WO2022146195A1 - Arn double brin interférant pour protéger les végétaux contre la phytofluorose - Google Patents

Arn double brin interférant pour protéger les végétaux contre la phytofluorose Download PDF

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WO2022146195A1
WO2022146195A1 PCT/RU2021/050444 RU2021050444W WO2022146195A1 WO 2022146195 A1 WO2022146195 A1 WO 2022146195A1 RU 2021050444 W RU2021050444 W RU 2021050444W WO 2022146195 A1 WO2022146195 A1 WO 2022146195A1
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pitg
dsrna
genes
seq
infestans
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PCT/RU2021/050444
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Russian (ru)
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Михаил Эммануилович ТАЛЬЯНСКИЙ
Наталья Олеговна КАЛИНИНА
Александр Михайлович ЧУЕНКО
Андрей Владимирович ХРОМОВ
Сергей Юрьевич МОРОЗОВ
Татьяна Павловна СУПРУНОВА
Андрей Геннадьевич СОЛОВЬЕВ
Николай Викторович МАРКИН
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Общество с ограниченной ответственностью "Международная лаборатория "Резистом"
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Priority claimed from RU2020143919A external-priority patent/RU2808216C2/ru
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Publication of WO2022146195A1 publication Critical patent/WO2022146195A1/fr

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/82—Solanaceae, e.g. pepper, tobacco, potato, tomato or eggplant
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)

