Fungal strains and growth conditions
F. oxysporum f. sp. cubense race 4 (FOC4) was obtained from Guangzhou, China, and F. oxysporum f. sp. cubense race 1 (FOC1) was obtained from Nanning, China. The pathotype of the isolates was periodically confirmed by plant assays in a growth chamber. The cultivars tested comprised Musa AAA Cavendish cv. Baxi, resistant to FOC1 and susceptible to FOC4; Musa AAB cv. Guangfen-1, susceptible to FOC1 and FOC4.
For pectinolytic enzyme production and extraction of RNA, fungal strains were grown in SM  supplemented with 1% [w/v] citrus pectin (Sigma). For extraction of DNA, mycelium was obtained from cultures grown for 5 d in potato dextrose broth (PDB) in Erlenmeyer flasks on a rotary shaker at 110 rpm and 25°C.
Purification of PGC2
Cultures of FOC4 grown in SM supplemented with 1% citrus pectin were centrifuged at 16,000 g for 20 min. Supernatant was collected and concentrated using an Amicon 8400 ultrafiltration system containing a 10 kDa MWCO membrane.
Concentrated extract filtrate was applied to a gel filtration column (Sephacryl S-100 16/60, Pharmacia) equilibrated and eluted with 50 mM sodium acetate buffer (pH 4.5) at a flow rate of 1 ml/min. Fractions containing PG activity were pooled and applied to a cation exchange column (Sepharose FF CM Hitrap, Pharmacia) equilibrated with 20 mM sodium acetate buffer (pH 4.5). The column was eluted with a gradient of NaCl (0-0.7 M) at a flow rate 2 ml/min.
PG activity and protein assays
PG activity was routinely determined by measuring the release of reducing groups from polygalacturonate (PGA; Sigma). The standard reaction mixture (1 ml total volume) contained 0.5% PGA (w/v), 50 mM sodium acetate buffer (pH 5.0), and various amounts of the enzyme preparation. After incubation at 45°C for 30 min, the reducing sugar content was determined by the methods previously reported . Appropriate controls without either enzyme or substrate were run simultaneously. The quantity of reducing sugar released was calculated from standards of D-galacturonic acid (GA; Sigma). One unit of enzyme activity was defined as the amount of enzyme that released 1 μmol of GA equivalent per minute under the above conditions.
Discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to Laemmli  using 10% acrylamide. After electrophoresis, gels were stained with Coomassie brilliant blue G250 (Sigma). Protein concentration was determined according to the Bradford method  using bovine serum albumin as a standard.
Biophysical characterization of the PGC2
The purified PGC2 was resolved on SDS-PAGE, blotted onto a PVDF membrane and analyzed by electrospray ionization-mass spectrometry (ESI-MS) using a triple quadrupole instrument from Applied Biosystems, API 3000 (PE Sciex, Canada). Samples were diluted in milli-Q water with a saturated solution of a-cyano-4-hydroxycinnamic acid containing 50% (v/v) acetonitrile and 0.1% (v/v) TFA. The spectra were acquired over a mass/charge (m/z) range of 400-2000 in direct and reflective mode and were interpreted using Voyager software v.5.0.
Biochemical characterization of PGC2
For analysis of hydrolysis products, the purified enzyme (0.02 units in 0.5 ml distilled water) was added to 1 ml of 0.5% (w/v) PGA in 50 mM sodium acetate buffer (pH 5), incubated at 45°C for 10, 20, 30, 40, 50, or 60 min, and then assayed for PG activity. Removal of N-linked carbohydrate was carried out using N-glycosidase F (Boehringer Mannheim, Mannheim, Germany). Deglycosylation protocols followed the manufacturer's instructions.
The standard assay was modified to test the effects of varying pH and incubation temperature. To determine the optimal pH, enzyme activity was assayed using 100 mM potassium phosphate buffer for pH values between 3 and 10 at 45°C and 0.5% (w/v) PGA as substrate. The effect of temperature on PGC2 activity was determined in the same buffer at pH 5 between 10°C and 90°C. One unit of PG activity was defined as one μmol/L of GA released by enzyme min-1.
To estimate pH stability, samples were incubated in 100 mM potassium phosphate buffer of different pH values at 4°C for 24 h. To evaluate the thermal stability, protein samples were incubated at different temperatures in the same buffer at pH 5 for 2 h. The residual activity was determined as previously described.
m and V
max values were determined from the Lineweaver-Burk double reciprocal plot of PG activity using PGA as substrates at concentrations between 2.5 mg ml-1 and 10 mg ml-1. The activity was assayed as previously described.
Cloning of fungal pgc2 genes
For RACE PCR, total RNA from FOC4 was reverse-transcribed into cDNA with murine leukemia-virus reverse transcriptase (TaKaRa). Two oligonucleotide primers were designed based on the N-terminal partial sequence of PGC2. The first PCR used the degenerate sense primer P1: 5'-GCNATHGAYYTNCCNGC-3', nest-PCR primer nP1: 5'-CCNGCNGGNGTNCCNMG-3', with the antisense primer Oligo(dT)20. Amplification cycles were as follows: one cycle of 3 min at 94°C, 35 cycles of 94°C for 30 s, 48°C for 30 s, and 72°C for 90 s, then 10 min for 72°C. The amplified fragments were gel-purified and cloned into pMD18-T (TaKaRa) and sequenced. Two primers (P2: 5'- GTAGTAGCGTTGATGTTG-3', nP2: 5'-GTAGCACGAGTCGATG-3') was designed for 5'-RACE in accordance with RT-PCR sequence, which was performed essentially as described for the 5' -Full RACE Core Set (TaKaRa).
