Suppression of RNA Silencing by a Plant DNA Virus Satellite Requires a Host Calmodulin-Like Protein to Repress <i>RDR6</i> Expression

<div><p>In plants, RNA silencing plays a key role in antiviral defense. To counteract host defense, plant viruses encode viral suppressors of RNA silencing (VSRs) that target different effector molecules in the RNA silencing pathway. Evidence has shown that plants also encode endogenous suppressors of RNA silencing (ESRs) that function in proper regulation of RNA silencing. The possibility that these cellular proteins can be subverted by viruses to thwart host defense is intriguing but has not been fully explored. Here we report that the <i>Nicotiana benthamiana</i> calmodulin-like protein Nbrgs-CaM is required for the functions of the VSR βC1, the sole protein encoded by the DNA satellite associated with the geminivirus <i>Tomato yellow leaf curl China virus</i> (TYLCCNV). <i>Nbrgs-CaM</i> expression is up-regulated by the βC1. Transgenic plants over-expressing <i>Nbrgs-CaM</i> displayed developmental abnormities reminiscent of βC1-associated morphological alterations. Nbrgs-CaM suppressed RNA silencing in an <i>Agrobacterium</i> infiltration assay and, when over-expressed, blocked TYLCCNV-induced gene silencing. Genetic evidence showed that <i>Nbrgs-CaM</i> mediated the βC1 functions in silencing suppression and symptom modulation, and was required for efficient virus infection. Moreover, the tobacco and tomato orthologs of <i>Nbrgs-CaM</i> also possessed ESR activity, and were induced by betasatellite to promote virus infection in these <i>Solanaceae</i> hosts. We further demonstrated that βC1-induced Nbrgs-CaM suppressed the production of secondary siRNAs, likely through repressing <i>RNA-DEPENDENT RNA POLYMERASE 6</i> (<i>RDR6</i>) expression. <i>RDR6</i>-deficient <i>N. benthamiana</i> plants were defective in antiviral response and were hypersensitive to TYLCCNV infection. More significantly, TYLCCNV could overcome host range restrictions to infect <i>Arabidopsis thaliana</i> when the plants carried a <i>RDR6</i> mutation. These findings demonstrate a distinct mechanism of VSR for suppressing PTGS through usurpation of a host ESR, and highlight an essential role for RDR6 in RNA silencing defense response against geminivirus infection.</p></div

Nbrgs-CaM positively regulated the symptom and accumulation of 10Aβ.

<p>(<b>A</b>) Symptoms of wild type (wt), overexpression line (35S:CaM) and RNAi line (35S:dsCaM) of Nbrgs-CaM transgenic <i>Nicotiana benthamiana</i> plants infected with 10Aβ at 30 dpi. (<b>B</b>) Southern blots of 10Aβ accumulation in systemic leaves of wt, 35S:CaM, 35S:dsCaM <i>N. benthamiana</i> infected with 10Aβ at 30 dpi. The DNA agarose gel was stained with ethidium bromide as loading control and then blotted using probes specific for 10A and betasatellite (10β). (<b>C</b>) Symptoms of rgs-CaM-silenced plants infected by 10Aβ. Partial fragments of <i>Nbrgs-CaM</i>, <i>Ntrgs-CaM</i> and <i>Slrgs-CaM</i> were cloned into the RNA2 of the TRV VIGS vector. <i>N. benthamiana</i> (<i>N.b</i>), <i>N. tabacum</i> (<i>N.t</i>), <i>Solanum lycopersicum</i> (<i>S.l</i>) plants at 4–5 leaf stage were infiltrated with <i>Agrobacterium</i> cultures carrying an empty vector (Mock), or pTRV1 with pTRV2-GFP, or with pTRV1 and respective pTRV2-CaM. Rgs-CaM silencing was confirmed in newly emerged leaves 7 days after TRV infiltration by RT-qPCR (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003921#ppat.1003921.s006" target="_blank">Fig. S6</a>). The systemic leaves of these plants were superinfected with 10Aβ and photographs were taken 20 dpi. (<b>D</b>) Southern blots of 10Aβ accumulation in systemic leaves of plants shown in (<b>C</b>) at 20 dpi. The DNA agrose gel was stained with ethidium bromide as a loading control. Viral single-stranded DNA (ssDNA) and supercoiled DNA (scDNA) are indicated.</p

Induction of <i>Nicotiana benthamiana</i> calmodulin-like protein Nbrgs-CaM by TYLCCNB βC1.