Definitions

  • the invention relates to the field of biotechnology, molecular biology, phytopathology and agriculture and is directed to the development of an effective means of biological protection of plants against infection by the oomycete Phytophthora infestans by the mechanism of RNA interference (RNAi), leading to a decrease/switching off the expression of the target gene.
  • RNAi RNA interference
  • the interference is based on the use of dsRNAs complementary to housekeeping genes and genes associated with pathogenesis (effector genes) of Phytophthora infestans.
  • 12 housekeeping genes essential for the phytophthora life cycle and 12 genes encoding effector proteins responsible for the pathogenicity and virulence of the oomycete have been identified. Selected 24 target gene fragments, those.
  • target genes are combined into blocks, including three segments in each cassette of eight genetic constructs, and can be used for the synthesis of dsRNA both in vitro and in vivo (Escherichia coli, Pseudomonas syringae, Saccharomyces cerevisiae) for subsequent processing of potato plants and other crops to protect them from Phytophthora infestans.
  • FIG. 1 Scheme of a plasmid carrying the sequences of the alpha-tubulin, 60S ribosomal protein L4 and cytochrome b5 genes (Hrl construct) of P. infestans (T30-4 strain).
  • the Hg2-Hg8 constructs differ only in the sequences of the genes included in the cloned fragment and are listed in Table. one.
  • FIG. 1 Electrophoresis of Phytophthora dsRNA synthesized in vitro in a non-denaturing agarose gel.
  • Figure 3 Phytophthora inhibition by dsRNA preparations of the constructs Hrl, Hg2, Hg3, Hg4, Hg5, Hgb, Hg7, Hg8 on days 5, 6 and 7 after infection (dpi) of the leaves of the variety 12-12-8 susceptible to late blight (selection of OOO DGT ”), the degree of leaf damage by the pathogen (the area of necrotic spots) are presented as the average values of 3 biological replicates ( ⁇ SE). In each variant of each experiment, 13-15 leaves were tested. Significant differences (between control and variants are indicated by "*" (p ⁇ 0.05) (ANOVA with Tukey's HSD pos-hoc test). Figure 4.
  • necrotic spots by 7 dpi) after treatment of late blight-infected potato leaves with dsRNA preparations of various designs (Hrl, Hr2, Hr3, Hr4, Hr5, Hrb, Hr7, Hr8).
  • Late blight caused by the oomycete Phytophthora infestans (Mont.) de Vagu is the most harmful and economically important disease of potatoes and tomatoes in most countries of the world.
  • late blight-resistant wild Mexican potato species were discovered and methods developed for crossing them with cultivated potatoes, it was not possible to create resistant varieties with permanent resistance to the pathogen, as new resistance genes from wild Mexican potatoes quickly began to lose effectiveness.
  • the use of the most effective contact chemicals containing phenylamides did not allow effective control of late blight, as their use led to the accumulation of strains resistant to phenylamides.
  • the increased epiphytological potential of P infestans has led to a decrease in the effectiveness of breeding, chemical and traditional methods of potato protection.
  • RNA interference can be used in practical terms to increase the resistance of agricultural plants to the pathogen P Infestans, significantly expanding the arsenal of narrowly targeted and safe plant protection products.
  • the aim of the present invention was to identify the target genes of P infestans in order to obtain a series of specific dsRNA preparations for plant protection against P infestans based on the mechanism of RNA interference.
  • the oomycete Phytophthora infestans infects many Solanaceae crop species, including potatoes (Solanum tuberosum) and tomatoes (Solanum lycopersicum).
  • P infestans is a hemibiotroph that has two stages of the infectious process: initial asymptomatic biotrophic and subsequent necrotrophic. During the biotrophic stage, P infestans successively forms appressoria, primary and secondary hyphae, and finally specialized structures called haustoria through which proteins and small molecules called effectors are delivered to the apoplast and plant cells (Dou et al., 2008). The subsequent necrotrophic stage is characterized by hyphae branching and host cell lysis (van Cap, 2006).
  • effector genes Lo Presti et al., 2015, Wang and Jiao, 2019
  • effector proteins allow pathogens to regulate metabolism and suppress host defense mechanisms (Abramovitch and Martin, 2004; Restrepo et al., 2005; Tian et al., 2007).
  • P infestans effector proteins mainly belong to two groups that target different compartments of the host plant. Apoplastic effectors are secreted into the extracellular space, while cytoplasmic effectors move inside the plant cell and interact with various subcellular compartments/structures (Kamoun, 2006; Whisson et al., 2007).
  • Cytoplasmic effectors can migrate to the nucleus, where they modulate plant signaling and immune/defense responses (Dou et al., 2008; Schornack et al., 2010).
  • the P infestans genome is one of the largest among oomycetes (240 MB) and contains a huge number of mobile elements (TEs) (Haas et al., 2009, Vetukuri et al., 2013).
  • TEs mobile elements
  • RNA interference was coined in 1998 when Tabara et al. (Tabara et al., 1998) showed that the process of gene expression inhibition can be initiated by incubation of nematodes in a solution of gene-specific double-stranded RNA fragments.
  • time clear indications of the role of complementary RNAs in the regulation of endogenous eukaryotic gene expression had already been obtained in transgenic plants and fungi (Jorgensen, 1990; Grierson et al., 1991; Romano and Macino, 1992).
