Primary and secondary siRNAs in geminivirus-induced gene silencing

In plants, RNA silencing-based antiviral defense is mediated by Dicer-like (DCL) proteins producing short interfering (si)RNAs. In Arabidopsis infected with the bipartite circular DNA geminivirus Cabbage leaf curl virus (CaLCuV), four distinct DCLs produce 21, 22 and 24 nt viral siRNAs. Using deep sequencing and blot hybridization, we found that viral siRNAs of each size-class densely cover the entire viral genome sequences in both polarities, but highly abundant siRNAs correspond primarily to the leftward and rightward transcription units. Double-stranded RNA precursors of viral siRNAs can potentially be generated by host RDR-dependent RNA polymerase (RDR). However, genetic evidence revealed that CaLCuV siRNA biogenesis does not require RDR1, RDR2, or RDR6. By contrast, CaLCuV derivatives engineered to target 30 nt sequences of a GFP transgene by primary viral siRNAs trigger RDR6-dependent production of secondary siRNAs. Viral siRNAs targeting upstream of the GFP stop codon induce secondary siRNAs almost exclusively from sequences downstream of the target site. Conversely, viral siRNAs targeting the GFP 3'-untranslated region (UTR) induce secondary siRNAs mostly upstream of the target site. RDR6-dependent siRNA production is not necessary for robust GFP silencing, except when viral siRNAs targeted GFP 5'-UTR. Furthermore, viral siRNAs targeting the transgene enhancer region cause GFP silencing without secondary siRNA production. We conclude that the majority of viral siRNAs accumulating during geminiviral infection are RDR1/2/6-independent primary siRNAs. Double-stranded RNA precursors of these siRNAs are likely generated by bidirectional readthrough transcription of circular viral DNA by RNA polymerase II. Unlike transgenic mRNA, geminiviral mRNAs appear to be poor templates for RDR-dependent production of secondary siRNAs

Sequencing of RDR6-dependent double-stranded RNAs reveals novel features of plant siRNA biogenesis

Biogenesis of trans-acting siRNAs (tasiRNAs) is initiated by miRNA-directed cleavage of TAS gene transcripts and requires RNA-dependent RNA polymerase 6 (RDR6) and Dicer-like 4 (DCL4). Here, we show that following miR173 cleavage the entire polyadenylated parts of Arabidopsis TAS1a/b/c and TAS2 transcripts are converted by RDR6 to double-stranded (ds)RNAs. Additionally, shorter dsRNAs are produced following a second cleavage directed by a TAS1c-derived siRNA. This tasiRNA and miR173 guide Argonaute 1 complexes to excise the segments from TAS2 and three TAS1 transcripts including TAS1c itself to be converted to dsRNAs, which restricts siRNA production to a region between the two cleavage sites. TAS1c is also feedback regulated by a cis-acting siRNA. We conclude that TAS1c generates a master siRNA that controls a complex network of TAS1/TAS2 siRNA biogenesis and gene regulation. TAS1/TAS2 short dsRNAs produced in this network are processed by DCL4 from both ends in distinct registers, which increases repertoires of tasiRNAs

Viral protein suppresses oxidative burst and salicylic acid-dependent autophagy and facilitates bacterial growth on virus-infected plants

Virus interactions with plant silencing and innate immunity pathways can potentially alter the susceptibility of virus-infected plants to secondary infections with nonviral pathogens. We found that Arabidopsis plants infected with Cauliflower mosaic virus (CaMV) or transgenic for CaMV silencing suppressor P6 exhibit increased susceptibility to Pseudomonas syringae pv. tomato (Pst) and allow robust growth of the Pst mutant hrcC-, which cannot deploy effectors to suppress innate immunity. The impaired antibacterial defense correlated with the suppressed oxidative burst, reduced accumulation of the defense hormone salicylic acid (SA) and diminished SA-dependent autophagy. The viral protein domain required for suppression of these plant defense responses is dispensable for silencing suppression but essential for binding and activation of the plant target-of-rapamycin (TOR) kinase which, in its active state, blocks cellular autophagy and promotes CaMV translation. Our findings imply that CaMV P6 is a versatile viral effector suppressing both silencing and innate immunity. P6-mediated suppression of oxidative burst and SA-dependent autophagy may predispose CaMV-infected plants to bacterial infection

Maps of vsRNAs from CaLCuV-infected wild type (Col-0) and <i>rdr1/2/6</i> triple mutant plants at single-nucleotide resolution.

