DNA sequencing

and a colourless upper aqueous phase. The upper aqueous phase in which RNA existsexclusively, was transferred to a fresh microfuge tube and RNA was precipitated byadding 0.5 ml of isopropyl alcohol for each ml of Trizol used. Samples were incubatedat 15 to 30ºC for 10-min and centrifuged at 12000 rpm for 10-min at 4ºC. RNA formeda gel like precipitate at the bottom of the tube. Supernatant was removed and RNA waswashed with 75% ethanol (by adding 1 ml of ethanol per ml of Trizolemployed). RNAcould be stored after this step in –20 or –70ºC for more than a year. RNA pellet was airdried for 15-to 30-min following which it was dissolved in nuclease free water. Theconcentrations and purity of RNA samples were determined spectroscopically as wellas by visual inspection on formaldehyde-agarose gel in MOPS buffer (Goodet al., 1996). Before loading onto the gel, RNA was mixed with loading buffer and heated at90ºC for 3-min

For isolation of RNA, cells were grown in minimal A medium supplemented with 0.2%glucose upto A600of 0.6. Cells were harvested by centrifugation and total RNA wasisolated by using Trizol (Invitrogen) according to manufacturer’s instructions. 1 ml ofTrizol was used to lyse cells equivalent of approximately 4 ml of overnight culture.Homogeneous lysis was achieved by gentle pipetting repeatedly. The homogenized samples were incubated at room temperature for 5-min to permit complete dissociationof nucleoprotein particles. Following homogenization, 0.2 ml of chloroform for each 1ml Trizol reagent was added and vigorously shaken with hand for 15-sec and incubatedfurther for 3-min at RT. It was then centrifuged at 12000 rpm for 10-min at 4ºC, whichseparates out the homogenate into lower phenol chloroform phase (red), an interphase

Isolation of total cellular RNA

require high fidelity,Taq DNA Polymerase from MBI Fermentas was used. However,for precise amplifications either Herculase Fusion or PfuDNA polymerasefrom Stratagene was used. Approximately, 10-20ng of plasmid or 100 to 200 ng ofchromosomal DNA was used as a template in a 50 μl reaction volume containing 200μM of each dNTP, 20 picomoleeach of forward and reverse primer and 1.5 units of DNA polymerase.In the case of colony PCR performed to examine multiple colonies for presence of the plasmid clones, E. coli cells from afreshly grown plate wereresuspended in 50 μl of sterile Milli-Q water to get a cell suspension (~109cells/ml)and 4 μl from this was usedas the source of DNA template. To verify various pMU575 clonesdescribed in this study, by colony PCR,the vector specific primer pairs JGJpMUF and JGJgalK were used. The expected amplicon for pMU575 alone is ~300-bp, while that carrying the cloned fragment would be >300-bp.For each PCR reaction, the samples were subjected to 30-cycles of amplification and the typical conditions were as follows (although there were slight alterations from one set of template/primerto another):The initial denaturation was carried out at 95°C for 4-min and the cycle conditionswere as given below:Annealing 45ºC to 50°C 1-minExtension 68°C (1-min/kb of DNA template to be amplified)Denaturation 95°C 1-minAfter 30 cycles of PCR, the final extension step was carried out again for 10-min at68°C

For amplification of short length (100-200-bp)DNA fragmentsor that do not

Polymerase chain reaction (PCR)

Molecular techniques

Gel-filtration chromatography was performed at room temperature on a BioLogic LP protein purification system (Biorad) with an in-house packed Sephadex G-100 column of size 1.5 X 43 cm; each protein sample was loaded in 0.8-ml volume, and the buffer used for chromatography was 20 mM Tris-Cl (pH 8) with 200 mM NaCl at a flow rate of 0.1 ml per min with 1.5-ml fractions being collected for analysis. Protein elution was detected by measurement of A295.The void volume, V0was determined using blue dextran (2X 106Daltons) and theelution parameter Kavfor each proteinwas calculated from elution volume Veand total bed volumeVtusing the equation:Kav= (Ve–V0)/(Vt–V0)Initially, acalibration curve was derived froma semilogarithmic plotof Kav of protein standardsalbumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen (25 kDa) and ribonuclease A (13 kDa) on the Y-axis against log10of their molecular masses on theX-axis. The Kavof the ArgPdproteins were calculated based on their elution volume and then the molecular masses were derived from the corresponding point on the calibration curve

Around 0.5 to 1 μg of DNA was regularly used for each restriction digestion. 2to 5units of restriction enzyme were used in the total reaction volume of 20 μl containing 2μl of the corresponding buffer supplied at 10 X concentration by the manufacturer. Thereaction was incubated for 2 hrs at the temperature recommended by the manufacturer.The DNA fragments were visualised by ethidium bromide staining after electrophoresison a 0.8 to 1% agarose gels. Commercially available DNA size markers were run alongwith the digestion samples to compare with and to estimate the sizes of the restrictionfragments

Restriction enzyme digestion and analysis

Thialysine or thiosine (S-Aminoethyl-L-cysteine)is a toxic analog of Lys. Strains were testedfor sensitivity/resistance to thialysine by streaking them on minimal A-glucose platessupplemented without and with100-200 μg/ml thialysine(Steffes et al., 1992)

Test for thialysine resistance

For testing ArgR+/–phenotype, the colonies werestreaked on minimal A-glucose plates containing uracil (40 μg/ml) and CAN(65 μg/ml). Uracil wasadded to the medium to sensitize an argR+strain to CAN. An argR+strain is inhibited at65 μg/ml CANon a uracil-containing plate, whereas on a plate without uracil, argR+would grow even at 700-800 μg/ml CAN. Uracil represses the carAB transcription, whichencodes the carbamoyl phosphate synthase enzyme (CarAB). This results in reducedamounts of carbamoyl phosphate, which is the common intermediate between pyrimidineand Arg biosynthetic pathways. Reduced carbamoyl phosphate levels would result indecreased flux through the Arg biosynthetic pathways. This in turn would result indecrease in Arg pools inside the cell. An argR mutant would be derepressed for the Argbiosynthetic pathway and is resistant even to 300 μg/ml CANin a uracil-containing plate

Test for ArgR+/–phenotype

Test for canavanine (CAN) sensitivity

CAN is a toxic analog of Arg and is an inhibitor of bacterial growth. Strains were tested for sensitivity/resistance to CAN by streaking them on minimal A-glucose platessupplemented withoutand with40 μg/ml CAN(or other concentrations as indicated) and 40 μg/ml uracil

The colonies to be tested were streaked on the surface of minimal A-glucose plates containing either 0.4-0.7 M NaCl with 1 mM glycine betaine, and incubated at 37oC. NaCl-tolerant strains grew toform single colonies in 36-60 hrs whereas NaCl-sensitive ones did not. As controls, MC4100 (WT) and other previously identified NaCl sensitive mutants were streakedfor comparison

NaCl-sensitivity testing

agar platesLac+colonies will appear dark pink colonies whereas Lac–will remain colourless

A. lacphenotype

Scoring for phenotypes

LB agarLB medium 1000 mlBacto-agar 15 gmZ broth (for P1 transduction)LB medium100 mlCaCl2(0.5 M) 0.5 mlZ agar (for P1 transduction)Z broth 100 mlBacto-agar0.75 gm

Amino acids when required, were added to a final concentration of 40 μg/ml. Whengrowth on other carbon sources was to be tested, glucose was substituted withappropriate sugar at 0.2%.Glucose-minimal A medium, pH 7.4This medium was same as Glucose-minimal A medium described above except for the difference in K2HPO4and KH2PO4which were as mentioned below:K2HPO414.0 gmKH2PO42.7 gmGlucose-minimal A medium, pH 5.8This medium was same as Glucose-minimal A medium described above except for the difference in K2HPO4andKH2PO4which wereas mentioned below:K2HPO41.5 gmKH2PO412.4 gmGlucose /Glycerol-minimal A 19 (18 or 17) amino acidmediumThis medium is essentially the same as glucose/glycerol-minimal A medium described above except that all 19 or 18 or 17 otherthan either Lys or Lys and Arg or Lys and Arg and His amino acids were added after autoclaving at a final concentration of 40μg/ml from autoclaved 4 mg/ml stock solutions.Minimal A agarIt contains 1.5% bacto-agar (Difco) in minimal A medium. The plates were pouredafter mixing double strength minimal A with 3% agarthat had been autoclaved separately.LB mediumTryptone 10.0 gmYeast Extract5.0 gmNaCl 10.0 gmWaterto1000 mlpH adjusted to 7.0 to 7.2 with 1 N NaOH

