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.
Table 1.
Pathogenicity of -nt substitution of chimeric TuMV-GFP viruses on amiR159-P69 plants.
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.
Table 2.
Additional mutations on the 21-nt target fixed during virus evolution.
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.