Massive production of small RNAs from a non-coding region of Cauliflower mosaic virus in plant defense and viral counter-defense

Abstract

To successfully infect plants, viruses must counteract small RNA-based host defense responses. During infection of Arabidopsis, Cauliflower mosaic pararetrovirus (CaMV) is transcribed into pregenomic 35S and subgenomic 19S RNAs. The 35S RNA is both reverse transcribed and also used as an mRNA with highly structured 600 nt leader. We found that this leader region is transcribed into long sense- and antisense-RNAs and spawns a massive quantity of 21, 22 and 24 nt viral small RNAs (vsRNAs), comparable to the entire complement of host-encoded small-interfering RNAs and microRNAs. Leader-derived vsRNAs were detected bound to the Argonaute 1 (AGO1) effector protein, unlike vsRNAs from other viral regions. Only negligible amounts of leader-derived vsRNAs were bound to AGO4. Genetic evidence showed that all four Dicer-like (DCL) proteins mediate vsRNA biogenesis, whereas the RNA polymerases Pol IV, Pol V, RDR1, RDR2 and RDR6 are not required for this process. Surprisingly, CaMV titers were not increased in dcl1/2/3/4 quadruple mutants that accumulate only residual amounts of vsRNAs. Ectopic expression of CaMV leader vsRNAs from an attenuated geminivirus led to increased accumulation of this chimeric virus. Thus, massive production of leader-derived vsRNAs does not restrict viral replication but may serve as a decoy diverting the silencing machinery from viral promoter and coding regions.

Figure 1.

Massive, RDR-independent generation of CaMV vsRNAs in infected Arabidopsis. (A) Total RNA from pools of CaMV-infected (+) or mock-inoculated (−) wild-type control plants (Col-0 and Col-gl1) and mutants defective in host sRNA biogenesis: dcl2-5, dcl3-1, dcl4-2, rdr2-1, rdr6-15 and rdr2 rdr6 (all in Col-0) and dcl1-8 (in Col-gl1). Plants were harvested 1-month post-inoculation or, in the case of dcl1-8, 2 months post-inoculation. Total RNA was separated by 15% PAGE and then stained with ethidium bromide (EtBr). Lower and upper portions of the gels are shown, the latter for RNA loading comparison. Positions of synthetic 21 and 24 nt RNA oligonucleotides and endogenous 102 nt U6 snRNA are indicated. (B) Illumina deep-sequencing of sRNAs from mock-inoculated and CaMV-infected Arabidopsis wild-type (Col-0) and mutants (rdr1/2/6, dcl1-8 and dcl2/3/4) confirms massive production of RDR-independent vsRNA, comparable to the entire complement of host sRNAs. The graph shows the percentage of Arabidopsis and vsRNAs in the pool of 20–25 nt reads mapped to the Arabidopsis and CaMV genomes with zero mismatches.

Figure 2.

Illumina deep-sequencing analysis of CaMV sRNAs and comparative mappings of viral RNA molecules. Bar graphs showing the percentages of (A) Arabidopsis sRNAs and (B) vsRNAs in the pool of 20–25 nt sRNA reads sequenced from CaMV-infected wild-type (Col-0) and mutant lines (rdr1/2/6, dcl1-8 and dcl2/3/4)—for number of reads, see Supplementary Table S1. (C) Genome-wide map of vsRNAs from CaMV-infected Col-0 at single-nucleotide resolution. The graph plots the number of 20–25 nt vsRNA reads at each position of the 8031-bp CaMV genome; the numbering starts at the 5′-terminus of the 35S RNA (genome position 7434) as depicted in panel D. Bars above the axis represent sense reads starting at each respective position; those below represent antisense reads ending at the respective position (Supplementary Data). (D) cRT–PCR mapping of CaMV 8S RNA from virus-infected Arabidopsis (Supplementary Figure S4 for experimental details). The circular CaMV genome organization is shown schematically with viral genes (boxes) and 35S and 19S promoters (dotted arrows) that drive Pol II transcription of two major transcripts: the pregenomic 35S RNA and subgenomic 19 RNAs (depicted below the genome). The position and termini of the CaMV leader region-derived 8S RNA species are indicated, as determined for sense and antisense polarities by cRT–PCR product sequencing. Regions surrounding the 35S RNA start site and the 3′-terminal part of the leader sequence preceding ORF VII are enlarged. The termini of sequenced 8S RNA clones are indicated by arrows above the sequence for sense RNAs (42 clones) and below the sequence for antisense RNAs (36 clones). The number of clones is given when more than one clone had the same 5′- or 3′-terminus. Thick arrows indicate the major transcription start site for sense 8S RNAs and the major 3′-terminus for antisense 8S RNAs, which fall on the same genome position.

Figure 3.

