DE102018001134A1 - Method for the targeted identification of highly efficient "small interfering RNAs" ("eRNAs") for use in plants and other target organisms - Google Patents

Abstract

RNA interference (RNAi) is a crucial component of the plant immune system and is used to fight infections caused by pathogens. The currently used methods of RNAi still have considerable disadvantages. These are in particular when used siRNAs that are either inefficient or show off-target effects, as they are usually proposed on very simple in silico predictions and tested for their effect in empirical studies ("try and error"). Evidence of the optimal and specific effect can not be provided. The object of the present invention is to carry out RNAi with siRNAs or siRNA-encoding ds RNAs which have a proven high specificity, which act efficiently antipathogenic and which require neither lengthy breeding nor the use of transgenic methods, moreover ecologically justifiable and quickly adaptable to newly emerging ( According to the invention, an experimental in vitro system has been established, the essential component of which is an extract from the cytoplasm of plant cells. An RNA selected as the target for RNA silencing (RNAi) is generated by in vitro transcription produced and transformed in the cytoplasm of plant cells (BYL) by the endogenous Dicer-like proteins (DCL) to small interfering RNAs (siRNAS). The method according to the invention identifies siRNAs which can recognize and inactivate target RNAs in or outside the plant in a highly efficient manner.

Description

RNA interference, RNA interference (RNAi) or RNA silencing, is a technology that can be used in cells of virtually all organisms (best characterized in higher eukaryotes) for silencing or modulation of gene expression (Kim and Rossi, 2008) Carthew and Sontheimer, 2009). It uses cellular functions to inactivate or modulate the function of so-called target ribonucleic acid molecules (target RNAs) via the activity of small ribonucleic acid molecules (small RNAs, sRNAs). The target RNAs may be pathogenic but also of cellular origin.

RNAi is based on an evolutionarily conserved defense mechanism of the cell. For example, RNAi is a major component of the immune response to pathogens in insects and plants ( Ding, 2010; Zvereva and Pooggin, 2012; Gammon and Mello, 2015 ). In the cell, RNAi is triggered by ribonucleic acid molecules that are double-stranded or have double-stranded regions (ds RNAs). This is best investigated in the case of viral infections: RNAi-triggering ds RNAs originate from structured regions of viral messenger RNAs (mRNAs), from viral RNA genomes, or from viral ds RNA replication intermediates. The RNAs are perceived in the cell as so-called pathogen-associated molecular pattern (PAMP) and thus as 'foreign'. Detectors of dsRNAs include cellular enzymes belonging to the family of type III ribonucleases called Dicer or Dicer-like proteins (DCLs) (Alyari and Ding, 2009, Fukudome and Fukuhara, 2017, Song and Rossi, 2017). In the antiviral RNAi immune response of the plant, DCL2 and DCL4, which were originally characterized by the model plant Arabidopsis thaliana (At), play a central role in this ( Bouche et al., 2006; Deleris et al., 2006; Parent et al., 2015 ). DCL2 and DCL4 bind ds RNAs and process them by endonucleolytic cleavage to so-called small interfering RNAs (siRNAs). SiRNAs are 5'-phosphorylated ds RNAs (RNA duplexes), which have lengths of 20-24 nucleotides (nt) and have a 2 nt-long overhang at the 3 'end ( Bernstein et al. 2001; Elbashir et al. 2001a, Elbashir et al., 2001b; Martinez et al., 2002 ). Antiviral activity in particular has the siRNAs, which have a length of 21nt or 22 nt (Deleris et al., 2006). The siRNAs are incorporated after generation by the DCLs in so-called RNA-induced silencing complexes (RISC). In the process, the RNA duplex is wound up and a strand, the passenger strand, is removed and degraded. The other strand of the siRNA duplex, the guide strand, is bound by the so-called Argonaute (AGO) proteins; these are a major component of the RISC ( Hammond et al., 2001; Master, 2013; Kobayashi and Tomari, 2016 ). At At 10 different AGO are known; they have different functions, some of which are not fully understood (Vaucheret, 2008). AGO1 and AGO2 but also AGO3, AGO5 and AGO7 have been shown to be involved in the plant's antiviral immune response (Carbonell and Carrington, 2015). The different AGO proteins have different binding priorities for siRNA guide strands. For example, high-priority AGO1 binds RNAs containing a 5 'U nucleotide, whereas AGO2 binds RNAs with a 5'A nucleotide prioritized (Mi et al., 2008, Takeda et al., 2008, Schuck et al., 2013 ). After binding of the siRNA guide strand by AGO proteins, the corresponding RISC are introduced to the target RNA. An essential role is played by the sequence complementarity of the siRNA guide strands to the target RNAs (Liu et al., 2014); However, there seem to be other criteria that have not yet been fully characterized (see below). Under natural circumstances, the target RNAs are RNA molecules that were originally recognized by the DCLs as PAMPS and processed into siRNAs (so-called "cognate RNAs"). In viral infections, these are correspondingly viral mRNAs or viral genomes. After association of the AGO / RISC to the siRNA guide strand complementary regions of the cognate RNA, these may be coding as well as non-coding regions (untranslated regions), there is an AGO protein-catalyzed endonucleolytic cleavage of the RNA; this occurs approximately in the middle of the siRNA guide strand (Elbashir et al., 2001b; Elbashir et al., 2001c; Wang et al., 2008). Alternatively, there is evidence that siRNA-containing AGO / RISC associated with target RNAs inhibit the translation of these RNAs (Brodersen et al., 2008, Iwakawa and Tomari, 2013). The result of the activity of these "primary siRNAs" in the RNAi process is correspondingly a temporary inhibition of gene expression. Target RNAs are cleaved and degraded, or the synthesis of proteins encoded by these RNAs is inhibited. The result of the RNAi directed against pathogens is thus an inhibition of the gene expression or the multiplication of the pathogen (Zvereva and Pooggin, 2012).
In antiviral RNAi in the plant, there is another mechanism that can significantly enhance the immune response. So-called RNA-dependent RNA polymerases (RDR) exist in plant cells. These can, for example, recognize the degradation products of the target RNAs as templates that have been formed by the AGO activity and then synthesize new ds RNAs. These ds RNAs are in turn recognized and processed by DCLs and there are further, so-called "secondary siRNAs" ( Wang et al., 2010; Wang et al., 2011 ). Primary as well as secondary siRNAs are mediated by the plasmodesmata of the plant cells or the phloem via mechanisms that have not yet been fully elucidated transported into the complete plant organism (Mermigka et al., 2016). As a result of the gradual accumulation of siRNAs, the antiviral RNAi response can be effective both locally and systemically ( ).

RNAi is used for the artificial "knock down" of gene expression. For this purpose, siRNAs or other related small RNAs such as micro RNAs (miRNAs) are introduced into the cell in order to influence the function of specific target RNAs (McManus and Sharp, 2002, Schwab et al., 2006). In the simplest case, gene expression can be suppressed with this method, as described above (PTGS, post-transcriptional regulation of gene expression). In turn, PTGS of a particular mRNA may favor or inhibit gene expression elsewhere; In this respect, monovalent and / or multivalent RNAi can also influence complex gene expression patterns in the cell. In addition to genome-wide investigations of gene functions in basic research, RNAi is also used in the plant in many applications. Thus, through the use of RNAi, crops could be developed whose nutritional value was e.g. through regulation of biomass, levels of toxins, minerals, vitamins, amino acids, fatty acids, fiber, allergens, etc. In addition, plants could be produced which have a higher resistance to abiotic stress (for example, dryness, salinity of the soil, etc.) but also to biotic stress (infection or damage by pathogens) (Saurabh et al., 2014). The latter aspect will be described in more detail below.

