Somatic and germinal mobility of the RescueMu transposon in transgenic maize

Abstract

RescueMu, a Mu1 element containing a bacterial plasmid, is mobilized by MuDR in transgenic maize. Somatic excision from a cell-autonomous marker gene yields >90% single cell sectors; empty donor sites often have deletions and insertions, including up to 210 bp of RescueMu/Mu1 terminal DNA. Late somatic insertions are contemporaneous with excisions, suggesting that "cut-and-paste" transposition occurs in the soma. During reproduction, RescueMu transposes infrequently from the initial transgene array, but once transposed, RescueMu is suitable for high throughput gene mutation and cloning. As with MuDR/Mu elements, heritable RescueMu insertions are not associated with excisions. Both somatic and germinal RescueMu insertions occur preferentially into genes and gene-like sequences, but they exhibit weak target site preferences. New insights into Mu behaviors are discussed with reference to two models proposed to explain the alternative outcomes of somatic and germinal events: a switch from somatic cut-and-paste to germinal replicative transposition or to host-mediated gap repair from sister chromatids.

Figure 1.

Structure of the RescueMu Vector. The 4.7-kb mobile element, RescueMu, consists of a plasmid inserted into an intact Mu1 nonautonomous element. RescueMu is inserted downstream of a cauliflower mosaic virus (CaMV) 35S promoter in the 5′ untranslated leader of maize Lc, a transcription factor of the R family required for anthocyanin production. Excision of RescueMu can restore tissue pigmentation. The integrated transgene locus is defined as Lc::RescueMu, and five independent Lc::RescueMu loci are presented in this article: R3-4, R3-8, R3-13, R3-15, and R3-17. Two RescueMu elements differ by the presence of unique 400-bp heterologous tags of Rhizobium meliloti DNA. These permit easier mutant allele–transposon cosegregation analysis in a background with multiple mobile RescueMu elements. There is no KpnI restriction site inside the RescueMu element, which is flanked by a unique BglII site in maize Lc. These restriction sites are used during plasmid rescue of new RescueMu insertion alleles. The pea rbcS 3′ region contains the polyadenylation sites. ORI, origin of replication; Amp, ampicillin.

Figure 2.

Developmental Timing of RescueMu Excisions in the Aleurone at Four Independent Lc::RescueMu Loci. Lc::RescueMu transgenic plants were crossed with plants express- ing the MuDR transposase in an r C1 background to score the size of revertant sectors. The size of each Lc revertant sector is an accurate indicator of RescueMu excision timing because Lc is a cell-autonomous marker (Ludwig et al., 1990). (A) Lc::RescueMu revertant aleurone sectors. Two independent transformants are shown, R3-13 and R3-17. Most purple sectors consist of single cells. (B) Quantitative analysis of Lc::RescueMu aleurone excision timing. The number of cells in 200 revertant sectors from four kernels was measured randomly for four independent Lc::RescueMu loci, R3-13, R3-17, R3-15, and R3-4. Despite different chromosomal locations and possible transgene position effects, all four lines exhibit the same late excision timing.

Figure 3.

Molecular Confirmation of RescueMu SEs and Analysis of DNA Repair Products. PCR primers flanking RescueMu were used to amplify empty CaMV35S-Lc sites in mature leaves. The wild-type allele and original Lc::RescueMu sequences are both shown at the top. The original 9-bp host duplication (TTTTGGGGA) is shown in outlined letters. Flanking this duplication is an overlapping 10-bp direct repeat (AAGCTTGGAT, underlined). After RescueMu excision, broken DNA ends appear to be digested variably by exonuclease followed by either blunt ligation (simple deletion) or DNA synthesis, resulting in a fill-in of Mu1 or other ectopic sequence (lowercase letters). Vector arrows indicate Mu1 TIR sequences. End points of long filler sequences are shown in parentheses. Nucleotide locations are relative to the original RescueMu insertion site. Underlined sequences indicate direct or inverted repeats. Next to the allele name (SE1 to SE45), the number of identical clones recovered and the plant source (a, b, or c; see below) are indicated in square brackets. The primers used for PCR amplification are indicated at the top. Clone SE45 was recovered by plasmid rescue, not PCR. Plant sources: a, MrG157.2; b, pooled MrG157, MrG158.2, MrG158.6, MrGH110-70, MrGH110-71, MrGH110-94, MrGH147-2, and MrGH148-2; c, MrG158.2. L, left; R, right.

