to hold 1.5 mL microfuge tubes.Acrylic shield was used through out to block the β radiation

Synthesis of 5[β-32P]IP7was conducted as described earlier (Azevedoetal., 2010). An acrylic box was placed at a 37°C in a hybridization oven before settingup the reaction

Synthesis of radiolabelled 5-IP7

GST fusions of yeast RNA Pol I subunits were purified as described in (Werneret al., 2010). Yeaststrainsover expressing GST tagged RNA Pol I proteins were grown overnight at 30°C in 10 mL of SC-Ura medium with 2% glucose medium. Cells were pelleted, washed in SC-Ura with galactose. Protein expression was induced by transferring the entire pellet into200 mL of SC-Ura with 2% galatose to give a final OD600of 0.8-1.0. For proteins A190 and A43 that express at very low levels, the overnight culture volume and induction volume were doubled. Cells were cultured at 30°C harvested at 3.0-5.0 OD and washedwith ice cold water. The cell pellet was suspended in 5 mL of ice cold Buffer A (Section 2.1.6.7), 750 μL ofcell suspensions were aliquoted into 1.5 mL microfuge tubes and to this 500 μL glass beads were added. Cells were lysed by bead beating using a vortex mixer (VortexGenei -2 with mix-mate attachment), and the lysate was centrifuged at high speed for 15 min at 4°C. Supernatants were dispensed into a 15 mL conical tube and Triton X-100 was added to a final concentration of 1%. Pre-swollen glutathionebeads were washed in Buffer B(Section 2.1.6.7)from which 200 μL of 1:1 bead suspension was added to approximately 5 mL of A34 and A43 expressing cell lysate and 100 μL of 1:1 bead suspension was added to 5 mL of A190 cell lysate and incubated for 2 h at 4°C on a rotary mixer. Lysates were centrifuged at 5000 xgfor 2 min and the beads were washed with ice cold Buffer C (Section 2.2.6.6) twice. Beads were further washed with ice cold Buffer B followed by ice cold 1X phosphate buffered saline (PBS) twice. Beads were suspended in an equal volume of 1X PBS with protease inhibitor cocktail (Sigma)

Protein expression and purification from yeast

recovery, cells were centrifuged at 2,500x gfor 4 min. The medium supernatant was discarded and cells were resuspended in 200 μLfresh sterile LB medium. Cells were plated on LB agar medium containing appropriate antibiotics. Plates were incubated at 37°C for 12-16 hto allow growh of individual colonies

E. coli DH5α strain was transformed with yeast plasmids carrying appropriate inserts that express S.cerevisiaeproteins (Sambrook and Russell, 2001). Ultracompetent cells stored at -80°C were thawed on ice for 5-10 min. 20μLligated plasmid was added to 100 μLultracompetent cells,and the cells were incubated on ice. After 30 min, competent cells were subjected to heat shock at 42°C for 90 seconds. Cells were immediately transferred to ice for 2-3 min. Next, 900 μLLB medium was added and cells were allowed to recover for 45 min on a shakerincubator set at 37°C. After the

Bacterial transformation

A single colony of E. coli DH5α strain was inoculated in 10 mL LB medium and incubated at 37°C overnight. Overnight culture was subculturedin 250 mL of LB medium of about 0.1 OD and incubated at 18°Cfor36 htillthe OD600reached approximately0.5. Cultures were centrifuged at 2,500 x gfor 10 min at 4 ̊C and harvested cells were washed gently with 80 ml ice-cold Inoue transformation buffer(Section 2.1.6.2). Cells were collected by centrifugation at 2,500x g for 10 min at 4 ̊C and gently resuspended in 20 mLice-cold Inoue transformation buffer. To this cell suspension, 1.5 ml sterile DMSO was added and swirled gently. The cell suspension waskept on ice for 30min,and 100 μLvolume was aliquoted into pre-chilled sterile microcentrifuge tubes. Cells were immediately snap-frozen in liquid nitrogen and stored at -80°C

Preparation of E. coli DH5α ultracompetent cells

and A190middle (557-1100 a.a)fragments were amplified from the plasmid pRS314RPA190gifted byDr. Herbert Tschochner with primers containing BamHI and XhoI (Table 2.4).The mutant versions of A34 and A190 fragments were generated by overlap extension PCR based method. p416GPD GST-RPA43 used in ChIPwas generated by extracting the GST-RPA43fragmentsfrom pYesGex6p2 GST-RPA43by restriction digestion with HindIII and XhoIfollowed by ligation into p416GPD. Clones were verified by bacterial colony PCR, sequencing and western blot. For colony PCR a small amount of bacterial colony was taken with the help of tooth pick and touch the bottom of the 0.2 mL PCR tube to which PCR reaction mix was added and performed PCR

