Local and systemic infection of <i>A</i>. <i>thaliana</i> single <i>ago</i> mutants by TuMV-GFP and TuMV-AS9-GFP.

<p>(A) Schematic representation of the TuMV and TuMV-GFP genomes showing insertion of GFP between P1 and HC-Pro, and the AS9 mutation on HC-Pro. (B) Visualization of local infection of inoculated rosette leaves. Pictures were taken at 7 days post inoculation (dpi). Col-0 infected by TuMV-GFP is shown for comparison. The histogram shows average (+ SE) infection efficiency of 14 plants, each with four inoculated leaves. Infection efficiency by TuMV-GFP or TuMV-AS9-GFP is expressed relative to Col-0 (9.8 ± 2 foci per leaf) or to <i>dcl2–1 dcl3–1 dcl4–2</i> (3.5 ± 1.4 foci per leaf), respectively. For each virus, bars with the same letter are not statistically different (Tukey’s test with α = 0.05). (C) TuMV-AS9-GFP coat protein (CP) accumulation in noninoculated cauline leaves and in inflorescence at 15 dpi is determined by immunoblotting and expressed relative to <i>dcl2–1 dcl3–1 dcl4–2</i>. The histogram shows average (+ SE) of four biological replicates. Bars with the same letter are not statistically different (Tukey’s test with α = 0.05). The experiment was repeated twice with similar results.</p

Profile of endogenous and TuMV-derived siRNAs in plants expressing HA-AGO2_DAD in an <i>ago2–1</i> background.

<p>Values are average and SE from two biological replicates normalized to reads per million. Inoculated rosette leaf and systemically infected cauline leaf samples were collected at 7 and 15 dpi, respectively. Inflorescence samples were collected at 10 dpi. (A) Panel I: number of reads by size, class, and polarity, for TuMV-derived siRNAs in input and HA-AGO2_DAD IP. Panel II: for 21 and 22 nt TuMV-derived siRNAs, enrichment in HA-AGO2_DAD IP. Enrichment is defined as immunoprecipitate (IP) reads/ input reads, expressed on a log₂ scale. Panel III: proportion (in percentage) of 5’ nt in 21 nt and 22 nt TuMV-derived siRNAs by fraction. Numbers were rounded to the nearest integer. (B) and (C) TuMV genome-wide distribution of 21 nt TuMV-derived siRNAs in input (B) and HA-AGO2_DAD IP (C). Panel I: TuMV-infected inflorescence. Panel II: TuMV-inoculated rosette leaves. Panel III: rosette leaves inoculated with TuMV-AS9. Panel IV: cauline leaves systemically infected with TuMV-AS9. Reads were plotted for each 1 nt position. The scale was capped at 150 reads.</p

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Profile of endogenous and TuMV-derived siRNAs in plants expressing HA-AGO1_DAH in an <i>ago2–1</i> background.

<p>Labels are as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004755#ppat.1004755.g003" target="_blank">Fig. 3</a>. Inflorescence samples were collected at 10 dpi. Inoculated rosette leaf and systemically infected cauline leaf samples were collected at 7 and 15 dpi, respectively.</p

Local and systemic infection of a selected group of double and triple <i>ago</i> mutants by TuMV-GFP and TuMV-AS9-GFP.

<p>(A) Local infection efficiency. Panel I: infection efficiency of TuMV-GFP or TuMV-AS9-GFP is expressed relative to Col-0 (19.6 ± 3.3 foci per leaf) or to <i>dcl2–1 dcl3–1 dcl4–2</i> (2.2 ± 0.7 foci per leaf), respectively. The histogram shows the average (+ SE) of 10 plants, each with four inoculated leaves. Panel II: local infection of inoculated rosette leaves for a selected group of mutants harboring <i>ago1–27</i>. The histogram shows average (+ SE) infection efficiency of 14 plants, each with four inoculated leaves. Infection efficiency of TuMV-GFP or TuMV-AS9-GFP is expressed relative to Col-0 (4.1 ± 1.2 foci per leaf) or to <i>dcl2–1 dcl3–1 dcl4–2</i> (2.8 ± 1.1 foci per leaf), respectively. Panel III: Representative leaves of <i>ago1–27</i> single and <i>ago1–27 ago2–1</i> double mutants showing TuMV-GFP local infection foci. <i>ago1–27 ago2–1</i>, but not <i>ago1–27</i>, was infected by TuMV-AS9-GFP. Col-0 is shown for comparison. Pictures were taken at 7 dpi under UV light. (B) Systemic infection. TuMV-AS9-GFP coat protein accumulation in noninoculated cauline leaves and in inflorescence at 15 dpi. Panel I: double mutants harboring <i>ago2–1</i>. Panel II: double and triple mutants harboring <i>ago1–27</i> and <i>ago10–5</i>. The histograms show average (+ SE) of four biological replicates, expressed relative to <i>dcl2–1 dcl3–1 dcl4–2</i>. Bars with the same letter are not statistically different (Tukey’s test with α = 0.05). Panel III: in double and triple mutants harboring <i>ago1–27</i>, inflorescence samples were collected only from clusters showing systemic GFP.</p