As discussed earlier, RNAi is a critical component of the plant immune system and infection by pathogens can be controlled by inducing the synthesis of primary and secondary siRNAs against pathogen-targeted RNAs. Thus, via artificial expression of siRNAs directed against viral RNAs, a plant can be protected against the virus in question. This applies to viruses with an RNA genome, but also to viruses with a DNA genome (Khalid et al., 2017) or plant-infecting bacteria whose mRNAs can be attacked by RNAi. Furthermore, applications against other plant pathogens and parasites are known which, together with the measures against viruses and bacteria, are summarized under the term host-induced gene silencing (HIGS). Thus, siRNAs which are directed against mRNAs of plant-damaging insects, worms or fungi can also be expressed in a crop plant (Price and Gatehouse, 2008, Nowara et al., 2010, Banerjee et al., 2017). Similarly, RNAi can also be used against parasitic plants (Tomilov et al., 2008, Bandaranayake and Yoder, 2013). While in viral and bacterial infections RNAi is active directly in the organism of the host plant, in the latter cases, the expressed siRNAs are transmitted to the pathogen or the parasite - in the simplest case on the uptake of the plant as food (eg in insects and worms) ( so). The technical possibilities of HIGS have been significantly expanded in recent years, after it has become possible to produce siRNAs or other sRNAs much cheaper and to use them by spray methods (Dalakouras et al., 2016).

The aim of HIGS is in any case the damage or killing of the pathogen and thus the protection of the plant. However, the technology carries risks. Thus, there is the possibility that RNAi in addition to the actual target RNA attacks other RNAs. In this case, one speaks of off-target effects (Jackson and Linsley, 2004, Senthil-Kumar and Mysore, 2011). This can e.g. then be the case when the off-target RNAs have similar sequence or structural compositions as the actual target RNAs (Jackson et al., 2006, Kamola et al., 2015). The problem of off-target effects is particularly evident in HIGS against plant-infecting complex organisms such as fungi or parasites, where it is of great importance to attack exclusively their target RNAs, but not the target RNAs of related species.

In the cells of most eukaryotic organisms, RNAi can be induced by various techniques. Thus, ds RNAs, single siRNAs, miRNAs or other sRNAs can either transiently be introduced or transgeneically expressed. In plants, transgenes have been established for about two decades. Various transgenic plant species, including cultivated plants, are already in use (Herrera-Estrella et al., 2005, Kamthan et al., 2016). However, the production of transgenes is complicated and tedious. In addition, the use of transgenic plants is controversial and in many countries, e.g. in the EU, prohibited in agricultural application (Lucht, 2015). In particular, the use of transgenic plants which are to have immunity to pathogens is problematic: it has become clear in many applications that new resistant pathogens are formed in a relatively short time due to the evolutionary pressure which the use of transgenic pathogen-resistant plants produces. Simon-Mateo and Garcia, 2006; Lin et al., 2009; Tabashnik et al., 2013; Kung et al, 2015). In transgenic plants, there is thus a similar problem as in traditionally obtained by breeding pathogen-resistant plants.

For the long-term management of plant diseases, the knowledge has now become established that new methods must be developed that are effective antipathogenically effective and that neither require lengthy breeding nor the use of transgenic methods. In addition, these methods should be ecologically sound and quickly adaptable to emerging or re-emerging pathogens.

Possible solutions are the already mentioned transient use of RNAi. However, the transient introduction of nucleic acids and especially of sensitive RNA molecules into the plant, especially due to the rigid plant cell walls, is a significant technical problem. Here are for a long time most different procedures in the testing. Practices from basic research such as biolistic methods (via gene gun) or the introduction of nucleic acids by foreign organisms (vectors) such as viruses or agrobacteria are not suitable for agricultural crop production for economic or safety reasons. The use of viruses always poses a latent security problem, solely because of the real possibility of recombination events of viral and cellular RNAs (Bujarski, 2013). In the case of DNA viruses and agrobacteria, there is also the risk that parts of the vector genome can be incorporated into the plant genome ( Harper et al., 2002; Ulker et al., 2008 ). Only recently there are approaches that make an efficient, non-transgenic use of RNA possible. This is due, on the one hand, to the fact that in the last 5 or so years processes have been developed which allow production of ds RNA on a gram or even kilogram scale (Robinson et al., 2014). For example, the price of one gram ds of RNA required for use in a small crop field has been reduced from over $ 100,000 to $ 2 (Le Page, 2017). In addition, there are indications that the RNA produced in E. coli and applied to plant leaves can also be absorbed and activated there (Tenllado et al., 2003). This process becomes particularly efficient by packaging the ds RNAs into ecologically fully degradable nanoparticles (Mitter et al., 2017).

The currently used methods of RNAi still have significant application disadvantages. These are in particular when used siRNAs that are either inefficient or show off-target effects. Various applications have already been carried out worldwide with individual siRNAs or siRNA pools (common use of a large number of different siRNAs) in plants. These siRNAs were mostly proposed via very simple in silico predictions and then tested for their effect in empirical studies ("try and error"). Accordingly, it has not been proven whether or to what extent these siRNAs really act in the most optimal manner and thus also specifically on the targeted RNA. Accordingly, when using antipathogenic siRNAs, it was frequently associated with observations that the protective effect was only short and / or that off-target effects occurred ( Qu et al., 2007; Kung et al., 2012 ; Casacuberta et al., 2015. Other forms of application use artificial ds RNA molecules (Robinson et al., 2014) , These ds RNAs consist either of completely complementary sequences of several hundred nucleotides of the target RNAs or of correspondingly complementary sequences which are connected by a 'spacer' (so-called 'hairpin' molecules). Upon entry into the plant cell, these ds RNAs are processed by the DCLs into a multitude (one pool) of different siRNAs. Antipathogenic ds RNAs generally show good activity; however, the likelihood of recombination events and off-target effects, which may have a deleterious effect on the host organism, is particularly high. Accordingly, the use of ds RNAs, in the use of which unspecified siRNAs can be generated, is also considered problematic ( Duan et al., 2012; Dalakouras et al., 2016 ).

The task is therefore to carry out RNAi with siRNAs or siRNA-encoding ds RNAs which have a proven high specificity. In this way high efficacy is achieved and the likelihood of off-target effects is minimized.

• - In BYL sind alle DCL-Proteine aktiv, die ds RNA in 21, 22 und 24 nt siRNAs prozessieren können. Entsprechend können mRNAs oder ds RNAs, die zuvor über Standardverfahren (in vitro Transkription) hergestellt wurden, in den BYL eingesetzt werden, und die RNAs werden dann entsprechend durch die endogenen DCLs zu siRNAs prozessiert.
• - Bei Zugabe von mRNAs, die für die AGO-Proteine kodieren, können AGO-Proteine eigener Wahl durch in vitro Translation generiert werden. Durch Einsatz leicht modifizierter AGOmRNAs, können die betreffenden AGO-Proteine mit einem tag, einer kurzen Peptidsequenz bspw. ein FLAG-tag aus dem Protein Flagellin, z.B. am N-Terminus des Proteins, hergestellt werden. Auf diese Weise lassen sich die AGO-Proteine so darstellen, dass sie anschließend mit einem Antikörper gegen das tag, z.B. einen Anti-FLAG-tag Antikörper, über ein Standardverfahren aus dem Extrakt herausextrahiert (immunopräzipitiert) werden können. Entscheidend bei diesem Verfahren ist, dass die im BYL auf beschriebene Weise hergestellten AGO-Proteine funktionell aktiv sind, d.h. sie können siRNA oder andere sRNA Duplexe erkennen und die betreffenden guide strands binden. Darüber hinaus bilden die AGO-Proteine funktionelle RISC Komplexe und sind in der Lage, über guide strands assoziierte RNA-targets endonukleolytisch zu spalten (slicing) bzw. deren Translation zu blockieren. Ein weiterer entscheidender Aspekt ist, dass die betreffenden in vitro hergestellten AGO/RISC mit verschiedensten siRNAs beladen (programmiert) werden können, z.B. einem siRNA pool, der nach Zufügen einer target RNA in den BYL durch die endogenen DCL erzeugt wurde. Alternativ kann aber auch während der Translation des entsprechenden AGO-Proteins ein einzelner, synthetischer siRNA Duplex in hoher Konzentration zugesetzt werden. Der guide strand dieser einzelnen siRNA wird dann bevorzugt in den generierten AGO/RISC eingebaut. In einem weiteren Schritt wird die target RNA zu dem BYL zugegeben. Somit können nun entweder die Aktivitäten eines siRNA pools oder aber einer einzelnen siRNA bezüglich ihrer Fähigkkeiten zum AGO-katalysierten slicing oder zur Translationsinhibition einer target RNA in vitro getestet werden.