Figure 4.

Evidence That RescueMu Elements Routinely Transpose to New Loci during Somatic Development. (A) The strategy to selectively plasmid rescue new somatic insertions of RescueMu (recipient loci) while preventing recovery of the original integrated transgene at Lc (donor locus). RescueMu is shown as purple triangles. Black bars represent flanking maize chromosomal DNA. Red bars represent the Lc::RescueMu allele. The strategy relies on using a unique BglII site flanking Lc::RescueMu. After genomic DNA is digested outside of RescueMu at KpnI sites, it is self-ligated to form circles. Before bacterial transformation, circles containing a BglII site are linearized to prevent replication in bacteria. (B) Tissue sources of genomic DNA used for plasmid rescue. Plants MrG157.2 and MrG158.2 are the F1 progeny of a cross between an Lc::RescueMu (no MuDR) plant and a MuDR transposase-containing (nontransgenic) plant. Therefore, these plants did not inherit RescueMu insertions from either parent. Only a small portion of a seedling leaf was used to isolate genomic DNA. Open (MrG158.2) and closed (MrG157.2) boxes are used in (B) to (E) to indicate the plant source. (C) Ethidium bromide–stained agarose gel of maize chromosomal DNA recovered as plasmids in bacteria on ampicillin-containing medium. Genomic DNA was subjected to the protocol shown in (A). Plasmids were digested with KpnI and HindIII. Plasmids range in size from ∼10 to 27 kb. Duplicate restriction patterns were not observed, suggesting that new RescueMu insertions occurred as small leaf sectors. (D) Sequence analysis of rescued plasmids from maize chromosomal DNA. The clone names (I-1 to I-20) correspond to the lane names in (C). PCR primers were used to sequence from the left and right borders of the RescueMu element. Each sequence carries a new 9-bp host duplication (boldface underlined letters), the hallmark of a new transposition event. (E) DNA gel blot analysis of the progeny of plant MrG157.2, a source of several somatic insertions. Pollen from plant MrG157.2 was outcrossed to a nontransgenic tester. The probe used hybridized to both RescueMu2 and RescueMu3 elements. Lane T0 corresponds to the primary transformant plant and shows a complex transgene array. In the progeny, only bands of the original transgene locus are present; no new bands are observed, indicating that none of the RescueMu insertions recovered in leaf 2 occurred early enough to be contained in the tassel.

Figure 5.

Evidence for RescueMu Duplicate Germinal Insertions in the F2 Progeny of Lc::RescueMu × Active MuDR Parents. (A) DNA gel blot evidence for new RescueMu insertions in the outcross progeny of a transgenic plant (allele R3-13). Novel bands are indicated by arrows. Insertions are not correlated with excisions, indicating that RescueMu elements duplicate in germinal cells. The single band present in lane 8 indicates that RescueMu inserted into a locus not linked to the original donor site. A rare deletion in lane 6 is indicated by an asterisk. (B) Sequence of I-55, a putative germinal insertion. DNA gel blot analysis showed the presence of a new RescueMu band in plant SH4713, which is not related to the plants shown in (A). After plasmid rescue, multiple colonies were found to have the same restriction fragment pattern. Two were sequenced and found to be identical. Because somatic insertions have been recovered only as unique colonies, the rescued I-55 allele was assumed to be germinal. Both the left and right flanking sequences showed strong homology with a rice panicle cDNA. (C) Confirmation of the germinal inheritance of RescueMu insertion allele I-55 in the progeny of plant SH4713. After sequencing of the rescued plasmid, flanking PCR primers were designed and used to generate an ∼520-bp left probe and an ∼400-bp right probe to RescueMu. Each probe was hybridized to the SH4713 parent and its outcross progeny. Lane 1, sibling of RescueMu parent; lane 2, parent plant SH4713; lanes 3 to 8, progeny (cross A188 × SH4713). In the left panel, the arrow shows the appearance of a new ∼4.5-kb band in the parent segregating in the progeny. Concurrently, as indicated by the asterisk, an ∼10.5-kb band is diminished. Genomic DNA was digested with XbaI. The decrease in band size is likely the result of RescueMu contributing a more proximal XbaI site present inside the element. The faint ∼10.5-kb band (asterisk) in lanes 2, 3, 6, and 7 is likely the intact wild-type homolog copy in combination with variable late somatic reversions of the mutant copy. Because all plants represent hybrid lines, other bands are found to be segregating. In the right panel, in the same progeny shown in the left panel, the right probe of plasmid I-55 detects the appearance of a novel ∼10-kb band, indicated by the arrow. This band is present in the SH4713 parent (lane 2) but not in its nontransgenic sibling (lane 1). As in the left panel, the appearance of the novel band coincides with a diminished high molecular weight band (∼11 kb, asterisk), although only in the parent (lane 2). The size decrease likely reflects the introduction of a more proximal HindIII site present within RescueMu. The genomic DNA was digested with HindIII. Because all of the progeny appear to inherit the ∼11-kb band (asterisk), the nontransgenic parent appears to be homozygous for this allele.

