En funksjonell studie av det essensielle amidotransferasekomplekset MurT/CobQ i Streptococcus pneumoniae

The bacterial cell wall is responsible for maintaining cell shape and gives protection from osmotic lysis caused by turgor pressure. The major component of the cell wall in Gram-positive bacteria is the structurally complex biopolymer peptidoglycan. Streptococcus pneumoniae (Pneumococcus) is a Gram-positive human pathogen responsible for an estimated 1-2 million deaths annually worldwide. Studies of its cell wall synthesis machinery are of high academic interest and it can contribute to drug target discoveries, which have the potential to improve treatments in the future. The recently discovered essential amidotransferase complex MurT/CobQ, encoded by the operon murTcobQ, is in S. pneumoniae responsible for the amidation the peptidoglycan precursor lipid II. The amidation of the second residue γ-glutamate to isoglutamine in lipid II has previously been shown to be necessary for the transpeptidase activity of the peptidoglycan synthesising proteins, known as penicillin binding proteins (PBPs). What biological role this amidation plays is currently not known. In the present work depletion of MurT/CobQ expression has been used extensively to study how low levels of amidated lipid II affects the phenotype of S. pneumoniae. The sensitivity against the β−lactam antibiotics cefotaxime and ampicillin did not appear to be significantly affected by MurT/CobQ depletion, and neither did lysozyme resistance. The non-essential PBP1a is the only PBP to have reported residual transpeptidase activity with non-amidated lipid II in vitro. This proved difficult to demonstrate in vivo, and as such the results of these experiments were inconclusive. It was shown that depletion of MurT/CobQ severely affected the pneumococcal cells ability to properly divide, with septal cell wall synthesis being inhibited. The cells were still able to synthesize cell wall peripherally, strongly indicating that there is a difference between the septal and peripheral cell wall synthesising machineries in their ability to utilize non-amidated lipid II as substrate. The depletion of MurT/CobQ also affected the ability of the muralytic fratricide protein CbpD to successfully lyse cells, further strengthening the existing theory that this enzyme attacks the septal region of dividing cells. Furthermore this work demonstrated that in vivo, the PBPs are able to cross-link the stem peptides of the cell wall using non-amidated lipid II as substrate. Here it was shown that while the cell walls of normal pneumococcal cells contained a small amount of non-amidated stem-peptide dimers, cells depleted of MurT/CobQ contained significantly higher amounts of non-amidated stem-peptide dimers. Den bakterielle cellveggen gir bakteriecellene sin form og beskytter dem fra osmotisk lysis. Hovedkomponenten i celleveggen hos grampositive bakterier den komplekse biopolymeren peptidoglykan. Streptococcus pneumoniae er en grampositiv, humanpatogen bakterie som er ansvarlig for mellom 1-2 millioner dødsfall årlig på verdensbasis, og studier av celleveggssyntesen kan potensielt lede til forbedrede behandlingsmetoder i fremtiden. Det nylig oppdagede essensielle amidotransferasekomplekset MurT/CobQ, kodet av operonet murTcobQ, er ansvarlig for amideringen av peptidoglykanforløperen lipid II i S. pneumoniae. Amideringen av aminosyren γ−glutamat til isoglutamin i lipid II er tidligere vist å være nødvendig for transpeptidaseaktiviteten til de peptidoglykansyntetiserende enzymene (PBPer). Hvilken biologisk rolle denne amideringen spiller er for øyeblikket ukjent. I dette arbeidet har depletion (underuttrykk) av MurT/CobQ uttrykk blitt brukt for å studere hvordan lave konsentrasjoner av amidert lipid II påvirker fenotype hos S. pneumoniae. Sensitiviteten mot β−laktam antibiotikaene cefotaxim og ampicillin, samt lysozym ble ikke signifikant påvirket av MurT/CobQ depletion. Det ikke-essensielle enzymet PBP1a er det eneste som tidligere har vist en viss aktivitet med uamidert lipid II in vitro. Dette viste seg å være vanskelig å demonstrere in vivo, og resultatene fra disse forsøkene var mangelfulle. Arbeidet har vist at depletion av MurT/CobQ påvirker streptokokk-cellenes evne til å dele seg ved at den septale celleveggssyntesen blir inhibert. Cellene evnet fremdeles å syntetisere ny cellevegg i lengderetningen, noe som indikerer at der er en forskjell mellom de septale og perifere celleveggssyntesemaskinerienes evne til å bruke uamidert lipid II. Depletion av MurT/CobQ førte også til at det muralytiske fratricidproteinet CbpD ikke klarer å lysere celler, noe som bidrar til å styrke den rådende teorien om at dette proteinet angriper septum hos pneumokokker i delingsfasen. Videre viser denne studien at in vivo så evner PBPene å inkorporere og kryssbinde uamidert lipid II til en viss grad i celleveggen. Det ble vist at mens celleveggen til normale celler inneholdt en liten mengde uamiderte peptid-dimerer, så inneholdt MurTCobQ-depleted celler et signifikant høyere nivå av uamiderte peptid-dimerer.M-M

