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

DNA sequencing

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

2μg of total RNA was reverse-transcribed using SuperScript III Reverse Transcriptase which is a commercially available version of M-MLVRT with reduced RNase H activity and increased thermal stability.According to manufacturer’s protocol1μg of RNA,1μl oligo(dT)(500ng),1μl 10mM dNTPand nuclease freewater was added to afinal volume of 13μlin a PCR tube.Thismixture was then incubated at 65°C for 5 minutesin a thermo cyclerand then quicklytransferredtoicefor 1minute. To this 4μl of 5X first strand buffer 1μl of 0.1MDTTand1μl ofRNaseOUT (40U/μl) were added. Then contents were then mixed and 1μl (200 units/μl) of SuperScript III RT was added. Themixture was then incubated at 50°C for 60 minutesin a thermo cycler.Lastlythe reaction was stopped byincubating the mixture at 70°C for 15 minutes. The cDNA thus prepared was then usedas a template for PCR

RT-PCR (Reverse Transcriptase PCR)

The quantity and purity of nucleic acids was determined by measuring the absorbance at 260 and 280 nm. The concentration of nucleic acids was calculated by considering the OD (λ260)= 1 corresponding to50μg/ml DNA and 40 μg/ml ofRNA. The purity of nucleic acids was checked by their A260/A280 ratioconsidering 1.8 for DNA and 2.0 for RNA. These measurements were done in NanoDrop 2000 UV-Vis Spectrophotometer

Quantification of nucleic acids

Total RNA was isolated by TRIzol method using the manufacturer’s protocol. Briefly, medium was removed from culture dish and recommended amount of TRIzol wasadded directly on to the dish and kept at room temperature for 5 minutes for lysis of cells. The cellular homogenate was then transferred to a 1.5ml microcentrifuge tube. For each mlof TRIzol, 200μl of chloroform was added and tubes were shaken vigorously for 10 seconds to completely dissociate the nucleoprotein complexes, followed by vortexing for about 30 seconds. The mixture was kept for 3-5 minutes at room temperature and then centrifuged at maximum speed of 12,000 rpm for 10 minutes. The upper aqueous phase was transferred into a fresh micro centrifuge tube and RNA was precipitated by adding 500μl of iso-propanol. The RNA pellet was obtainedby centrifugation at 12,000 rpm for 30 minutes at 4°C. The pellet was washed with 1ml of chilled 70% ethanol followed by centrifugation at 12,000 rpmfor 5minutes. The supernatant was removed and the pellet air-dried for about 5 minutes. The pellet was resuspendedin 30-50μl RNase free deionisedwater and dissolved at 55ºC followed by quantificationusingnanodrop spectrophotometerfor further use.The RNA integrity was checked by evaluating the 18S and 28S rRNA signals by running 1μl of total RNA on denaturing agarose gel stained with ethidium bromide

Total RNA isolation from cultured cells

Molecular techniques

Themixture is incubated in a water bath at 37⁰C for 15 min and afterwards transferred on ice and 4μl of DNA loading buffer is added. The samples were then run on a polyacrylamide gel electrophoresis which had been pre-run for 30 min. Electrophoresis was carried out at 4⁰C for 3h till the bromophenol blue migrated to 2cm above the bottom of gel. The gel was taken out and kept on Whatman filter paper sheet and covered by saran wrap followed by drying in a gel dryer at 80⁰C for 1h under suction. The dried gel was exposed to phosphoimager screen by keeping in phosphoimager cassette overnight

A binding reaction mixture was prepared by adding the following components to a microcentrifuge tube on ic

Binding reaction

Cells were seeded in replicates of five @ 3X103cells per wellinfive different 96well cell culture platesand grown in complete media. The method described earlier was slightly modified and followed (Gillies et al., 1986). After every 24h of seeding, one plate was stained with 0.2% crystal violet in 2% ethanolfor 15 minutestill 4thday i.e. 96h.One plate was stained just after the cells get attached to use as 0h time point. Excess dye was removed from the plates by washing with ample amount of water. Crystal violet dye incorporated in the cells was extracted using 0.1% SDS solution by shaking for 10 minutes on a shaker. Absorbance of the extracted dye was then determined at 570 nm in a spectrophotometer. The experiment was repeated at least three times and the average absorbance was plotted for each time point to generate a growth curve

Cell growth Assay

Malachite green reagent

Reaction Buffer

Cell lysis Buffer

Calcineurin phosphatase assay

DNA staining solution

Fixative

For cell cycle analysis by flow cytometry

Inoue buffer

For preparation of Ultra competent cells

DNA loading dye

Agarose gel

TAE

For DNA electrophoresis

Neutralization solution(Solution III)

Lysissolution(Solution II)

Resuspension solution(Solution I)

For Plasmid isolation

Binding Buffer (10X)

EMSA Buffer

Nuclear lysis buffer

Polydeoxy (Inosinate-cytidylate) (Poly dI-dC)

For Electrophoretic mobility shift assay (EMSA)

Nuclear extractionbuffer (without protease inhibitors)

Cytoplasmic extractionbuffer (without protease inhibitors)

For Cell fractionation

Blocking buffer: 2% BSA

Permeabilization buffer: 0.2% Triton X100

Fixative : 4% Formaldehyde

For Immunofluorescence

Stripping Buffer

Blocking Buffer

TBST

Transfer Buffer

Running Buffer

Stacking and resolving AcrylamidegelsResolving gel (10 ml)

6X protein loading buffer (Laemmlibuffer)

Cell lysis buffer(RIPA Buffer)

For Immunoblotting

Tris Buffered Saline (TBS)

Phosphate Buffered Saline (PBS)

General Buffers

Ammonium persulfate(APS)

Acrylamide (29:1)

Phenylmethylsulfonyl fluoride (PMSF)

Benzamidine

Aprotinin

Leupeptin

NP-40ComponentsFinal concentrationFor 10 mlNP-4010%1mlH2O9ml

Dithiothreitol (DTT)ComponentsFinal concentrationFor 5 mlDTT1.0M0.7725gH2Oq.s

Ethylenediamine tetraacetic acid (EDTA), pH 8.0ComponentsFinal concentrationFor 500 mlEDTA0.5M93.05gH2Oq.sThe pH is adjusted to 8.0 using 10M NaOH

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

Potassium Chloride (KCl)ComponentsFinal concentrationFor 100 mlKCl2M14.91gH2Oq.s

Sodium Chloride (NaCl)ComponentsFinal concentrationFor 100 mlNaCl5M29.22gH2Oq.s

Potassium Chloride (KCl)

HEPES pH 7.9ComponentsFinal concentrationFor 100 mlHEPES1M23.83gH2Oq.sThe pH wasadjusted to 7.9 using 10M NaOH