According to the invention, an experimental in vitro system has been established and optimized, the essential component of which is an extract from the cytoplasm of tobacco cells (BY-2 cells from Nicotiana tabacum, Nt). This extract form was originally described by Komoda et al. published ( Komoda et al., 2004 ). In the system according to the invention, culture-grown tobacco cells are digested by biochemical methods and the cytoplasm of the cells (BY2 lysate or BYL) is recovered in evacuated form (free of constituents of the plant vacuoles) (Komoda et al., 2004). In this system, on the one hand, mRNAs in the extract are translated in vitro, ie in the Eppendorf tube (test tube). This makes it possible to produce proteins of choice in the extracts. Furthermore, the inhibition of the translation of mRNAs can be measured by means of the mRNA-encoded reporter proteins, eg, firefly luciferase. Second, the complete replication process of RNA viruses (viruses with one (+) RNA genome) can be reproduced in vitro ( Komoda et al., 2004 ; Gursinsky et al., 2009 ). Third, the RNA silencing (RNAi) process can be readjusted in the test tube ( Iki et al., 2010; Schuck et al., 2013 ).
This third aspect is an essential part of the method according to the invention and shown in detail below.

• - In BYL, all DCL proteins are active that can process RNA in 21, 22 and 24 nt siRNAs. Accordingly, mRNAs or ds RNAs, which were previously prepared by standard methods (in vitro transcription), can be inserted into the BYL, and the RNAs are then correspondingly processed by the endogenous DCLs to form siRNAs.
• Upon addition of mRNAs encoding the AGO proteins, AGO proteins of their own choice can be generated by in vitro translation. By using slightly modified AGO mRNAs, the AGO proteins in question can be produced with a tag, a short peptide sequence, for example a FLAG tag from the protein flagellin, for example at the N-terminus of the protein. In this way, the AGO proteins can be displayed in such a way that they can subsequently be extracted (immunoprecipitated) from the extract with an antibody against the tag, for example an anti-FLAG tag antibody, using a standard method. Crucial to this method is that the produced in BYL in the manner described AGO proteins are functionally active, ie they can recognize siRNA or other sRNA duplexes and bind the relevant guide strands. In addition, the AGO proteins form functional RISC complexes and are capable of endonucleolytically cleaving (slicing) or blocking the translation of RNA targets associated with guide strands. Another crucial aspect is that the relevant in vitro produced AGO / RISC can be loaded (programmed) with a wide variety of siRNAs, for example a siRNA pool, which was generated by the endogenous DCL after adding a target RNA to the BYL. Alternatively, however, a single, synthetic siRNA duplex can be added in high concentration during translation of the corresponding AGO protein. The guide strand of this single siRNA is then preferably incorporated into the generated AGO / RISC. In a further step, the target RNA is added to the BYL. Thus, either the activities of a siRNA pool or of a single siRNA can now be tested for their abilities for AGO-catalyzed slicing or for the translation inhibition of a target RNA in vitro.

1. 1. Über einen im Folgenden als DCL-Assay bezeichneten Ansatz die endogenen DCL-Aktivitäten des BYL genutzt um, aus einer beliebigen RNA, die ds Bereiche enthält, siRNAs zu generieren.
2. 2. Durch in vitro Translation können AGO-Proteine eigener Wahl (z.B. die zehn verschiedenen bekannten AGO-Proteine aus At) hergestellt werden. Auch mit tag sind die AGO-Proteine funktionell, d.h. sie können sRNA guide strands binden und aktive RISC Komplexe bilden.
3. 3. Die in vitro translatierten AGO-Proteine können mit bereits im Extrakt vorliegenden/bereits aus RNA-Substraten über die endogenen DCLs generierten siRNAs (einem siRNA pool) beladen werden. Zwecks Identifizierung einzelner AGO-assoziierte siRNAs, können die RISC Komplexe über den AGO-assoziierten tag aus den Extrakten präpariert/immunopräzipitiert werden. Dieses Verfahren wird nachfolgend als AGO-IP bezeichnet.
4. 4. Alternativ zu den unter Ziffer 2 und 3 beschriebenen Verfahren kann bereits während der in vitro Translation des betreffenden AGO-Proteins eine synthetische siRNA hinzugefügt werden und das so generierte AGO-Protein damit präferenziell mit dieser siRNA beladen/programmiert werden.
5. 5. Fügt man den gemäß Ziffer 3 und 4 behandelten experimentellen Systemen eine target RNA hinzu, so kann die Aktivität der entsprechend generierten AGO/RISC auf dieser target RNA untersucht werde. Die verwendete target RNA wird dazu entweder radioaktiv markiert oder sie enthält ein Reportergen. In diesem, im Folgenden als Slicer-Assay bezeichneten Verfahren, kann man entsprechend die Spaltung der target RNA verfolgen und deren Effizienz auch quantifizieren. Alternativ kann die Inhibition der Translation des Reportergens bestimmt bzw. auch quantifiziert werden.

According to the invention

1. 1. Using an approach called DCL assay, the endogenous DCL activities of the BYL are used to generate siRNAs from any RNA containing the domains.
2. 2. By in vitro translation AGO proteins of their own choice (eg the ten different known AGO proteins from At) can be produced. Also tagged, the AGO proteins are functional, ie they can bind sRNA guide strands and form active RISC complexes.
3. 3. The in vitro translated AGO proteins can be loaded with siRNAs (a siRNA pool) already present in the extract / already generated from RNA substrates via the endogenous DCLs. In order to identify individual AGO-associated siRNAs, the RISC complexes can be prepared / immunoprecipitated from the extracts via the AGO-associated tag. This method will be referred to as AGO-IP hereinafter.
4. 4. As an alternative to the methods described under Nos. 2 and 3, a synthetic siRNA can already be added during the in vitro translation of the relevant AGO protein and thus the AGO protein thus generated can preferably be loaded / programmed with this siRNA.
5. 5. If a target RNA is added to the experimental systems treated according to points 3 and 4, the activity of the correspondingly generated AGO / RISC on this target RNA can be investigated. The target RNA used is either radioactively labeled or contains a reporter gene. In this method, hereinafter referred to as the slicer assay, it is possible to follow the cleavage of the target RNA and also to quantify its efficiency. Alternatively, the inhibition of translation of the reporter gene can be determined or quantified.

The BYL system has been shown to surprisingly produce only a very limited number of cleavage products when performing slicer assays with a siRNA pool (batch 3) and viral target RNAs ( ). This revealed that of several hundred or a thousand siRNAs generated from a ds RNA by the DCLs, only very few are actually active on the target RNA.

1. (i) Dass die DCL aus einer bestimmten viralen RNA nur eine bestimmte Zahl von siRNAs, davon einige mit hoher Präferenz, generieren ( ).
2. (ii) Dass von den DCL-generierten siRNAs wiederum nur eine bestimmte Anzahl in AGO1/RISC bzw. AGO2/RISC angereichert werden ( und ).
3. (iii) Dass von den in den AGO1/RISC bzw. AGO2/RISC eingebauten siRNA guide strands wiederum nur eine Minorität aktiv ist ( ).

Furthermore it could be shown:

1. (i) That DCLs from a given viral RNA generate only a certain number of siRNAs, some of which are highly preferential ( ).
2. (ii) That only a certain number of the DCL-generated siRNAs are enriched in AGO1 / RISC or AGO2 / RISC ( and ).
3. (iii) That only one minority of the siRNA guide strands installed in the AGO1 / RISC or AGO2 / RISC is active again ( ).

1. a) verschiedene Bereiche einer target RNAs unterschiedliche Zugänglichkeiten (Substratpräferenzen) für die DCLs aufweisen
2. b) die Effizienz des Einbaus von siRNA guide strands in die AGO/RISC nicht, wie bisher angenommen, ausschließlich durch die 5'Nukleotide der RNAs bestimmt wird
3. c) verschiedene Bereiche einer target RNA wiederum unterschiedliche Zugänglichkeiten (Substratpräferenzen) für die siRNA-beladenen AGO/RISC aufweisen

The data obtained by means of the method according to the invention indicate that

1. a) different regions of a target RNAs have different accessibilities (substrate preferences) for the DCLs
2. b) the efficiency of incorporation of siRNA guide strands into the AGO / RISC is not, as previously thought, exclusively determined by the 5 'nucleotides of the RNAs
3. c) different regions of a target RNA in turn have different accessibilities (substrate preferences) for the siRNA-loaded AGO / RISC

In summary, it can again be stated that of a large number of siRNAs which are processed by DCLs from a target RNA or which are incorporated by AGO / RISC, only a small number are efficiently active in the RNAi process on the target RNA.

