Retrotransposon BARE-1: expression of encoded proteins and formation of virus-like particles in barley cells

Retrotransposons are ubiquitous and major components of plant genomes, and are characteristically retroviral-like in their genomic structure and in the major proteins encoded. Nevertheless, few have been directly demonstrated to be transcribed or reverse transcribed. The BARE-1 retrotransposon family of barley (Hordeum vulgare) is highly prevalent, actively transcribed, and contains well conserved functional regions. Insertion sites for BARE-1 are highly polymorphic in the barley genome. Here we show that BARE-1 is translated and the capsid protein (GAG) and integrase (IN) components of the predicted polyprotein are processed into polypeptides of expected size. Some of the GAG sediments as virus-like particles together with IN and with BARE-1 cDNA. Reverse transcriptase activity is also present in gradient fractions containing BARE-1 translation products. Virus-like particles have also been visualized in fractions containing BARE-1 components. Thus BARE-1 components necessary for carrying out the life cycle of an active retrotransposon appear to be present in vivo, and to assemble. This would suggest that post-translational mechanisms may be at work to prevent rapid genome inflation through unrestricted integration.

Retrotransposon BARE-1 and Its Role in Genome Evolution in the Genus Hordeum.

The replicative retrotransposon life cycle offers the potential for explosive increases in copy number and consequent inflation of genome size. The BARE-1 retrotransposon family of barley is conserved, disperse, and transcriptionally active. To assess the role of BARE-1 in genome evolution, we determined the copy number of its integrase, its reverse transcriptase, and its long terminal repeat (LTR) domains throughout the genus Hordeum. On average, BARE-1 contributes 13.7 x 10(3) full-length copies, amounting to 2.9% of the genome. The number increases with genome size. Two LTRs are associated with each internal domain in intact retrotransposons, but surprisingly, BARE-1 LTRs were considerably more prevalent than would be expected from the numbers of intact elements. The excess in LTRs increases as both genome size and BARE-1 genomic fraction decrease. Intrachromosomal homologous recombination between LTRs could explain the excess, removing BARE-1 elements and leaving behind solo LTRs, thereby reducing the complement of functional retrotransposons in the genome and providing at least a partial "return ticket from genomic obesity."

The core domain of retrotransposon integrase in Hordeum: predicted structure and evolution.

Propagation of long terminal repeat (LTR)-bearing retrotransposons and retroviruses requires integrase (IN, EC 2.7.7.-), encoded by the retroelements themselves, which mediates the insertion of cDNA copies back into the genome. An active retrotransposon family, BARE-1, comprises approximately 7% of the barley (Hordeum vulgare subsp. vulgare) genome. We have generated models for the secondary and tertiary structure of BARE-1 IN and demonstrate their similarity to structures for human immunodeficiency virus 1 and avian sarcoma virus INs. The IN core domains were compared for 80 clones from 28 Hordeum accessions representative of the diversity of the genus. Based on the structural model, variations in the predicted, aligned translations from these clones would have minimal structural and functional effects on the encoded enzymes. This indicates that Hordeum retrotransposon IN has been under purifying selection to maintain a structure typical of retroviral INs. These represent the first such analyses for plant INs.

Gypsy-like retrotransposons are widespread in the plant kingdom.

Retrotransposons propagate via an RNA intermediate which is then reverse-transcribed and packaged into virus-like particles. They are either copia- or gypsy-like in coding domain order and sequence similarity, the gypsy-like elements sharing their organization with the retroviruses but lacking retroviral envelope domains. Copia-like retrotransposons, or at least their reverse transcriptase domains, appear broadly distributed in higher plants, but gypsy-like elements have been reported only for scattered species. The authors have exploited the difference in domain order between these groups to amplify and clone segments bridging the reverse transcriptase-integrase region of specifically gypsy-like retrotransposons. Species representative of the diversity of higher plants yielded products whose sequences establish that gypsy-like transposons are dispersed throughout the plant genomes. This class of plant elements has been named romani retrotransposons. The presence of both types ubiquitously in the fungi, plants and animals support their existence as ancient distinct lineages and subsequent, vertical radiation.

BARE-1 insertion site preferences and evolutionary conservation of RNA and cDNA processing sites.