Stock solution

Buffers and solutions

Transfection of plasmid DNAin cellswas performed using Lipofectamine 2000 reagent as per manufacturer’s protocolprovided with the reagent. Briefly, 0.5 to 1 million cells were seeded in a 60mm or 100mm tissue culture dish. After 12h of seeding,transfections were performed. 6-12 μg DNA was mixed in 500-1500 μl of Opti-MEM in one polypropylene tube and simultaneously, 15-30 μl of Lipofectamine 2000 was mixed in similar volumes of Opti-MEM in another tube and incubated at room temperature for 10minutes. Opti-MEM containingDNA and Lipofectamine 2000 were then mixed and incubated for 30 minutes at room temperature for the formation of DNA-lipid complex. Meanwhile, the cells were washed with sterile PBS and 4-10ml of Opti-MEM was addedin the plate. DNA-lipid complexes were then added to each dish for 6h. After that, the medium containing complexes was removed and complete medium (DMEM containing FBS) was added. Expression of transgene was evaluated 24-48hafter transfectioneitherby immunoblottingor immunofluorescence or by RT-PCR followed by PCR

Transient transfection in adherent cells

Extraction buffer

10XBinding buffer

Agarose gel

Nuclear lysis buffer (without protease inhibitors

Permeabilisation buffer: 0.2% Triton X100

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)

(b) Tris Buffered Saline (TBS)

obtained from Gibco, Invitrogen(Carlsbad, CA, USA). For cell culture transfections, Lipofectamine-2000 and Opti-MEM were alsoobtainedfrom Life Sciences, Invitrogen(Carlsbad, CA, USA).Commonly used chemicals in cell culture based experiments such asall-trans retinoic acid (ATRA), arabinoside cytosine (AraC),carbobenzoxy-Leu-Leu-Leucinal (MG-132), cycloheximide (CHX),DMSO, doxorubicin, hydrogen peroxide (H2O2),lipopolysaccharide (LPS, Escherichia coli055:B5), okadaicacid (OA), oleandrin,paclitaxel, phorbolmyristate acetate (PMA), vinblastine and vincristine wereobtained from SigmaAldrichChemicals.Benzofuran was synthesized as reported earlier (Manna et al., 2010).Recombinant human TNFα, IL-1and IL-8 were obtained from PeproTech Inc.(Rocky Hill, NJ, USA).Growth media for bacteria culture,Luria Broth (LB) and Agar were obtained from HiMedia laboratories (Mumbai, India). Bacterial strain DH5was used to make ultra-competent cells for transformation and plasmid isolation. Antibiotics, such as Ampicillin and Kanamycin used for selection of transformed colonies and culture were obtained from Sigma AldrichChemicals

The cell lines used in the present study, HuT-78 (human T-cell lymphoma), MDA-MB-231 (human breast cancer) and MDA-MB-468 (human breast cancer) were obtained from American Type culture collection (Manassas, VA, USA). Human colon carcinoma cell lines HCT-116 (wild-type, p53+/+) and HCT-116 (null, p53-/-) were a kind gift fromProf. B. Vogelstein (Johns Hopkins Oncology Center, Baltimore, MD). Cells were cultured in DMEM or RPMI medium containing 10% FBS, penicillin (100 U/ml), and streptomycin (100 μg/ml). Cells were maintained in humidified incubator at 37ºC in 5% CO2-95% air. Media for mammalian cell culture (DMEM and RPMI),fetal bovine serum (FBS)and other reagentsused in cell culture such as, PBS, Trypsin-EDTA, Antibiotic-antimycotic, Freezing medium, Geniticin, L-Glutamine, HEPES, etc. were

Cell cultureand Media

For growth analysis of S. cerevesiae strain, single colony was inoculated in appropriate broth medium and grown over night. This culture was used to inoculate the test medium to an initial OD600 of 0.1. Cultures were transferred to a shaker incubator set at 30°C and 200 rpm. Absorbance of cultures was measured using Ultraspec 2100 pro UV/visible spectrophotometer (Amersham Biosciences) at 600 nm at regular time-intervals till 72 h. Absorbance values were plotted with respect to time and the generation time was calculated from the logarithmic phase of the growth curve, by plotting A600vs.timeon a semi-logarithmic scale, using GraphPad Prism5 for curve fitting analysis

Growth analysis and determination of generation time

2 mM EDTA5 mM DTT1% Triton-XYeast protease inhibitor cocktail and phosphatase inhibitor cocktail (added fresh to the buffer B)

20 mM HEPES pH 6.8100 mM NaCl

Buffer B

30%GlycerolMade in 100 mL.RNA sample loading buffer (10X)50% glycerol10mM EDTA 0.025% Bromophenol blue 0.025% Xylene cyanolInoue transformation buffer, pH 6.7(125 mL, prepared just before use)10 mM PIPES 15 mM CaCl2.2H2O 250 mM KCl 55 mM MnCl2.4H2O (1.361 g is dissolved in 10 mL of water separately)PIPES(0.307 g), CaCl2.2H2O (0.275 g) and KCl (2.325 g)were added to 80 mL ofsterile water while mixing with a magnetic stirrer and the pH was adjusted to 6.8with 1 N KOH. After attaining the appropriate pH, MnCl2solution wasadded slowly in aliquotes of 300 μL over 10 min,while stirring to avoidabrown precipitate.MOPS buffer(10X)0.2 M MOPS, pH 7.220 mM CH3COONa10 mM EDTABuffer was made in DEPC treated waterYeast transformation reagents1 M Lithium acetate 50% Polyethylene glycol2 mg/mLSalmon sperm carrier DNA Dimethyl sulfoxide (DMSO) Zymolyase cocktail buffer for yeast colony PCR 2.5 mg/mLZymolyase (ZymoResearch)1.2 M SorbitolZymolyase buffer was prepared in 1X PBS

Yeast lysis buffer for genomic DNA extraction50 mM Tris-HCl,pH 8.010 mM EDTA 150 mM NaCl 1% Triton-X 1% SDSAE buffer for RNA extraction50 mMSodium acetate,pH 5.31 mMEDTA,pH 8.0Solution was made in DEPC treated water. 0.2%diethyl pyrocarbonate (DEPC)was added to the water and stirred for 12 h. To remove DEPC,water was autoclaved twice. DNA sample loading buffer (6X)15.25 mg Bromophenol blue15.25 mg Xylene cyanol

Buffers for extraction and analysis of genomic DNA and RNA

0.5% Yeast Extract 1% Tryptone 1% NaClLB-ampicillin plates LB medium 100 μg/mL ampicillin 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.For yeast and bacterial growth, plates were preparedby adding 2% to the medium before autoclaving

Luria-Bertani (LB) medium forbacterialgrowth

Antibodies used in this study are listed in Table 2.3

Antibodies

Plasmids containing the shRNA of interestwere either transfected transiently or were stably transfected. Transient transfection of shRNA was performed using eitherLipofectamine 2000 or PEI (as per the method explained before). Stable integration of shRNA was performed by transfecting shRNA along with retroviral packaging vector PCL-Ampho into BOSC23 packaging cells. The supernatantcontaining the packed viruses (viral medium)was collected at 48 and 72 hours of transfection. The viral mediumwas then added to thetarget cells in the presence of polybrene (8μg/mL). Two days later, cells were cultured in medium containing puromycin for the selection of stable clones.The clones stably expressing the desiredshRNA were identifiedandverified through western blotting and immunostaining using specificantibodies. A similar protocol was used to generate stable cell lines that expressed control shRNA