1. a) Rekombinationsereignissen und
2. b) off-target Effekten so gering wie möglich zu halten.

The fact that only a small minority of the siRNAs generated from a ds target RNA is active in AGO / RISC sets the task of developing a method that is capable of producing exactly those siRNAs that are referred to below as highly efficient siRNAs, eRNAs , to be designated, to determine purposefully. All currently used methods for the determination of RNAi-common siRNAs are based on empirical studies or in silico predictions that consider almost exclusively the sequence homologies of siRNA guide strands and target RNAs, thus none of the essential aspects described above such as incorporation rate in AGO / RISC and Consider activity in AGO / RISC. The task of making effectively effective eRNAs identifiable arises from the need, as described above, to minimize the number of siRNAs used in an RNAi approach by the probabilities of

1. a) recombination events and
2. b) to keep off-target effects as low as possible.

1. (i) Durch die Kombinierbarkeit verschiedener eRNAs bzw. von eRNAs, die in verschiedene AGO/RISC Komplexe eingebaut werden, kann die Spezifität des RNAi maximal gesteigert werden.
2. (ii) Bei Pathogenen, die neu auftreten, kann eine RNAi Immunantwort durch eine Neukombination von anti-pathogenen eRNAs schnell und spezifisch angepasst werden.
3. (iii) Durch eine ökonomische Produktion von eRNAs sollte sich die Akzeptanz des Einsatzes von RNAi in der Pflanzenproduktion deutlich erhöhen und der Einsatz transgener Ansätze in der Pflanzenzucht deutlich vermindern lassen.

The use of effectively effective eRNAs also offers the following additional application advantages:

1. (i) The combinability of different eRNAs or eRNAs that are incorporated into different AGO / RISC complexes, the specificity of the RNAi can be maximally increased.
2. (ii) In new pathogens, an RNAi immune response can be rapidly and specifically adapted by recombination of anti-pathogenic eRNAs.
3. (iii) An economic production of eRNAs should significantly increase the acceptance of the use of RNAi in plant production and significantly reduce the use of transgenic approaches in plant breeding.

The method according to the invention identifies siRNAs which can recognize and inactivate target RNAs in or outside the plant in a highly efficient manner. An example of the use of this technology is the production of plants that are temporarily or lifelong protected against the infestation of pathogens such as viruses. Others include the production of plants that are temporarily or lifelong protected against the infestation of pathogens such as bacteria, worms, fungi, insects and parasitic plants. Still others include the production of plants that have temporarily or lifelong improved growth and consumer properties.

1. 1. Eine RNA, die als target für RNAi ausgewählt wurde, wird durch in vitro Transkription hergestellt und in BYL durch die endogenen DCL zu siRNAs umgesetzt (DCL-Assay). Bei dieser RNA kann es sich um eine mRNA beliebiger Art (d.h. aus Organismen beliebiger Art), eine genomische virale RNA, oder aber ein virales ds RNA Replikationsintermediat handeln. Aus der betreffenden RNA entsteht ein DCL-generierter siRNA pool. Die in diesem pool enthaltenen siRNA können durch RNA seq. Analyse ermittelt werden.
2. 2. Dem BYL wird die wiederum über in vitro Transkription synthetisierte mRNA eines AGO-Proteins zugefügt. Die mRNA ist so aufgebaut, dass sie für das AGO-Protein mit einem tag kodiert. Über in vitro Translation entstehen AGO-Proteinmoleküle mit tag. Diese AGO Moleküle bilden RISC Komplexe. Diese werden mit den vorliegenden DCL-generierten siRNAs beladen/programmiert. Die Beladung richtet sich nach der Verfügbarkeit der durch die DCL-Aktivität entstandenen siRNAs bzw. nach bekannten und bisher unbekannten Beladungsprioritäten der betreffenden AGO/RISC.
3. 3. Über den tag werden siRNA-beladene AGO/RISC aus den BYL immunopräzipitiert (AGO-IP). Die gebundenen siRNA guide-strands werden durch RNA seq. Analyse ermittelt. Es werden solche siRNAs, die besonders stark in AGO/RISC angereichert sind, durch eine RNA seq. Analyse identifiziert.
4. 4. Die Schritte (1) und (2) werden in einem Parallelansatz wiederholt. In diesen Ansatz wird dann die target RNA (markiert) eingesetzt und die Spaltung in einem Slicer-Assay über Detektion der Spaltprodukte gemessen. Es entstehen bei der RNAi-mediierten Umsetzung einer target RNA aufgrund der nur begrenzten Zahl funktioneller siRNA aus einem siRNA pool nur eine begrenzte Zahl an Spaltprodukten. Über Korrelation der Größe der entstandenen Spaltprodukte und der in (1-3) erhaltenen RNA seq. Daten lassen sich entsprechend siRNAs ermitteln, die einerseits in AGO/RISC hochangereichert und andererseits hochaktiv sind.
5. 5. So ermittelte siRNA-Kandidaten werden synthetisch hergestellt und ihre Funktionalität wiederum in einem Slicer-Assay mit markierter target RNA getestet.
6. 6. Die so ermittelten eRNAs können dann transgen oder transient angewendet werden. Entsprechend der im Slicer-Assay ermittelten Effizienz der eRNAs, werden diese einzeln oder in Kombination eingesetzt. In Kombination bedeutet, dass entweder verschiedene einzelne siRNAs kombiniert und eingesetzt werden oder dass ds RNAs generiert und eingesetzt werden, von denen erwartet werden kann, dass sie in der Pflanze zu eRNAs prozessiert werden.
7. 7. Mit den behandelten („eRNA-vakzinierten“) Pflanzen werden Virusbeslastungstests durchgeführt, um ihre Wirksamkeit in einem proof of concept abzusichern.

According to the invention, the process is carried out as follows:

1. 1. An RNA selected as the target for RNAi is prepared by in vitro transcription and converted into siRNAs in BYL by the endogenous DCL (DCL assay). This RNA can be an mRNA of any kind (ie of any kind), a genomic viral RNA, or a viral ds RNA replication intermediate. From the RNA in question, a DCL-generated siRNA pool is generated. The siRNAs contained in this pool can be determined by RNA analysis.
2. 2. The BYL is added to the mRNA of an AGO protein synthesized again via in vitro transcription. The mRNA is designed to encode the AGO protein with a tag. About in vitro translation arise AGO protein molecules with tag. These AGO molecules form RISC complexes. These are loaded / programmed with the present DCL-generated siRNAs. The loading depends on the availability of siRNAs generated by the DCL activity or on known and previously unknown loading priorities of the relevant AGO / RISC.
3. 3. During the day, siRNA-loaded AGO / RISC are immunoprecipitated from BYL (AGO-IP). The bound siRNA guide-strands are determined by RNA seq. Analysis. Such siRNAs, which are particularly strongly enriched in AGO / RISC, are identified by RNA seq. Analysis.
4. 4. Steps (1) and (2) are repeated in a parallel approach. In this approach, the target RNA (labeled) is then used and the Cleavage in a slicer assay measured by detection of the cleavage products. Due to the limited number of functional siRNAs from a siRNA pool, RNAi-mediated conversion of a target RNA results in only a limited number of cleavage products. By correlating the size of the resulting cleavage products and the RNA seq. Data obtained in (1-3), it is possible to determine correspondingly siRNAs which on the one hand are highly enriched in AGO / RISC and, on the other hand, highly active.
5. 5. SiRNA candidates determined in this way are synthesized and their functionality is tested in a sliced assay with labeled target RNA.
6. 6. The thus-determined eRNAs can then be applied transgenically or transiently. According to the efficiency of the eRNAs determined in the slicer assay, these are used individually or in combination. In combination, this means that either different single siRNAs are combined and used, or that the RNAs are generated and used that can be expected to be processed into eRNAs in the plant.
7. 7. Virus control tests are performed on the treated ("eRNA-vaccinated") plants to ensure their effectiveness in a proof of concept.

The process according to the invention described above is described in more detail by the following application examples.

