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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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Identification schemes for T-DNA junction types in M. oryzae. TAIL-PCR products from transformants with a single T-DNA insertion (A), multiple insertions at different locations (B), or multiple tandem or inverted insertions (C) were sequenced. Resulting sequences were subjected to blast searches (black vertical arrow) and analysed for junction type (see Experimental procedures). Those that produced a single fragment in TAIL-PCR and matched a T-DNA border (yellow) and a flanking genomic region (grey) without a gap were considered ‘precise junctions’ (type i) (51). In some cases, multiple fragments were amplified in TAIL-PCR at two different genomic regions (type iii; grey and dark grey). The transformant was regarded as having mixture of ‘precise’ and ‘imprecise’ junctions (types i and ii respectively), and treated as two TTLs. The imprecise junction was defined as the sequence matched to both a border and a flank with a gap (type ii) (51). When multiple T-DNA (blue bar with yellow borders) were integrated into the same location with tandem or inverted arrays, multiple flanking regions could appear (C). According to the concentration of co-amplified PCR products, sequencing results were classified as type ii (similar in both dark and light grey products), type iii (more dark grey product), type iv (similar in both grey and blue product), type v (more blue product) or type vi (more grey products). In the blast search, different locations or kinds of matching results were shown by the different line levels and colours.
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fig02: Identification schemes for T-DNA junction types in M. oryzae. TAIL-PCR products from transformants with a single T-DNA insertion (A), multiple insertions at different locations (B), or multiple tandem or inverted insertions (C) were sequenced. Resulting sequences were subjected to blast searches (black vertical arrow) and analysed for junction type (see Experimental procedures). Those that produced a single fragment in TAIL-PCR and matched a T-DNA border (yellow) and a flanking genomic region (grey) without a gap were considered ‘precise junctions’ (type i) (51). In some cases, multiple fragments were amplified in TAIL-PCR at two different genomic regions (type iii; grey and dark grey). The transformant was regarded as having mixture of ‘precise’ and ‘imprecise’ junctions (types i and ii respectively), and treated as two TTLs. The imprecise junction was defined as the sequence matched to both a border and a flank with a gap (type ii) (51). When multiple T-DNA (blue bar with yellow borders) were integrated into the same location with tandem or inverted arrays, multiple flanking regions could appear (C). According to the concentration of co-amplified PCR products, sequencing results were classified as type ii (similar in both dark and light grey products), type iii (more dark grey product), type iv (similar in both grey and blue product), type v (more blue product) or type vi (more grey products). In the blast search, different locations or kinds of matching results were shown by the different line levels and colours.

Mentions: Whereas 70 flanking sequences (3.5% of 2026) matched with repetitive sequences such as MsR02/RETRO7, MAGGY and MGLR3/MG-SINE transposons (see Experimental procedures), the majority (96.5%) of TTLs corresponded to single-copy regions of the genome. Although in most transformants, a single copy of T-DNA was inserted at one locus (82% in Table S2; Fig. 2A), integrations of T-DNA at multiple loci and an insertion of tandem-repeated T-DNAs in one locus were also observed (Fig. 2B and C respectively, and Table S2).


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)

Identification schemes for T-DNA junction types in M. oryzae. TAIL-PCR products from transformants with a single T-DNA insertion (A), multiple insertions at different locations (B), or multiple tandem or inverted insertions (C) were sequenced. Resulting sequences were subjected to blast searches (black vertical arrow) and analysed for junction type (see Experimental procedures). Those that produced a single fragment in TAIL-PCR and matched a T-DNA border (yellow) and a flanking genomic region (grey) without a gap were considered ‘precise junctions’ (type i) (51). In some cases, multiple fragments were amplified in TAIL-PCR at two different genomic regions (type iii; grey and dark grey). The transformant was regarded as having mixture of ‘precise’ and ‘imprecise’ junctions (types i and ii respectively), and treated as two TTLs. The imprecise junction was defined as the sequence matched to both a border and a flank with a gap (type ii) (51). When multiple T-DNA (blue bar with yellow borders) were integrated into the same location with tandem or inverted arrays, multiple flanking regions could appear (C). According to the concentration of co-amplified PCR products, sequencing results were classified as type ii (similar in both dark and light grey products), type iii (more dark grey product), type iv (similar in both grey and blue product), type v (more blue product) or type vi (more grey products). In the blast search, different locations or kinds of matching results were shown by the different line levels and colours.
© Copyright Policy
Related In: Results  -  Collection

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fig02: Identification schemes for T-DNA junction types in M. oryzae. TAIL-PCR products from transformants with a single T-DNA insertion (A), multiple insertions at different locations (B), or multiple tandem or inverted insertions (C) were sequenced. Resulting sequences were subjected to blast searches (black vertical arrow) and analysed for junction type (see Experimental procedures). Those that produced a single fragment in TAIL-PCR and matched a T-DNA border (yellow) and a flanking genomic region (grey) without a gap were considered ‘precise junctions’ (type i) (51). In some cases, multiple fragments were amplified in TAIL-PCR at two different genomic regions (type iii; grey and dark grey). The transformant was regarded as having mixture of ‘precise’ and ‘imprecise’ junctions (types i and ii respectively), and treated as two TTLs. The imprecise junction was defined as the sequence matched to both a border and a flank with a gap (type ii) (51). When multiple T-DNA (blue bar with yellow borders) were integrated into the same location with tandem or inverted arrays, multiple flanking regions could appear (C). According to the concentration of co-amplified PCR products, sequencing results were classified as type ii (similar in both dark and light grey products), type iii (more dark grey product), type iv (similar in both grey and blue product), type v (more blue product) or type vi (more grey products). In the blast search, different locations or kinds of matching results were shown by the different line levels and colours.
Mentions: Whereas 70 flanking sequences (3.5% of 2026) matched with repetitive sequences such as MsR02/RETRO7, MAGGY and MGLR3/MG-SINE transposons (see Experimental procedures), the majority (96.5%) of TTLs corresponded to single-copy regions of the genome. Although in most transformants, a single copy of T-DNA was inserted at one locus (82% in Table S2; Fig. 2A), integrations of T-DNA at multiple loci and an insertion of tandem-repeated T-DNAs in one locus were also observed (Fig. 2B and C respectively, and Table S2).

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