ShRNA

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

Plasmid transfection using PEI

PCR products or plasmids were analyzed by agarose gel electrophoresis. The samples were mixed with 6X loading dye (0.25% bromophenol blue and 0.25% xylene cyanoland 30% glycerol in water) and loaded onto a pre-cast gel, the percentage of gel ranged from 0.7 to 3 %, depending on the size of the DNA sample. Ethidium bromide at 1 μg/ml was included in the gel. The gel was visualized by fluorescence under UV-light

Agarose gel electrophoresis

All the antibodies used in the present study are mentioned in the table 1.Table 1: Antibodies used in the study

Antibodies

developer solution for appropriate time and immediately kept in fixer solution to see the protein band. For alkaline phosphatase method, blot was incubated with 5 ml of BCIP/NBT solution (Amresco) under dark condition. After incubation, blot was washed with water to see the blue-violet color protein band

volume of 50 mM acetate buffer (pH-5.4), and dialyzed overnight with 10 mM Tris buffer, pH 7.5. Pellet was used for dilution plating for calculating CFUs. For whole cell protein isolation, bacterial pellet was dissolved in 50 mM sodium acetate buffer (pH-5.4) and sonicated for 30 min (1 min on and off, Amplitude 32) by adding phenylmethylsulfonyl fluoride (PMSF) at a final concentration of 1 mM in ice-cold solution. Both extracellular proteins and whole cell lysate fractions were aliquoted in 1.5 ml microcentrifuge tube, and protein quantification was performed using a Pierce BCA protein assay kit (Thermo Scientific) as per manufacturer’s instructions using bovine serum albumin as standard and stored at -80°C for further use. Cell normalized extracellular and whole cell lysate proteins fractions from different strains were resolved on 12% SDS-PAGE gel at 90 V till the dye front reached the bottom. One gel was processed for silver staining (Sambrook et al., 1989), and other for western-blot analysis by using anti-GFP antibody. For western blot analysis, resolved proteins were transferred to Hybond-ECL membrane (Amersham biosciences) at 35 V for overnight in the cold room. Transfer of the proteins were visually confirmed by examining marker’s lane and membranes were incubated in small box for 2-3 h in 5% fat free milk prepared in 1X PBST for blocking. Blocking solutions were discarded, and primary antibody, appropriately diluted in 5% fat free milk prepared in 1X PBST, was added to the box containing membrane. After 2-3 h incubation in primary antibody, membranes were washed thrice with 1X PBST for 10 min. Membranes were incubated for 2 h in appropriate secondary antibody (anti-Rabbit antibody)diluted in 5% fat free milk prepared in 1X PBST. Blots were either developed by chemiluminescence based ECL-plus western detection system or alkaline phosphatase method. For HRP based chemiluminescence method, detection was performed using the ECL plus kit (Amersham biosciences) and incubated for 3 min. Blot was exposed to the film and developed i

For protein extraction, Xanthomonas oryzaepv. oryzaestrains with eGFP plasmid were grown for 24-30 h in PS medium to an OD of 0.8 as described above and centrifuged at 12,000 g for 10 min. The supernatant was taken as extracellular fraction and protein was extracted as described previously (Ray et al., 2000). Extracellular proteins were precipitated from this fraction by constantly adding 50% (wt/vol) ammonium sulphate at 4°C. After precipitation, the solution was kept on ice for 15-20 min and centrifuged at 12,000 g for 30 min at 4°C. The pellet was dissolved in s

Protein extraction and immunoblotting

respectively. The resulting constructs pRR14 and pRR15 were transferred in E.coliDH5α. Through triparental mating using pRK600 helper plasmid the construct were transferred in E.coliS17-1. After confirming pRR14 and pRR15 constructs by sequencing, the constructs were then introduced into BXOR1 strain through biparental mating using E. coliS17-1. X. oryzaepv. oryzicolaGUS and GFP reporter strains were selected on PS medium plates containing suitable antibiotics. Since pVO155 cannot replicate in X. oryzaepv. oryzae, ampicillin and kanamycin-resistant colonies were obtained upon chromosomal integration of the plasmid using the cloned DNA sequence as a region of homology. pProbeGTcan replicate independently in Xanthomonasand report for the gene expression

Glucuronidase (GUS) reporter gene fusion and GFP reporter fusion was created by using the suicide plasmid pVO155 having a promoterless gusAgene (Oke and Long, 1999), and pProbeGThaving a promoterless GFP (Miller et al., 2000). To construct the xsuA::gusAand xsuA::gfptranscriptional fusion, a 611-bp DNA fragment containing the putative promoter of the xssoperon (+213 to −398) was amplified by using the SCRsid_ pProbeGFP_F and SCRsid_ pProbeGFP_R primers (Table 2.2). This promoter fragment was subsequently digested with HindIII and BamHI,and directionally cloned upstream of the promoterless gusAand gfpgene in pVO155 and pProbeGTplasmids to create the xsuA::gusAand xsuA::pProbeGT(gfp) reporter constructs pRR1

Construction of xsuA::gusAand xsuA::gfp strains in X. oryzaepv. oryzicola background

(SCR65/ SCR66, SCR63/ SCR64 and SCR61/SCR62, respectively) designed from the neighbourhood region of the deleted gene.Replacement of ΔrpfFdeletion mutant with the point mutant allele (E141A and E161A: Glutamate to Alanine) (rpfF*) was carried out by transforming XocΔrpfFmutant with pbsks suicide vector harbouring full length rpfF* allele. The DNA fragment carrying the rpfF* allele was constructed by overlap PCR as described previously (Ionescu et al., 2013)using two 21 and 28 bp complementary primers for E141A-F/R and E161A-F/R, respectively; harbouring GAA to GCA substitution (Table 2.2). The mutated rpfF* allele was amplified by using the end primers only (SC14 and SC17) and cloned into pbsks vector with HindIII and XhoI restriction sites. The resulting suicide vector (pRR16 and pRR17) was transformed into ΔrpfFmutant and single recombinants were selected on PSA medium with kanamycin and ampicillin. Colonies were screened for integration of rpfF* (E141A or E161A) allele through homologous recombination with the flanking region of deleted rpfF allele