Based on the nucleotide sequence of the RACE PCR, first-strand cDNAs were synthesized from total RNA of FOC1 and FOC4 using reverse transcriptase XL (TaKaRa) by reverse transcription polymerase chain reaction (RT-PCR) using the forward primer (5'-GTGGAATTCATGAAGGTCTCGAGCTTCTTCT-3') and the reverse primer (5'- CGGTCTAGATTAACCGTTGAACTTTCTAACC-3'). The EcoRI and XbaI restriction sites are underlined. The following PCR conditions were used: 35 cycles with denaturation at 94°C for 30 s, annealing at 49°C for 30 s and extension at 72°C for 90 s. An initial denaturation step of 4 min at 94°C and a final elongation step at 72°C for 10 min were performed. The RT-PCR products were cloned into pMD18-T (TaKaRa) and verified by nucleotide sequencing analysis, and then they were digested with EcoRI and XbaI and subcloned into the recombinant eukaryotic expression vector pPICZaA (Invitrogen) digested with the same enzymes to generate pPICZaA-pgc2-FOC1 and pPICZaA-pgc2-FOC4. The presence of the inserted pgc2-FOC1 and pgc2-FOC4 genes was verified by nucleotide sequencing analysis.
Genomic DNA was extracted from fungal mycelium using the E.Z.N.A Fungal DNA Kit (OMEGA) according to the manufacturer's instructions. DNA sequences were sequenced using DNA isolated from FOC1 and FOC4 as the template and primers described above.
The complete nucleotide sequences of pgc2 genes from FOC1 and FOC4 were deposited in the GenBank database under accession numbers GU224636 and GU224637.
Expression and purification of PGC2 in P. pastoris
Yeast transformation was performed according to manufacturer's instructions. The recombinant plasmids pPICZaA-pgc2-FOC1 and pPICZaA-pgc2-FOC4 were linearized by digestion with SacI and transformed into P. pastoris SMD1168 strain by electroporation. SMD1168 strains transformed with pPICZaA plasmid and SMD1168 strains without transformation served as negative controls. Cells were incubated on yeast extract peptone dextrose (YPD) plates containing 1% yeast extract, 2% peptone, 2% dextrose, and 100 μg/ml of zeocin at 28°C for 48 h. Integration of the pgc2 gene into the genome of P. pastoris was determined by PCR using 5'AOX1 and 3'AOX1 primers. The presence of the pgc2 gene in transformants was confirmed by gene sequencing of PCR products.
Yeast transformants were grown in 30 ml of BMGY medium (1% yeast extract, 2% peptone, 1.34% yeast nitrogen base, 100 mM potassium phosphate, 4 × 10-5% biotin and 1% glycerol) at 28°C for 24 h. Cell pellets were harvested and resuspended in 200 ml BMMY (1% yeast extract, 2% peptone, 1.34% yeast nitrogen base, 100 mM potassium phosphate, 4 × 10-5% biotin and 0.5% methanol). The cultures were returned to the incubator and grown under the same conditions, and samples were collected at 1, 2 and 3 d. 1 ml of 100% methanol was added every 24 h to maintain a final concentration of 0.5%, assuming that the methanol was completely utilized in 24 h.
The cultures were centrifuged at 10,000g for 20 min at 4°C, and supernatants were filtered through membranes (0.22 μm, Millipore). The supernatant was fractionated by salting out with solid ammonium sulfate at 0-80% (w/v) saturation. The precipitate formed was collected by centrifugation at 10,000g for 20 min at 4°C, dissolved in 50 mM sodium acetate buffer (pH 5.0), and then dialyzed overnight against the same buffer at 4°C.
After centrifugation, recombinant r-FOC1-PGC2 and r-FOC4-PGC2 were further purified using a gel filtration column (Sephacryl S-100 16/60, Pharmacia) equilibrated and eluted with 50 mM sodium acetate buffer (pH 5.0) at a flow rate of 1 ml/min. Fractions containing PG activity were collected.
Tissue maceration and necrosis assayed with recombinant PGC2
To evaluate tissue maceration, the cultivars tested comprised Musa AAA Cavendish cv. Baxi, resistant to FOC1 and susceptible to FOC4; Musa AAB cv. Guangfen-1, susceptible to FOC1 and FOC4. 1 cm lengths of tissue (0.5 g) were taken from the healthy stems of the four leaf stage banana and placed in test tubes. A mixture of 1 unit of purified enzyme with 1 ml of 50 mM sodium acetate buffer, pH 5.0, was inoculated with the sterilized banana tissue, and maceration was evaluated after 48 h. Control tubes contained the same buffer without enzyme. Released reducing sugar was calculated from standards of GA after incubation 48 h at 45°C. The reducing sugar content was determined by the method of Somogyi .
For the tissue necrosis assay, 1 unit of enzyme was applied to the stems of healthy banana plant by injection. For each treatment, stems were cut (vertical-sectioned) 5 d later to observe vascular necrosis, ten replicates. Sterile double distilled water and 50 mM sodium acetate buffer pH 5.0 were used as controls.