Nucleic acid active agents against various plant pathogens

Abstract

The present invention relates to newly identified nucleic acids, ribonucleic acids (RNA) and desoxyribonucleic acids (DNA), in particular esiRNA / ERNA (effective small interfering RNA) and RNA derived therefrom, as well as eASO (effective antisense DNA oligonucleotides), collectively referred to as eNA (effective nucleic acid), which can be used in RNA silencing / RNAi or RNA silencing / antisense methods as active agents against various variable plant pathogens. To identify these eNAs, a screening method (also hereinafter referred to as' eNA screening ') is first applied in standardized form to the target RNA of various plant pathogens. As a result, a new class of active agents against these pathogens was identified and successfully used against these pathogens. The invention also relates to the construction of a double-stranded ribonucleic acid edsRNA (efficient double-stranded RNA) comprising the nucleotide sequence of the identified esiRNA / ERNA or the related RNA derived therefrom, and can be successfully used as active agents against these variable plant pathogens in plant protection in RNA silencing / RNAi methods.

Description

Technical Field

[0001] The present invention relates to newly identified nucleic acids, ribonucleic acids (RNA) and deoxyribonucleic acids (DNA), specifically esiRNA / ERNA (effective small interfering RNA) and sRNA derived therefrom, as well as eASO (effective antisense DNA oligonucleotides), collectively referred to as eNA (effective nucleic acid), which can be used in RNA silencing / RNAi or RNA silencing / antisense methods as active agents against various variable plant pathogens. To identify eNAs, a screening method (WO2019001602A1), hereinafter also referred to as "eNA screening", was first applied in a standardized form to target RNAs of various plant pathogens. As a result, a new class of active agents against these pathogens was identified and successfully used to combat these pathogens.

[0002] The present invention also relates to the construction of double-stranded ribonucleic acids edsRNA (effective double-stranded RNA) comprising the nucleotide sequence of the identified esiRNA / ERNA or related RNAs derived therefrom, and which can be successfully used as active agents in RNA silencing / RNAi methods in plant protection against these same variable plant pathogens*.

[0003] As used herein, the term "plant pathogen" generally refers to plant-infecting viruses, as well as plant-infecting organisms such as nematodes and fungi, that have destructive effects on plants. Background Art

[0004] RNA interference (RNAi) is a mechanism that is best characterized in higher eukaryotes, but it is active in cells of virtually all organisms. The RNAi mechanism is used to shut down (RNA silencing) or regulate gene expression, and as will be explained below, can also be used for these purposes in a targeted manner (see Shabalina and Koonin 2008; Carthew and Sontheimer 2009). In this process, cellular factors (described in detail below) inactivate various forms of small RNA molecules, collectively referred to herein as small RNAs ("sRNAs"), targeting RNA molecules (hereinafter referred to as "target RNAs") or regulating the function of these target RNAs, such as translation. The term small RNA includes, for example, small interfering RNA (siRNA) and, for example, microRNA (miRNA), but also includes other small RNA forms that can induce RNA silencing (see, for example, Borges and Martienssen, 2015; Zhan and Meyers, 2023). The target RNA originates from a pathogen that infects a foreign cell, or directly from a cell that is part of the organism, including an organism that is a pathogen, in which RNAi is effective.

[0005] RNAi likely originated as an evolutionarily conserved cellular defense mechanism. RNAi is a major component of the immune response to pathogens in plants, as well as in insects, nematodes, oomycetes, and fungi (see Ding 2010; Zvereva and Pooggin 2012; Gammon and Mello 2015; Guo et al. 2019). The following will focus on plant cells and further describe the RNAi process in conjunction with its function as a defense mechanism, as this is one of the best-studied aspects, particularly with regard to its antiviral effects (see the schematic for a diagram). Figure 1 ). After modification, RNAi is also active in similar forms in nematodes, oomycetes, fungi, and insect cells.

[0006] RNAi is triggered by RNA molecules containing double-stranded (ds) regions—that is, two nucleotide strands from different or the same RNA molecule that pair (hybridize) through complementary base pairing. RNA molecules with nucleotide building blocks capable of forming double strands over large regions (potentially spanning hundreds or thousands of nucleotides) are called "dsRNA." The induction of RNAi in viral infections has been well studied: the trigger can be a specifically structured double-stranded region of viral messenger RNA (mRNA) or the viral RNA genome. dsRNA replication intermediates are particularly effective in triggering RNAi. These intermediates are produced during RNA viral replication and consist of complementary RNA duplexes spanning hundreds or thousands of base pairs. Cells can recognize double-stranded regions of RNA as "pathogen-associated molecular patterns" (PAMPs), thus treating them as "foreign." Detector proteins include cellular enzymes belonging to the type III ribonuclease family, known as Dicer or Dicer-like proteins (DCLs) (Fukudome and Fukuhara 2017; Song and Rossi 2017). The proteins DCL2 and DCL4, first identified in the model plant Arabidopsis thaliana (A. thaliana), play a central role in the plant's antiviral RNAi immune response (Deleris et al. 2006; Parent et al. 2015). DCL2 and DCL4 bind to dsRNA and process it into siRNAs through endonucleolytic hydrolysis (cleavage). siRNAs are short RNA molecules, 21-25 nucleotides (nt) in length, composed of two complementary single-stranded RNA components. These "RNA duplexes" are phosphorylated at the 5' end and have a 2nt-long single-stranded overhang and a 3' hydroxyl group at the 3' end (Elbashir et al. 2001a; Elbashir et al. 2001b). siRNAs of 21 or 22 nt in length have particularly potent antiviral effects (Deleris et al. 2006). After being generated by DCL, one strand of the predominantly double-stranded siRNA (see above), the “guide strand,” becomes active in the RNA-induced silencing complex (RISC). Key components of RISC are “argonaute” (AGO) proteins; these proteins, similar to RNase H enzymes, also possess endonucleolytic activity. AGO binds to the guide strand (gs) of the siRNA duplex, while the other strand of the duplex (the passenger strand, ps) is removed and disassembled (Meister 2013; Kobayashi and Tomari 2016)( Figure 1). The Arabidopsis genome encodes 10 different AGO proteins (AGO1-10); they have different functions, some of which are not yet fully understood. However, it has been well documented that AGO1 and AGO2 proteins play a central and crucial role in the plant's antiviral immune response (Carbonell and Carrington 2015). AGO proteins have different binding preferences for siRNA guide strands. For example, AGO1 preferentially binds to RNAs containing a 5'-terminal U nucleotide, while AGO2 preferentially binds to RNAs containing a 5'-terminal A nucleotide (Mi et al. 2008; Takeda et al. 2008; Schuck et al. 2013). After AGO binds to the siRNA guide strand, the corresponding RISC binds to the target RNA. Sequence complementarity between the bound siRNA guide strand and the sequence on the target RNA to which it can hybridize (referred to herein as the target site) plays an important role (Liu et al. 2014). Sequence complementarity with the target RNA from which the siRNA was originally generated is naturally the highest. Therefore, under natural circumstances, the target RNA is also the RNA molecule that is initially processed into the siRNA ("homologous RNA"). In viral infections, these are correspondingly viral mRNA, viral genome, or viral replication products; in other pathogens, these are usually mRNA. After AGO / RISC binds to the region of the homologous target RNA that is complementary to the siRNA guide strand (these regions can be coding regions or non-coding (untranslated) regions), the target RNA can undergo endonucleolytic hydrolysis (cleavage or "scissoring") catalyzed by the AGO protein ( Figure 1 ); this occurs between nucleotides 10 and 11 in the target RNA opposite to the siRNA guide strand (Elbashir et al. 2001a; Elbashir et al. 2001c; Wang et al. 2008). Furthermore, there is evidence that siRNA-containing AGO / RISC binding to target RNAs inhibits translation of these RNAs (Brodersen et al. 2008; Iwakawa and Tomari 2013). Thus, in RNAi, the result of siRNA activity is either a temporary inhibition of gene expression: the target RNA is cleaved and degraded, or the synthesis of the protein encoded by this RNA is inhibited. Thus, RNAi targeting pathogens results in the suppression of pathogen replication by inhibiting gene expression (RNA silencing) (Zvereva and Pooggin 2012). Accordingly, RNAi has been used to protect plants against pathogens such as viruses (Khalid et al. 2017; Pooggin 2017).

[0007] dsRNAs derived from pathogen target RNA sequences can therefore be used as antipathogen active agents: they are processed by the Dicer enzyme and produce siRNAs, some of which (see below) can then act in the manner described above. In order to achieve an antiviral effect, dsRNA must be used in plants. Accordingly, dsRNA active agents and the siRNAs formed thereby, produced in the RNAi immune system of plants, nematodes, fungi, oomycetes, and insects, act against the RNA (usually mRNA) of these pathogens themselves and can be used as herbicides, nematicides, fungicides, or insecticides in and on plants. As an alternative to dsRNA, sRNAs such as siRNAs, miRNAs, or other forms of small RNAs that are effective in the RNA interference process (e.g., piRNAs, tasiRNAs, vasiRNAs, etc.) can also be used directly as antipathogen active agents (see Tomilov et al. 2008; Banerjee et al. 2017; Majumdar et al. 2017; Price and Gatehouse 2008). As explained in more detail below, all of these sRNAs work by the same principle as siRNAs: essentially, the single-stranded components of these sRNAs are incorporated into RISC in a similar manner and exert their activity on the target RNA by either cleavage or inhibition of translation in the process of RNA silencing / RNAi.

[0008] As previously mentioned, RNAi is used to artificially "knock down" gene expression by introducing siRNA or other related sRNAs or sRNAs derived therefrom (such as miRNA) as active agents into cells to affect the function of the target RNA in a targeted manner. For example, as described, cellular gene expression and gene expression of pathogens can be inhibited or regulated at the mRNA level. In addition, the replication of viruses whose genomes are RNA molecules can be inhibited by silencing these genomes.

[0009] For the purpose of artificially inducing RNAi, organisms themselves can produce (express) RNA active agents. This has already been applied in plants. In this case, "host-induced gene silencing" (HIGS) is involved: plants express dsRNA, siRNA or other sRNA (such as miRNA) in the form of transgenes (Herrera-Estrella et al. 2005; Dong and Ronald 2019; Koch and Wassenegger 2021) to achieve protection against pathogens such as viruses and nematodes, fungi, insects, oomycetes or other parasitic plants. This can be permanent (and only permanent protection is meaningful) by stably integrating the corresponding nucleic acid expressing the exogenous gene into the genome of the organism to be protected. The process of obtaining transgenics is complex, not feasible for all plants, and not suitable for many applications. The latter is particularly relevant in combating rapidly changing pathogens: genetically modified crops, developed over many years, may still be susceptible to infection by new variants of these pathogens (Jan et al. 2000; Savenkov and Valkonen 2001; Simón-Mateo and García 2006; Tabashnik et al. 2013; Kung et al. 2015). Furthermore, in many countries, the use of genetically modified organisms (GMOs) in agriculture and horticulture is prohibited (Lucht 2015).

[0010] Alternatively, RNA agents can be applied topically or transiently. This involves applying dissolved RNA (e.g., in the simplest case, as a spray) to a target organ or cell in an organism. RNA is partially taken up by mechanisms that are not yet fully elucidated. Improving the uptake of negatively charged RNA molecules into target cells (currently inefficient) is a subject of intensive research. Due to the biodegradability of RNA, "spray-induced gene silencing" (SIGS), in which RNA is applied to or into plants in solution (e.g., as irrigation water or a spray), is much less ecologically problematic than HIGS and therefore an attractive approach for administering RNA agents (Dalakouras et al. 2020). Furthermore, over the past five years or so, methods have been developed that enable the production of RNA on a gram or even kilogram scale (Robinson et al. 2014; Kaur et al. 2018). For example, the price of one gram of dsRNA, needed to treat a small plot of farmland, has dropped from over $100,000 to less than $2 (Le Page 2017).

[0011] As mentioned above, in the context of plant protection, the purpose of topical / transient application of RNA is to allow the plant to internalize the RNA agent, for example to inhibit viral replication; or to allow invasive pathogens such as fungi, nematodes, insects or parasitic plants to take up the RNA agent through the surface of the protected plant, where it then acts in the RNAi system of the relevant organism against important target RNAs of the pathogen (see also Tomilov et al. 2008; Banerjee et al. 2017; Majumdar et al. 2017; Price and Gatehouse 2008 above).

[0012] However, the use of RNA agents in localized / transient applications has not yet fully matured for a variety of reasons. One reason is the aforementioned inefficiency of RNA entry into target cells. Consequently, extensive research efforts are underway to explore methods for stabilizing RNA agents through chemical modifications of their nucleotide building blocks and / or for delivering them to their site of action in the cytoplasm of target cells via physical or biochemical methods (Dowdy 2017; Setten et al. 2019; Dalacouras et al. 2020). However, significant differences exist between different application scenarios and between different organisms.

[0013] Another reason is related to the fact that the natural and artificially induced RNA silencing / RNAi process is generally not very efficient. A large amount of data has fully demonstrated that the main reason is that although a large number ("pool") of siRNAs are generated from the target RNA through the activity of Dicer / DCI, only a few siRNAs in this final "pool" are actually effective against the target RNA ( Figure 1 This is mainly because target RNA is usually highly structured, and only a few "accessible sites" (called "a sites") can enable sRNA / AGO / RISC to effectively bind to the complementary target site and achieve silencing.

[0014] This is especially true for target RNAs from pathogens: target RNAs of pathogens often undergo adaptive changes (attenuation) in their structure through co-evolution with the target organism in order to escape silencing / RNAi as much as possible. In this article, an a site is defined as a region on the target RNA that is accessible to a nucleic acid active agent. An a site can correspond to a "target site". In this article, a target site is defined as a sequence of a target RNA to which a single-stranded nucleic acid (such as an siRNA guide strand) or an antisense oligonucleotide (see below) can hybridize by complementary base pairing. An a site can also comprise a larger region of the target RNA, for example, an RNA structural motif that is particularly accessible and comprises one or more single-stranded nucleic acid target sites.

[0015] To date, double-stranded forms of target RNAs, such as mRNA or dsRNA forms of viral genomes, have been primarily used for RNAi SIGS (Robinson et al. 2014). It is important to note that all dsRNAs used for RNAi to date contain the complete sequence of the corresponding target RNA on one strand, or longer continuous segments (typically several hundred nucleotides) (see also in Figure 9 、 10 , 11, and 15). The second RNA strand is a second molecule with a completely complementary sequence. Alternatively, a "hairpin" structure can be used. In this case, the two complementary RNA strands are located in the same molecule and separated by a "spacer," a largely unstructured single-stranded sequence of arbitrary nucleotide composition and length. This creates an incomplete duplex, a "hairpin." Once inside the plant cell, the dsRNA or dsRNA hairpin is processed by DCL into a pool of siRNAs, just like normal target RNA.

[0016] However, this pool faces the aforementioned problem: only a very small number of siRNAs generated from these “conventional dsRNAs” are effective (see also Figure 1 、 Figure 10 and Figure 15 Schematic description in ).

[0017] "Conventional dsRNA" refers to these RNAs in which one strand consists of an exact copy of the targeted target RNA, which then hybridizes with a complementary RNA strand: as explained, dsRNAs constructed according to this principle are currently used in RNAi-mediated plant protection.

[0018] In contrast, the remaining siRNAs in the pool, representing the vast majority of siRNAs, nonspecifically saturate cellular AGO / RISC (thus, they act as "decoys" for AGO / RISC), which can even inhibit RNA silencing / RNAi using conventional dsRNAs or dsRNA hairpins. There is also the risk that siRNAs in the pool will trigger silencing of non-target RNAs ("off-target effects"), for example, through incomplete complementary base pairing of the guide strand with other RNAs that is still sufficient to form a functional RISC (Jackson and Linsley 2004; Jackson et al. 2006; Senthil-Kumar and Mysore 2011; Casacuberta et al. 2015; Kamola et al. 2015). In summary, the use of dsRNAs that generate large quantities of poorly characterized siRNAs that are inactive against their target RNAs is problematic (Qu et al. 2012; Dalacouras et al. 2016). This is also true for dsRNAs that generate other forms of sRNA.

[0019] Until recently, it has been impossible to reliably identify and use the small number of siRNAs that are effective in RNAi, referred to herein as "esiRNAs" or "ERNAs," and collectively referred to as "esiRNAs / ERNAs" below, from a pool of siRNAs. Consequently, many siRNA or sRNA agents have been designed to target regions of target mRNAs encoding conserved protein motifs, or based on unreliable in silico predictions of α sites in the target RNA, and then tested for their effectiveness by "trial and error" in very complex empirical studies (Birmingham et al. 2007; Cerritelli and Crouch 2009; Miozzi et al. 2013; Fakhr et al. 2016; Carbonell et al. 2018; Eastman et al. 2018; Han et al. 2018; Qureshi et al. 2018; Setten et al. 2019).

[0020] Only in recent years, mainly through the work of our laboratory, has an experimental screening method called "eNA screening" been established, which can reliably detect a sites in a variety of target RNAs in a short time (Schuck et al. 2013; Gago-Zachert et al. 2019; WO2019001602 A1; WO 2022 / 200407; see for example Figure 1Therefore, knowledge of the a site enables the identification of esiRNAs / ERNAs that function reliably and efficiently against relevant target RNAs during RNA silencing / RNAi processes.

[0021] The terms "reliable" and "efficient" can be precisely defined: "eNA screening" is a multi-stage in vitro method (see further description below), in which the efficiency of the hydrolysis of the target RNA by the identified siRNA is ultimately measured in a "slicer" (spaltung) assay. According to the invention, siRNAs which hydrolyze at least 25% of the amount of target RNA used in a standardized and stringent slicing assay are considered to be effective and are therefore designated as esiRNA / ERNA (see also Tables 1, 2, 7 and 8). It could be demonstrated that these esiRNA / ERNA reliably display a high degree of antipathogen efficacy in the respective in vivo systems, which in most cases correlates with the ability to hydrolyze the target RNA in the slicing assay (Gago-Zachert et al. 2019; see, for example, Figure 2-6 ).

[0022] The ability to specifically target target RNA with esiRNA / ERNA, thereby combating pathogens, significantly increases the efficiency of the RNA silencing / RNAi process. Furthermore, the use of esiRNA / ERNA increases the specificity of RNAi: the likelihood of off-target effects on untargeted RNA molecules, and thus, the potential for adverse and undesirable side effects of RNA-active agents, is significantly reduced.

[0023] As previously mentioned, esiRNA / ERNA can be defined by identifying the a site in the target RNA. In other words, there is an accessible sequence (target site) in the a site through which the esiRNA / ERNA guide strand can bind to the target RNA (hybridization by complementary base pairing), and then, through the activity of RISC, the target RNA is inactivated in the manner described (i.e., by endonuclease cleavage or inhibition of translation, see above). Similar effects can also be achieved by related sRNAs (such as miRNAs), whose sequences can be derived from the sequences of esiRNA / ERNAs, which can then be used in similar RNA silencing / RNAi processes as esiRNA / ERNA.

[0024] Only recently has it been demonstrated that by identifying α sites in target RNAs and defining esiRNAs / ERNAs (whose guide strands can bind to target sites within these α sites), it is also possible to identify antisense deoxyribonucleic acid (DNA) oligonucleotides (ASOs) that can bind to these target sites (WO 2022 / 200407). Using similar terminology, ASOs derived from esiRNAs / ERNAs are referred to as eASOs. eASOs have homologous DNA sequences to the single strands of esiRNAs / ERNAs (i.e., deoxynucleotides rather than ribonucleotides; thymidine rather than uridine) and are accordingly able to hybridize to the corresponding target sites within the α sites of target RNAs, similarly causing RNA silencing. Like sRNAs, ASOs can be used transiently; they act in an “antisense manner” through mechanisms distinct from sRNAs. In a given context, two of these mechanisms are crucial: first, the formation of a DNA:RNA heteroduplex between the single-stranded ASO and the target RNA can inhibit RNA translation in the cytoplasm. Secondly, the formation of DNA:RNA heteroduplexes can activate RNase H endonucleases in the nucleus or cytoplasm (RNase H1 and / or RNase H2 in eukaryotic cells), which then catalyze the degradation of the target RNA, guided by the binding of the single-stranded ASO to the complementary RNA, similar to AGO / RISC (Shen and Corey 2018; Bennett 2019; WO 2022 / 200407; Wdowikowska and Janicka, 2021, Crooke et al., 2021). The optimally active ASO length is 12-20 nucleotides (Crooke et al., 2021). Therefore, eASOs can be easily derived from esiRNA / eRNAs with lengths of 21, 22, 23, or 24 nt.

[0025] The following text is primarily limited to the description of esiRNA / ERNA activity. Similar findings also apply to other related sRNAs (such as miRNAs and eASOs), whose RNA or DNA sequences can be derived from the sequences of identified esiRNA / ERNAs: due to sequence matching, single strands of such sRNAs or eASOs are able to bind to the same target site in the corresponding target RNA and can therefore also be used in RNA silencing / RNAi or RNA silencing / antisense methods to control pathogens (WO 2022 / 200407). The esiRNA / ERNA identified by "eNA screening" and the related sRNAs and eASOs derived therefrom are collectively referred to as eNAs (effective nucleic acids) below.

[0026] It should also be noted here that RNA and DNA active agents can be used in chemically modified form: chemical modifications, conjugates or structures, especially as "gapmers" or "mixed polymers", can significantly increase the potential of siRNA- or ASO-based active agents in in vivo RNAi or antisense approaches (Setten et al. 2019; Wdowikowska and Janicka, 2022; Crooke et al., 2021; see also claims).

[0027] Knowledge of the large number of target RNA a-sites (and thus target sites) is particularly important in approaches aimed at combating highly variable pathogens. Specifically, viruses with RNA genomes and RNA-dependent RNA polymerases (RdRp) as the primary enzymes of viral replication exhibit a high degree of plasticity (variability), and therefore have the potential to rapidly develop resistance to antiviral agents. Viral RdRp possesses no or only inefficient editing functions that correct errors that occur when nucleotides are incorporated into newly synthesized daughter nucleic acid chains. Consequently, RNA viruses experience a significant rate of mutation and evolution during replication, a process known as "antigenic drift." These rates are particularly high for viruses with segmented genomes. When cells are co-infected with different viruses, these segments can recombine into novel combinations—a process known as "reassortment." This results in "antigenic shift," a major genetic change that can lead to the emergence of viruses with novel properties and, since an effective immune response may not exist against these viruses ("viral escape"), poses a significant threat to the host.