electroporation. Single Kmr recombinants were selected on PSA plate containing kanamycin. Insertion of the pK18mob vector in xssAgene was confirmed with PCR and sequencing. To further confirm the mutation in the siderophore biosynthetic gene, we did siderophore production assay on Peptone-sucrose agar (PSA)-chrome azurol sulfonate (CAS) (Schwyn and Neilands, 1987). PSA-CAS plate assay indicated that the xssA mutnat of Xocwas deficient in production of secreted siderophore.Deletion of the chromosomal rpfG, rpfC andclpgene of the X. oryzaepv. oryzicolawas accomplished by allelic exchange, following homologous recombination, utilizing the suicide vector pK18mobsacB harboring 5’ region and 3’ regions of the gene of interest (Katzen et al., 1999). 5’ and 3’ regions of rpfG and rpfC andclp gene were first amplified from the BXOR1by PCR using primers indicated in Table no. 2.2 and products were ligated together. After restriction digestion of ligated PCR products and the pK18mobsacB vector with appropriate restriction enzymes, they were ligated to get the plasmids pRR9, pRR10 and pRR11, respectively. These plasmids were then transformed into E. coliDH5α cells. The transformed E. colicells were selected on the LB agar plates containing nalidixic acid and kanamycin. The positive colonies carrying vector with correct inserts were further selected by colony PCR. These donor cells carrying pRR9, pRR10 and pRR11 containing 5’ and 3’ regions of the gene of interest were then transformed into electrocompetent BXOR1 wild-typecells. First crossover (single crossover) was achieved by culturing the cell mixture on Nutrient agar(NA) containing rifampicin and kanamycin,after transformation. The second crossover was allowed by passaging the cells with single crossover in nutrient brothmedium and then selecting on PSAplates containing rifampicin and 5% sucrose. BXOR1with deletion of the rpfG,rpfCand clp genes by double crossover was identified by PCR using pri

Two fragments, each approximately 300 bp in length corresponding to 5’and 3’ end of the rpfFgene were amplified using genomic DNA of Xocwild-type strain BXOR1, and cloned in pBSKS vector to obtain pRR7 (Table S1 and S5). pRR8 was obtained after ligation of Kmrcassette (EZ::Tn5TM<Kan-2>; Madison, WI) in the HindIII site of pRR7. The resulting plasmid (pRR8) was introduced into XocBXOR1 strain by electroporation. Doublerecombinants (Kmrand Aps) were screened on PSA plates containing appropriate antibiotics. Deletion of rpfF(76 amino acids) in the ∆rpfF mutant strain was confirmed by PCR and sequencing. For complementation analysis, full lengthrpfF gene was amplifiedfrom genomic DNA of Xoc Wild-typeBXOR1 strain with HindIII and EcoRI restriction sites and cloned into stable broad host range vector pHM1 (Hopkins et al., 1992)downstream to lacZpromoter to obtain pSC9. The pSC9 plasmid harboring the wild-typerpfFallele was introduced into ∆rpfF mutant strain by electroporation.To obtain the insertional nonpolar mutant in the xssA(xanthomonas siderophore synthesis A), a 321 bp internal fragment of the xssAgene containing the XbaI and HindIII sites was cloned inpK18mob suicide vector, in which the lacZpromoter drives the expression of downstream gene (Schäfer et al., 1994; Windgassen et al., 2000)to obtain pRR12. The resulting plasmid (pRR12) was introduced into Xoc BXOR1 strain by

Construction of mutants in X. oryzaepv. oryzicolaand rescue of the mutation

confirmed through PCR (by using primers SC11 and SC10) and squencing. Double mutant was complemented for DSF production by cloning whole rpfFgene of Xoo, cloned in HindIII and EcoRI sites of pHM1 (a broad host range vector for Xanthomonas) to get pSC6 plasmid. The resultant pSC6 plasmid was introduced into double mutant by electroporation

To obtain the insertional nonpolar mutant in the motA (encodes flagellar motor stator protein)andfliC (flagellin)genes, a 321 bp internal fragment of the motAgene and a 450 bp internal fragment of fliC containing the EcoRI and XbaI site were amplified using respective primer listed in Table 2.2. These fragments were cloned in pk18mob suicide vector, in which the lacZpromoter drives the expression of downstream gene (Schäfer et al., 1994; Windgassen et al., 2000), to obtain pRR1 and pRR2,respectively (Table 2.2). The resulting plasmid (pRR1& pRR2) was introduced into XooBXO43 strain by electroporation. Single Kmrrecombinants were selected on PSA plate containing kanamycin. Insertion of the pK18mob vector in motA andfliCgene was confirmed with PCR and sequencing. To further confirm the mutation in the flagellar genes, we did swimming motility assay on 0.1% peptone-sucrose agar (PSA). Swimming plate assay indicated that both motA and fliCmutant of Xoowas deficient in motility. Further, to obtain motAand fliC insertional knock out mutants in rpfFbackground, we cloned spectinomycin cassette obtained from pUC1318Ω plasmid, into the HindIII site of pRR1 and pRR2 plasmid to obtain pRR3 and pRR4. The resulting plasmid (pRR3& pRR4) were transformed in rpfF. Single specrrecombinants were selected on PSA plate containing kanamycin and spectinomycin. Insertion of the vector was further confirmed by PCR and sequencing. T2SSrpfFdouble mutant was constructed by transforming the plasmid with rpfF::Tn7Kanamycin cassette in the T2SS (xpsF) mutant background, and Kmrrecombinants were selected on PSA plates containing kan

Construction of mutants in X. oryzaepv. oryzae

insert of 1:3 for sticky end ligations. Ligation mix was incubated either at 22°C for 30 min or 16°C for 14-16 h. After incubation, T4DNA ligase was inactivated at 65°C for 20 min

After restriction enzyme digestion, digested products were resolved on agarose gels, and desired DNA fragments were extracted from the gel. Otherwise digested DNA fragments were precipitated by Phenol-choloroform-isoamyl alcohol method. Concentration of gel extracted or precipitated fragments were determined using spectrophotometer and ligation reactions were set up using a molar ratio

Ligation

For DNA precipitation after digestion, 500 μl nuclease free water was added to the digested DNA fragment. Equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) was added to the mixture and centrifuged at 13,000 g for 10 minat RT. Upper aqueous phase containing DNA fragment was transferred to fresh microcentrifuge tube and DNA was precipitated by adding 0.7 volume of iso-propanol and 1/10thvolume of sodium acetate. Precipitated DNA was washed with 70% ethanol, pellet was air dried for 20-30 min at RT and dissolved in nuclease-free water

DNA precipitation

QIAGEN QIAquick Gel extraction kit containing required buffers, spin columns and collection tubes was used to extract and purify DNA from agarose gels. Digested DNA samples and PCR products were resolved on 1% agarose gel and gel piece containing desired fragment was cut on an UV-transilluminator. DNA fragment was purified following manufacturer’s instructions

Gel extraction of DNA

strain or the ∆rpfFmutantharboring the Wild-typeallele in plasmid (pSC9).Genes that were significantly up regulated by 0.6 or more or down regulated by -0.6 or less fold (log2–fold change) were identified.The microarray data have been deposited in the NCBI Gene Expression Omnibus (GEO) under the GEO series accession number GSE53255

