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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| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
| Published: |
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 |
| Subjects: | |
| Online Access: | https://www.academax.com/doi/10.3785/1008-9209.2006.04.0360 |
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| Summary: | 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. |
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| ISSN: | 1008-9209 2097-5155 |