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Genome-wide analysis of T-DNA integration into the chromosomes of Magnaporthe oryzae.

Choi J, Park J, Jeon J, Chi MH, Goh J, Yoo SY, Park J, Jung K, Kim H, Park SY, Rho HS, Kim S, Kim BR, Han SS, Kang S, Lee YH - Mol. Microbiol. (2007)

Bottom Line: We identified a total of 1110 T-DNA-tagged locations (TTLs) and processed the resulting data via TAP.Analysis of the TTLs showed that T-DNA integration was biased among chromosomes and preferred the promoter region of genes.Our results support the potential of ATMT as a tool for functional genomics of fungi and show that the TAP is an effective informatics platform for handling data from large-scale insertional mutagenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Agricultural Biotechnology, Center for Fungal Genetic Resources, and Center for Agricultural Biomaterials, Seoul National University, Seoul 151-921, Korea.

ABSTRACT
Agrobacterium tumefaciens-mediated transformation (ATMT) has become a prevalent tool for functional genomics of fungi, but our understanding of T-DNA integration into the fungal genome remains limited relative to that in plants. Using a model plant-pathogenic fungus, Magnaporthe oryzae, here we report the most comprehensive analysis of T-DNA integration events in fungi and the development of an informatics infrastructure, termed a T-DNA analysis platform (TAP). We identified a total of 1110 T-DNA-tagged locations (TTLs) and processed the resulting data via TAP. Analysis of the TTLs showed that T-DNA integration was biased among chromosomes and preferred the promoter region of genes. In addition, irregular patterns of T-DNA integration, such as chromosomal rearrangement and readthrough of plasmid vectors, were also observed, showing that T-DNA integration patterns into the fungal genome are as diverse as those of their plant counterparts. However, overall the observed junction structures between T-DNA borders and flanking genomic DNA sequences revealed that T-DNA integration into the fungal genome was more canonical than those observed in plants. Our results support the potential of ATMT as a tool for functional genomics of fungi and show that the TAP is an effective informatics platform for handling data from large-scale insertional mutagenesis.

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Patterns of T-DNA integration. A. A single T-DNA integration. T-DNA is shown as a blue bar enclosed with yellow borders. The expected restriction enzyme sites are indicated by triangles. Southern blot analysis of transformant ATMT0879C5 (lane H) revealed a single T-DNA integration, resulting in a ∼2.3 kb hybridizing band. A pair of flanking primers (arrow; a and b) amplified a 2.5 kb band in the transformant (lane 2), compared with wild-type (lane 1). B. Multiple T-DNA integration at one site. Southern blot analysis confirmed the insertion of three T-DNAs in one location (ATMT0060C3), based on two restriction enzymes that cut inside (lane H) or outside (lane A) the T-DNA. A pair of flanking primers (c and d) amplified a 4.0 kb band in wild-type (lane 1) but no band in the transformant (lane 2). Additionally, the combination of border and flanking primers (c-rb and d-rb) proved that both sides of the repeated T-DNAs ended with RB (lanes 3 and 4). C. Chromosomal rearrangement. The translocation between chromosomes 1 and 3 occurred in transformant ATMT0879C6 as a result of T-DNA integration (red hexagon). This rearrangement was confirmed by PCR amplification using mixed combination of deduced primers specific for flanking areas (lanes 1 and 2, 1f/1r; lanes 3 and 4, 3f/3r; lanes 5 and 6, 1f/3r; lanes 7 and 8, 3f/1r). Wild-type DNA (lanes 1, 3, 5 and 7) was compare with the transformant (lanes 2, 4, 6 and 8). In all figures, H and A indicate HindIII and ApaI respectively. The unit of number of markers and length indicator is kilobase.
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fig06: Patterns of T-DNA integration. A. A single T-DNA integration. T-DNA is shown as a blue bar enclosed with yellow borders. The expected restriction enzyme sites are indicated by triangles. Southern blot analysis of transformant ATMT0879C5 (lane H) revealed a single T-DNA integration, resulting in a ∼2.3 kb hybridizing band. A pair of flanking primers (arrow; a and b) amplified a 2.5 kb band in the transformant (lane 2), compared with wild-type (lane 1). B. Multiple T-DNA integration at one site. Southern blot analysis confirmed the insertion of three T-DNAs in one location (ATMT0060C3), based on two restriction enzymes that cut inside (lane H) or outside (lane A) the T-DNA. A pair of flanking primers (c and d) amplified a 4.0 kb band in wild-type (lane 1) but no band in the transformant (lane 2). Additionally, the combination of border and flanking primers (c-rb and d-rb) proved that both sides of the repeated T-DNAs ended with RB (lanes 3 and 4). C. Chromosomal rearrangement. The translocation between chromosomes 1 and 3 occurred in transformant ATMT0879C6 as a result of T-DNA integration (red hexagon). This rearrangement was confirmed by PCR amplification using mixed combination of deduced primers specific for flanking areas (lanes 1 and 2, 1f/1r; lanes 3 and 4, 3f/3r; lanes 5 and 6, 1f/3r; lanes 7 and 8, 3f/1r). Wild-type DNA (lanes 1, 3, 5 and 7) was compare with the transformant (lanes 2, 4, 6 and 8). In all figures, H and A indicate HindIII and ApaI respectively. The unit of number of markers and length indicator is kilobase.

