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Cloning of insertion site flanking sequence and construction of transfer DNA insert mutant library in Stylosanthes colletotrichum.

Chen H, Hu C, Yi K, Huang G, Gao J, Zhang S, Zheng J, Liu Q, Xi J - PLoS ONE (2014)

Bottom Line: We selected 1230 transformants, and obtained 23 strains with pathogenic defects, namely, 18 strains with reduced pathogenicity and five strains with lost pathogenicity.The flanking sequence of T-DNA was compared with the Colletotrichum genome by BLAST, and a sequence of 401 bp was found in Contig464 of the Colletotrichum genome.By predicting the function of the flanking sequence, we discovered that T-DNA insertion in the promoter region of the putative gene had 79% homology with the aspartate aminotransferase gene in Magnaporthe oryzae (XP_003719674.1).

View Article: PubMed Central - PubMed

Affiliation: Institute of Tropical Bioscience and Biotechnology, Key Laboratory of Tropical Crop Biotechnology, Ministry of Agriculture, Chinese Academy of Tropical Agricultural Sciences, Haikou, China.

ABSTRACT
Stylosanthes sp. is the most important forage legume in tropical areas worldwide. Stylosanthes anthracnose, which is mainly caused by Colletotrichum gloeosporioides, is a globally severe disease in stylo production. Little progress has been made in anthracnose molecular pathogenesis research. In this study, Agrobacterium tumefaciens-mediated transformation was used to transform Stylosanthes colletotrichum strain CH008. The major factors of the genetic transformation system of S. colletotrichum were optimized as follows: A. tumefaciens' AGL-1 concentration (OD(600)), 0.8; concentration of Colletotrichum conidium, 1 × 10(6) conidia/mL; acetosyringone concentration, 100 mmol/L; induction time, 6 h; co-culture temperature, 25 °C; and co-culture time, 3 d. Thus, the transformation efficiency was increased to 300-400 transformants per 106 conidia. Based on the optimized system, a mutant library containing 4616 mutants was constructed, from which some mutants were randomly selected for analysis. Results show that the mutants were single copies that could be stably inherited. The growth rate, spore amount, spore germination rate, and appressorium formation rate in some mutants were significantly different from those in the wild-type strain. We then selected the most appropriate method for the preliminary screening and re-screening of each mutant's pathogenic defects. We selected 1230 transformants, and obtained 23 strains with pathogenic defects, namely, 18 strains with reduced pathogenicity and five strains with lost pathogenicity. Thermal asymmetric interlaced PCR was used to identify the transfer DNA (T-DNA) integration site in the mutant that was coded 2430, and a sequence of 476 bp was obtained. The flanking sequence of T-DNA was compared with the Colletotrichum genome by BLAST, and a sequence of 401 bp was found in Contig464 of the Colletotrichum genome. By predicting the function of the flanking sequence, we discovered that T-DNA insertion in the promoter region of the putative gene had 79% homology with the aspartate aminotransferase gene in Magnaporthe oryzae (XP_003719674.1).

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Effect of Agrobacterium concentration on transformation efficiency.Note: In the same series (four series), the same capital letters indicate no significant difference (p>0.01), whereas different capital letters indicate significant differences (p<0.01).
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pone-0111172-g002: Effect of Agrobacterium concentration on transformation efficiency.Note: In the same series (four series), the same capital letters indicate no significant difference (p>0.01), whereas different capital letters indicate significant differences (p<0.01).

Mentions: Results of the differences in concentrations of A. tumefaciens and Colletotrichum spore liquid were analyzed by ANOVA (similar to Duncan’s new multiple range method below). According to the related results, the difference in experimental results was significant at p<0.01. Based on Figure 2, each OD value had a corresponding and appropriate spore liquid concentration within an appropriate range of OD600 (0.4–0.8). A high concentration of spore liquid resulted in a high transformation efficiency. In particular, when Agrobacterium OD600 was equal to 0.8 and the number of Colletotrichum CH008 spores was 106 spores/mL, the results were significantly higher than the other combinations, and the transformation efficiency peaked. This result may be related to the growth cycle of Agrobacterium and features of Colletotrichum CH008 spores. When the OD600 values of Agrobacterium were 1.0 and 1.2, the transformation efficiency showed a reducing trend with false-positive results.


