Role of laccase as a virulence factor in the infection of grapes by Botrytis cinerea

Botrytis cinerea is a fungal pathogen that is responsible for grey mould in several horticultural crops, including grapevines. A range of enzymes are produced by B. cinerea as it infects plant tissues, and some, such as laccases, are inducible. Laccases can be induced by gallic acid and copper. The laccase induced by gallic acid is a 32 kDa protein while the laccase induced by copper is a 75 kDa protein. There are three laccase-coding genes (LAC1, LAC2, LAC3), but no gene-enzyme linkage has been established and no virulence determinants are known. This thesis focused on predicting the structural and functional properties of laccases, to investigate gene diversity and to characterise the expression of three laccase genes during the infection of B. cinerea in grapevines and with respect to different inducers.
B. cinerea was isolated from different wine growing regions. DNA was extracted and LAC1 and LAC2 genes were amplified and sequenced. Sequences were aligned and phylogenetic trees were constructed separately for LAC1 and LAC2. Aside from intraspecies variations, LAC1 and LAC2 were found to be highly conserved amongst the isolates. The functional domains of the genes were predicted to be the cupredoxin domains, the characteristic protein motif in multi-copper oxidases (MCOs) and EFG-like domain signature 2 with unequal distribution among isolate. Furthermore, they were predicted that they would be localised extracellularly associated with the cell wall, and also to contain signal peptides cleavage sites, which suggests they could readily access host tissues during plant attack and have a role in the development of grey mould disease. The presence of signal peptides and cupredoxin domains in LAC2 were predicted to be common among all of the isolates studied, but in LAC1, they were limited indicating the importance of LAC2 in the pathogen’s physiological functions, particularly virulence.
To elucidate the distinct role of laccases as a virulence factor/ during pathogenicity, the expressions of three genes (LAC1, LAC2 and LAC3) were characterised by transcript quantification during grey mould development in Chardonnay and Thompson Seedless grapes. Transcript quantification was performed by RT-qPCR, dependent on cDNA quality. Commercially available RNA extraction kits which do not provide more hazardous chemicals have often failed in plant systems, particularly in plant tissues such as grapes due to the high amount of polyphenolics present that interfere with RNA extraction. RNA extraction from Botrytis-infected grapes was thus optimised to overcome the problems associated with polyphenolics. The protocol of a commercial kit (Bioline) was modified for effective RNA recovery from infected grapes so that it was suitable for RT-qPCR analysis.
Transcript quantification of different laccases resulted in consistent expression of LAC3 and inconsistent expression of LAC1 and LAC2, indicating the involvement of LAC3 in the initiation and the progression of grey mould disease. To the best of the author’s knowledge, this is the first study demonstrating LAC3 as a potential virulence determinant in the development of grey mould by B. cinerea. The corresponding enzyme was purified from infected berries and identified by ESI/MS/MS analysis. This revealed a 26 kDa protein with the predicted domain ‘Glyco-hydro 12 superfamily’. Proteins from this family are involved in polysaccharide hydrolysis, which are important in the fungal degradation of plant cell walls during lesion expansion.
Laccase activity, total protein and pH were measured in infected berry extracts during grey mould development concurrently. Laccase activity was first detected three days post-inoculation. Total protein diminished gradually during infection, demonstrating the complex interaction that occurs among polyphenolics, laccases and proteins (fungal and plant origin) in the berry environment that affects protein solubility. Similarly, pH decreased with infection, indicating pathogen-mediated pH modulation, which may be correlated with the induction of laccase. Although pH modulation is a general mechanism of fungal pathogens that regulates the expression of virulence factors, its effect in this context is yet to be elucidated.
Laccase induction was investigated by measuring laccase activity in the culture filtrate of B. cinerea in a laccase-inducing medium supplemented with the inducers, gallic acid and copper sulphate for up to three days post-inoculation. Laccase activity increased at two days post-inoculation. When laccase activity was measured separately with gallic acid and copper after two days of growth in liquid culture, it was found that laccase was induced by copper but not gallic acid. Transcript quantification of different laccases (LAC1, LAC2 and LAC3) in the fungal mycelium concurrently, led to copper-induced LAC2 expression. The optimum copper concentration required for laccase induction was 0.6 mM. Copper at this concentration was not found to be inhibitory to fungal growth. Laccase was further purified by protein precipitation with ammonium acetate in cold methanol from the liquid filtrate of cultures induced with copper, and then subjected to ESI/MS/MS analysis. This resulted in a 63 kDa protein with greatest homology for LAC2, confirming that copper is able to induce the LAC2-encoding 63 kDa enzyme.
In conclusion, despite being conserved, there appears to be an intra-species range of laccases variants with differing functional and structural properties, and are potential virulence factors. Two enzymes are encoded by two genes, which are induced by different inducers. This is the first study to establish the copper-inducible laccase codes for the 63 kDa enzyme (LAC2) as the link between gene, enzyme and inducer for B. cinerea. The LAC2 gene does not seem to be involved in the disease development, and LAC1 is constitutively expressed with no evidence of an involvement in the infection of grapes during grey mould development. Although B. cinerea has different laccases, only LAC3 appears to determine the development of grey mould in grapes, and could be the gallic acid-inducible laccase. This is also the first study to report LAC3 to be a virulence determinant in B. cinerea. The underlying mechanism of LAC3 induction during infection and the biochemical characterisation of the LAC3 enzyme warrants further investigation, because this information may provide new insights into the development of different enzyme assays and control strategies for laccases in wineries.