<p>(<b>A</b>) RT-qPCR analyses of <i>Nbrgs-CaM</i> mRNA levels in <i>N. benthamiana</i> plants infected by TYLCCNV (10A) alone or together with betasatellite (10Aβ), or with a<i>grobacterium</i> carrying an empty vector (Mock) (left bar graph) at 21 dpi, and in βC1 transgenic plants (βC1) and nontransgenic control plants (wt) at 6 to 7-leaf period (right bar graph). Each mean value was derived from three independent experiments (<i>n</i> = 9) and normalized to mRNA of <i>NbGAPDH</i> that served as an internal standard. The relative mRNA levels of <i>Nbrgs-CaM</i> in mock plants (left bar graph) or wt plants (right bar graph) is arbitrarily set as 1. Values are means ± SD. The data were analyzed using one-way ANOVA followed by Student's <i>t</i> test. Asterisk indicates significant differences between genotypes or treatments (**P≤0.01). (<b>B</b>) Northern blot analyses of the <i>Nbrgs-CaM</i> and <i>βC1</i> mRNA levels in plants described in (<b>A</b>). Ethidium bromide staining of 25S ribosomal RNA (rRNA) was used to confirm equal RNA loading. (<b>C</b>) Pairwise alignment of rgs-CaMs from <i>N. benthamiana</i> (Nbrgs-CaM), <i>N. tabacum</i> (Ntrgs-CaM) and <i>Solanum lycopersicum</i> (Slrgs-CaM) using ClustalX2 software. The N-terminal extension specific for rgs-CaM is denoted by a solid line above the sequences, whereas the three EF-hand motifs characteristic of calmodulins are illustrated by the dotted lines. (<b>D</b>) Phylogenetic tree showing the relationship between the three rgs-CaMs and selective calcium-binding proteins from <i>Arabidopsis</i> (At) and tobacco (Nt). Numbers represent systemic names or GenBank accession numbers for the corresponding genes. Sequence alignments were performed in ClustalX2 and tree construction was conducted in MEGA5.1 using the Neighbour Joining method. The bootstrap values are shown near the internal nodes. CaM: calmodulin; CML: calmodulin-like protein; CBL: calcineurin B-like protein. The scale bar represents the number of changes per site.</p

RDR6 mediates nonhost defense against TYLCCNV in <i>Arabidopsis</i>.

<p>(<b>A</b>) Symptoms of wt (Col-0) and <i>rdr6-11 Arabidopsis</i> plants infected by 10A and 10Aβ at 30 dpi. (<b>B</b>) Southern blot analysis of viral DNAs of 10A and 10β in systemically infected leaves of Col-0 and <i>rdr6-11</i> plants at 30 dpi. The total DNA agarose gel was stained with ethidium bromide to show the equal loading. Viral single-stranded DNA (ssDNA) and supercoiled DNA (scDNA) forms are indicated.</p

Nbrgs-CaM suppresses PTGS of GFP and VIGS of an endogenous gene.

<p>(<b>A</b>) Suppression of GFP silencing in <i>Nicotiana benthamiana</i> line 16c. Leaf patches were co-infiltrated with <i>Agrobacterium tumefaciens</i> cultures expressing <i>GFP</i> (35S:GFP) and vector control (Vec), <i>Nbrgs-CaM</i>, <i>Ntrgs-CaM</i>, <i>Slrgs-CaM</i>, TYLCCNB <i>βC1</i> or TBSV <i>p19</i>, and GFP fluorescence were photographed under UV light at 4 dpi. (<b>B</b>) Protein and RNA gel blot analyses of GFP silencing in agroinfiltrated leaf samples. GFP or βC1 specific monoclonal antibody was used in immunoblottings. [α-³²P]-labeled <i>GFP</i>- or <i>Nbrgs-CaM</i>-specific probe was used in the large RNA blot. Note that the <i>Nbrgs-CaM</i>-specific probes also cross-reacts to <i>Ntrgs-CaM</i> and <i>Slrgs-CaM</i> mRNAs in Northern blots owing to their high sequence similarities. [γ-³²P] ATP-labeled GFP or U6 oligonucleotides were used as probes in the small RNA blots. The sizes of the 21-, 22- and 24-nt RNAs are indicated to the right of the small RNA panel. Ethidium bromide staining of 25S rRNA and Coomassie blue staining of the large subunit of Rubisco served as loading controls for RNA and protein gels, respectively. (<b>C</b>) Phenotypes of <i>Su</i> VIGS. Wt, 35S:CaM or 35S:dsCaM <i>N. benthamiana</i> plants were infected with the TYLCCNV-derived VIGS vector carrying a portion of the endogenous <i>Su</i> gene (10A+2mDNA1-<i>NbSu</i>) and photographed at 30 dpi. (<b>D</b>) RT-qPCR analysis of <i>Su</i> silencing. The relative levels of <i>Su</i> mRNA in plants shown in (<b>C</b>) were normalized to mRNA of <i>NbGAPDH</i> that served as an internal standard. Mock represents relative <i>Su</i> mRNA in wt plants infected by the VIGS vector without <i>Su</i> insertion (10A+2mDNA1). The relative level of <i>Su</i> mRNA in mock plants is arbitrarily set as 1. Error bars represent SD of nine individual plants. Asterisks indicate P values compared with mock-treated wild type plants: *P≤0.05, **P≤0.01 (Student's <i>t</i> test). (<b>E</b>) Southern blot analysis of viral DNAs in the VIGS vector-infected plants shown in (<b>C</b>). Total DNAs were blotted with α-³²P-labeled probes specific for TYLCCNV (10A) and alphasatellite (DNA1). The migrations of viral supercoiled DNA (scDNA) are indicated to the right of the panel. Total DNA agarose gel was stained with ethidium bromide to show the equal loading.</p