<p>The graphs plot the number of 20–25 nt vsRNA reads at each nucleotide position of the 2583 bp DNA-A (<b>A</b>) and the 2513 bp DNA-B (<b>B</b>); Bars above the axis represent sense reads starting at each respective position; those below represent antisense reads ending at the respective position (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002941#ppat.1002941.s009" target="_blank">Tables S2</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002941#ppat.1002941.s010" target="_blank">S3</a>). The genome organizations of DNA-A and DNA-B are shown schematically above the graphs, with leftward (AC1, AC4, AC2, AC3 and BC1) and rightward (AV1 and BV1) ORFs and common region (CR) indicated. The predicted rightward and rightward mRNAs are shown as respectively blue and red solid lines with arrowheads. Potential readthrough transcripts are shown as dotted thin lines.</p

VIGS phenotypes and accumulation of primary and secondary siRNAs in L2 transgenic plants infected with CaLCuV::GFP viruses targeting the <i>GFP</i> transcribed region.

<p>(<b>A</b>) The L2 T-DNA region containing the 35S-GFP transgene is shown schematically. Positions of the duplicated CaMV 35S enhancer and core promoter elements, <i>GFP</i> mRNA elements including 5′UTR, translation start (AUG) and stop (UAA) codons and 3′UTR with poly(A) signal (AAUAAA), and 35S terminator sequences indicated. Numbering is from the T-DNA left border (LB). The VIGS target sequences, inserted in the CaLCuV::GFP viruses <i>Lead</i>, <i>CodB, CodM, CodE, Trail</i> and <i>polyA</i>, are indicated with dotted boxes; (<b>B</b>) Pictures under UV light of L2 transgenic plants infected with the above viruses; (<b>C</b>) Blot hybridization analysis of total RNA isolated from plants shown in Panel B. The blot was successively hybridized with short DNA probes specific for CaLCuV <i>AC4</i> gene (AC4_s) and 35S::GFP transgene sequences inserted in the CaLCuV::GFP viruses (<i>Lead, CodB, CodM, CodE, Trail</i> and <i>polyA</i>), the <i>GFP</i> mRNA 3′UTR non-target sequence (3′UTR) and <i>Arabidopsis</i> miR173 and Met-tRNA (the latter two serve as loading control).</p

Primary and secondary siRNAs in CaLCuV::Chl virus-infected wild type (Col-0) plants.

<p>(<b>A</b>) The 2300 bp region of the <i>Arabidopsis</i> genome, which contains <i>Chlorata I/CH42</i> gene (<i>ChlI</i>), is shown schematically with positions of <i>ChlI</i> promoter, pre-mRNA with two introns, and terminator sequences indicated; numbering starts 500 nucleotides upstream of the transcription start site. The VIGS target sequence (inserted in CaLCuV::Chl virus) is highlighted with grey. The graph plots the number of 20–25 nt siRNA reads at each nucleotide position of the <i>ChlI</i> gene; Bars above the axis represent sense reads starting at each respective position; those below represent antisense reads ending at the respective position (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002941#ppat.1002941.s011" target="_blank">Table S4</a>). (<b>B</b>) The left bar graph shows the total numbers of 20–25 nt primary (CaLCuV::Chl-derived) and secondary siRNAs derived from <i>ChlI</i> sequences outside of the VIGS target region, while the right bar graph shows the number of primary siRNAs for each size class and polarity.</p

VIGS phenotypes and primary siRNA accumulation in L2 transgenic plants infected with CaLCuV::GFP viruses that target the <i>GFP</i> promoter and terminator elements.

<p>(<b>A</b>) The L2 T-DNA region containing the 35S-GFP transgene is shown schematically. Positions of the duplicated CaMV 35S enhancer (<i>Enh</i>) and core promoter (<i>Core</i>) elements (CAAT and TATA boxes and transcription start <i>Plus1</i>), the <i>GFP</i> mRNA elements (5′UTR, AUG and UAA codons and 3′UTR, and 35S terminator are indicated. Numbering is from the T-DNA left border (LB). The VIGS target sequences, inserted in the CaLCuV::GFP viruses <i>ProFL, Enh, CAAT, TATA, Plus1, CodFL, Trail</i> and <i>Post</i> are indicated with dotted boxes; (<b>B</b>) and (<b>C</b>) Blot hybridization analysis of total RNA isolated from L2 plants infected with the above viruses. The two blots were successively hybridized with short DNA probes specific for CaLCuV <i>AC4</i> gene (AC4_s) and the 35S::GFP transgene sequences inserted in CaLCuV::GFP viruses and <i>Arabidopsis</i> miR173 and Met-tRNA (the latter two serve as loading control). (<b>D</b>) Pictures under UV light of L2 transgenic plans infected with the CaLCuV::GFP viruses (names indicated).</p