  • RNAi or RNA silencing a sequence-specific mechanism of post-transcriptional gene silencing mediated by small interfering RNAs (siRNAs) or microRNAs, plays an important role in protection against viral infection, development and maintenance of genome integrity (Ding, 2000; Vance and Vaucheret, 2001; Baulcombe , 2004; Chen, 2012).
  • siRNAs small interfering RNAs
  • microRNAs RNAs
  • DCL Dicer-like proteins
  • AGO Argonauts
  • RDRs RNA-dependent RNA polymerases
  • DCL proteins are type III RNases that process dsRNA or hairpin RNA to form siRNA or microRNA, respectively, 20-24 nucleotides in length.
  • AGO proteins are endonucleases that form an RNA-induced silencing complex (RISC) with siRNA or microRNA.
  • RISC can bind to target/targeted mRNA or non-coding RNA according to the principle of complementarity through the included in the complex miRNA/miRNA.
  • the silencing (switching off) of the expression of the target gene is carried out by cutting and degrading the target mRNA (RNA interference) or by attracting cofactors and inhibiting the translation of this mRNA or by modifying histones (methylation) and inhibiting the transcription of the target gene (transcriptional silencing).
  • RDR proteins transcribe single-stranded RNA to form dsRNA, which is further processed to form miRNA with the participation of DCL proteins.
  • dsRNA production is a common feature of silencing mechanisms
  • artificially synthesized dsRNAs can mimic RNA silencing in plants by regulating gene expression and thus providing an approach to improve crop properties (downregulation of cellular gene function) as well as inhibiting the reproduction or virulence of pathogens (Dalakouras et al. ., 2020).
  • the silencing signal (ds miRNA) travels through the plant within days of initiation and is generally directed from photosynthetic sources (i.e. leaves) to roots and growth points (Lough and Lucas, 2006; Voinnet et al., 1998 ).
  • photosynthetic sources i.e. leaves
  • grisea has been achieved by suppressing two genes, namely OsFAD7 and Os FAD f, which encode fi-3-fatty acid desaturase proteins (Yara et al., 2007). Moreover, suppression of genes controlling lignin production resulted in increased resistance of soybean plants to the phytopathogen Sclerotinia sclerotiorum (Peltier et ah, 2009). A decrease in symptoms of powdery mildew in barley or wheat plants was found with HIGS-mediated suppression of the AvralO effector protein gene and a decrease in the number of functional haustoria inside epidermal cells.
  • Whisson et ah, 2005 and Grenville-Briggs et ah, 2008 describe the first use of in vitro synthesized dsRNA to trigger P infestans gene silencing upon delivery to pathogen protoplasts.
  • the studies used dsRNAs to the infl and cdcl4 genes (Whisson et al., 2005) and CesA (Grenville-Briggs et al., 2008).
  • INFI Presents is a secreted putative sterol carrier protein with a mol mass of 10 kDa. The Cdcl4 protein is required for sporulation of the pathogen P infestans.
  • CesA genes are activated during cyst germination and subsequent formation of appressors. Silencing of the CesAl-4 genes significantly reduces the amount of cellulose in the appressor cell wall, which leads to a decrease in the formation of appressors. Later studies of PsCdcl4, a homologue of the Cdcl4 gene of P.
  • RNAi suppressor PSR2 Phytophthora RNA silencing suppressor 2
  • PSR2 is a conserved effector protein, one of the few present in most Phytophthora species.
  • the highest level of gene expression is observed in the biotrophic phase of the pathogen, which suggests that PSR2 is important at the early stage of infection.
  • PSR2 has been shown to increase Phytophthora virulence, making it a promising target for pathogen control (Xiong et al., 2014; Sophie de Vries et al., 2017).
  • RNA biogenesis The other two effectors PsAvhl8 and PsAvhl46, which are expressed in P sojae during soybean infection, inhibit small RNA biogenesis. Ectopic expression of these RNA sales suppressors increases plant susceptibility to Phytophthora, indicating that some pathogens contain virulence proteins that target host plant RNA biogenesis suppression processes to promote infection (Qiao et al., 2013).
  • RNA interference can be effectively used to achieve the desired resistance of agricultural crops to pathogens by manipulating the gene expression of viruses, bacteria, fungi, nematodes, and insects (Koch and Kogel, 2014; Kamthan et al., 2015; Morozov et al. . 2019).
  • GMOs genetically modified organisms
  • a promising method of using dsRNA is spraying plants - spray-induced gene silencing (SIGS). dsRNAs can either be directly taken up by the pathogen or first transferred to plant cells and then transferred to pathogen cells (Dubrovina and Kiselev, 2019, Morozov et al., 2019).
  • a close analogue to the claimed invention is a nucleic acid-based preparation for combating the larvae of the gray metalloid, using a short (18-membered) single-stranded antisense fragment of the baculovirus genome from a conservative domain RING of the IAP gene of the gray metalloid nuclear polyhedrosis virus 5'-CGACATGACCGCAAGGTA-3'.
  • a significant insecticidal effect is manifested (Oberemok V.V. RU 2645258).
  • dsRNAs that are complementary to sections of individual pathogen genes, in particular, to a section of the HXT1 gene of soybean rust fungi, have a similar effect.
  • dsRNA in this case, is a nucleotide sequence identical to the 18 nucleotide mRNA sequence transcribed from the HXT1 gene.
  • To control soybean rust, dsRNA is applied to the soil where the plants grow or are able to grow, to the leaves and/or fruits/seeds of soybean plants. (Bayer Kyu A., RU 2018106970).