8x15k (AMADID: 25096) custom Agilent platform comprised of coding sequences for the three strains of Xanthomonas-X. oryzaepv. oryzae(KACC10331), X. oryzaepv. oryzicola(BLS256) and X. axonopodispv. citri 306 gathered from National Center for Biotechnology Information (NCBI). A total of 8113 probes were designed wherein 2120 probes corresponding to genes of interest replicated three times on Agilent platform. Feature extraction software GeneSpring GX version 10.5.1 of Agilent and GeneSpring GX percentile shift normalization was used for data analysis. Genes that were significantly up or down regulated by more than 1.5 fold and less than 0.5 fold were identified. Hierarchical clustering was performed for the differentially regulated genes and classified based on functional category. Data are the average of two hybridizations from biological replicates of each sample andraw data sets for this study are available at the Gene Expression Omnibus database (Accession number –GSE217809). Likewise, Microarray analysis for Xanthomonas oryzaepv. oryzicolawas performed by isolating RNA from the strains grown under low-ironcondition. The labeled cRNA samples were hybridized on to a Genotypic Technology Private Limited designed 8x15k (AMADID: 41087) Agilent platform. Data extraction from Images was done using Feature Extraction software v 10.7 of Agilent. Data normalization was done in GeneSpring GX using 75thpercentile shift and normalization to specific samples. Differentially regulated genes were clustered hierarchically to identify significant gene expression patterns.Genes were classified based on functional category. Hierarchical clustering of DSF regulated genes in X. oryzaepv. oryzicola grown under low-iron conditions is based on similar expression profiles in ∆rpfFmutant vs either the Wild-typeBXOR1 strain or ∆rpfF(pSC9). Clustering analysis was performed using GeneSpring GX Software using Average Linkage rule with pearson uncentered distance metric. log2–fold change differences between the ∆rpfFmutant with either the Wild-typeBXOR1

Xanthomonas oryzae pv. oryzae strains grown in PS medium to an OD600of 1, were collected, washed once with 150 mM sodium chloride (NaCl) solution to remove excess EPS. RNA isolation was performed using Trizol method described above. After isopropanol precipitation, RNA was frozen at -80°C. Quality of RNA was examined by determining the RNA integrity number (RIN) before microarray analysis. Microarray experiments were performed at Genotypic Technology Pvt. Ltd., Bangalore.Briefly, a

Microarray analysis

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

Quantitative real-time PCR

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)

work were autoclaved twice and dried at 80°C for overnight before use. RNA was isolated from Xanthomonasculture using Trizol method. Xanthomonascells were harvested at 12,000 g for 5 min at 4°C, resuspended in approximately 1 ml Trizol (Invitrogen),mixed properly and incubated at room temperature (RT) for 5 min. 200 μl chloroform was added to the tube, shaken for 15 seconds and incubated at RT for 2-15 seconds. Next, tubes were centrifuged at 13,000 g for 15 min at 4°C. Aqueous phase was transferred to new 1.5 ml microcentrifuge tube and RNA was precipitated by adding 500 μl isopropanol and incubated for 5-10 min at RT. Precipitated RNA was collected by centrifugation at 10,000 gfor 10 min at 4°C. RNA pellet was washed with 70% ethanol and resuspended in 20 μl nuclease-free water. RNA concentration was determined by measuring absorbance at 260 nm. Quality of RNA was examined by gel electrophoresis on 0.8% agarose gel with TAE buffer prepared in DEPC treated water

For RNA experiments, all solutions were prepared in RNase free diethylpyrocarbonate (DEPC) treated water. Microcentrifuge and tips u

RNA extraction

Xanthomonasstrains were grown overnight in 3 ml PS medium. Cells were harvested at 12,000 g for 5 min, resuspended in RNase added P1 buffer and were transferred to 2 ml microcentrifuge tube. Cells were lysed by adding 40 μl lysozyme followed by adding 80 μl 10% SDS and incubated at 50°C for 10 min. Further, proteins were removed by treating the cell suspension with 16 μl proteinase K and incubated at 37°C for overnight. Next day, 200 μl CTAB/NaCl was added and cell suspension was heated at 65°C for 10 min. Next, 1 ml chloroform-isoamyl alcohol was added to the cell suspension and tubes were vortexed for 2-3 min. After centrifugation at maximum speed for 10 min at room temperature, aqueous phase was carefully transferred to a fresh microcentrifuge tube. To further remove cell debris, previous step was repeated with 1 ml of phenol-chloroform-isoamyl alcohol and aqueous phase containing DNA was taken out carefully. Genomic DNA from the aqueous phase was precipitated by adding 700 μl isopropanol and 170 μl sodium acetate (3M, pH-7). Next, DNA pellet was washed with 70% ethanol and dried at room temperature for 20 min. Genomic DNA pellet was dissolved in 50 μl nuclease free water and stored at -20°C. Quality of extracted genomic DNA was checked on 0.7% agarose gel by electrophoresis

Xanthomonasstrains were grown overnight in 3 ml PS medium. Cells were harvested at 12,000 g for 5 min, resuspended in RNase added P1 buffer and were transferred to 2 ml microcentrifuge tube. Cells were lysed by adding 40 μl lysozyme followed by adding 80 μl 10% SDS and incubated at 50°C for 10 min. Further, proteins were removed by treating the cell suspension with 16 μl proteinase K and incubated at 37°C for overnight. Next day, 200 μl CTAB/NaCl was added and cell suspension was heated at 65°C for 10 min. Next, 1 ml chloroform-isoamyl alcohol was added to the cell suspension and tubes were vortexed for 2-3 min. After centrifugation at maximum speed for 10 min at room temperature, aqueous phase was carefully transferred to a fresh microcentrifuge tube. To further remove cell debris, previous step was repeated with 1 ml of phenol-chloroform-isoamyl alcohol and aqueous phase containing DNA was taken out carefully. Genomic DNA from the aqueous phase was precipitated by adding 700 μl isopropanol and 170 μl sodium acetate (3M, pH-7). Next, DNA pellet was washed with 70% ethanol and dried at room temperature for 20 min. Genomic DNA pellet was dissolved in 50 μl nuclease free water and stored at -20°C. Quality of extracted genomic DNA was checked on 0.7% agarose gel by electrophoresis

Genomic DNA isolation

200 rpm in LBbroth supplemented with appropriate antibiotics (plasmid antibiotic marker). Cells were harvested by centrifugation at 12,000 g for 5 min. Plasmids were extracted using Qiagen plasmid miniprep ormidiprep kit following the manufacturer’s instructions. Concentration of the extracted plasmid DNAs was measured using spectrophotometer at 280 nm and stored at -20°C

E.colistrains carrying plasmids were inoculated and grown overnight at 37°C and

Plasmid DNA purification

All standard molecular biology and genetics were performed as described previously (Sambrook et al. 1989)

