanisms of the transgene silencing and silencing of plant own genes induced by external treatments with RNA solutions. First, this project is intended to study the diversity of small RNAs produced after treatment with the double-stranded RNA precursors and to study the nature of the target suppression distribution (local or systemic silencing, peculiarities of the penetration and distribution of the exogenous RNAs in plant tissues). In addition, the project is also devoted to a comparison of the new and traditional approaches for target gene silencing, namely the effectiveness of exogenous treatment of plants with RNA solutions (the developed approach) and the generation of transgenic plants expressing target silencing-inducing structures (the traditional approach). Active research in this direction is extremely relevant for the study of the possibility to regulate the expression of plant genes and regulation of plant properties a desired direction. Development of the means to influence plant gene expression without obtaining transgenic plants is highly relevant for agriculture.



Annotation of the results obtained in 2021
1) The data revealed that exogenous dsRNAs and siRNAs applied to the foliar plant surfaces induce suppression of target endogenous plant genes (AtCHS, AtMybL2 and AtANAC032) and changes in the amount of the final product – anthocyanins. Direct treatment of the leaf surface of A. thaliana with aqueous solutions of AtCHS-dsRNA and AtCHS-siRNA led to effective suppression of AtCHS gene expression and significantly reduced accumulation of anthocyanins. Exogenous AtMYBL2-dsRNA and AtANAC032-dsRNA significantly reduced the mRNA levels of the genes, activated the expression of AtCHS and increased the content of anthocyanins. The observed suppressing effect of the gene-specific dsRNAs persisted for 2, 7, 14, and 21 days after treatment. The results have been published "External dsRNA Downregulates Anthocyanin Biosynthesis-Related Genes and Affects Anthocyanin Accumulation in Arabidopsis thaliana" (International Journal of Molecular Sciences 2021, 22, 6749). 2) We evaluated the effectiveness of simultaneous inhibition of five genes encoding important proteins involved in regulation of anthocyanin biosynthesis and metabolism in A. thaliana (AtCPC, AtCBP60g, AtMybL2 and AtANAC032 and AtBAN). We analyzed the effect of processing plant foliar surface with the gene-specific dsRNAs separately and in combination. It has been shown that the treatment of plants with the five dsRNAs simultaneously activated anthocyanin accumulation of these substances three times more efficiently as treatment against any one of these factors separately. 3) Using high-throughput small RNA sequencing by Illumina technology and subsequent bioinformatic data analysis, it was established that exogenous application of both AtCHS-dsRNA and NPTII-dsRNA led to the generation of a large number of AtCHS- or NPTII-specific small RNAs that have not been present after control water treatments. We analysed the composition of the sequenced small RNA fraction, the distribution by length and coverage of the corresponding genes. The data revealed that the exogenous AtCHS-dsRNAs led to a significant increase in the level of 21-nt small RNAs, while the content of 23-nt and 24-nt small RNAs was significantly reduced. It has been shown that exogenous AtCHS-dsRNAs were processed to siRNAs and induced RNA interference gene silencing, which led to AtCHS gene silencing, while NPTII-dsRNAs were presumably degraded. sRNA library sequences were deposited in NCBI under registration number PRJNA827691 and in the database of the Laboratory of Biotechnology of the Federal Research Center for Biodiversity of the FEB RAS ( 4) It has been shown that none of the EGFP-dsRNA doses led to a significant decrease in the expression of the NPTII transgene in NPTII-transgenic plants, while the specific NPTII-dsRNA significantly reduced the expression of NPTII in a dose-dependent manner. Long DNAs imitating dsRNAs and short DNA oligonucleotides imitating siRNA had no significant effect on the expression of the NPTII transgene. The analysis showed that exogenously applied NPTII-dsRNA did not affect the expression of four non-target A. thaliana genes, including AtGAPDH, AtCHS, AtUBQ and AtCML80 at three different concentrations, which confirms the specificity of the action of exogenous dsRNAs. Thus, exogenous NPTII-dsRNAs induced a sequence-specific and RNA-specific transgene-suppressing effect. Based on the data obtained, we published the article "The Specificity of Transgene Suppression in Plants by Exogenous dsRNA" (Plants 2022, 11, 715). 5) We compared two methods of RNA interference-based silencing of target genes, namely, plant exogenous treatment with dsRNA and the transgenic plants expressing artificial microRNAs using the regulation of the AtCHS gene in A.thaliana and VaMyb1 gene in V. amurensis. It has been shown that amiRNA-AtCHS-transgenic plants of A. thaliana accumulated considerably lower amounts of anthocyanins and this effect persisted for 21 days. Transgenic callus cultures of V. amurensis actively expressed the artificial amiRNA-VaMyb1 and were characterized by a significant reduction in expression of VaMyb1 and a sharp activation of stilbene biosynthesis. The effect persisted for 3 months and then disappeared. Spraying of nontransgenic callus cultures with the VaMyb1-dsRNA reduced VaMyb1 expression and contributed to increased accumulation of stilbenes. Treatment of the V. amurensis leaves with the VaMyb1-dsRNA led to a considerable suppression of VaMyb1 and increased production of stilbenes. Thus, it is possible to significantly reduce the expression of a target plant gene using both approaches. The data were partially published in “Application of RNA interference methods to regulate gene expression in grapes” (Viticulture and Winemaking 2021, 50:32-35). 6) Bisulfite sequencing was used to analyze the amount of methylated cytosines in the DNA coding sequence of the AtCHS gene (3’-terminal region with a length of 292 bp) after water treatment (negative control), specific exogenous AtCHS-dsRNA and non-specific NPTII-dsRNA treatments. The obtained results of bisulfite sequencing suggested that the treatment of A.thaliana plants with the AtCHS-dsRNA construct led to a decrease in the level of methylated cytosines in the 3’-coding region of the AtCHS gene, and at the same time, the treatment of plants with non-specific NPTII-dsRNA did not affect the level of methylation of the AtCHS gene. However, the resulting decrease in the level of AtCHS methylation did not statistically differ from the level of methylation of samples treated with water and NPTII construction. 7) A review of the scientific literature on the topic “Novel RNA interference-based tools for plant improvement and pathogen control” has been prepared, where we have described and analyzed modern approaches based on the phenomenon of RNA interference to regulate gene expression in plants and plant pathogens. The review included data analysis on three approaches: (1) spray-induced gene silencing or RNA "vaccination" of plants based on the use of exogenous dsRNA and siRNA; (2) "virus-induced gene suppression" or VIGS and (3) "host-induced gene silencing" or HIGS. The main attention was paid to the regulation of plant gene expression using exogenous dsRNAs.