Anvende og fundamentale perspektiver på nedbrytningsmekanismer hos brunråtesopp

Woody biomass is an important material for the growing bioeconomy, and has gained significant attention as a feedstock for second-generation biorefineries. Wood has traditionally been used as a building material for millennia, but due to its biogenic nature is susceptible to degradation by wood decaying fungi. The biochemistry used by these fungi to degrade wood is of interest, both from a wood protection perspective, and as potential bioprocessing tools. In Nature, wood-degrading basidiomycetes, which can be grouped as white- or brown-rot fungi, are the only organism known to fully degrade the polysaccharides of lignified woody biomass. Brown-rot fungi are unique, in that they successfully remove holocellulose without the mineralization of lignin, unlike white-rot fungi, which degrade both holocellulose and lignin. The objective of this thesis is the study of fundamental brown-rot fungal decay mechanisms for applied utilization. This thesis describes studies on brown-rot decay from three perspectives; 1) the oxidative non-enzymatic early decay mechanisms as potential pretreatment of wood, 2) the expression of brown-rot decay associated genes on modified wood and 3) the interplay of cellulose-oxidizing lytic polysaccharide monooxygenases with hydrogen peroxide and reductants. In Paper I the early decay mechanisms of brown-rot fungi was studied as a potential pretreatment for Norway spruce wood. We show that Norway spruce pretreated with two species of brown-rot fungi yielded more than 250% increases in glucose release when subsequently treated with a commercial enzyme cocktail. A series of experiments were performed that aimed at mimicking the brown-rot pretreatment, using a modified version of the Fenton reaction. After pretreatment, where the aim was to generate reactive oxygen species within the wood cell wall matrix, a small increase in digestibility was observed, Further experiments were performed to assess the possibility of performing pretreatment and saccharification in a single system to avoid loss of solubilized sugars, but the results indicated the need for a complete separation of oxidative pretreatment and saccharification. We conclude that a biomimicking approach to pretreatment of softwoods using brown-rot fungal mechanisms is possible, but that there are additional factors of the system that need to be known and optimized before serious advances can be made to compete with already existing pretreatment methods. In Paper II, the aim was to determine the effect of acetylation of Pinus radiata wood (a type of wood modification), on the expression of genes involved in wood decay by brown-rot fungus Rhodonia placenta. The initiation of decay was delayed as a result the degree of acetylation, and gene expression analysis using qRT-PCR captured incipient to advanced decay stages. Once decay was established, the rate of degradation in acetylated samples was similar to that of unmodified wood. This suggests a delay in decay, rather than an absolute protection threshold at higher acetylation levels. In accordance with previous studies, the oxidative system of R. placenta was more active in wood with higher degrees of acetylation and expression of hydrolytic enzymes was delayed in acetylated samples compared to untreated samples. Enzymes involved in hemicellulose and pectin degradation have previously not been the focus of studies on degradation of acetylated wood. Interestingly, we observed that a CE16 carbohydrate esterase assumed to be involved in deacetylation of carbohydrates was expressed significantly higher in untreated samples compared to highly acetylated samples. We hypothesize that this enzyme might be regulated through a negative feedback system, where acetic acid suppresses the expression. The up-regulation of two expansin genes in acetylated samples suggests that their function, to loosen the cell wall, is needed more in acetylated wood due the physical bulking of the cell wall. In this study, we demonstrate that acetylation affects the expression of specific target genes not previously reported, resulting in delayed initiation of decay. In Paper III we purified and characterized a recombinant family AA9 lytic polysaccharide monooxygenase from Gloeophyllum trabeum, GtLPMO9B, which is active on both cellulose and xyloglucan. Activity of the enzyme was tested in the presence of three different reductants: ascorbic acid, gallic acid and 2,3-dihydroxybenzoic acid (2,3-DHBA). When using standard aerobic conditions typically used in LPMO experiments, the former two reductants could drive LPMO catalysis whereas 2,3-DHBA could not. In agreement with the recent discovery that H2O2 can drive LPMO catalysis, we show that gradual addition of H2O2 allowed LPMO activity at very low, sub-stoichiometric (relative to products formed) reductant concentrations. Most importantly, we found that while 2,3-DHBA is not capable of driving the LPMO reaction under standard aerobic conditions, it can do so in the presence of externally added H2O2. At alkaline pH, 2,3-DHBA is able to drive the LPMO reaction without externally added H2O2 and this ability overlaps entirely with