Retrotransposon vectors for gene delivery in plants.

BACKGROUND: Retrotransposons are abundant components of plant genomes, and although some plant retrotransposons have been used as insertional mutagens, these mobile genetic elements have not been widely exploited for plant genome manipulation. In vertebrates and yeast, retrotransposons and retroviruses are routinely altered to carry additional genes that are copied into complementary (c)DNA through reverse transcription. Integration of cDNA results in gene delivery; recombination of cDNA with homologous chromosomal sequences can create targeted gene modifications. Plant retrotransposon-based vectors, therefore, may provide new opportunities for plant genome engineering. RESULTS: A retrotransposon vector system was developed for gene delivery in plants based on the Tnt1 element from Nicotiana tabacum. Mini-Tnt1 transfer vectors were constructed that lack coding sequences yet retain the 5' and 3' long terminal repeats (LTRs) and adjacent cis sequences required for reverse transcription. The internal coding region of Tnt1 was replaced with a neomycin phosphotransferase gene to monitor replication by reverse transcription. Two different mini-Tnt1 s were developed: one with the native 5' LTR and the other with a chimeric 5' LTR that had the first 233 bp replaced by the CaMV 35 S promoter. After transfer into tobacco protoplasts, both vectors undergo retrotransposition using GAG and POL proteins provided in trans by endogenous Tnt1 elements. The transposition frequencies of mini-Tnt1 vectors are comparable with native Tnt1 elements, and like the native elements, insertion sites are within or near coding sequences. In this paper, we provide evidence that template switching occurs during mini-Tnt1 reverse transcription, indicating that multiple copies of Tnt1 mRNA are packaged into virus-like particles. CONCLUSIONS: Our data demonstrate that mini-Tnt1 vectors can replicate efficiently in tobacco cells using GAG and POL proteins provided in trans by native Tnt1 elements. This suggests that helper Tnt1 constructs can be developed to enable a Tnt1-based two-component vector system that could be used in other plant species. Such a vector system may prove useful for gene delivery or the production of cDNA that can serve as a donor molecule for gene modification through homologous recombination.

High-frequency homologous recombination in plants mediated by zinc-finger nucleases.

Homologous recombination offers great promise for plant genome engineering. This promise has not been realized, however, because when DNA enters plant cells homologous recombination occurs infrequently and random integration predominates. Using a tobacco test system, we demonstrate that chromosome breaks created by zinc-finger nucleases greatly enhance the frequency of localized recombination. Homologous recombination was measured by restoring function to a defective GUS:NPTII reporter gene integrated at various chromosomal sites in 10 different transgenic tobacco lines. The reporter gene carried a recognition site for a zinc-finger nuclease, and protoplasts from each tobacco line were electroporated with both DNA encoding the nuclease and donor DNA to effect repair of the reporter. Homologous recombination occurred in more than 10% of the transformed protoplasts regardless of the reporter's chromosomal position. Approximately 20% of the GUS:NPTII reporter genes were repaired solely by homologous recombination, whereas the remainder had associated DNA insertions or deletions consistent with repair by both homologous recombination and non-homologous end joining. The DNA-binding domain encoded by zinc-finger nucleases can be engineered to recognize a variety of chromosomal target sequences. This flexibility, coupled with the enhancement in homologous recombination conferred by double-strand breaks, suggests that plant genome engineering through homologous recombination can now be reliably accomplished using zinc-finger nucleases.

Identification of Arabidopsis rat mutants.

Limited knowledge currently exists regarding the roles of plant genes and proteins in the Agrobacterium tumefaciens-mediated transformation process. To understand the host contribution to transformation, we carried out root-based transformation assays to identify Arabidopsis mutants that are resistant to Agrobacterium transformation (rat mutants). To date, we have identified 126 rat mutants by screening libraries of T-DNA insertion mutants and by using various "reverse genetic" approaches. These mutants disrupt expression of genes of numerous categories, including chromatin structural and remodeling genes, and genes encoding proteins implicated in nuclear targeting, cell wall structure and metabolism, cytoskeleton structure and function, and signal transduction. Here, we present an update on the identification and characterization of these rat mutants.

Length and sequence heterogeneity in 5S rDNA of Populus deltoides.

The 5S rRNA genes and their associated non-transcribed spacer (NTS) regions are present as repeat units arranged in tandem arrays in plant genomes. Length heterogeneity in 5S rDNA repeats was previously identified in Populus deltoides and was also observed in the present study. Primers were designed to amplify the 5S rDNA NTS variants from the P. deltoides genome. The PCR-amplified products from the two accessions of P. deltoides (G3 and G48) suggested the presence of length heterogeneity of 5S rDNA units within and among accessions, and the size of the spacers ranged from 385 to 434 bp. Sequence analysis of the non-transcribed spacer (NTS) revealed two distinct classes of 5S rDNA within both accessions: class 1, which contained GAA trinucleotide microsatellite repeats, and class 2, which lacked the repeats. The class 1 spacer shows length variation owing to the microsatellite, with two clones exhibiting 10 GAA repeat units and one clone exhibiting 16 such repeat units. However, distance analysis shows that class 1 spacer sequences are highly similar inter se, yielding nucleotide diversity (pi) estimates that are less than 0.15% of those obtained for class 2 spacers (pi = 0.0183 vs. 0.1433, respectively). The presence of microsatellite in the NTS region leading to variation in spacer length is reported and discussed for the first time in P. deltoides.