Mentions: Several abnormal patterns of T-DNA integration were observed. In 70 transformants with a known T-DNA copy number and sequences flanking both sides of the T-DNA, 57 (81%) had single T-DNA integration (Fig. 6A) and the remaining 13 harboured more than two copies of T-DNA. Abnormal patterns were found in 9 out of 70 (13%) transformants, where four were confirmed to have direct or inverted repeats of T-DNA, and five single-copy transformants exhibited translocation, fragment insertion or readthrough patterns.


Genome-wide analysis of T-DNA integration into the chromosomes of Magnaporthe oryzae.

Choi J, Park J, Jeon J, Chi MH, Goh J, Yoo SY, Park J, Jung K, Kim H, Park SY, Rho HS, Kim S, Kim BR, Han SS, Kang S, Lee YH - Mol. Microbiol. (2007)

Patterns of T-DNA integration. A. A single T-DNA integration. T-DNA is shown as a blue bar enclosed with yellow borders. The expected restriction enzyme sites are indicated by triangles. Southern blot analysis of transformant ATMT0879C5 (lane H) revealed a single T-DNA integration, resulting in a ∼2.3 kb hybridizing band. A pair of flanking primers (arrow; a and b) amplified a 2.5 kb band in the transformant (lane 2), compared with wild-type (lane 1). B. Multiple T-DNA integration at one site. Southern blot analysis confirmed the insertion of three T-DNAs in one location (ATMT0060C3), based on two restriction enzymes that cut inside (lane H) or outside (lane A) the T-DNA. A pair of flanking primers (c and d) amplified a 4.0 kb band in wild-type (lane 1) but no band in the transformant (lane 2). Additionally, the combination of border and flanking primers (c-rb and d-rb) proved that both sides of the repeated T-DNAs ended with RB (lanes 3 and 4). C. Chromosomal rearrangement. The translocation between chromosomes 1 and 3 occurred in transformant ATMT0879C6 as a result of T-DNA integration (red hexagon). This rearrangement was confirmed by PCR amplification using mixed combination of deduced primers specific for flanking areas (lanes 1 and 2, 1f/1r; lanes 3 and 4, 3f/3r; lanes 5 and 6, 1f/3r; lanes 7 and 8, 3f/1r). Wild-type DNA (lanes 1, 3, 5 and 7) was compare with the transformant (lanes 2, 4, 6 and 8). In all figures, H and A indicate HindIII and ApaI respectively. The unit of number of markers and length indicator is kilobase.
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Related In: Results  -  Collection

