Yuh-Chyang Charng

An inducible transposon system to terminate the function of a selectable marker in transgenic plants

Since the maize transposon Ac can move to a new location within the genome, it has been used in removing selectable markers in transgenic plants. In this paper, we developed an inducible transposon system to truncate a selectable marker in transgenic plants. In this system, the marker gene was accompanied by the inducible transposon, but one end of the transposon was located in the intron of the marker gene. As an example of a marker gene, we isolated the rice 5-enolpyruvylshikimate-3-phosphate synthase (epsps) and modified it for glyphosate tolerance. The transposon contained Ac transposase, which fused with the promoter of the inducible gene for pathogenesis-related protein 1a (PR-1a). This construct was engineered into an expression vector pCAMBIA1300, harboring a hygromycin-resistant gene. The construct was first transformed into rice calli, and transformed plants were selected on hygromycin-containing medium. The stably transformed calli underwent determination for normal transcripts and tolerance to glyphosate. The results were applied to a rice transformation with the same construct, but using glyphosate as the selective agent. By determining the transformation efficiency, T-DNA copy patterns, we demonstrate that the modified epsps could be a suitable selectable marker to create transgenic rice. Furthermore, after obtaining stable transgenic plants and inducing transposition by salicylic acid, the transposon was excised, the marker gene became truncated, and its expression was terminated. This strategy could be applicable to yield self-stabilizing transposon by locating the transposon’s end in the transposase gene’s intron.

Transposable elements ofXanthomonas campestris pv.citri originating from indigenous plasmids

SummaryAfter introduction of the broad host range plasmid RP4 inXanthomonas campestris pv.citri strain XAS4501 twoXanthomonas transposable elements, ISXC4 and ISXC5, were isolated. These elements were found to be capable of transposition inEscherichia coli. Restriction analysis, DNA hybridization and heteroduplex experiments revealed that ISXC4 and ISXC5 are about 5.55 and 6.95 kb in size, respectively, possess inverted repeats about 50±18 bp in length and share DNA homology in their left (5.0 kb) and right (0.6 kb) ends. ISXC4 and ISXC5 were found to originate from plasmids pXW45N and pXW45J, which are indigenous replicons inX. campestris pv.citri strain XW45.

The firefly luciferase gene as a reporter for in vivo detection of Ac transposition in tomato plants

Abstract The maize transposable element Ac has been introduced into many plant species by Agrobacterium -mediated gene transfer, and evidence of transposition has been obtained by applying various reporter gene systems which are mostly based on antibiotic resistance or destructive enzyme assays. Therefore, it has not been possible so far to monitor transposon activity during the development of living plants. We used the firefly luciferase gene to develop a reporter gene system amenable for in vivo detection of Ac transposition events. The construct pBinLucA was introduced into tomato, and luciferase activity was analysed during the regeneration and development of transgenic tomato plants. By Southern blot analyses and in vitro luciferase enzyme assays, the highest frequencies of Ac transposition (90%) were found at the callus stage of plant regeneration and in the cotyledons of the progeny from selfed primary transformants. In addition, we were able to follow excision of the transposon Ac in vivo at different developmental stages of individual plants. Therefore, the luciferase reporter gene system seems to be particularly suited to detect somatic excision of transposable elements in transgenic plants allowing the rescue of independent transposition events from a single plant.

Identification and nucleotide sequence analysis of an open reading frame involved in high-frequency conversion of turbid to clear plaque mutants of filamentous phage Cf1t

Clear plaque mutants (Cf1c) isolated from the temperate filamentous phage Cf1t occurred at a frequency of approximately 10(-3). The pahge yield from Cf1c-infected cells was higher than that from Cf1t-infected cells. Results of spot complementation tests implied that the turbid plaque phenotype is dominant. DNA fragment substitution studies indicated that a NcoI/KpnI fragment of 591 bp was responsible for the determination of plaque turbidity. Sequence data from four Cf1c isolates revealed base pair alterations and a deletion located in the upstream region of an open reading frame (ORFII) which might encode a 18.2-kDa protein. When the ORFII in Cf1t was disrupted by a frameshift mutation, this recombinant phage formed clear plaques. These observations suggest that ORFII may participate in the formation of turbid plaques. ORFII does not show significant homology with the sequence of f1 gpII, gpV, or other known phage proteins.

Ds transposon is biased towards providing splice donor sites for exonization in transgenic tobacco

Insertion of transposed elements into introns can lead to their activation as alternatively spliced cassette exons, an event called exonization, which can enrich the complexity of transcriptomes and proteomes. In this study, the first exonization event was detected when the modified rice EPSPS marker gene was inserted with the Ac transposon 5′ end, which provided a splice donor site to yield abundant novel transcripts. To assess the contribution of splice donor and acceptor sites of transposon sequences, we inserted a Ds element into each intron of the EPSPS marker gene. This process yielded 14 constructs, with the Ds transposon inserted in the forward and reverse direction in each of the 7 introns of the EPSPS marker gene. The constructs were transformed into tobacco plants, and novel transcripts were identified by RT-PCR with specific primers. Exonization of Ds in EPSPS was biased towards providing splice donor sites of the inserted Ds sequence. Additionally, when the Ds inserted in reverse direction, a continuous splice donor consensus region was determined by offering 4 donor sites in the same intron. Information on these exonization events may help enhance gene divergence and functional genomic studies.

