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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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Characteristics of T-DNA insertion sites. A. Distribution of TTL frequency and GC ratio in the genic region (blue and red lines respectively). The profile of TTL frequency was summed for every 50 bp unit, and that of the GC ratio was calculated for 50 bp windows at 5 bp intervals. The position 0 on the x-axis indicates the CDS start and end points. Negative numbers indicate upstream regions of the CDS start or end, whereas positive numbers indicate areas downstream from those points. On the y-axis, the left side presents the GC ratio and the right side indicates the TTL frequency. B. GC ratio profile around T-DNA insertion sites. The GC ratio was analysed for the 800 bp region flanking T-DNA insertion sites. Centred on the T-DNA insertion site, the profile is plotted as a red line. It was compared with the control profile of GC ratio generated from randomly selected locations (green line). Each point shows an average GC ratio of the 50 bp window around the point. The position 0 indicates the T-DNA insertion site. Negative and positive numbers on the x-axis indicate upstream and downstream of T-DNA insertion sites respectively. C. The bendability profile around T-DNA insertion sites. Bendability of the 800 bp region flanking T-DNA insertion sites was analysed. The window moved at 5 bp intervals through this region. High bendability indicates a more flexible region for T-DNA integration. The scale of the x-axis is the same as that of the axis in B.
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fig04: Characteristics of T-DNA insertion sites. A. Distribution of TTL frequency and GC ratio in the genic region (blue and red lines respectively). The profile of TTL frequency was summed for every 50 bp unit, and that of the GC ratio was calculated for 50 bp windows at 5 bp intervals. The position 0 on the x-axis indicates the CDS start and end points. Negative numbers indicate upstream regions of the CDS start or end, whereas positive numbers indicate areas downstream from those points. On the y-axis, the left side presents the GC ratio and the right side indicates the TTL frequency. B. GC ratio profile around T-DNA insertion sites. The GC ratio was analysed for the 800 bp region flanking T-DNA insertion sites. Centred on the T-DNA insertion site, the profile is plotted as a red line. It was compared with the control profile of GC ratio generated from randomly selected locations (green line). Each point shows an average GC ratio of the 50 bp window around the point. The position 0 indicates the T-DNA insertion site. Negative and positive numbers on the x-axis indicate upstream and downstream of T-DNA insertion sites respectively. C. The bendability profile around T-DNA insertion sites. Bendability of the 800 bp region flanking T-DNA insertion sites was analysed. The window moved at 5 bp intervals through this region. High bendability indicates a more flexible region for T-DNA integration. The scale of the x-axis is the same as that of the axis in B.

Mentions: We analysed the distribution of TTLs in the genic and intergenic regions. More TTLs were observed in the genic region than in the intergenic region of the M. oryzae genome (799 and 311 respectively; Table 2). As supported by the chi-squared test (Table 2), the observed numbers represented 94% and 120% of the expected numbers, suggesting that T-DNA integration seemed to be slightly biased towards the intergenic region in M. oryzae. Within the genic region, a higher frequency of T-DNA insertions was observed in the promoter region (defined as 1 kb upstream of the transcriptional starting point; 415 of 799) than the coding or 3′ untranslated regions (UTRs, defined as 500 bp downstream of end codon; 256 and 128 respectively; Table 2). The observed number of TTLs in the promoter region was twofold higher than the expected number of TTLs, suggesting a strong bias towards T-DNA insertions in this region. Among the 256 TTLs in coding sequences (CDS), 196 (77%) and 60 (23%) were found in the exon and intron regions respectively; the observed frequencies were negatively correlated with the expected value (43% and 76% respectively). To further investigate the nature of bias in the genic region, TTL frequencies around the start and the end of CDS were examined (blue line in Fig. 4A). At the start of CDS, more TTLs were found in the promoter region (average 25.7) than in the CDS region (average 11.3). A similar tendency of TTL distribution was found around the end of CDS (12.7 in 3′ UTR and 10.4 in CDS on average of 500 bp region). To determine whether this TTL pattern exhibited any relationship with base composition, the GC ratio was analysed. The GC ratio (red line in Fig. 4A) was plotted over the TTL frequencies, which showed dramatic changes at the start and end of CDS. Near the CDS start, the GC ratio increased sharply from 47% to 56% within 100 bp, and the number of TTLs decreased from 23 to 15. Near the CDS end, the overall tendency was also negatively correlated (Fig. 4A). In summary, a high GC ratio appeared to be negatively correlated with T-DNA integration, suggesting that T-DNA preferred AT-rich regions.