[0028] However, high variability can also be found in other organisms, particularly those subject to strong selective pressures. This is the case with plant pathogens that are routinely treated with antipathogenic substances. These mutants mutate under the influence of these substances and develop resistant forms. This applies, for example, to nematodes and fungi that are routinely treated with nematicides and fungicides in agriculture and horticulture.

[0029] However, by using broad-spectrum eNAs identified or derived by "eNA screening" in HIGS or SIGS, the possibility of developing pathogenic forms resistant to anti-pathogen methods (escape) in various organisms can be greatly reduced. Broad spectrum means using two or more eNAs to target a target RNA, or using two or more eNAs to target different target RNAs in a target organism. The same approach can also achieve a broad spectrum effect against pathogen variants (such as those that may appear during a viral epidemic). Finally, using different eNAs to target various target RNAs of different pathogens can achieve broad-spectrum protection against different pathogens.

[0030] Therefore, the use of effective eNA yields the following application advantages:

[0031] (i) The combinability of various esiRNA / ERNA makes it possible to maximize the specificity and efficiency of RNA silencing / RNAi methods (see schematic diagram Figure 1 ). This is also true for other sRNAs whose sequences are derived from esiRNA / ERNA. This is also true for eASOs whose sequences are derived from esiRNA / ERNA and used in antisense approaches.

[0032] (ii) Novel antipathogen esiRNA / eRNA combinations enable rapid and specific adaptation of RNAi immune responses to pathogens that may have undergone significant changes due to antigenic drift or antigenic shift, or to pathogens that have emerged in novel forms. This also applies to other sRNAs derived from esiRNA / eRNA. This also applies to eASOs derived from esiRNA / eRNA that can be used in antisense approaches.

[0033] (iii) Economical production of nucleic acid-based active agents should significantly increase the acceptability of RNAi or antisense approaches in, for example, plant production. SIGS approaches can be implemented more efficiently and the use of transgenic approaches can be significantly reduced.

[0034] (iv) In order to optimally achieve the simultaneous use of esiRNA / ERNA and simultaneously improve the usability of RNA in HIGS and SIGS methods, these nucleic acids are preferably used in the form of dsRNA. The "edsRNA" referred to herein should include the nucleotide sequences of several esiRNA / ERNA (or other sRNA derived therefrom) identified by "eNA screening" (schematically shown in FIG. Figure 1 As an overall advantage, dsRNA has a significantly reduced degradation rate (and extended half-life) in HIGS and SIGS methods compared to siRNA with a 3'-terminal single-stranded sequence overhang (Bachman et al. 2020), thus having obvious application advantages.

[0035] Problem / Purpose

[0036] In plant protection, when combating highly variable pathogens, there is the problem of using adaptable and effective nucleic acid active agents to inactivate the target RNA of these pathogens and thus inhibit their replication. Therefore, the technical problem underlying the present invention is to protect plants from a variety of variable pathogens using a sustainable nucleic acid-based method.

[0037] To address this issue, the present invention provides a variety of nucleic acid-based active agents (eNAs) that are effective against three important plant pathogens using "eNA screening" (RNAi or antisense), as well as methods for preparing and using these agents. As mentioned above, the term "effective" is clearly defined as a common technical feature of the eNAs used.

[0038] In one aspect, the present invention relates to RNAi methods using one or more esiRNAs / ERNAs or related sRNAs, or antisense methods using one or more eASOs whose sequences are derived from esiRNAs / ERNAs. Active esiRNAs / ERNAs or other derived sRNAs or eASOs (collectively referred to as eNAs) should have reliably high antipathogen efficacy and, in the combination used, should be directed against a variety of target RNAs or various regions of target RNAs (target sites) so as to be able to reliably combat highly variable pathogens.

[0039] In another aspect, the present invention relates to an RNAi method using edsRNA, which is a completely new form of dsRNA designed according to the present invention, which comprises the nucleotide sequence of esiRNA / ERNA or sRNA derived therefrom and is processed into the esiRNA / ERNA or sRNA during the RNA silencing / RNAi process.

[0040] The nucleic acid-based active agents of the present invention can be prepared or used to target representatives of various types of variable or highly variable pathogens that infect host organisms, plants. As previously mentioned, the most important variable plant pathogens include viruses, nematodes and fungi. The present invention is particularly advantageous because the nucleic acid-based active agents of the present invention can be prepared or used to target representatives of these pathogens that are particularly economically important. A representative species of a highly variable viral pathogen of economic importance in plants is cucumber mosaic virus (CMV). A representative species of a variable nematode pathogen of economic importance in plants is the southern root-knot nematode (M. incognita). A representative species of a variable fungal pathogen of economic importance in plants is Botrytis cinerea (B. cinerea).

[0041] The present invention provides a solution to this problem by providing a wide range of effective eNAs that can be used alone or in combination as active agents in plant protection against these important pathogens.

[0042] The "eNA screening" method used

[0043] To identify eNAs, a previously published method described in a patent application (WO 2019 / 001602), referred to herein as "eNA screening", was employed: for the first time, it was applied in a new standardized and stringent format to target RNAs of the following: Cucumber mosaic virus, Meloidogyne incognita and Botrytis cinerea, allowing for the first identification of a class of effective eNAs in RNA silencing against these pathogens, namely esiRNA / ERNA and the sRNAs, eASOs and edsRNAs derived therefrom (see description below).

[0044] The eNA screening method was performed in three steps.

[0045] (i) Target RNA (which can be genomic RNA, mRNA, or dsRNA) is added to a cytoplasmic extract from plant cells (Nicotiana tabacum BY-2 cells), which is called "BYL" (L stands for "lysate"). The (endogenous) DCL present in the extract generates an siRNA pool from the target RNA, which contains 21, 22, 23, and 24 nt siRNAs as the main products. The entire siRNA pool is captured by next-generation RNA sequencing (NGS, RNA-Seq).

[0046] (ii) In the extract, RISC is reconstituted with the siRNA pool from (i) and the selected AGO protein. To this end, AGO protein is in vitro translated from the added mRNA in the extract. The formed AGO / RISC is immunoprecipitated, and siRNA chains enriched by binding to AGO are again identified by RNA-Seq. Here, the RNA Seq data from step (i) are used for comparison to determine enrichment in the relevant AGO / RISC.

[0047] (iii) Finally, for the siRNAs captured in (ii), a further in vitro assay (hereinafter and in the figures referred to as "cleavage assay") is used, in which the respective AGO / RISC used is tested for endonuclease cleavage (cleavage), thereby identifying those siRNAs that have previously been determined to be enriched in the relevant AGO / RISC and effectively induce target RNA cleavage. As previously described, if the siRNA induces hydrolysis of 25% or more of the amount of target RNA used in a standardized and rigorous in vitro cleavage assay using the corresponding AGO / RISC, these siRNAs are defined as esiRNA / ERNA.

[0048] Subsequently, further validation steps tested the efficacy of each esiRNA / eRNA in vivo. Different methods tailored to each target pathogen were used. Other sRNAs and eASOs with similar nucleotide sequences were derived from the esiRNA / eRNA identified in this way. These can also be tested in BYL using a cleavage assay. The active component of eASOs is the RNAse H enzyme (e.g., see WO 2022 / 200407).

[0049] Target organisms

[0050] Plant pathogen cucumber mosaic virus (CMV)

[0051] CMV, a type-defining virus in the genus Cucumber mosaic virus (Bromoviridae family), is an economically important plant pathogen (Scholthof et al., 2011; Rybicki, 2015; Gallitelli, 2000). CMV has a tripartite, segmented, single-stranded (ss), positive-sense (+) strand RNA genome. The three genomic components, RNA1 (3.3 kb), RNA2 (3.0 kb), and RNA3 (2.2 kb), are encapsidated in three distinct viral capsids that only function together to cause infection. These capsids contain a cap structure at the 5' end and a tRNA-like structure at the 3' end. Due to the segmented nature of its genome, CMV is a reassortant virus with a high mutation rate: in addition to antigenic drift caused by the viral RdRP, the virus also exhibits antigenic shifts. After entering the host cell, the three genomic RNAs function as mRNAs due to their positive (positive) orientation (positive, similar to mRNA). RNA1 and RNA2 encode the "1a" protein (111 kDa) and the "2a" protein (97 kDa), respectively. The 2a protein is an RdRp; together with the 1a protein, it forms the viral component of the replicase, which catalyzes genome replication and transcription of subgenomic (sg) RNAs.

[0052] During RNA replication (not described in detail here), complementary (-) RNA copies of the viral RNA are transcribed by the replicase. These serve as templates for the synthesis of new (+) RNA molecules, as well as for the synthesis of subgenomic sgRNAs. The sgRNA, sgRNA4 (1.1 kb), is transcribed from the (-) RNA copy of RNA 3 produced during replication and is also packaged into the capsid with it. RNA 3 itself encodes the 30 kDa "movement protein 3a"; while sgRNA4 encodes the 24 kDa "capsid protein CP". During infection, 3a and CP are both essential for the intercellular and systemic movement of the virus in the plant. Another subgenomic RNA, sgRNA 4A (0.7 kb), is transcribed from the (-) RNA copy of RNA 2. It encodes the viral RNA silencing suppressor (VSR), the "2b" protein (15 kDa). The 2b protein interferes with the RNA silencing / RNAi process by sequestering (high affinity binding) the siRNAs produced in this process. These five gene products all influence virus transmission in plants in a host-specific manner, thereby affecting virulence. During infection, additional satellite RNAs can be produced during viral RNA replication, and their presence can significantly affect pathogenicity (Gallitelli, 2000; Garcia-Arenal et al., 2008; Jaquemond, 2012; Roossinck et al., 2001; Roossinck et al., 2002; Palukaitis, 2016; Nouri et al., 2014; Mochizuki and Ohki, 2012).

[0053] CMV strains can be broadly divided into two subgroups, subgroup 1 (I) and subgroup 2 (II). Subgroup I strains are further subdivided into two subgroups (A and B) (see also below and Figure 7). While sequence similarity within a subgroup is high (subgroup I: >88%; subgroup II: >96%), sequence similarity between subgroups is only 70–75%. Subgroup IA and subgroup II CMV strains are distributed worldwide; subgroup IB strains are primarily found in East Asia (Garcia-Arenal et al., 2008; Jaquemond, 2012; Nouri et al., 2014; Mochizuki and Ohki 2012). The serological differentiation index (SDI) between CMV subgroups is approximately 1–2. This is in contrast to other cucumoviruses (PSV (peanut stunt virus) and TAV (tomato sterility virus), which have SDIs of 6–7). Like other segmented RNA viruses, CMV is highly variable due to the accumulation of mutations during antigenic drift and shift (Scholthof et al., 2011; Rybicki, 2015; Gallitelli, 2000).

[0054] Unlike other members of the Bromoviridae family, CMV strains have a very broad overall host range and infect more than 1,200 plant species from more than 100 families in monocots and eudicots / dicots. This includes important fruits, vegetables, and ornamentals such as Fabaceae, Cucurbitaceae, Convolvulaceae, and Solanaceae. Thus, CMV has the broadest host range of any plant virus and infects important agricultural crops such as beans, sugar beets, carrots, celery, lettuce, peppers, melons, squash, tomatoes, and spinach (Scholthof et al., 2011; Garcia-Arenal et al., 2008; Jaquemond, 2012; Mochizuki and Ohki 2012). CMV infection, in particular, causes severe systemic mosaic symptoms, leaf deformation, systemic necrosis, chlorosis, stunting, and fruit lesions (Garcia-Arenal et al., 2008; Jaquemond, 2012; Holeva et al., 2021). CMV can interact synergistically with potato virus Y, tobacco mosaic virus, and potato virus X (PVX) in Solanaceae plants, as well as with potato virus Y in cucurbit hosts (Scholthof et al., 2011; Gallitelli, 2000; Jaquemond, 2012).

[0055] CMV particles can be transmitted by more than 80 aphid species from 33 genera through a non-persistent "stylet-borne" method. Furthermore, depending on the plant species, they can be transmitted through infected seeds (Jaquemond, 2012; Ali and Kobayashi, 2010; O'Keefe et al., 2007). There is also evidence that the virus can overwinter in seeds, which then become an important source of infection early in the growing season.

[0056] Due to its broad host plant range, global distribution, and non-persistent transmission by numerous aphids, CMV is considered one of the most economically important plant viruses and is ranked among the most important viruses of annual crops worldwide (Scholthof et al., 2011; Rybicki, 2015). Annual crop losses vary significantly across regions and are difficult to quantify, especially in the presence of mixed infections. Estimates from the 1990s and 2000s provide some guidance; for example, in China, tomato yield losses of 25%–50% were estimated, or in Spain, losses of 60%–80% were estimated for melon and 80% for pepper. When certain CMV strains express necrotizing satellite RNA, losses of 80%–100% of tomato plants have been documented across 70% of the cultivated areas in Spain and Italy (Gallitelli, 2000). Significantly, new CMV hosts and CMV-induced plant diseases are being discovered each year. Increased aphid activity in northern temperate regions due to climate change is expected to lead to further outbreaks (Scholthof et al., 2011; Nicaise, 2014). However, CMV is also becoming increasingly important in tropical and subtropical regions, particularly in areas with mixed crop cultivation.

[0057] Control measures against aphids that rely solely on the use of insecticides are not very effective (Gallitelli, 2000). However, plant protection methods using genetically modified plants or RNA-based methods have also been less successful so far, largely due to the high variability of the virus (Nicaise, 2014). Summary of the Invention

[0058] To address this issue, within the scope of the present invention, an eRNA screening method can be used to characterize esiRNAs / ERNAs against various genomic CMV RNAs with reliable high antiviral efficacy. In addition to having universal high antiviral efficacy against homologous target RNAs, these esiRNAs / ERNAs or variants of these esiRNAs / ERNAs should also have antiviral effects on various CMV variants. In addition, dsRNAs, referred to as "edsRNAs," are generated, which are preferably used in HIGS and / or SIGS methods, from which large quantities of esiRNAs / ERNAs are produced during RNA silencing / RNAi processes, thus also having reliable and effective antiviral effects. From the esiRNAs / ERNAs characterized in this manner, other sRNAs or eASOs can also be derived, which can also be used in RNA silencing / RNAi methods or RNA silencing / antisense methods against CMV.

[0059] Meloidogyne incognita

[0060] Nematodes are evolutionarily the oldest multicellular worms. Across their diverse range of moist habitats, they are often the largest group of metazoans in terms of both individual numbers and species diversity (Wikipedia). Their structure is very simple: their bodies are limbless, cylindrical, elongated, and smooth, surrounded by a flexible cuticle. The cuticle, secreted by layers of epidermal cells, forms the nematode's exoskeleton (Bird and Bird, 1991a). The cuticle is permeable to ions and water and regulates the hydrostatic pressure within the nematode's body. Most nematodes undergo four molts during their development from the larval stage (J1 to J4) to the adult stage. During this process, the cuticle is completely shed or, in the case of Meloidogyne, partially resorbed (Perry and Moens, 2011). Beneath the cuticle, muscles run longitudinally along the inner side of the body and are activated by two longitudinal nerves located dorsally and ventrally, which are connected by a nerve ring (Bird and Bird, 1991b). The nematode's head contains sensory organs and a "mouth" leading to a muscular pharynx. This pharynx acts as a pump, drawing food into the connected intestine. The intestine leads to a long, simple, muscleless intestinal cavity, ultimately to an anus located near the end of the body. Nematodes lack a vascular system for transporting digested food, nor a respiratory system for absorbing or transporting oxygen. Instead, nutrients and metabolic waste are distributed within a pseudocoelom, the contents of which are regulated by excretory ducts running along each side of the body (Bird and Bird, 1991c).

[0061] A subgroup of nematodes is the plant-parasitic nematodes (PPNs). PPNs infect many plant species and cause significant crop losses worldwide (Blok et al., 2008). Although their lifestyles and feeding strategies vary widely, all PPNs possess a hollow, protruding stylet that pierces plant cell walls and injects secretions and / or enzymatically active proteins that facilitate infection and nutrient uptake. These secretions or proteins are produced by three esophageal "salivary glands," the cuticle, and chemoreceptor organs (Perry, 1996; Semblat et al., 2001; Curtis, 2007).

[0062] Among PPNs, those known as "resident endoparasites" cause the greatest economic losses. These include root-knot nematodes (RKN), of which the species Meloidogyne (Meloidogyne incognita) is the most important plant pathogen (Trudgill and Blok, 2001), responsible for an estimated €10 billion in crop losses worldwide annually. RKN are particularly widespread in temperate and tropical regions of the world (Blok et al., 2008; Abad and Williamson, 2010). They infect thousands of plant species, including nearly all crops, and cause characteristic root deformations (nodules), which weaken the plant and reduce yield.

[0063] The life cycle of RKN lasts 3–10 weeks, depending on the nematode species and environmental conditions. Second-stage (J2) worm-like larvae hatch from eggs and burrow into the soil to infect the host's roots. During the early parasitic phase, J2s typically penetrate the root tissue behind the root tip by physically piercing root cells with their stylets. Simultaneously, they release enzymes that modify the cell wall, enabling them to migrate further into the root tissue and move between cells toward the root tip. From there, they migrate into the plant's vascular cylinder (Perry and Moens, 2011) and induce the formation of specialized feeding sites in the form of giant cells (GCs). GCs are highly hypertrophic and multinucleated. They form through repeated nuclear division and cell growth without cell division (Jones and Payne, 1978; Caillaud et al., 2008) and serve as the nematode's sole source of nutrition. The nematode pierces these cells to gain access to the cytoplasm, while secretions alter the cell's hydrostatic pressure, facilitating nutrient uptake (Abad and Williamson, 2010). Division of the vascular cells and feeding cells surrounding the nematode leads to the formation of a typical root nodule. After establishing a feeding site, the J2 nematode settles and then undergoes three more molts (parasitic J3 and J4) to mature into an adult female or male nematode. The female nematode settles, while the male nematode becomes mobile again, migrating from the root into the soil. RKN reproduce by mitotic parthenogenesis (Castagnone-Sereno et al., 2013); sex is determined by environmental conditions: under poor nutritional conditions, the number of males increases (Papadopoulou and Triantaphyllou, 1982). At the end of development, female RKN become pear-shaped. They produce hundreds to thousands of eggs in a protective gelatinous matrix on the outer surface of the root, which are released directly into the root's surroundings. Inside the egg, the first-stage larvae become J2 after embryogenesis, and then hatch under favorable conditions to continue their life cycle in a suitable host (Hussey and Mims, 1991; Chitwood and Perry, 2009; Curtis et al., 2009).

[0064] Various chemicals, such as methyl bromide and carbamates, have long been used as nematicides. However, due to potential environmental and health risks to consumers, most are now banned. An alternative method for controlling PPNs is crop rotation, in which nematode-resistant plants are grown continuously in different seasons. However, this approach has limitations: some nematodes, such as root-knot nematodes, have a wide host range, making it difficult to select suitable crops to interrupt the infestation cycle. Biological control of PPNs involves the use of one or more organisms, such as nematode-feeding fungi or certain species of bacteria. These organisms are introduced into the soil, where they attack the nematodes without affecting plant growth (Evans et al., 1993). However, these "nematode predators" are difficult to manage on a large scale. Furthermore, environmental factors such as soil conditions, moisture, temperature, and pH significantly affect the survival of biocontrol agents (Chen and Dickson, 2004; Stirling, 2014), which is detrimental to their use. For nematodes such as Caenorhabditis elegans (C. elegans) and M. incognita, it has been reported that they take up siRNA and dsRNA via the above-mentioned nutrient uptake mechanism, and siRNA can then become active in the animal via the nematode's RNA silencing / RNAi system via uptake into cells, for example, in a nematicidal manner by inactivating the mRNA of key proteins (Arguel et al., 2012; Bakhetia et al., 2005; Banakar et al., 2020; Chaudhary et al., 2019; Dalzell et al., 2010a; Dalzell et al., 2010b; Danchin et al., 2013; Dong et al., 2014). al., 2014; Dong et al., 2016; Dutta et al., 2015; Huang et al., 2006; Iqbal et al., 2020; Niu et al., 2012; Papolu et al., 2013; Shivakumara et al., 2016). Summary of the Invention

[0066] Similar to the above-mentioned CMV, esiRNA / ERNA and edsRNA have been characterized, and various variants thereof have reliably high nematicidal efficacy against the southern root-knot nematode (M. incognita). Starting from the esiRNA / ERNA characterized in this way, other sRNAs or eASOs can also be derived, which can also be used in RNA silencing / RNAi methods or RNA silencing / antisense methods against the southern root-knot nematode.

[0067] Plant pathogenic fungus Botrytis cinerea

[0068] Botrytis cinerea (sexual stage: Botryotinia fuckeliana), the primary causative agent of gray mold, is an aggressive necrotrophic fungal pathogen that infects a large number of plant species (over 200) (Elad, 1997; van Kan, 2006; Choquer et al., 2007; Williamson et al., 2007; Nakajima & Akutsu, 2014). B. cinerea secretes nonspecific phytotoxins that kill a broad spectrum of plant cells (Pinedo et al., 2008). Economic losses from this disease can reach $10–100 billion annually worldwide, and without chemical control, greenhouse and field crop losses can reach as high as 40%, making B. cinerea one of the ten most important fungal plant pathogens (Dean et al., 2012; Pedras et al., 2011; Villa Rojas et al., 2012). The use of chemical fungicides has caused significant environmental damage (Malhat et al., 2015; Oliveira et al., 2015; Tomenson and Matthews, 2009) and is also limited by the nutritional diversity of Botrytis cinerea. Furthermore, there have been several reports of fungicide resistance in Botrytis cinerea (Leroux, 2007).

[0069] Taxonomically, Botrytis cinerea belongs to the phylum Ascomycota, class Hammerglomerata, and family Sclerotiniaceae (Garfinkel, 2021). Two phylogenetic species groups have been proposed: Group I and Group II. Group I strains (also known as "otrytis pseudocinerea") and Group II ("Botrytis cinerea sensu stricto") differ in ecology and fungicide resistance patterns (Fournier et al., 2003, 2005). The genomes of two strains (B05.10 and T4) have been fully sequenced (Choquer et al., 2007).

