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Title: Linking genes and secondary metabolites in Mycosphaerella graminicola
Author: Khalid , Rozida Mohd
Awarding Body: University of Bristol
Current Institution: University of Bristol
Date of Award: 2011
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Mycosphaerella graminicola is an important pathogenic fungus of the wheat that causes Septoria tritici blotch. The pathogenesis of M graminicola differs from most plant pathogenic fungi because M graminicola mostly infects the host via stomata, not by formation of appresorium. The latent and biotrophic period can last up to three weeks until a rapid collapse of mesophylls and the nectrotropic stage begins, indicating that toxins are being released. Attempts to isolate the secondary metabolites from four strains of M graminicola: IP0323, STII, STl6 and ST93, that could be associated with the toxin production has led to the isolation of MG 1, MG2 and anthranillic acid. It was noted that the strains produced a low amount of yield and low amount of metabolites, indicating that the genes are tightly regulated. Epigenetic modifiers 5-azacytidine and suberohydroxamic acid were treated to IP0323, with no new gene expression. The genome of M graminicola has been sequenced, and indicated that M graminicola has 10 polyketide synthase and 1 polyketide-nonribosomal peptide synthetase hybrid, which could be involved in pathogenicity. Three PKS were selected: MgPKS2, MgPKS8 and MgPKS9 for heterologous expression in Aspergillus oryzae. The heterologous expression with eGFP fused to the respective genes indicated that: MgPKS2 was not expressed in A. oryzae, probably due to the inability of A. oryzae to splice the five introns of MgPKS2; MgPKS8 was expressed, due to the positive eGFP result, however there was no new metabolite detected in the LCMS; MgPKS9 was not expressed, probably the same reason as MgPKS2. MgPKS9 was used to check the intron splicing ability of A. oryzae because there are only two putative introns in MgPKS9. It was observed that removal of both introns gave positive eFGP results and that the presence of Intron 1 halted MgPKS9 expression in A. oryzae. LCMS analysis of positive transformants of MgPKS9 without both introns however showed no new metabolite production. This indicated heterologous expression of M graminicola in A. OIyzae is not favourable probably due to these reasons: problems in introns splicing, incorrect protein folding, protein being degraded, lack of special ACPS and lack of unique starter unit.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available