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)

Characteristics of T-DNA insertion sites. A. Distribution of TTL frequency and GC ratio in the genic region (blue and red lines respectively). The profile of TTL frequency was summed for every 50 bp unit, and that of the GC ratio was calculated for 50 bp windows at 5 bp intervals. The position 0 on the x-axis indicates the CDS start and end points. Negative numbers indicate upstream regions of the CDS start or end, whereas positive numbers indicate areas downstream from those points. On the y-axis, the left side presents the GC ratio and the right side indicates the TTL frequency. B. GC ratio profile around T-DNA insertion sites. The GC ratio was analysed for the 800 bp region flanking T-DNA insertion sites. Centred on the T-DNA insertion site, the profile is plotted as a red line. It was compared with the control profile of GC ratio generated from randomly selected locations (green line). Each point shows an average GC ratio of the 50 bp window around the point. The position 0 indicates the T-DNA insertion site. Negative and positive numbers on the x-axis indicate upstream and downstream of T-DNA insertion sites respectively. C. The bendability profile around T-DNA insertion sites. Bendability of the 800 bp region flanking T-DNA insertion sites was analysed. The window moved at 5 bp intervals through this region. High bendability indicates a more flexible region for T-DNA integration. The scale of the x-axis is the same as that of the axis in B.
© Copyright Policy
Related In: Results  -  Collection

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

fig04: Characteristics of T-DNA insertion sites. A. Distribution of TTL frequency and GC ratio in the genic region (blue and red lines respectively). The profile of TTL frequency was summed for every 50 bp unit, and that of the GC ratio was calculated for 50 bp windows at 5 bp intervals. The position 0 on the x-axis indicates the CDS start and end points. Negative numbers indicate upstream regions of the CDS start or end, whereas positive numbers indicate areas downstream from those points. On the y-axis, the left side presents the GC ratio and the right side indicates the TTL frequency. B. GC ratio profile around T-DNA insertion sites. The GC ratio was analysed for the 800 bp region flanking T-DNA insertion sites. Centred on the T-DNA insertion site, the profile is plotted as a red line. It was compared with the control profile of GC ratio generated from randomly selected locations (green line). Each point shows an average GC ratio of the 50 bp window around the point. The position 0 indicates the T-DNA insertion site. Negative and positive numbers on the x-axis indicate upstream and downstream of T-DNA insertion sites respectively. C. The bendability profile around T-DNA insertion sites. Bendability of the 800 bp region flanking T-DNA insertion sites was analysed. The window moved at 5 bp intervals through this region. High bendability indicates a more flexible region for T-DNA integration. The scale of the x-axis is the same as that of the axis in B.
Mentions: We analysed the distribution of TTLs in the genic and intergenic regions. More TTLs were observed in the genic region than in the intergenic region of the M. oryzae genome (799 and 311 respectively; Table 2). As supported by the chi-squared test (Table 2), the observed numbers represented 94% and 120% of the expected numbers, suggesting that T-DNA integration seemed to be slightly biased towards the intergenic region in M. oryzae. Within the genic region, a higher frequency of T-DNA insertions was observed in the promoter region (defined as 1 kb upstream of the transcriptional starting point; 415 of 799) than the coding or 3′ untranslated regions (UTRs, defined as 500 bp downstream of end codon; 256 and 128 respectively; Table 2). The observed number of TTLs in the promoter region was twofold higher than the expected number of TTLs, suggesting a strong bias towards T-DNA insertions in this region. Among the 256 TTLs in coding sequences (CDS), 196 (77%) and 60 (23%) were found in the exon and intron regions respectively; the observed frequencies were negatively correlated with the expected value (43% and 76% respectively). To further investigate the nature of bias in the genic region, TTL frequencies around the start and the end of CDS were examined (blue line in Fig. 4A). At the start of CDS, more TTLs were found in the promoter region (average 25.7) than in the CDS region (average 11.3). A similar tendency of TTL distribution was found around the end of CDS (12.7 in 3′ UTR and 10.4 in CDS on average of 500 bp region). To determine whether this TTL pattern exhibited any relationship with base composition, the GC ratio was analysed. The GC ratio (red line in Fig. 4A) was plotted over the TTL frequencies, which showed dramatic changes at the start and end of CDS. Near the CDS start, the GC ratio increased sharply from 47% to 56% within 100 bp, and the number of TTLs decreased from 23 to 15. Near the CDS end, the overall tendency was also negatively correlated (Fig. 4A). In summary, a high GC ratio appeared to be negatively correlated with T-DNA integration, suggesting that T-DNA preferred AT-rich regions.

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