CaMV vsRNAs and DNA/RNA titers detected in quadruple dcl-mutants. (A) Analysis of low-molecular weight RNA extracted from pools of CaMV-infected (+) or mock-inoculated (−) plants: wild-type (Col-0), triple dcl-mutant (dcl2/3/4) and two quadruple dcl-mutants carrying weak dcl1 alleles (dcl1/2/3/4-sin, dcl1/2/3/4-caf). Size-fractionated RNA was analyzed by 18% PAGE followed by blot hybridization. The membrane was successively hybridized with sense and antisense DNA oligonucleotide probes for the CaMV leader region, and probes for host sRNAs: miR173 (22 nt), miR168 (21 nt), siR255 (21 nt), siR1003 (24 nt). Met-tRNA (72 nt) serves as a loading control. Sizes are indicated on each data image. (B) Viral titers in CaMV-infected wild-type (Col-0), dcl2/3/4 and dcl1/2/3/4-caf were measured using quantitative real-time PCR (qPCR) for viral DNA and qRT–PCR for polyadenylated 35S and 19S transcripts (see ‘Materials and Methods’ section). In both cases, PCR primers specific for the CaMV leader-region and the CaMV TAV region (Supplementary Table S3) were used in parallel for each sample. As internal controls, we performed qPCR and qRT–PCR on the same DNA and cDNA samples with primers specific for 18S rDNA and ACT2 gene, respectively (Supplementary Table S3). The mean of the normalized levels for viral DNA and RNA (the sum of 35S and 18S RNAs) is shown; the CaMV titer in wild-type (Col-0) plants was set to one in each case.

Figure 4.

Preferential loading of CaMV leader-derived vsRNAs into AGO1 and the effect of CaMV infection on accumulation of AGO1 protein and AGO1-associated miRNA. The upper panel shows RNA blot hybridization analysis of total sRNAs (input) and sRNAs associated with AGO1 protein in mock-inoculated (−) or CaMV-infected (+) plants following immunoprecipitation with AGO1-specific antibodies (IP:AGO1) or normal rabbit serum (IP:NRS) as a negative control. The membrane was successively hybridized with mixtures of non-overlapping sense and antisense DNA probes specific for the CaMV leader region (CaMV leader mix) or the CaMV 35 promoter and ORF VII/I regions (CaMV promoter + ORF mix) and the probe specific for miR173 (22 nt) (Supplementary Table S3). The lower panel shows western blot analysis of AGO1 protein accumulation in the input and the IP:AGO1 fractions using AGO1-specific antibodies (@AGO1). The blot was also stained with antibodies specific for CaMV TAV protein (@TAV).

Figure 5.

Ectopic expression of CaMV leader-derived vsRNAs from an attenuated geminivirus slightly increases viral DNA accumulation. (A) Viral DNA titers in Arabidopsis plants infected with a CaLCuV vector carrying the CaMV leader sequence (CaLCuV-CamvL) versus the vector control virus (CaLCuV-vec) or the vector carrying a non-viral insert (CaLCuV-GFP) were measured by semi-quantitative PCR on serial dilutions (5-fold each) of total DNA isolated from pools of three to four infected or mock-inoculated plants. 18S ribosomal DNA amplification is an internal control. The titers were also confirmed by qPCR. (B) vsRNAs were analyzed by RNA blot hybridization using total RNA from pools of three to four virus-infected (CaMV, CaLCuV-vec, CaLCuV-CamvL and CaLCuV-GFP) or mock-inoculated plants. The membrane was successively hybridized with DNA oligonucleotide probes for the CaMV leader region and CaLCuV AC4 gene. Met-tRNA is shown as a loading control. Note that two independent clones of the CaLCuV-CamvL construct were analyzed.

Figure 6.

Model for CaMV interactions with the Arabidopsis sRNA-generating machinery. Viral DNA is released from the virion into the nucleoplasm. Gaps in this DNA left by RT during the previous replication cycle are repaired by host DNA repair enzymes to create covalently closed molecules. Closed viral DNA is transcribed by Pol II into pregenomic 35S RNA, which is a polycistronic mRNA for several viral proteins including RT and a template for RT. Viral DNA is also transcribed by Pol II into subgenomic 19S RNA, which is the mRNA for the viral transactivator/viroplasmin protein (TAV). Abrupt termination of Pol II-driven transcription, potentially caused by an unrepaired DNA gap (Met-tRNA gap), results in production of aberrant 8S RNAs lacking poly(A) tails. This 8S RNA matches the 35S RNA leader sequence, and is predicted to form a viroid-like secondary structure, which may be converted to dsRNA by Pol II. We hypothesize that 8S RNA-derived dsRNA serves as a decoy to engage all four DCLs in massive production of 21, 22 and 24 nt vsRNAs. These leader-region vsRNAs would compete with vsRNAs derived from other regions for AGO proteins. Resulting antisense vsRNAs would potentially guide AGO-meditated silencing of 35S RNAs, but the structured nature of the leader would likely hinder vsRNA pairing. Sporadic cleavage products of 35S or 19S RNA enter sRNA biogenesis pathways (evidenced by low-level vsRNA biogenesis from these regions), but host RDR6-dependent processes are suppressed by viral TAV protein (19,20).