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Endogenous Pararetroviral Sequences in Tomato (Solanum Lycopersicum) and Related Species

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Endogenous Pararetroviral Sequences in Tomato (Solanum Lycopersicum) and Related Species

Christina Staginnus et al. BMC Plant Biol.

Abstract

Background: Endogenous pararetroviral sequences (EPRVs) are a recently discovered class of repetitive sequences that is broadly distributed in the plant kingdom. The potential contribution of EPRVs to plant pathogenicity or, conversely, to virus resistance is just beginning to be explored. Some members of the family Solanaceae are particularly rich in EPRVs. In previous work, EPRVs have been characterized molecularly in various species of Nicotiana including N.tabacum (tobacco) and Solanum tuberosum (potato). Here we describe a family of EPRVs in cultivated tomato (Solanum lycopersicum L.) and a wild relative (S.habrochaites).

Results: Molecular cloning and DNA sequence analysis revealed that tomato EPRVs (named LycEPRVs) are most closely related to those in tobacco. The sequence similarity of LycEPRVs in S.lycopersicum and S.habrochaites indicates they are potentially derived from the same pararetrovirus. DNA blot analysis revealed a similar genomic organization in the two species, but also some independent excision or insertion events after species separation, or flanking sequence divergence. LycEPRVs share with the tobacco elements a disrupted genomic structure and frequent association with retrotransposons. Fluorescence in situ hybridization revealed that copies of LycEPRV are dispersed on all chromosomes in predominantly heterochromatic regions. Methylation of LycEPRVs was detected in CHG and asymmetric CHH nucleotide groups. Although normally quiescent EPRVs can be reactivated and produce symptoms of infection in some Nicotiana interspecific hybrids, a similar pathogenicity of LycEPRVs could not be demonstrated in Solanum L. section Lycopersicon [Mill.] hybrids. Even in healthy plants, however, transcripts derived from multiple LycEPRV loci and short RNAs complementary to LycEPRVs were detected and were elevated upon infection with heterologous viruses encoding suppressors of PTGS.

Conclusion: The analysis of LycEPRVs provides further evidence for the extensive invasion of pararetroviral sequences into the genomes of solanaceous plants. The detection of asymmetric CHH methylation and short RNAs, which are hallmarks of RNAi in plants, suggests that LycEPRVs are controlled by an RNA-mediated silencing mechanism.