[0070] Unlike many other plant pathogens, Botrytis cinerea persists year-round and under a wide range of environmental conditions (Nair et al., 1995). The infection / vegetative process of Botrytis cinerea can generally be described as follows: Conidia produced on sclerotia or plant debris from infected plants attach to the surface of a new host and form germ tubes. This develops into appressorium, which facilitates penetration of the host surface. To overcome the host cuticle barrier, Botrytis cinerea also secretes cell wall-degrading enzymes (CWDEs). Epidermal and mesophyll cells die before the infecting hyphae penetrate. Some metabolites and proteins secreted by the fungus induce symptoms of programmed cell death (PCD) or have been shown to cause cell death (Choquer et al., 2007; Nakajima and Akutsu, 2014).

[0071] HIGS have been used to control Botrytis cinerea. Knockout mutants provide a basis for the study of pathogenicity or virulence genes (Nakajima and Akutsu, 2014). Thus, potential target genes can also be identified, some of which are used in the examples of this application described in more detail below (ten Have et al., 1998; Li et al., 2019; Liu et al., 2018; Nerva et al., 2020; Qiao et al., 2021; Schumacher et al., 2008; Segmüller et al., 2008; Soulie et al., 2006; Ren et al., 2018; Yang et al., 2013; Zheng et al., 2000). In addition to HIGS, localized / transient application of dsRNA has been considered as an alternative approach (Wang et al., 2016; Weiberg et al., 2013), and some studies have demonstrated efficacy in various fungi (McLoughlin et al., 2018; Gebremichael et al., 2021). Current scientific understanding is that the success of this approach depends largely on the ability of the fungus to take up exogenous RNA (Qiao et al., 2021). Summary of the Invention

[0073] Similar to the description above for CMV and incognita, esiRNAs / ERNAs and edsRNAs with reliable high fungicidal efficacy against various variants of Botrytis cinerea have been characterized. From the esiRNAs / ERNAs characterized in this manner, other sRNAs or eASOs can be derived, which can also be used in RNA silencing / RNAi approaches or RNA silencing / antisense approaches against Botrytis cinerea. DETAILED DESCRIPTION

[0074] The problem solved by the present invention is by nucleic acids for protecting plants against the plant pathogens cucumber mosaic virus, southern root-knot nematode and Botrytis cinerea, wherein

[0075] a. The nucleic acid is a small interfering RNA (siRNA), the small interfering RNA comprising a fully or partially complementary nucleic acid consisting of 21, 22, 23 or 24 base pairs and containing two single-stranded RNAs selected from a guide strand and a passenger strand, wherein the guide strand and the passenger strand are selected from the nucleic acids having SEQ ID NO: 1-4, 6-11, 14-17, 21-25, 27-30, 32-37, 40-43, 47-51, 53, 55-66, 69-70, 73, 75-86, 89-90, 93-120, 124, 126-132, 134-138, 140-150, 154, 156-162, 164-168 and 170-180; or

[0076] b. The nucleic acid is an siRNA of group a, wherein at least one of the single-stranded RNAs selected from the guide strand and the passenger strand has a modification at 1-7 positions in the nucleotide sequence; or

[0077] c. The nucleic acid is a small RNA (sRNA) selected from siRNA and microRNA (miRNA), wherein the RNA duplex of the small RNA consists of complementary or partially complementary nucleic acids of group a and / or group b; or

[0078] d. the nucleic acid is a double-stranded RNA containing at least two siRNA or sRNA nucleotide sequences of group a, b or c; or

[0079] e. The nucleic acid is a single-stranded DNA consisting of 12-25 nucleotides and containing a sequence of 12 or more nucleotides that is homologous to one of the nucleotide sequences of the guide strand and passenger strand of the single-stranded RNA of group a or b (deoxyribonucleotides instead of ribonucleotides); or

[0080] f. The nucleic acid is a single-stranded DNA of group e, which has a modification at position 1-7 in the nucleotide sequence;

[0081] The nucleic acid is provided for protection against plant pathogens using a method for targeted identification of effective small interfering RNAs (esiRNA / ERNA) and sRNAs derived therefrom, as well as effective antisense DNA oligonucleotides (eASOs) of varying lengths, collectively referred to as effective nucleic acids (eNAs), comprising the following steps:

[0082] (i) RNA selected as a target for RNA silencing (RNAi) is produced using in vitro transcription and converted into small interfering RNA (siRNA) by endogenous Dicer-like protein (DCL) in cytoplasmic extracts of plant cells;

[0083] (ii) forming a siRNA pool generated from the used RNAs and determining the siRNAs contained in the pool by RNA-seq analysis;

[0084] (iii) adding messenger RNA (mRNA) of argonaute (AGO) protein to cytoplasmic plant cell extract, wherein the mRNA is synthesized by in vitro transcription and is constructed to encode the AGO protein of interest with a tag;

[0085] (iv) using in vitro translation to form AGO protein molecules, which form RNA-induced silencing complexes (RISCs) with siRNA produced by the presence of DCL;

[0086] (v) immunoprecipitating tagged siRNA-loaded AGO / RISC from BYL and determining the bound siRNA guide strand by RNA-seq analysis;

[0087] (vi) using the RNA-seq data to compare with the RNA-seq data from step (ii) to determine those siRNAs enriched in AGO / RISC;

[0088] (vii) then synthetically producing and testing its functionality in a cleavage assay using the labeled target RNA; and

[0089] (viii) identifying esiRNA / ERNA in this way, and determining sRNA and eASO derived therefrom, collectively referred to as eNA;

[0090] It is characterized in that

[0091] I. To form the RISC in step (vii), 0.5 pmol of the mRNA of the AGO protein to be used is translated in the presence of 10-100 nM of the synthetic siRNA to be characterized and a 10-fold excess (0.1-1 μM) of a competing siRNA (e.g., siR gf698, selected from SEQ ID NOs: 205, 206, 207, and 208; Iki et al., 2010) in a reaction solution containing 50% (v / v) BYL;

[0092] II. After incubation at 25°C for 2.5 h in each batch in step I, 3.4 pmol of non-specific mRNA (e.g., encoding firefly luciferase protein SEQ ID NO: 209, Schuck et al., 2013) was added as a further competitor RNA, and 10 fmol of target RNA was added, the target RNA was labeled, and the reaction batch was incubated again at 25°C for 15 min;

[0093] III. During the incubation period in step II, the target RNA is cleaved by the formed AGO / RISC;

[0094] IV. After gel electrophoresis of the extracted RNA, the remaining amount of target RNA or the resulting cleavage product is quantified compared to a control reaction (performed without siRNA) by measuring the band intensity (ImageQuantTL or ImageJ);

[0095] V. Classifying each RISC formed with the siRNA as esiRNA / ERNA based on its measured cleavage activity (cleavage activity) on the target RNA;

[0096] VI. selecting an esiRNA / ERNA that endonuclease converts at least 25% or more of the amount of the target RNA initially used in step II of the method into cleavage products; and

[0097] VII. Optionally, other sRNAs and eASOs derived from the sequence of the esiRNA / ERNA are identified in this manner.

[0098] In step 1, BYL preferably has a defined protein content and translation activity: the protein amount is determined using a conventional Bradford assay (Wikipedia) and should be 7-12 mg / ml extract (Gursinsky et al., 2009). Translation activity is determined under translation conditions of 85 fmol of firefly luciferase mRNA: under these conditions, the measurable activity of the translated luciferase should be at least 10% of the converted substrate (e.g., luciferin; Wikipedia). 6 RLU (Relative Light Unit) (Gursinsky et al., 2009; Schuck et al., 2013; Gago-Zachert et al., 2019).

[0099] In step I, the amount of siRNA to be tested is preferably adjusted to the activity of each AGO protein with siRNA siR gf698 (guide and passenger strands selected from SEQ ID NOs: 205, 206, 207 and 208) against its mRNA target (encoding GFP (green fluorescent protein)): in the cleavage assay, the measurable cleavage activity of siR gf698 and GFP mRNA target is typically 90%, that is, 90% of the amount of target RNA used is converted into cleavage products.

[0100] In step II, the target RNA can be labeled by any method known to those skilled in the art. For example, the target RNA can be fluorescently labeled or radioactively labeled. Preferably, the target RNA in step II is radioactively labeled.

[0101] esiRNA / eRNA with an in vitro cleavage efficiency of at least 25% (step VI) exhibited significant, measurable antipathogenic effects in vivo compared to control siRNA (see the following exemplary embodiments and Tables 1, 2, and 7). Thus, the present invention advantageously provides eNAs that can be used in RNA silencing / RNAi or RNA silencing / antisense approaches as active agents against a variety of plant pathogens.

[0102] The esiRNA / ERNA having an in vitro cleavage efficiency of at least 25% or higher is, for example, a siRNA of Group a, consisting of 21, 22, 23 or 24 nucleotides, and containing two nucleic acids selected from a guide strand and a passenger strand, selected from the nucleic acids having SEQ ID NO: 1-4, 6-11, 14-17, 21-25, 27-30, 32-37, 40-43, 47-51, 53, 55-66, 69-70, 73, 75-86, 89-90, 93-120, 124, 126-132, 134-138, 140-150, 154, 156-162, 164-168 and 170-180.

[0103] According to the present invention, esiRNA / ERNA with an in vitro cleavage efficiency of at least 50% or higher is preferred. The esiRNA / ERNA with an in vitro cleavage efficiency of at least 50% or higher is, for example, a siRNA of Group a, consisting of 21, 22, 23, or 24 nucleotides, and containing two nucleic acids selected from a guide strand and a passenger strand, selected from the nucleic acids having SEQ ID NO: 1, 2, 4, 6-11, 14-17, 21-25, 27, 28, 30, 32-37, 40-42, 47-51, 55-66, 75-86, 93-120, 128-132, 134, 135, 138, 140-150, 158-162, 164, 165, 168, and 170-180.

[0104] According to the present invention, esiRNA / ERNA having an in vitro cleavage efficiency of at least 75% or higher is preferred. The esiRNA / ERNA having an in vitro cleavage efficiency of at least 75% or higher is, for example, an siRNA of Group a, consisting of 21, 22, 23 or 24 nucleotides, and containing two nucleic acids selected from a guide strand and a passenger strand, selected from the nucleic acids having SEQ ID NO: 2, 4, 6, 8, 10, 11, 14-16, 21, 22, 24, 25, 28, 30, 32, 34, 36, 37, 40-42, 47, 48, 50, 51, 55, 60-63, 65, 69, 75, 80-83, 85, 89, 94-101, 103-106, 108-115, 117-120, 130, 134, 140, 143, 145, 146, 160, 164, 170, 173, 175 and 176.

[0105] According to the present invention, esiRNA / ERNA having an in vitro cleavage efficiency of at least 90% or higher is preferred. The esiRNA / ERNA having an in vitro cleavage efficiency of at least 90% or higher is, for example, a siRNA of Group a, consisting of 21, 22, 23, or 24 nucleotides, and containing two nucleic acids selected from a guide strand and a passenger strand, selected from the nucleic acids having SEQ ID NOs: 4, 6, 10, 11, 14-16, 21, 22, 24, 30, 32, 37, 40-42, 47, 48, 50, 61, 81, 96, 97, 99, 101, 104-106, 110, 111, 113, 115, and 118-120.

[0106] According to the present invention, esiRNA / ERNA having an in vitro cleavage efficiency of at least 95% or higher is preferred. The esiRNA / ERNA having an in vitro cleavage efficiency of at least 95% or higher is, for example, a siRNA of Group A, consisting of 21, 22, 23, or 24 nucleotides and containing two nucleic acids selected from a guide strand and a passenger strand, selected from the nucleic acids having SEQ ID NOs: 6, 10, 32, 36, 97, and 11.

[0107] The sequences of the guide strand and the associated passenger strand are listed in Tables 1, 2, 7, and 8, respectively.

[0108] Method steps (i) to (vii) are described in WO2019001602 A1. WO2019001602 A1 is incorporated herein by reference.

[0109] Process steps I to VII are novel and are based on exemplary embodiments of the present application.

[0110] According to group a, the nucleic acid is an siRNA consisting of 21, 22, 23 or 24 nucleotides and containing two nucleic acids selected from a guide strand and a passenger strand, selected from the nucleic acids having SEQ ID NO: 1-4, 6-11, 14-17, 21-25, 27-30, 32-37, 40-43, 47-51, 53, 55-66, 69-70, 73, 75-86, 89-90, 93-120, 124, 126-132, 134-138, 140-150, 154, 156-162, 164-168 and 170-180. Single-stranded nucleic acids having SEQ ID NOs: 1-4, 6-11, 14-17, 21-26, 27-30, 32-37, 40-43, 47-51, 53, 55-66, 69-70, 73, 75-86, 89-90, 93-120, 124, 126-132, 134-138, 140-150, 154, 156-162, 164-168 and 170-180 are newly identified components of effective nucleic acids (single-stranded components, such as guide strands and passenger strands) within the scope of the present invention, which can be used as active agents in RNA silencing / RNAi plant protection methods against various variable plant pathogens in the form of small RNAs (sRNAs).

[0111] According to group b, nucleic acid of the present invention is siRNA, it comprises two single-stranded RNAs selected from guide strand and passenger strand of group a, and wherein guide strand and / or passenger strand have modification at 1-7 position of nucleotide sequence.Therefore, the nucleic acid of group b represents the variant of the nucleic acid of group a.In the meaning of the present invention, " modification " means " base substitution ", and the base that is selected from adenine (A), uracil (U), guanine (G) and cytosine (C) can be replaced by any other base.Such base substitution can occur at 1-7 position of the nucleotide sequence of the nucleic acid of group a.Base substitution is preferably independent of each other at each position, and each in 1-7 position in the nucleotide sequence of the nucleic acid of group a can be replaced by any other base independently of each other.

[0112] According to group c, the nucleic acid of the present invention is a small RNA (sRNA, such as small interfering RNA (siRNA), or such as microRNA (miRNA)), whose RNA duplex is composed of completely or partially complementary nucleic acids of group a and / or group b.

[0113] Small interfering RNAs, or siRNAs, are short, double-stranded, non-coding RNA molecules, 20-25 base pairs in length, phosphorylated at the 5' end, and possessing a 2-nucleotide (nt) single-stranded overhang at the 3' end. As described above, siRNAs are naturally produced within cells from double-stranded regions of RNA molecules, typically non-self RNA; however, they can also be synthesized and used in targeted RNA silencing / RNAi approaches. Aside from the aforementioned 3' overhang, the two component strands of the siRNA are fully base-paired (i.e., along the entire length of the RNA) through complementary base pairing (typically adenine (A) with uracil (U), and guanine (G) with cytosine (C)), forming an RNA duplex. During the RNA silencing / RNAi process, one of the two single-stranded components of the siRNA binds to a complementary single-stranded region (target site) of another RNA molecule, referred to herein as the target RNA. These are primarily homologous RNAs from which the siRNA was originally generated. Therefore, the function of the target RNA can be inhibited or modulated in various ways. For example, in eukaryotes, siRNA can be used to inhibit the replication of pathogens such as viruses or to suppress the expression of cellular genes at the post-transcriptional level.

[0114] MicroRNA, referred to as miRNA, is a short non-coding RNA encoded in the form of precursor molecules by the cell genome. After transcription and processing, the complementary region of the miRNA composed of an RNA chain of 21-23nt in length forms a double chain by base pairing. Unlike siRNA, these double-stranded regions in miRNA can be interrupted by single-stranded regions in the form of "mismatches" (related to a single nucleotide) and / or "loops" and / or "protrusions" (related to several nucleotides). Similar to siRNA, miRNA inactivates or regulates target RNA by binding (hybridization) to the complete or incomplete complementary region of the target RNA, and its function is determined by the RNA silencing / RNAi process. Therefore, miRNA plays an important role in the post-transcriptional regulation of cellular gene expression, and, like siRNA, can also be used for artificial regulation of cellular gene expression.

[0115] As explained above, two nucleic acid chains are said to be "complementary" if their nucleotides can base pair with each other. For DNA, the pairing rules were originally defined based on the traditional pairing rules of nucleotide bases by Watson and Crick, where adenine (A) on one nucleic acid chain can interact with thymine (T) on the other nucleic acid chain through hydrogen bonds (base pairing), and guanine (G) on one nucleic acid chain can interact with cytosine (C) on the other nucleic acid chain (base pairing). Therefore, DNA forms a double strand, so to speak, with each strand being a negative version of the other.

[0116] RNA molecules can form double strands in a similar pattern, i.e., through hydrogen bonds between adenine (A) and uracil (U) (RNA molecules do not contain thymine, but rather uracil) and hydrogen bonds between guanine (G) and cytosine (C), but can also form double strands through other base pairings not described herein. These double strands can be formed intermolecularly, i.e., between different RNA molecules, or intramolecularly, i.e., inside an RNA molecule. As explained above, the double-stranded region of RNA can contain many nucleotide building blocks (possibly hundreds or thousands), and thus contain a corresponding number of base pairs. In this case, as explained above, the term "dsRNA" is used. The double strands formed within the molecule are an important determinant of the complex structure formed inside the RNA. Complementary RNA and DNA molecules may also form double strands, which are called DNA:RNA hybrids or DNA:RNA heteroduplexes.

[0117] "Fully complementary" generally defines that two nucleic acid chains are capable of forming base pairs over their entire length and over the total number of nucleotide building blocks, thereby forming a duplex. As previously mentioned, functional siRNAs and other sRNAs have overhangs of one or more nucleotides at their ends. Similarly, dsRNAs may have overhangs of one or more nucleotides at their ends, or may have spacers (see definition above). In the meaning of the present invention, the term "fully complementary" refers to the nucleotide sequences of Group A, Group B, Group C, and Group D nucleic acids, respectively, but does not include these overhangs or spacers.

[0118] In the meaning of the present invention, "partial complementarity" accordingly defines nucleic acid chains that are not completely complementary and therefore cannot form base pairing over the entire length and over the full number of nucleotide building blocks. The double-stranded regions of these nucleic acids are interrupted by single-stranded regions such as mismatches, loops and / or bulges.

[0119] Particularly preferably, in group c, when the nucleic acid is an siRNA, its RNA double strand (duplex) consists of a completely complementary nucleic acid of group a and / or group b. Within the scope of the present invention, these are siRNAs newly identified by "eNA screening" and, according to the above definition, induce hydrolysis of the corresponding target RNA in a standardized and stringent in vitro cleavage assay in the corresponding AGO / RISC by 25% or more, preferably 50% or more, more preferably 75% or more, particularly preferably 90% or more, particularly preferably 95% or more of the amount used, and are therefore classified as esiRNA / ERNA (effective siRNA) and can be used as active agents in RNA silencing / RNAi plant protection methods against a variety of variable plant pathogens.

[0120] Also preferably, in group c, when the nucleic acid is an sRNA, such as an miRNA, its RNA duplex consists of partially complementary nucleic acids of group a and / or group b. These are sRNAs whose nucleotide sequences can be derived from the above-mentioned esiRNA / ERNA and can also be used as active agents against various variable plant pathogens in RNA silencing / RNAi plant protection methods.

[0121] According to group d, the nucleic acids of the present invention are double-stranded RNAs comprising the nucleotide sequences of at least two siRNAs or sRNAs of group c. Within the scope of the present invention, these are newly designed and constructed double-stranded ribonucleic acids (edsRNA, effectively double-stranded RNAs) comprising the nucleotide sequences of the identified esiRNAs / ERNAs or other sRNAs derived therefrom (e.g., miRNAs) and can be used as active agents in RNA silencing / RNAi plant protection methods against various variable plant pathogens.

[0122] In the meaning of the present invention, this means that the RNA duplex of these nucleic acids comprises the nucleotide sequences of at least two nucleic acids of group a, group b or group c.

[0123] In a preferred embodiment, the nucleic acid of group D comprises the nucleotide sequences of at least two small RNAs (sRNAs, such as small interfering RNAs (siRNAs), or microRNAs (miRNAs)), and the RNA duplex thereof consists of nucleic acids that are completely complementary or partially complementary to those of group A and / or group B. In a further preferred embodiment, the nucleic acid of group D comprises the nucleotide sequences of at least two siRNAs, and the RNA duplex thereof consists of nucleic acids that are completely complementary to those of group A and / or group B. In yet another preferred embodiment, the nucleic acid of group D comprises the nucleotide sequences of at least two sRNAs (e.g., miRNAs), and the RNA duplex thereof consists of nucleic acids that are partially complementary to those of group A and / or group B.

[0124] In a further preferred embodiment, the nucleic acid of group d comprises 2, 3, 4, 5 ... up to an unlimited number of siRNA and / or sRNA nucleotide sequences of group a, group b or group c. Preferably, the nucleic acid of group d comprises 2-100 siRNA and / or sRNA nucleotide sequences of group a, group b or group c. Particularly preferably, the nucleic acid of group d comprises 2-90, 2-80, 2-70, 2-60, 2-50, 2-40 or 2-30 siRNA and / or sRNA nucleotide sequences of group a, group b or group c. Specifically, preferably, the nucleic acid of group d comprises 2-20 or 2-10 siRNA and / or sRNA nucleotide sequences of group a, group b or group c. In another embodiment of the present invention, preferably, the nucleic acid of group d comprises more than 2 siRNA and / or sRNA nucleotide sequences of group a, group b or group c, i.e., at least 3, 4, 5, 6, 7, 8, 9, 10 or more (up to 100) sRNAs of group a, group b or group c.

[0125] According to group e, the nucleic acid is a single-stranded DNA comprising a sequence consisting of 12 or more nucleotides which is homologous to one of the nucleotide sequences of the single-stranded RNA of group a or group b (deoxyribonucleotides instead of ribonucleotides).

[0126] Sequence homology refers to the similarity of nucleotide or amino acid sequences due to the identical chemical building blocks of more or less substantial segments within the molecular chains of peptides, RNA, or DNA. If the molecular chains are identical, there is 100% sequence homology. In the context of the present invention, homology means that 12 or more nucleotides contained in a single-stranded DNA have 100% sequence homology with one of the single-stranded RNAs of Group A or Group B, where DNA is composed of deoxyribonucleotides and RNA is composed of ribonucleotides.

[0127] Group e comprises antisense deoxyribonucleic acid (DNA) oligonucleotides (ASOs) provided by the present invention, the sequences of which can be derived from esiRNA / ERNA identified within the scope of the present invention. Using a nomenclature similar to the term esiRNA / ERNA, ASOs derived from the sequences of the single-stranded RNA components of group a and group b esiRNA / ERNA are referred to as eASOs accordingly. eASOs comprise DNA sequences homologous to the single-stranded RNA components of group a and group b esiRNA / ERNA (i.e., deoxynucleotides replace non-ribonucleotides; thymidine replaces non-uridine), and therefore can also hybridize to the respective target sites on the a site of the target RNA through corresponding base pairing and exert activity. The mechanism of action of ASOs is similar to, but not identical to, sRNA. As described in detail above, ASOs bind to a fully or partially complementary region (target site) of the target RNA through base pairing (hybridization). However, the inactivation mode of ASOs on target RNAs does not occur through RNAi like sRNAs, but rather occurs through an antisense process, such as inhibition of translation or endonucleolytic degradation by RNase H-type RNases (see above).