Ecotopic expression of <i>Nbrgs-CaM</i> in <i>Nicotiana benthamiana</i> phenocopys βC1 transgenic plants.

<p>(<b>A</b>) Parallel comparisons of phenotypes in Nbrgs-CaM and βC1 transgenic <i>N. benthamiana</i> plants. Representative leaves of type I, type II and type III 35S:CaM transgenic plants with varied phenotypic severities were shown on the left panel with the close-up views of the adaxial and abaxial side of upward-curled leaves. βC1 transgenic plants were shown in parallel on the right panels. The arrows indicate outgrowth tissues on the abaxial leaf surfaces of Nbrgs-CaM and βC1 transgenic plants. (<b>B</b>) Analysis of <i>Nbrgs-CaM</i> mRNA and protein levels in nontransgenic (wt) and three <i>Nbrgs-CaM</i> transgenic lines (35S:CaM). Equal loading of proteins and total RNA were confirmed by Coomassie blue staining of Rubisco large subunit and ethidium bromide staining of 25S rRNA, respectively. (<b>C</b> and <b>D</b>) Phenotype of 35S:dsCaM transgenic <i>N. benthamiana</i> (<b>C</b>) and levels of <i>Nbrgs-CaM</i> analyzed by RT-qPCR in 35S:dsCaM-1, 35S:dsCaM-2 transgenic lines at 6–7 leaf period (<b>D</b>). Values are means ± SD. Nine individual plants per genotype were used in each of the measurements. The asterisk indicates significant differences between genotypes analyzed by Student <i>t</i> test (**P≤0.01).</p

<i>Nbrgs-CaM</i> is genetically required for the functions of βC1 in PTGS suppression and symptom modulation.

<p>(<b>A</b>) GFP fluorescence in leaves of wt and 35S:dsCaM <i>Nicotiana benthamiana</i> plants co-infiltrated with 35S:GFP and 35S:FP, together with indicated suppressors. Photographs were taken under UV light at 3 and 5 dpi. (<b>B</b>) Protein and RNA gel blot analyses of GFP silencing in agroinfiltrated leaf samples shown in (<b>A</b>) at 5 dpi. GFP or βC1 specific monoclonal antibody was used in immunoblotting. Coomassie blue staining of the large subunit of Rubisco served as loading controls. In large RNA blot, [α-³²P]-labeled DNA fragments of <i>GFP</i> and <i>Nbrgs-CaM</i> were used as probes and ethidium bromide staining of 25S rRNA confirmed the equal loading. In the small RNA blot, [γ-³²P] ATP-labeled GFP or U6 oligonucleotides were used as probes. The sizes of the 21-, 22- and 24-nt marker RNAs are indicated to the right of the small RNA panel. (<b>C</b>) Symptoms of wt and dsCaM transgenic <i>N. benthamiana</i> plants infected with PVX-βC1 at 15 dpi. (<b>D</b> and <b>E</b>) Analyses of the RNA and protein levels of PVX coat protein (CP) and βC1 in wt and dsCaM transgenic plants infected with PVX-βC1. Total RNAs were extracted from infected plants at 15 dpi and blotted with probes specific for PVX <i>CP</i> or <i>βC1</i> (<b>D</b>), and total protein samples were blotted with specific PVX-CP and βC1 antibodies in a Western blot (<b>E</b>). Ethidium bromide staining of 25S rRNA and Coomassie staining are shown to indicate equal loading. The migrations of genomic RNA (gRNA), subgenomic RNA1 (sgRNA1), CP subgenomic RNA (CPsgRNA) and βC1 subgenomic RNA (βC1sgRNA) were indicated.</p

Compromised VIGS efficiency and enhanced susceptibility to TYLCCNV infection in RDR6-deficient <i>Nicotiana benthamiana</i> plants.