endogenous generation of H2O2 by GtLPMO9B-catalyzed oxidation of 2,3-DHBA. These findings support the notion that H2O2 is a co-substrate of LPMOs, and provide insight into how LPMO reactions depend on, and may be controlled by, the choice of pH and reductant.Biomasse fra tre er et viktig materiale for den gryende bioøkonomien, og har tiltrukket seg betydelig oppmerksomhet som et råstoff for 2. generasjons bioraffinerier. Tre har tradisjonelt blitt brukt som byggemateriale i årtusener, men er på grunn av sitt biologiske opphav utsatt for angrep av vednedbrytende sopp. Biokjemien benyttet av disse soppene til å bryte ned tre er av interesse, både fra et trebeskyttelsesperspektiv, og som potensielle bioprosesseringsverktøy. I naturen er basidiomycete brun- og hvitråtesopp de eneste som bryter ned alle polysakkaridene i lignifisert plantemateriale. Brunråtesoppene er unike i at de fjerner holocellulose uten å mineralisere lignin, mens hvitråtesoppene bryter ned både lignin og holocellulose. Målet ved denne avhandlingen er å studere fundamentale brunråtesoppmekanismer for anvendte øyemed. Denne avhandlingen beskriver brunråtenedbrytning fra tre perspektiver: 1) oksidative ikke-enzymatiske nedbrytningsmekanismer som forbehandling av tremasse, 2) genuttrykk av nedbrytningsassosierte gener under vekst på modifisert tre, og 3) samspillet mellom celluloseoksiderende lytisk polysakkaridmonooksygenaser, hydrogenperoksid og reduktanter. I Paper I var de tidlig nedbrytningstrinn hos brunråtesopp studert som en potensiell forbehandling for gran (Picea abies). Vi viser at ved å forbehandle gran med to brunråtesopparter, kan enzymatisk hydrolyse med en kommersiell enzymcocktail forbedres, og fikk en over 250% økning i glukosefrigivelse. Vi utførte deretter en rekke eksperimenter, hvor målet var å mimikere brunråteforhåndsbehandlingen, ved bruk av en modifisert Fenton reaksjon. Her fikk vi en marginal økning i fornøyelighet etter forhåndsbehandling, hvor hensikten var å generere reaktive oksygenarter inne i treets cellevegg. Videre eksperimenter ble utført for å undersøke mulighetene for å gjøre forhåndsbehandling og sakkarifisering i ett og samme system, og resultatene her indikerer et behov for komplett seperasjon av forhåndsbehandling og sakkarifisering, da kjemikaliene i forhåndsbehandlingen viste seg å være skadelige for enzymene. Vi konkluderer med at en biomimetisk tilnærming til forhåndsbehandling av gran er teoretisk mulig, men at systemet trenger optimalisering før videre arbeid kan gjøres. I Paper II var målet å bestemme hvordan acetylering (trebeskyttelse) av Pinus radiata påvirket uttrykk av nedbrytningsgener hos brunråtesoppen Rhodonia placenta. Genuttrykk ble analysert ved bruk av qRT-PCR og fanget både tidlige og sene nedbrytningstrinn. Initieringen av nedbrytning ble forsinket som et resultat av acetylering. Når nedbrytningen først var etablert i acetylert tre var raten sammenlignbar med umodifisert tre, noe som indikerer en hemning av nedbrytning og ikke en total beskyttelse. I samsvar med tidligere studier var det oksidative nedbrytningssystemet hos R. placenta mer aktivt i tre med høy grad av acetylering, og uttrykk av hydrolytiske gener var forsinket sammenlignet med umodifisert tre. Vi studerte uttrykk av gener involvert i hemicellulose og pektin nedbrytning som ikke tidligere er beskrevet i studier på nedbrytning av acetylert tre. Vi observerte at en karbohydratesterase (CE16) som er antatt å være involvert i deacetylering av hemicellulose var nedregulert i acetylert tre, og fremsetter en hypotese om at dette genet er regulert via en negativ feedback mekanisme. Oppreguleringen av to expansin-gener i acetylert tre indikerer at denne modifiseringen øker behovet for å løsne cellevegginteraksjoner som en konsekvens av økte massetettheten. I denne studien demonstrerer vi at acetylering påvirker uttrykk av en rekke gener ikke tidligere studert under disse forholdene, og resulterer i forsinket nedbrytning. I Paper III har vi renset og karakterisert en rekombinant familie AA9 lytisk polysakkaridmonooksygenase (LPMO, GtLPMO9B) fra brunråtesoppen Gloeophyllum trabeum, som er aktiv på både cellulose og xyloglucan. Enzymaktivitet ble testet med tre forsjellige reduktanter: ascorbic acid (AscA), gallic acid (GA) og 2,3-dihydroxybenzoic acid (2,3-DHBA). Under reaksjonsforhold vanligvis brukt i LPMO reaksjoner, var enzymet katalytisk aktivt med AscA og GA, man var det ikke med 2,3-DHBA. I samsvar med den nylige oppdagelsen at LPMO-katalyse kan drives av H2O2, viser vi at gradvis tilføring av H2O2 tillater LPMO aktivitet ved svært lave, sub-støkiometriske (relativt til produkt) reduktantkonsentrasjoner. Viktigst, så vi viser at, mens 2,3-DHBA ikke kunne drive LPMO reaksjonen under standard aerobe forhold, så kan den det i nærvær av tilført H2O2. Ved alkalisk pH (8.0-9.0), ble aktivitet med GtLPMO9B observert med 2,3-DHBA (uten ekstern tilførsel av H2O2), noe som overlappet 100% med endogen H2O2 produksjon via GtLPMO9B-katalysert oksidering av 2,3-DHBA. Disse funnen støtter teorien om at H2O2 er et kosubstrat for LPMOer, og tilfører ny kunnskap om hvorledes LPMO reaksjoner er avhengige, og potensielt kan kontrolleres med bruk av forskjellige reduktanter.NIBI