Profile of endogenous and TuMV-derived siRNAs in plants expressing HA-AGO10.

<p>Labels are as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004755#ppat.1004755.g003" target="_blank">Fig. 3</a>. Catalytically active HA-AGO10_DDH and catalytic mutant HA-AGO10_DAH were expressed in a wild-type Col-0 (<i>AGO2</i>) or <i>ago2–1</i> background, respectively. Inflorescence samples were collected at 10 dpi. Inoculated rosette leaf and systemically infected cauline leaf samples were collected at 7 and 15 dpi, respectively.</p

Profile of TuMV-derived siRNAs in plants infected with TuMV-HIS or TuMV-HIS-AS9.

<p>(A) Panel I: schematic representation of the TuMV genome and modified clones with an AS9 mutation and a 6xHIS tag (TuMV-HIS). Coordinates correspond to wild-type TuMV. The 6xHIS tag fused in frame to HC-Pro is underlined. Panel II: representative blot CP and HC-Pro accumulation in inflorescence of Col-0 at 10 dpi. (B) CP and HC-Pro accumulation in input and HC-Pro (wild-type and AS9) immunoprecipitation from cauline leaves of <i>ago2–1</i> plants. Samples from plants infected with TuMV-HIS or TuMV-HIS-AS9 were collected at 10 and 15 dpi, respectively. 6.25 μg of total protein or 10 μl of immunoprecipitate (IP) were loaded for TuMV-HIS input and IP samples, respectively. Amounts were doubled for TuMV-HIS-AS9 input and IP. (C) Panel I: number of reads by size, class, and polarity, for TuMV-derived siRNAs in input and wild-type or AS9 HC-Pro IP. Panel II: enrichment in HC-Pro IP as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004755#ppat.1004755.g003" target="_blank">Fig. 3</a>. Panel III: proportion (in percentage) of 5’ nt in 21 nt and 22 nt TuMV-derived siRNAs by fraction. Panel IV: bars show the enrichment of TuMV-derived siRNAs by 5’ nt and polarity. (D) and (E) TuMV genome-wide distribution of 21 nt TuMV-derived siRNAs in input (D) and HC-Pro IP (E). Reads were plotted for each 1 nt position. The scale was capped at 150 reads.</p

TuMV-GFP and TuMV-AS9-GFP infection in selected <i>ago2–1</i> based double mutants^a.

<p>^a Number of plants showing local and systemic infections were scored by GFP fluorescence under UV illumination. Local infection foci were counted at 7 days post-inoculation (dpi). All other data is from plants at 15 dpi.</p><p>TuMV-GFP and TuMV-AS9-GFP infection in selected <i>ago2–1</i> based double mutants<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004755#t002fn001" target="_blank">^a</a>.</p

A model for direct action of <i>A</i>. <i>thaliana</i> AGO proteins in anti-TuMV defense.

<p>AGO-mediated antiviral silencing is suppressed through sequestration of TuMV-derived siRNAs by silencing suppressor HC-Pro (left panels), in both inoculated leaves and inflorescences. In the absence of active HC-Pro (right panels), AGO2, AGO10 and, to a lesser extent AGO1, associate with TuMV-AS9-derived siRNAs to potentially repress TuMV RNAs through slicing or translational repression. AGO2 protects leaves from TuMV infection and movement, with non-additive contributions by AGO10, AGO5 and AGO7. Redundant activities of AGO10 and AGO1 protect inflorescence from TuMV infection, with an additive contribution by AGO2.</p

Additional file 3: of Metagenomic analysis of viruses associated with maize lethal necrosis in Kenya

Figure S2. Size and frequency of de novo-assembled representative contigs with high similarity to known viruses. In total 68 samples were sequenced and analyzed. Contig size is represented in 0.5 Kb increments in the X axis. The Y axis represents the number of contigs per size class. For each virus, the number of samples categorized as infected is indicated. a MCMV. b SCMV. c MSV. d MYDV-RMV. (PDF 278 kb