Figure 6.

Plasmid Size Distribution of 58 Transposed RescueMu Alleles Recovered in E. coli.

Figure 7.

Models Proposed for RescueMu and MuDR/Mu Element Insertion Activities in Somatic and Germinal Lineages. (A) RescueMu elements excise just before or after the last cell division in the aleurone. Red circles indicate an excision event. The diagram shows the developmental lineage of the aleurone after fertilization (Levy and Walbot, 1990). Numbers to the right of each cell population are with respect to the zygote (cell 1). (B) A model of Mu somatic transposition. Because both RescueMu excisions and insertions occur developmentally late, we propose that Mu transposes by a cut-and-paste mechanism in terminally dividing somatic cells. When transposition reactions start, double-strand breaks are subject to exonuclease and/or blunt ligation. Before the last S-phase, the homologous Mu1 template on the sister chromatid (red triangles) may be used to fill in some or perhaps all of the missing Mu1 sequence. Hence, few revertants of two or more cell sectors are seen. After the last S-phase, because a sister chromatid is not present, single cell revertants are abundant. An alternative model to explain late excision timing is that cell cycle factors may bind to the TIRs to prevent transposition during cell proliferation (Raizada et al., 2001a). Some excised RescueMu elements may not reinsert because the TIRs are damaged. These could exist as extrachromosomal circles before degradation (Sundaresan and Freeling, 1987). Those Mu elements that do reinsert are associated with a 9-bp host duplication (yellow bars). (C) RescueMu and other Mu elements insert but rarely excise in premeiotic, meiotic, and postmeiotic germinal cells. Red circles indicate an insertion event. Shown are cell lineages from the zygote to sperm nuclei located within pollen. After meiosis, each haploid nucleus divides mitotically to produce a vegetative cell nucleus and a generative cell nucleus. The generative cell further divides to produce two sperm nuclei. The majority of Mu insertions occur late during development. Up to 20% of Mu insertions occur after the last postmeiotic mitosis (data summarized from Robertson, 1981, 1985; Robertson and Stinard, 1993). Insertions occur after the last gamete S-phase, but germinal revertants are rare. (D) A gap repair model to explain how Mu elements insert in germinal cells but generate no reversions at the donor allele. Mu continues to transpose by a cut-and-paste mechanism as in the soma. However, there is enhanced and more efficient sister chromatid–dependent DNA synthesis (gap) repair to completely replace the missing MuDR/Mu element at the empty donor site in germinal cells (summarized from Donlin et al., 1995; Hsia and Schnable, 1996). Transposition is inhibited in individual sperm, which lack a sister chromatid to use as the template for gap repair. (E) An alternative replicative transposition model to explain the lack of germinal revertants. Mu elements switch from a cut-and-paste transposition mechanism in somatic cells to a replicative mechanism in pregerminal and postmeiotic cells. Hence, no excisions occur, because only a single strand of the donor allele is transferred to the new insertion site. DNA synthesis at the donor and recipient sites generates the complementary strands, followed by ligation of the transposon to the host chromosome (reviewed in Craig, 1995).