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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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Conidial germination and appressorium formation after 8 h.
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pone-0111172-g011: Conidial germination and appressorium formation after 8 h.

Mentions: The results show that wild-type conidia and 10 randomly selected transformant spores could germinate after some time. The time that most of the transformants took to germinate was shorter than that of the wild-type strains, and the average length of germ tube growth in some transformants was longer than that of the wild-type strains. Three mutant strains demonstrated both low germination and appressorium formation rates, namely, t-960, t-604, and t-2327. By contrast, seven mutant spores (t-2393, t-2515, t-906, t-888, t-1130, t-2416, and t-3616) showed no significant difference from wild-type strains. In addition, wild-type strains and most transformants grew one to two germinal tubes from both ends of spores, and 80% of them demonstrated appressorium formation. Spore germination of mutant t-906 was abnormal; this mutant had three to four germinal tubes and its germination rate was high, but it germinated few appressoria (Table 3 and Figure 11).


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)

Conidial germination and appressorium formation after 8 h.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111172-g011: Conidial germination and appressorium formation after 8 h.
Mentions: The results show that wild-type conidia and 10 randomly selected transformant spores could germinate after some time. The time that most of the transformants took to germinate was shorter than that of the wild-type strains, and the average length of germ tube growth in some transformants was longer than that of the wild-type strains. Three mutant strains demonstrated both low germination and appressorium formation rates, namely, t-960, t-604, and t-2327. By contrast, seven mutant spores (t-2393, t-2515, t-906, t-888, t-1130, t-2416, and t-3616) showed no significant difference from wild-type strains. In addition, wild-type strains and most transformants grew one to two germinal tubes from both ends of spores, and 80% of them demonstrated appressorium formation. Spore germination of mutant t-906 was abnormal; this mutant had three to four germinal tubes and its germination rate was high, but it germinated few appressoria (Table 3 and Figure 11).

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