APPLIED AND ENVIRONMENTAL MICROBIOLOGY. June 1989, p. 1457-1465 Vol. 55, No. 6 0099-2240/89/061457-09$02.00/0 Copyright C) 1989, American Society for Microbiology Detection of Lignin Peroxidase and Xylanase by Immunocytochemical Labeling in Wood Decayed by Basidiomycetest R. A. BLANCHETTE,'* A. R. ABAD,1 R. L. FARRELL,2 AND T. D. LEATHERS3 Department of Plant Pathology, University of Mintnesota, St. Paiul, Minnesota 551081; Repligen-Sandoz Research Corp., Lexington, Massachusetts 021732; and Northernl Regional Research Ceniter, Agricultutiral Research Service, U.S. Department of Agriculture, Peoria, Illiniois 616043 Received 6 February 1989/Accepted 19 March 1989 The white rot fungi used in this study caused two different forms of degradation. Phanerochaete chrysosporium, strain BKM-F-1767, and Phellinus pini caused a preferential removal of lignin from birch wood, whereas Trametes (Coriolus) versicolor caused a nonselective attack of all cell wall components. Use of polyclonal antisera to H8 lignin peroxidase and monoclonal antisera to H2 lignin peroxidase followed by immunogold labeling with protein A-gold or protein G-gold, respectively, showed lignin peroxidase extra- and intracellularly to fungal hyphae and within the delignified cell walls after 12 weeks of laboratory decay. Lignin peroxidase was localized at sites within the cell wall where electron-dense areas of the lignified cell wall layers remained. In wood decayed by Trametes versicolor, lignin peroxidase was located primarily along the surface of eroded cell walls. No lignin peroxidase was evident in brown-rotted wood, but slight labeling occurred within hyphal cells. Use of polyclonal antisera to xylanase followed by immunogold labeling showed intense labeling on fungal hyphae and surrounding slime layers and within the woody cell wall, where evidence of degradation was apparent. Colloidal-gold-labeled xylanase was prevalent in wood decayed by all fungi used in this study. Areas of the wood with early stages of cell wall decay had the greatest concentration of gold particles, while little labeling occurred in cells in advanced stages of decay by brown or white rot fungi. White rot basidiomycetes produce extracellular enzymes exist. All are glycoproteins and appear to have similar that degrade all cell wall components of wood. The sequence catalytic activity and molecular masses (29, 31). Two iso- and rate in which lignin, cellulose, and hemicellulose are zymes, H8 and H2, dominate in the extracellular culture removed from wood varies among different species of fungi. fluid of P. chrvsosporiumn. Lignin peroxidase is also pro- Selective delignification is characteristic of decay by certain duced by other white rot fungi, and current investigation strains of Ganoderuna applanatlum, Phanerochaete chr so- suggests that lignin peroxidase is a key enzyme of white rot sporiuim, Phellinuts pini, Phlebia tremellosa, and many oth- fungi produced during the ligninolytic growth phase (32). ers (2, 10, 11, 14, 17, 41). These fungi preferentially degrade Other enzymes are also undoubtedly involved in the process large amounts of lignin from wood, with only slight or no loss of lignin degradation (22, 32, 45). Mn-dependent peroxidase of cellulose and moderate losses of hemicellulose (8. 42). is a heme protein that acts as a classical peroxidase (45). Other white rot fungi, such as Trametes versicolor, some Multiple molecular forms of this enzyme also have been strains of P. ch,ysosporiuim, Phlebia slubseriialis, and others, found that oxidize Mn(II) to Mn(III), resulting in oxidation are not selective and remove all cell wall components of various substrates (37). The role of Mn(II)-dependent simultaneously (8, 10, 40, 42). Lignin may be removed peroxidases in lignin degradation, however, is not com- slightly in advance of cellulose degradation, but extensive pletely understood. loss of cellulose and hemicellulose usually accompanies or The use of polyclonal and monoclonal antibodies and immediately follows lignin removal (13). There are also other immunocytochemistry has allowed the detection and local- forms of white rot degradation that result in different pat- ization of fungal metabolites within substrates (43). Fluores- terns of cell wall attack depending on the type of substrate, cence immunohistochemical detection in wood of two white chemical composition of the wood, and species of white rot rot that cause severe root Armillaria mellea and fungus involved (11, 30, 41). Brown rot fungi, in contrast, fungi rot, depolymerize cellulose rapidly, and extensive degradation of Heterobasidioni annosuim, has been successful with poly- cellulose and hemicellulose occurs (9, 39). Lignin loss is clonal antisera to homogenized mycelia (52). Detection of a slight, but a considerable amount of lignin modification takes brown rot fungus, Postia placenta, with polyclonal antibod- place (25, 33). ies to extracellular fractions and cell wall material of the Enzymes involved in degrading lignin have been discov- fungus has also been shown (23). Polyclonal antisera of this ered (35, 50). Two major