Applied and fundamental perspectives on brown-rot decay mechanisms

Woody biomass is an important material for the growing bioeconomy, and has gained significant attention as a feedstock for second-generation biorefineries. Wood has traditionally been used as a building material for millennia, but due to its biogenic nature is susceptible to degradation by wood decaying fungi. The biochemistry used by these fungi to degrade wood is of interest, both from a wood protection perspective, and as potential bioprocessing tools. In Nature, wood-degrading basidiomycetes, which can be grouped as white- or brown-rot fungi, are the only organism known to fully degrade the polysaccharides of lignified woody biomass. Brown-rot fungi are unique, in that they successfully remove holocellulose without the mineralization of lignin, unlike white-rot fungi, which degrade both holocellulose and lignin. The objective of this thesis is the study of fundamental brown-rot fungal decay mechanisms for applied utilization. This thesis describes studies on brown-rot decay from three perspectives; 1) the oxidative non-enzymatic early decay mechanisms as potential pretreatment of wood, 2) the expression of brown-rot decay associated genes on modified wood and 3) the interplay of cellulose-oxidizing lytic polysaccharide monooxygenases with hydrogen peroxide and reductants. In Paper I the early decay mechanisms of brown-rot fungi was studied as a potential pretreatment for Norway spruce wood. We show that Norway spruce pretreated with two species of brown-rot fungi yielded more than 250% increases in glucose release when subsequently treated with a commercial enzyme cocktail. A series of experiments were performed that aimed at mimicking the brown-rot pretreatment, using a modified version of the Fenton reaction. After pretreatment, where the aim was to generate reactive oxygen species within the wood cell wall matrix, a small increase in digestibility was observed, Further experiments were performed to assess the possibility of performing pretreatment and saccharification in a single system to avoid loss of solubilized sugars, but the results indicated the need for a complete separation of oxidative pretreatment and saccharification. We conclude that a biomimicking approach to pretreatment of softwoods using brown-rot fungal mechanisms is possible, but that there are additional factors of the system that need to be known and optimized before serious advances can be made to compete with already existing pretreatment methods. In Paper II, the aim was to determine the effect of acetylation of Pinus radiata wood (a type of wood modification), on the expression of genes involved in wood decay by brown-rot fungus Rhodonia placenta. The initiation of decay was delayed as a result the degree of acetylation, and gene expression analysis using qRT-PCR captured incipient to advanced decay stages. Once decay was established, the rate of degradation in acetylated samples was similar to that of unmodified wood. This suggests a delay in decay, rather than an absolute protection threshold at higher acetylation levels. In accordance with previous studies, the oxidative system of R. placenta was more active in wood with higher degrees of acetylation and expression of hydrolytic enzymes was delayed in acetylated samples compared to untreated samples. Enzymes involved in hemicellulose and pectin degradation have previously not been the focus of studies on degradation of acetylated wood. Interestingly, we observed that a CE16 carbohydrate esterase assumed to be involved in deacetylation of carbohydrates was expressed significantly higher in untreated samples compared to highly acetylated samples. We hypothesize that this enzyme might be regulated through a negative feedback system, where acetic acid suppresses the expression. The up-regulation of two expansin genes in acetylated samples suggests that their function, to loosen the cell wall, is needed more in acetylated wood due the physical bulking of the cell wall. In this study, we demonstrate that acetylation affects the expression of specific target genes not previously reported, resulting in delayed initiation of decay. In Paper III we purified and characterized a recombinant family AA9 lytic polysaccharide monooxygenase from Gloeophyllum trabeum, GtLPMO9B, which is active on both cellulose and xyloglucan. Activity of the enzyme was tested in the presence of three different reductants: ascorbic acid, gallic acid and 2,3-dihydroxybenzoic acid (2,3-DHBA). When using standard aerobic conditions typically used in LPMO experiments, the former two reductants could drive LPMO catalysis whereas 2,3-DHBA could not. In agreement with the recent discovery that H2O2 can drive LPMO catalysis, we show that gradual addition of H2O2 allowed LPMO activity at very low, sub-stoichiometric (relative to products formed) reductant concentrations. Most importantly, we found that while 2,3-DHBA is not capable of driving the LPMO reaction under standard aerobic conditions, it can do so in the presence of externally added H2O2. At alkaline pH, 2,3-DHBA is able to drive the LPMO reaction without externally added H2O2 and this ability overlaps entirely with endogenous generation of H2O2 by GtLPMO9B-catalyzed oxidation of 2,3-DHBA. These findings support the notion that H2O2 is a co-substrate of LPMOs, and provide insight into how LPMO reactions depend on, and may be controlled by, the choice of pH and reductant