The BARE-1 copia-like retrotransposon constitutes nearly 7% of the barley (Hordeum vulgare L.) genome as a family of more than 2 x 10(4) mostly full-length copies dispersed on all chromosomes. BARE-1 elements are transcribed in barley tissues from promoters within the LTR (long terminal repeat). The predicted, translated polyprotein contains conserved domains for GAG, aspartic proteinase, integrase, reverse-transcriptase, and RNase H. Here, we have used inverse PCR with LTR-based primers to establish the consensus sequences for the terminal region of the LTR, the external dinucleotides of the cDNA integration intermediate, and the minus- and plus-strand priming sites. These key functional entities are well-conserved in the BARE-1 family, including wheat Wis2, but differ from those of other plant retrotransposons. The target site duplication was established as 5 bp. Of the 13 integration sites identified here, 8 were other BARE-1 elements and 1 another retrotransposon; 59% of the total 17 identified BARE-1 insertion sites are retrotransposons. This nested insertion pattern may represent a basic feature of plant retrotransposons.

The BARE-1 retrotransposon is transcribed in barley from an LTR promoter active in transient assays.

The BARE-1 retrotransposon occurs in more than 10(4) copies in the barley genome. The element is bounded by long terminal repeats (LTRs, 1829 bp) containing motifs typical of retrotransposon promoters. These, the presence of predicted priming sites for reverse transcription, and the high conservation for all key functional domains of the coding region suggest that copies within the genome could be active retrotransposons. In view of this, we looked for transcription of BARE-1 within barley tissues and examined the promoter function of the BARE-1 LTR. We demonstrate here that BARE-1-like elements are transcribed in barley tissues, and that the transcripts begin within the BARE-1 LTR downstream of TATA boxes. The LTR can drive expression of reporter genes in transiently transformed barley protoplasts. This is dependent on the presence of a TATA box functional in planta as well. Furthermore, we identify regions within the LTR responsible for expression within protoplasts by deletion analyses of LTR-luc constructs. Similarities between promoter regulatory motifs and regions of the LTR were identified by comparisons to sequence libraries. The activity of the LTR as a promoter, combined with the abundance of BARE-1 in the genome, suggests that BARE-1 may retain the potential for propagation in the barley genome.

Retrotransposon BARE-1 is a major, dispersed component of the barley (Hordeum vulgare L.) genome.

The barley BARE-1 is a transcribed, copia-like retroelement with well-conserved functional domains, an active promoter, and a copy number of at least 3 x 10(4). We examined its chromosomal localization by in situ hybridization. The long terminal repeat (LTR) probe displayed a uniform hybridization pattern over the whole of all chromosomes, excepting paracentromeric regions, telomeres, and nucleolar organizer (NOR) regions. The integrase probe showed a similar pattern. The 5'-untranslated leader (UTL) probe, expected to be the most rapidly evolving component, labeled chromosomes in a dispersed and non-uniform manner, concentrated in the distal regions, possibly indicating a targe site preference.

High frequency of conjugation versus plasmid segregation of RP1 in epiphytic Pseudomonas syringae populations.

The maintenance and transfer of the broad host-range plasmid RP1 in epiphytically growing populations of Pseudomonas syringae was monitored in the phyllosphere of bush bean (Phaseolus vulgaris). When foliage was inoculated with plasmid-containing bacteria, the plasmid was lost from the majority of the cells within 2 d but was stably maintained in 0.8% of the population. A high frequency of conjugation between added donors and recipients was observed under high humidity conditions. In 1 d, the number of transconjugants rose to 10(-1) of the donors and the proportional level of transconjugants continued to increase until 3 d after inoculation. Under these conditions the proportion of plasmid-containing bacteria stabilized at about 0.8% of the total population. The conjugation rate appeared to be in equilibrium with plasmid loss and the slower growth of the plasmid-carrying cells. A factor that influenced the high conjugation frequency observed was the available nutrients provided by the leaf and also, to a lesser extent, the leaf surface itself. Transfer of the plasmid from added donors to indigenous bacteria was also studied, using a donor-specific bacteriophage for counterselection of the donor. Transfer was observed to 10 different species of Gram-negative epiphytically growing bacteria. The bean leaf surface appears to function as a hotspot at least for intraspecific transfer of plasmids in high humidity. The frequency of transfer was higher than in soil or in rhizosphere habitats. This is likely to be the result of an environment that is nutritionally rich in combination with a limited colonizable surface area which permits close contact between the bacterial cells.