[0128] According to the f group, nucleic acid of the present invention is a single-stranded DNA, which has the modification of the 1-7 position in the nucleotide sequence. Therefore, the nucleic acid of the f group represents the variant of the e group nucleic acid. In the meaning of the present invention, "modification" means "base substitution", i.e., the base selected from adenine (A), thymine (T), guanine (G) and cytosine (C) can be replaced by any other base. Such base substitutions can occur at the 1-7 position of the e group nucleic acid nucleotide sequence. Base substitutions are preferably independent of each other at each position, i.e., each of the 1-7 positions in the nucleotide sequence of the nucleic acid of the e group can be replaced independently of each other by any other base.

[0129] According to one aspect of the invention, the nucleic acid is a nucleic acid for protecting plants against the plant pathogen Cucumber Mosaic Virus (CMV).

[0130] Preferably, the nucleic acid for the protection against CMV is a nucleic acid for a target RNA for CMV. Particularly preferably, the nucleic acid for the protection against CMV is a nucleic acid for a target RNA for CMV, wherein the target RNA for CMV is selected from the target RNA with SEQ ID NO: 189 and 190.

[0131] Preferably, the nucleic acid against a target RNA of CMV selected from the group consisting of SEQ ID NOs: 189 and 190 is a ribonucleic acid or a deoxyribonucleic acid comprising or consisting of at least one nucleic acid selected from the group consisting of the nucleic acids having SEQ ID NOs: 1-92.

[0132] The target RNA of CMV having SEQ ID NO: 189 is also referred to below as “CMV RNA2.” The target RNA of CMV having SEQ ID NO: 190 is also referred to below as “CMV RNA3.”

[0133] As described in Example 1, nucleic acids were identified against CMV RNA 2 (SEQ ID NO: 189), most of which induced efficient hydrolysis of the target RNA (CMV RNA 2) in a cleavage (cleavage) assay (at least 25%, preferably at least 50%, more preferably at least 75%, particularly preferably at least 90%, very particularly preferably at least 95% of the original amount used in the assay in question) and protected plants from CMV infection (see also Figure 2-4 Screening was performed using AGO1 (L form; Gursinsky et al., 2015) and AGO2 from Nicotiana benthamiana (Nb).

[0134] As described in Example 1, nucleic acids against CMV RNA3 (SEQ ID NO: 190) were identified, some of which induced efficient hydrolysis of the target RNA (CMV RNA 3) in cleavage (cleavage) assays (at least 25% of the original amount used in the assay in question) and protected plants from CMV infection (see also Figure 5 and 6 Screening was performed using AGO1 (L form; Gursinsky et al., 2015) and AGO2 from Nicotiana benthamiana (Nb).

[0135] The nucleic acid against the target RNA (CMV RNA2) of CMV having SEQ ID NO: 189 is preferably a ribonucleic acid or a deoxyribonucleic acid comprising at least one nucleic acid selected from the nucleic acids having SEQ ID NO: 1 to 52, or consisting thereof. Particularly suitable nucleic acids against the target RNA (CMV RNA2) of CMV having SEQ ID NO: 189 are ribonucleic acids or deoxyribonucleic acids comprising at least one nucleic acid selected from the nucleic acids having SEQ ID NO: 1 to 4, 6 to 11, 14 to 17, 21 to 25, 27 to 30, 32 to 37, 40 to 43 and 47 to 51, or consisting thereof.

[0136] The nucleic acid identified in the screening using AGO1 against the target RNA of CMV having SEQ ID NO: 189 (CMV RNA 2) is preferably a ribonucleic acid or deoxyribonucleic acid comprising or consisting of at least one nucleic acid selected from the group consisting of nucleic acids having SEQ ID NOs: 1-12 and SEQ ID NOs: 27-38, preferably nucleic acids having SEQ ID NOs: 1-4, 6-11, 27-30 and 32-37.

[0137] The nucleic acid identified in the screening using AGO2 against the target RNA of CMV with SEQ ID NO: 189 (CMV RNA2) is preferably a ribonucleic acid or deoxyribonucleic acid comprising at least one nucleic acid selected from the group consisting of nucleic acids with SEQ ID NOs: 13-26 and SEQ ID NOs: 39-52, preferably with SEQ ID NOs: 14-17, 21-25, 40-43 and 47-51, or consists thereof.

[0138] The nucleic acid against the target RNA (CMV RNA3) of CMV having SEQ ID NO: 190 is preferably a ribonucleic acid or a deoxyribonucleic acid, which comprises or consists of at least one nucleic acid selected from the nucleic acids having SEQ ID NOs: 53-92.

[0139] The nucleic acid identified in the screening using AGO1 against the target RNA of CMV having SEQ ID NO: 190 (CMV RNA 3) is preferably a ribonucleic acid or deoxyribonucleic acid comprising at least one nucleic acid selected from the group consisting of nucleic acids having SEQ ID NOs: 53-55, 59, 63, 64, 66, 67, 70-75, 79, 83, 84, 86, 87, 90-92, preferably SEQ ID NOs: 53, 55, 59, 63, 64, 66, 70, 73, 75, 79, 83, 84, 86 and 90, or consists thereof.

[0140] The nucleic acid identified in the screening using AGO2 against the target RNA of CMV having SEQ ID NO: 190 (CMV RNA 3) is preferably a ribonucleic acid or deoxyribonucleic acid comprising at least one nucleic acid selected from the group consisting of nucleic acids having SEQ ID NOs: 56-58, 60-62, 65, 68, 69, 76-78, 80-82, 85, 88, 89, preferably SEQ ID NOs: 56-58, 60-62, 65, 69, 76-78, 80-82, 85 and 89, or consists thereof.

[0141] According to another aspect of the invention, the nucleic acid is a nucleic acid for protecting plants against the plant pathogen Meloidogyne incognita.

[0142] Preferably, the nucleic acid for protection against the incognita root-knot nematode is a nucleic acid for a target RNA of the incognita root-knot nematode. Particularly preferably, the nucleic acid for protection against the incognita root-knot nematode is a nucleic acid for a target RNA of the incognita root-knot nematode, wherein the target RNA of the incognita root-knot nematode is selected from the target RNAs having SEQ ID NO: 191, 192 and 193.

[0143] The target RNA of the incognita root-knot nematode having SEQ ID NO: 191 is also referred to as "SPF" hereinafter. The target RNA of the incognita root-knot nematode having SEQ ID NO: 192 is also referred to as "INT" hereinafter. The target RNA of the incognita root-knot nematode having SEQ ID NO: 193 is also referred to as "ACT" hereinafter.

[0144] As described in Example 3, nucleic acids against SPF (SEQ ID NO: 191) were identified, most of which induced efficient hydrolysis of the target RNA (SPF) in vivo (at least 25% of the original amount used in the assay in question) after nematode ingestion in a cleavage (cleavage) assay and protected plants from infection by the southern root-knot nematode (see also Figure 16 A and 16B). Screening was performed using the AGO2 protein from Nicotiana benthamiana (Nb).

[0145] As described in Example 3, nucleic acids were identified against INT (SEQ ID NO: 192), most of which induced efficient hydrolysis of the target RNA (SPF) in vivo after nematode ingestion in a cleavage (cleavage) assay (at least 25% of the original amount used in the assay in question) and protected plants from infection by the southern root-knot nematode (see also Figure 17 A and 17B). Screening was performed using the AGO2 protein from Nicotiana benthamiana (Nb).

[0146] As described in Example 3, nucleic acids were identified against ACT (SEQ ID NO: 193), most of which induced efficient hydrolysis of the target RNA (SPF) in vivo after nematode ingestion in a cleavage (cleavage) assay (at least 25% of the original amount used in the assay in question) and protected plants from infection by the southern root-knot nematode (see also Figure 17 A and 17B). Screening was performed using the AGO2 protein from Nicotiana benthamiana (Nb).

[0147] The nucleic acid against the target RNA of the incognita root-knot nematode selected from SEQ ID NOs: 191, 192 and 193 is preferably a ribonucleic acid or a deoxyribonucleic acid, which comprises or consists of at least one nucleic acid selected from the nucleic acids having SEQ ID NOs: 93-120.

[0148] The nucleic acid for the target RNA (SPF) of the southern root-knot nematode having SEQ ID NO: 191 is preferably a ribonucleic acid or a deoxyribonucleic acid, which comprises at least one nucleic acid selected from the nucleic acids having SEQ ID NO: 93-95 and 107-109, or consists of the same.

[0149] The nucleic acid for the target RNA (INT) of the incognita root-knot nematode having SEQ ID NO: 192 is preferably a ribonucleic acid or a deoxyribonucleic acid, which comprises or consists of at least one nucleic acid selected from the nucleic acids having SEQ ID NOs: 96-99 and 110-113.

[0150] The nucleic acid targeting RNA (ACT) against the incognita root-knot nematode having SEQ ID NO: 193 is preferably a ribonucleic acid or a deoxyribonucleic acid, which comprises or consists of at least one nucleic acid selected from the nucleic acids having SEQ ID NOs: 100-106 and 114-120.

[0151] According to another aspect of the invention, the nucleic acid is a nucleic acid for protecting plants against the plant pathogen Botrytis cinerea.

[0152] Preferably, the nucleic acid for protection against Botrytis cinerea is a nucleic acid against a target RNA of Botrytis cinerea. Particularly preferably, the nucleic acid for protection against Botrytis cinerea is a nucleic acid against a target RNA of Botrytis cinerea, wherein the target RNA of Botrytis cinerea is selected from the target RNAs having SEQ ID NO: 194, 195, 196, 197, 198 and 199.

[0153] The target RNA of Botrytis cinerea with SEQ ID NO: 194 is also referred to as "VDS" hereinafter. The target RNA of Botrytis cinerea with SEQ ID NO: 195 is also referred to as "DCTN" hereinafter. The target RNA of Botrytis cinerea with SEQ ID NO: 196 is also referred to as "SAC" hereinafter. The target RNA of Botrytis cinerea with SEQ ID NO: 197 is also referred to as "ERG" hereinafter. The target RNA of Botrytis cinerea with SEQ ID NO: 198 is also referred to as "EF" hereinafter. The target RNA of Botrytis cinerea with SEQ ID NO: 199 is also referred to as "CHS" hereinafter.

[0154] As described in Example 4, nucleic acids were identified against VDS (SEQ ID NO: 194), most of which induced efficient hydrolysis of the target RNA (VDS) in cleavage (cleavage) assays (at least 25% of the original amount used in the assay in question) and protected plants from infection with Botrytis cinerea (see also Figure 18 、 Figure 19A and Figure 19B The screening was performed using the AGO1 protein of Colletotrichum graminicula (Cg).

[0155] As described in Example 4, nucleic acids were identified against DCTN (SEQ ID NO: 195), most of which induced efficient hydrolysis of the target RNA (DCTN) in cleavage (cleavage) assays (at least 25% of the original amount used in the assay in question) and protected plants from infection with Botrytis cinerea (see also Figure 18 、 Figure 19A and Figure 19B The screening was performed using the AGO1 protein of Colletotrichum graminearum (Cg).

[0156] As described in Example 4, nucleic acids were identified against SAC (SEQ ID NO: 196), most of which induced efficient hydrolysis of the target RNA (SAC) in cleavage (cleavage) assays (at least 25% of the original amount used in the assay in question) and protected plants from infection with Botrytis cinerea (see also Figure 18 、 Figure 19A and Figure 19B The screening was performed using the AGO1 protein of Colletotrichum graminearum (Cg).

[0157] As described in Example 4, nucleic acids were identified against ERG (SEQ ID NO: 197), most of which induced efficient hydrolysis of the target RNA (ERG) in cleavage (cleavage) assays (at least 25% of the original amount used in the assay in question) and protected plants from infection with Botrytis cinerea (see also Figure 18 、 Figure 19A and Figure 19B The screening was performed using the AGO1 protein of Colletotrichum graminearum (Cg).

[0158] As described in Example 4, nucleic acids were identified against EF (SEQ ID NO: 198), most of which induced efficient hydrolysis of the target RNA (ERG) in a cleavage (cleavage) assay (at least 25% of the original amount used in the assay in question) and protected plants from infection with Botrytis cinerea (see also Figure 18 、 Figure 19A and Figure 19B The screening was performed using the AGO1 protein of Colletotrichum graminearum (Cg).

[0159] As described in Example 4, nucleic acids were identified against CHS (SEQ ID NO: 199), most of which induced efficient hydrolysis of the target RNA (CHS) in cleavage (cleavage) assays (at least 25% of the original amount used in the assay in question) and protected plants from infection with Botrytis cinerea (see also Figure 18 、 Figure 19A and Figure 19B The screening was performed using the AGO1 protein of Colletotrichum graminearum (Cg).

[0160] The nucleic acid against a target RNA of Botrytis cinerea selected from the group consisting of SEQ ID NOs: 194, 195, 196, 197, 198 and 199 is preferably a ribonucleic acid or deoxyribonucleic acid comprising or consisting of at least one nucleic acid selected from the group consisting of nucleic acids having SEQ ID NOs: 121 to 180, preferably SEQ ID NOs: 124, 126-132, 134-138, 140-150, 154, 156-162, 164-168 and 170-180.

[0161] The nucleic acid against the target RNA (VDS) of Botrytis cinerea having SEQ ID NO: 194 is preferably a ribonucleic acid or a deoxyribonucleic acid comprising or consisting of at least one nucleic acid selected from the group consisting of nucleic acids having SEQ ID NOs: 121, 122, 151 and 152.

[0162] The nucleic acid against the target RNA (DCTN) of Botrytis cinerea having SEQ ID NO: 195 is preferably a ribonucleic acid or deoxyribonucleic acid comprising or consisting of at least one nucleic acid selected from the group consisting of the nucleic acids having SEQ ID NOs: 123-131 and 153-161, preferably selected from the group consisting of SEQ ID NOs: 124, 126-131, 154 and 156-161.

[0163] The nucleic acid against the target RNA (SAC) of Botrytis cinerea having SEQ ID NO: 196 is preferably a ribonucleic acid or deoxyribonucleic acid comprising or consisting of at least one nucleic acid selected from the group consisting of nucleic acids having SEQ ID NOs: 132-140 and 162-170, preferably selected from the group consisting of SEQ ID NOs: 132, 134-138, 140, 162, 164-168 and 170.

[0164] The nucleic acid against the target RNA (ERG) of Botrytis cinerea having SEQ ID NO: 197 is preferably a ribonucleic acid or deoxyribonucleic acid comprising or consisting of at least one nucleic acid preferably selected from the nucleic acids having SEQ ID NOs: 141-149 and 171-179.

[0165] The nucleic acid against the target RNA (EF) of Botrytis cinerea having SEQ ID NO: 198 is preferably a ribonucleic acid or deoxyribonucleic acid comprising or consisting of at least one nucleic acid preferably selected from the nucleic acids having SEQ ID NOs: 150 and 180.

[0166] In another aspect of the invention, the nucleic acid is a double-stranded RNA, which preferably contains a nucleotide sequence consisting of a so-called "pseudo-siRNA sequence" and at least two and up to an unlimited number of sequences of sRNAs of group C. Preferably, the double-stranded RNA comprises a nucleotide sequence consisting of a pseudo-siRNA sequence and 2-10,000 sequences of sRNAs of group C. Particularly preferably, the double-stranded RNA comprises a nucleotide sequence consisting of a pseudo-siRNA sequence and 2-5,000 or 2-2,500 sequences of sRNAs of group C. Very particularly preferably, the double-stranded RNA comprises a nucleotide sequence consisting of a pseudo-siRNA sequence and 2-1,000, 2-500, 2-250, 2-100 or 2-50 sequences of sRNAs of group C. As described above and in Example 2, these are nucleic acids of group D, "edsRNA".

[0167] Within the meaning of the present invention, what are called "pseudo-siRNA sequences" or "pseudo-siRNAs" are double-stranded ribonucleotide sequences of any composition, which, depending on the intended processing of the respective edsRNA by the Dicer enzyme or DCL, are 21, 22, 23 or 24 nt in length (see also Example 2). The name "pseudo-siRNA sequence" or "pseudo-siRNA" was chosen to indicate the fact that these are double-stranded ribonucleotide sequences that are constructed to resemble siRNA, but do not actually function as siRNA, but rather have other functions as described below. Pseudo-siRNA sequences are placed at the ends of the double-stranded RNA (edsRNA) designed according to the invention. Their presence forces a "synchronous processing" of the Dicer or DCL that is active on this RNA. As described in detail in Example 2, synchronous processing means that, depending on the position and length of the pseudo-siRNA sequence, and accordingly on the activity of the Dicer / DCL involved, the endonucleolytic cleavage of the double-stranded RNA occurs in such a way that the sRNA preferably produced in this process has the same length as the pseudo-siRNA. According to the invention, it has been demonstrated that this occurs in this way ( Figure 14 ). In addition, functional elements within the meaning of the present invention can be incorporated into the pseudo-siRNA sequence: these can be elements that are important for the transcription and processing of the RNA of interest. For example, these can be regions of transcription promoters or terminators, but they can also be transport signals or parts of ribozymes that generate the correct 5' or 3' end of the RNA of interest by self-splicing (autocatalytic cleavage).

[0168] A transcription promoter is a signal sequence in a double-stranded DNA, one of which (the template strand) encodes an RNA molecule. The transcription promoter is recognized by the DNA-dependent RNA polymerase complex, and due to binding to the promoter sequence, the RNA polymerase complex is able to initiate transcription (synthesis) of an RNA molecule whose nucleotide sequence is complementary to the DNA template strand. Transcription activators that bind to the RNA polymerase complex and / or other DNA sequences can specifically induce transcription. Examples of transcription promoters include viral promoters, such as the promoter of T7 bacteriophage (T7 promoter). Examples of cellular promoters include Pol I and Pol II promoters. Other common viral promoter examples include: T3 promoter (promoter of T3 bacteriophage), SP6 promoter (promoter of SP6 bacteriophage), CMV promoter (promoter of human cytomegalovirus). Other common cellular promoter examples include: GAL promoter, LAC4 promoter, actin promoter, Pol III promoter. Those skilled in the art are familiar with other suitable transcription promoters.

[0169] Similar to transcription promoters, transcription terminators are nucleotide sequences encoded by the DNA encoding RNA. When these sequences are transcribed by the RNA polymerase complex, protein-RNA complexes are formed, which cause the RNA polymerase complex to terminate transcription. An example of a transcription terminator is the transcription terminator of vesicular stomatitis virus (VSV). Other suitable transcription terminators are well known to those skilled in the art.

[0170] Transport signals in RNA molecules need to be bound to proteins that enable targeted transport of these RNA molecules into or out of specific cellular compartments. An example is a nuclear RNA export signal that enables RNA molecules to be exported from the nucleus into the cytoplasm. Other transport signals are well known to those skilled in the art.

[0171] Ribozymes are catalytically active RNA molecules that catalyze chemical reactions like enzymes. Examples include hammerhead (HH) ribozymes (Meyer and Masquida 2014) or hepatitis D virus (HDV) ribozymes (Avis et al. 2012). These ribozymes are able to independently catalyze the endonucleolytic cleavage (self-cleavage or self-splicing) of the RNA molecules of which they are a component.

[0172] Therefore, the structural rationale for edsRNA designed within the meaning of the present invention is as follows (see also Figures 8-11 ):

[0173] The sequence contains at least one pseudo-siRNA sequence, which, as mentioned above, can achieve synchronous processing by Dicer / DCL. The sequence of edsRNA also contains some "continuous" esiRNA / ERNA from 5' to 3' end or other sRNA (such as miRNA) derived from esiRNA / ERNA. These sequences can be derived from different "eNA screenings", and accordingly, esiRNA / ERNA or sRNA derived therefrom can be active at different AGO / RNA. Therefore, the esiRNA / ERNA or sRNA derived therefrom constituting edsRNA can be directed against different target RNAs derived from one or different organisms.

[0174] EdsRNA can be produced in two ways: by two independently transcribed complementary RNA molecules, or by a single transcribed RNA molecule containing two complementary segments ( Figure 8 ). In the latter case, the two complementary segments of the transcribed RNA are connected to each other by a spacer and form a hairpin. The spacer is a sequence of any composition with a minimum length of 4 nucleotides (see definition above), but for the specific purpose of edsRNA construction, it can include functional regions, such as ribozymes (such as HH or HDV ribozymes), transcription promoters or transcription terminators, transport signals or splice sites.

[0175] Splice sites are sequence motifs in regions of precursor RNA molecules, such as introns, which are recognized by the cell's splicing machinery. The splicing machinery catalyzes the complete or partial removal of intronic sequences. In addition to the function of connecting the two complementary single-stranded components of dsRNA, spacer sequences also have other roles due to the presence of these elements: for example, if splice sites are included, spacers can be truncated by the cell's splicing machinery during RNA expression in vivo. Unlike double-stranded RNA regions, spacers are sensitive to ribonucleases present in the cell, such as the single-strand-specific RNases T1 or A, and therefore the sequence can also be completely removed by these RNases (see also ). Figure 8 ). Examples of spacers are sequences from introns of mRNA precursor molecules ("pre-mRNA"), which contain all the recognition sequences required for splicing. These recognition sequences are well known to those skilled in the art.

[0176] RNA transcription can occur in vitro or in vivo. Double strands are obtained by hybridization of complementary RNA strands ( Figure 8 ). Transcription can be carried out by a variety of promoters (see e.g. Figure 11 ). Transcription termination can be carried out by any type of transcription terminator (such as the vesicular stomatitis virus (VSV) transcription terminator, see e.g. Figure 11 ).

[0177] Depending on how it is produced, edsRNAs can be blunt-ended or contain overhangs. ( Figure 15 ). It can be generated in various ways, for example by "run-off transcription" ("disconnecting the RNA polymerase complex from the DNA template"), or by terminating the associated RNA polymerase complex through transcription terminators, or by utilizing the self-splicing activity of ribozymes.

[0178] The sequences of the two strands are designed so that during DCL processing, each true sRNA guide strand and passenger strand sequence is generated. The presence of the pseudo-siRNA sequence and the associated "synchronous processing" by DCL / Dicer ensure that processing primarily produces constitutive esiRNA / ERNA or sRNA derived therefrom (see Examples 2 and Figures 8-14 ).

