Differential expression of laccases in Botrytis cinerea in the presence of inducers and during the infection of grapes

Buddhika UVA, Savocchia S, Schmidtke LM and Steel CC

National Wine and Grape Industry Centre, Charles Sturt University, School of Agriculture and Wine Sciences, Wagga Wagga, NSW, 2678.

Botrytis cinerea (grey mould) produces oxidative enzymes, such as laccases, during the infection of plant tissues. Laccases are induced by gallic acid and copper, however, the responsive genes are not known. Although three laccase encoding genes (LAC1, LAC2, LAC3) have been reported, no virulence determinants have been identified in B. cinerea to date. This study characterised the expression patterns of laccases from B. cinerea infected grapes with respect to different inducers. Laccase activity in the culture filtrate of B. cinerea was measured and laccase transcripts were quantified in a laccase-inducing medium supplemented with inducers (gallic acid, CuSO4). Increased laccase activity occurred 2-days post-inoculation along with increased expression of LAC2 mRNA in response to copper. The purified enzyme was confirmed as LAC2, 63kDa in size by ESI/MS/MS analysis. The expression of laccase mRNA transcripts was analysed from B. cinerea infected Vitis vinifera berries (cvs Thompson Seedless and Chardonnay) during disease development and resulted in consistent expression of LAC3 and inconsistent expression of LAC1 and LAC2 suggesting involvement of LAC3 in initiation and the progression of disease in grapes. To confirm the expression of LAC3 during disease development, ESI/MS/MS analysis of the purified protein from infected grapes was performed, following bioinformatics analysis. The resulting protein was 26kDa in size and harbored the functional group ’Glyco-hydro 12 superfamily’, responsible for the hydrolysis of polysaccharides. The presence of this domain indicates the protein function in plant cell wall and membrane degradation during pathogen penetration and in the disease progression, although ESI/MS/MS analysis did not confirm the protein as LAC3. In conclusion, LAC3 is potentially a determinant of virulence while LAC2 is copper inducible. LAC3 expression is presumably induced by gallic acid. Further experiments are warranted to confirm LAC3 as the determinant of virulence and its induction by gallic acid.