All media and buffers were sterilised by autoclaving at 121ºC for 15 mins. Mediaand buffers used in this study are given below:Glucose /Glycerol-minimal A mediumK2HPO410.5 gmKH2PO44.5 gm(NH4)2SO41.0 gmSodium citrate, 2H200.5 gmWater to 1000mlAfter autoclaving the following solutions were addedMgSO4(1M) 1 mlGlucose (20%) 10 mlOr Glycerol (80%)5 mlVitamin B1 (1%) 0.1 ml

Media

Log-phase yeastcells were collected, washed and suspendedin 10 mM Tris-HCl (pH 7.5) containing 50 mg/ml zymolyase-20T. Cell suspension was incubated at room temperature and absorbance was monitored at 600 nm every10mininterval. Initial absorbance of the cultures at 0 minwas normalized to 100%and the graph was plottedas%decrease in the absorbance with respect to time

Zymolyasedigestion assay

OD600of 0.5and transferred to a 1.5 ml microcentrifuge tube. Probe loading was carried out by adding freshly-prepared CFDA-SE solution (0.01 M stock in DMSO) tocell suspension to a final concentration of 160 μM. Cell suspension was mixed on vortex mixerfor 10 secand incubated at 37 ̊C for 1 hwith shaking at 300 rpmon thermo mixer.Cells were harvested, washed twice with 1 ml 50 mM CP buffer to remove unloaded probe,resuspendedin 250 μl CP buffer andwereincubated at 30 ̊C for 30 minwith shaking to recover from the stress induced during probe loading. Afterincubation,fluorescent intensitywasdetermined with spectrofluorophotometer (Varioskan flash-3001, Thermo Scientific) by excitation at 430 nm (pH-independent) and 490 nm (pH-dependent) with emission at 525 nm. Background fluorescence of the probe was removed by subtracting the fluorescence intensity of the probe in CP buffer from the fluorescence intensity of the probe-loaded cells

Intracellular pH(pHi)in yeast cells was determinedusing fluorescent 5,(6)-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE; Molecular Probes) asdescribed previously (Bracey et al.1998). For pHiprobe estimation,YNB medium-grown log-phase cells were inoculatedin YNB, YNB-pH 2.0 or YNB medium supplemented with acetic acid and incubated at 30 ̊C for different time points.Log-phase C. glabratacells were harvested and washed twice with 50 mM citric-phosphate (CP) buffer (pH 4.0). Washed cells were resuspendedin 1ml 50 mM CP buffer to an

Measurementof intracellular pH (pHi)

stranded DNA. Final reaction volume was adjusted to 20 μl with DEPC-treated waterandamplificationreaction was carried out usingthese parameters: initial denaturation at 95 ̊C for 5 min followed by 40 cycles of denaturationat 95 ̊C for 30 sec, annealing at 55 ̊C-57 ̊C for 30 sec, elongation at 72 ̊C for 40 sec and final extension at 72 ̊C for 10 min. Transcript levelswerequantified with an end-point value known as Ct (cycle threshold). The Ctdefines the number of PCR cycles required forthe fluorescent signal of SYBR green dye to cross more than the background level. The Ctvalue isinversely proportional to the amount of nucleic acid product. Ctvalues were obtained during exponential phase of amplification and used forcalculation of relative-fold change in gene expression after normalization to Ctvalues ofeither housekeeping gene ACT1 (gene encoding actin)orTDH3 (gene encoding Gapdh)with the help of the following formula. Fold change in expression = 2-∆∆Ct∆∆Ct= ∆Cttreated -∆Ctuntreated∆Cttreated = Ctvalue forgene of interest under test/treatedcondition -Ctvalue forinternal controlgene(ACT1/TDH3) under test/treatedcondition∆Ctuntreated = Ctvalue forgene of interest under untreatedcondition -Ctvalue forinternal control (ACT1/TDH3)gene under untreatedcondition

Todeterminethe expression level of a specific gene, quantitative real-time polymerase chain reaction (qRT-PCR/qPCR)was performed oncDNA usinggene specific primers. Primers for qPCR weredesigned in such a way so as to get amplification products in a size range of 150 to 300 bp. Optimalprimer and cDNA concentrationswere standardized and qPCR was performed in ABI Prism 7000/7500 Real time PCR Machine (Applied Biosystems). Briefly, 0.4 μl cDNA was mixed with 0.1 to 0.2 picomolesof gene specific forward and reverse primers and 10 μl 2X MESA GREEN qPCR™Mastermix Plus containing SYBR green dye (Eurogentec) in awell of a96-well PCR plate (Axygen). SYBR green is a dye that specifically binds to double

Quantitative real-time polymerase chain reaction (qRT-PCR)

C. glabratastrains were grown overnighteither in YPDor YNBliquid mediumat 30 ̊C with shaking at 200 rpm. Cells were harvested and suspended in 1X PBS to a final OD600of 1.0.Five 10-fold serial dilutions of cell suspension wereprepared in PBS and3-4μlwasspotted on YPD/YNBplates containing various test compoundsusing a multi-channel pipette.Plates were incubated at 30 ̊C and growth profileswererecorded after2-4days

Serial dilution spot assay

10mM EDTA0.1% SDS 1 M ureaToluidine blue staining solution:0.05% Toluidine blue20% Methanol2% GlycerolSolution was prepared in H2O.Destaining solution for polyphosphate gels:20% Methanol2% GlycerolSolution was prepared in H2O.Spheroplast buffer:50 mM Potassium phosphate (pH 7.5)0.6M Sorbitol0.2 X YPD mediumPS(PIPES-Sorbitol)buffer:10 mM PIPES-KOH (pH 6.8)200mM Sorbitol1 X protease inhibitor cocktail (Roche Cat # 04693159001)**To be added fresh before use

Citric-Phosphate buffer:0.5 M citric acid0.5 M dibasic sodium phosphatepH was adjusted to 5.0 with phosphoric acid and filter-sterilized.MES/TEA buffer:1 mM MES(2-(N-morpholino)ethanesulfonic acid)pH was adjusted to pH 5.0 with TEA(triethanolamine).Plasma membrane suspension buffer:50 mM Tris-HCl(pH 7.5)0.1mM EDTA0.1 mM Dithiothreitol 20% GlycerolPolyphosphate extraction buffer:50 mM HEPES (pH 7.2)

Genomic DNAisolation buffersBuffer A:50 mM Tris-HCl10mM EDTA150 mM NaCl 1% Triton-X 1% SDSBuffer B:50 mM Tris-HCl (pH 7.5)10 mM EDTA1.1 M Sorbitol50 mM β-mercaptoethanol(To be added just before use

Other buffers

15% Acetic acidTris-Borate Saline(TBS):25 mM Tris150 mM NaClpH was adjusted to 7.4withHCl.This was prepared as 10 X stock solution and used at 1 X concentration.Blocking and wash buffers(PBS-T and TBS-T):5% Fat-free milk 0.1% Tween-20 Volume was made to 100 ml either with 1 X PBS(PBS-T)or 1 X TBS(TBS-T)

0.02% Bromophenol blue 2% DTT This was prepared as a 4 X stock solution and used at a 1 X concentration.SDS-PAGE running buffer:0.25 M Tris-HCl (pH 8.0) 1.92 M Glycine 1% SDS This was preparedas a 10 X stock solution and used at a 1 X concentration.Coomassie brilliant blue (CBB) staining solution:50% Methanol10% Acetic acid0.1% Coomassie brilliant blue-R250Western blotTransfer buffer:0.25 M Tris-HCl (pH 8.0) 1.92 M Glycine 1% SDS Thiswas preparedas a 10 X stock solution and used ata 1 X concentration.1X Transfer buffer (1litre):200 ml of methanol 100 ml of 10 X transfer buffer 700ml of waterPonceau 3S staining solution:0.25% Ponceau 3S40% Methanol