Example of Use: Identification of Tomato Bushy Stunt Virus (TBSV) ERNAs

Tomato bushy stunt virus (TBSV) is a (+) RNA virus of the Tombusviridae family (White and Nagy, 2004). Most plant-infecting viruses are (+) RNA viruses. In these viruses, the genome is a (+) RNA; it acts directly as mRNA after entry into the host cell. The translation-generated viral proteins, including the viral RNA-dependent RNA polymerase, form a replication complex that generates new progeny (+) RNA molecules in a two-step process via a (-) RNA intermediate (Gunawardene et al., 2017). The resulting replication intermediate is ds RNA. TBSV was used here as a model virus.

DCL assay

• - genomic (+) TBSV RNA and (-) TBSV RNA were prepared by in vitro transcription, (ds) TBSV RNA was prepared by annealing equimolar amounts of (+) TBSV RNA and (-) TBSV RNA
• - BYL was incubated with (+) TBSV RNA or (ds) TBSV RNA under translation conditions for 2 h (50% BYL, ATP, GTP, creatine-P, creatine kinase, 25 ° C)
• Total RNA was isolated from the batches (proteinase K reaction, phenol-chloroform extraction, ethanol precipitation)
• Total RNA was characterized by RNA seq analysis (Illumina)

From the TBSV RNAs was produced by DCL activity in the BYL siRNA pools. The performed RNA seq. Analysis indicated the enrichment of siRNA species generated from genomic (+) TBSV RNA or (ds) TBSV RNA according to the substrate preference of DCL contained in BYL ( )

AGO IP

• - AGO1 mRNA (from Nicotiana tabacum, Nt) or AGO2 mRNA (from Arabidopsis thaliana, At), each with sequence for N-terminal FLAG tag, were translated in BYL for 2 h. The translation was performed in the presence of (+) TBSV RNA or (ds) TBSV RNA to allow loading of the AGO / RISC at the moment of their formation with the siRNA pools generated from the TBSV RNAs by the DCLs.
• The reaction was stopped by addition of anti-FLAG M2 affinity gel (anti-FLAG antibody coupled to agarose) and immunoprecipitated overnight at 4 ° C
• Unbound proteins and RNAs were removed by several washes
• Total RNA was isolated from the batches (proteinase K reaction, phenol-chloroform extraction, ethanol precipitation)
• Total RNA was characterized by RNA seq analysis (Illuma)

The AGO proteins were generated by in vitro translation; at the same time, BYL's own DCLs processed viral RNA into vsiRNAs. SiRNA duplexes with high affinity for AGO proteins were incorporated into AGO1 / RISC or AGO2 / RISC; one strand (guide strand) remained in the RISC, the other strand (passenger strand) was removed. By immunoprecipitation and subsequent RNA seq. Analysis, the siRNA guide-strands specially enriched in AGO1 or AGO2 RISC were identified. It could be shown that in comparison to the distribution pattern of the siRNAs (related to the total TBSV genome) after DCL assay ( ), different distribution patterns are obtained in AGO1 / RISC and AGO2 / RISC ( ; ). This allowed a specific accumulation of siRNAs in the AGO / RISC compared to the original siRNA pool. The enrichment was also followed by Mi et al. (2008), ie preference was given to siRNAs with a 5'-U in AGO1 / RISC, and preferential siRNAs to a 5'-A in AGO2 ( ).

Slicer assay with siRNA pool

• - The TBSV genome or a part of the TBSV genome to be tested (s. ) was produced single- or double-stranded (unlabelled) by in vitro transcription
• AtAGO2 mRNA was translated in BYL in the presence of the appropriate TBSV RNA (2 h incubation). The resulting AGO / RISC complexes were loaded / programmed at the moment of formation with the siRNAs produced by the DCL activity in the same extract
• Subsequently, the identical, but this time marked, single-stranded TBSV RNA was added as a target for the corresponding siRNA-loaded RISC and the incubation continued for 20 minutes
• Total RNA was isolated from the batches (proteinase K reaction, phenol-chloroform extraction, ethanol precipitation)
• The slicing products of the slicing reaction were analyzed by denaturing polyacrylamide gel electrophoresis (PAGE with 8 M urea) and subsequent autoradiography (Phosphor Imager).

From the shown TBSV fragment A (5'-terminal 740 nt), despite a large number of siRNAs generated from the fragment, only a few main cleavage products were generated in the slicer assay ( ). This result showed that only a minority of siRNAs, which are basically generated from an RNA, are efficient in RNAi.

Slicer assay with selected siRNAs (1)

Selected 21 nt siRNAs whose guide strands are in AGO-IPs in RNA Seq. were detected very frequently (see also heat map in ) were synthesized by a commercial supplier and tested in slicer assays. It is known that during viral replication only the actual genomic RNA or (+) RNA molecules which are newly formed during replication can be attacked by AGO / RISC (Schuck et al., 2013). Correspondingly, siRNAs identified by the RNA analysis were used whose sequence corresponds to the (-) strand of the TBSV, ie which are complementary to the (+) RNA of TBSV (denoted by "m" for "minus"). As a passenger strand, the corresponding complementary sequence was used. The slicer assay was carried out with the individual, synthetic siRNAs and AtAGO1 / RISC on complete, labeled genomic TBSV RNA ( ; Sequence of the assay shown, also compare with ).

Of a series of siRNAs previously identified by RNA seq analysis, four RNAs, 176m, 186m, 3243m and 3939m (the latter not in shown) as efficiently cleaving siRNAs. This result showed the general applicability of the method for the identification of eRNAs. It also showed that again only one minority of siRNA guide strands associated with AGO / RISC is effective. In addition, it also became clear that the identification of siRNA guide strands that accumulate in AGO / RISC is not sufficient as the sole method to identify eRNAs. In accordance with a further step, namely the correlation of the data obtained in the RNA seq. Analyzes with the data of the slicer assay, which was carried out with the siRNA pool ( ).

Slicer assay with selected siRNAs (2)

The RNA seq. Data showed the accumulation of siRNAs generated from (+) TBSV RNA and ds (TBSV) RNA, respectively, in AGO2 / RISC. At the same time, however, it became clear with the data described above that high accumulation rates of siRNA guide strands (indicated by the heat map in ) in RNA seq. did not necessarily indicate eRNAs (see above). Accordingly, the accumulation rates of the RNA seq. Analyzes were as a result of the slicer assay with the RNA pool ( ). The size of the cleavage products of the viral RNA allowed conclusions to be drawn about the respective siRNAs involved. So in the application shown in and For example, several 200 nt long, dominant cleavage products determined that originated from the investigated TBSV genome area after implementation with the siRNA pool. After correlation with the RNA seq. Data it became clear that in fact there was a high accumulation of siRNAs in AGO2 / RISC, which could be presumed to cause the cleavage of the TBSV RNA into 200 nt long cleavage products. These siRNAs (207m, 209m, 221m, 228m) were synthesized and assayed in the slicer assay with AGO2 / RISC and the corresponding TBSV RNA genome region (labeled) (assay comparable to ). To further test the efficiency of these siRNAs, the slicer assay was also performed under altered conditions. Here, the quantity of the siRNAs tested was significantly reduced and, in addition, a 10-fold molar excess of a non-specific control siRNA was added. In addition, a mix of different siRNAs was used.

It could be shown that the correlation of the RNA seq. Analysis with the in and Slice assay with the siRNA pool, which allows direct identification of eRNAs: Virtually all identified eRNAs showed extremely high slicing efficiency; siRNA 209m was identified as the most efficient anti-TBSV siRNA by the competition approach. In close agreement with the data from the slicer assay (1), it became clear again that the level of accumulation rates of siRNAs in AGO / RISC, which can be determined by RNA seq. Analyzes, does not allow conclusions to be drawn regarding the ultimate efficiency of the respective siRNAs. In the executed combination, however, both methods are optimally meaningful. The method according to the invention for the identification of eRNAs is thus based on the correlation of data obtained on the one hand from RNA analysis of AGO-IPs and on the other hand from slicer assays with the respective AGO proteins. The starting material used in each case is a pool of DCL-generated siRNAs of a target RNA.