Acetylation of Pinus radiata delays hydrolytic depolymerisation by the brown-rot fungus Rhondonia placenta

Acetylation of wood can provide protection against wood deteriorating fungi, but the exact degradation me- chanism remains unclear. The aim of this study was to determine the effect of acetylation of Pinus radiata wood (weight percent gain 13, 17 and 21%) on the expression of genes involved in decay by brown-rot fungus Rhodonia placenta. Gene expression analysis using qRT-PCR captured incipient to advanced decay stages. As expected the initiation of decay was delayed as a result the degree of acetylation. However, once decay was established, the rate of degradation in acetylated samples was similar to that of unmodi fied wood. This suggests a delay in decay rather than an absolute protection threshold at higher acetylation levels. In accordance with previous studies, the oxidative system of R. placenta was more active in wood with higher degrees of acetylation and expression of cellulose active enzymes was delayed for acetylated samples compared to untreated samples. The reason for the delay in the latter might be because of the slower diffusion rate in acetylated wood or that partially acetylated cellobiose may be less effective in triggering production of saccharification enzymes. Enzymes involved in hemicellulose and pectin degradation have previously not been focused on in studies of degradation of acetylated wood. Surprisingly, CE16 carbohydrate esterase, assumed to be involved in deace- tylation of carbohydrates, was expressed significantly more in untreated samples compared to highly acetylated samples. We hypothesise that this enzyme might be regulated through a negative feedback system, where acetic acid supresses the expression. The up-regulation of two expansin genes in acetylated samples suggests that their function, to loosen the cell wall, is needed more in acetylated wood due the physical bulking of the cell wall. In this study, we demonstrate that acetylation affects the expression of specific target genes not previously re- ported, resulting in delayed initiation of decay. Thus, targeting these degradation mechanisms can contribute to improving wood protection systems.Acetylation of Pinus radiata delays hydrolytic depolymerisation by the brown-rot fungus Rhondonia placentaacceptedVersio

Wood modification by furfuryl alcohol caused delayed decomposition response in Rhodonia (Postia) placenta