SDS-PAGE30% Acrylamidesolution29 g Acrylamide1 g Bis-acrylamideDissolved in 100 ml H2O.10% Sodium Dodecyl Sulfate (SDS):10 g SDS in 100 mlH2OResolving gel mix (12%) (15 ml): 4.89 ml H2O6 ml 30% acrylamide:bisacrylamide (29:1) mix3.8 ml 1.5 M Tris-HCl (pH 8.8) 150 μl 10% SDS 150 μl 10% APS 10 μl TEMEDStacking gel mix (3 ml):1.689 ml H2O500 μl 30% acrylamide:bisacrylamide (29:1) mix380 μl 1 M Tris-HCl (pH 6.8) 30 μl 10% SDS 30 μl 10% APS 10 μl TEMEDSDS loading buffer:130 mM Tris-HCl (pH 8.0) 20% (v/v)Glycerol 4.6% (w/v) SDS

Whole cell lysis buffer(Homogenizing buffer):50 mM Tris-HCl(pH 7.5)2 mM EDTA10 mM sodium fluoride*1 mM sodium orthovanadate*1 X protease inhibitor cocktail (Roche Cat # 04693159001)**To be added fresh before use

Protein isolation and SDS-PAGE(sodium dodecyl sulfate polyacrylamide gel electrophoresis)

Protein isolation and SDS-PAGE(sodium dodecyl sulfate polyacrylamide gel electrophoresis)

Buffer C:100 mM Tris-HCl (pH 7.5)10 mM EDTA10% SDSRNA isolation bufferAE buffer: 3 M Sodium acetate0.5 M EDTA(pH 8.0)Phenol:Chloroform:Isoamyl Alcohol (25:24:1)solution:25 volume of Phenol24 volume of Chloroform1 volume of Isoamyl alcholDNA sampleloading buffer:0.25% Bromophenol blue0.25% Xylene cyanol15% Ficoll

Genomic DNA and RNA isolation buffers

15 mM CaCl2.2H2O 250 mM KCl 55 mM MnCl2.4H2O pH was adjusted to 6.7 with 1 N KOH. MnCl2needsto beaddedseparately,drop by drop with stirring, tothe buffer. PIPES goes into solutionwhenpH is greater than 6.7. The solution, after pH adjustment to 6.7 was filter-sterilized and stored at -20ºC.Reagents for yeast transformation:1 M Lithium acetate (LiOAc)50% Polyethylene glycol10 mg/ml Carrier DNADimethylsulfoxide (DMSO)

INOUE transformation buffer:For bacterial DH5α ultra-competent cells preparation10 mM PIPES (free acid)

Transformation-related solutions

10 mM Tris-HCl (pH 8.0)1 mM EDTA Tris-Acetic acid EDTA (TAE) buffer:40 mM Tris base 0.5 M EDTApH was adjusted to 8.5 with glacial acetic acid.This was prepared as a 50 X stock solution and used at a 1 X concentration. Tris-Borate EDTA (TBE) buffer:90 mM Tris-borate 2 mM EDTA (pH 8.0) pH was adjusted to 8.3withHCl.This was prepared as a 10 X stock solution and used at a 1 X concentration.Both TAE and TBE were used asstandard gel electrophoresis buffers.HEPES buffer:This was used to prepare YNB medium of different pH.1M HEPESpH was adjusted to 7.5withNaOH.Bufferwas filter-sterilized and stored in an amber-coloured bottle. Citrate buffer(0.1M, pH 5.5):4.7 volume of 0.1 M Citric acid 15.4 volume of 0.1 M Sodium citrate

Phosphate-Buffered Saline (PBS):137 mM NaCl 2.7 mM KCl10 mM Na2HPO42 mM KH2PO4pH was adjusted to 7.3.This was prepared as a 10 X stock solution andused at a 1 X concentration.Tris-HCl buffer:0.5 M TrizmaBase pH was adjusted to7.6 using concentrated HCl.This was prepared as a 10 X stock solution andused at a 1 X concentration.Tris-EDTA (TE)buffer:

Common buffers

Buffersand solutions

activated TLC silicagel-60plate and transferred to theTLC chamber. After the solvent had migratedupwards (1.5 cm fromthetop), TLC plate was removed, air dried behind perspex shield, wrapped with cling plastic wrap and was exposed to phophorimager screenfor 2 h. Phosphorimager screen was scanned usingaFugi-FLA 9000 scanner

To resolvephospholipids,a TLC chamber was prepared by pouring50 ml developing solution and sealing the chamber with aluminium foil so that developing solution can generate vapor. TLC silicagel-60plate(Merck)was incubated at 80ºCfor 4 h for activation. After 30 min of TLC chamber preparation, phospholipidsextracted from C. glabratacells werespotted at thebottom (1.5 cm fromthelowerend) of the

Seperation of phospholipids by thin layer chromatography(TLC)

PI-3kinase reaction was set up ina total volume of50μlin a 1.5 ml microcentrifuge tube as described below.PI-3 kinase reaction buffer = 25 μlSpheroplast lysate = 20 μl (equivalent to 10 μg protein)Sonicated phosphatidyl inositol = 5 μlReaction mix was incubated at 25ºC for 20 min and enzyme reaction was stopped by adding 80μlHCL (1N) solution. To extract phospholipids, 160 μl chloroform:methanol (1:1) was added to the reaction mix withcontinuous mixing. Organic phase containing phopholipidswas separated fromaqueous phase by centrifugation at 7,500g for 4 min at 4ºC and transferred to a new vial. Using vacuum evaporator apparatus, solvent was evaporated and phospholipidsweredissolved in 10 μl chloroform

PI-3 kinase reaction set up and phopsholipid extraction

10 mg phosphatidylinositol-sodium salt(from Glycine max)was dissolved in 2 ml chloroform to prepare a 5 mg/ml stock solution. This solution was prepared in a small glass vial aschloroformis known to reactwith polypropylene. Small aliquots of stock solution were madeand stored at -20ºC till further use. To avoid spillage due to vapor pressure, vials containing phosphatidylinositol-sodium salt solutionwereopened very carefully.To prepare sonicated phosphatidylinositolfor one PI-3 kinase reaction, 2 μlof the stock phosphatidylinositolsolution (10 μg) wastransferredtoanew1.5 ml microcentrifuge tube. Using vacuum evaporator apparatus, chloroformwas evaporated from the solution and phosphatidylinositol-sodium saltwas resuspended in 5 μl sonication buffer.For sonication, a total of 20 pulses, each of 30 sec with30 sec resting time weregiven on ice

Preparation and sonication of phosphatidylinositol-sodium salt solution

A single colonyof desired C. glabratastrainwas inoculated in YPD-liquid mediumand grown for 14-16 h. 50 μl overnight culture was inoculated inYPD-liquid mediumfor 4 h. Log-phase-grownyeast cells were harvested,washedwith PBSandwereinoculated atinitial OD600of 2 and 4,into YNB-dextrose and YNB-sodium acetate liquid medium,respectively.After 4 hincubation,yeast cells were harvested by centrifugation at 2,500g for 5 minand treated with 1.2 M zymolyasefor 1 hto obtain spheroplasts.Post zymolyase treatment, spheroplasts were resuspended in 100 μl resuspension bufferandanequal amount of 0.25 mm glass beadswasadded to lyse the spheroplasts. Using bead beater apparatus, spheroplasts were lysed and protein concentration in spheroplast lysateswas determined usingbicinchoninic acid assay (BCA) method and samples were stored at -20ºC till further use

Preparation of cell lysate

In vitroPI-3 kinase reactions wereset up to measure PI-3P synthesized as described earlier(Whitman et al., 1988)

Phosphatidyl inositol-3 kinase (PI-3 kinase assay)

Colony blot assay was performed to analyse secretion of carboxypeptidase Y(CPY)as described previously (Roberts et al., 1991). Single colony of a C. glabratastrain was inoculated in YPD medium andculture was grown till stationary phase. 0.1 OD600equivalent cellsfrom this culture were spotted on CAA medium,overlaidwith a nitrocellulose membrane and plate was incubated at30 ̊C for 18-20 h.Afterincubation, nitrocellulose membranewas washed with water to remove cells and membrane-bound CPYwas detected by immunoblotting with polyclonal anti-CPY antibody at a dilution of 1:10,000

Colonyblot assay

was washed three times with PBS to remove non-adherantC. glabratacellsand Lec-2 cells were lysed in 5% SDS. Lysates were transferred totubes containing scintillation fluidand radioactive counts obtained were considered as ‘output values’. Percentage adherence wasdetermined using following formula

