A new level of RNA-based plant protection: dsRNAs designed from functionally characterized siRNAs highly effective against Cucumber mosaic virus

Abstract

RNA-mediated crop protection increasingly becomes a viable alternative to agrochemicals that threaten biodiversity and human health. Pathogen-derived double-stranded RNAs (dsRNAs) are processed into small interfering RNAs (siRNAs), which can then induce silencing of target RNAs, e.g. viral genomes. However, with currently used dsRNAs, which largely consist of undefined regions of the target RNAs, silencing is often ineffective: processing in the plant generates siRNA pools that contain only a few functionally effective siRNAs (esiRNAs). Using an in vitro screen that reliably identifies esiRNAs from siRNA pools, we identified esiRNAs against Cucumber mosaic virus (CMV), a devastating plant pathogen. Topical application of esiRNAs to plants resulted in highly effective protection against massive CMV infection. However, optimal protection was achieved with newly designed multivalent 'effective dsRNAs' (edsRNAs), which contain the sequences of several esiRNAs and are preferentially processed into these esiRNAs. The esiRNA components can attack one or more target RNAs at different sites, be active in different silencing complexes, and provide cross-protection against different viral variants-important properties for combating rapidly mutating pathogens such as CMV. esiRNAs and edsRNAs have thus been established as a new class of 'RNA actives' that significantly increase the efficacy and specificity of RNA-mediated plant protection.

Graphical Abstract

Figure 1.

Identification and characterization of esiRNA candidates from CMV Fny RNAs 2 and 3 (eNA screens, steps 1 and 2). (A) Schematic of the siRNA pool generation and characterization procedure (see text for details). (B) Size distribution of siRNAs generated by the BYL-endogenous DCLs from dsRNA versions of CMV RNAs 2 and 3. Bars above and below the axis represent siRNAs derived from viral (+) and (−) strand RNA, respectively. Data represent the mean of three experiments. (C) Schematic representation of the procedure to identify AGO-bound siRNAs (see text for details). (D) Size distribution of CMV siRNAs isolated from AGO immunoprecipitations (IP). Data represent the mean of three experiments, except for AGO1 IP with CMV RNA 2 siRNAs (two experiments). (E) Relative abundance of the respective 5′ terminal nucleotides of the AGO1- and AGO2-associated 21 nt siRNA guide strands. The abundance was compared with the nucleotide compositions of the respective CMV RNAs (CMV), and the relative abundance of the 5′ terminal nucleotides of all sequenced 21 nt CMV siRNAs generated in BYL by endogenous DCLs [DCL; see panels (A) and (B)]. Identified esiRNA candidates from CMV RNA 2 and CMV RNA 3 are listed in Tables 1 and 2, respectively.

Figure 2.

RNA silencing activity of esiRNA candidates in vitro (eNA screens step 3). Slicer assays with the esiRNA candidates identified in step 2 of the eNA screen and CMV RNAs 2 or 3 (Fig. 1) were performed as shown schematically in Supplementary Fig. S1C and described in the text. The numbers of the esiRNA candidates correspond to the designations given in the text, tables, and figures below. Asterisks (*) denote the cleavage products. The fact that in some cases only one cleavage product was detected can be explained by co-migration of larger cleavage products with the target RNA, weak labeling of smaller cleavage products and/or by different stabilities (especially of the 3′ cleavage products) with respect to further degradation by RNases. Determined slicing efficiencies are summarized in Tables 1 and 2. (A) Representative slicer assays performed with esiRNA candidates, CMV RNA 2 and AGO1/RISC or AGO2/RISC. Six esiRNA candidates (three active with AGO1, three active with AGO2) that were further investigated are shown in bold. (B) Schematic representation of the binding sites of these siRNAs on CMV RNA 2. (C) Representative slicer assays performed with esiRNA candidates, CMV RNA 3 and AGO1/RISC or AGO2/RISC. esiRNA candidates that were further investigated are shown in bold. (D) Binding sites of these siRNAs on CMV RNA 3.

Figure 3.

Identified esiRNAs protect plants efficiently against CMV infection. Nicotiana benthamiana plants were mechanically co-inoculated with the individual synthetic esiRNA candidates and with the genomic CMV RNAs 1, 2, and 3. The concentration of the viral RNAs was chosen to achieve a maximal successful challenge with the pathogen (see text). siRNA gf698 targeting GFP mRNA was used as a negative control. Plants were monitored for the appearance of CMV-specific symptoms for at least 28 dpi. (A, C) Representative plant images illustrate the differences between asymptomatic and symptomatic individuals at 35 dpi (RNA 2-targeting siRNAs) or at 28 dpi (RNA 3-targeting siRNAs). The percentage of plants remaining asymptomatic is given for each siRNA. (B) Percentage of asymptomatic plants over the entire course of the experiment with RNA 2-targeting siRNAs. Results are from three independent experiments with 4–5 plants each, the total number of plants is indicated (n = 12–15). (D) Percentage of asymptomatic plants over the entire course of the experiment with RNA 3-targeting siRNAs. Results are from three independent experiments with 3–5 plants each, the total number of plants is indicated (n = 9–15). An esiRNA directed against CMV RNA 2 (siR359) was used as an additional control.