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fig06: Patterns of T-DNA integration. A. A single T-DNA integration. T-DNA is shown as a blue bar enclosed with yellow borders. The expected restriction enzyme sites are indicated by triangles. Southern blot analysis of transformant ATMT0879C5 (lane H) revealed a single T-DNA integration, resulting in a ∼2.3 kb hybridizing band. A pair of flanking primers (arrow; a and b) amplified a 2.5 kb band in the transformant (lane 2), compared with wild-type (lane 1). B. Multiple T-DNA integration at one site. Southern blot analysis confirmed the insertion of three T-DNAs in one location (ATMT0060C3), based on two restriction enzymes that cut inside (lane H) or outside (lane A) the T-DNA. A pair of flanking primers (c and d) amplified a 4.0 kb band in wild-type (lane 1) but no band in the transformant (lane 2). Additionally, the combination of border and flanking primers (c-rb and d-rb) proved that both sides of the repeated T-DNAs ended with RB (lanes 3 and 4). C. Chromosomal rearrangement. The translocation between chromosomes 1 and 3 occurred in transformant ATMT0879C6 as a result of T-DNA integration (red hexagon). This rearrangement was confirmed by PCR amplification using mixed combination of deduced primers specific for flanking areas (lanes 1 and 2, 1f/1r; lanes 3 and 4, 3f/3r; lanes 5 and 6, 1f/3r; lanes 7 and 8, 3f/1r). Wild-type DNA (lanes 1, 3, 5 and 7) was compare with the transformant (lanes 2, 4, 6 and 8). In all figures, H and A indicate HindIII and ApaI respectively. The unit of number of markers and length indicator is kilobase.
Mentions: Several abnormal patterns of T-DNA integration were observed. In 70 transformants with a known T-DNA copy number and sequences flanking both sides of the T-DNA, 57 (81%) had single T-DNA integration (Fig. 6A) and the remaining 13 harboured more than two copies of T-DNA. Abnormal patterns were found in 9 out of 70 (13%) transformants, where four were confirmed to have direct or inverted repeats of T-DNA, and five single-copy transformants exhibited translocation, fragment insertion or readthrough patterns.

Bottom Line: We identified a total of 1110 T-DNA-tagged locations (TTLs) and processed the resulting data via TAP.Analysis of the TTLs showed that T-DNA integration was biased among chromosomes and preferred the promoter region of genes.Our results support the potential of ATMT as a tool for functional genomics of fungi and show that the TAP is an effective informatics platform for handling data from large-scale insertional mutagenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Agricultural Biotechnology, Center for Fungal Genetic Resources, and Center for Agricultural Biomaterials, Seoul National University, Seoul 151-921, Korea.

ABSTRACT
Agrobacterium tumefaciens-mediated transformation (ATMT) has become a prevalent tool for functional genomics of fungi, but our understanding of T-DNA integration into the fungal genome remains limited relative to that in plants. Using a model plant-pathogenic fungus, Magnaporthe oryzae, here we report the most comprehensive analysis of T-DNA integration events in fungi and the development of an informatics infrastructure, termed a T-DNA analysis platform (TAP). We identified a total of 1110 T-DNA-tagged locations (TTLs) and processed the resulting data via TAP. Analysis of the TTLs showed that T-DNA integration was biased among chromosomes and preferred the promoter region of genes. In addition, irregular patterns of T-DNA integration, such as chromosomal rearrangement and readthrough of plasmid vectors, were also observed, showing that T-DNA integration patterns into the fungal genome are as diverse as those of their plant counterparts. However, overall the observed junction structures between T-DNA borders and flanking genomic DNA sequences revealed that T-DNA integration into the fungal genome was more canonical than those observed in plants. Our results support the potential of ATMT as a tool for functional genomics of fungi and show that the TAP is an effective informatics platform for handling data from large-scale insertional mutagenesis.

Show MeSH
Related in: MedlinePlus