Restriction endonuclease

DNaseI, Glucose-1-phosphate, dTTP, dTDP-D-glucose, malachite green reagent, NADPH, phenylmethanesulfonyl fluoride (PMSF), pyrophosphatase, UTP and UDP-glucose were obtained from Sigma-Aldrich (St. Louis MO), dTDP-4-keto-6-deoxy-glucose came from Carbosynth (Berkshire, UK), novobiocin and ampicillin were obtained from Duchefa Biochemie (Haarlem, The Netherlands), while restriction endonucleases were purchased from Promega (Madison, WI).

Soybean flour extract was prepared following the method of previous report with some modifications [14] . The soybean flour was defatted in three volumes of n-hexane for 30 min with agitation at room temperature, centrifuged at 13,000 g for 20 min, and air-dried to a fine powder.
The substrates cellobiose, carboxymethyl cellulose sodium (CMC-Na), Avicel, laminiarin, lichenin, amygdalin, daidzin, genistin, glycitin, daidzein, genistein, glycitein, birchwood xylan, beechwood xylan, sophorose, gentiobiose, salicin, lactose, p-nitrophenyl β-d-glucopyranoside (pNPG) and other p-nitrophenyl glycosides, and Novozyme 188 (cellobiase from Aspergillus niger) were purchased from Sigma-Aldrich (St. Louis, MO). Other substrates including barley β-glucan and xyloglucan were obtained from Megazyme (Wicklow, Ireland). T4 DNA ligase and restriction endonucleases were purchased from Promega (Madison, WI).
The Fungal DNA Isolation Kit and RNeasy Plant Mini Kit were obtained from Omega Bio-tek (Norcross, GA) and QIAGEN (Hilden, Germany), respectively. The DNA Purification Kit, LA Taq DNA polymerase and restriction endonucleases were purchased from TaKaRa (Otsu, Japan). All other chemicals were of analytical grade and commercially available.

RPMI 1640 medium, Opti-MEM® I Reduced-Serum Medium and fetal bovine serum, Lipofectamine™ 2000 Transfection Reagent, TRIzol RNA isolation reagent and primers were from Invitrogen (Grand Island, NY). Cell Counting Kit-8 (CCK-8), melphalan, myrecitin, synthetic siRNA against APE1 and MDR1 were from Sigma-Aldrich (St Louis, MO). Tetrahydrofuran-containing oligonucleotides, biotin-conjugated oligos and all other regular oligos were synthesized from Takara (Dalian, China). T4 polynucleotide kinase (T4 PNK), T4 ligase, restriction endonucleases, and high-fidelity Pfu DNA polymerase were from Promega (Madison, WI). Halt protease inhibitor cocktail, LightShift chemiluminescent EMSA kit, Super Signal West Pico chemiluminescent reagents, horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG antibodies were from Pierce (Rockford, IL). The chemo-sensitive RPMI-8226 and U266 cell line were purchased from American Type Culture Collection (Manassas, VA) and their melphalan-resistant sublines RPMI-8226/LR5 and U266/LR6 were obtained from Dr. William S. Dalton (Lee Moffitt Cancer Center, Tampa, FL). All MM cell lines were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin (Hyclone, Logan, UT), and maintained at 37°C in a humidified atmosphere in the presence of 5% CO₂-95% air.

DNA manipulations followed the procedures described by Sambrook and associates50 . Plasmids were transformed into cells of E. coli and Xanthomonas spp. by electroporation or conjugation described by Turner and associates51 . The restriction endonucleases, T4 DNA ligase, and pfu polymerase were provided by Promega (Shanghai, China). The total RNAs from Xanthomonas spp. were extracted with a total-RNA extraction kit (Promega), and reverse transcription was performed using a cDNA synthesis kit (Fermentas Co., Vilnius, Lithuania). Each kit was used according to the manufacturer’s instructions.
Western blotting was carried out as previously described21 (link). Briefly, bacterial proteins were separated by 12% (w/v) SDS-PAGE and transferred onto PVDF (polyvinylidene difluoride) membrane (Millipore Corporation, Billerica, MA, USA). After blocking, the 1:2500 diluted anti-His-tag mouse monoclonal antibody (Qiagen, Shanghai, China) was used as the primary antibody, and the 1:2500 diluted horseradish peroxidase conjugated goat antimouse IgG (Bio-Rad, Hercules, CA, USA) was used as secondary antibody.

The DNA manipulations followed the procedures described by Sambrook et al.42 . Conjugation between the Xcc and E. coli strains was performed as described by Turner et al.43 . The restriction endonucleases, T4 DNA ligase and pfu polymerase were provided by Promega (Shanghai, China). The total RNAs were extracted from the cultures of the Xcc strains using a total-RNA extraction kit (Promega) according to the manufacturer’s instructions.
To assay the transcription level of gumB, real-time quantitative PCR (qRT-PCR) was carried out as previously described44 . qRT-PCR was conducted with the total RNA extracted from the Xcc strains grown in NY medium containing 2% glucose for 12 and 48 h. The synergy brand (SYBR) green-labelled PCR fragments were amplified using the primer set gumNF/R (Supplementary Table 1), which was designed from the transcribed region of gumB. The relative mRNA level was calculated with respect to the level of the corresponding transcript in the wild-type strain 8004 (equalling 1). The expression level of the 16S rRNA gene was used as an internal standard. The qRT-PCR tests were performed in triplicate.

The DNA manipulations followed the procedures described by Sambrook et al. (1989). Conjugation between the Xcc and E. coli strains was performed as described by Turner et al. (1985). The restriction endonucleases, T4 DNA ligase and pfu polymerase were provided by Promega (Shanghai, China). The total RNAs were extracted from the cultures of the Xcc strains with a total‐RNA extraction kit (Invitrogen, Waltham, MA, USA) and cDNA generated using a cDNA synthesis kit (Invitrogen). For Semi‐quantitative reverse‐transcription PCR (RT‐PCR), the obtained cDNA was diluted and used as a template with selected primers (see Supporting Information Table S5) for target genes.
To assay the transcription level of certain genes (e.g. gumB, xcsC, pelB, egl), quantitative real‐time PCR (qRT‐PCR) was carried out as previously described (Li et al., 2014). The synergy brand (SYBR) green‐labelled PCR fragments were amplified using the corresponding primer set (Supporting Information Table S5). The relative mRNA level was calculated with respect to the level of the corresponding transcript in the wild‐type strain 8004 (equalling 1). The expression level of the 16S rRNA gene was used as an internal standard. The qRT‐PCR tests were performed in triplicate.