[0179] Thus, in one embodiment of the present invention, the double-stranded RNA of the present invention may have blunt ends, while in another embodiment, it may have overhanging ends.

[0180] In another embodiment of the present invention, the double-stranded RNA may comprise a spacer. In yet another embodiment of the present invention, the pseudo-siRNA and / or spacer in the nucleic acid of the present invention comprises an element selected from the group consisting of a transcription promoter, a transcription terminator, a transport signal, a splice site, and a ribozyme.

[0181] In a particularly preferred embodiment of the present invention, the double-stranded RNA of the present invention is selected from the nucleic acids having SEQ ID NOs: 181, 182, 185, 186 and 200-204.

[0182] According to the present invention, the functionality of this class of novel edsRNAs has been demonstrated, as well as significantly enhanced protection against pathogens compared to conventional dsRNAs (see, e.g., Figure 14 and Figure 15 ).

[0183] In another embodiment, the nucleic acids of the invention have one or more chemical modifications, wherein the chemical modifications are selected from conjugates such as GalNac; base modifications such as 5-methylcytosine; 2' sugar modifications such as 2'-O-methyl, 2'-fluoro, 2'-O-methoxyethyl (2'-MOE), cETBNA (bicycloethyl (S)-linked); other sugar modifications such as "locked" (LNA) or "unlocked" (UNA); "backbone" modifications such as phosphorothioate (PS) or "peptide nucleic acid" (PNA), and sugar-phosphate modifications such as morpholino / PMO (phosphodiamidate morpholino). Other modifications are well known to those skilled in the art.

[0184] In another aspect, the present invention relates to a composition comprising at least one, and optionally a plurality, of the nucleic acids of the invention as described herein.

[0185] The nucleic acids of the present invention can be used in transgenic form, for example, for the HIGS method for pathogen control, or for targeted transcription and post-transcriptional regulation of gene expression. They are particularly suitable for pathogen control in plants / crops. Therefore, in one embodiment, the present invention relates to the use of nucleic acids of the present invention or compositions in plants for preventing and / or treating infestation and / or infection by pathogens, particularly for preventing and / or treating infestation and / or infection by pathogens selected from cucumber mosaic virus, southern root-knot nematode and botrytis cinerea.

[0186] Transgenic means that the genetic information encoding at least one, optionally multiple, nucleic acid of the present invention is stably introduced into the genome of a host organism, preferably a plant, a microorganism or one of the mentioned pathogens, and is produced (expressed) in the organism in an inductive or non-inductive manner via a corresponding promoter. If the organism is a plant, the plant is thus able to develop resistance to invasion and / or infection by one or more of the corresponding pathogens. If the organism is a microorganism such as a bacterium or yeast, the corresponding nucleic acid can be produced in these microorganisms.

[0187] The nucleic acids of the present invention can also be used in transient (non-transformed) forms, for example, for the SIGS method for pathogen control, or for targeted transcription and post-transcriptional regulation of gene expression. They are particularly suitable for pathogen control in plants / crops. Therefore, in one embodiment, the present invention relates to the use of nucleic acids or compositions of the present invention in plants for preventing and / or treating infestation and / or infection by pathogens, particularly for preventing and / or treating infestation and / or infection by pathogens selected from CMV, southern root-knot nematode and Botrytis cinerea.

[0188] In topical / transient applications, one or more nucleic acids of the invention are applied to target organs of plants, such as leaves, stems, or roots. These nucleic acids are then taken up by the plant, for example to inhibit the replication of infectious viruses, or attacking (infectious) pathogens (such as nematodes and fungi) take up nucleic acids through the surface of the plant to be protected, whereupon these nucleic acids are active against essential target RNAs of the pathogen in the RNA silencing / RNAi or RNA silencing / antisense mechanisms of the relevant organism.

[0189] In one embodiment, the present invention accordingly relates to a composition comprising at least one nucleic acid according to the invention and optionally one or more carrier substances and / or auxiliaries, said composition being suitable for application in / on plants.

[0190] The composition is preferably a solution, which can be applied in direct form, for example as a nutrient solution or an aerosol / spray. This makes it particularly easy to prevent or treat plant / crop diseases.

[0191] Preferably, the composition comprises at least one physiologically compatible carrier, diluent, and / or adjuvant. The nucleic acid of the present invention can be contained in a pharmaceutically compatible carrier, for example, in a conventional medium such as an aqueous saline medium or a buffered solution, as an aerosol / spray composition. Such a medium may also contain conventional adjuvants, such as salts for regulating osmotic pressure, buffers, preservatives, nanoparticles, etc.

[0192] Other suitable compatible carrier substances are known to those skilled in the art, for example, from Remington's Practice of Pharmacy, 13th edition, and J. of. Pharmaceutical Science & Technology, vol. 52, no. 5, Sept-Oct., pages. 238-311.

[0193] In a particularly preferred embodiment of the present invention, the nucleic acids of the invention are used in a transgenic or transient form in an RNA silencing / RNAi method, wherein the nucleic acids of the invention are in the form of double-stranded RNA molecules of group d, characterized in that they are constructed according to claims 8 to 12 and processed by Dicer or a Dicer-like enzyme to obtain the relevant sRNA. This use is described in Example 2.

[0194] The problems listed are solved by the invention described below and by the described embodiments.

[0195] Newly identified nucleic acid active agents, esiRNA / ERNA and sRNA and eASO (eNA) derived therefrom, can be used in plants as active agents for protection against the pathogens cucumber mosaic virus, southern root-knot nematode or Botrytis cinerea in various applications (RNA silencing / RNAi or RNA silencing / antisense approaches).

[0196] The present invention also relates to the construction of double-stranded ribonucleic acids (edsRNA (effective double-stranded nuclear RNA)), which construct the identified esiRNA / ERNA and / or sRNA derived therefrom and can be used as active agents in RNA silencing / RNAi methods to provide protection in plants against the above-mentioned pathogens.

[0197] The present invention will be described in more detail below with reference to 19 drawings, 8 tables and 4 examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0198] Figure 1: Effective esiRNAs / ERNAs and edsRNAs. Left: Schematic diagram of the RNA silencing / RNAi process, in which a natural siRNA pool, for example, is generated by viral dsRNA through DCL activity: the siRNA pool contains only a small amount of esiRNAs / ERNAs (marked with "e"); therefore, RNA silencing / RNAi is inefficient. Center: RNA silencing / RNAi using esiRNAs / ERNAs identified through "eNA screening": RNA silencing / RNAi is effective. Right: Sequence information of the identified esiRNAs / ERNAs is used to generate edsRNAs; RNA silencing / RNAi using these edsRNAs and the esiRNAs / ERNAs generated therefrom is also effective. DCL - Dicer-like protein; AGO - argonaute protein; RISC - RNA-induced silencing complex. For conventional siRNAs, the siRNA guide strand is highlighted in black; for esiRNAs / ERNAs, it is highlighted in red.

[0199] Figure 2: Cleavage assay for CMV RNA 2 (SEQ ID NO: 189) performed with identified esiRNA / ERNA. As described in the Examples, "eNA screening" was performed with CMV RNA 2 (Fny type, here a double-stranded version of RNA). In the final step shown here, the obtained siRNA candidates were subjected to a cleavage assay. It should be noted that, as shown here and in all the cleavage assays shown below, all tested siRNAs were directed against the target RNA in the (+) direction. In the presence of the siRNA to be tested, AGO1 or AGO2 mRNA was translated in BYL (Gago-Zachert et al., 2019). Therefore, the RISC formed according to the method is programmed by the siRNA to be characterized, and the endonucleolytic hydrolysis of a given amount of radiolabeled CMV RNA 2 (target RNA) is detected by gel electrophoresis and autoradiography. The asterisk (*) marks the cleavage product of the target RNA produced by the cleavage activity of AGO / RISC. Cleavage efficiency can be quantified by autoradiography, comparing the amount of target RNA initially used with a negative control and the cleavage products formed (Image OutTL; also see Table 1). As described herein, and as shown in all figures below, a newly established standardized and stringent form of cleavage assay was used: each siRNA to be tested (10 nM) was subjected to AGO / RISC formation (also see Examples) in the presence of a 10-fold excess (100 nM) of nonspecific competing siRNA ("siR gf698" directed against the mRNA of green fluorescent protein (GFP)). This standardized and stringent type of cleavage assay was used to ultimately define esiRNA / ERNA: in a cleavage assay performed in this manner, siRNAs that induced AGO / RISC-mediated hydrolysis of at least 25% of the target RNA initially used in the assay were designated as effective and were esiRNA / ERNA (also see Table 1). (A) siRNA candidates from "eNA screening" were tested with AGO1 / RISC. (B) siRNA candidates from "eNA screening" were tested with AGO2 / RISC. All identified esiRNAs / ERNAs are summarized in Table 1. (C) Schematic representation of the binding sites of the most potent siRNAs on CMV RNA 2. Protein coding regions are indicated by gray boxes. The numbers of the siRNA candidates correspond to the names given in the figures, tables, and text (abbreviated as siR or siRCV2).

[0200] Figure 3: Characterization of the protective efficacy of CMV RNA 2-specific esiRNA / ERNA in plants. 4-5 week-old Nicotiana benthamiana plants were mechanically inoculated with genomic CMV RNAs 1, 2, and 3 generated from infectious cDNA, as well as individual synthetic siRNA candidates, and monitored for the appearance of CMV-specific symptoms over 35 days (dpi = days post-inoculation). (A) Representative images of individual plants show the difference between asymptomatic and symptomatic plants 35 days after inoculation. The number of plants that remained asymptomatic is indicated. (B) The graph shows the percentage of asymptomatic plants throughout the experiment: the trend for AGO1-specific siRNA is shown in black, and the AGO2-specific siRNA and siR gf698 control are shown in gray. The results are from three independent plant experiments. The number of plants used is indicated (n=12-15). As shown, esiRNA / ERNA is very effective in protecting plants against CMV infection. (C) An agarose gel of RT-PCR used to detect viral infection at the RNA level is shown. Leaf discs from asymptomatic and symptomatic plants were collected at 35 days post-infection, RNA was extracted, and cDNA was synthesized using reverse transcriptase. PCR was then performed to detect conserved sequences within Cucumber mosaic virus RNAs 1, 2, and 3. CMV-specific sequences were amplified using plasmids containing the cDNA sequences of the corresponding viral RNAs (positive control) and samples from symptomatic plants. As can be seen, no PCR products derived from CMV RNA were obtained from the cDNA of asymptomatic plants. Therefore, RT-PCR confirmed the visual classification of symptomatic and asymptomatic plants. Numbers correspond to the plant numbers used in the infection experiment. (–)RT assays (no reverse transcriptase added during the reaction) and "water controls" (water added instead of cDNA during the PCR reaction) served as negative controls. (M = GeneRuler 100bp DNA ladder, Thermo Scientific). The numbers of the siRNA candidates correspond to the designations given above and below in the figures, tables, and text (abbreviated as siR or siRCV2).

[0201] Table 1. esiRNA / ERNA candidates targeting CMV RNA 2 (SEQ ID NO: 189). Listed are 21nt-long siRNAs from CMV RNA 2 identified within the "eRNA Screen." The corresponding single-stranded guide strands and complementary passenger strands are listed. The siRNAs are designated siRCV (CV stands for CMV) based on their respective target RNAs (CMV RNA 2) and the position of the identified guide strand's 5' end. As previously described, siRNAs were classified using cleavage assays: the percentage of esiRNA / ERNA cleavage of the corresponding target RNA in standardized and stringent cleavage assays is given. EsiRNAs / ERNAs identified in the "eRNA Screen" according to the definitions (see text) are highlighted in bold: black for selected AGO1; gray for selected AGO2. Other siRNAs identified in the screen that did not meet the esiRNA / ERNA definition are shown in regular font. Some of these siRNAs (such as siRCV21844 and 2634) were used as negative controls in the Nicotiana benthamiana infection experiments and are therefore listed here. For the siRNAs tested in planta, the efficacy of the Nicotiana benthamiana protection experiment is given (na = not available); the proportion of symptom-free plants 35 days after infection is given. Patent protection is accordingly preferred for the siRNAs highlighted in bold and their variants (see Table 6), as are esiRNA / ERNA or sRNAs derived therefrom or eASO (eNA) formats for use against CMV. The corresponding SEQ ID NOs are given and listed separately as "Appendix to Table 1."

[0202] Figure 4 . Efficacy of 21nt and 22nt long esiRNA / ERNA against CMV RNA 2 in vitro and in planta. (A) Cleavage assay using 21nt long esiRNA / ERNA and its derived 22nt long variant examples ( Figure 2(A standardized and stringent format as described). As shown, both variants have high cleavage activity. In particular, the cleavage activity of AGO1 and AGO2 in combination with 21nt-long esiRNA / ERNA is slightly higher than that of 22nt-long esiRNAs / ERNA. In contrast, the cleavage activity of AGO2 in combination with 21nt or 22nt-long esiRNA / ERNA is minimally different. Asterisks (*) mark the cleavage products produced by the cleavage activity of AGO / RISC. (B) Comparison of the protective effect of 21nt and 22nt-long siRNAs (examples) in plants. 4-5 week-old Nicotiana benthamiana plants were mechanically co-inoculated with 21nt and 22nt-long synthetic esiRNA / ERNA and genomic CMV RNA. The percentage of symptom-free plants is shown (dpi = days after inoculation). Both forms of esiRNA / ERNA have significant antiviral effects. (C) Representative plant images 28 days after mechanical co-inoculation. The percentage of symptom-free plants for each siRNA is given. 9 plants were used for each CMV-specific siRNA and 3 plants were used for each siR gf698 control. All other controls were referenced to Figure 3 The numbers of the siRNA candidates correspond to the names given above and below in the figures, tables and text (abbreviated as siR or siRCV).

[0203] Figure 5 Cleavage assay of esiRNA / eRNA identified against CMV RNA 3 (SEQ ID NO: 190). As described in the examples of the present application, "eNA screening" was performed against the double-stranded form of CMV RNA 3 (Fny type), and in the final step, a cleavage assay was performed on the obtained siRNA candidates (see also Figure 2). In BYL, AGO1 or AGO2 mRNA is translated in the presence of the siRNA to be tested. Thus, the respective RISC is programmed by the siRNA to be characterized, and endonucleolytic hydrolysis of radiolabeled CMV target RNA 3 is detected by agarose gel electrophoresis and autoradiography. Asterisks (*) mark cleavage products resulting from the cleavage activity of AGO / RISC. A standardized and stringent form of the cleavage assay is used to definitively define esiRNAs / ERNAs: in the cleavage assay performed in this manner, siRNAs that induce AGO / RISC-mediated hydrolysis of at least 25% of the amount of target RNA initially used in the assay are designated as potent and are esiRNAs / ERNAs (see also Table 2). (A) siRNA candidates from the AGO1 screen. (B) siRNA candidates from the AGO2 screen. All identified esiRNAs / ERNAs are summarized in Table 2. (C) Schematic diagram of the binding sites of the most potent esiRNAs / ERNAs on CMV RNA 3. Protein coding regions are indicated by gray boxes. The numbers of the siRNA candidates correspond to the designations given above and below in the figures, tables and text (abbreviated as siR or siRCV).As shown and described, a close correlation between in vitro RNA cleavage activity and in vivo antiviral activity was observed.

[0204] Table 2. esiRNA / ERNA candidates against CMV RNA 3 (SEQ ID NO: 190). 21nt long siRNAs from CMV RNA 3 identified within the "eRNA screen" are listed. The corresponding single-stranded guide strand and complementary passenger strand are listed. siRNAs corresponding to the respective target RNA (CMV RNA 3) and the position of the identified guide strand 5' end are named siRCV (CV stands for CMV). As previously described, siRNAs were classified using cleavage assays: the percentage of esiRNA / ERNA cleavage of the corresponding target RNA in standardized and stringent cleavage assays is given. According to the definitions (see above and Figure 2) The esiRNAs / ERNAs identified in the "eNA Screen" are highlighted in bold: black for selected AGO1; gray for selected AGO2. Other siRNAs identified in the screen that did not meet the esiRNA / ERNA definition are shown in regular font. Some of these siRNAs (e.g., siRCV32061, 1132) were used as negative controls in the Nicotiana benthamiana infection assay. For siRNAs tested in planta, the efficacy in the Nicotiana benthamiana protection assay is given (na = not available): the proportion of symptom-free plants 35 days post-infection is given. Patent protection is accordingly preferred for the siRNAs highlighted in bold and their variants (see Table 6), esiRNAs / ERNAs or sRNAs or eASO (eNA) derived therefrom for use against CMV. The corresponding SEQ ID NOs are given and listed separately as "Appendix to Table 2." As shown and described, a correlation between in vitro RNA cleavage activity and in vivo antiviral activity was observed. However, this correlation was less pronounced than for the esiRNAs / ERNAs identified against CMV RNA 2 (see also the text).

[0205] Figure 6 Characterization of the protective efficacy of esiRNA / ERNA against CMV RNA 3 in plants. 4-5 week old Nicotiana benthamiana plants were mechanically inoculated with genomic CMV RNA and individual synthetic esiRNA / ERNA (see Figure 3 ) and monitored for the appearance of CMV-specific symptoms over 28 days (dpi = days post inoculation). (A) The graph shows the percentage of symptom-free plants during the experimental period. The trend for AGO1-specific esiRNA / ERNA is shown in black, and the trend for AGO2-specific esiRNA / ERNA and siR gf698 control is shown in gray. (B) Representative plant images showing the difference between symptom-free and symptomatic plants 28 days after inoculation. Results are from three independent plant experiments. The percentage of symptom-free plants and the number of plants used are given (n = 9-15). All other controls are similar to Figure 3 The numbers of the siRNA candidates correspond to the names given above and below in the figures, tables and text (abbreviated as siR or siRCV).

[0206] Figure 7. Comparison of the target sites of CMV (Fny)-specific esiRNA / ERNA in RNA 2 with the corresponding potential target sites of these esiRNA / ERNA in RNA 2 molecules of other CMV strains. (A) This figure ("Phylogenetic Tree") summarizes exemplary CMV strains and the specific subgroups (II, IB, IA) to which they belong. The virus Fny from subgroup IA used in the screening is highlighted with an arrow (modified according to Roossinck, 1999). (B) An overview shows exemplary AGO1-specific (top) and AGO2-specific (bottom) esiRNA / ERNAs from CMV RNA 2 and their sequence matches with the target sites in the target RNAs of different CMV strains. The "expectation" value is an indicator of the mismatch between the siRNA guide strand and the complementary target site. The higher the value, the lower the complementarity of the siRNA with the target site in CMV RNA 2 of the CMV strain of interest (https: / / www.zhaolab.org / psRNATarget / help#maxexpectation). The listed esiRNA / ERNA therefore has a very high complementarity with the genomic RNA of other CMV strains.

[0207] Table 3. CMV (Fny) specific esiRNA / ERNA for RNA 2 and 3 fully matches RNA 2 and 3 (complete match of corresponding target sites) of other CMV strains. (A) Table lists esiRNA / ERNA identified from CMV (Fny) (black is from AGO1 screening; gray is from AGO2 screening), and its target site fully matches the potential target site of the selected CMV strain of IA subgroup (also referring to Figure 7). Numbering corresponds to the corresponding siR or siRCV in the previous figures and tables. (B) Table lists esiRNA / ERNA identified from CMV (Fny) (as highlighted above), and its target site fully matches the potential target site of the selected CMV strain of IB subgroup. The esiRNA / ERNA identified from CMV (Fny) is not fully matched to the potential target site in RNA 2 and 3 of the selected CMV strain of II subgroup (see Table 6). The numbering of siRNA candidates corresponds to the names given above and below in the figures, tables and text (abbreviated as siR or siRCV). The information is similar for other sRNAs and eASOs whose sequences are derived from these esiRNAs / ERNAs.

[0208] Table 4. is for the protection potential of CMV 2RNA.The esiRNA / ERNA that highlights with (+) has the target site of complete complementarity in the genomic RNA molecule of selected CMV strain.According to the data of Fny strain, use the grey highlighting that identifies from CMV RNA2 and the esiRNA / ERNA that highlights with (+) can realize the special broad spectrum protection that infects for CMV, because each in these esiRNA / ERNA has the target site of complete complementarity in the 5-6 CMV strain that is selected for comparison.Therefore, in protection experiment, the combination of different esiRNA / ERNA makes it possible to provide protection for all CMV strains (and other representative strains of these subgroups possible) of IA and IB subgroup considered herein.Correspondingly change esiRNA / ERNA, promptly replace one or more (see Table 6) in shown nucleotide and also make it possible that the esiRNA / ERNA of identification can be used for the strain for II subgroup.Information is similar for other sRNA and eASO that sequence is derived from these esiRNA / ERNA.

[0209] Table 5. is for the protection potential of CMV 3RNA.The siRNA with (+) highlights has the target site of complete complementarity in selected CMV strain.Use the grey highlighting from CMV RNA 3 identification and with (+) highlighting esiRNA / ERNA can realize to infect special broad spectrum protection for CMV, because each in these esiRNA / ERNA has the target site of complete complementarity in the 5-6 CMV strain being selected for comparison.Therefore, in protection experiment, the combination of different esiRNA / ERNA makes it possible to provide protection for all CMV strains (and other representative strains of these subgroups possible) of IA and IB subgroup considered herein.Correspondingly change esiRNA / ERNA, promptly replace one or more (see Table 6) in shown in nucleotide and also make that the esiRNA / ERNA of identification can be used for the strain for II subgroup.Information is similar to other sRNA and eASO that sequence is derived from these esiRNA / ERNA.

[0210] Table 6. Matching of target sites of CMV(Fny)-specific esiRNAs / ERNAs with potential target sites of these esiRNAs / ERNAs in genomic RNAs of other CMV strains. (A) The table shows esiRNAs / ERNAs identified from CMV(Fny) (numbered corresponding to the corresponding siR or siRCV in the previous figure and table) whose target sites partially match those of selected CMV strains from subgroup IA. Respective mismatches are given in parentheses. (B) The table shows esiRNAs / ERNAs identified from CMV(Fny) whose target sites partially match those of selected CMV strains from subgroups IB and II. Specific binding of sRNAs to the target RNA's target site is guaranteed with up to five mismatches; however, in cases where the complementary RNA strands have higher melting points, binding specificity is generally maintained even with at least two more mismatches (Liu et al., 2014). As shown in Table 6, except for the four strains with a large number of mismatches, all other strains can still provide potential protection in RNA silencing methods through the identified esiRNA / ERNA, even if there is a mismatch of 7 with the target site. Based on this, the scope of protection of the claims (see below) supports eNAs with modifications at positions 1-7. The candidate numbers correspond to the names given above and below in the figures, tables and text (abbreviated as siR or siRCV). This information has similar applicability for other sRNAs and eASOs whose sequences are derived from these esiRNAs / ERNAs. The information is similar for other sRNAs and eASOs whose sequences are derived from these esiRNAs / ERNAs.

