Cladosporium fulvum is the causative agent of tomato leaf mould and has at least 6 hydrophobins named HCf-I to -6. To investigate the function of the six hydrophobins, three approaches were carried out: 1) Analysis of the expression of the six hydrophobins using quantitative PCR performed in vitro; 2) The immunolocalisation of the six hydrophobins in vitro and in plan/a; 3) RNAi silencing to silence up to six hydrophobin genes. The main results generated from the expression and localisation studies of the hydrophobins showed that not only are the hydrophobins produced at different times, but most unexpectedly, that they were located in different portions of the mycelium. Thus HCf-I, HCf-2 and HCf-3 were found in patches on the conidia, and on the aerial hyphae that produce the conidia. HCf-4 appeared in small areas both in the mycelium that is submerged in the agar and in the aerial hyphae. HCf-5 was visible only on the aerial hyphae at the time of sporulation, but was not found on the conidia. HCf-6 was secreted by the young hyphae 24 hours after germination and was evident as a layer on the surface of the growth medium, not associated with fungal structures. As HCf-6 accumulates at the periphery of the hyphae during tissue invasion, it was hypothesised that it might serve a role in adhesion. Assays were carried out to measure the adhesion of hyphae of the wild type and four ~HCf-6 strains to glass slides. The result show that hyphae of mCf-6 adhere significantly better than those of the wild type strain. Thus, HCf-6 does not act as an adhesion factor but more as a surfactant. Hydrophobin-less mutants by targeted single gene deletion were created but did not have evident phenotypes (Whiteford and Spanu, 2001). In order to investigate the possibility that several hydrophobin genes together contribute to fulfil a function or complement each other in knock-out strains, RNAi-based multigene silencing constructs were prepared and transformed into C. fulvum. Results show that the gene silencing constructs cause a reduction (up to 98%) of mRNA levels for the targeted hydrophobin genes. The silencing seems to be happening throughout the development of the fungus for single silenced strains, but not for multiple silenced strains. Complementation was observed between hydrophobins as the silencing of one hydrophobin affected the expression of the non-targeted hydrophobins. The silencing of all hydrophobins simulteanously in one strain showed to be less vigorous than the wild type, indicating that although the six hydrophobins ate not individually necessary for the fungus and can complement each other for certain functions, if all the six hydrophobins are disminished then the fungus is affected.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:486347 |
| Date | January 2007 |
| Creators | Lacroix, Helene Anne |
| Publisher | Imperial College London |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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