1. Kiselev K.V., Suprun A.R., Aleynova O.A., Ogneva Z.V., Kalachev A.V., Dubrovina A.S. External dsRNA Downregulates Anthocyanin Biosynthesis-Related Genes and Affects Anthocyanin Accumulation in Arabidopsis thaliana International Journal of Molecular Sciences, 22(13):6749 (year - 2021).

2. Kiselev K.V., Suprun A.R., Aleynova O.A., Ogneva Z.V., Kostetsky E.Y., Dubrovina A.S. The Specificity of Transgene Suppression in Plants by Exogenous dsRNA Plants (Basel), 2022, 11(6), 715 (year - 2022).

3. Nityagovskiy N.N., Suprun A.R., Ananev A.A., Tyunin A.P., Kiselev K.V., Dubrovina A.S. Применение методов РНК-интерференции для регуляции экспрессии генов винограда ВИНОГРАДАРСТВО И ВИНОДЕЛИЕ, Т. 50. С. 32-35. (year - 2021).

4. Nityagovsky, N.N.; Kiselev, K.V.; Suprun, A.R.; Dubrovina, A.S. Exogenous dsRNA induces RNA interference of a chalcone synthase gene in Arabidopsis thaliana International Journal of Molecular Sciences, 23(10), 5325 (year - 2022).