Figure 4.

CMV edsRNAs: composition, processing and in vitro silencing activity. (A) Exemplary edsRNA and control dsRNAs. Top: Composition of an edsRNA generated from two complementary RNA transcripts. The edsRNA dsCMV6-21o contains a 21 nt long pseudo-siRNA at each end [symbolized by asterisks (*)] and six 21 nt long esiRNA sequences that have been shown to be effective against CMV RNA 2 in vitro and antivirally protective in planta (numbering according to Fig. 2 and Table 1). Guide strands (gs) are shown as arrows pointing in the 5′–3′ direction. The AGO1-specific gs are located on one RNA strand, the AGO2-specific gs are located on the other RNA strand. The example RNA shown here has 2 nt 3′ overhangs (dsCMV6-21o); however, edsRNAs with blunt ends (dsCMV6-21) were also generated and tested. Middle: Control RNA dsCMV consists of pseudo-siRNA sequences at the ends and a 126 nt-long fragment of a ds version of CMV RNA 2 (corresponding to a length of six 21 nt-long siRNAs). By chance, the dsCMV also contains the sequences of two siRNAs that were identified as esiRNAs in the screen against CMV RNA 2. Bottom: Control RNA dsGFP consists of pseudo-siRNA sequences at the ends and a 126 nt long fragment of a ds version of GFP mRNA. The exact sequences of the dsRNAs shown are given in Supplementary Fig. S5. (B) Processing of an edsRNA by DCLs in vitro. Labeled dsCMV6-21o was added to BYL and DCL-mediated processing analyzed over 24 h (see Supplementary Materials and methods) (M = 21 nt siRNA as marker). (C) Slicer assays with individual AGO1- and AGO2-specific esiRNAs from CMV RNA 2 and with the analogous esiRNAs generated from an edsRNA in BYL. AGO1- or AGO2/RISC were reconstituted with individual esiRNAs, with an appropriate mix of these esiRNAs or with esiRNAs processed from the edsRNA dsCMV6-21o in BYL by the DCLs present there. Endonucleolytic hydrolysis of labeled CMV RNA 2 target was detected by gel electrophoresis and autoradiography. Asterisks (*) indicate the generated cleavage products.

Figure 5.

The esiRNA constituents are generated at high proportion from edsRNA in BYL. dsCMV6-21 (blunt ends) and dsCMV6-21o (2 nt 3′ overhangs) were processed in BYL by the endogenous DCLs and the small RNA fraction analyzed by RNA-seq (see also scheme in Fig. 1A). (A) Size distribution of the 20–25 nt reads mapping to the edsRNA sequences. (B) Proportion of guide and passenger strand reads among all 21 nt reads that mapped to the edsRNA sequences; the peaks indicate the position of the 5′ nucleotide of reads with respect to the edsRNA. Peaks corresponding to the pseudo-siRNA and esiRNA sequences are specifically colored. In the case of dsCMV6-21 there are no mapping 21 nt reads for the ‘passenger’ strands of the pseudo siRNAs, as the two 3′ nucleotides (position 20 and 21) are missing.

Figure 6.

Comparison of the protective effect of different dsRNAs in planta. N. benthamiana plants were mechanically co-inoculated with different dsRNAs (see text) and with the genomic CMV RNAs 1, 2, and 3 and monitored for the appearance of CMV-specific symptoms for 35 dpi. Results are from two independent experiments; the total number of plants tested is indicated (n = 15). (A) Representative plant images 35 days after co-inoculation. The percentage of asymptomatic plants at 35 dpi is indicated for each dsRNA. (B) Percentage of asymptomatic plants over the entire course of the experiment.

Figure 7.

Identifying esiRNAs and designing edsRNAs for significantly improved RNA-based crop protection. Upper/left parts: Upon infection of a plant with an RNA virus, viral dsRNA (e.g. replication intermediates) is detected by Dicer-like proteins (DCLs) and processed into a pool of siRNAs. The same happens with ds elements of other target RNAs and also with artificial dsRNAs generated from target RNAs. Of the produced siRNAs, only a few mediate efficient slicing of the target RNAs by AGO protein containing RISC resulting in inefficient RNA silencing. Middle/bottom parts: Functional siRNAs, esiRNAs, are characterized by a high affinity to antiviral AGO proteins and high accessibility of the respective complementary target sites in the target RNA. The eNA screen reliably identifies esiRNAs from siRNA pools, leading to an effective RNA silencing process. Right/bottom parts: The esiRNAs, which provide highly effective protection, e.g. against a viral infection, can be used to design edsRNAs. The edsRNAs are essentially composed of the sequences of the functionally characterized esiRNAs and are preferentially processed by the DCLs into these esiRNAs. Multivalent edsRNAs thus have the potential to significantly increase the efficiency of RNA silencing-mediated protection of plants against pathogens such as RNA viruses.