1. 1) Generierung transgener eRNA-exprimierender Pflanzen, die gegen Infektionen durch das Tomato bushy stunt virus geschützt sind. Mit aktueller (state of the art) Technologie ( Prado et al., 2014 ; Cillo and Palukaitis, 2014 ; siehe auch Anwendung (2)) wurden transgene Solanum lycopersicum (Sl) Tomatenpflanzen generiert, die in persistenter Form identifizierte anti-TBSV eRNAs (209m, 221m, 3243m und 3939m) exprimieren. In Kontrollexperimenten wurde die Expression der siRNAs sichergestellt. So hergestellte Sl Pflanzen wurden über verschiedene Verfahren, wie TBSV-exprimierende Agrobakterien (siehe unten) oder rub-in inokuliertem TBSV belastet. Die Virusreplikation in den Pflanzen wurde durch RT-PCR Verfahren überprüft. Im Gegensatz zu Kontrollpflanzen, die nicht antiviral-wirkende siRNAs exprimierten, konnte bei den eRNA-behandelten Pflanzen eine vollständige und langfristig auch gegen mehrfach folgende Belastungen erhaltene Protektion gegen das Virus demonstriert werden. D.h. das Virus wurde 1-2 Wochen nach challenge aus den Pflanzen entfernt (Nachweis über RT-PCR nicht mehr möglich). Die Generierung transgener Pflanzen diente als Kontrolle (proof of concept), dass der hier erfindungsgemäß etablierte eRNA Ansatz funktionell ist.
2. 2) Transienter Schutz von Pflanzen durch eRNAs gegen Infektionen durch das Tomato bushy stunt virus. Transiente Anwendungen erfolgten über verschiedene Verfahren.

Application examples for the use of eRNAs for the generation of plants which are protected against infections by the Tomato bushy stunt virus.

1. 1) Generation of transgenic eRNA-expressing plants which are protected against infections by the Tomato bushy stunt virus. With current (state of the art) technology ( Prado et al., 2014 ; Cillo and Palukaitis, 2014 ; see also application (2)) transgenic Solanum lycopersicum (Sl) tomato plants were generated expressing in a persistent form identified anti-TBSV eRNAs (209m, 221m, 3243m and 3939m). In control experiments, the expression of the siRNAs was ensured. Sl plants prepared in this way were loaded by various methods, such as TBSV-expressing Agrobacteria (see below) or rub-in inoculated TBSV. The virus replication in the plants was checked by RT-PCR method. In contrast to control plants that did not express antiviral siRNAs, in the eRNA-treated plants a complete protection against the virus was obtained, which in the long term was also protected against multiple exposures. This means that the virus was removed from the plants 1-2 weeks after challenge (detection via RT-PCR is no longer possible). The generation of transgenic plants served as a control (proof of concept) that the here established according to the invention eRNA approach is functional.
2. 2) Transient protection of plants by eRNAs against infections by the Tomato bushy stunt virus. Transient applications were made using different methods.

In the form of artificial microRNAs (amiR) intermediates.

Agrobacteria expressed eRNAs in the form of amiRs in Nicotiana benthamiana (Nb) tobacco plants. For this, the siRNA sequences were integrated into the precursor structure of At miR390a instead of the miRNA sequences (vector pMDC32B-AtMIR390a-B / c, Carbonell et al., 2014) ( ). The siRNAs are expressed by the CaMV 2x 35S promoter and nos terminator. The pMDC32B asiRNA constructs as well as control constructs (containing nonspecific siRNAs) were each transformed into Agrobacterium tumefaciens (GV3101). 4-6 week old Nb plants were infiltrated with the transformed agrobacteria (2 leaves per plant, ca. 1 ml agrobacteria suspension). In control experiments (Northern blots) the expression of the respective siRNAs in the respective plants was ensured. The plants were allowed to grow for 48 h after inoculation (14 h light at 23 ° C, 10 h dark at 21 ° C). In parallel, a plasmid (pBGW-TBSV-Rib1: 35S-TBSV cDNA-HDV-ribozyme) encoding the TBSV RNA genome was transformed into Agrobacteria as a control mCherry (reporter gene) -coding plasmid (pBGW-mCherry). The TBSV-expressing Agrobacteria were then diluted 1: 1000 with mCherry-expressing Agrobacteria and the Agrobacterium mixture inoculated into the pretreated leaves (with amiRNA expression): 200 μl per plant (4 inoculation sites with 50 μl, 2 per leaf). The Nb plants were then grown again under the same conditions (14 h light at 23 ° C, 10 h dark at 21 ° C) and examined for symptom formation up to 40 days after inoculation ( ). In a series of independent infection experiments with Nb plants, a high and statistically highly significant protective effect of the previously identified by the inventive method eRNAs against TBSV infection was found ( ). Thus, it was found that even with the treatment with individual eRNAs such as 209m protection of up to 90% of the plants was achieved. In other words, the appropriate treatment with the siRNA 209m provided lasting protection of the plants (over the entire experimental period) against TBSV infection. Like other eRNAs, 209m was protective in both AGO1-compatible (5'U) and AGO2-compatible (5'A) forms. In the other unprotected plants, the infection was significantly delayed, ie the first symptoms of the viral infection were observed only after an average of about 20 days, while this was already after about 9 days in the Control plants was the case ( ). By producing and using a mix of different eRNAs (consisting of siRNAs 179m, 186m, 207m, 209m, 221m, 228m, and 238m), protection against a single eRNA could not be increased - this was probably related to a lower availability of the individual eRNAs Mix together - however, the protection of the unprotected plants could be extended to an average of about 25 days. In addition, when using a siRNA mix, the probability of the emergence of resistant virus variants is significantly reduced. As another important finding, it was shown that the individual eRNAs' activity correlated exactly with their efficiency in the in vitro slicer assay. For example, the two siRNAs 3701m and 3722m showed no slicer activity with AGO2 or AGO1 ( ), and they also had no protective effect. The siRNAs 179m, 3243m and 3939m, on the other hand, showed slicer activity, which was weaker compared to 209m; accordingly, these eRNAs had a protective effect, but was less pronounced than that of 209m ( ).

(ii) In the form of eRNA-encoding ds RNAs.

Plasmid constructs containing a promoter (e.g., the T7 promoter) in front of an integrated eRNA-encoding cDNA were prepared. The cDNA consisted of two sequence sequences of several eRNAs and a spacer (for example the DNA template of the intron of pyruvate orthophosphate dikinase mRNA). The cDNAs were constructed in such a way that the sequence sequence of several (i.d.R.> 5) eRNAs began immediately after the T7 promoter. The sequence of an eRNA was immediately followed by the sequence of the next eRNA, etc. At the end of the second sequence of sequences, the cDNA encoded a transcription terminator, e.g. the T7 terminator. Transcription of the cDNA, e.g. via the T7 RNA polymerase, accordingly generated a ds hairpin RNA consisting of a region encoding only the selected eRNAs. The ds region was interrupted by a largely dsRNA-free loop. The eRNA coding regions were organized to encode either 21 nt siRNAs or 22 nt siRNAs. Other plasmid variants contained eRNA coding regions that encoded only eRNAs with a 5'U or 5'A. In this way ds RNA hairpins were generated which, after uptake by the plant, are either predominantly processed by DCL4 (producing 21 nt siRNAs) or DCL2 (producing 22 nt siRNAs). Alternatively, eRNA-encoding ds RNAs were generated, in which case, after uptake by the plant and processing by DCLs, the resulting siRNA guide strands were preferably integrated into AGO1 / RISC or AGO2 / RISC.

• (a) die flexible Kombinierbarkeit mehrerer eRNAs,
• b) die flexible Kombinierbarkeit von 21 und 22 nt eRNAs,
• c) die flexible Kombinierbarkeit AGO1 und AGO2 inkorporierender eRNAs. Mit diesen Experimenten wurde der proof of concept erbracht, dass ein sehr kostengünstiger und nichttransgener Einsatz von eRNAs möglich ist.