Figures

Figure 1
Figure 1
Genomic organization of EPRV sequences in the genus Solanum subsection Lycopersicon. DNA preparations from five species of the genus Solanum subsection Lycopersicon and Solanum tuberosum were restricted with XbaI and hybridized to a 5.5 fragment of NsEPRV covering ORF 2 to 4 and the IGR. Similar data (lanes 1 to 5) have been shown previously [47].
Figure 2
Figure 2
A. Analysis of cloned LycEPRV sequences and flanking sequences. Alignment of cloned EPRV sequences from S.lycopersicum (Le1-5) and S.habrochaites (Lh1-9) to the structure of TVCV-like EPRVs comprising four ORFs (upper bar): coat protein (CP), cell-to-cell movement protein (MP), polyprotein (POL) and transactivator domain (TAV). Rearranged coding regions are indicated by extra boxes and arrows for a deviating orientation of the reading frames. Nine clones contain parts of the intergenic region (IGR) marked by grey boxes with a white square for the position of the conserved 272 to 282 bp-box. Black bars indicate flanking sequences unrelated to EPRVs. Survey of sequences flanking the EPRVs in S.lycopersicum and S.habrochaites is given by coloured boxes. The majority represents repetitve elements (orange, red and blue boxes) most of which belong to retrotransposons (orange and red boxes), especially the LTR regions (red boxes). Arrows point towards the end of similar LTRs which is marked by a bracket. A description of the flanking sequences is listed in Table 1 according to the numbers. B. Sequence conservation within a 272to 282 bp box of the IGR from differentSolanaceae EPRVs. Alignment of the respective region of three LycEPRVs (Lh1, Lh2, Lh5) to three different tobacco EPRVs (TVCV, [2]; NsEPRV, [1]; NtoEPRV, [3]) and to two Solanum tuberosum EPRV copies (SoTuI-2, SoTuI-10; AJ564214, AJ564220; [4]). Next to a remarkable overall sequence homogeneity within the IGR region several shorter motives are highly conserved between EPRVs from all three species (red frames).
Figure 3
Figure 3
Chromosomal localization of LycEPRVs. Double target fluorescent in situ hybridization was carried out on root tip metaphases and male meiotic pachytene cells of S.lycopersicum (A-F) and S.habrochaites (G-I). Biotin labelled pooled probes of LycEPRVs from S.lycopersicum (LycEPRV-Sl, A-F) and S.habrochaites (LycEPRV-Sh, G-I), respectively, that cover most of LycEPRV sequence (for clone combinations see Table 2) were detected by red Alexa-594 fluorescence and hybridized together with digoxigenin labelled repeated DNA probes detected by green FITC fluorescence. Chromosomes were counterstained with DAPI (blue fluorescence). A-C) Metaphase chromosomes of S.lycopersicum (2n = 24). LycEPRV-Sl sequences (red in B and magenta in the overlay with blue DAPI staining in A) are located at the centromeres of most chromosomes with variable intensity, but are absent from the NOR region (green rDNA probe in A) and reduced on four chromosomes (arrows in B). In C the LycEPRVs are shown to co-localize with the retroelement sequence U30 from S.lycopersicum (green) that shows dispersed signals on all chromosomes. D-F) Pachytene chromosomes of S.lycopersicum are much more extended than metaphase chromosomes and show differentiation with DAPI into strongly stained heterochromatin and weakly stained euchromatin (D). The red LycEPRV signal is almost exclusively seen in the pericentromeric heterochromatic regions and intercalary chromocentre (arrowheads in D and E), but not at the NOR region (green in E, F; DAPI is shown as grey image with the probe signal falsely coloured red and green, respectively). G-I) Metaphase chromosomes of S.habrochaites (2n = 24). LycEPRV-Sh sequences (red in H, magenta in the overlay with blue DAPI staining in G, I) are located near the centromeres of most chromosomes showing stronger signal in some. No signal is visible in the NOR regions (green rDNA probe in G, arrow heads in I). Bar 10 μm.
Figure 4
Figure 4
Analysis of cytosine methylation in LycEPRV sequences. DNA of parental plants (flanking) and interspecific hybrids (central) was restricted with XbaI (lane 2 to 4 each) and either ScrFI (S) and BstNI (B) to detect CHG methylation (A, B) or Sau3aI (Sa) and NdeI (N) for asymmetric cytosine sites (C, D), the first enzyme of each pair being methylation sensitive. The first lane each contains undigested DNA (un). A, C. DNAs were hybridized to a 1.3 kb fragment of the CP/MP reading frame (E1) of a S.lycopersicum EPRV copy (Le1) and B, D. to a 580 bp fragment of the IGR (H7) of a S.habrochaites clone (Lh7).
Figure 5
Figure 5
Identification of transcripts homologous to Lycopersicon EPRVs. A. Survey over a selection of homologous ESTs of the genus Solanum subsection Lycopersicon and their position (grey boxes) in relation to the LycEPRV structure. Details about the ESTs (according to the numbers) are given in Table 3. Arrows mark the position of primers used for RT-PCR. B. PolyA+-enriched RNA of S.lycopersicum, S.habrochaites and an interspecific hybrid was used for RT-PCR with primer pairs of the CP/MP and TAV ORFs and the IGR as indicated in (A). The first strand DNA template was prepared from polyA+ enriched RNA from leaves of S.lycopersicum, S.habrochaites and an interspecific hybrid (lane 1–3 each). To detect possible genomic DNA contaminations an actin sequence spanning an intron was amplified in parallel. Water controls are indicated by a dash (lane 4 each). C, D. Unrooted dendrograms showing the genetic distance between genomic and cDNA sequences of the TAV region (C) and the IGR (D) of S.lycopersicum (red boxes), S.habrochaites (green boxes) and an interspecific hybrid (white boxes). cDNA sequences are indicated by a square, circles mark genomic sequences. The horizontal bar represents percent divergence (/100).
Figure 6
Figure 6
Analysis of short RNAs homologous to LycEPRV. The short RNA fraction of S.lycopersicum leaves (1, 2), S.lycopersicum flowers (3), S.habrochaites leaves (4) and leaves of an interspecific hybrid (5) was hybridized to riboprobes derived from three different TAV clones (top) and from a clone carrying the conserved part of the IGR (bottom). Ethidium bromide staining of the major RNA on the gel is shown as a loading control below each blot.
Figure 7
Figure 7
Short LycEPRV RNAs after heterologous virus infection. The short RNA fraction of S.lycopersicum leaves derived from the cultivar "Moneymaker" (MM), a transgenic line of "Moneymaker" (CS3, [63]) and the cultivar "MicroTom" (MT) was hybridized to TAV (B, D) and IGR (A, C) riboprobes after infecting the plants with PVY (Potyvirus Y; in A, B) or TBSV (Tomato bushy stunt virus; in C, D) that express suppressors of PTGS. Individual plants may show different reactions to virus infection therefore several individuals were infected in each assay. Since a general trend became visible only one representative plant is shown here. C0: bulked leaves harvested before infection; C-: mock infection; TBSV+: infected with Tomato bushy stunt virus; PVY+: infected with Potyvirus Y. Ethidium bromide staining of the major RNA on the gel is shown as a loading control below each blot.

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