The aim of this study was to investigate differential expression profiles of the brown rot fungus Rhodonia placenta (previously Postia placenta) harvested at several time points when grown on radiata pine (Pinus radiata) and radiata pine with three different levels of modification by furfuryl alcohol, an environmentally benign commercial wood protection system. The entire gene expression pattern of a decay fungus was followed in untreated and modified wood from initial to advanced stages of decay. The results support the current model of a two-step decay mechanism, with the expression of genes related to initial oxidative depolymerization, followed by an accumulation of transcripts of genes related to the hydrolysis of cell wall polysaccharides. When the wood decay process is finished, the fungus goes into starvation mode after five weeks when grown on unmodified radiata pine wood. The pattern of repression of oxidative processes and oxalic acid synthesis found in radiata pine at later stages of decay is not mirrored for the high-furfurylation treatment. The high treatment level provided a more unpredictable expression pattern throughout the incubation period. Furfurylation does not seem to directly influence the expression of core plant cell wall-hydrolyzing enzymes, as a delayed and prolonged, but similar, pattern was observed in the radiata pine and the modified experiments. This indicates that the fungus starts a common decay process in the modified wood but proceeds at a slower pace as access to the plant cell wall polysaccharides is restricted. This is further supported by the downregulation of hydrolytic enzymes for the high treatment level at the last harvest point (mass loss, 14%). Moreover, the mass loss does not increase during the last weeks. Collectively, this indicates a potential threshold for lower mass loss for the high-furfurylation treatment

Wood modification by furfuryl alcohol caused delayed decomposition response in Rhodonia (Postia) placenta

The aim of this study was to investigate differential expression profiles of the brown rot fungus Rhodonia placenta (previously Postia placenta) harvested at several time points when grown on radiata pine (Pinus radiata) and radiata pine with three different levels of modification by furfuryl alcohol, an environmentally benign commercial wood protection system. The entire gene expression pattern of a decay fungus was followed in untreated and modified wood from initial to advanced stages of decay. The results support the current model of a two-step decay mechanism, with the expression of genes related to initial oxidative depolymerization, followed by an accumulation of transcripts of genes related to the hydrolysis of cell wall polysaccharides. When the wood decay process is finished, the fungus goes into starvation mode after five weeks when grown on unmodified radiata pine wood. The pattern of repression of oxidative processes and oxalic acid synthesis found in radiata pine at later stages of decay is not mirrored for the high-furfurylation treatment. The high treatment level provided a more unpredictable expression pattern throughout the incubation period. Furfurylation does not seem to directly influence the expression of core plant cell wall-hydrolyzing enzymes, as a delayed and prolonged, but similar, pattern was observed in the radiata pine and the modified experiments. This indicates that the fungus starts a common decay process in the modified wood but proceeds at a slower pace as access to the plant cell wall polysaccharides is restricted. This is further supported by the downregulation of hydrolytic enzymes for the high treatment level at the last harvest point (mass loss, 14%). Moreover, the mass loss does not increase during the last weeks. Collectively, this indicates a potential threshold for lower mass loss for the high-furfurylation treatment.publishedVersio

Wood modification by furfuryl alcohol caused delayed decomposition response in Rhodonia (Postia) placenta

The aim of this study was to investigate differential expression profiles of the brown rot fungus Rhodonia placenta (previously Postia placenta) harvested at several time points when grown on radiata pine (Pinus radiata) and radiata pine with three different levels of modification by furfuryl alcohol, an environmentally benign commercial wood protection system. The entire gene expression pattern of a decay fungus was followed in untreated and modified wood from initial to advanced stages of decay. The results support the current model of a two-step decay mechanism, with the expression of genes related to initial oxidative depolymerization, followed by an accumulation of transcripts of genes related to the hydrolysis of cell wall polysaccharides. When the wood decay process is finished, the fungus goes into starvation mode after five weeks when grown on unmodified radiata pine wood. The pattern of repression of oxidative processes and oxalic acid synthesis found in radiata pine at later stages of decay is not mirrored for the high-furfurylation treatment. The high treatment level provided a more unpredictable expression pattern throughout the incubation period. Furfurylation does not seem to directly influence the expression of core plant cell wall-hydrolyzing enzymes, as a delayed and prolonged, but similar, pattern was observed in the radiata pine and the modified experiments. This indicates that the fungus starts a common decay process in the modified wood but proceeds at a slower pace as access to the plant cell wall polysaccharides is restricted. This is further supported by the downregulation of hydrolytic enzymes for the high treatment level at the last harvest point (mass loss, 14%). Moreover, the mass loss does not increase during the last weeks. Collectively, this indicates a potential threshold for lower mass loss for the high-furfurylation treatment