[0211] Figure 8 .Schematic diagram of the organizational form of edsRNA. EdsRNA is produced by: (a) hybridization of two different complementary RNA molecules; or (b) hybridization of complementary regions of one RNA molecule. In the case of (b), the RNA molecule contains a spacer (defined in the text). The spacer can be reduced or completely removed (processed) by different mechanisms (such as splicing or nuclease activity), thereby forming other forms of RNA hairpin molecules or dsRNA molecules with similar structures, as in the case of (a). In the simplest case, the edsRNA in (a) is obtained by hybridization of complementary RNA molecules generated independently of each other (see also the Examples and Figure 9 and Figure 10). Black and grey boxes: pseudo (p)-siRNAs with variable sequences at the 5' and 3' ends, respectively (see text for definitions); (s) sense, (as) antisense. A, B...Y...Z represent 1-n (or n-1 in reverse order) esiRNA / ERNA sequences identified by screening with AGO protein (A) or other AGO proteins (B..., Y, Z) and inserted into the edsRNA sequence; (s) sense, (as) antisense. R: Hammerhead or hepatitis D virus (HDV) ribozyme. Ribozymes are used to generate transcript ends by "self-splicing". After the corresponding edsRNA is generated, the ribozyme is no longer functional. Examples of edsRNAs constructed according to a) are shown in Figure 9 and Figure 10 Examples of cDNA construct compositions that can be used to generate edsRNAs designed according to a) and / or b) are given in Figure 11 .

[0212] Figure 9 .Exemplary edsRNA and control dsRNA. Figure 8 A) Scheme Structure of edsRNA generated from two transcripts. The edsRNA contains a 21nt long esiRNA / ERNA sequence, which has been shown to be effective in RNA silencing / RNAi against CMV RNA2 and to provide antiviral protection in plants (numbering refers to Figure 2 and Table 1). In addition, two conventionally constructed dsRNAs are shown as controls, i.e., they are not constructed from esiRNA / ERNA sequences, but rather from uninterrupted (continuous) regions of the target RNA. (A) Exemplary edsRNA 'dsCMV6si21'. It consists of a 21nt-long pseudo-siRNA at both ends (indicated by asterisks (*)) and six 21nt-long esiRNAs / ERNA sequences against CMV RNA2, which are active in plant AGO1 / RISC or AGO2 / RISC. The guide strands (gs) are each indicated by an arrow pointing in the 5'-3' sense direction. The AGO1-specific gs is located on one RNA strand and the AGO2-specific gs is located on the other RNA strand. The example RNAs shown here have blunt ends, i.e., no nucleotide overhangs at the ends. However, RNAs with overhanging ends were also produced and tested. The two forms of edsRNA achieved the same results in terms of their protection efficiency. Figure 8 B or Figure 11The protection efficiency of edsRNA generated by the protocol was also similar (not shown). (B) Control dsRNA 1. dsCMV consists of a terminal pseudo-siRNA sequence and a 126 nt long double-stranded segment from CMV RNA 2 (corresponding to six 21 nt long siRNAs) and a complementary sequence corresponding to this segment of CMV RNA 2. Importantly, dsCMV also contains the sequences of two siRNAs identified as esiRNA / eRNA in the eNA screen against CMV RNA 2 (see above) (see Figure 10 (C) Control dsRNA 2. dsGFP also consists of a terminal pseudo-siRNA sequence and a 126nt long double-stranded segment from GFP mRNA (mRNA encoding green fluorescent protein) and a complementary sequence corresponding to this segment of GFP mRNA. The exact sequence of the dsRNA shown is shown in Figure 10 .

[0213] Figure 10 . Structure / sequence of exemplary edsRNA and control dsRNA used (eg Figure 9 Two edsRNAs constructed from pseudo siRNA sequences (located at the ends) and esiRNAs / ERNA sequences, dsCMV6si21 and dsCMV6si22, are shown; control dsRNAs, dsCMV and dsGFP, are also shown. Figure 9 The ends consist of a pseudo-siRNA sequence, and the remainder is derived from a continuous region of CMV RNA 2 or GFP mRNA, respectively (the sense and antisense strands are shown in each case). The edsRNA contains a 21nt or 22nt long esiRNA / ERNA sequence, which has been shown to be effective in RNA silencing / RNAi against CMV RNA 2 and to provide antiviral protection in plants ( Figure 2-Figure 4). dsCMV6si21 consists of a 21nt-long pseudo-siRNA at each end and six 21nt-long CMV-specific esiRNAs / ERNAs (three of which are active in AGO1 / RISC and three are active in AGO2 / RISC). The AGO1-specific guide strand (gs) is located on one RNA strand, and the AGO2-specific gs is located on the other RNA strand. Since the gs of one siRNA on the dsRNA overlaps with the 2nt 3' overhang of the corresponding subsequent siRNA passenger strand, some modifications are made to the two 3'-terminal nucleotides of the passenger strand at these sites to achieve complete complementarity with the edsRNA. dsCMV6si22 is constructed according to the same principle and consists of a 22nt-long pseudo-siRNA at each end and six 22nt-long CMV-specific esiRNAs / ERNAs (three of which are active in AGO1 / RISC and three are active in AGO2 / RISC). The AGO1-specific gs is located on one RNA strand, and the AGO2-specific gs is located on the other RNA strand.

[0214] The different segments of RNA are identified as follows:

[0215] dsCMV6si21 and 22 (5'-3'): bold black - mock siRNA, dark grey - siRCV21172 gs, light grey - siRCV2 1489 gs, black italics - siRCV2359 gs. gs – guide strand

[0216] dsCMV6si21 and 22 (3'-5'): bold black - mock siRNA, light grey - siRCV2380 gs, black italic - siRCV2 2041 gs, dark grey - siRCV21020 gs

[0217] The ends of the dsCMV were also composed of pseudo-siRNA sequences and contained a 126-nt double-stranded segment from CMV RNA2 (corresponding to six 21-nt siRNAs). Importantly, incidentally, the segment selected from CMV RNA2 also contained two siRNAs identified as esiRNAs / ERNAs in the eNA screen (see above) (underlined siRCV2557 and italicized siRCV2540, respectively).

[0218] The ends of the dsGFP also consist of a dummy siRNA sequence and contain a 126 nt long double-stranded segment of GFP mRNA. This dsRNA contains the sequence of the control siRNA used in the previous experiment, siR gf698 (lowercase letters).

[0219] Each SEQ ID NO is annotated.

[0220] Figure 11 . Exemplary structures of cDNA constructs / templates with different structural edsRNAs generated in vitro or in vivo by various means. The cDNAs shown in the figure contain different exemplary promoters (bacteriophage T7 RNA polymerase, Pol II and Pol I promoters (e.g., Pol II and Pol I promoters from Saccharomyces cerevisiae)) by which the edsRNAs of these cDNAs can be transcribed. The generated edsRNAs contain at least one pseudo-siRNA sequence and an esiRNA sequence, which here, for example, corresponds to an esiRNA sequence identified as being directed against CMV ( Figure 9 and Figure 10 As noted, the esiRNA sequences used are exemplary; that is, correspondingly different but otherwise similarly constructed cDNA constructs can contain entirely different pseudo-siRNA sequences or esiRNA / ERNA sequences (see Figure 8 (See also the text). In addition to the complementary pseudo-siRNA or esiRNA sequence, the cDNA encodes a further spacer sequence, here, as an example, encoding a cellular intron (actin 1) and can be spliced by the cellular splicing machinery or degraded by cellular RNAases. In addition, the cDNA encodes a ribozyme (HH ribozyme or HDV ribozyme), which generates one or both ends of the edsRNA by self-splicing after transcription. They also encode a transcription terminator (here, for example, a VSV or cellular Pol terminator) and restriction sites for cloning purposes. The individual sequences, which may also partially overlap, were identified by various means (see above). The individual SEQ ID NOs are noted.

[0221] edsRNA produced by T7 RNA polymerase

[0222] Construct 1.1. edsRNA is produced as a hairpin transcript. The construct contains: a T7 RNA polymerase promoter (T7 promoter); an esiRNA sequence and a pseudo-siRNA sequence located at one end of the esiRNA sequence; an actin-1 intron as a spacer, a hepatitis delta virus (HDV) ribozyme; a vesicular stomatitis virus (VSV) transcription terminator; and restriction sites (Spe I / Xba I) for cloning.

[0223] Construct 1.2. edsRNA is produced as a hairpin transcript after further processing. The construct contains: a T7 promoter; an esiRNA sequence and two pseudo-siRNA sequences at either end of the esiRNA sequence; an actin-1 intron as a spacer; a hepatitis delta virus (HDV) ribozyme; a VSV transcriptional terminator; and restriction sites (Spe I / Xba I) for cloning.

[0224] edsRNA produced by RNA polymerase II (Pol II)

[0225] Construct 2. edsRNA is produced as a further processed hairpin transcript. The cDNA is cloned downstream of a cellular Pol II promoter (which can be inducible, such as the Gal1 promoter of Saccharomyces cerevisiae) and terminated by a 3'-flanking Pol II terminator (e.g., the CYC1 terminator of Saccharomyces cerevisiae). The construct contains: a hammerhead ribozyme (HH ribozyme); an esiRNA sequence and a pseudo-siRNA sequence located at one end of the esiRNA sequence; an actin-1 intron as a spacer; a hepatitis delta virus (HDV) ribozyme; and restriction sites (Hind III / Xba I) for cloning.

[0226] edsRNA produced by RNA polymerase I (Pol I)

[0227] Construct 3.1. edsRNA is produced as a hairpin transcript. The construct contains: a cellular Pol I promoter; an esiRNA sequence and a pseudo-siRNA sequence located at one end of the esiRNA sequence; an actin-1 intron as a spacer; a hepatitis delta virus (HDV) ribozyme; a minimal Pol I terminator; and restriction sites (Spe I / Xba I) for cloning.

[0228] Construct 3.2. edsRNA is produced as a hairpin transcript that undergoes further processing. The construct contains: a Pol I promoter; an esiRNA sequence and two pseudo-siRNA sequences located at the 5' and 3' ends of the esiRNA sequence; an actin-1 intron as a spacer; an HDV ribozyme; a Pol I terminator; and restriction sites (Spe I / Xba I) for cloning.

[0229] Figure 12In vitro edsRNA processing by DCLs. Radiolabeled dsCMV6si21 was added to BYL, and the processing of endogenously present DCL4, DCL2, and DCL3 in the extract was monitored over 24 hours. Samples were taken from BYL at the indicated time points after dsRNA addition, separated by PAGE, and visualized by autoradiography (M = 21nt siRNA as a marker). The clear banding pattern indicates that the edsRNA is clearly processed by DCL proteins. This results in a significant proportion of 21nt siRNA.

[0230] Figure 13 Cleavage assays were performed using single AGO1-specific and AGO2-specific esiRNAs / ERNAs from CMV RNA2, as well as similar esiRNAs / ERNAs generated from edsRNA in BYL. In BYL, AGO1 / RISC and AGO2 / RISC were tested using single esiRNAs / ERNAs, corresponding mixtures of these esiRNAs / ERNAs, or esiRNAs / ERNAs reconstructed from the edsRNA 'dsCMV6si21' by DCL present in BYL. Endonucleolysis of radiolabeled target RNAs was detected by agarose gel electrophoresis and autoradiography. Asterisks (*) mark cleavage products generated by the cleavage activity of AGO / RISC. Both single siRNAs and siRNAs processed from edsRNAs resulted in efficient cleavage of the target RNA into the expected cleavage products.

[0231] Figure 14 NGS RNA-Seq analysis of siRNAs produced from edsRNAs (containing 21nt esiRNA / ERNA sequences) in BYL. The proportion of 21nt esiRNA / ERNA reads relative to all 21nt total reads is shown. (A) The proportion of esiRNA / ERNA guide and passenger strand reads based on the position of the edsRNA dsCMV6si21 used herein. (B) The proportion of guide and passenger strand reads for each esiRNA / ERNA. All CMV-specific esiRNAs / ERNAs were detected. Therefore, it was demonstrated that they were processed by the corresponding edsRNAs via DCL4 contained in BYL. (C) Comparison of the detectable proportion of guide and passenger strands for CMV-specific siRNAs produced from edsRNAs. Clearly, the proportion of esiRNAs / ERNAs used was high (approximately 60% of reads), indicating that they were preferentially produced from edsRNAs by DCL4.

[0232] Figure 15Comparison of the protective effect of different dsRNAs in plants. 4-5 week old Nicotiana benthamiana plants were mechanically inoculated with different dsRNAs or single-stranded components of specific dsRNAs. Genome-infectious CMV RNA, which triggers infection in the absence of mediated protection, was simultaneously applied (co-inoculation). The figure shows the percentage of symptom-free Nicotiana benthamiana plants in each case over a period of 35 days (dpi = days post inoculation). (A) Application Percentage of symptom-free plants after dsCMV and dsGFP. (B) Representative plant images of the experiments shown in (A). A CMV-specific esiRNA / ERNA consisting of six 21nt long RNAs was used. Provides very effective (100%) protection against CMV infection. Using a similar construct consisting of 22nt long variants of the same esiRNA / ERNA, plants were less protected from CMV infection (30%). The protection provided by the conventionally constructed dsCMV (containing two esiRNA / ERNA sequences) was even more significantly reduced, while dsGFP did not provide protection. (C) Comparison of the protective effects of dsRNA and single-stranded RNA corresponding to the constituent strands of the dsRNA (see the text of the Examples). As shown, double-stranded RNA consisting only of multiple CMV-specific esiRNA / ERNA Provide very effective protection for CMV infection. In contrast, all single-stranded components of conventionally constructed dsCMV and dsRNA do not provide any protection. This shows that the protection of edsRNA for viral infection is based on its processing into esiRNA / ERNA by plant DCL (see the text of the examples).

[0233] Table 7. Sequences and activities of esiRNAs / ERNAs against three different mRNA targets identified in various screens against M. incognita. siRNAs (again, each single-stranded guide strand and complementary passenger strand are listed) were identified using the "eNA screen" approach described in the Examples and classified as esiRNAs / ERNAs using cleavage assays: the percentage of the corresponding target RNA cleaved by the esiRNAs / ERNAs in both standardized and stringent cleavage assays is given. As highlighted (bold), all listed siRNAs met the corresponding criteria of at least 25% cleavage efficiency of the target RNA used. siRNAs are designated siRMI (MI stands for M. incognita) and correspond to the respective target RNAs (SPF - splicing factor, SEQ ID NO: 191; INT - integrase, SEQ ID NO: 192; ACT - actin, SEQ ID NO: 193) and the position of the 5' end of the identified guide strand. Whether the esiRNA / ERNA has nematicidal activity in planta (yes / not tested) is also indicated. Finally, the activity of esiRNA / ERNA after in vivo treatment (immersion) of animals is given: Here the normalized expression ratio (NER) of the corresponding mRNA determined by qRT-PCR is given (see also Figure 14 ). The values (expressed as percentages) reflect the proportion of cleavage products: for example, in the case of siRMISPF 441, 90% of the mRNA in vivo was cleaved after treatment (compared to 0% with siR gf698). As shown and described, a close correlation between in vitro and in vivo RNA cleavage activity was observed. The RNAs and their variants are listed, which are claimed for patent protection and which are used in the form of esiRNAs / ERNAs or sRNAs or eASOs (eNAs) derived therefrom against the southern root-knot nematode. The corresponding SEQ ID NOs are given and are listed separately as "Appendix to Table 7".

[0234] Figure 16.Silencing effect of the identified esiRNA / ERNA on splicing factor mRNA of southern root-knot nematode in vivo and in planta. A) In vivo: Normalized expression ratio (NER) of splicing factor mRNA (SPF SEQ ID NO: 191) was determined by qRT-PCR (showing the amount of mRNA that could still be measured), which was performed after J2 stage nematode larvae (J2s) were incubated in siRNA solution (siRMISPF 166, siRMISPF 220 and siRMISPF 441) for 24 hours. According to standard methods, about 500 ng of total RNA or 1 / 5 diluted cDNA was used as PCR template and cDNA was synthesized by gene-specific reverse transcription (RevertAid reverse transcriptase). MI 18S rRNA (HE667742) was used as a reference gene in quantitative RT-PCR. Normalized expression ratio (NER) was calculated according to the mathematical method 2 -ddCt Calculation was performed using the average Ct value of the reference gene. Two representative experiments are shown, each with two replicates (water - water control without siRNA; siR GFP (or siR gf698) - negative control: the measured mRNA amount is 100%). B) The number of eggs in the plant / tomato roots (eggs - J2 s). J2 stage larvae were incubated in water for 24 hours in the presence of control siRNA (siR GFP or siR gf698) or test siRNA. The ability of nematodes to establish infection and complete their life cycle in tomato plant roots (two weeks old, n = 15) was analyzed by counting the number of eggs (eggs J2) 56 days after infection. Error bars: standard deviation of the mean (SDM). Asterisks indicate statistical differences from the control group, determined using a two-tailed Student's t-test, p ≤ 0.05 (*), p ≤ 0.01 (**) and p ≤ 0.001 (***). Representative experiments are shown.

[0235] Figure 17 In vivo silencing of actin 4 and integrase mRNA. Representative experiments shown here were performed using a similar Figure 17 A: Normalized expression ratio (NER) of actin 4 mRNA (ACT SEQ ID NO: 193), determined by qRT-PCR (see Figure 15 ), which was performed after incubating J2 worm larvae (J2s) in siRNA solutions (siR 154, siR 200, siR 303, siR 419, siR 433, siR 435, and siR 661) for 24 hours. B: Normalized expression ratio (NER) of integrase mRNA (INT SEQ ID NO: 192) determined by qRT-PCR (see Figure 15) were performed after incubating J2 nematode larvae (J2) for 24 hours in siRNA solutions (siR 135, siR 273, siR 423, and siR 444). Error bars: standard error of the mean (SDM). Asterisks indicate statistical differences from the control group (two-tailed Student's t-test): p ≤ 0.05 (*), p ≤ 0.01 (**), and p ≤ 0.001 (***).

[0236] Table 8. Sequences of esiRNAs / ERNAs against different mRNA targets identified in corresponding screens against Botrytis cinerea. siRNAs (each single-stranded guide strand and complementary passenger strand are listed) were identified by the "eNA screen" approach described in the Examples and classified as esiRNAs / ERNAs by cleavage assays: the percentage of the corresponding target RNA cleaved by the esiRNA / ERNA in the standardized and stringent cleavage assays is given here. The siRNAs are designated siRBC (BC stands for Botrytis cinerea) and correspond to the respective target RNAs (VDS-VDS51 SEQ ID NO: 194; DCTN-DCTN1 SEQ ID NO: 195; SAC-Sac1 SEQ ID NO: 196; ERG-ERG27 SEQ ID NO: 197; EF-EF2 SEQ ID NO: 198; CHS-CHS1 SEQ ID NO: 199) and the position of the 5' end of the identified guide strand. EsiRNA / ERNA highlighted in bold: The claims preferably seek patent protection for these siRNAs highlighted in bold and their variants (see Table 6), which are used in the form of esiRNA / ERNA or sRNA or eASO (eNA) derived therefrom for use against Botrytis cinerea. The corresponding SEQ ID NOs are given and listed separately as "Appendix to Table 8".

[0237] Figure 18 Cleavage assay of esiRNAs / eRNAs identified against various Botrytis cinerea mRNAs siRNAs were identified by applying the "eNA screening" approach described in the Examples and classified by standardized and stringent cleavage assays.

[0238] The cleavage assay was performed as described above using AGO1 from C. graminicula (see text). The translation reaction of C. graminicula AGO1 was carried out in the presence of the synthetic siRNA duplex to be tested, resulting in the incorporation of the desired siRNA into the AGO / RISC complex. Radiolabeled mRNA was then added as target RNA. Total RNA was isolated from the batch and the cleavage products were analyzed by PAGE and autoradiography. The target RNA used (VDS represents VDS51; DCTN represents DCTN1; SAC represents Sac1; ERG represents ERG27; EF represents EF2) and the resulting cleavage products are highlighted in each case. The siRNAs are named as shown in Table 8. For comparison, siRNAs (257, 470, 653, 808) obtained by computer prediction (https: / / www.zhaolab.org / pssRNAit / ) were used in each case in the assay. Cleavage assays performed without siRNA (-) served as controls. The originally used dsRNA (ds) and siRNA pool were applied to the gel as further controls.

[0239] Figure 19. Inhibition of Botrytis cinerea growth by topical application of esiRNA / ERNA directed against specific target RNAs. (A) Bar graph showing Botrytis cinerea lesions formed on leaves of Arabidopsis thaliana (wild type; Col-0). Plants were inoculated with a suspension of Botrytis cinerea spores and a combination of the following siRNAs: i) a mixture of six esiRNA / ERNAs directed against Erg27 mRNA (siRBCERG); ii) a mixture of four esiRNA / ERNAs directed against Erg27 mRNA, two esiRNA / ERNAs directed against Sad mRNA, and one esiRNA / ERNA directed against Ef2 mRNA (siRBCMIX); and iii) one siRNA directed against GFP-mRNA (siR gf698 or siR GFP). Twelve plants were used for each treatment, with three leaves per plant inoculated (leaf 8, 9, and 10). A drop of suspension containing spores and 400 ng (siR gf698 or siR GFP), 2400 ng (siRBCERG), or 2800 ng (siRBCMIX or siR GFP) RNA was added to each leaf. A suspension without RNA ("water") served as a fungal growth control. Three days post-inoculation (dpi), leaf lesion area was determined using ImageJ software (see example image below) and categorized into five sizes: i) >50 mm 2 (+++), ii) 20-50mm 2 (++), iii) 10-20mm 2 (+), iv) 1-10mm 2(+ / -) and v)0mm 2 (-). Different categories are expressed as percentages. Representative images of leaves with different categories of lesions (bottom). (B) Erg27 mRNA levels in vivo (during fungal infection) were determined by qRT-PCR at 3 dpi. Total RNA was extracted from leaf fungal lesions and cDNA was synthesized according to standard protocols. For subsequent PCR amplification, two sets of primers were used to cover the regions targeted by various esiRNA / ERNA (top). Erg27 mRNA levels were normalized to the endogenous levels of Botrytis cinerea actin mRNA ("housekeeping gene"). Normalized expression ratios (NER, see top figure) are given. The bar graph represents the mean of four biological replicates (each containing six lesions from independent leaves), error bars and SDM (standard deviation of the mean, bottom figure). Statistical differences from the water control ("water") were determined using a two-tailed Student's t-test: *p≤0.05, **p≤0.01, ***p≤0.001.