Fusion of the inducible promoter of the PR-1a gene to the Activator transposase gene can transactive excision of a non-autonomous transposable element by external and by internal stimuli

Abstract The open reading frame coding for the transposase gene of the maize transposon Activator ( Ac ) was expressed in transgenic tobacco plants under the control of the promoter of the inducible gene for pathogenesis-related protein 1a (PR-1a). Excision of a non-autonomous transposable element ( Ds ) from chimeric β-glucuronidase (GUS) and luciferase reporter gene constructs was employed to analyze the induction of the Ac transposase by external and by internal stimuli. Applying the GUS histochemical assay, Ds excision events were detected in leaves, stems, and roots after treatment of regenerating shoots with salicylic acid (SA). Varying the SA induction procedure led to different Ds excision patterns in leaves and in roots. Furthermore, Ds excision events were also observed in non-treated, older transgenic plants in the green leaves, but not in germinal cells. Thus, the PR-1a promoter/ Ac transposase gene fusion, together with the improved methods for induction of this chimeric gene, may provide a valuable tool for studying basic mechanisms of Ac transposition and for developing modified transposable element systems suitable for gene tagging in higher plants.

A 200-bp constructed inducible PR-1a promoter fusion to the Ac transposase gene drives higher transposition of a Ds element than the native PR-1a promoter fusion drives

Abstract An inducible transposon tagging technique in plants with a large genome was accessed. The open reading frame coding for the transposase gene of the maize transposon Activator ( Ac ) was expressed in transgenic tobacco plants under the control of the promoter of the inducible gene for pathogenesis-related protein 1a (PR-1a). Based on the native PR-1a promoter, several inducible promoters were constructed in order to induce variability of the Ac transposase expression level. Excision of a non-autonomous transposable element ( Ds ) from the chimeric β -glucuronidase gene construct was employed to analyze the induction of the Ac transposase by salicylic acid (SA). Applying the β -glucuronidase histochemical assay, Ds excision efficiency was determined in the regeneration calli after treatment of a tobacco leaf disc with SA. A 111-bp regulatory element, located between nucleotides −699 to −588 of the PR-1a promoter, was fused to the 89-bp CaMV35S core promoter to serve as the inducible promoter, PRΔD. When the PRΔD promoter was fused with the Ac transposase gene and induced by SA, it triggered a 26-fold higher Ds excision efficiency than the native PR-1a promoter fusion. Furthermore, Ds excision events occurred mainly in the regeneration calli at day 8 after induction with SA. Because Ds excision events could be induced in such a short period, an alternative gene tagging strategy is suggested.

Genome-Wide Survey of Ds Exonization to Enrich Transcriptomes and Proteomes in Plants

Insertion of transposable elements (TEs) into introns can lead to their activation as alternatively spliced cassette exons, an event called exonization which can enrich the complexity of transcriptomes and proteomes. Previously, we performed the first experimental assessment of TE exonization by inserting a Ds element into each intron of the rice epsps gene. Exonization of Ds in plants was biased toward providing splice donor sites from the beginning of the inserted Ds sequence. Additionally, Ds inserted in the reverse direction resulted in a continuous splice donor consensus region by offering 4 donor sites in the same intron. The current study involved genome-wide computational analysis of Ds exonization events in the dicot Arabidopsis thaliana and the monocot Oryza sativa (rice). Up to 71% of the exonized transcripts were putative targets for the nonsense-mediated decay (NMD) pathway. The insertion patterns of Ds and the polymorphic splice donor sites increased the transcripts and subsequent protein isoforms. Protein isoforms contain protein sequence due to unspliced intron-TE region and/or a shift of the reading frame. The number of interior protein isoforms would be twice that of C-terminal isoforms, on average. TE exonization provides a promising way for functional expansion of the plant proteome.

A one-time inducible transposon for creating knockout mutants

The maize transposon Ac can move to a new location within the genome to create knockout mutants in transgenic plants. In rice, Ac transposon is very active but sometimes undergoes further transposition and leaves an empty mutated gene. Therefore, we developed a one-time transposon system by locating one end of the transposon in the intron of the Ac transposase gene, which is under the control of the inducible promoter (PR-1a). Treatment with salicylic acid induced transposition of this transposon, COYA, leading to transposase gene breakage in exons. The progeny plants inheriting the transposition events become stable knockout mutants, because no functional transposase could be yielded. The behavior of COYA was analyzed in single-copy transgenic rice plants. We determined the expression of the modified transposase gene and its ability to trigger transposition events in transgenic rice plants. The COYA element thus exhibits potential for development of an inducible transposon system suitable for gene isolation in heterologous plant species.

Analysis of New Functional Profiles of Protein Isoforms Yielded by Ds Exonization in Rice

Insertion of transposable elements (TEs) into introns can lead to their activation as alternatively spliced cassette exons, an event called exonization. Exonization can enrich the complexity of transcriptomes and proteomes. Previously, we performed a genome-wide computational analysis of Ds exonization events in the monocot Oryza sativa (rice). The insertion patterns of Ds increased the number of transcripts and subsequent protein isoforms, which were determined as interior and C-terminal variants. In this study, these variants were scanned with the PROSITE database in order to identify new functional profiles (domains) that were referred to their reference proteins. The new profiles of the variants were expected to be beneficial for a selective advantage and more than 70% variants achieved this. The new functional profiles could be contributed by an exon–intron junction, an intron alone, an intron–TE junction, or a TE alone. A Ds-inserted intron may yield 167 new profiles on average, while some cases can yield thousands of new profiles, of which C-terminal variants were in major. Additionally, more than 90% of the TE-inserted genes were found to gain novel functional profiles in each intron via exonization. Therefore, new functional profiles yielded by the exonization may occur in many local regions of the reference protein.