Annotation of the results obtained in 2019
- It has been shown that leaf spraying and treatment of Arabidopsis with brushes using water solutions of transgen-coding double-stranded RNA precursors (dsRNA) reduced expression of the target neomycin phosphotransferase (NPTII) transgene with a higher efficiency as compared to the other tested methods, including infiltration, inoculation with carbonudrum, treatment with needle and pipetting. We found that the time of day critically affects the effectiveness of external palnt treatments with the dsRNA solutions targeting the target. External plant treatments with dsRNA were highly effective only when treated in the evening or at night, while treatment in the morning had no effect at all, and treatment in the afternoon had only some limiting effect. We also found that the degree of soil moisture has a significant stimulating effect on the degree of reduction of transgene expression. Cold treatment, heat treatment, and salt stress did not exhibit a considerable and stable effect. In addition, that the data revealed that the age of plants is an important factor, since the expression of target transgenes decreased more actively in younger plants. - It has been shown that the expression level of the NPTII and enhanced green fluorescent protein (EGFP) transgenes being under the control of the double CaMV 35S promoter significantly varied between different organs of A. thaliana even without any additional RNA or other treatments. Both transgenes were significantly more active in cotyledons, the first appearing leaves and roots, gradually reducing their expression to the "older" leaves and leaves on the stem. The lowest expression of transgenes was observed in stems and inflorescences. In addition, we found that the level of expression of transgenes gradually increased over time in plants. Furthermore, it was found that the level of expression of the transgenes can vary during the day in plants without additional effects. However, the observed differences in the expression of transgenes during the day were in most cases relatively small and/or unstable. When cultivating Arabidopsis under drought conditions, the expression of transgenes was significantly reduced as compared to that under normal conditions. The data revealed that cold stress, on the contrary, significantly increased expression of the transgenes. Analysis of the NPTII and EGFP protein content in various organs of Arabidopsis and under the influence of cold stress confirmed the data obtained. Wounding, heat and salt stress did not exhert a significant and stable effect on the expression of transgenes. - We studied the effects of external plant treatments with solutions of modified and unmodified transgene-coding RNA oligonucleotides, DNA oligonucleotides and compared to that of the long dsRNA and long dsRNA-mimicking DNAs targeting expression of the NPTII transgene in A. thaliana. It was confirmed that external treatment of plants with synthetic RNA oligonucleotides significantly reduced the expression of the target transgene. However, treatment of plants with similar synthetic modified and unmodified short DNA oligonucleotides or long DNAs did not lead to any significant and stable effect. Western blotting demonstrated that external RNA oligonucleotide application to the plant surfaces significantly reduced the accumulation of the target transgene protein product. Bisulfite sequencing showed that cytosine methylation of the transgene considerably increased after external plant treatments with RNA oligonucleotides. It has been shown that transgene suppression caused by external treatment of plants with dsRNA solutions was sequence-specific. For this purpose, NPTII-transgenic plants were treated with different concentrations of both specific transgene-encoding NPTII-dsRNA and separately with EGFP-dsRNA. - During the implementation of the grant, it was decided to expand the list of candidate target genes for subsequent artificial suppression of their expression. Four candidate target genes were identified that are directly related to the biosynthesis of stilbenes. Further analysis of the expression of 4 target genes, VaWRKY53, VaCHI3, VaRPP13 and VaCAMTA4, and that of small non-coding RNAs regulating their expression revealed a complete correspondence, expressed in the inverse relationship between the expression of target genes and small RNAs in V. amurensis cells. The obtained data on the complete nucleotide sequence of cDNA of the desired transcripts allowed us to proceed to experiments aimed at artificially suppressing their expression in V. amurensis cells.



1. Dubrovina AS, Aleynova OA, Suprun AR, Ogneva ZV, Kiselev KV. Transgene suppression in plants by foliar application of in vitro-synthesized small interfering RNAs. Applied Microbiology and Biotechnology, 104(5):2125-2135. (year - 2020).

