Molecular characterization of a fusarium graminearum lipase gene and its promoter
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A triglyceride lipase gene FgLip1 was identified in the genome of Fusarium graminearum strain PH-1. Yeast cells overexpressing FgLip1 showed lipolytic activity against a broad range of triglyceride substrates. Northern blot analyses revealed that expression of FgLip1 was activated in planta during the fungal infection process and under starvation conditions in vitro. FgLip1 expression was strongly induced in minimal medium supplemented with wheat germ oil, but only weakly induced by olive oil and triolein. Saturated fatty acids were the strongest inducers for FgLip1 expression and this induction was proportionally suppressed by the presence of unsaturated fatty acids. To determine the potential function of FgLip1, gene replacement was conducted on strain PH-1. When compared to wild-type PH-1, ∆FgLip1 mutants showed greatly reduced lipolytic activities at the early stage of incubation on minimal medium supplemented with either saturated or unsaturated lipid as the substrate, indicating that FgLip1 encodes a secreted lipase for exogenous lipid hydrolysis. The ∆FgLip1 mutants exhibited growth deficiency on both liquid and solid minimal media supplemented with the saturated triglyceride tristearin as the sole carbon source, suggesting that FgLip1 is required for utilization of this substance. No variation in disease symptoms between the ∆FgLip1 mutants and the wild-type strain was observed on susceptible cereal hosts including wheat, barley and corn. To delineate the promoter region responsible for the specific regulation of FgLip1 expression, a series of deletions of FgLip1 5’ upstream region were fused with the open reading frame of a green florescent protein (GFP) gene and the constructs were introduced into F. graminearum. GFP expression in the resulting transformants indicated that a 563-bp FgLip1 promoter sequence was sufficient to regulate expression of the FgLip1 gene and regulatory elements responsible for gene induction were located within the 563-372 bp region. To further investigate the regulatory elements, putative cis-acting elements within the 563-372 bp region were mutated using a linker-scanning mutagenesis approach. A CCAAT box, a CreA binding site, and a fatty acid responsive element (FARE) were identified and confirmed to be responsible for FgLip1 basal expression, glucose suppression and fatty acid induction, respectively.
DegreeDoctor of Philosophy (Ph.D.)
SupervisorHughes, Geoffrey R.; Hucl, Pierre J.; Wei, Yangdou
CommitteePozniak, Curtis J.; Fobert, Pierre R.; Coulman, Bruce E.
Copyright DateFebruary 2007