Immunocytochemical localization of laccase L1 in wood decayed by Rigidoporus lignosus

Abstract

The cellular distribution of laccase Li during degradation of wood chips by Rigidoporus lignosus, a tropical white rot fungus, was investigated by using anti-laccase Li polyclonal antisera in conjunction with immunolabeling techniques. The enzyme was localized in the fungal cytoplasm and was associated with the plasmalemma and the fungal cell wall. An extracellular sheath, often observed around fungal cells, often contained laccase molecules. Diffusion of laccase within apparently unaltered wood was seldom observed. The enzyme penetrated all degraded cell walls, from the secondary wall toward the primary wall, including the middle lamella. Xylem cells showing advanced stages of decay were sometimes devoid of significant labeling. These data suggest that the initial attack on wood was not performed by laccase Li of R. lignosus. Previous alteration of the lignocellulose complex may facilitate the movement of laccase within the wood cell walls. This immunogold study revealed that laccase localization during wood degradation seems limited not in space but in time.

A Cytochemical Study of Extracellular Sheaths Associated with Rigidoporus lignosus during Wood Decay

“…It has been suggested that early formation of slime layers might facilitate subsequent hyphal penetration of the cell wall (37,50). EFS of R. lignosus were seldom described in vivo during the interaction with rubber tree roots (42, 44) but were commonly observed on wood fragments inoculated in vitro (43). There * Corresponding author.…”

Enzymatic interactions between Gremmeniella abietina var. abietina, European race, and two resistant hosts, Pinus banksiana and P. contorta

“…Sections were blocked with 0.01 M PBS-1% BSA-0.05% T20 for 10 min, floated on an anti-laccase polyclonal antibody raised against the laccase L1 obtained from Rigidoporus lignosus (Klotsch) Imazeki (Nicole et al 1992) using dilutions between 1 : 800 and 1 : 1200, rinsed in the same buffer and incubated on 0.02 M Tris-HCl-1% BSA-0.05% T20 (pH 8.2) for 10 min before placing the sections on a drop of goat-anti-rabbit-gold (15 nm) for 1 h. Laccases are often reported as oxidative enzymes involved in lignin degradation (Nicole et al 1992). Specificity of the labelling was tested by omitting the primary antibody, by incubating with pre-immune serum or by adsorbing the primary antibody with a commercial laccase (Sigma-Aldrich, Oakville, ON, Canada).…”

“…We suggest that peroxidases and glucanases first alter the ligno-cellulose complex to facilitate laccase activities. Nicole et al (1992) used the same antibody to localize laccase in wood degraded by the white rot fungus Rigidoporus lignosus (Klotsch) Imazeki. As indicated in their paper, laccase was produced in quantity during wood degradation but, apparently, it was not directly involved in the first steps leading to wood alterations.…”

“…It was suggested that the initial diffusion of this enzyme was facilitated by previous enzymatic alterations. As the anti-laccase used in the present study was raised against the laccase L1 of the basidiomycete R. lignosus (Nicole et al 1992), it is possible that the binding affinity of this antibody was not as effective with G. abietina as in the system studied by Nicole et al Thus, we might have underestimated the amount of laccase secreted by GAA, but as its spatial distribution was positively followed in our sections, we can still suggest that the initial attack was not mainly performed by this laccase.…”

Extracellular laccase activity and transcript levels of putative laccase genes during removal of  the xenoestrogen technical nonylphenol by the aquatic hyphomyceteClavariopsis aquatica

“…Intracellular and also extracellular laccase activities remaining associated with cell surfaces have been detected in numerous fungi (Linden et al, 1991;Schlosser et al, 1997;Klonowska et al, 2001and Tetsch et al, 2005, 2006Valaskova & Baldrian, 2006). Extracellular laccase located within extracellular glucan sheaths has been detected in the basiomycete Rigidoporus lignosus (Nicole et al, 1992(Nicole et al, , 1993 and in the ascomycete Botrytis cinerea (Gil-ad et al, 2001). In aquatic fungi, cell-associated laccase activities have not been investigated up to now.…”