Nucleic acid active agents against various plant pathogens

Nucleic acid for the protection of plants against the plant pathogens cucumber mosaic virus, Meloidogyne incognita and Botrytis cinerea, wherein

• a. the nucleic acid is a small interfering RNA (siRNA) that consists of 21, 22, 23 or 24 base pairs and contains 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 group consisting of the nucleic acids having SEQ ID NOs:
• 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 according to group a., wherein at least one of the single-stranded RNAs selected from a guide strand and a passenger strand has modifications at 1 to 7 positions in the nucleotide sequence;
• or
• c. the nucleic acid is a small RNA selected from an siRNA and a micro RNA (miRNA), wherein the RNA double strand thereof 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 containing nucleotide sequences of at least two siRNAs or sRNAs of groups a., b., or c.;
• or
• e. the nucleic acid is a single-stranded DNA that consists of 12 to 25 nucleotides and contains a sequence of 12 or more nucleotides which are homologous to one of the nucleotide sequences of the single-stranded RNAs of groups a. or b.;
• or
• f. the nucleic acid is a single-stranded DNA according to group e. which has modifications at 1 to 7 positions in the nucleotide sequence,
• wherein the nucleic acid is provided for protection against plant pathogens using a method for the targeted identification of effective small interfering RNAs (esiRNAs/ERNAs) and sRNAs derived therefrom, and also effective antisense DNA oligonucleotides (eASO) of different lengths, collectively referred to as effective Nucleic Acids (eNAs), comprising the steps of
• (i) an RNA which was selected as a target for RNA silencing (RNAi) is produced using in vitro transcription and converted to small interfering RNAs (siRNAs) in cytoplasmic extracts of plant cells by means of endogenous Dicer-like proteins (DCL);
• (ii) a DCL-generated siRNA pool being formed from the RNA used and the siRNAs contained in this pool being determined by means of RNA-seq analysis;
• (iii) a messenger RNA (mRNA) of an argonaute (AGO) protein, which mRNA was synthesized by in vitro transcription, is added to the cytoplasmic plant cell extract, the mRNA being constructed such that it encodes the AGO protein in question with a tag;
• (iv) AGO protein molecules are formed using in vitro translation, said AGO protein molecules forming RNA-induced silencing complexes (RISC) with the DCL-generated siRNAs that are present;
• (v) tagged siRNA-loaded AGO/RISC are immunoprecipitated from BYL and the bound siRNA guide strands are determined by means of RNA-seq analysis;
• (vi) those siRNAs that are enriched in AGO/RISC are determined using the comparison of the RNA-seq data with the RNA-seq data from step (ii);
• (vii) subsequently produced synthetically and tested for their functionality in a Slicer assay with labeled target RNA, and
• (viii) in this way, esiRNAs/ERNAs are identified, and sRNAs, derived therefrom, as well as eASO, collectively referred to as eNAs, are determined;
• wherein
• I. in order to form the RISC in step (vii), 0.5 pmol of the mRNA of the AGO protein to be used is translated in a reaction solution containing 50% (v/v) BYL, 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 competitor siRNA (e.g., siR gf698, selected from the group consisting of SEQ ID NOs:
• 205, 206, 207, and 208);
• II. after an incubation time of 2.5 h at 25° C. per batch in step I., 3.4 pmol of a nonspecific mRNA (e.g., encoding the Firefly luciferase protein, SEQ ID NO:
• 209, Schuck et al., 2013) is added as a further competitor RNA, and also 10 fmol of the target RNA is added, the target RNA being labeled, and the reaction batches are incubated again for 15 min at 25° C.;
• III. during the incubation in step II., the target RNA is cleaved by the AGO/RISC formed;
• IV. after gel electrophoresis of the extracted RNA, the remaining quantity of target RNA or the resulting cleavage products compared to a control reaction (carried out without siRNA) is quantified by measuring the band intensities (ImageQuantTL or ImageJ);
• V. classification as esiRNAs/ERNAs takes place using the cleavage activity (slicer activity) on the target RNA, measured in this way, of each RISC formed with this siRNA,
• VI. esiRNAs/ERNAs which endonucleolytically convert at least 25% or more of the quantity of target RNA originally used in method step II. to cleavage products are selected.