Transformations of grapevine pathogens Eutypa lata and Phaeomoniella chlamydospora : a one year project thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University, Palmerston North, New Zealand
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Date
2005
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Massey University
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Abstract
A transformation system has been developed for the grapevine pathogenic fungi Eutypa lata and Phaeomoniella chlamydospora using a positive selection system based on the Escherichia coli hygromycin B phosphotransferase gene (hph). The system developed could give large, stable transformants at frequencies between 0.7 and 6.5 transformants per µg of DNA. A second type of colony also grew on the selective media. These were believed to be abortive transformants. The first type of transformants were characterized using classical molecular biological technologies such as PCR and Southern hybridization, and the transformation was shown to be successful. Plasmids (pBCH-gfp and pCT74) containing a gfp reporter gene were also transformed into these two fungal species. Expression of the gfp gene was checked using a fluorescence microscope and gfp-expressing E. lata transformants were inoculated onto the host plants blackcurrant and grapevine. Confocal observation of the movement of fungal mycelia in wood tissues was performed but its interaction with host plant was not established in the time available. Purified gfp-expressing P. chlamydospora transformants were also obtained. A vector containing a fragment of the P. chlamydospora putative toxin gene moxY was constructed and transformed into P. chlamydospora. Putative moxY gene disruption transformants were screened with PCR followed by Southern hybridization. The putative moxY gene disruption transformants were spore purified and further confirmed with Southern hybridization. Whilst both PCR and Southern hybridization confirmed disruption of the moxY gene, clear evidence for the presence of an additional wild type moxY was also seen in the same transformants. This led to the suggestions that either P. chlamydospora is a natural diploid, or that moxY is essential for growth and that selective pressures led to the formation of a wild type: moxY-hph diploid.
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Eutypa lata, Molecular genetics, Phaeomoniella chlamydospora, Phytopathogenic fungi, Fungal diseases plants, Diseases and pests of New Zealand grapes