DNA fragments to be used for specific purposes like ligation or radioactive labeling were eluted from the agarose gel after electrophoresis. The gel piece containing thedesired band was sliced out from the gel and the DNA was purified using commerciallyavailable purification kits for this purpose. The efficiency of elution was determined bychecking a small aliquot of DNA sample on the gel

Purification of DNA by gel elution

and colony purified on CAA plate. 15% glycerol stocks were made for two independent transformants and stored at -80 ̊C

C. glabrataCgYPS7ORFwas cloned in a self-replicating pGRB2.2plasmidwhich contains C. glabrata CEN-ARS, S. cerevisiaeURA3gene, S. cerevisiaePGK1promoter and C. glabrataHIS3-3′ untranslated region. For cloning CgYPS7in pGRB2.2,CgYPS7ORF (1.764 kb) was PCR-amplified from the wild-type genomic DNA with high fidelity Platinum PfxDNA polymeraseusing primers carrying restriction sites for XbaIand XhoI. The1.764 kb amplifiedPCR product waspurified with QIAquick PCR purification kit (Qiagen # 28104),digested with XbaI and XhoI and cloned in the pGRB2.2plasmid at XbaI–XhoI sites in the multiple cloning site (MCS)region downstream of the PGK1promoter.Positiveclones were verified by PCR, sequencing and complementation analysesofCgyps7∆mutant. Yeast transformantsobtained by lithiumacetate methodwere selected on plates lacking uracil

Single colony of C. glabratastrains wasinoculated in 10ml YPD-liquid medium and grown at 30°C with constant shaking at 200 rpm for 14-16 h. Overnight culture was used to inoculate 10 ml YPD broth to an initial OD600of 0.1 and culture was grown for 4-5 h to obtain log-phase culture. Log-phase C. glabratacells were harvested in 15 ml sterile polypropylene tubesby centrifugation at 4,000 rpm for 5 min. Harvested cells were washed with10ml sterile water,resuspendedin 1 ml sterile water and transferred to a 1.5 ml microcentrifuge tube. Cells were harvested at 4,000 rpm for 5 minand resuspended in 100 μl of100mM lithium acetate solution.Yeast transformation cocktail was prepared in a 1.5 ml microcentrifuge tube by mixing 240 μlpolyethylene glycol(50%), 36μl lithium acetate(1 M) and25μlheat-denatured single stranded carrier DNA(2 mg/ml). 50 μlC. glabratacell suspension and 50 μltransforming DNAwas added to the transformation cocktail, mixed well andincubatedat 30 ̊C for45 min. 43 μlDMSO was added and cells were subjected to heat shock at 42 ̊Cfor 15 min. After the heat shock, cells were transferred to ice for 10-15 seconds, centrifuged at 4,000 rpm for 5 min and supernatantwas removed.Cells wereresuspended in 200 μlsterile water andspread platedonappropriate selectionmedium. Plates wereincubatedat30 ̊Cfor 2-3 days

Automated DNA sequencing on plasmid templates or on PCR products was carried out with dye terminator cycle sequencing kits from Perkin-Elmer on an automated sequencer (model 377, Applied Biosystems), following the manufacturer’s instructions

The plasmid-DNA/PEI mixture was incubated for 15 minutesat room temperature.The mixture was added to cells,andmixed properlyby rocking the culture plate back and forth. Cells were incubated at 37°C in a CO2 incubator.The transfected cells were harvested at 24-48 hours post-transfection

Cells were plated inthe cell culture dishes one day before transfection in RPMI1640 supplemented with FBS and penstrep (complete medium). All the reagents were brought to room temperature before starting transfection. Plasmid-DNA was diluted in serum-free medium and PEI was added(Table 9)Table 9: PEI plasmid-transfection methodology

BXOR1, ΔrpfFand ΔrpfF(pSC9) strains were grown to OD600of 1 in rich media (PS), PS + 50 μM 2,2’-dipyridyl (DP) and PS + DP + 30 μM FeSO4. RNA was isolated by Trizol (Invitrogen) method as described above. Optimal primer and cDNA concentrations were standardized, and qRT-PCR was performed using ABI 7500 Fast Real-Time PCR system (Applied Biosystems). In brief, 1 μl cDNA, 0.25 picomoles of gene specific primers and 10 μl 2X SYBR GREEN qPCR Mastermix (Qiagen)were mixed in the wells of 96-well PCR plate (Axygen). Final reaction volume was adjusted to 20 μl with nuclease-free water. Transcript levels were quantified with an end-point value known as Ct(cycle thresold) value. Expression of 16S rRNA was used as an internal control. The Ct values defines the number of PCR cycles required for the fluorescent signal of SYBR green dye to cross beyond the background level. Fold-change in transcript expression was determined using following formula.Fold change in expression = 2-ΔΔCtΔΔCt= ΔCt treated-ΔCt untreatedΔCttreated = Ctvalue for the gene of interest under treated condition -Ct value for the internal control gene (16S rRNA) under treated conditionΔ Ctuntreated = Ct value for thegene of interest under untreated condition -Ct value for the internal control (16S rRNA) gene under untreated condition

Primers for real-time PCR analysis were designed using Primer3 plus software and are listed in Table 2.2.For RNA isolation, X. oryzaepv.oryzaewild-type, rpfFmutant, rpfF/CG8 complemented strains were grown in PS medium at 28°C for 28 h at 200 rpm. Similarly, for RNA isolation from X. oryzaepv. oryzicola, the Wild-type