  • dsRNA is also used to inhibit the expression of one or more target genes in the beetle parasite Diabrotica spp., resulting in inhibition of its growth (Monsanto Technology LLC (US), RU2478710 C2).
  • the closest analogue to the disclosed solution is the invention according to patent EA028662, which provides a group of inventions related to imparting resistance to oomycetes to a plant by inhibiting the saccharopine dehydrogenesis gene.
  • the difference of the disclosed solution is the use of other genes in the expression cassettes, respectively, producing dsRNA of a different sequence.
  • dsRNA in the prior art, there is practically no generalized information that allows predicting effective combinations of target genes, processing conditions and possible effect size.
  • the application of dsRNA to the complex of genes responsible both for various pathogenetic properties of phytophthora and directly to housekeeping genes for protecting plants from P Infestans is unknown from the prior art and has not been used previously to create protective compositions.
  • dsRNAs Upon entering the cell, such dsRNAs are recognized by the components of the plant's RNA interference system, resulting in the formation of a set of diverse short miRNAs that are complementary to the target RNA, which fully reproduces the process occurring in natural conditions. The probability of the presence of highly effective miRNAs in such a set is significantly higher than when artificially synthesized miRNAs are used.
  • DCL Dicer-Like
  • siRNA small interfering RNA
  • the inventors conducted a bioinformatic analysis of the P Infestans genome. And they selected the genes of P. infestans to create expression constructs.
  • the selection criteria were (1) the presence of data on the need for the protein encoded by the gene for the life cycle, infectivity and pathogenicity of P. infestans and (2) the availability of experimental data on the decrease in the infectivity/pathogenicity of P. infestans with a decrease in the level of expression of this gene.
  • Conservative genes selected pathogen necessary for its growth or virulence, which reduces the likelihood of mutations in these important genes necessary to maintain the vital functions of the pathogen.
  • Inhibition of the development of the pathogen P infestans on plant leaves was achieved by selecting a series of effective target genes of two types, namely, housekeeping genes essential for the phytophthora life cycle, and genes responsible for pathogenicity and virulence (effector protein genes), cloning fragments of selected genes as part of a single sequence/cassette, three fragments for each type of genes, in vitro synthesis of specific dsRNAs, and spraying the resulting preparations with selected concentrations of leaves of plants infected with P infestans.
  • Spraying the leaves of potato plants of line 12-12-8 with an aqueous solution of dsRNA in the amount of 100 ⁇ l of the drug at a concentration of 100 ng/ ⁇ l per 1 leaf leads to inhibition of the development of the pathogen P infestans previously applied to the leaf, which was estimated by the area of damage to the leaves in the range from 2.0 to 10 times compared with the control leaves.
  • the value of 100 ng/ ⁇ l is obviously excessive, the minimum required concentration of dsRNA in the composition may vary depending on the direction of the dsRNA, while the minimum value to maintain efficiency can be achieved by simply titrating the composition with an assessment of its effectiveness.
  • ssRNA single-stranded RNA
  • ssRNA RNA-derived transcripts
  • ssRNA inactivated DNase by phenol/chloroform extraction (or heating to 70° C. for 10 min).
  • the resulting transcripts (ssRNA) were purified by reprecipitation with ethanol in the presence of sodium acetate. After RNA precipitation, washing and drying of the RNA precipitate in air, it was dissolved in an appropriate volume of nuclease-free water.
  • Example 1 Influence of dsRNA on the development of Phytophthora infection on the leaves of a susceptible potato line.
  • the inhibitory effect of dsRNA was tested on separated leaves of the potato breeding line 12-12-8 susceptible to late blight (selection of OOO DGT).
  • the leaves of the 3rd-4th tier were separated from the plant, placed in a wet box in a thermostat at a temperature of P°C, where they were cooled for 2-3 hours, after which they were inoculated with a suspension of phytophthora (100 ⁇ l of suspensions of phytophthora with a concentration of 10 4 ' 5 zoosporangia / ml).
  • Leaves infected with phytophthora were incubated in a wet box for one day at 11°C, after which the leaves were treated with dsRNA preparations (100 ⁇ l of the drug at a concentration of 100 ng/ ⁇ l was applied to 1 leaf).
  • Potato leaves treated with dsRNA preparations were incubated in a wet box at room temperature until symptoms of infection appeared.
  • the inhibitory effect of dsRNA was assessed visually and by quantitative analysis of digital images of leaves using the Leaf Doctor software. 15-0319-RE in dynamics by the area of necrotic spots developing on the surface of potato leaves on days 5, 6 and 7 after infection (dpi), expressed as a percentage of the total leaf area. In each variant, 13-15 leaves were tested.
  • FIG. 3 shows the results of the experiment (mean values of 3 biological replicates).
  • a statistically significant decrease in the area of leaf damage by late blight compared with the control was found in all variants where the leaves were treated with dsRNA constructs.
  • Figure 4 illustrates the appearance of the leaves on the 7th day of the experiment.