[0240] Example

[0241] Example 1: Nucleic acid active agent against cucumber mosaic virus CMV

[0242] The "eNA screening" method was first applied in this format to two RNA segments of the CMV genome (Fny strain) as target RNAs: RNA 2 (SEQ ID NO: 189) encodes the 2a protein, and subgenomic RNA 4A encodes the RNA-silencing viral suppressor protein (VSR) 2b; RNA 3 (SEQ ID NO: 190) encodes the 3a protein, and subgenomic RNA 4 encodes the capsid protein (CP) ( Figure 2 D and Figure 5 Screening was performed using AGO1 (L form; Gursinsky et al., 2015) and AGO2 from Nicotiana benthamiana (Nb). The target RNA used in the screening was in double-stranded form.

[0243] Therefore, a series of siRNAs classified as esiRNA / ERNA were identified using NbAGO1L and NbAGO2 against CMV RNA 2 ( Figure 2; Table 1). As described above, in the final step of the "eNA screening," esiRNA / eRNA sorting was performed by a standardized and stringent cleavage assay (protocol modified from WO 2019 / 001602, WO 2022 / 200407, and Gago-Zachert et al., 2019; see also the methods described above): To form RISC, 0.5 pmol of the mRNA for the AGO protein of interest was translated in a reaction solution containing 50% (v / v) BYL (protein amount and translation activity determined) (Gago-Zachert et al., 2019) in the presence of 10-100 nM of the synthetic siRNA to be characterized and a 10-fold excess (0.1-1 μM) of a competing siRNA (siR gf698). The amount of the siRNA to be tested was adjusted to the activity of the AGO protein of interest using the effect of siR gf698 on its GFP mRNA target. siRgf698 meets the criteria for esiRNA / eRNA targeting GFP mRNA (Schuck et al., 2013). After incubation at 25°C for 2.5 hours, 3.4 pmol of nonspecific mRNA (encoding firefly luciferase protein) was added per batch as a further competitor RNA, along with 10 fmol of radiolabeled target RNA, and the reaction batch was incubated at 25°C for an additional 15 minutes. During this incubation, the target RNA may be cleaved by the formed AGO / RISC. Further conditions and analysis of the cleavage reaction were as described by Gago-Zachert et al., 2019: After gel electrophoresis of the extracted RNA, the amount of remaining target RNA or generated cleavage products was quantified by measuring band intensities (ImageQuantTL or ImageJ) compared to a control reaction (performed without siRNA). Based on the cleavage activity of the corresponding RISC formed by the siRNA against the target RNA (cleavage activity), which was measured in this way, the esiRNA / ERNA classification was performed (see also the table): if at least 25% of the amount of target RNA originally used in the assay was converted into cleavage products by endonuclease means under standardized and stringent (competitive) conditions, the siRNA was considered effective, i.e., an esiRNA / ERNA. The 25% cleavage efficiency threshold was set based on earlier data (Gago-Zachert et al. 2019) and the data obtained herein, which showed that esiRNAs / ERNAs with this characteristic in vitro had a clearly measurable antipathogenic effect in vivo compared to control siRNAs (see below). Based on this, esiRNAs / ERNAs that specifically had cleavage activity against CMV RNA2 together with AGO1 were characterized.For example, siRCV2359, siRCV21172, and siRCV21489 (the candidate is named "siR," "CV" stands for CMV, and the position of the 5' nucleotide of the complementary viral (+) RNA of the respective RNA and siRNA guide strand). siRCV2149, siRCV2 186, siRCV21613, siRCV21982, siRCV22441, siRCV22562, and siRCV22727 also have high cleavage activity. The in vitro functional data of these siRNAs are as follows. Figure 2 As shown; their sequences are summarized in Table 1 (the corresponding SEQ ID NOs are listed in the Appendix of Table 1).

[0244] Similarly, siRCV2 380, siRCV2 1020, and siRCV2 2041 were identified as RNA agents with particularly high cleavage activity in the RISC complex with AGO2. siRCV2407, siRCV2449, siRCV2540, siRCV2557, siRCV21054, and siRCV21248 also had high cleavage rates ( Figure 2 ; Table 1). Figure 2 C shows a schematic diagram of the binding site of the siRNA with particularly high cleavage activity (most potent) located on RNA2.

[0245] For example, the in vitro identified esiRNA / ERNA siRCV21020, 1172, 359, 1489, 380, and 2041 were used in in planta protection experiments. For this purpose, a statistically representative number of Nicotiana benthamiana plants (n=12-15) were co-inoculated per plant using silicon carbide according to a standard protocol ("rub-in") with 150 pmol (~1 μg) of the siRNA to be tested (obtained from the company for synthesis) and 20 fmol of each genomic CMV RNA (1-3; generated by in vitro transcription of "infectious cDNA" (obtained from Prof. Fernando García-Arenal Rodríguez (Polytechnic University of Madrid) and Prof. John Carr (University of Cambridge); Rizzo and Palukaitis 1990) (RNA dissolved in a 15 mM KH2PO4, 25 mM glycine solution). The infection with CMV RNA was performed so that, when inoculated in Nicotiana benthamiana without any additives, 100% of the corresponding RNA amount resulted in infection and the appearance of clear symptoms (so-called "maximum challenge"). Nonspecific siR Gf698 or siRNAs with no or only slight cleavage activity against the target RNA in previous screening (siRCV21844 and siRCV22634) were used as controls. The plants were examined for symptom development 35 days after infection (dpi). Figure 3 A shows representative images of plants treated in the described manner. Figure 3 B shows the overall trend of multiple experiments (three biological replicates). It is clear that siRCV2 359 and siRCV21489 each achieved 93% protection against CMV infection, and siRCV21020 and siRCV21172 each achieved 100% protection against CMV infection: in other words, under the maximum attack used, all or the vast majority of the plants treated in this way remained asymptomatic. siRCV2380 and siRCV2 2041 achieved 60% protection against CMV infection: in other words, 40% of these plants developed symptoms during the attack. The various negative control siRNAs provided almost or no protection: only 0-7% of the plants treated in this way remained asymptomatic during the attack. This protective effect can also be confirmed by the fact that 35 days after treatment with the corresponding protective esiRNA / ERNA (dpi), in plants identified as asymptomatic "by eye", genomic CMV RNA could no longer be detected by RT-PCR (standard procedure) ( Figure 3 C).

[0246] Plant protection against CMV infection can also be achieved using the 22nt version of the corresponding esiRNA / ERNA. Figure 4 As shown in the example, in the in vitro cleavage assay, the 22nt esiRNA / ERNA formats showed similar or occasionally slightly lower cleavage activity against the target RNA in the corresponding AGO / RISC. A similar trend was observed in the plant protection experiment: the protection of 22nt siRNA was comparable to that of 21nt esiRNA / ERNA, or slightly lower (e.g. Figure 4 Cleavage assays and protection experiments using 24 nt long versions of the corresponding esiRNA / ERNAs showed similar results to those performed with 22 nt esiRNA / ERNAs (not shown).

[0247] The same approach was used to identify esiRNAs / ERNAs against CMV RNA 3; NbAGO1L and NbAGO2 were used again. A series of siRNAs were also identified that induced very high (siRCV3239, siRCV3507, siRCV3985) or high (siRCV3 151, siRCV3988, siRCV31098) cleavage activity against CMV target RNA 3 when bound to AGO1, and very high (siRCV3593, siRCV31019, siRCV31569) or high (siRCV3358, siRCV3478, siRCV3496, siRCV3592, siRCV3733, siRCV31394) cleavage activity against CMV target RNA 3 when bound to AGO2. Figure 5 A. Figure 5 B) The position of the most effective siRNA guide strand on RNA 3 in the cleavage assay. Figure 5 C. Table 2 summarizes the screening results of esiRNA / ERNA against CMV RNA 3 (the corresponding SEQ ID NOs are listed in the Appendix of Table 2).

[0248] In contrast, esiRNAs / ERNAs characterized against CMV RNA 2 tended to exhibit higher cleavage activity than those identified against RNA 3 (see Tables 1 and 2). This may be due to a slightly higher stability or a more fundamentally compact structure (lower a-site accessibility) of CMV RNA 3 compared to CMV RNA 2 (data not shown).

[0249] In plant protection experiments, the 21nt and 22nt esiRNA / ERNA characterized against CMV RNA 3 showed effective protection against the virus. As expected from in vitro experiments, this was slightly lower than the case of esiRNA / ERNA agents against CMV RNA 2 ( Figure 6 Cleavage assays and protection experiments performed using 24 nt long versions of the corresponding esiRNA / ERNA showed similar results to those performed with 22 nt esiRNA / ERNA (not shown).

[0250] Sequence comparison shows that many esiRNA / ERNAs identified from CMV Fny strains herein are also effective on the similar RNA segments of other CMV strains, because there is complete complementarity between the sequence of each guide strand of these esiRNA / ERNA and the corresponding target site on viral RNA.As shown in Figure 7, as an example, 6 kinds of esiRNA / ERNAs have shown the coupling (according to Roossinck, 1999 revision) of complementary target site on sequence and CMV RNA 2 genome segments.Also shown in the figure is the calculated expected rate (when siRNA guide strand is combined with target site, the base pairing of one or more positions in nucleic acid is missing).Based on these data, table 3 provides the complete overview of the esiRNA / ERNA identified, and its guide strand is attached to the target site in RNA2 and RNA 3 of corresponding CMV strain in the mode of complete complementarity (i.e., no mispairing).Therefore, as shown in the table, when these esiRNA / ERNA are used alone or in combination, in fact, all CMV strains for IA and IB groups provide protection.Table 4 and table 5 have been illustrated this.

[0251] As shown in Tables 3-5, the esiRNA / ERNA identified are not bound to the corresponding target sites in RNA2 and RNA 3 of the CMV strains of the II subgroup without mismatching. As described above and in prior applications (WO 2022 / 200407), esiRNA / ERNA indicates a so-called a-site, i.e., a region containing these esiRNA / ERNA target sites in the target RNA. Although the target RNA has a complex structure, it can still be approached by RISC or other cell complexes containing endonucleases (WO 2022 / 200407). Therefore, by adapting the sequence of the esiRNA / ERNA identified herein to the target site in the target RNA, i.e., avoiding mismatching, the silencing of the genomic RNA2 and RNA3 of the strains of the II subgroup can still be achieved. This is summarized in Table 6, which shows the number of mismatches when the guide strand of the esiRNA / ERNA identified is bound to the corresponding target sites in RNA 2 and RNA3 of various CMV strain subgroups. The table shows all forms of mismatches. This can involve the so-called siRNA seed region (the most important region for the interaction between siRNA and target RNA in AGO / RISC-mediated silencing; (Jackson and Linsley, 2010)) or the 5' end of the RNA, which has been shown not to be involved in siRNA binding. Specific binding of the sRNA to the target RNA site is guaranteed when the number of mismatches does not exceed 5; however, at higher melting temperatures of the complementary RNA strands, binding specificity is generally maintained even when the number of mismatches exceeds this limit by at least 2 nucleotides (Liu et al., 2014). As shown in Table 6, except for the four strains with a higher number of mismatches, all other strains provided potential protection in RNA silencing approaches using the identified esiRNA / eRNA, even with up to 7 mismatches to the target site.

[0252] Summarizing these data, the esiRNA / ERNA population identified herein can be used in an unmodified form (e.g., for most members of the 1a and 1b subgroups) or in a form varying in 1 to a maximum of 7 nucleotide positions for the purpose of protecting most CMV strains (Table 6). As is clear from the table, according to the present invention, almost 95% of all CMV strains compared can be targeted by mapped esiRNA / ERNA, even if there is a variation in the range of 7 positions. Under the same background, it is important that esiRNA / ERNA can be used alone or in an RNAi approach much more effectively as a combination (mixing) (see also below). In this way, even if there is a mismatch in individual esiRNA / ERNA, escape by antigenic drift or transfer can be particularly effectively prevented. Similar findings apply to other sRNAs and eASOs (collectively referred to as eNAs), the sequences of which can be derived from corresponding esiRNA / ERNAs.

[0253] Example 1 Summary: The above-mentioned problems have been solved. According to the present invention, esiRNA / eRNA agents or, in particular, derived eNA agents have been identified that have antiviral activity against CMV and can be used for plant protection against CMV. The identified eNAs can be used in unmodified form or modified at 1-7 nucleotide positions for protection against 95% of known CMV variants. The eNAs can be used alone for plant protection or, even more effectively, in combination (mixture) in RNA silencing methods against CMV.

[0254] Example 2: Double-stranded RNA active agents containing esiRNA / ERNA sequences or other related sRNA sequences

[0255] The goal is to construct double-stranded RNA (called edsRNA) that is optimized for practical use of esiRNAs / ERNAs or related sRNAs in RNA silencing / RNAi processes. Therefore, edsRNA should contain multiple esiRNAs / ERNA sequences and / or especially derived sRNA sequences (see Figure 1 ). When used in a target organism, the edsRNA should be processed by the DCL / Dicer present therein to produce a high proportion of the original constituent esiRNA / ERNA or sRNA, and these should be active against the target RNA in the corresponding RISC. In order to produce a maximum effective silencing response against one or even multiple target RNAs by these edsRNAs, as a further characteristic, these edsRNAs should consist of sequences of esiRNAs / ERNAs or related sRNAs that are active in the RNA silencing / RNAi process of various AGO proteins and the corresponding RISC.

[0256] According to the present invention, the design of edsRNA is based on a hypothesis that has not yet been fully verified. This hypothesis assumes that DCL / Dicer can be active at both ends (termini) of dsRNA. This hypothesis also includes that the presence of so-called "pseudo-siRNA sequences" at the ends of the dsRNA can force DCL / Dicer to perform "co-processing". Therefore, within the meaning of the present invention, a pseudo-siRNA sequence is a double-stranded ribonucleotide sequence of any composition, whose length is 21, 22, 23 or 24 nt according to the predetermined processing of the corresponding edsRNA by DCL / Dicer. The pseudo-siRNA sequence should be located at the end of the double-stranded RNA accordingly, and its presence should force DCL / Dicer, which is active on the dsRNA, to perform co-processing.

[0257] Synchronous processing means that, depending on the position and length of the pseudo-siRNA sequence and therefore on the activity of the DCL / Dicer involved, the endonuclease cleavage of the dsRNA takes place in such a way that the sRNA produced in this process preferably has the same length as the pseudo-siRNA. For example, if the pseudo-siRNA sequence is 21 nt in length and DCL4 is the processing enzyme (DCL4 preferably produces 21 nt siRNAs from dsRNA), then 21 nt sRNAs are preferably produced from the edsRNA constructed in this way and in the order in which they appear in the sequence following the pseudo-siRNA in the edsRNA. DCL4 acts in a serial manner: starting from the pseudo-siRNA sequence at the end of the edsRNA, the enzyme performs the endonuclease cleavages in sequence. If the edsRNA is constructed such that, for example, a 21 nt long pseudo-siRNA sequence is directly followed by a 21 nt long esiRNA / ERNA sequence (see Figures 8-11 ), 21nt long esiRNA / ERNA should be formed preferentially.

[0258] The pseudo-siRNA sequence may also contain elements that are important for the successful transcription and processing of the corresponding edsRNA: these may be regions of the corresponding transcription promoter or terminator; or they may be parts of ribozymes that generate the correct 5' or 3' end of the corresponding RNA (see Figure 8 and Figure 11 The latter is applicable, for example, to the HH ribozyme; an example used in this example. Here, the pseudo-siRNA sequence comprises a given number of nucleotides complementary to the 5' end of the ribozyme and can form helix I by hybridization, thereby forming its functional structure ( Figure 11 ).

[0259] The basic principle of constructing the edsRNA designed in this way is as follows ( Figure 8 and below):

[0260] - The sequence contains at least one pseudo-siRNA sequence as described above, which enables simultaneous processing by Dicer / DCL. The sequence of the edsRNA also contains the sequences of multiple esiRNAs / ERNAs or other sRNAs (such as miRNAs derived from esiRNAs / ERNAs) "contiguously arranged" from 5' to 3', Figure 8 These sequences can be derived from various "eNA screens" and thus the esiRNA / ERNA or sRNA derived therefrom can be active in different AGO / RISCs ( Figure 8 Ideally, the esiRNA / ERNA constituting the edsRNA or the sRNA derived therefrom can also be directed against different target RNAs from one or more different organisms. Figure 8 Where n is a value from 2 to infinity, preferably 2-100, or as described in claim 1d.

[0261] -edsRNA can be produced in two ways: from two complementary RNA molecules or from a single transcribed RNA molecule containing two complementary segments ( Figure 8 ). In the latter case, the two complementary segments of the transcribed RNA are connected to each other via a spacer and form a hairpin. A spacer is a sequence of any composition with a minimum length of 4 nucleotides (its definition and that of a hairpin are described above), but for the specific purpose of edsRNA construction, it may contain functional regions such as ribozymes, transcription promoters or transcription terminators, transport signals or splicing sites. Therefore, in addition to the function of connecting the two complementary single-stranded components of dsRNA, the spacer sequence also plays other roles: for example, if it contains a splicing signal, the spacer can be truncated by the splicing machinery of the cell during RNA expression in vivo. Since, unlike double-stranded RNA regions, spacers are sensitive to ribonucleases (such as single-stranded-specific RNase T1 or A), this sequence can also be completely removed by these RNases (see also Figure 8 ).

[0262] -RNA transcription can be performed in vitro or in vivo. Double strands are obtained by hybridization of complementary RNA strands ( Figure 8 ). Transcription can be carried out by a variety of promoters (see, for example Figure 11 ). Transcription termination can be performed by any type of transcription terminator (see, for example Figure 11 ).

[0263] - Depending on how it is produced, edsRNA can be blunt-ended or contain overhangs It can be produced in various ways, such as by "run-off transcription" or by using ribozymes (such as the HH or HDV ribozymes used in the examples of this application) to terminate the relevant polymerase through self-splicing activity.

[0264] - The sequences of the two strands are designed so that during DCL processing, each true sRNA guide and passenger strand sequence is generated. The presence of the pseudo-siRNA sequence and the associated "synchronous processing" by DCL / Dicer ensure that processing primarily generates constitutive esiRNA / ERNA or sRNA derived therefrom (see above and Figures 8-14 ).

[0265] According to the present invention, i.e., by adopting the principle of iterative trial and error, the "synchronous processing" hypothesis and the functions of various edsRNA constructs constructed based on this principle were verified and confirmed. Figure 9 and Figure 10Examples of simple designed edsRNA compositions that can be produced by hybridization between two complementary RNA molecules are given (SEQ ID NOs: 181 and 185 and 182 and 186). Such edsRNAs can be produced by in vitro transcription of conventionally constructed cDNAs (consisting of a promoter, coding sequence, and terminator / run-off). Figure 11 Examples of the invention of compositions of edsRNAs that can be produced in vitro and in vivo are given by showing basic cDNA constructs (SEQ ID NOs: 200, 201, 201, 203 and 204). The latter edsRNAs all contain a spacer, which can remain in place when used, or can be truncated or decomposed as described above.

[0266] Figure 12-15 The functionality of the edsRNA designed in this way was demonstrated. Figure 9 、 Figure 10 and Figure 15 ; SEQ ID NO: 181 and 185) as an example, which contains 6 different 21nt long esiRNA / ERNA sequences targeting CMV RNA2. Three of these esiRNAs / ERNAs were confirmed in Example 1 to be active against CMV RNA2 in plant AGO1 / RISC; the other three of these esiRNAs / ERNAs have previously been confirmed to be active against CMV RNA2 in plant AGO2 / RISC. In this case, the functionality of the edsRNA is independent of whether it is produced in vitro or in vivo ( Figure 11 and not shown); or whether the RNA is blunt-ended or has overhangs The functionality of edsRNAs is also independent of whether they are constructed from 21-, 22-, or 24-nt-long pseudo-siRNAs and esiRNAs / ERNAs.

[0267] First, it has been demonstrated that in BYLs shown to contain active forms of DCLs 4, 2, and 3 (Schuck et al., 2013), edsRNAs are processed to produce siRNAs of 21 nt, 22 nt, and 24 nt in length, respectively. Thus, the fundamental processibility of edsRNA constructs designed by Dicer / DCLs has been demonstrated ( Figure 12 ).

[0268] By using edsRNA in an in vitro cleavage assay of the target RNA, it can be confirmed that the expected cleavage products resulting from the acquired esiRNA / ERNA activity are generated from the target RNA (e.g., CMV RNA 2) ( Figure 13Subsequent experiments further confirmed this. By NGS (RNA-seq), we determined the siRNAs generated from the edsRNA, dsCMV6si21, in BYL (through its presence and active DCL): It is clear that a high proportion of pseudo-siRNAs and esiRNAs / ERNAs (identifiable by detectable guide or passenger strands) are generated from the edsRNA processing ( Figure 14 A+B; 21 nt reads are shown in each case).

[0269] Thus, the hypothesis proposed by the present invention, which led to the defined structure of edsRNA, turned out to be correct: from dsRNA constructed in this way, DCL4 preferentially generates 21 nt pseudo-siRNAs and esiRNAs / ERNAs, which together account for approximately 60% of all 21 nt siRNAs generated ( Figure 14 C) According to the present invention, it has been demonstrated that the processing of DCL occurs as a "synchronous processing" from both ends of the dsRNA.

[0270] Finally, plant protection experiments demonstrated high efficacy (protection) of edsRNA against CMV challenge (infection with a lethal concentration of CMV), thus conclusively demonstrating the functionality of edsRNA ( Figure 15 ).

[0271] Several important aspects are clear from the exemplary experiments:

[0272] - First, it was shown that compared with nonspecific dsRNA (control dsRNA 2, dsGFP; composed of sequences similar to GFP mRNA and its complementary RNA), edsRNA constructed from 21nt esiRNA / ERNA (dsCMV6si21 or ) had a 100% protection efficiency. In other words, in the CMV challenge experiment, all plants treated with edsRNA remained symptom-free compared to non-specifically constructed dsRNA ( Figure 15 ).