<p>(<b>A</b>) Phenotype of <i>Su</i> silencing in the wt and <i>NbRDR6</i> RNAi line (dsRDR6) induced by the 10A+2mDNA1-<i>NbSu</i> VIGS vector at 30 dpi. (<b>B</b>) RT-qPCR analysis of levels of <i>Su</i> mRNA in silenced plants shown in (<b>A</b>). Mock indicates wt plants infected by the empty VIGS vector (10A+2mDNA1) and the relative <i>Su</i> mRNA value in mock plants was set as 1. Error bars: ± SD. Asterisk indicates P value compared with mock-treated wild type plants: *P≤0.05 (Student's <i>t</i> test). (<b>C</b>) Southern blot analysis of 10A and DNA1 accumulation in systemic leaves of <i>Su</i>-silenced plants shown in (<b>A</b>) at 30 dpi. (<b>D</b>) Symptoms of wt and dsRDR6 <i>N. benthamiana</i> plants infected with 10A or 10Aβ at 30 dpi. (<b>E</b>) Southern blot analysis of viral DNA accumulation in systemically infected leaves of wt and dsRDR6 at 30 dpi shown in (<b>D</b>). Total DNA agarose gels were stained with ethidium bromide to show the equal loading. Viral single-stranded DNA (ssDNA) and supercoiled DNA (scDNA) forms are indicated. (<b>F</b>) RNA blot hybridization analysis of viral small RNA accumulations in VIGS vector-infected plants shown in (<b>A</b>) and wt virus-infected plants shown in (<b>D</b>). The blots were hybridized to single-stranded DNA oligonucleotide probes specific for 10A, 10β, <i>NuSu</i> and U6 small RNA.</p

The role of co-opted ESCRT proteins and lipid factors in protection of tombusviral double-stranded RNA replication intermediate against reconstituted RNAi in yeast

<div><p>Reconstituted antiviral defense pathway in surrogate host yeast is used as an intracellular probe to further our understanding of virus-host interactions and the role of co-opted host factors in formation of membrane-bound viral replicase complexes in protection of the viral RNA against ribonucleases. The inhibitory effect of the RNA interference (RNAi) machinery of <i>S</i>. <i>castellii</i>, which only consists of the two-component <i>DCR1</i> and <i>AGO1</i> genes, was measured against tomato bushy stunt virus (TBSV) in wild type and mutant yeasts. We show that deletion of the co-opted ESCRT-I (<u>e</u>ndosomal <u>s</u>orting <u>c</u>omplexes <u>r</u>equired for <u>t</u>ransport I) or ESCRT-III factors makes TBSV replication more sensitive to the RNAi machinery in yeast. Moreover, the lack of these pro-viral cellular factors in cell-free extracts (CFEs) used for <i>in vitro</i> assembly of the TBSV replicase results in destruction of dsRNA replication intermediate by a ribonuclease at the 60 min time point when the CFE from wt yeast has provided protection for dsRNA. In addition, we demonstrate that co-opted oxysterol-binding proteins and membrane contact sites, which are involved in enrichment of sterols within the tombusvirus replication compartment, are required for protection of viral dsRNA. We also show that phosphatidylethanolamine level influences the formation of RNAi-resistant replication compartment. In the absence of peroxisomes in <i>pex3Δ</i> yeast, TBSV subverts the ER membranes, which provide as good protection for TBSV dsRNA against RNAi or ribonucleases as the peroxisomal membranes in wt yeast. Altogether, these results demonstrate that co-opted protein factors and usurped lipids are exploited by tombusviruses to build protective subcellular environment against the RNAi machinery and possibly other cellular ribonucleases.</p></div

Elevated phospholipid level in yeast decreases the sensitivity of replicating tombusvirus RNA to the reconstituted RNAi.

<p>(A-B) Induction of RNAi in <i>opi1Δ</i> yeast inhibits TBSV repRNA accumulation less effectively than in wt yeast. Top panels: Replication of the TBSV repRNA was measured by Northern blotting 24 h after initiation of TBSV replication in wt BY4741 or <i>opi1Δ</i> yeast strains. Note that AGO1 and DCR1 were continuously expressed during the entire yeast culturing process to enhance RNAi activities in both strains. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006520#ppat.1006520.g001" target="_blank">Fig 1A</a> for further details. (C) Scheme of the CFE-based TBSV replication assay with in vitro reconstituted VRCs. MNase treatment (0.05 U/μl) was performed for 15 min, as shown, followed by inactivation of the MNase with EGTA. Each CFE-based assay lasted for three hours to accomplish maximum level of TBSV repRNA accumulation. (D) Decreased sensitivity of viral dsRNA products to MNase treatment (0.05 U/μl) in the <i>opi1Δ</i> CFE-based TBSV replication assay. Non-denaturing PAGE analysis of the ³²P-labeled TBSV repRNA products obtained in the CFE-based assay programmed with <i>in vitro</i> transcribed TBSV DI-72 (+)repRNA and purified recombinant MBP-p33 and MBP-p92^pol replication proteins of TBSV. The CFEs were prepared from BY4741 or <i>opi1Δ</i> yeast strains. Each experiment was repeated.</p