Annotation of the results obtained in 2020
1a) We studied the influence of various physiological conditions on the neomycin phosphotransferase II (NPTII) transgene silencing in Arabidopsis thaliana after plant treatment with exogenous NPTII-coding double-stranded RNA (dsRNA). The results revealed a high importance of appropriate plant age (4 weeks), time of day, and low soil moisture at the time of exogenous dsRNA treatment for effective inhibition of the target gene. Significantly higher NPTII suppression was found when dsRNA was applied in the evening and at night. Treatment of the adaxial and abaxial leaf surface with soft sterile brushes, spraying, and pipetting showed higher suppression of NPTII than infiltration and mechanical inoculation. The results have been published (Kiselev et al. Plants (Basel) 2021, 10, 264). 1b) We analyzed expression of NPTII and EGFP transgenes driven by the CaMV 35S double promoter in A. thaliana plants without exogenous RNA application. It has been found that the transcript levels of NPTII and EGFP were significantly higher in cotyledons, young leaves and roots than in inflorescences, stems and adult leaves of three independent NPTII and EGFP-transgenic lines of A. thaliana. The transcript levels of NPTII and EGFP varied throughout the day and gradually increased with the plant age. Drought and cold stresses significantly affected the expression of transgenes, while heat stress, high salinity, and injury had no significant effect. Thus, this study showed that the transcriptional activity of transgenes driven by the CaMV 35S can significantly vary depending on the plant organ and tissues, plant age, time of day, and in response to abiotic stresses. The results have been published (Kiselev et al. 2021 Molecular Biology Reports 48: 2235-2241). 2) The specificity of the observed transgene-suppressing effect caused by exogenous NPTII - and EGFP-coding dsRNAs was investigated. None of the EGFP-dsRNA doses resulted in a significant decrease in NPTII expression in NPTII-transgenic plants, while treatment of these plants with the specific NPTII-dsRNA significantly reduced NPTII expression. DNA-oligonucleotides and long DNA mimicking the NPTII-dsRNA did not have a significant effect. Thus, exogenous dsRNA induced a sequence-specific and RNA-specific transgene-suppressing effect. The work is being prepared for publication. 3) New scientific information was obtained on the effect of exogenous synthetic dsRNA and siRNA on the expression of target endogenous genes in A. thaliana plants and Vitis amurensis cell cultures. In this work, we targeted the genes important for the biosynthesis of anthocyanins in A. thaliana (AtCHS, AtMybL2, AtANAC032) and stilbenes in V. amurensis (VaSTS, VaMyb1). Direct foliar application of AtCHS-coding dsRNA resulted in a significant and effective reduction of the AtCHS mRNA levels and suppression of anthocyanin accumulation in the A. thaliana leaves. A. thaliana was incubated both under standard conditions (+ 22°C, 16 h day/8 h night) and under conditions of modulation of anthocyanin biosynthesis (+7oC and constant light 24 h) for 7 days. Direct application of AtCHS-coding siRNAs (short 21-nt modified RNA oligonucleotides, 5' - phosphorylation, 2ʹ-O-methyl at the 3 ' end) resulted in the same effects. Targeting the AtMybL2 and ANAC032 genes by the same approach significantly reduced their mRNA levels and at the same time led to a pronounced activation of the AtCHS gene. After treatment with MybL2-dsRNA, a significant increase in the content of anthocyanins was observed. The work is being prepared for publication. Treatment of V. amurensis callus cell cultures with exogenous VaSTS-and VaMyb1-dsRNA led to a decrease in the mRNA level of the VaSTS and VaMyb1 genes that are important for stilbene biosynthesis. Exogenous VaSTS-dsRNA reduced the content of stilbenes, while exogenous VaMybL2-dsRNA, on the contrary, significantly increased. 4) The content and diversity of produced small RNAs were studied before and after treatment of plants with exogenous dsRNAs encoding the endogenous AtCHS gene and the NPTII transgene (using illumina sequencing of the small RNA fraction). We obtained 526,174,120 reads. Two biological repeats were sequenced for each type of exposure, i.e. water, AtCHS-dsRNA, and NPTII-dsRNA (6 samples of the small RNA fraction). It has been established that plants treated with AtCHS-dsRNA contained significantly higher levels of AtCHS-coding small RNAs than plants treated with water or non-specific NPTII-dsRNA. Moreover, a large number of NPTII-coding small RNAs were found in plants treated with NPTII-dsRNA, while no such small RNAs were detected in plants treated with water and AtCHS-dsRNA. The obtained results indicated that the foliar plant dsRNA treatment unduces RNA interference-mediated AtCHS and NPTII gene silencing. Bioinformatic analysis is being further prepared. 5) Confocal microscopy was used to analyze the uptake and spreading of AtCHS- and NPTII-coding dsRNA. The in vitro synthesized AtCHS-dsRNA and NPTII-dsRNA were labeled with Cy3 dye and applied to the leaf surface. Leaf examination 1 day post-treatment revealed the presence of the Cy3-labeled dsRNA in the leaf vessels, leaf parenchyma cell and stomata of the treated leaves of A. thaliana. The data obtained indicated the dsRNA uptake and spreading into the leaf tissues and plant individual cells. 6) We obtained the vector constructs, transgenic plants of A. thaliana and cell cultures of V. amurensis to compare two methods of silencing target genes (CHS in A. thaliana and Myb1 in V. amurensis). At present, the first data obtained showed an activation of the total stilbene content in 5-15 times in the transgenic V. amurensis callus expressing two types of amiRNA-VaMyb1. We treated the control non-transformed calli with the VaMyb1-coding dsRNA. The preliminary results revealed that the observed effects (decreased VaMyb1 expression and increased anthocyanin content) were less pronounced and less significant than in the amiRNA-transgenic calli. The work is being continued. 7) We analysed the level of AtCHS cytosine methylation before and after treatment with exogenous AtCHS-dsRNA using the bisulfite sequencing. The results showed a slight decrease in the level of cytosine methylation within the AtCHS in contrast to the previously published data for NPTII and EGFP methylation, which was increased (Dubrovina et al. 2019 IJMS).



1. Kiselev K.V., Suprun, A.R., Aleynova O.A., Ogneva Z.V., Dubrovina A.S. Physiological Conditions and dsRNA Application Approaches for Exogenously induced RNA Interference in Arabidopsis thaliana Plants (Basel), 10(2):264 (year - 2021).

2. Kiselev K.V., Aleynova O.A., Ogneva Z.V., Suprun A.R., Dubrovina A.S. 35S promoter-driven transgenes are variably expressed in different organs of Arabidopsis thaliana and in response to abiotic stress Molecular Biology Reports, 48:2235–2241 (year - 2021).