The respective ds RNA hairpins were first prepared by in vitro transcription and the correct processing into the respective eRNAs or their incorporation into the respective AGO / RISC in the BYL in vitro system (see above) was checked. With dsRNAs composed of the siRNA sequences of 179m, 209m, 221m, 3243m and 3939m (one variant with 5'U in all siRNAs, one variant with 5'A), it could be shown that the construction scheme outlined above of the transcriptional plasmids outlined above for the particular dsRNA hairpins did indeed result in the generation of the desired eRNAs. Thus, it was also generally demonstrated that it is possible to produce eRNA-specific ds RNAs. The respective eRNA-encoding hairpin ds RNAs were subsequently produced in E. coli strain HT115. This strain is RNase III deficient and expresses the T7 RNA polymerase. The expression of the RNA could be induced by induction of IPTG (isopropyl β-D-1-thiogalactopyranoside) and the RNA purified by ion exchange chromatography from the bacterial cells ( Nwokeoji et al., 2017 ). Purified RNA packaged in either nanoparticles or, alternatively, with the complete ds RNA-containing E. coli extract were analyzed according to published protocols ( Tenllado et al, 2003; Mitter et al., 2017 ) treated the leaves of Nb plants and then the plants according to the patterns described above then loaded with TBSV. Both treatment methods, based on eRNA-encoding hairpin ds RNAs, provided similarly effective protection of plants against TBSV as was possible with Agrobacterium-expressed eRNAs. It proved extremely effective

• (a) the flexible combinability of multiple eRNAs,
• b) the flexible combinability of 21 and 22 nt eRNAs,
• c) the flexible combinability of AGO1 and AGO2 incorporating eRNAs. With these experiments, the proof of concept was demonstrated that a very cost-effective and non-transgenic use of eRNAs is possible.

(iii) in the form of purified eRNAs.

Using a completely new process, eRNAs could be produced and purified in yeast. In yeast of the type Kluyveromyces lactis foreign proteins ( Arnold et al., 2012 ) but also the RNAs are produced. The latter is possible because K. lactis RNAi is deficient, ie, neither DCLs nor AGO or RDR proteins express themselves (Drinnenberg et al., 2009). About a previously developed process ( Krijger et al., 2012 ), K. lactis strains were prepared which express either DCL4 (from At) the DCL4 co-factor DRB4 (from At) or, alternatively, DCL2 (from At). Additionally expressed these yeast strains are the ds RNA hairpins described above. The plasmids used were similar to the T7 transcription plasmids described above, except that the T7 promoter was replaced by the K. lactis P _LAC4 promoter and the terminator by the K. lactis TT terminator. The P _LAC4 promoter allows induced expression via the addition of lactose into the yeast growth medium. The functioning of the generated strains was performed by detecting the expression of DCL4, DRB4 or DCL2 in the yeast, the proof of the correct processing of eRNA-encoding ds RNAs in the desired siRNAs was again with the BYL. In yeast, the respective expressed eRNA-encoding ds RNAs were processed by the present DCLs into the eRNAs, and the eRNAs were purified from the yeast cells by chromatographic methods. Protective experiments with Nb plants and TBSV loading were carried out in purified form or re-packed with nanoparticles, and again a sustainable protection of the plants against the virus could be demonstrated. According to the proof of concept, these experiments enabled the efficient use of RNAi in non-transgenic applications, even with purified eRNAs.

list of figures

• Antiviral RNA silencing Double-stranded (ds) viral RNA is detected in the cytoplasm of the infected cell by Dicer-like proteins (DCL) and processed into small interfering RNAs (siRNAs). These siRNAs are incorporated into RNA-induced silencing complexes (RISC). The siRNA duplex is wound up and a strand (guide strand) bound by the Argonaut (AGO) protein, the main component of the RISC. The other strand of the duplex (passenger strand) is removed and dismantled. Viral RNAs that have complementary sequences to the siRNA are recognized by the loaded RISC and destroyed by AGO-catalyzed endonucleolytic cleavage. Of the 10 different AGO proteins identified in Arabidopsis thaliana, AGO1 and AGO2 in particular have antiviral activity. The antiviral immune response can be significantly enhanced by plant RNA-dependent RNA polymerases (RDR). These enzymes can use the degradation products of viral RNA as a template to produce new ds RNAs. These are again detected by DCLs and processed into secondary siRNAs.
• Generation of TBSV siRNAs by DCL Activities in BYL Genomic (+) TBSV RNA or (ds) TBSV RNA was incubated in BYL and processed into siRNAs by BYL's own DCLs. From these approaches, total RNA was isolated and characterized by RNA Seq analysis (Illumina). Shown here is the relative abundance of sequenced 21 nt vsiRNA species whose sequence either corresponded to the (+) TBSV RNA (green), or was identical to the (-) TBSV RNA (red). The signals shown in the diagram correspond to the position of the 5 'nucleotide of an siRNA on the (+) TBSV genome, and particularly common siRNAs were numbered.
• Enrichment of AGO-associated siRNAs Genomic (+) TBSV RNA in BYL processed into siRNAs. In the same approach, either AGO1 or AGO2 protein with N-terminal FLAG tag was generated by in vitro translation of corresponding mRNAs. Subsequently, the AGO / RISC loaded with TBSV siRNAs were isolated by immunoprecipitation with the aid of an anti-FLAG antibody. The siRNAs were purified from the complexes and characterized by RNA-Seq analysis (Illumina). Shown here is the relative abundance of sequenced 21 nt vsiRNA species (see ).
• Enrichment of AGO-associated siRNAs (ds) TBSV RNA was processed into siRNAs in BYL. In the same approach, either AGO1 or AGO2 protein with N-terminal FLAG-tog was generated by in vitro translation of corresponding mRNAs. Subsequently, the AGO / RISC loaded with TBSV siRNAs were isolated by immunoprecipitation with the aid of an anti-FLAG antibody. The siRNAs were purified from the complexes and characterized by RNA-Seq analysis (Illumina). Shown here is the relative abundance of sequenced 21 nt vsiRNA species (see ).
• Properties of the BYL-DCL Generated and AGO-Associated VSiRNAs Shown is the relative abundance of the four nucleotides A, C, G and U in the TBSV genome and at the 5 'position of the siRNAs generated by BYL's own DCLs. subsequently isolated by AGP-IP. In the AGO1-IP siRNAs were enriched, which have a 5'-U, in AGO2-IPs, however siRNAs with a 5'-A.
• Slice assay with siRNA pool A region of the (+) TBSV genome (fragment A) was added to BYL and processed into siRNAs by BYL's own DCLs. In the same approach, AGO2 protein was generated in parallel by in vitro translation and loaded with the TBSV siRNAs formed. Subsequently, the same TBSV fragment, this time radioactively labeled, was added and incubated as a target for the thus programmed RISC. To the To detect cleavage of the target RNA, total RNA was isolated from the batches and analyzed by denaturing PAGE and autoradiography. The main split products are marked with an asterisk in the illustration.
• Slicer assay with selected TBSV siRNAs (1) Selected 21 nt siRNAs whose guide strands were enriched in AGO-IPs were tested in the slicer assay with genomic (+) TBSV RNA. To do this, AGO1 protein was generated in BYL by in vitro translation. The translation reaction was carried out in the presence of the synthetic siRNA oligonucleotides to be tested so that incorporation of the desired siRNAs into the AGO / RISC occurred. Thereafter, radiolabeled (+) TBSV RNA was added as a target and incubated. From these approaches, total RNA was isolated and analyzed for cleavage products by denaturing PAGE and autoradiography. Some siRNAs proved to be active (eg siRNA179m, siRNA186m), in other cases (eg siRNA3722m, siRNA3722) no cleavage fragments could be detected
• Slicer assay with selected TBSV siRNAs (2) By comparison of the cleavage products from the slicer assay with siRNA pool ( ) with the RNA Seq data, the number of siRNA candidates that might be responsible for observed fission fragments was significantly limited. The heat map presented above shows the relative abundance of (-) vsiRNAs generated from the relative region of the TBSV genome (single- or double-stranded) and their accumulation in the AGO-IP. The frequency of an siRNA is indicated by the colored representation of the position of its 5 'nucleotide from black (no siRNA or small number) via green (medium number) to red (high number). The siRNAs 207m, 209m, 221m and 228m were selected for further analysis as they were detected in the AGO2-IP with (+) TBSV RNA and / or (ds) TBSV RNA (green arrows under the heat map) and their Position matches the estimated size of the fission fragments. These siRNAs were used as synthetic oligonucleotides in a slicer assay with TBSV fragment A (see FIG. In parallel, the slicer assay was carried out with siRNA pools generated from (+) TBSV fragment A or (ds) TBSV fragment A (see FIG. ). It was found that all siRNAs tested were active and that the fission fragments generated by siRNA209m and siRNA221m each corresponded to a fission fragment caused by the siRNA pool (left gel pattern). In order to determine which of the siRNAs identified so far from TBSV fragment A acts most efficiently, the slicer assay was repeated under altered conditions. The amount of siRNA oligonucleotides was reduced to 10% and 10-fold excess of non-specific competitor siRNA was added. In addition, a mix of different siRNAs was used (right gel picture). With this experiment, siRNA209m was identified as the most efficient of the siRNAs tested.
• System for expression of siRNAs as artificial microRNAs The expression of TBSV siRNAs in Nicotiana benthamiana was carried out in the form of artificial microRNAs (amiRNA) using a method developed by Carbonell et al. (2014) developed system (plasmid pMDC32B-AtMIR390a-B / c). In the process, the siRNA sequences to be expressed are integrated into the precursor structure of the Arabidopsis thaliana microRNA390a in such a way that exactly the sequences of the miRNA guide strand (highlighted in blue) and the passenger strands (miRNA *, highlighted in green) are replaced. Expression is under the control of a CaMV 2x35S promoter and a nos terminator. After transcription in the plant cell, the siRNA is cut out of the primary transcript by Dicer-like 1 (DCL1) and incorporated into AGO / RISC.
• TBSV infection experiment with siRNA-expressing N. benthamiana plants. Two leaves of 4-6 week old N. benthamiana plants were infiltrated with Agrobacterium tumefaciens cultures containing siRNA-encoding plasmids (see p. ) contained. In the example shown here, the control plants were given a mix of agrobacteria expressing two different control siRNAs (not TBSV specific) while the test plants received a mix of agrobacteria containing the TBSV siRNAs 179m, 186m, 207m, 209m, 221m , 228m and 238m. Two days later, the same leaves were infiltrated with Agrobacteria expressing the genomic TBSV RNA under control of a 35S promoter. By fusion with an HDV ribozyme sequence (rib), it was ensured that after transcription an infectious genomic TBSV RNA with the correct 3 'end is formed. This second infiltration occurred within the area of the first infiltration. Subsequently, the plants were observed for at least 5 weeks to document the development of typical symptoms of TBSV infection.
• Infection Experiment, Example 1 6 plants expressing non-specific control siRNAs showed clear symptoms at the latest 15 days after infiltration with TBSV RNA-expressing Agrobacteria (15 dpi) a viral infection. Of the 6 plants expressing a mix of TBSV siRNAs, only one showed symptoms.
• Infection Experiment, Example 2 Sixteen plants expressing nonspecific control siRNAs showed clear symptoms of viral infection no later than 13 days after infiltration with TBSV RNA-expressing Agrobacteria (13 dpi). Of the 16 plants expressing a mix of TBSV siRNAs, only two showed symptoms and were much later than the control plants.
• Summary of results with N. benthamiana plants treated with various siRNAs and infected (burdened) with TBSV. Given are the respective siRNAs that have been expressed via amiRNA and Agrobakterieninfektion; These are described in the text. The siRNA gf698 is directed against the mRNA of green fluorescent protein (GFP) and was used as a control. The type of 5'-nucleotide of the respective siRNAs is indicated. It also indicates how many of the plants used in the experiments after exposure to TBSV (via TBSV RNA-expressing Agrobacteria) remained symptom-free (also in%) and when the first symptoms were observed in the unprotected plants.

