**2.9.2 Incubation experiment**

398 Gel Electrophoresis – Advanced Techniques

Total RNA was extracted from mycelia after varying incubation periods under MnP- and Lcc- induced conditions using TRIzol Reagent (Invitrogen, CA). cDNA was synthesized from total RNA using an RNA PCR Kit Ver.3.0 (Takara Bio, Japan), and amplified using the primer set LeMnP2En5f (5'-TCCGACAGTGTCAATGACCTCGCTC) and LeMnP2En13r (5'**-** GTCAGTGGTGAGATTTGGGAAGGGC), which were designed based on the highly conserved *lemnp2* region (DDBJ Acc. No. AB306944; Sakamoto et al., 2009). A fragment measuring approximately 700 bp was then extracted from the 1% agarose−formaldehyde gel, purified, and sequenced using an ABI Prism 3100-*Avant* Genetic Analyzer (Applied Biosystems, CA) according to the manufacturer's instructions. Fragments with sequences matching *lemnp2* were then labeled using a PCR-based digoxigenin (DIG)-dUTP labeling kit (PCR DIG Probe Synthesis Kit, Roche Diagnostics) according to the manufacturer's instructions. The resulting DIG-labeled probe, *lemnp2*N, was then used for Northern hybridization following blotting of 10 μg of total RNA onto a Hybond-N+ membrane (GE

Healthcare, Switzerland) using an established protocol (Sambrook et al., 1989).

Genomic DNA was extracted from mycelia using cetyltrimethylammonium bromide (CTAB) isolation buffer (J. J. Doyle & J. L. Doyle, 1987, as cited in Milligan, 1998). To prepare the DIG-labeled probe, genomic DNA was used as a template for PCR with the primer set, LeMnP2En5f2 (5'**-**TCAGGAAAATTCCCGACTAT) and LeMnP2En12r (5'-GAACCTCGATG CCATCAA); this primer set was designed to amplify the region from exon 5 to exon 12 of *lemnp2*, including introns (DDBJ Acc. No. AB306944; Sakamoto et al., 2009), and the resulting probe was named *lemnp2*S. In addition, to examine cross-hybridization between *lemnp2* and a relative of the manganese peroxidases LeMnP1 coded by *lemnp1*, a probe for *lemnp1* was also prepared as described above using the primer set LeMnP1f1 (5'- GATTCCTGAGCCTTTCG) and LeMnP1r (5'-TTCGGGACGGGAATAAC); this primer set was designed to amplify the regions from exon 7 to exon 15 of *lenmp1* including introns (DDBJ Acc. No. AB241061; Nagai et al., 2007) and the resulting probe was named *lemnp1*S.

These two probes were then used for Southern blot analysis (Sambrook et al., 1989).

To assay the abilities of MnP and Lcc to degrade the β-*O*-4 lignin model compound, 4 ethoxy-3-methoxyphenylglycerol- β-guaiacyl ether (Fig. 2; β-*O*-4 compound, hereafter) was synthesized according to the method previously described (Umezawa & Higuchi 1985).

To prepare media containing the β-*O*-4 compound to media, 300 µg of the β-*O*-4 compound was diluted in 50 µl acetone and added to 30 ml MYPG-S. Whole liquid culture media was collected 14, 21 and 42 days after inoculation and assayed for phenol-oxidizing enzyme activity. The β-*O*-4 compound was then recovered from the liquid culture media by the addition of two volumes of ethyl acetate to separate the aqueous phase, before evaporating the ethyl acetate off and then precipitating the compound. To improve subsequent chromatographic analysis, the recovered β-*O*-4 compound was silylated using N-*O*-bis- (trimethylsilyl) trifluoroacetamide (BSTFA) to form a trimethylsilyl (TMS) derivative. This

**2.9 Degradation assay of β-***O***-4 lignin model compound** 

**2.9.1 Culture experiment** 

**2.7 RNA isolation and Northern blot analysis** 

**2.8 DNA isolation and Southern blot analysis** 

Degradation of the β-*O*-4 compound was also examined by incubation with the extracellular enzyme solution, which was prepared using the same procedures used for electrophoresis (section 2.4) with the following slight modifications. The extracellular enzyme solutions were diluted with 0.2 M sodium tartrate buffer (pH 5.0, final concentration of 0.1 M) and distilled water to bring the volume to 10 ml and keep the activity of MnP, Lcc and the mixed solution (MnP+Lcc) at 17 U/ml. For the MnP and MnP+Lcc reactions, additional H2O2 (final concentration 0.1 mM) and MnSO45H2O (final concentration 0.1 mM) were added to the reaction for Lcc. Then, 500 µg of the β-*O*-4 compound in 50 µl of acetone was added to the 10 ml enzyme solution and incubated at 37°C with agitation at 100 rpm for up to 10 days. The rate of degradation of the β-*O*-4 compound was evaluated using GC-MS as above.