Molecular biology methods

For growth analysis of Xanthomonasstrains, a loopful of bacterial colony was inoculated in appropriate broth medium and grown for 14-16 h. 0.2% of overnight grown culture was used to inoculate the test medium (for iron limitation, PS with 50 or 100 μM of 2,2’-dipyridyl, and for iron supplementation, different concentrations of either FeCl3or FeSO4was added). Cultures were transferred to a shaker incubator set at 28°C and 200 rpm. Absorbance of cultures was measured using Ultraspec 2100 pro UV/visible spectrophotometer (Amersham Biosciences)at 600 nm at regular time-intervals till 48 h. Absorbance values were plotted with respect to time and generation time was determined from the logarithmic (log) phase of bacterial growth using the following formula.G = Generation time (h)T1= Initial time point taken for analysisT2= Final time point taken for analysisNf = Absorbance at time T2(Final OD)Ni= Absorbance at time T1(Initial OD)

Growth analysis and determination of generation time

0.5% DEPC Added in H2O, stirred vigorusly and autoclaved prior to use.DNA sample loading buffer0.25% Bromophenol blue0.25% Xylene cyanol30% GlycerolDNA sample loading buffer was prepared in water

10 g of SDS (Sodium Dodecyl Sulfate) was dissolved in 80 ml of H2O, and volume was adjusted to 100 ml with H2O.CTAB/NaCl solution10% CTAB 0.7 M NaCl10 g of CTAB was dissolved in 80 ml 0.7 M NaCl solution by stirring it on a hot magnetic stirrer. Volume was adjusted to 100 ml with 0.7 M NaC1 solution.Lysozyme solution100 mg of lysozyme was dissolved in 1 ml of H2O (100 mg/ml).Proteinase K solution10 mg of proteinase K was dissolved in 1 ml of H2O (10 mg/ml).5 M Sodium chloride (NaCl) 292.2 g of Sodium chloride (NaC1; M.W. 58.44) was dissolved in 800 ml of H2O. Volume was adjusted to 1 liter with H2O. Sterilized by autoclaving.3 M Sodium acetate (NaOAc)(pH 5.2 and 7.0) 24.6 g sodium acetate anhydrous (CH3COONa; M.W. 82) was dissolved in 80 ml H2O. pH was adjusted to 5.2 with glacial acetic acid or 7.0 with dilute acetic acid. Volume was adjusted to 100 ml with H2O. Sterilized by autoclaving.Phenol:Chloroform:Isoamyl alcohol (25:24:1) solution25 ml Tris-equilibrated phenol24 ml Chloroform1 ml Isoamyl alcoholDEPC (diethyl polycarbonate) treated water

50 mM Tris-HCl (pH 8.0)10 mM EDTA (pH 8.0)100 μg/ml RNaseVolume was adjusted to 100 ml with sterile H2O.10% SDS

Buffers and solutions for extraction and analysis of genomic DNA and RNAResuspension buffer (P1)

Oligonucleotides

Oligonucleotides used in this study were designed either by freely available online tool Primer3plus (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi/) or Generunner software. Oligonucleotides were commercially synthesized at Eurofins MWG operons, Bangalore, India. Oligonucleotides used in this study are listed in Table 2.2

For bacterial isolates, a single colony from a nutrient agar slant was inoculated into 50 ml of nutrient broth in a 250 ml Erlenmeyer flask. These flasks were incubated at 37±1°C in a incubator shaker till an optical density of 0.6 at 660nm. Now these cultures were used to inoculate 50 ml of the tannase production medium in 250 ml Erlenmeyer flasks using 2% v/v inoculum. These flasks were incubated at 37±1°C in an incubator shaker (Multitron AG-27; Switzerland) at 200 rpm for 72h. The experiments were carried out in triplicates. Samples (2.0 ml for bacteria and same for fungi) were withdrawn at regular intervals of 12h upto 72 h. The samples thus obtained were centrifuged at 10,000 rpm in a refrigerated centrifuge (SIGMA 4K15 Germany) for 10 min at 4°C. The supernatant/s were analyzed for tannase activity

Microorganisms were isolated from the above mentioned sources using direct plating method. Serial dilution of the different soil samples with normal saline was carried out and the different dilutions were spread plated on to potato dextrose agar (PDA) for isolation of fungi and on to nutrient agar (NA) for the isolation of bacteria. The plates were incubated at either 30 or 37±1°C in a bacteriological incubator so that the different organisms could grow and form visible colonies. The different fungal and bacterial colonies isolated by the procedure mentioned above were purified by subculturing on respective media, and subsequently screened for tannase production. The new isolates, alongwith different cultures obtained from laboratory stock culture collection, were revived on potato dextrose agar (PDA) slants. These cultures were regularly subcultured and stored at 8±1°C in a BOD incubator. Their purity was periodically checked by microscopic examination.

Isolation of bacteria and fungi from the samples

1N Hydrochloric acid (HCl)* One normal hydrochloric acid was prepared by adding 1.0 ml concentrated HCl to 10.0 ml of double distilled water. •1N Sodium hydroxide (NaOH)* One normal sodium hydroxide was prepared by dissolving 4.0 g of NaOH in 100 ml of double distilled water. *: These were used for adjusting the pH of the medium. •Tween-80Tween-80 used as surfactant was prepared by adding 100 μl of concentrated Tween-80 to 100 ml of double distilled water and autoclaved.

tock Solution

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

microcentrifuge tube. For precipitation of RNA, 1/10thvolume of 3 M sodium acetate (pH 5.3) and 2.5 volume of 100% ice-coldethanol was added. In order to facilitate precipitation, tubes werekept at -20°C for 20 min. Tubes were centrifuged at 13,000 rpm for 10 min in a refrigerated centrifuge. The RNA pellet was washed with 70% ethanol,resuspendedin 100-200 μl of nuclease-free water and stored at -20°C untiluse.Care was taken to keep allreagents and tubes on ice to maintain the cold temperature throughout theRNA extractionprocess

All reagents required for RNA extraction were preparedin DEPC-treated water. RNasecontamination from non-autoclavable items wasremoved by wiping them with RNaseZap® (Ambion). Total RNA from yeast cells was extractedusing acid phenolextractionmethod. Briefly, yeast cells were grown underappropriate conditions and at suitabletime points,cells were harvested by centrifugation at 4,000 rpm for 5 min. The cell pellet was washed twice with ice-cold DEPC-treated water, resuspended in 350 μl of AE buffer and transferred toa1.5 ml microcentrifuge tube. To this,40 μl of 10% SDS and 400 μl of acid phenol (pH 4.3) was added. The cell suspension was mixed well by vortexing thrice, short pulsesof10 seconds each,and incubated at 65°C for 15 min with continuous agitation at 800 rpm. Post incubation, cells were kepton ice for 5 min and centrifuged at 13,000 rpm in a refrigerated centrifuge set at 4°C for 10 min. After centrifugation, aqueous layer was transferred to a new1.5 ml microcentrifuge tube and 400 μl of chloroform was added. Tubes were mixed well by gentlyinverting them 4-5 times and centrifuged at 13,000 rpm for 10 min. The aqueous layer was separated and transferred to a new1.5 ml