[0273] - a conventional dsRNA constructed with a ds sequence from CMV RNA 2 of similar length to dsCMV6si21 (control dsRNA 1, dsCMV; consisting of a CMV RNA 2 region of the same length and its complementary RNA; see Figure 9 and Figure 10 ) compared to edsRNA, which has significantly more efficient antiviral efficacy:

[0274] *“Conventional dsRNA” means that one strand of these RNAs consists of an exact copy of the targeted target RNA, which subsequently hybridizes with a complementary RNA strand: dsRNAs constructed according to this principle are currently used for RNAi-mediated plant protection.

[0275] Although the conventionally constructed dsCMV (control dsRNA 1) showed some protection compared to dsGFP (control dsRNA 2), this protection did not persist throughout the entire 35 dpi test period. In contrast, the edsRNA constructed from esiRNA / ERNA maintained 100% protection throughout the test period ( Figure 15 ). It is noteworthy that the control dsCMV also contained two of the sequences previously characterized herein as esiRNAs / ERNAs (e.g. Figure 9 Thus, this control reflects well the situation of conventional dsRNA treatment in which only a few, if any, esiRNA / ERNA sequences are present (see schematic diagram for Figure 1 ).

[0276] - The edsRNAs that produced 21nt esiRNA / ERNA had the highest protection against CMV; the edsRNAs that produced the same edsRNA but in a 22nt form had lower protection: compared to the 21nt edsRNA, the 22nt edsRNA did not show 100% protection throughout the test period. Nevertheless, the protection provided by the edsRNAs that produced 22nt esiRNA / ERNA was still significantly better than that provided by the conventionally constructed dsCMV ( Figure 15 The same is true for edsRNA that produces 24nt esiRNA / ERNA (not shown).

[0277] - When dsRNAs (dsCMV6si21 or and dsCMV) was found to offer no protection ( Figure 15 C). This experiment, performed as a further control, demonstrated that the edsRNA used is actually only active in double-stranded form. Therefore, the observed protective effect must be based on (synchronous) processing of the edsRNA by the plant DCL in vivo to produce esiRNA / eRNA. In other words, it can be ruled out that during the experiment, 21-, 22-, or 24-nt-long segments of the single-stranded dsRNA component accidentally bind to the target RNA, triggering AGO / RISC-mediated cleavage and thus conferring protection.

[0278] Summary of Example 2: According to the present invention, a novel designed and constructed edsRNA active agent was developed. It can be easily prepared by in vitro or in vivo transcription and can be processed by Dicer / DCL to generate a large number of various constitutive sRNAs. The edsRNA constructed in this way can be used as an effective antipathogen active agent.

[0279] Example 3: Nucleic acid active agent against southern root-knot nematode

[0280] In BYL, the described approach for the AGO2 protein of Nicotiana benthamiana (Nb) was used to identify esiRNAs / ERNAs against three mRNAs of the incognita target genes (SEQ ID NOs: 191-193): these target genes encode the "splicing factor," "actin-4," and "integrase" proteins. Among other factors, each of these proteins is believed to have a key function in the life cycle of the incognita (see also above).

[0281] The corresponding gene or target RNA sequence (see Appendix) was determined as follows.

[0282] Actin-4: The sequence was determined using the accession number for actin-4 from Caenorhabditis elegans at https: / / wormbase.org / / #012-34-5. Sequence comparison (Blast N) was then performed in the database https: / / meloidogyne.inrae.fr / . In this way, the predicted cDNA sequence of the southern root-knot nematode (M. incognita) was determined. The derived protein sequence was further confirmed by sequence comparison (Blast P) in the NCBI database (National Center for Biotechnology Information) and cloned. For splicing factors and integrase: EST (Expressed Sequence Tag) sequences of southern root-knot nematode (M. incognita) from the NCBI database (AW828516 and AW871671, respectively) were used here. These were then subjected to Blast N in the database https: / / meloidogyne.inrae.fr / . After finding the predicted cDNA sequence, Blast P was performed using the derived protein sequence to confirm homology with the splicing factor and integrase sequences of C. elegans. Accordingly, these cDNAs were cloned.

[0283] The esiRNA / ERNAs summarized in Table 7 (SEQ ID NOs in the Appendix to Table 7) were identified and classified based on the characteristics defined above. The nomenclature is as follows: "siR", "MI" for Meloidogyne incognita, the corresponding mRNA (SPF splicing factor; INT integrase; ACT actin 4), and the position of the RNA to which the 5' nucleotide of the siRNA guide strand complements. They were validated by various methods (see Figure 16 and Figure 17 ).

[0284] - Standardized cleavage assay according to the above protocol. esiRNA / ERNA and edsRNA derived therefrom ( Figure 8 and Figure 11 ) The cleavage (cleavage) activity of the respective target RNAs was verified in vitro with NbAGO2 in a cleavage assay. All 14 siRNAs identified using "eNA screening" showed induction of target RNA hydrolysis, which was greater than 25% of the amount of these target RNAs originally used (summarized in Table 7).

[0285] -in vivo. In addition, the efficiency of cutting the corresponding mRNA after siRNA uptake was tested in vivo (i.e., in animals). To this end, J2 southern root-knot nematode animals were obtained, and 10,000 were incubated (soaked) in 40 μl of water containing 50-200 ng / μl RNA for 24 hours to allow them to take up RNA. siRNA siR gf698 (siR GFP) was used as a negative control. The nematodes were then digested using standard methods, total RNA was extracted, and the corresponding primers and quantitative real-time PCR (qRT-PCR) were used to detect the cutting of the corresponding target mRNA. The gene-specific reverse transcription (RevertAid Reverse Transcriptase, ThermoFisher) was used for cDNA, also according to standard methods, using about 500 ng of total RNA or 1 / 5 diluted cDNA as a PCR template. The reference gene used was the southern root-knot nematode GAPDH gene (Minc3s07075g40689). The normalized expression ratio (NER) reflects the measurable amount of target mRNA originally used in the assay that remains after cleavage (see also Figure 14 ), which is based on the mathematical method 2 -ddCt The average Ct value of the reference genes was used for calculation.

[0286] - in vivo / in planta. Finally, the ability of esiRNA / ERNA to inhibit the complete life cycle of nematodes in planta was tested. In the first form of these experiments, J2 animals were placed (immersed) in water-soluble RNA or control RNA for 24 hours as described above. Subsequently, two-week-old tomato plants were infected with nematodes treated in this way according to a standard protocol (500 animals per plant), and it was investigated whether this treatment resulted in a reduction in egg laying after infection or a reduction in pathogenicity (especially root knot formation) of the infected plants.

[0287] In the second form of these experiments, infection research is carried out on fresh seedlings. For this reason, the tomato seed surface is sterilized by immersion in 70% ethanol. After removing the ethanol, for further treatment, the seeds are treated with a solution containing 30% (v / v) NaOCl and 0.02% Triton X-100 and incubated for 20-30 minutes. Wash with sterile distilled water subsequently, and be placed in a culture dish containing about 6mm of 0.6% Phytagel (pH 6.4), in 1 / 4MS (Murashige and Skoog medium), which contains 0.5% sucrose. 10 seeds are randomly distributed in each culture dish, and the culture dish is then sealed with Nescofilm and maintained at 28 ℃, 16 hours of light / 8 hours of dark cycle. 7-10 days after sowing, 100-200 sterilized and soaked J2 are inoculated into each seedling. To this end, the surface of J2 animals was sterilized in a solution containing 0.004% mercuric chloride, 0.004% sodium azide, and 0.002% Triton X-100 for 5 minutes, washed with sterile water, and suspended in agarose (0.1%). The immersion treatment was as described above. The inoculated seedlings were cultured under the above conditions. The infection process was monitored by checking for root knot formation three weeks after inoculation. The immersion was performed as described above. The inoculated seedlings were grown under the above conditions. The infection process was monitored by checking for root knot formation three weeks after inoculation.

[0288] In the test, the esiRNA / ERNA listed in Table 7 showed significant silencing or nematode killing effects (see Figure 16 and Figure 17 ).

[0289] Example 3 Summary: The above problems are solved. According to the present invention, esiRNA / ERNA active agents or eNA active agents derived therefrom that have nematicidal effects on southern root-knot nematodes are identified, and they can be used for plant protection against southern root-knot nematodes. The identified eNA can be unmodified or used in a form modified at 1-7 nucleotides for protection against all known southern root-knot nematode variants. In the RNA silencing method for southern root-knot nematodes, eNA can be used alone for plant protection, or in combination (mixing), which is much more effective. It is also possible to use edsRNA containing the sequence of the identified esiRNA / ERNA or other sRNA derived therefrom for plant protection against southern root-knot nematodes.

[0290] Example 4: Nucleic acid active agent against Botrytis cinerea

[0291] In BYL, the AGO1 protein method of Colletotrichum graminearum (C. graminearum, like Botrytis cinerea, is a plant pathogenic fungus) was used to identify and classify esiRNA / ERNAs against various mRNAs of Botrytis cinerea target genes. Three of the target RNAs (SEQ ID NOs: 197, 199, 198) were selected because the proteins they encode, "cytochrome P450 monooxygenase" (Erg27), "chitin synthase 1" (CHS1), and "elongation factor 2" (EF2), are known targets of various fungicides (see the introduction to Botrytis cinerea). Another three target RNAs (SEQ ID NOs: 194, 195, 196) were selected because the proteins they encode, "vacuolar protein sorting 51" (VPS51), "dynamin activator" (DCTN1), and "actin inhibitor" (SAC1), are involved in the vesicle trafficking pathway in fungi and are key virulence factors in the interaction of Botrytis cinerea with its plant host.

[0292] cDNAs for these genes were generated and cloned from mRNA preparations prepared from Arabidopsis thaliana plants infected with Botrytis cinerea according to standard methods.

[0293] The esiRNA / ERNAs summarized and highlighted in Table 8 were identified (SEQ ID NOs in the "Appendix to Table 8"). The candidate designations are as follows: "siR," "BC" for Botrytis cinerea, the corresponding RNAs (ERG - Erg 27; CHS - chitin synthase 1; EF - elongation factor 2; VPS - vacuolar protein sorting 51; DCTN - dynein activator protein 1; SAC - actin inhibitor), and the position of the RNA to which the 5' nucleotide of the siRNA guide strand is complementary. Validation was performed as follows.

[0294] - Standardized cleavage assay according to the above-mentioned protocol. For this purpose, RNAs were tested in vitro for their cleavage (cleavage) activity against the corresponding target RNA using Colletotrichum graminearum AGO1: 24 of a total of 30 siRNAs identified using the "eNA screen" showed induction of hydrolysis of the corresponding target RNA, which was higher than 25% of the amount of target RNA originally used ( Figure 18 and Table 8).

[0295] - In vitro fungal growth experiment. Cultivate Botrytis cinerea B05.10 on the fruit. After the fungus is able to produce spores for five days, collect the spores and dilute them in the corresponding culture medium to a final concentration of 1x10 5 Spores / ml (standard method). Spore germination was then initiated by adding phosphate. To ensure uniformity, 10 μl of 1 M phosphate buffer at pH 6.4 was added to 990 μl of spore solution. The effect of esiRNA / ERNA on the growth of Botrytis cinerea was tested in vitro on potato dextrose agar (PDA) plates. A final concentration of 1×10 5 10 μl of spore solution at 400 μg / ml was added to the center of the plate. For treatment, 400-8000 ng of RNA was added to the spore solution and continued every 12 hours. In addition to the Botrytis cinerea-specific esiRNA / ERNA and edsRNA, siRgf698 (siR GFP) and water were added to the spores as controls. Growth dynamics under the influence of RNA relative to controls were assessed by measuring fungal colony diameters (ImageJ software) at 1, 2, 3, and 5 days after inoculation.

[0296] - In vivo / in planta fungal growth assay: Here, the ability of esiRNA / ERNA to inhibit the complete life cycle of Botrytis cinerea in planta was tested. For this purpose, a final concentration of 400-8000 ng of RNA was added to the induced spore solution (1×10 5 / ml, see above) and incubated at room temperature for 1.5 hours. For infection, Arabidopsis leaves were moistened with 10 μl drops of incubation solution. The plants were then kept in a high humidity chamber for two days, after which the size of the lesions caused by the fungus was compared with control plants infected with siR gf698 control spores or water (ImageJ software) (see also Figure 19). To further determine fungal growth, leaf section tissue was removed and Botrytis cinerea DNA was quantified by qPCR using a standard curve method. The Botrytis cinerea actin gene and the control gene of the plasmid preparation incorporated into the plant sample were also detected. The plasmid control gene was used to estimate the efficiency of the DNA extraction process and to normalize the amplification of cut A. Finally, the amount of Botrytis cinerea DNA was determined by interpolating the normalized cut A value on the standard curve.

[0297] - By transcript analysis: To evaluate the silencing of target RNA in Botrytis cinerea, mRNA expression analysis was performed by quantitative real-time PCR (qRT-PCR). 5 Spores were placed in 2 mL of liquid culture medium and RNA was added (see above). The samples were then grown at room temperature with shaking for 24, 48, and 72 hours. RNA was extracted from the fungal samples using TRIzol according to the manufacturer's instructions. qRT-PCR was performed as described in Example 3 (see also FIG19 ).

[0298] The esiRNAs / eRNAs listed in Table 8 exhibited significant silencing effects ( FIG. 19 ) or fungicidal effects in vitro ( FIG. 19 ). It is also apparent from FIG. 19 that even nonspecific NA treatment in vivo had a mild fungicidal effect; however, the fungicidal effect was significantly stronger when eNA treatment was specific for the target RNA.

[0299] Example 4 Summary: The above problems are solved. According to the present invention, esiRNA / ERNA active agents or eNA active agents derived therefrom with fungicidal effects against Botrytis cinerea are identified, which can be used for plant protection against Botrytis cinerea. The identified eNA can be unmodified or modified at 1-7 nucleotides for protection against all known Botrytis cinerea variants. In the RNA silencing method against Botrytis cinerea, eNA can be used alone for plant protection, or in combination (mixture), which is much more effective. It is also possible to use edsRNA containing the sequence of the identified esiRNA / ERNA or other sRNA derived therefrom for plant protection against Botrytis cinerea.

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Claims

1. A nucleic acid for protecting plants against the plant pathogens cucumber mosaic virus, southern root knot nematode (Meloidogyne incognita) and Botrytis cinerea, characterized in that a. The nucleic acid is a small interfering RNA (siRNA) consisting of 21, 22, 23 or 24 base pairs and containing two single-stranded RNAs selected from a guide strand and a passenger strand, wherein the guide strand and the passenger strand are selected from the nucleic acids having SEQ ID NO: 1-4, 6-11, 14-17, 21-25, 27-30, 32-37, 40-43, 47-51, 53, 55-65, 69-70, 73, 75-85, 89-90, 93-120, 124, 126-132, 134-138, 140-150, 154, 156-162, 164-168 and 170-180; or b. The nucleic acid is an siRNA of group a, wherein at least one of the single-stranded RNAs selected from the guide strand and the passenger strand has a modification at 1-7 positions in the nucleotide sequence; or c. The nucleic acid is a small RNA selected from siRNA and microRNA (miRNA), wherein the RNA duplex of the small RNA consists of complementary or partially complementary nucleic acids of group a and / or group b; or d. the nucleic acid is a double-stranded RNA comprising at least two siRNA or sRNA nucleotide sequences of group a, b or c; or e. The nucleic acid is a single-stranded DNA consisting of 12-25 nucleotides and containing a sequence of 12 or more nucleotides homologous to one of the nucleotide sequences of the single-stranded RNA of group a or b; or f. The nucleic acid is a single-stranded DNA of group e, which has a modification at position 1-7 in the nucleotide sequence; The nucleic acid is provided for protection against plant pathogens using a method for targeted identification of effective small interfering RNA (esiRNA / ERNA) and sRNA derived therefrom, and effective antisense DNA oligonucleotides (eASO) of varying lengths, collectively referred to as effective nucleic acids (eNA), the method comprising the following steps: (i) RNA selected as a target for RNA silencing (RNAi) is produced using in vitro transcription and converted into small interfering RNA (siRNA) by endogenous Dicer-like protein (DCL) in cytoplasmic extracts of plant cells; (ii) generating a pool of siRNAs from a DCL formed from the RNAs used and identifying the siRNAs contained in the pool by RNA-seq analysis; (iii) adding messenger RNA (mRNA) of argonaute (AGO) protein to cytoplasmic plant cell extract, wherein the mRNA is synthesized by in vitro transcription and is constructed to encode the AGO protein of interest with a tag; (iv) using in vitro translation to form AGO protein molecules, which form RNA-induced silencing complexes (RISCs) with siRNA produced by the presence of DCL; (v) immunoprecipitating tagged siRNA-loaded AGO / RISC from BYL and determining the bound siRNA guide strand by RNA-seq analysis; (vi) using the RNA-seq data to compare with the RNA-seq data from step (ii) to determine those siRNAs enriched in AGO / RISC; (vii) then synthetically producing and testing its function in a cleavage assay using the labeled target RNA; and (viii) identifying esiRNA / ERNA in this way, and determining sRNA and eASO derived therefrom, collectively referred to as eNA; It is characterized by: I. To form the RISC in step (vii), 0.5 pmol of the mRNA of the AGO protein to be used is translated in the presence of 10-100 nM of the synthetic siRNA to be characterized and a 10-fold excess (0.1-1 μM) of a competing siRNA (e.g., siR gf698, selected from SEQ ID NOs: 205, 206, 207, and 208) in a reaction solution containing 50% (v / v) BYL; II. After incubating each batch at 25° C. for 2.5 h in step I, 3.4 pmol of nonspecific mRNA (e.g., encoding firefly luciferase protein SEQ ID NO: 209, Schuck et al., 2013) was added as a further competitor RNA, and 10 fmol of target RNA was added, the target RNA was labeled, and the reaction batch was incubated again at 25° C. for 15 min; III. During the incubation period in step II, the target RNA is cleaved by the formed AGO / RISC; IV. After gel electrophoresis of the extracted RNA, the remaining amount of target RNA or the resulting cleavage product is quantified compared to a control reaction (performed without siRNA) by measuring the band intensity (ImageQuantTL or ImageJ); V. Classifying each RISC formed with the siRNA as esiRNA / ERNA based on its measured cleavage activity (cleavage activity) on the target RNA; VI. Selecting an esiRNA / ERNA that endonuclease-converts at least 25% or more of the amount of target RNA initially used in method step II into cleavage products.

2. The nucleic acid according to claim 1, characterized in that The pathogen is cucumber mosaic virus (CMV) and contains ribonucleotides or deoxyribonucleotides of at least one nucleic acid against a target RNA of CMV, wherein the target RNA of CMV is selected from the target RNA having SEQ ID NO: 189 and 190.

3. The nucleic acid according to claim 1 or 2, characterized in that The pathogen is cucumber mosaic virus (CMV) and contains a ribonucleotide or deoxyribonucleotide sequence of at least one nucleic acid selected from the group consisting of nucleic acids having SEQ ID NOs: 1-4, 6-11, 14-17, 21-25, 27-30, 32-37, 40-43, 47-51, 53, 55-66, 69-70, 73, 75-86, 89-90.

4. The nucleic acid according to claim 1, characterized in that The pathogen is the southern root-knot nematode and contains a ribonucleotide or deoxyribonucleotide sequence of at least one nucleic acid against the target RNA of the southern root-knot nematode, wherein the target RNA of the southern root-knot nematode is selected from the target RNAs having SEQ ID NO: 191, 192 and 193.

5. The nucleic acid according to claim 1 or 4, characterized in that The pathogen is Meloidogyne incognita and contains a ribonucleotide or deoxyribonucleotide sequence of at least one nucleic acid selected from the group consisting of nucleic acids having SEQ ID NOs: 93-120.

6. The nucleic acid according to claim 1, characterized in that The pathogen is Botrytis cinerea and contains a ribonucleotide or deoxyribonucleotide sequence of at least one nucleic acid against a target RNA of Botrytis cinerea, wherein the target RNA of Botrytis cinerea is selected from the target RNAs having SEQ ID NO: 194, 195, 196, 197, 198 and 199.

7. The nucleic acid according to claim 1 or 6, characterized in that The pathogen is Botrytis cinerea and contains a ribonucleotide or deoxyribonucleotide sequence of at least one nucleic acid selected from the group consisting of nucleic acids having 124, 126-132, 134-138, 140-150, 154, 156-162, 164-168, and 170-180.

8. The nucleic acid according to claim 1, characterized in that The nucleic acid is a double-stranded RNA, and the double-stranded RNA contains a nucleotide sequence consisting of a pseudo-siRNA sequence and sequences of at least two siRNAs or sRNAs according to claim 1a, b or c.

9. The nucleic acid according to claim 8, characterized in that The nucleic acid has blunt ends or overhanging ends.

10. The nucleic acid according to claim 8 or 9, characterized in that The nucleic acid contains a spacer.

11. The nucleic acid according to any one of claims 8 to 10, characterized in that The pseudo-siRNA sequence and the spacer in the nucleic acid contain an element selected from the group consisting of a transcription promoter, a transcription terminator, a transport signal, a splice site, and a ribozyme.

12. The nucleic acid according to any one of claims 8 to 11, characterized in that The nucleic acid is selected from the group consisting of nucleic acids having SEQ ID NOs: 181, 182, 185, 186, and 200-204.

13. The nucleic acid according to claim 1, which has one or more chemical modifications, characterized in that The chemical modifications are selected from conjugates, such as GalNac; base modifications, such as 5-methylcytosine; 2' sugar modifications, such as 2'-O-methyl, 2'-fluoro, 2'-O-methoxyethyl (2'-MOE), cETBNA (ethylbicyclo (S)-bonded); other sugar modifications, such as "locked" (LNA) or "unlocked" (UNA); "backbone" modifications, such as phosphorothioate (PS) or "peptide nucleic acid" (PNA), and sugar phosphate modifications, such as morpholino / PMO (phosphodiamidate morpholino).

14. A composition for controlling pests of plants, comprising one or more nucleic acids according to any one of claims 1 to 13 and optionally one or more carrier substances and / or adjuvants.

15. Use of a nucleic acid or a composition according to any one of claims 1 to 14 for preventing and / or treating infestation and / or infection by pathogens in plants.

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