groups of lignin-degrading en- type, however, were not species specific, and cross-reac- zymes, lignin peroxidases (also referred to as ligninases) and tions to other fungi occurred. manganese-dependent peroxidases, have been characterized The first investigations with immunogold cytochemical (22, 29, 35, 45). Multiple isozyme forms of lignin peroxidase investigations by transmission electron microscopy and polyclonal lignin peroxidase antisera have shown that the enzyme is not released extracellularly by P. chrvsosporium * Corresponding author. (21, 38). Lignin peroxidase was only found intracellularly in t Published as contribution no. 16,913 of the series of the Minne- the cytoplasmic area close to and bound with the fungal cell sota Agricultural Experiment Station. plasmalemma. These results were difficult to interpret since 1457 1458 BLANCHETTE ET AL. Appt- ENVIRON. MICROBIOL. ligninase could be obtained from extracellular culture fluids The sound and decayed wood blocks were cut into small of white rot fungi (33), and therefore ligninase also should be segments (approximately 1 by 1 by 0.4 mm) and fixed in 1%1 found outside of the hyphal cell wall. In addition, if this glutaraldehyde in 0.1 M sodium cacodylate buffer at pH 7.4 enzyme were important in lignin degradation, it should be for 2 h. Samples were rinsed three times for 20 min each and found within cell walls of wood attacked by white rot fungi. dehydrated through a graded acetone series. Embedding Many factors may affect the cytochemical localization of procedures with Quetol 651 resin (34) were as described specific enzymes resulting in negative labeling with gold previously (1). Samples in resin were cured at 74°C for 8 h complexes (7, 48). One important aspect is the effect of and cut with a diamond knife. Sections (100 to 120 nm) were tissue processing (i.e., washing, fixation, type of resin, and collected on nickel grids and used for immunocytochemical conditions of embedding), which can introduce changes in studies. Additional samples were cut from the wood blocks tissue components (7). Culture conditions for the fungus and fixed with 2%/ KMnO4 in distilled water, followed by within the substrate must be conducive to lignin peroxidase dehydration in Quetol 651 and embedment (1). production. In addition, antisera have to be specific for the Polyclonal antiserum against a highly purified xylanase particular antigen of interest. Recently, Daniel et al. (16) from Alur-eobasidiia,n pl/lliilaitns Y-2311-1 (T. D. Leathers, J. showed extracellular localization of lignin peroxidase in Indus. Microbiol., in press) was obtained from New Zealand slime layers around fungal hyphae and along areas of cell White rabbits. Xylanase has shown amino acid sequence wall erosion in decayed wood by using polyclonal antisera homology to xylanases from taxonomically distant sources and immunogold labeling techniques. The strain of P. c/hvy- (36). Polyclonal anti-lignin peroxidase H8 was also raised in sosporiutm used in the study caused a nonselective attack on White rabbits and partially purified by ammonium sulfate cell wall components. This type of white rot resulted in an precipitation (31). Monoclonal antibodies to lignin peroxi- erosion of the cell wall from the lumen toward the middle dase H2 from three distinct cell lines were also used. lamella. Lignin peroxidase was found localized along the For the indirect gold labeling used in this study, a colloidal edge of the eroded cell wall but not within the wall. To gold solution, with a gold particle size of 13 to 15 nm, was determine how far enzymes of P. c/-yVsosporiu,n would prepared by the methods of Frens (20). The collodial gold penetrate the cell wall, Strebotnik et al. (48) infiltrated was coupled to protein A (5, 19) for use with sections treated decayed pine wood with a concentrated culture filtrate with polyclonal antisera or to protein G (6) for use with the followed by immunogold labeling for lignin peroxidase. monoclonal antisera. Lignin peroxidase was found on the surface of the cell wall Samples fixed in glutaraldehyde were used for the immu- or within areas that were degraded extensively. Enzymes nolabeling studies. Sections on nickel grids were washed in from the culture fluid did not diffuse into nondecayed areas 0.01 M phosphate-buffered saline (PBS) for 10 min at room of the cell wall. temperature, followed by incubation in one of the primary Another important aspect of lignocellulose degradation is antibodies for 1.5 h. Sections were washed three times in the role of hemicellulases during lignin degradation. Lignin is 0.01 M PBS, incubated for 45 min in the protein A-gold or intimately associated with hemicellulose in the woody cell protein G-gold solution, and rinsed in distilled water. Anti- wall (28, 44), and hemicellulose loss usually accompanies sera were diluted in various concentrations to determine extensive lignin removal (8, 14, 42). Improved techniques for optimum labeling with 100 mM PBS containing 1.4% NaCl, labeling hemicellulose in wood have been developed (9, 27, 0.1% Tween 80, and 1% fish gelatin.