RNA silencing of host transcripts by cauliflower mosaic virus requires coordinated action of the four Arabidopsis Dicer-like proteins

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   Fig. 1.

   CaMV infection in B. rapa and Arabidopsis triggers accumulation of vsRNAs derived from the 35S RNA leader sequence. (A) Organization of the double-stranded circular DNA genome of CaMV and the two 35S and 19S viral RNAs. The leader sequence is in the 5′ end of the 35S RNA. (B) RNA blot analyses of low-molecular-weight RNAs from CaMV-infected plants. The probe used covered the entire CaMV genome. 5S, ethidium bromide staining of 5S ribosomal RNA. (C) Schematics of the 35S and 19S viral RNAs and RNA blot analyses of low-molecular-weight RNAs derived from different regions of the CaMV genome (nucleotide coordinates are indicated) by using DNA probes. Only a 1,500-nt fragment between nucleotides 4700 and 6264 was not probed. The probe against the leader sequence (1b) allowed detection of mainly 24-nt vsRNAs at short exposures (≈2 h). Longer exposures (≈20 h) revealed 21-nt vsRNA accumulation. (D) Predicted strong secondary structure of the 35S RNA leader (Vienna RNA folding package). Col-0, Columbia.

   
   
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   Fig. 2.

   DCL dependencies of vsRNA patterns and CaMV accumulation in Arabidopsis silencing mutants. (A) Northern blots of low-molecular-weight RNA accumulating in CaMV-infected silencing mutants of Arabidopsis. Probe was specific for the entire leader sequence. The numbers of inoculated plants showing systemic infection are from two separate experiments involving five plants each. (B) Same as in A, but the probe was an end-labeled oligonucleotide corresponding to the sRCC1 vsRNA (sequence on top) identified through the procedure described in Fig. 3. (C) Same as in B, but in the dcl1–9 and hen1 mutants. (D) Same as in C, but with two independent alleles of dcl1. The blot was exposed longer to allow detection of vsRNA accumulation (Lower). (E) Immunoblot of CaMV CP (a product of the 35S RNA) accumulation in various RNA silencing mutants and combinations thereof. Proteins were extracted from the samples used in A–D. 5S, ethidium bromide staining of 5S ribosomal RNA; Tot prot: total protein revealed by Coomassie staining. (F) Northern blot analysis of high-molecular-weight RNA extracted from the samples used in A–D. (Right) Composite comparing effects of dcl1–9 and triple dcl2-dcl3-dcl4 mutations.

   
   
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   Fig. 3.

   The leader-derived sRCC1 vsRNA down-regulates accumulation of an Arabidopsis transcript and acts as a bona fide siRNA in infected turnip. (A) Discrete, leader-derived sRNAs, as detected by Northern blot of low-molecular-weight RNA from CaMV-infected Arabidopsis. Probes used were oligonucleotides complementary to indicated sequences. sRNAs in black were not found to exhibit significant complementarity to Arabidopsis transcripts as assessed by BLAST searches. sRNAs in blue were found to exhibit near-perfect complementarity to Arabidopsis transcripts (BLAST search results only). (B) Schematic of predicted sRCC1 target position in At1g76950. (C) sQPCR analysis of At1g76950 transcript accumulation in infected (I) and mock-inoculated (M) Arabidopsis. No RT, control reaction performed without reverse transcriptase; UBI, amplification control using ubiquitin-specific primers. (D) sRCC1 accumulation as assessed by Northern blot of low-molecular-weight RNA in CaMV-infected and mock-inoculated turnip leaves. (E) Schematic of sRCC1 sensor transgene constructs and their transient expression in CaMV-infected and noninfected (Mock) turnip leaves at 15 dpi. LB and RB, left and right T-DNA borders, respectively. (F) Immunoblot analysis of GFP accumulation in the tissues depicted in E. e.v., empty vector used as a negative control in infiltrations.

   
   
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   Fig. 4.

   Tens of Arabidopsis transcripts are potentially targeted by sRCC1 and other leader-derived vsRNAs. (A) sRCC1 accumulation in infected tissues of the At1g76950 T-DNA insertion mutant, as assessed by Northern blot of low-molecular-weight RNA. The last panel shows CaMV CP accumulation assessed by immunoblot. (B) sQPCR analysis of At1g75330 and At3g52500 transcript levels in leaves of CaMV-infected (I) and mock-inoculated (M) plants at 21 dpi (33 amplification cycles, Upper). (Lower) QPCR analysis of At1g75330 transcript levels in WT and dcl2-dcl3-dcl4 mutant backgrounds. For each sample, mRNA levels were normalized to that of Actin2 (At3g18780). Error bars represent standard deviation from three independent experiments involving triplicate PCRs. (C) The sRCC1 sequence was queried for complementarity to Arabidopsis transcripts by using miRU settings with decreasing stringencies. Mism, mismatch; G:U, G:U wobbles allowances. The transcript sets retrieved under high stringency conditions were always contained within those obtained under lower stringency settings. The target transcript number reached a plateau of 90. Sequence alignments and identities of predicted targets are in Table 1 and Data Set 1, which are published as supporting information on the PNAS web site.