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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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Distribution of 1110 TTLs across M. oryzae chromosomes. The frequency of TTLs (blue bar) and expected (based on the random insertion) T-DNA insertions (green dot) in every 200 kb are plotted along the length of each chromosome. Gene density on each chromosome is indicated as a red line. Frequencies of expected insertions were estimated by Monte Carlo simulations. The unassigned area includes the TTLs that matched the 2.01 Mb of unmapped genome sequences. The length of each chromosome is indicated on the x-axis, and frequencies of genes and TTLs are indicated on the left and right y-axes respectively.
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fig03: Distribution of 1110 TTLs across M. oryzae chromosomes. The frequency of TTLs (blue bar) and expected (based on the random insertion) T-DNA insertions (green dot) in every 200 kb are plotted along the length of each chromosome. Gene density on each chromosome is indicated as a red line. Frequencies of expected insertions were estimated by Monte Carlo simulations. The unassigned area includes the TTLs that matched the 2.01 Mb of unmapped genome sequences. The length of each chromosome is indicated on the x-axis, and frequencies of genes and TTLs are indicated on the left and right y-axes respectively.

Mentions: To confirm whether T-DNAs were evenly distributed, 10 000 simulations using Monte Carlo methods were performed based on a purely random model (Fig. 3). The distribution of simulated samples (green dots) showed no significant correlation to that of observed TTLs (blue bars), indicating that the TTL distribution in this organism did not follow the purely random model (r = 0.154, P < 0.05). When analysed at the chromosomal level, 44% of the total TTLs were observed on chromosomes 1 and 2 (one in every 30 kb), which was higher compared with other chromosomes, and exceeded the expected numbers by more than 20% (Table 1). In contrast, chromosomes 4, 5 and 7 contained less than 80% of the expected T-DNA insertions (one insertion in every 50 kb; Table 1). These tendencies were significant in chi-squared tests as indicated by the resulting P-values (Table 1). In addition, TTL frequency had no specific association with the following characters of the genome: gene density (red lines in Fig. 3), GC ratio, transposable elements and microsatellites (data not shown; see Experimental procedures).


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)

Distribution of 1110 TTLs across M. oryzae chromosomes. The frequency of TTLs (blue bar) and expected (based on the random insertion) T-DNA insertions (green dot) in every 200 kb are plotted along the length of each chromosome. Gene density on each chromosome is indicated as a red line. Frequencies of expected insertions were estimated by Monte Carlo simulations. The unassigned area includes the TTLs that matched the 2.01 Mb of unmapped genome sequences. The length of each chromosome is indicated on the x-axis, and frequencies of genes and TTLs are indicated on the left and right y-axes respectively.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169514&req=5

fig03: Distribution of 1110 TTLs across M. oryzae chromosomes. The frequency of TTLs (blue bar) and expected (based on the random insertion) T-DNA insertions (green dot) in every 200 kb are plotted along the length of each chromosome. Gene density on each chromosome is indicated as a red line. Frequencies of expected insertions were estimated by Monte Carlo simulations. The unassigned area includes the TTLs that matched the 2.01 Mb of unmapped genome sequences. The length of each chromosome is indicated on the x-axis, and frequencies of genes and TTLs are indicated on the left and right y-axes respectively.
Mentions: To confirm whether T-DNAs were evenly distributed, 10 000 simulations using Monte Carlo methods were performed based on a purely random model (Fig. 3). The distribution of simulated samples (green dots) showed no significant correlation to that of observed TTLs (blue bars), indicating that the TTL distribution in this organism did not follow the purely random model (r = 0.154, P < 0.05). When analysed at the chromosomal level, 44% of the total TTLs were observed on chromosomes 1 and 2 (one in every 30 kb), which was higher compared with other chromosomes, and exceeded the expected numbers by more than 20% (Table 1). In contrast, chromosomes 4, 5 and 7 contained less than 80% of the expected T-DNA insertions (one insertion in every 50 kb; Table 1). These tendencies were significant in chi-squared tests as indicated by the resulting P-values (Table 1). In addition, TTL frequency had no specific association with the following characters of the genome: gene density (red lines in Fig. 3), GC ratio, transposable elements and microsatellites (data not shown; see Experimental procedures).

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