Cloning of insertion site flanking sequence and construction of transfer DNA insert mutant library in Stylosanthes colletotrichum.

Chen H, Hu C, Yi K, Huang G, Gao J, Zhang S, Zheng J, Liu Q, Xi J - PLoS ONE (2014)

Effect of Agrobacterium concentration on transformation efficiency.Note: In the same series (four series), the same capital letters indicate no significant difference (p>0.01), whereas different capital letters indicate significant differences (p<0.01).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215998&req=5

pone-0111172-g002: Effect of Agrobacterium concentration on transformation efficiency.Note: In the same series (four series), the same capital letters indicate no significant difference (p>0.01), whereas different capital letters indicate significant differences (p<0.01).
Mentions: Results of the differences in concentrations of A. tumefaciens and Colletotrichum spore liquid were analyzed by ANOVA (similar to Duncan’s new multiple range method below). According to the related results, the difference in experimental results was significant at p<0.01. Based on Figure 2, each OD value had a corresponding and appropriate spore liquid concentration within an appropriate range of OD600 (0.4–0.8). A high concentration of spore liquid resulted in a high transformation efficiency. In particular, when Agrobacterium OD600 was equal to 0.8 and the number of Colletotrichum CH008 spores was 106 spores/mL, the results were significantly higher than the other combinations, and the transformation efficiency peaked. This result may be related to the growth cycle of Agrobacterium and features of Colletotrichum CH008 spores. When the OD600 values of Agrobacterium were 1.0 and 1.2, the transformation efficiency showed a reducing trend with false-positive results.

Bottom Line: We selected 1230 transformants, and obtained 23 strains with pathogenic defects, namely, 18 strains with reduced pathogenicity and five strains with lost pathogenicity.The flanking sequence of T-DNA was compared with the Colletotrichum genome by BLAST, and a sequence of 401 bp was found in Contig464 of the Colletotrichum genome.By predicting the function of the flanking sequence, we discovered that T-DNA insertion in the promoter region of the putative gene had 79% homology with the aspartate aminotransferase gene in Magnaporthe oryzae (XP_003719674.1).

View Article: PubMed Central - PubMed

Affiliation: Institute of Tropical Bioscience and Biotechnology, Key Laboratory of Tropical Crop Biotechnology, Ministry of Agriculture, Chinese Academy of Tropical Agricultural Sciences, Haikou, China.

ABSTRACT
Stylosanthes sp. is the most important forage legume in tropical areas worldwide. Stylosanthes anthracnose, which is mainly caused by Colletotrichum gloeosporioides, is a globally severe disease in stylo production. Little progress has been made in anthracnose molecular pathogenesis research. In this study, Agrobacterium tumefaciens-mediated transformation was used to transform Stylosanthes colletotrichum strain CH008. The major factors of the genetic transformation system of S. colletotrichum were optimized as follows: A. tumefaciens' AGL-1 concentration (OD(600)), 0.8; concentration of Colletotrichum conidium, 1 × 10(6) conidia/mL; acetosyringone concentration, 100 mmol/L; induction time, 6 h; co-culture temperature, 25 °C; and co-culture time, 3 d. Thus, the transformation efficiency was increased to 300-400 transformants per 106 conidia. Based on the optimized system, a mutant library containing 4616 mutants was constructed, from which some mutants were randomly selected for analysis. Results show that the mutants were single copies that could be stably inherited. The growth rate, spore amount, spore germination rate, and appressorium formation rate in some mutants were significantly different from those in the wild-type strain. We then selected the most appropriate method for the preliminary screening and re-screening of each mutant's pathogenic defects. We selected 1230 transformants, and obtained 23 strains with pathogenic defects, namely, 18 strains with reduced pathogenicity and five strains with lost pathogenicity. Thermal asymmetric interlaced PCR was used to identify the transfer DNA (T-DNA) integration site in the mutant that was coded 2430, and a sequence of 476 bp was obtained. The flanking sequence of T-DNA was compared with the Colletotrichum genome by BLAST, and a sequence of 401 bp was found in Contig464 of the Colletotrichum genome. By predicting the function of the flanking sequence, we discovered that T-DNA insertion in the promoter region of the putative gene had 79% homology with the aspartate aminotransferase gene in Magnaporthe oryzae (XP_003719674.1).

Show MeSH
Related in: MedlinePlus