The identification and characterization of four laccases from the plant pathogenic fungus Rhizoctonia solani

Abstract

Four distinct laccase genes, lcc1, lcc2, lcc3 and lcc4, have been identified in the fungus Rhizoctonia solani. Both cDNA and genomic copies of these genes were isolated and characterized. Hybridization analyses indicate that each of the four laccase genes is present in a single copy in the genome. The R. solani laccases can be divided into two groups based on their protein size, intron/exon organization, and transcriptional regulation. Three of these enzymes have been expressed in the fungus Aspergillus oryzae. Two of the recombinant laccases, r-lcc1 and r-lcc4, as well as the native lcc4 enzyme were purified and characterized. The purified proteins are homodimeric, comprised of two subunits of approximately 66kDa for lcc4 and 50-100kDa for the recombinant lcc1 protein. These laccases have spectral properties that are consistent with other blue copper proteins. With syringaldazine as a substrate, lcc4 has optimal activity at pH7, whereas lcc1 has optimal activity at pH6.

Heterologous expression of laccase cDNA from Ceriporiopsis subvermispora yields copper-activated apoprotein and complex isoform patterns

“…To our knowledge, this is the first report of expression of a white-rot fungus laccase in A. nidulans and the second instance of using A. niger (Record et al, 2002) as a host. Enzyme titres were substantially lower than those reported for other laccases in Aspergillus oryzae systems (Wahleithmer et al, 1995;Yaver et al, , 1999Record et al, 2002;Berka et al, 1997).…”

“…Laccase isoenzyme multiplicity is a phenomenon observed in several fungi, and in the case of Pleurotus sajor-caju (Soden & Dobson, 2001), the basidiomycete CECT 20197 (Mansur et al, 1997), Pleurotus ostreatus (Palmieri et al, 2000), Trametes villosa , Agaricus bisporus (Perry et al, 1993) and Rhizoctonia solani (Wahleithmer et al, 1995), families of structurally related genes have been described. In the case of C. subvermispora, over four isoforms are observed, with different substrate specificity, but only one gene, lcs-1, has been identified Salas et al, 1995).…”

Tandem organization and highly disparate expression of the two laccase genes lcc1 and lcc2 in the cultivated mushroom Agaricus bisporus

“…These are present at low constitutive levels and are further suppressed by addition of p-anisidine to the cultures. The mRNA for the fourth laccase is present at higher basal levels and its transcription is increased in response to p-anisidine (Wahleithner et al, 1996).…”

“…This is sometimes simply due to allelic differences as found in Neurospora crassa (Germann et al, 1988) and the laccase genes (PO1 and P 0 2 ) of Coriolus hirsutus (Kojima et al, 1990). In contrast, of the four laccase genes isolated from Rhizoctonia solani, three are closely linked in tandem (and the fourth is an allele of one of the others ; Wahleithner et al, 1996). Five non-allelic laccase genes have been characterized from the white-rot basidiomycete Trarnetes uillosa (Yaver et al, 1996 ;Yaver & Golightly, 1996).…”

“…More recently, four distinct laccase genes have been characterized from the plant-pathogenic fungus Rhizoctonia solani (Wahleithner et al, 1996) and five from the white-rot fungus Trametes villosa (Yaver et al, 1996;Yaver & Golightly, 1996), and although only a single laccase gene has been isolated from the filamentous ascomycete Podospora anserina, Southern analysis and restriction mapping suggest the presence of at least three others (Fernandez-Larrea & Stahl, 1996).…”