Adherence of C. glabratacells toLec-2 epithelial cells wasmeasured as described previously(Cormack et al., 1999).Lec2cells were seeded ina 24-well tissue culture plate at a seeding density of 5X105cells per well and allowed to adhere for 12 h. After 12 h,medium supernatant was discarded by inverting the plate in a reservoir and cells were washed thrice with PBS. Lec2 cells were fixed in 3.7% para-formaldehyde for 15 minfollowed by 2 PBS washes. PBS containing antibiotics, penicillin and streptomycin,was added toeach well of the 24-well plate and Lec-2 cellswere stored at 4°C.For adherence measurement,strains were taken out either on YPD or CAA mediumandgrown at 30°C for 2 days. Single colony of a C. glabratastrain wasinoculated in 10 ml CAA medium ina 100 ml culture flaskand allowed to grow at 30°C for 16-20 h. 100 μlyeast culture wasreinoculated in fresh 5 ml CAA liquid medium in a 15 ml polypropylene tube. 200 μCi of S35(Met:Cys-65:25) INVIVO PROTWIN labelmix(JONAKI, India) was added to thetube and cultures were grown at 30°C for 16-20 h for radiolabeling of C. glabratacells. C. glabratacells from 1 ml culture were harvested and washed threetimes with PBS to remove residual S35(Met:Cys-65:25) labeling mix from medium supernatant. Next,cells were resuspended in 1 ml PBS. OD600was measured and cell suspensions of 0.5 OD600were prepared. PBS was aspirated out of the wells of 24-well plate containing fixed Lec-2 cells. 200 μl of S35(Met:Cys-65:25)-labeled C. glabratacell suspensions were added to each well. To determine the total amount of radioactivity present in labeled C. glabratacell suspension, 200 μl of S35(Met:Cys-65:25)-labeledC. glabratacell suspensions were transferred to a scintillation vial containing scintillation fluid. Radioactive counts present in this fraction were considered as ‘input values’. For measurement of yeast adherence to Lec-2 cells, plates were centrifuged at 1,000g for 5 min and incubated for 30 min at room temperature. Following incubation, each wel

Adherence assay

20 mg protein samples, isolated from RPMI-grown and macrophage-internalized yeast, were usedto measure KDAC activityusing HDAC Fluorimetric Assay/Drug Discovery Kit (EnzoLifeScience) as per manufacturer’s instructions

Lysine deacetylase (KDAC) activity measurement

For protein extraction, yeast cells were suspended in 50-100μl protein extraction buffer containing 320 mM (NH4)2SO4, 200 mM Tris-Cl (pH 8), 20 mM EDTA (pH 8), 10 mM EGTA (pH 8), 5 mM MgCl2, 1 mMDTT, 10% glycerol and protease inhibitorsand disrupted using glass beads.Cell lysate was centrifuged at 7,500g and4oC for 15 min. 30 μg of total protein was resolved on a 15% SDS-PAGE gelat 32 mA till the dye front reachedthe bottom. Resolved proteins were transferred to Hybond-P membrane at 350 mA for 1.5 h in the cold room.Transfer of the proteins was visually confirmed by examining marker’s lane and membranes wereincubated in a small box for 2 h in 5% fat free milkprepared in 1X TBST for blocking. Blocking solutions were discarded and primary antibody, appropriately diluted in 5% fat free milkprepared in 1X TBST,was added to the box containing membrane. After overnight incubation in primary antibody, membranes were washed thrice with 1X TBST for 10 min. Membranes wereincubated for 2 h inappropriate secondary antibodydiluted in 5% fat free milkprepared in 1X TBST. Blots were either developedby chemiluminescence based ECL-Plus western detection system orChemidocTMgel imagingsystem. CgGapdhwas used as a loading control. To exclude the possibility of any contribution of THP-1 proteins tocell extracts prepared frommacrophage-internalized yeast, two control experiments wereperformed. First, we probedthe blots with antibodies specific for mammalian tubulin and actin.As expected, we neitherdetectedanysignal for mammalian actin nor formammalian tubulin. In the second control experiment, we treated macrophage lysates with proteinase-K prior to the yeast pellet disruptionand probed yeast lysates for different histone modifications.This proteinase-K treatmentdid not alter the epigenetic signature of C. glabratacells.Together, these data indicate that yeast protein samples were devoid ofany mammalian protein contamination

Protein extraction and immunoblotting

homogenizedin 1 ml PBS and fungal burden was assessed by plating appropriate dilutions of tissue homogenate on YPD plates containing penicillin and streptomycinantibiotics (100units/mlpenicillin and 100μg/mlstreptomycin). All mice experiments were repeated twice with a set of 7-8 mice per strain in each experiment

Experiments involving mice were conducted at VIMTA Labs, Hyderabad.100 l YPD-grown C.glabratacellsuspension(4 X 107cells)was injected into female BALB/c mice (6-8 weeks old) through tail vein. Seven dayspost infection, mice weresacrificedand kidneys, liver,spleenand brainwere harvested. Organs were

Mouse infection assay

Experiments involving mice were conducted at VIMTA Labs Limited, Hyderabad in strict accordance withguidelines of The Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India. The protocol was approved by the Institutional Animal Ethics Committee (IAEC) of the Vimta Labs Ltd. (IAEC protocol approval number: PCD/OS/05). Procedures used in this protocol were designed to minimizeanimalsuffering

Ethics statement

Othermethods

After14-16 hincubation, hybridization buffer was decanted to a radioactive liquid waste container.Membraneswere washedtwice with 2X SSC (saline-sodium citrate) containing 0.1% SDS for 15 min at 55°C followed by two washes with 1X SSC containing 0.1% SDS for 15 min at room temperature. Post washes,membranes were rinsed with 1XSSC buffer at room temperature and exposed to phosphorimager screen for 2-3 h

Post-hybridization washes

Identified mutants were phenotypically characterized in 96-well plate format. Mutant cultures were grown in YPD medium for overnight, diluted 150-fold in PBS and 5 μl of cell suspension was spotted on different plates with a 96-pin replicator. Growth was recorded after 1-2 daysof incubation at 30°C

Phenotypic profiling

THP-1 monocytes were treated with phorbol myrsitylacetate (PMA) to differentiate them to macrophages(Tsuchiya et al., 1982). For PMA treatment, THP-1 cells grown upto 70-80% confluencewere harvested from the culture dishes at 1,000 rpm for 3 min. Harvested THP-1 cells were resuspended in 5-10 ml fresh and prewarmed complete RPMI medium. 100μlof thiscell suspensionwasappropriatelydilutedinPBS and numberof viable cells was determined by trypan blue stainingusing hemocytometer. Cell suspension was diluted with prewarmed RPMI medium to a final density of 106cells/ml. PMA was added to this THP-1 cell suspension to a final concentration of 16 nM and mixedwell.PMA-treated THP-1 cellswere seeded either in 24-well cell culture plate or culture dishes and transferred to the incubator set at 37°C and 5%CO2.After 12 hincubation, medium was replaced with fresh prewarmed medium and cells wereallowed to recover for 12 h

Treatmentof THP-1 monocytic cells with phorbol myrsityl acetate

Spheroplast resuspension buffer0.1M KCl15 mM HEPES (pH 7.5)3 mM Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid(EGTA)10% GlycerolPhosphatidylinositol sonication buffer10 mM HEPES (pH 7.5)1 mM EGTA PI3-kinase reaction buffer40 mM HEPES (pH 7.5)20 mM MgCl280 μM ATP5 μCi γ-P32ATPDeveloping solution for thin layer chromatography(120.2 ml)Chloroform –60 mlMethanol –47 mlAmmonia –4.4 mlWater –8.8 ml

Reagents for PI3-kinase assay

Table 2.4: List of the oligonucleotides used to confirm deletion of C. glabrataORFs