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited non-patent literature

• Ding, 2010; Zvereva and Pooggin, 2012; Gammon and Mello, 2015 [0002]
• Bouche et al., 2006; Deleris et al., 2006; Parent et al., 2015 [0002]
• Bernstein et al. 2001; Elbashir et al. 2001a, Elbashir et al., 2001b; Martinez et al., 2002 [0002]
• Hammond et al., 2001; Master, 2013; Kobayashi and Tomari, 2016 [0002]
• (Mi et al., 2008, Takeda et al., 2008, Schuck et al., 2013 [0002]
• Wang et al., 2010; Wang et al., 2011 [0002]
• Harper et al., 2002; Ulker et al., 2008 [0008]
• Qu et al., 2007; Kung et al., 2012 [0009]
• Casacuberta et al., 2015. Other forms of application use artificial ds RNA molecules (Robinson et al., 2014) [0009]
• Duan et al., 2012; Dalakouras et al., 2016 [0009]
• Komoda et al., 2004 [0011]
• Gursinsky et al., 2009 [0011]
• Iki et al., 2010; Schuck et al., 2013 [0011]
• Prado et al., 2014 [0030]
• Cillo and Palukaitis, 2014 [0030]
• Nwokeoji et al., 2017 [0033]
• Tenllado et al, 2003; Mitter et al., 2017 [0033]
• Arnold et al., 2012 [0034]
• Krijger et al., 2012 [0034]

Claims (16)

Method for the targeted identification of highly efficient small interfering RNAs (eRNAs) of different lengths, characterized in that a) an RNA selected as a target for RNA silencing (RNAi) is produced by in vitro transcription and converted into cytoplasmic extracts of plant cells by the endogenous Dicer-like proteins (DCL) to small interfering RNAs (siRNAS); b) wherein a DCL-generated siRNA pool is formed from the RNA used and the siRNAs contained in this pool are determined by RNA analysis; c) the cytoplasmic plant cell extract is supplemented with a messenger RNA (mRNA) of an Argonaute (AGO) protein synthesized by in vitro transcription, wherein the mRNA is designed to encode a tag for the AGO protein in question; d) via in vitro translation AGO protein molecules are formed which form RNA-induced silencing complexes (RISC) complexes with the present DCL-generated siRNAs; e) immunoprecipitated from the BYL siRNA-loaded AGO / RISC by day and the bound siRNA guide-strands determined by RNA seq analysis; f) via the alignment of the RNA seq. Data with cleavage products, which are preferably generated in a slicer assay with the siRNA pool from the target RNA, such siRNAs are determined, which are efficient on the one hand and on the other hand enriched in AGO / RISC; g) subsequently synthesized and tested for functionality in a further sliced assay with labeled target RNA, and h) thus determined eRNAs transgene or transiently be used individually or in combination. Method according to Claim 1 for the identification of eRNAs from coding or non-coding RNAs, characterized in that these eRNAs, individually or in combination, inactivate target RNAs highly efficiently in the cellular RNA interference / silencing (RNAi) process without off-target effect. Method according to Claim 1 and 2 characterized in that a cytoplasmic extract of plant cells for the implementation of DCL assays, AGO-IPs, the RNA sequencing of DCL-generated and AGO-associated siRNAs and for performing slicer assays is used. Method according to Claim 1 and 2 characterized in that a cytoplasmic extract of Nicotiana tabacum BY2 cells (BYL) is used to perform DCL assays, AGO-IPs, RNA sequencing of DCL-generated and AGO-associated siRNAs and to perform slicer assays. Method according to Claim 1 to 4 characterized in that Argonaute (AGO) proteins of various organisms and / or different AGO proteins of an organism are used for the purposeful specification of eRNA activities on target RNAs in the target organism. Method according to Claim 1 to 5 characterized in that it can be used for target RNAs of all forms of organisms. Method according to Claim 6 characterized in that it can be used for target RNAs of all forms of viroids, virusoids, viruses, prokaryotes and eukaryotes. Method according to Claim 6 characterized in that it can be used for target RNAs of all forms of phytopathogenic organisms. Method according to Claim 6 characterized in that it can be used for target RNAs of all forms of phytopathogenic viroids, virusoids, viruses, bacteria, oomycetes, fungi, nematodes, insects and parasitic plants. Highly efficient small interfering RNAs (eRNAs) after Claim 1 for transient or transgenic application in cells or complete organisms. Transient or transgenic application of multiple eRNAs Claim 10 in the form of ds RNAs consisting predominantly of eRNA sequences. Transient or transgenic application of eRNAs after Claims 10 and 11 for efficient gene knockdown, eg for the investigation of gene functions Transient or transgenic application of eRNAs after Claims 10 and 11 to improve stress resistance, growth and / or consumer properties of beneficial organisms. Transient or transgenic application of eRNAs after Claims 10 and 11 to improve stress resistance, growth and / or consumer properties of crops. Method according to one or more of the preceding claims, which can be used for a temporary or lifelong protection of target organisms with RNAi immune system against pathogens with an RNAi immune system, wherein the protection can be done by host induced gene silencing (HIGS). Method according to one or more of the preceding claims, which can be used for a temporary or lifelong protection of plants against pathogenic viroids, virusoids, viruses, bacteria, oomycetes, fungi, nematodes, insects and other parasitic plants, wherein the protection by host induced gene silencing (HIGS) can take place.

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