Total RNA isolation

This method was used to isolate highly pure genomic DNA. Briefly, 10 ml overnight grownC. glabratacultures were spun downandwashed with 10 ml sterile water. Washed cells wereresuspended in500 μl sterile water and transferred toa1.5 ml microcentrifuge tube. Tubes were spundownat 4,000 rpm for 5 min, supernatant was discarded andcell pellet was resuspended in 500 μl of buffer containing 100 mM EDTA and 5% β-mercaptoethanol and incubatedat 42°C for 10 min. Post incubation, cells were spun down at 4,000 rpm for 5 min and resuspended in freshly prepared Buffer B. To this, one tip-full of lyticase (Sigma, L4025) was added and incubated at 37°C for 1 h.After incubation, spheroplasts were collected by spinning downtubes at 6,000 rpm for 5 min, supernatant was discarded and the pellet was resuspended in 500 μl of Buffer C. DNA was extracted twice with 500 μl of PCI (25:24:1) solution and the aqueous layer was transferred toa new1.5 ml microcentrifuge tube. To this, 2.5 volume of absolute ethanol and 1/10thvolume of 3 M sodium acetate (pH 5.3) wereadded. Tubes were spundownat 13,000 rpm for 10 min, DNA pellet was resuspended in 200 μl of 1X TE buffer containing0.3 μl of RNase cocktail (Ambion) and incubated at 37°C for30 min. DNA was precipitated again by adding absolute ethanol and sodium acetate as mentioned above. DNA pellet was washed once with 70% ethanol, centrifuged at 13,000 rpm for 10 min, air-dried at room temperature and was resuspended in 100-200 μl of 1X TE buffer by gently tapping the tube. DNAwas stored at -20°C until use

Spheroplast lysis method

Bacterial transformation

microcentrifuge tube. For precipitation of RNA, 1/10thvolume of 3 M sodium acetate (pH 5.3) and 2.5 volume of 100% ice-coldethanol was added. In order to facilitate precipitation, tubes werekept at -20°C for 20 min. Tubes were centrifuged at 13,000 rpm for 10 min in a refrigerated centrifuge. The RNA pellet was washed with 70% ethanol,resuspendedin 100-200 μl of nuclease-free water and stored at -20°C untiluse.Care was taken to keep allreagents and tubes on ice to maintain the cold temperature throughout theRNA extractionprocess

All reagents required for RNA extraction were preparedin DEPC-treated water. RNasecontamination from non-autoclavable items wasremoved by wiping them with RNaseZap® (Ambion). Total RNA from yeast cells was extractedusing acid phenolextractionmethod. Briefly, yeast cells were grown underappropriate conditions and at suitabletime points,cells were harvested by centrifugation at 4,000 rpm for 5 min. The cell pellet was washed twice with ice-cold DEPC-treated water, resuspended in 350 μl of AE buffer and transferred toa1.5 ml microcentrifuge tube. To this,40 μl of 10% SDS and 400 μl of acid phenol (pH 4.3) was added. The cell suspension was mixed well by vortexing thrice, short pulsesof10 seconds each,and incubated at 65°C for 15 min with continuous agitation at 800 rpm. Post incubation, cells were kepton ice for 5 min and centrifuged at 13,000 rpm in a refrigerated centrifuge set at 4°C for 10 min. After centrifugation, aqueous layer was transferred to a new1.5 ml microcentrifuge tube and 400 μl of chloroform was added. Tubes were mixed well by gentlyinverting them 4-5 times and centrifuged at 13,000 rpm for 10 min. The aqueous layer was separated and transferred to a new1.5 ml

Total RNA isolation

This method was used to isolate highly pure genomic DNA. Briefly, 10 ml overnight grownC. glabratacultures were spun downandwashed with 10 ml sterile water. Washed cells wereresuspended in500 μl sterile water and transferred toa1.5 ml microcentrifuge tube. Tubes were spundownat 4,000 rpm for 5 min, supernatant was discarded andcell pellet was resuspended in 500 μl of buffer containing 100 mM EDTA and 5% β-mercaptoethanol and incubatedat 42°C for 10 min. Post incubation, cells were spun down at 4,000 rpm for 5 min and resuspended in freshly prepared Buffer B. To this, one tip-full of lyticase (Sigma, L4025) was added and incubated at 37°C for 1 h.After incubation, spheroplasts were collected by spinning downtubes at 6,000 rpm for 5 min, supernatant was discarded and the pellet was resuspended in 500 μl of Buffer C. DNA was extracted twice with 500 μl of PCI (25:24:1) solution and the aqueous layer was transferred toa new1.5 ml microcentrifuge tube. To this, 2.5 volume of absolute ethanol and 1/10thvolume of 3 M sodium acetate (pH 5.3) wereadded. Tubes were spundownat 13,000 rpm for 10 min, DNA pellet was resuspended in 200 μl of 1X TE buffer containing0.3 μl of RNase cocktail (Ambion) and incubated at 37°C for30 min. DNA was precipitated again by adding absolute ethanol and sodium acetate as mentioned above. DNA pellet was washed once with 70% ethanol, centrifuged at 13,000 rpm for 10 min, air-dried at room temperature and was resuspended in 100-200 μl of 1X TE buffer by gently tapping the tube. DNAwas stored at -20°C until use

Spheroplast lysis method

E. coliDH5α ultra-competent cells were used for all bacterial transformations. Briefly, frozen DH5α ultra-competent cells were taken out from -80°C freezerandthawed on ice for 15 min. DNA to be transformed was added to the bacterial cell suspension and incubated on ice for 30 min. For transforming ligation mixtures and plasmids,5-10 μl and 100-500 ng of DNA was used, respectively. Followed by 30 min incubation on ice, heat shock was given for 60-90 sec at 42°C in a water bath and cells were immediately kept back onice for 2 min. To this,1 ml of sterile LB medium was added and tubes were incubated inashaker incubator set at 37°C, 200 rpm for 45 min.Next, cells were spun down and resuspended in 500 μl of LB medium. About 100-200 μl of resuspended cells were plated on LB-agar medium containing appropriate antibiotics and incubated for 12-16 h at 37°C. Transformants were purified on LB-agar plates containing appropriate antibiotics andpositive transformantscarrying desired DNAwere verified by PCR, restriction digestion and sequencing analyses