Table 2.3: List of the oligonucleotides used in the study

Table2.2: List of the antibodies used in the study

Table 2.1: List of strains and plasmidsused in the study

methanol, acetic acid, potassium dihydrogen orthrophosphate, dipotassium hydrogen phosphate, disodium hydrogen orthrophosphate, acetone and citric acid were purchased from Qualigen chemicals. Fluconazole was procured from Ranbaxy.Lysotracker-Red DND 99 and FM 4-64 were obtained from Molecular Probes. Hybond-N and Hybond-P membranes for nucleic acid and protein transfer, respectively, were purchased from Amersham Biosciences. SYBR-green kit for real-time PCR was procured from Eurogentech. Superscipt SS-III RT kit and Pfu polymerase were obtained from Invitrogen. Different restriction enzymes used for cloning and knock-out generation were purchased from New England Biolabs (NEB). High fidelity DNA Pfx polymerase waspurchased fromFinnzymes. Plasmid DNA purification, PCR purification, gel extraction and reaction clean up kits were procured from Qiagen.Medium components for C. glabrataand bacterial culture viz.,yeast extract, peptone, tryptone, cassamino acid hydrolysate, yeast nitrogen base, yeast nitrogen base without ammonium sulphate, yeast nitrogen base without ammonium sulphate and amino acids and yeast carbon basewere purchased from BD (Becton, Dickinson and Company, USA). Animal cell culture media RPMI-1640, DMEM and α-MEM were procured from Hyclone. Fetal bovine serum, glutamine and antibiotics for cell culture medium were obtained from Gibco-Invitrogen

Agarose, phenol, dimethyl sulphoxide (DMSO), sodium acetate, sodium chloride, sodium hydroxide, sodium carbonate, sodium bicarbonate, trizma base, sodium dodecyl sulphate (SDS), formamide, calcium chloride, ethylenediaminetetraacetic acid(EDTA), glycerol, polyethylene glycol, ficoll, diphenyleneiodinium (DPI), methyl methanesulphonate (MMS), camptothecin, hydroxyurea, ammonium persulphate, TEMED, acrylamide, bis-acrylamide, coomassie brilliant blue (CBB), chloroform, formaldehyde, glycine, lithium chloride, lithium acetate, menadione, isopropanol, phorbol myrsityl acetate (PMA), nuclease free water, wortmannin, bafilomycin-A, diethylpyrocarbonate (DEPC), orthrophenylenediamine (OPD), tween-20, acid washed glass beads, trypan blue, Taq DNA Polymease, trisodium citrate dihydrate and uracil were purchased from Sigma Chemicals. β-mercaptoethanol was obtained from GE Biosciences.Protease inhibitor tablets were procured from Roche. Dextrose, sucrose, agar, ammonium sulphate, potassium chloride, caffeine, magnesium chloride and sorbitol were obtained from Himedia.Hydrogen peroxide, hydrochloric acid, sulphuric acid,

Chemicals, kits and culture medium components

The immunoblots were quantified by densitometry software ImageJ 1.17 developed by Wayne Rasband, NIH Bethesda, MD (http://rsb.info.nih.gov/nih-image).All experiments were done at least in triplicates and results were expressed as mean ±s.e.m. A two tailedStudent’s t-test was done in Graph pad to arrive at p values and differences were considered statistically significant when p-value was less than 0.05 (*p≤ 0.05), highly significant(**p≤ 0.01)andextremely significant (***p≤ 0.001)

Quantification of blots and statistical analysis

A total of 100-200ng of DNA was used in each ligation reaction. Vector to insert ratio of 1:3 to 1:5was maintained. The reaction volume was generally maintained at 10μl containing 1μl of 10X ligation buffer (provided by the manufacturer) and 0.05 Weissunit of T4-DNA ligase. The reaction was carried outat 16ºC for 14-to 16-hrs or at room temperature for 4hours

Ligation of DNA

Agarose gels were preparedby boiling appropriate amount of agarose in TAEbuffer. After dissolution, it was cooled and then poured in a casting tray containing a comb for desired number of wells. The gel was allowed to solidify and then shifted to horizontal electrophoresis tank containing TAE buffer. The DNA samples were mixed with appropriate volumes of 6X DNA loading dye, loaded on the gel andelectrophoresedat appropriate voltage and current conditions (generally 80 V,400 mA). The gel was stained in ethidium bromide solution(1 μg/ml)for 15-min at room temperature and visualisedby fluorescence under UV-light in a UV-transilluminator

Agarose Gel Electrophoresis

For preparation ofcellular homogenate from adherent cell culture, the medium was first removed and cells were washed with ice cold 1X PBS. The cells were then scraped in 1X PBS and pellet down by gentle centrifugation (4000 rpm for 2 minutes) at 40C. Cell lysis buffer was then added to the cell pellets and lysis was allowed for 30 minutes on a rotor at 4⁰C. Post lysis, cellswere centrifuged at 13000 rpm for 10min at 4°C. The pellet was discarded and supernatantwascollectedas cell homogenate

Extraction of total cellular protein

Binding Buffer (10X)

Blocking buffer: 2% BSA

Permeabilization buffer: 0.2% Triton X100

Fixative : 4% Formaldehyde

For Immunofluorescence

Running Buffer

Ethylene Glycol Tetraacetic acid (EGTA), pH 7.0ComponentsFinal concentrationFor 50 mlEGTA0.1M1.902gH2Oq.sThe pH is adjusted to 7.0 using 10M NaOH

Wound healing assay(Liang et al., 2007) was employed to study the difference in migration rates ofprofilin-stable compared to parentalcells. Briefly, cells were cultured upto 90-95% confluent monolayer and a scratch was created through the cell monolayer with sterile needle. Cell debris was then removed by washing with PBS before adding the media. Images of the open gap created by this “wound” were then captured at three random locations immediately (0 h) and then at the same locations after regular interval using phase contrast microscopy untilthey are closed by migrating cells. Captured images were then used to quantify wound closure by the percentage change in the wound area per unit time and averaged for three locations for each experimental condition. During the course of the experiment, cells were maintained in 0.1% FBS containing DMEM media to ensure that wound closurewas due to the migration of cells rather thandivisonof cells

Wound healing assay

Extraction buffer

MTT reagent

For Cytotoxicity assays

6XEMSA sample loading dye

5X EMSA buffer

Native EMSA PAGE

10XBinding buffer

For Electrophoretic Mobility Shift Assay (EMSA)

For preparation of Ultra competent cells

Inoue buffer

6X DNA loading dye

Agarose gel

TAE

For DNA electrophoresis

Nuclear lysis buffer (without protease inhibitors

Cytoplasmic extraction buffer (without protease inhibitors)

For Cell fractionation

Blocking buffer: 2% BSA

Permeabilisation buffer: 0.2% Triton X100

4% Formaldehyde fixative

For Immunofluorescence(IF)

Stripping buffer

Blocking buffer

TBS-T

Transfer buffer

(f) Running buffer

(e) Stacking polyacrylamide gel

(d) Resolvingpolyacrylamide gel

(c) 6X Protein loading buffer (Lammeli buffer)

(b) Celllysis buffer B(For IB)

Cell lysis bufferA(For IP)

II. For Immunoprecipitation(IP)and Immunoblotting(IB)

Table 2.1: Commonly used buffers and solutionsI. General buffers(a)Phosphate Buffered Saline (PBS)

(b) Tris Buffered Saline (TBS)

For DNA isolation and purification, various kitssuch as Miniand midi-prep plasmid isolation, Gel extraction, PCR purification,etc., wereprocured fromQiagen(Hilden, Germany) or HiMedia(India). For RNA extraction, TRIzol wasobtained from Gibco BRL(Grand Island, NY). cDNA was made from RNA byeither Reverse transcriptase (SuperScript III, Invitrogen) or One step Access RT-PCR kit (Promega, Madison, WI). Reagents for PCR such as PCR 10X buffer, dNTPs, MgCl2, Taqpolymerase or AccuTaq were obtained from Fermentas or Sigma Aldrich. Recombination enzymes such as Restriction Endonucleases and DNA ligaseused for recombinant DNA experiments (Bam-H1, Hind-III, Xho-I, Eco-RI, Not-I, and Sal-I) were obtained from New England Biolabs(Ipswich, MA, USA). Oligonucleotidesusedfor various Gel shift assays viz.AP-1, NF-κB, p53 and Sp-1 were commercially synthesizedfrom XCelris(Ahmedabad, India).For protein extraction, protease inhibitors such as aprotinin, leupeptin, PMSF, NaF, NaVO4,etc. were obtained from Sigma Aldrich.Bradford reagent for estimation of protein concentration wasobtained from Bio-Rad(Rockford Illinois, USA).ForImmunoblotting, PVDF membrane, X-ray films andchemi-luminiscentdetection reagent (ECL prime) were obtained from GE Healthcare(Little Chalfont, UK). For Immunofluorescence, vectashield-mountingmedium with DAPIand Propidium Iodide (PI)were obtained from Molecular Probes, Invitrogen.For detection of cytotoxicity, MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) dye, SDS and DMF (Dimethylformamide) wereobtained from SigmaAldrich. Live and dead cell assay kit was obtained from Molecular Probes.Various chemicals required for preparation of regular buffers and solutionsviz. Tris, Glycine, SDS, Sodium Chloride, Potassium Chloride, HEPES, Disodium Phosphate, Nonidet P-40, Tween 20, TritonX100, Formaldehyde, Glycerol, Agarose, Acrylamide,Bis-acrylamide,APS, TEMED, BSA,etc. were obtained from SigmaAldrich.The procedure of preparation of buffers and reagents usedin the present studied are described below:

Reagents and Buffers

Analysis of sensitivity to translation inhibitors was conducted in theDDY1810 S. cerevisiaestrain background, whichdoes not contain the kanrselection marker. Sensitivity to 6-azauracil (6AU) was monitored in the DDY1810, BY4741 or NOY222 strain backgrounds (Table 2.1). As uracil is a competitive inhibitor of 6AU, the plasmid p416GPD, carrying the URA3gene (Mumberget al., 1995)was introduced into BY4741-derived strainswhereas DDY1810 derived yeast strains were supported by the pYesGex plasmid carrying the URA3gene.Yeast strains were grown in YPD or SC-Ura medium,for 14-16 h at 30°C under continuous shaking at 200 rpm. Cultures were diluted to 0.25OD600, followed by 5 fold serial dilutions, and 3μL of each dilution was spotted on a YPD-agar plate containing the translation inhibitors G418 (8 μg/mL), paromomycin (100μg/mL or 200μg/mL), or hygromycin B (8 μg/mL), or an SC-Ura agar plate, containing 6AU 50 μg/mL or100 μg/mL and growth was monitored at 30°C or 37°C for 2-3 days. To perform an analysis of 6AU sensitivity with yeast carrying pYesGex6p2 plasmid, cells were grown overnight in SC-Uramedium and the serial dilutions were plated on SC-Ura medium containing 6AU, with galactose instead of glucose to express proteins under the GAL4promoter

Drug sensitivity analysis

Volume was adjusted with water to 1 L and solution was sterilized by autoclaving.Pre-hybridization/hybridization buffer (Modified Church and Gilbert buffer)0.5 M phosphate buffer (134g of Na2HPO4.7H2O,4 mL of 85%H3PO4), pH7.27% (w/v) SDS10 mM EDTA Volume was adjusted to 1 L with DEPC treated sterile water. Buffer was aliquoted into 50 mL RNase free conical tubes (Corning) and stored in -20oC.Post hybridization wash buffersWash buffer 1 2X SSC 0.1% SDS Wash buffer 2 1X SSC 0.1% SDSWash buffer 3 0.5X SSC0.1% SDSBuffers were prepared with sterile DEPC treated water

TMN buffer10mM Tris-HCl, pH 7.45 mM MgCl2100 mM NaCl Permeabilization buffer950 μLof coldwater50 μLof 10% (wt/vol)sodium N-lauroyl sarcosineTranscription assay buffer(100 μL)50mM Tris-HCl, pH 7.4100mMKCl5mM MgCl21mM MnCl22 mM dithiothreitol 0.5mM ATP 0.25 mM GTP0.25mM CTP10mM phosphocreatine2.4 units creatine phosphokinase100μCi [α-32P] UTP (3,000Ci/mmol)Alkaline denaturing solution for DNA for membrane preparation0.5 M NaCl 0.25 M NaOH Volume was adjusted to 20 mLwith sterile water. Saline Sodium Citrate (SSC) buffer (20X) 3.0 M Sodium chloride 0.3 M Sodium citrate

Buffers fortranscription run on analysis

Taq polymerase was from ThermoScientific. Plasmid DNA purification, PCR purification, gel extraction and reaction clean up kits were procured from Qiagen. Medium components for growth of S. cerevisiae,namely, YPD, yeast nitrogen base, and yeast nitrogen base without ammonium sulphate were purchasedfrom BD (Becton, Dickinson and Company, USA).Yeastsyntheticdropoutmediasupplementwithouturacil/histidinewereobtainedfromSigma-Aldrich

Agarose, phenol, dimethyl sulphoxide (DMSO), sodium acetate, sodium chloride, sodium carbonate, sodium bicarbonate, sodium dodecyl sulphate (SDS), formamide, calcium chloride, ethylenediaminetetraacetic acid (EDTA), glycerol, polyethylene glycol, ammonium persulphate, N,N,N′,N′-Tetramethylethylenediamine (TEMED), acrylamide,dithiothreitol (DTT),bis-acrylamide, chloroform, formaldehyde, lithium chloride, lithium acetate,isopropanol, nuclease free water, diethylpyrocarbonate (DEPC), Tween-20, acid washed glass beads, trisodium citrate dehydrate, β-mercaptoethanol, 0.4% trypan blue solution, yeast protease inhibitor cocktail, magnesium chloride, manganese chloride and phosphatase inhibitors were purchased from Sigma-Aldrich Chemicals. Agar, uracil, leucine, lysine, histidine, tryptophan, methionine, yeast extract, peptone, tryptone, and sorbitol were obtained from HiMedia. Dextrose, sucrose, potassium chloride, sodium hydroxide, hydrochloric acid, Tris and glycine were from Fisher Scientific. [14C]-labelled uracil was from Ogene Systems.γ[32P]ATP, [35S]Met/Cysin vivoprotein twin label mix, α[32P]UTP and Taq DNA polymease were from JONAKI/BRIT,Ultimaflow liquid scintillation fluid was obtained from Perkin-Elmer.Hybond-N+and Hybond-P membranes for nucleic acid and protein transferrespectively, and protein A beads were purchased from GE Life Science. NuPAGE gradient gels, MES running buffer and 4X LDS sample buffer were purchased from Invitrogen. Super Signal West pico chemiluminiscent substrate was from Thermo Scientific. Different restriction enzymes used for cloning and knock-out generation were purchased from New England Biolabs (NEB). High-fidelity Phusion

Chemicals, kits and culture medium components

1. The composition of different types of gels run during the course of this study is

Media

The LR reaction mixturewas incubated at room temperature for 1hr, followed by proteinase K treatment (2μL) at 37°C for 10 min. 5uL of the reaction mix was transformed into DH5α competent cells. Bacterial cells were spread on LB agar plates containing antibiotic ampicillin (50ug/ml). Plates were incubated at 37°C for overnight. The bacterial colonies were inoculated into 5mL LB broth containing 5uL ampicillin and incubated overnight in shaking incubator at 220 rpm. Next day, Plasmids were prepared using Plasmid miniprep kit (QIAprep miniprep). The destination plasmids obtained by LR reaction were given for plasmid-DNA sequencing to confirm thepositive clones. The positive clones were amplified through DH5α transformation and plasmid DNA maxiprep (Invitrogen). The expression plasmids generated through LR were used for studies in the mammalian and the bacterial cells

BP reaction mix was incubated at room temperature for 1hr, followed by proteinase Ktreatment (2μL) at 37°C for 10 min. 5uL of the reaction mix was transformed into DH5α competent cells (Transformation into DH5α competent cells; plasmid constructs added into DH5α competent cells and incubated on ice for 30 min. followed by heat shock at 42°C for 1 min. 800μL LB broth was added and transformed DH5α cells were incubated at 37°C in shaking incubator at220 rpm for 1hr.;after 1 hr. cells were centrifuged at 6000 rpm for 1 min.;the supernatant was discarded,and the pellet was resuspended in 100μL of LB broth). Bacterial cells were spread on LB agar plates containing antibiotic kanamycin (30ug/ml). Plates were incubatedat 37°C for overnight. The bacterial colonieswereinoculated into 5mL LB broth containing5uL kanamycinand incubated overnight in shaking incubator at 220 rpm. Next day, Plasmids were prepared using Plasmid miniprep kit(QIAprep miniprep). The donor plasmids generatedby BP reaction were given for plasmid-DNA sequencing to confirm the positive clones.The donor plasmids obtained by BP reaction were cloned into the Gateway destination vectors by LR reaction (Table 7).Table 7: LR reaction mixture

Gateway cloning is the highly efficient gene cloning technology. It comprises two primary steps of cloning; the BP reaction and the LR reaction. The PCR products of gene of interest were cloned into the Gateway donor vector by BP reaction(Table 6).Table 6: BP reaction mixture

