Agrobacterium tume faciens-mediated transformation of Colletotrichum lagenarium, the causal agent of cucumber anthracnose
To elucidate the molecular mechanism of pathogenicity in Colletotrichum lagenarium, the casual agent of cucumber anthracnose, conditions for T-DNA insertional mutagenesis using Agrobacterium-mediated transformation approach were established and optimized. Binary vector BIG2RHPH2-GUS-GFP was used, wh...
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| Language: | English |
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Zhejiang University Press
2006-07-01
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| Series: | 浙江大学学报. 农业与生命科学版 |
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| Online Access: | https://www.academax.com/doi/10.3785/1008-9209.2006.04.0360 |
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| author | FANG Li LIU Hai-qing SONG Feng-ming ZHENG Zhong |
| author_facet | FANG Li LIU Hai-qing SONG Feng-ming ZHENG Zhong |
| author_sort | FANG Li |
| collection | DOAJ |
| description | To elucidate the molecular mechanism of pathogenicity in Colletotrichum lagenarium, the casual agent of cucumber anthracnose, conditions for T-DNA insertional mutagenesis using Agrobacterium-mediated transformation approach were established and optimized. Binary vector BIG2RHPH2-GUS-GFP was used, which carried the bacterial hygromycin B phosphotransferase gene cassette under the control of Aspergillus nidulans trpC promoter as a selection marker and a GFP-GUS fusion cassette driven by the A. nidulans trpC promoter enabling detection of GFP microscopically and GUS stain in the resultant transformants. In average, a total of 150-200 hygromycin-resistant transformants could be obtained for transforming 10<sup>6</sup> spores of C. lagenarium under the optimized conditions. All hygromycin-resistant transformants contained T-DNA insertions in their genomes which were detected by PCR amplification of the GUS gene in the T-DNA cassette with genomic DNA as templates. These hygromycin-resistant transformants also showed GFP fluorescence visualized under microscope and GUS activity detected by staining. The T-DNA inserted transformants appeared to be stable as they showed hygromycin resistance, GFP fluorescence and GUS activity after 4 generations. Preliminary results from pathogenicity test of randomly selecting 36 hygromycin-resistant transformants on leaves of two-week-old cucumber seedlings identified two transformants with reduced pathogenicity or loss-of-pathogenicity, respectively, except that the majority of the transformants showed wild-type pathogenicity. The establishment and optimization of the conditions for Agrobacterium-mediated transformation system facilitate the construction of the T-DNA inserted mutant population, screening of mutants with altered pathogenicity and identification of pathogenicity-related genes in C. lagenarium. |
| format | Article |
| id | doaj-art-e3700a59f18846039da018bb5a550c28 |
| institution | Kabale University |
| issn | 1008-9209 2097-5155 |
| language | English |
| publishDate | 2006-07-01 |
| publisher | Zhejiang University Press |
| record_format | Article |
| series | 浙江大学学报. 农业与生命科学版 |
| spelling | doaj-art-e3700a59f18846039da018bb5a550c282025-08-20T03:58:19ZengZhejiang University Press浙江大学学报. 农业与生命科学版1008-92092097-51552006-07-013236036610.3785/1008-9209.2006.04.036010089209Agrobacterium tume faciens-mediated transformation of Colletotrichum lagenarium, the causal agent of cucumber anthracnoseFANG LiLIU Hai-qingSONG Feng-mingZHENG ZhongTo elucidate the molecular mechanism of pathogenicity in Colletotrichum lagenarium, the casual agent of cucumber anthracnose, conditions for T-DNA insertional mutagenesis using Agrobacterium-mediated transformation approach were established and optimized. Binary vector BIG2RHPH2-GUS-GFP was used, which carried the bacterial hygromycin B phosphotransferase gene cassette under the control of Aspergillus nidulans trpC promoter as a selection marker and a GFP-GUS fusion cassette driven by the A. nidulans trpC promoter enabling detection of GFP microscopically and GUS stain in the resultant transformants. In average, a total of 150-200 hygromycin-resistant transformants could be obtained for transforming 10<sup>6</sup> spores of C. lagenarium under the optimized conditions. All hygromycin-resistant transformants contained T-DNA insertions in their genomes which were detected by PCR amplification of the GUS gene in the T-DNA cassette with genomic DNA as templates. These hygromycin-resistant transformants also showed GFP fluorescence visualized under microscope and GUS activity detected by staining. The T-DNA inserted transformants appeared to be stable as they showed hygromycin resistance, GFP fluorescence and GUS activity after 4 generations. Preliminary results from pathogenicity test of randomly selecting 36 hygromycin-resistant transformants on leaves of two-week-old cucumber seedlings identified two transformants with reduced pathogenicity or loss-of-pathogenicity, respectively, except that the majority of the transformants showed wild-type pathogenicity. The establishment and optimization of the conditions for Agrobacterium-mediated transformation system facilitate the construction of the T-DNA inserted mutant population, screening of mutants with altered pathogenicity and identification of pathogenicity-related genes in C. lagenarium.https://www.academax.com/doi/10.3785/1008-9209.2006.04.0360<italic>Colletotrichum lagenarium</italic><italic>Agrobacterium</italic>-mediated transformationT-DNA insertional mutagenesis |
| spellingShingle | FANG Li LIU Hai-qing SONG Feng-ming ZHENG Zhong Agrobacterium tume faciens-mediated transformation of Colletotrichum lagenarium, the causal agent of cucumber anthracnose 浙江大学学报. 农业与生命科学版 <italic>Colletotrichum lagenarium</italic> <italic>Agrobacterium</italic>-mediated transformation T-DNA insertional mutagenesis |
| title | Agrobacterium tume faciens-mediated transformation of Colletotrichum lagenarium, the causal agent of cucumber anthracnose |
| title_full | Agrobacterium tume faciens-mediated transformation of Colletotrichum lagenarium, the causal agent of cucumber anthracnose |
| title_fullStr | Agrobacterium tume faciens-mediated transformation of Colletotrichum lagenarium, the causal agent of cucumber anthracnose |
| title_full_unstemmed | Agrobacterium tume faciens-mediated transformation of Colletotrichum lagenarium, the causal agent of cucumber anthracnose |
| title_short | Agrobacterium tume faciens-mediated transformation of Colletotrichum lagenarium, the causal agent of cucumber anthracnose |
| title_sort | agrobacterium tume faciens mediated transformation of colletotrichum lagenarium the causal agent of cucumber anthracnose |
| topic | <italic>Colletotrichum lagenarium</italic> <italic>Agrobacterium</italic>-mediated transformation T-DNA insertional mutagenesis |
| url | https://www.academax.com/doi/10.3785/1008-9209.2006.04.0360 |
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