Abstract

La présente invention concerne un complexe de gènes à usage domestique et de gènes de protéines-effecteurs Phytophthora infestans, qui peuvent être utilisés en qualité de cibles pour inhiber l'expression selon le mécanisme ARN d'interférence (ARNi), ce qui permet de protéger des végétaux contre un pathogène. La présente invention concerne 24 gènes-cibles: 12 gènes domestiques et 12 gènes de protéines-effecteurs, 8 variantes d'ARN double brin étant synthétisées in vitro sur la base des séquences combinées de ces derniers. L'aspersion de feuilles de de plants de pomme de terre avec une solution aqueuse de la composition contenant un complexe des ARN double brin synthétisés entraîne une inhibition de la multiplications de l'oocmycète Phytophthora infestans.
PCT/RU2021/050444 2020-12-30 2021-12-21 Arn double brin interférant pour protéger les végétaux contre la phytofluorose WO2022146195A1 (fr)

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RU2020143919 2020-12-30
RU2020143919A RU2808216C2 (ru) 2020-12-30 Композиция на основе интерферирующей дцРНК для защиты растений от фитофтороза

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EA028662B1 (ru) * 2011-10-04 2017-12-29 Байер Интеллекчуал Проперти Гмбх Рнк-интерференция для борьбы с грибами и оомицетами путем ингибирования гена сахаропиндегидрогеназы

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EA028662B1 (ru) * 2011-10-04 2017-12-29 Байер Интеллекчуал Проперти Гмбх Рнк-интерференция для борьбы с грибами и оомицетами путем ингибирования гена сахаропиндегидрогеназы

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DUBROVINA ALEXANDRA S., KISELEV KONSTANTIN V.: "Exogenous RNAs for Gene Regulation and Plant Resistance", INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, vol. 20, no. 9, pages 2282, XP055953611, DOI: 10.3390/ijms20092282 *
RANDALL THOMAS A. ET AL.: "Large-scale gene discovery in the oomycete Phytophthora infestans reveals likely components of phytopathogenicity shared with true fungi", MOLECULAR PLANT-MICROBE INTERACTIONS, vol. 18, no. 3, 2005, pages 229 - 243, XP002588625, DOI: 10.1094/MPMI -18-0229 *
XIONG QIN, YE WENWU, CHOI DUSEOK, WONG JAMES, QIAO YONGLI, TAO KAI, WANG YUANCHAO, MA WENBO: "Phytophthora Suppressor of RNA Silencing 2 Is a Conserved RxLR Effector that Promotes Infection in Soybean and Arabidopsis thaliana", MOLECULAR PLANT-MICROBE INTERACTIONS, vol. 27, no. 12, 1 December 2014 (2014-12-01), US , pages 1379 - 1389, XP055953608, ISSN: 0894-0282, DOI: 10.1094/MPMI-06-14-0190-R *

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