Gateway cloning

SiRNAWWP2 siRNA described earlier [216]and prevalidated siRNAs for PPM1G (catalog numbers S102658684 and S102658691) were purchased from Qiagen. ShRNAWWP2 shRNA (shRNA1, 5=-CAGGAUGGGAGAUGAAAUAUU-3=;shRNA2, 5= ACAUGGAGAUACUGGGCAAUU-3=)WWP1 shRNA (shRNA1, 5=-ATTGCTTATGAACGCGGCT-3=; shRNA2, ACAACACACCTTCATCTCC-3=)Both WWP2 and WWP1 shRNA were purchased from Open Biosystem.2.1.5Cell linesHeLa cells, HEK293T, and BOSC23celllineswere used in the present study wherever indicated. All the cells werecultured and maintained in RPMI 1640 supplemented with 10% serum and 1% antibiotic (penicillin-streptomycin)at 37° C with 5%CO2.2.2 Buffers and mediaThe buffers and media used in the present study is mentioned in the table 3.Table 3: Buffers and media used in the study

SiRNAandshRNA

biosensor strain 8523/KLN55was inoculated in fresh medium, and grown with the ethyl acetate extract isolated from the test strain as described earlier. After 30 h of growth, cells were pelleted by centrifugation, washed once with sterile water and resuspended in sterile miliQ waterfor measuring the GFP fluorescence intensity at excitation and emission wavelength of 472 and 512 nm, respectively. 1 DSF unit is equivalent to increase in fluorescence by 1 arbitary unit in DSF biosensor strain

For DSF extraction, X. oryzaepv. oryzicolastrains were grown in PS media to an OD600 of 1.2 as described earlier. Supernatant was collected by pelleting down the cells at 7000 g for 10 min. Next, water-saturated ethyl acetate was added to the cell-free culture supernatant in a ratio of 2:1, and mixed properly for 5-10 min. The mixture was centrifuged at 5000 g to separate the DSF containing organic phase. The ethyl acetate layer (organic phase) was evaporated at 37°C, remaining residue was dissolved in methanol, and assayed for DSF by using Xccbiosensor strain 8523/KLN55 (Newman et al., 2004). Biosensor strain is a DSF minus strain comprised of DSF responsive endoglucanase promoter fused to promoterless gfpand expressed through plasmid (Peng::gfp). To check the DSF production by a particular strain, 0.2% inoc

Isolation and detection of DSF

Complementary-DNA synthesis was performed using reverse transcriptase enzyme (Invitrogen) and random hexamers (Qiagen). For this, 1 μg good quality RNA was treated with 1 μl (1 unit) DNase I (Invitrogen) for 20 min to remove DNA contamination. Next, Superscript III Reverse Transcriptase kit (Invitrogen) was used to synthesize cDNA according to the manufacturer’s instructions. cDNA synthesized was further confirmed by using it as a template for amplification in PCR. cDNA was stored at -20°C till further use

Synthesis of complementary DNA (cDNA)

A single colony of E.coliDH5α strain was inoculated in 5 ml LB medium and incubated at 37°C for overnight. 1% of overnight grown culture was inoculated in 500 mlfresh LB medium and incubated at 37°C for 2-3 h till the OD600 reached to 0.4-0.5. Culture was chilled on ice for 5 min followed by centrifugation at 3000 g for 15 min at 4°C. Harvested cells were washed gently with 200 ml ice-cold TFb-I buffer. Cells were collected by centrifugation at 3000 g for 5 min at 4°C and gently resuspended in 20 ml ice-cold TFb-II buffer. Bacterial cell suspension was kept on ice for 15 min and was aliquoted in 100 μl volumes in chilled sterile microcentrifuge tubes. Cells were immediately snap-frozen in liquid nitrogen and stored at -80°C

Preparation of E.coliultracompetent cells

50 mM Phosphate citrate buffer (pH-6.8)0.1M Citric acid0.2M dibasic Sodium phosphate16.9 ml Citric acid (0.1 M) and 33.1 ml Sodium phosphate (0.2 M) was mixed and volume was adjusted to 100 ml with H2O.Lipase assay0.1M Tris-HCl buffer (pH-8.2)pH was adjusted to 8.2 with HCl. 0.5 mM p-Nitrophenol standard solution8.69 mg p-Nitrophenol was dissolved in Tris-HCl buffer (0.1M) and volume was adjusted to 25 ml to make a final concentration of 25 mM.1volume of the above solution (25 mM) was diluted with 49 volume of 0.1 M Tris-HCl buffer to get a final concentration of 0.5 mM p-Nitrophenol standard solution.p-Nitrophenyl butyrate solution (420 μM)7.3 μl p-Nitrophenol butyrate (F.W. 209.2) 11 mg SDS650 μL Triton-X-100Volume was adjusted to 100 ml with H2O. Mixture was heated to 65°C in a water bath for 15 min, mixed well, and cooled down to room temperature prior to use. It can be stored upto 3 days at 4°C.Xylanase assay5 mg/ml RBB-xylan0.05 M di-Sodium hydrogen phosphate (Na2HPO4)

5 mg/ml RBB-Xylan was dissolved in 0.05 M Na2HPO4pH-7.5

Buffers for enzyme assaysCellulase assay

10 mM Ferric ethylenediaminetetraacetic acid (Fe(III)EDTA)100 mM Magnesium chloride (MgCl2)Working solution 0.18% Xylose 670 μM L-Methionine10 mM Sodium glutamate14.7 mM Potassium dihydrogen phosphate (KH2PO4)40 μM Mangenese sulphate (MnSO4)240 μM Ferric ethylenediaminetetraacetic acid (Fe(III)EDTA)5 mM Magnesium chloride (MgCl2)1.2% AgarLuria Bertani (LB)0.5% Yeast extract1% Tryptone1% Sodium cholride (NaCl)Media and solutions were sterilized either by routine autoclaving at 121°C and 15 psi for 20 min or by filtration through membrane of 0.22 μM porosity

Peptone Sucrose (PS)1 % Peptone1 % SucroseFor preparing plates, 1.2 % agar was added to the medium before autoclaving.Minimal Medium (MM9)Stock SolutionMinimal Salt (2X) for 250 mL 5.25 g di-Potassium hydrogen phosphate (K2HPO4) 2.25 g Potassium dihydrogen phosphate (KH2PO4)0.5 g Ammonium sulphate (NH4)2SO40.25 g Tri-Sodium citrate (Na3citrate)1 M Magnesium sulphate heptahydrate (MgSO4.7H2O) -250 μl 25 mg/mL L-Methionine-1 ml25 mg/mL Nicotinic acid-1 ml10 mg/mL Glutamic acid-25 ml20% Glucose-12.5 ml3% Agar-100 mlPlant mimicking medium (XOM2)Stock preparation100 mM L-Methionine1 M Sodium glutamate1 M Potassium dihydrogen phosphate (KH2PO4)10 mM Manganese sulphate (MnSO4)

Bacterial media

Media

was observed by the formation of a clear zone of hydrolysis around the bacterial/fungal colony. Tannase production, in terms of the diameter of the zone of hydrolysis around the colony, was measured (in mm) after 24 (bacteria) and 48 hours (fungi) of incubation. The diameter of the hydrolytic zone was measured at three points and the average was calculated. The microorganisms showing a zone of tannic acid hydrolysis were considered as tannase producers. The potent tannase producers were further tested quantitatively for the amount of enzyme produced in broth.

The procedure of Bradoo et al. (1996), involving point inoculation of the microorganisms on tannic acid agar plates was followed. The plates were incubated at 37 and 30±1°C for bacterial and fungal isolates. The presence of tannase activity

Qualitative screening for tannase producer/s

Nutrient agar (NA) medium The composition per litre of the medium is as follows: Peptone : 5.00 g Sodium chloride : 8.00 g Beef extract : 1.50 g Yeast extract : 1.50 g Agar-agar : 20.0 g Double distilled water : to make the final volume 1000 ml (iii) Tannic acid agar (TAA) medium This medium was used for screening of tannase producers. The composition per litre of the medium is as follows: Tannic acid : 10.00 g Agar-agar : 30.00 g Citrate phosphate buffer : to make the final volume 1000 ml (0.1M, pH 5.0) 30.0 g of agar-agar was melted and subsequently autoclaved. Citrate phosphate buffer and 0.1% (w/v)tannic acid, filter sterilized through 0.22μmembrane filters, were added to the sterilized molten agar and the final volume was made 1.0 L. (IV) Czapek Dox minimal medium (modified for tannase production)The composition per litre of the medium is as follows: Ingredients Fungi Bacteria Tannic acid : 10.00 g 10.00 g D-Glucose : 10.00 g 0.50 g NaNO3 : 6.00 g – NH4Cl : – 1.0 g KH2PO4 : 1.52 g 0.50 g K2HPO4 : – 0.50 g KCl : 0.52 g – MgSO4.7H2O : 0.52 g 0.50 g CaCl2 : – 0.01 g Cu(NO3)2.3H2O : trace – FeSO4.7H2O : trace – ZnSO4.7H2O : trace – Double Distilled water : to make 1.0 L to make 1.0 L pH : 5.0±0.2 5.0±0.