Bacterial transformation

E. colibacterial strain DH5α was taken out on LB-agar mediumfrom -80°C freezer and incubated at 37°C for 14-16 h. To obtain the starter culture,single bacterial colony was inoculated in 25 ml of SOB medium ina25 ml flask. The flask was incubatedfor 6-8 hat 37°C with continuous shaking at 200 rpm. Next, 2, 4 and 10 ml of the starter culture was inoculated in three different 1 litre flasks each containing 250 ml of SOB medium. Cultures were incubated overnightat 18°C with continuous shaking at 200 rpm. After overnight incubation, OD600 of allthree cultures were monitored after every 45 min interval till OD600of any of the three cultures reached 0.55. These cells werekept onice for 10 min and the other two cultures were discarded.Cells were harvested by centrifugation at 2,500g in a Sorvall GSA rotor for 10 min at 4°C. Supernatant was poured offcompletely andcells were gently resuspended in 80 ml of ice-cold Inoue transformation buffer by swirling the tubes (pipetting was avoided at this step). Followingresuspension, cells were spun down by centrifugation at 2,500g in a Sorvall GSA rotor for 10 min at 4°C and the supernatant was discarded completely. The cell pellet was resuspended gently in 20 ml of ice-cold Inoue transformation buffer by swirling.1.5 ml of DMSO was added to the cell suspension and incubated on ice for 10 min. 50 μl aliquotsof cell suspensionwere dispensed in pre-chilled 1.5 ml microfuge tubes, snap-frozen in liquid nitrogen and stored in -80°C freezer till further use

Preparation of ultra-competent E. colicells

ml YPD broth at an initial OD600of 0.1. Cultures were allowed to grow for 4-5 hin a shaker incubator setat 30°C, 200 rpm until the OD600of the cultures reached 0.4-0.6. Next,cells were harvested ina15 ml centrifuge tube by centrifugation, washed twice with 10 ml of sterile water, resuspended in 1 ml of sterile water and were transferred to a 1.5 ml microfuge tube. Cells were harvested by centrifugation at 4,000 rpm for 5 min,resuspended in 50 μl of100 mM lithium acetate solution and transformation mixture was added. Transformation mixture consisted of 240 μl polyethylene glycol (50%), 36 μl of lithium acetate (1 M), 5 μl of heat denatured single stranded carrier DNA (10 mg/ml), 500 ng to 1 μg of transforming DNA and final volume was made to 360 μl with sterile water. The tubes were incubated at 30°C for 45 min. To this, 43 μl of sterile DMSO was added and heat shock was given at 42°C for 15 min. Next, tubeswere transferred to ice for 10-15 sec, centrifuged at 4,000 rpm and transformation mixture reagents wereremoved completely by pipetting. Cells were resuspended in 200 μl of sterile water and spread-plated on appropriate selection medium. Plates were incubated at 30°C for 24-48 h

Yeast transformation was performed as described previously (Gietz et al., 1992) with fewmodifications. Briefly, overnight grown C. glabratacultures were freshly inoculated in 10

Yeast transformation

To phenotypically characterize C. glabratamutants,serial dilution spot growth assays were performed. Briefly, the optical density of overnight-grown C. glabratacultures wasnormalized to OD600of 1.0andnormalized cultures were further diluted 10-fold in 1X sterile PBS five times. 3 μl of serially diluted culture were spotted on test plates. Plates were incubated at 30°C (unless mentioned otherwise) for 24-48hand growth was recorded by capturing plate images. For experiments involvingchecking theability of mutants to utilize non-fermentable carbon sources,growth was scoredafter 6-7 days of incubation

Serial dilution spot growth assay

For time course growth analysis,C. glabratacells were inoculated in appropriate medium and grown for 14-16 h. Followed by overnight growth,yeast cells were sub-cultured in test medium at an initial OD600of 0.1 and growth was monitored by recording the absorbance of the culture at 600 nm at regular time-intervals till 72h. Absorbance values were plotted with respect to time and generation time of yeast strains were calculated between 4-8 h of growthwhen cells were in logarithmic phase of growth using following equation.Generationtime(G)=T2−T1Xlog2logNf/NiG = Generation time (h)T1 = Initial time point taken for analysisT2 = Final time point taken for analysisNf= Number of cells at time T2 (1 OD600of C. glabratacorresponds to 2 X 107cells)Ni = Number of cells at time T1 (calculated from OD600 value as mentioned above)For CFU-based viability assays, appropriate dilutions of yeast cultures were prepared in 1X sterile PBS and suitable volume of diluted cultures wasplated on YPD-agar medium. Plates were incubated at 30°C for 24-48 h and viable colonies that appeared on YPD-agar plate were counted.To obtain logarithmic phase cells, overnight-grown C. glabratacell suspension was inoculated in appropriate medium at an OD600of 0.1 and grown ina shakerincubator set at 30°C, 200 rpm for approximately 4 htill the culture density reached OD600of 0.4-0.6

Time course growth analysis and determination of generation time

C. glabratastrains were routinely grown in rich YPD medium or synthetically defined YNB medium, or YNB medium supplemented with CAA, unlessstatedotherwise.To obtain overnight grown liquid cultures, C. glabratacells were inoculated in appropriate medium and incubated at 30°C under constant agitation (200 rpm) to maintain proper aeration.To revive the frozenstocks,about one tipfull of frozen culture was streaked either on YPD-agar or on CAA-agar medium. In general, frozen stocks of C. glabratastrains were revived on YPD-agar medium.However,C. glabratastrains harbouring plasmidscontainingURA3as a selectable marker were revived on CAA-agar medium. After streaking, plates were allowed to grow for 24-48 h at 30°C and were stored at 4°C for a maximum period of two weeks. For long term storage, freezer stocks of C. glabratastrainswere prepared in 15% glycerol and stored at -80° C.Escherichia colistrain DH5αwas revived on LB-agar medium from frozenstock and incubated at 37°C for 14-16 h. DH5α strainwas used for transformation purpose and maintaining plasmids. Bacterial strains harbouring plasmids containing selection markerswere revived on LB-agar medium supplemented with appropriate antibiotics.Bacterial liquid cultures were either grown in LB broth or LB broth containing suitable antibioticsand incubatedin a shakerincubator set at 37°C, 200 rpm for 14-16 h. For preparation of bacterial frozenstocks, 1 ml overnight grown bacterial culture was added to500 μl of 50% glycerolto obtain final concentration of ~16 % glyceroland stored at -80°Cuntil use

Strains and culture conditions

Microbiological methods

For cryopreservation of THP-1 and Lec-2 cells, 5-6 million cells wereresuspendedin 0.5 ml of eithercommercially procuredcell preservation medium from GIBCO(12648010)or complete medium supplemented with 10 % fetal bovine serum and 10 % DMSO.Cells were initially kept inanisopropanol bath and werelatertransferred to -70°C freezer. After 2-3 days, frozencells were transferred to liquid nitrogen container till further use. To revive the cells, frozenstockswere taken out of the liquid nitrogen container and immediately transferred to water bath set at 37°Cfor thawing. When freezing medium has thawed completely, cells were transferred to a 100 mm cell culture dishcontaining 12 ml completemedium and incubated under tissue culture conditions at 37°C and 5% CO2for 12 h. Afterincubation, medium was replaced by 12 ml fresh pre-warmed medium and incubated under tissue culture conditions till they reached 70-80% of confluencebefore splitting