Nicotiana small RNA sequences support a host genome origin of cucumber mosaic virus satellite RNA

Abstract

Satellite RNAs (satRNAs) are small noncoding subviral RNA pathogens in plants that depend on helper viruses for replication and spread. Despite many decades of research, the origin of satRNAs remains unknown. In this study we show that a β-glucuronidase (GUS) transgene fused with a Cucumber mosaic virus (CMV) Y satellite RNA (Y-Sat) sequence (35S-GUS:Sat) was transcriptionally repressed in N. tabacum in comparison to a 35S-GUS transgene that did not contain the Y-Sat sequence. This repression was not due to DNA methylation at the 35S promoter, but was associated with specific DNA methylation at the Y-Sat sequence. Both northern blot hybridization and small RNA deep sequencing detected 24-nt siRNAs in wild-type Nicotiana plants with sequence homology to Y-Sat, suggesting that the N. tabacum genome contains Y-Sat-like sequences that give rise to 24-nt sRNAs capable of guiding RNA-directed DNA methylation (RdDM) to the Y-Sat sequence in the 35S-GUS:Sat transgene. Consistent with this, Southern blot hybridization detected multiple DNA bands in Nicotiana plants that had sequence homology to Y-Sat, suggesting that Y-Sat-like sequences exist in the Nicotiana genome as repetitive DNA, a DNA feature associated with 24-nt sRNAs. Our results point to a host genome origin for CMV satRNAs, and suggest novel approach of using small RNA sequences for finding the origin of other satRNAs.

Figure 1. The 35S-GUS:Sat fusion transgenes show repressed expression in N. tabacum in comparison to the 35S-GUS transgene.

(A) Schematic diagrams of the transgene constructs. (B) MUG assay of independent primary (T0) transformants. (C) MUG assay of second (T1) and third-generation (T2) transformants, plus non-transgenic (NT) control plants. (D) mRNA northern blot analysis of the plants shown in (C). Note: the different units in the Y-axis of the two MUG assay histograms are due to the use of different fluoroscan machines and different ways of calculation.

Figure 2. The 35S-GUS:Sat transgene is transcriptionally repressed.

(A) Three of the four 35S-GUS:Sat transgenic lines analysed show no detectable accumulation of 21-nt PTGS-associated GUS or Y-Sat-specific siRNAs, indicating that PTGS is not the main cause of 35S-GUS:Sat transgene repression. M, 21 and 24-nt small RNA size markers. (B) and (C) The highly expressed 35S-GUS line (GUS-3) (B) generates much stronger nuclear run-on transcript signals than the two repressed 35S-GUS:Sat lines (C), indicating that the reduced expression of the 35S-GUS:Sat transgenes results from transcriptional repression. FKS1 and EF1α are negative control and internal reference gene sequences, respectively.

Figure 3. Methylation analysis of the 35S-GUS:sSat transgene in transgenic N. tabacum using McrBC PCR.

(A) and (B) McrBC PCR of 35S promoter, different regions of GUS coding sequence, and Y-Sat. (C) qPCR quantification of McrBC digestion of the Y-Sat sequence shown in (B) (nt. 1-214) (left) and MUG assay of GUS activity in the transgenic plants used for the McrBC PCR analysis (right). M, DNA size marker.

Figure 4. Bisulfite sequencing shows strong DNA methylation in the Y-Sat sequence of the 35S-GUS:sSat transgene in all cytosine contexts, particularly at the CG and CHG sites.

The methylation status of cytosines in CG, CHG and CHH contexts is presented separately in three line graphs. Each point on the line represents a cytosine in a sequential 5′ to 3′ order along the bisulfite-sequenced Y-Sat sequence, with the Y-axis showing the percentage of methylated cytosines (based on the ratio of cytosine peak height in the trace file) at each of these positions. The top panel shows the methylation pattern of two F1 sibling plants derived from a cross between WT N. tabacum (as the maternal parent) and a 35S-GUS:sSat transgenic line. The bottom panel shows the methylation pattern of two independent 35S-GUS:sSat transgenic lines. Note that in both cases the plant showing a higher level of GUS expression (F1-1 or GUS:sSat-2, shown in red) displays a lower degree of CHG and CHH methylation in the Y-Sat sequence.

Figure 5. sRNAs of 24 nt in size are readily detectable in Nicotiana plants using Y-Sat probe.

(A) The 24 nt siRNAs are detected in the flowers of both WT and 35S-GUS, 35S-GUS:sSat, 35S-GUS:asSat transgenic N. tabacum plants. M21+24nt, 21 and 24-nt small RNA size marker. (B) 24 nt siRNAs are detectable in leaf tissues of three different Nicotiana species, N. tabacum (tab), N. clevelandii (cle), and N. benthamiana (ben). “Y-Sat+” is a RNA sample derived from a Y-Sat-infected N. tabacum plant, which was under-exposed to show the dominant ∼21-nt Y-Sat-derived siRNAs. M24nt, 24-nt small RNA size marker. Both hybridized blots were treated with RNase A before exposure.

Figure 6. Alignment of sRNAs to three CMV satRNA genome sequences at the E-value of 1e⁻³ (A and B) and 1e⁻⁵ (C).

(A) sRNAs from uninfected N. tabacum Xanthi plants. (B) sRNAs from SD-CMV△satR-infected N. tabacum Xanthi plants. sRNAs matching the satRNA plus-strand and minus-strand are shown as short thin lines above and below the arrow-headed black lines, respectively. sRNAs mapping to the two most conserved regions of CMV satRNAs (nt. 60–80 and nt. 280–300; see S5 Fig.) are boxed. The five sRNAs perfectly matching to SD-satRNA, or nearly perfectly matching (for three of the five sRNAs) to Y-Sat and satCMV110, are shown as color-coded sequences and their respective nucleotide positions in the satRNA genome indicated. (C) Sequence and location of the 14 sRNAs identified from CMVΔsatR-infected Nt-Xanthi plants that map to the Y-Sat genome (the relevant part of the Y-Sat genome sequence, from nt. 1–90 and from nt. 200–328, is also shown).

Figure 7. Multiple DNA bands are detectable in Nicotiana species that show homology to Y-Sat.

(A) One or multiple bands are detected in BamHI-digested DNA of N. tabacum (tab), N. clevelandii (cle), and N. benthamiana (ben). (B) Digestion of N. tabacum DNA with different enzymes gives different band patterns. M, DNA size marker (1 kb DNA ladder).