Molecular evolution of a viral non-coding sequence under the selective pressure of amiRNA-mediated silencing

Abstract

Plant microRNAs (miRNA) guide cleavage of target mRNAs by DICER-like proteins, thereby reducing mRNA abundance. Native precursor miRNAs can be redesigned to target RNAs of interest, and one application of such artificial microRNA (amiRNA) technology is to generate plants resistant to pathogenic viruses. Transgenic Arabidopsis plants expressing amiRNAs designed to target the genome of two unrelated viruses were resistant, in a highly specific manner, to the appropriate virus. Here, we pursued two different goals. First, we confirmed that the 21-nt target site of viral RNAs is both necessary and sufficient for resistance. Second, we studied the evolutionary stability of amiRNA-mediated resistance against a genetically plastic RNA virus, TuMV. To dissociate selective pressures acting upon protein function from those acting at the RNA level, we constructed a chimeric TuMV harboring a 21-nt, amiRNA target site in a non-essential region. In the first set of experiments designed to assess the likelihood of resistance breakdown, we explored the effect of single nucleotide mutation within the target 21-nt on the ability of mutant viruses to successfully infect amiRNA-expressing plants. We found non-equivalency of the target nucleotides, which can be divided into three categories depending on their impact in virus pathogenicity. In the second set of experiments, we investigated the evolution of the virus mutants in amiRNA-expressing plants. The most common outcome was the deletion of the target. However, when the 21-nt target was retained, viruses accumulated additional substitutions on it, further reducing the binding/cleavage ability of the amiRNA. The pattern of substitutions within the viral target was largely dominated by G to A and C to U transitions.

Figure 1. Schematic representations of infectious clones of chimeric Turnip mosaic virus (TuMV).

(A) Schematic representation of TuMV-GFP infectious clone carrying a GFP gene inserted between the NIb and CP genes. (B) Arrows represent the positions and orientations of primers used for RT-PCR. The primer sets PTuNIb-8671/MTuCP-8982 and PXFP-532/MTuCP-8982 were used to amplify the NIb-CP and GFP-CP regions, respectively. (C,D) Schematic diagrams showing the 21-nt sequence of P69 and P69m in the chimeric viruses TuMV-GP69 and TuMV-GP69m, respectively. Predicted base pairing of the 21-nt target RNA sequence (top strand) and amiR¹⁵⁹-P69 (bottom strand) are shown below the amino acid sequence of the TuMV-GFP poly-protein.

Figure 2. A 21-nt sequence targeted by amiRNA is necessary and sufficient to confer virus resistance.

(A) amiR¹⁵⁹-P69 and amiR¹⁵⁹-HC-Pro transgenic Arabidopsis plants were mock-inoculated or inoculated with TuMV-GFP, TuMV-GP69, or TuMV-GP69m. As controls, the same transgenic lines were inoculated with TuMV-GFP. Photographs were taken at 12 dpi. Bar = 0.5 cm. (B) GFP fluorescence of systemic leaves of plants infected with chimeric viruses. Leaves were examined by fluorescence microscopy. Leaves of sensitive amiR¹⁵⁹-P69 plants displayed green fluorescence due to replication of GFP-virus, whereas no green fluorescence was detected in leaves of resistant amiR¹⁵⁹-HC-Pro or amiR¹⁵⁹-P69 plants. Bar = 2 cm.

Figure 3. Transgenic N. benthamiana plants expressing amiR¹⁵⁹-P69 are resistant to infection by chimeric TuMV virus.

(A) amiR¹⁵⁹- P69 expression levels of transgenic N. benthamiana plants carrying 35S-pre-amiR¹⁵⁹-P69. Four independent lines (# 1, 2, 3, and 4) were analyzed. 5S rRNAs were used as a loading control. (B) Early infection of chimeric TuMV viruses monitored by UV excitation. (C) amiR¹⁵⁹-P69 N. benthamiana plants were resistant to TuMV-GP69 but susceptible to TuMV-GP69m or TuMV-GFP.

Figure 4. Scanning mutagenesis of the amiR¹⁵⁹-P69 target site on TuMV-GP69 chimeric virus.

(A) A schematic representation of the 21 scanning mutants with substitution of single nucleotide within the 21-nt sequence targeted by amiR¹⁵⁹-P69. (B) Representative amiR¹⁵⁹-P69 N. benthamiana plants displaying different degree of breakdown when inoculated with the scanning mutants. The ratio in each panel indicates the number of susceptible amiR¹⁵⁹-P69 plants amongst 20 plants challenged. (C) A summary of critical positions within the amiR¹⁵⁹-P69 target site. The 21-nt RNA sequence is shown on the x-axis. Numbers below the sequence indicate the positions of amiR¹⁵⁹-P69 starting from the 5′ end. The degree of resistance breakdown was represented as the percent of inoculated plants with viral disease symptoms. Red bars represent critical positions for resistance; yellow bars represent positions of moderate importance; green bars represent positions of minimal influence in resistance-breakdown.

Figure 5. Sequence analysis of chimeric TuMV viruses recovered from susceptible amiR¹⁵⁹-P69 transgenic plants.

(A) Representative RT-PCR results of chimeric TuMV viruses derived from susceptible transgenic plants infected with m1, m2, and m3. The NIb-CP (top panel) and GFP-CP regions (bottom panel) of scanning mutants virus TuMV-GP69m1 (m1; lane 1), TuMV-GP69m2 (m2; lanes 2–7), and TuMV-GP69m3 (m3; lanes 8–14) were checked for deletion of the 21-nt target sequence by RT-PCR. (B) Representative results of chimeric TuMV viral sequences with deletion in the 21-nt target site. The sequence of TuMV-GP69 was used as the standard sequence (gray box), and the 21-nt target site was underlined. Representative sequences of three scanning mutant viruses, TuMV-GP69m5-13, 15, and 19, from susceptible plants were aligned. Nucleotide mutation in position 5 is in bold and indicated with an arrow. Additional mutations are marked by asterisks. (C) Frequency of additional mutation on the 21-nt target site. The x-axis shows the 21-nt sequence on TuMV-GP69. Numbers below indicate the positions of amiR¹⁵⁹-P69 starting from the 5′ end. Bars show frequency of additional mutations in scanning mutant viruses recovered from susceptible plants.

Figure 6. A working model to explain breakdown of amiRNA-mediated resistance by virus mutation.

(A) Complete sequence complementarity between the 21-nt target site and the amiRNA. (B) TuMV-GP69m9 is a mutant virus with a single mutation (underlined) on position 9 of the target site. As this position is critical, the mutation causes a decrease in the cleavage efficiency of TuMV-GP69m9 viral RNAs, allowing some viral RNAs to escape the amiRNA-mediated surveillance. The surviving TuMV-GP69m9 virus rapidly undergoes evolution, collecting additional mutations on the target site. The next generation of mutated viruses with additional mutations can overcome the amiRNA-mediated resistance.