Additional mutations on the 21-nt target fixed during virus evolution.

1<p>m2, m3, m4, …, m18 = mutant virus that single-mutated on targeted 21-nt seq.</p>2<p>A.M.F. = frequency of additional mutation.</p>3<p>The original mutation generated by PCR mutagenesis is in bold.</p

Schematic representations of infectious clones of chimeric <i>Turnip mosaic virus</i> (TuMV).

<p>(A) Schematic representation of TuMV-GFP infectious clone carrying a GFP gene inserted between the <i>NIb</i> and <i>CP</i> 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.</p

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

<p>(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.</p

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

<p>(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 <i>N. benthamiana</i> 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 <i>x</i>-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.</p

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

<p>(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 <i>x</i>-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.</p

Pathogenicity of -nt substitution of chimeric TuMV-GFP viruses on amiR¹⁵⁹-P69 plants.

*<p>Pathogenicity values significantly greater than zero.</p

Mutated NSs proteins for analyzing RNA silencing suppression function.

<p><b>(A)</b> The maps of different mutants with individual deletions of the highly conserved regions (ΔCR1-ΔCR 6) or the common epitope NSscon (ΔCE) of the NSs protein of <i>Watermelon silver mottle virus</i> (WSMoV). The aa positions of the individual deleted regions are indicated. <b>(B)</b> The aa positions locating on each conserved region chosen for alanine-mutagenesis in this study.</p

Expression levels of deleted and point-mutated NSs proteins analyzed by co-infiltration with potyviral suppressor HC-Pro.

<p><b>(A)</b> Expression levels of protein and mRNA of deleted NSs mutants, following co-infiltration of the empty vector (EV) or HC-Pro (HC), detected at 4 day post agroinfiltration (dpa) by anti-NSs MAb (left panel) or PAb (right panel) and α-<b>32</b>P labeled NSs-probe, respectively. <b>(B)</b> Expression levels of point-mutated NSs proteins, following co-infiltration with the empty vector or HC-Pro, detected at 4 dpa by anti-NSs MAb (left panel) or PAb (right panel). <b>(C)</b> Time-course detection of the protein expression levels of the NSs mutants, G180A and Y398A, at different hours post infiltration (hpi) with EV or HC, detected by anti-NSs MAb (left panel) or anti-HC-Pro PAb (right panel). <b>(D)</b> Expression levels of G180A, R181A and Y398A NSs proteins, mRNA and barstar^R mRNA following co-infiltration of EV or HC construct, detected at 4 dpa. Coomassie Blue stained-RuBisCO proteins or 18S rRNA were used as loading controls.</p

Symptoms on squash plants after inoculation with <i>Zucchini yellow mosaic virus</i> (ZYMV) recombinant viruses carrying different point-mutated NSs proteins.

<p><b>(A)</b> Symptoms on squash plants inoculated with individual ZYMV recombinants at 14 days post-infection (dpi). <b>(B)</b> Detection of mutated NSs proteins expressed by individual ZYMV recombinants at 14 dpi using anti-NSs MAb or anti-ZYMV CP PAb. Coomassie blue-stained RuBisCO protein was used as loading controls.</p

Two Novel Motifs of <i>Watermelon Silver Mottle Virus</i> NSs Protein Are Responsible for RNA Silencing Suppression and Pathogenicity

<div><p>The NSs protein of <i>Watermelon silver mottle virus</i> (WSMoV) is the RNA silencing suppressor and pathogenicity determinant. In this study, serial deletion and point-mutation mutagenesis of conserved regions (CR) of NSs protein were performed, and the silencing suppression function was analyzed through agroinfiltration in <i>Nicotiana benthamiana</i> plants. We found two amino acid (aa) residues, H113 and Y398, are novel functional residues for RNA silencing suppression. Our further analyses demonstrated that H113 at the common epitope (CE) (¹⁰⁹KFTMHNQ¹¹⁷), which is highly conserved in Asia type tospoviruses, and the benzene ring of Y398 at the C-terminal <i>β</i>-sheet motif (³⁹⁷IYFL⁴⁰⁰) affect NSs mRNA stability and protein stability, respectively, and are thus critical for NSs RNA silencing suppression. Additionally, protein expression of other six deleted (ΔCR1-ΔCR6) and five point-mutated (Y15A, Y27A, G180A, R181A and R212A) mutants were hampered and their silencing suppression ability was abolished. The accumulation of the mutant mRNAs and proteins, except Y398A, could be rescued or enhanced by co-infiltration with potyviral suppressor HC-Pro. When assayed with the attenuated <i>Zucchini yellow mosaic virus</i> vector in squash plants, the recombinants carrying individual seven point-mutated NSs proteins displayed symptoms much milder than the recombinant carrying the wild type NSs protein, suggesting that these aa residues also affect viral pathogenicity by suppressing the host silencing mechanism.</p></div