Potato dextrose agar (PDA) medium The composition per litre of the medium is as follows: Ingredients g/l Peeled and sliced potatoes : 200.0 Dextrose : 20.0 Agar-agar : 20.0 Double Distilled water : to make the final volume 1000 ml pH was adjusted to 6.2 ± 0.2 using 1N NaOH / HCl

Medium Compositio

Overnight-grown C. glabratacells were freshly inoculated either in YNBmedium or YNBmedium supplemented with BPS (50 μM) or FeCl3(500 μM) and allowed to grow for 4 h at 30°C, 200 rpm. After 4 h growth, cells were spun down at 4,000 rpm for 5 min in a refrigeratedcentrifuge set at 4°C and total protein was isolated. For estimation of histone deacetylase (HDAC) activity, 40 μg of protein samples were taken and HDAC Fluorometric Activity Assay Kit (#10011563; Cayman Chemical Company, Ann Arbor, MI, USA) was used as per manufacturer’s instructions. Fluorescence intensity values obtained inthepresence of the HDAC inhibitor, trichostatin A, were subtracted from those of the samples without inhibitorand plotted as relative arbitrary fluorescence units

Estimation of histone deacetylase (HDAC) activity

spectro-photometrically at 340 nm. For wild-type cells,mitochondrial aconitae activity was normalized to 100 % and for mutants the relative aconitase activity percentages were calculated

To determine aconitase activity, mitochondria were isolated as described by Meisinger et al. Briefly, YPD-grown C. glabratacells (500 OD600) were subjected to spheroplasting followed by homogenization (15 strokes) with glass Teflon homogenizer. To collect mitochondria, homogenate was centrifuged at 13200 g for 20 min in a refrigerated centrifuge set at 4°C. The mitochondrial pellet was resuspended in SEM buffer (250 mM sucrose, 1 mM EDTA, 10 mM Mops-KOH, pH 7.2) and stored at -80°C until further use. Mitochondrial aconitase activity was estimated by using method as described by Bulteau et al. Mitochondrial protein samples (5 μg) were prepared in KH2PO4buffer (25 mM, pH 7.2) containing 0.05 % Triton X-100. The samples were incubated with sodium citrate (1 mM), MnCl2(0.6 mM), NADP (0.2 mM) and isocitrate dehydrogenase (1 U/ml) for 20 min at room temperature. Isocitrate dehydrogenase catalysed reduction of NADP was recorded

Measurement of aconitase activity

For quantification of intracellular iron content, BPS-based colorimetric method as described by Tamarit et al.,was used. Briefly, overnight grown C. glabratacells were inoculated in fresh YPDmedium and allowed to grow at 30°Cand200 rpm for 6 h. Cultures were spun down and 50 OD600cells were subjected to nitric acid digestion (500 μl, 3%) for 16 h at 96°C.Next, lysates were spun down at 13,000 rpm to remove the cell debrisand 400 μl of the lysatewas incubated with 126 μl of ammonium acetate (1 M), 320 μl of BPS (1.7 mg/ml) and 160 μl of sodium ascorbate (38 mg/ml)for 5 min at room temperature. Absorbance of the samples was takenagainst the reagent blank at 535 nm and 680 nm which correspond to BPS-Fe-specific and BPS-Fe-non-specific absorbance, respectively. Non-specific absorbance was subtracted from specific absorbance and the iron content in each sample was calculated from the standard curve prepared using FeCl3and expressed as μM per OD600cells. In each experiment performed, total iron content of wild-typecellswas normalized to 100% and the iron content of mutants were calculated with respect tothe iron content of wild-type cells

Estimation of total iron

Radioactive counts measured in2x106labelled C. glabratacells and lysates were considered as ‘input’ and ‘output’ values, respectively. Percentage adherence was calculated by following equation.%Adherence=Output radioactive countsInput radioactive countsX 100

Adherence of C. glabratacells to Lec2, Chinese hamster ovarian (CHO) cells, wasdetermined as described previously (Cormack et al., 1999). Briefly, Lec2 cells were seeded at a cell density of 5x105cells per wellin a 24-well tissue culture plate.Cells were incubated in a cell culture incubator (Thermo Scientific) set at 37°C and 5%CO2for 12 h. Post incubation, the medium was discarded in a reservoir and Lec2 monolayer was washed thrice with sterile 1X PBS without disturbing the monolayer. Lec2 cells were fixed with 3.7% para-formaldehyde for15 min followed by twoPBS washes. 1 mlof 1X PBS containing antibiotics, penicillin (100 units/ml) and streptomycin (100 μg/ml), was added to each well, plates were sealed with PARAFILM, Cole-Parmer(PM-996) and stored at 4°C until use.C. glabrata cells,to be tested for their adherence potential, were grown in CAAmedium for 24 h.100 μl of 24 h-grownculture was re-inoculated in fresh 5 ml CAAmedium containing 200 μCi of S35(Met:Cys-65:25) INVIVO PROTWIN label mix (JONAKI, India)in a 15 ml polypropylene tube.Cultures were allowed to grow for 16-20 h at 30°C with shakingat200 rpm to radiolabel the cells. Radiolabelled C. glabratacells were harvested by spinning down1 ml of labelled yeast cultures,andthe cell pellet was washed thrice with sterile 1X PBS to remove any residual S35(Met:Cys-65:25) labelling mix from the medium. Post washes, the pellet was resuspended in 1 ml PBS, OD600was measured andcell suspension of 0.4 OD600wasprepared.Next, 24well plates containing fixed Lec2 cells were taken out from 4°C and PBS from the wells wasdiscarded by inverting the plates. Wells were washed once with PBS and 2x106labelled yeast cells were added to eachwell, andincubatedfor 30 min at room temperature.Post incubation, plates were centrifuged at 1,000 rpm and the wells were washed thrice with 1X PBS to remove non-adherent C. glabratacells. Lec2 cells were lysed with 5% SDS in PBS by scraping the wells, lysates were collected and transferred to a vial containing scintillation fluid

Adherence assay

For mouse infection assay, 10 ml YPD medium was inoculated with different C. glabratastrains and allowed to grow at 30°C for 12-16 h. After growth,cultures were washed twice in sterile 1X PBS and the cell pellet was resuspended in appropriate volume of 1X PBSto obtain a cell density corresponding to20OD600. 100 μlcell suspension(4x107yeast cells)was injected into female BALB/c mice (6-8 weeks old) through tailvein. Seven days post

infection, mice were sacrificed and kidneys, liver, brain and spleen were harvested. Organs were homogenised in 1 ml PBS and appropriate dilutions of tissue homogenate were plated onYPD-agar mediumsupplemented with penicillin and streptomycin antibiotics (100 units/ml penicillin and 100 μg/ml streptomycin). Plates were incubated at 30°C for 24-48 h and CFUs were counted. Fungal burden in different organs wasdetermined by multiplying the CFUsobtainedwithanappropriate dilution factor

For mouse infection assay, 10 ml YPD medium was inoculated with different C. glabratastrains and allowed to grow at 30°C for 12-16 h. After growth,cultures were washed twice in sterile 1X PBS and the cell pellet was resuspended in appropriate volume of 1X PBSto obtain a cell density corresponding to20OD600. 100 μlcell suspension(4x107yeast cells)was injected into female BALB/c mice (6-8 weeks old) through tailvein. Seven days post

Mouse infection assay

Experiments involving mice were performed at the CDFD animal facility, VIMTA Labs Ltd., Hyderabad, India(www.vimta.com) in strict accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India. The protocol was approved by Institutional Animal Ethics Committee (IAEC) of the Vimta Labs Ltd. (IAEC protocol approval number: PCD/CDFD/05). Procedures used in this protocol were designed to minimize animal suffering