Copper induction of laccases in the plant pathogenic fungus, Botrytis cinerea

UVA Buddhika1, S Savocchia1, P Strappe2, LM Schmidtke1 and CC Steel1

  1. National Wine and Grape Industry Centre, School of Agricultural and Wine Sciences
  2. School of Biomedical Sciences,Charles Sturt University, Wagga Wagga, NSW 2678

Botrytis cinerea, a fungal pathogen of grapevines, produces oxidative enzymes including laccases, during the infection of plant tissues. Laccases are reported to be induced by copper however it is not known if this is the case for B. cinerea. Three laccase genes are known (LAC1, LAC2 and LAC3) but gene specific inducers have not been fully investigated. This study investigates the expression of laccases in response to copper. Liquid cultures of B. cinerea were prepared in potato dextrose broth by inoculation of the media with 100 μL of spore suspension (105/ml). After 5 days mycelia were harvested and placed in a laccase inducing medium with a range of CuSO4 concentrations (0–0.8 mM). Laccase activities in culture filtrates were determined after 2 days. Laccase activity was dependent upon copper concentration up to 0.6 mM and decreased at 0.8 mM indicating 0.6 mM is the optimum concentration of maximum laccase production. Mycelia were harvested for mRNA transcript analysis. LAC2 gene expression was similarly correlated with the copper concentrations in the culture medium with a 5-fold increase at 0.6 mM CuSO4. There were no significant changes in LAC1 and LAC3 gene expression indicative that LAC2 is a copper-inducible laccase gene. Proteins from culture filtrates were concentrated using ultra centrifugal filters (30 KDa) and separated by SDS-PAGE. A single band corresponding to a 75 KDa protein was observed which may correspond to a copper inducible laccase. This is the first time laccase gene expression in response to copper has been investigated in B. cinerea.