The nomenclature system for RNA Pol I subunits, gene and protein name is given in Table 2.4 and The S.cerevisiaeRNA Pol I subunits Uaf30, A34.5, A43 full length and fragmentsof A135 (1-112 a.a) and A190 (1101-1664 a.a) were PCR amplified from genomic DNA of the wild-type strain using high fidelity DNA Taq polymerase, with primers carrying restriction sites for BamHI and NotI or BamHI and XhoI (Table 2.4). Amplified fragments were cloned downstream of the GAL4promoter in the pYesGex6p2 plasmid (Werneret al., 2010).These plasmids were generated by a colleague,Mr. Unnikannan CP,in the laboratory. Fragments and mutant versions of full length RNA Pol I subunits A34.5 and A43 weregenerated using above plasmids as templates (Table 2.3).A190 N-terminal (1-556 a.a)Calculation of adjusted input-Volume of cell lysate: 500 uL-Volume set aside as input: 10 uL, eluted into a final volume of 40 uL-Volume of lysate taken for IP: 490 uL; eluted into 100 uL after IP, of which 90 uL was eluted into a final volume of 40 uL. Therefore, volume of lysate for IP corresponds to (90/100)*490 = 440 uL (approx. 500 uL)1μLof input sample, and 3 μLof immunoprecipitated sample was taken from40μLof eluted volumes, for q-PCR reaction.The input sampletaken for q-PCR = 1/40x10 uL= 0.25 uLThe immunoprecipitated DNAtaken for q-PCR = 3/40x 500=37.5uLTherefore, the Ct values obtained for input samples (Ct input) were from 0.25 μL out of 37.5 uLoftotal lysateAdjusted Ct=[Ct(Input)-log2(input dilution factor)]The dilution factor forinput sample was 37.5/0.25 = 150Adjusted Ct = [Ct(Input)-log2(150)] = Ct(Input)-7.23Table. 2.6 The gene name and correspondingprotein name for RNA Pol Isubunits.RPA represents RNA polymerase I and protein name starts with A which represents that the subunit is present in RNA pol I

Cloning of S.cerevisiaeORFs

Calculation of adjusted input-Volume of cell lysate: 500 uL-Volume set aside as input: 10 uL, eluted into a final volume of 40 uL-Volume of lysate taken for IP: 490 uL; eluted into 100 uL after IP, of which 90 uL was eluted into a final volume of 40 uL. Therefore, volume of lysate for IP corresponds to (90/100)*490 = 440 uL (approx. 500 uL)1μLof input sample, and 3 μLof immunoprecipitated sample was taken from40μLof eluted volumes, for q-PCR reaction.The input sampletaken for q-PCR = 1/40x10 uL= 0.25 uLThe immunoprecipitated DNAtaken for q-PCR = 3/40x 500=37.5uLTherefore, the Ct values obtained for input samples (Ct input) were from 0.25 μL out of 37.5 uLoftotal lysateAdjusted Ct=[Ct(Input)-log2(input dilution factor)]The dilution factor forinput sample was 37.5/0.25 = 150Adjusted Ct = [Ct(Input)-log2(150)] = Ct(Input)-7.23

PCR reactions were set up with the immunoprecipitated DNA by using the primers thatamplify the 5’ETSand promoter regions. Real-time PCR was performed using Mesa Green 2X PCR MasterMix (Eurogentec)in a 20 μL reactionvolume using 1 μL from the input sample and 3 μL from the immunoprecipitated samplein an RT-PCR mechine (Applied Biosystems). Ct values of the immunoprecipitated samples were normalised to the adjusted Ct values of the inputsample, andthedata were plotted as ‘immunoprecipitated DNA as a percentage of input DNA (% Input)’, as described in (Haringet al., 2007; Livak and Schmittgen, 2001).Calculation of %InputThe amount of DNA taken for q-PCR analysis is inversely proportional to the Ct value% Input = 2-Ct(IP) / 2-Ct(Input)x 100, (% of gDNA immunoprecipitated from the total amount of gDNA taken)Ct(IP) = Ctvalue of immunoprecipitated DNACt(Input) = Ctvalue of Input DNAIn this experiment,10 μL of this lysate was taken as input and this has to be adjusted to the total volume of the lysate. Therefore, the formula would be,

Quantitative PCR (q-PCR) reaction

Immunoprecipitation of chromatin was performed by incubating the lysate with 3 μg of anti-GST antibody overnight at 4°C followed by50 μL of 1:1 suspension of Protein A beads for 4 h. Beads were washedtwice each in wash buffer I, wash buffer II, and TE buffer(Section 2.1.6.5). Each wash was performed for 15 min at 4oC by rotating on a Lab-net end over mixer. Chromatin was eluted in 100 μL of elution buffer(Section 2.1.6.5) by rigorous mixing on a Thermo mixer (Eppendorf) for 30 min at 65oC. 90 μLof this eluted sample was incubated overnight at 65oCto reverse the cross linking. 10 μLlysate taken as input was diluted to 90 μLwith elution buffer and incubated overnight at 65oC to reverse the crosslinking.DNA was extractedfrom the input and immunoprecipitated samplesintoafinal volumeof40μLusing aPCR purification kit (Qiagen)

Yeast strains carrying p416GPD GST-RPA43were grown in SC-Ura medium overnight and sub cultured at 0.2 OD600.45 mL of mid-log phase yeast cultures were subjected to cross linking with 1% formaldehyde for 15 min at room temperature(Szijgyartoet al., 2011). Cross linking was quenched by adding glycine to a final concentration of 0.1 M. Cells were washed in ice cold Tris-buffered saline and were lysed in 500 μL of ice cold lysis buffer (Section 2.1.6.5) by bead beating. Chromatin was fragmented using a Diagenode bath sonicator, 15 sec on time and 30 sec off time, for 15 min. Cell lysates were centrifuged at high speed and the supernatant was pre-cleared with 3 μg of normal rabbit IgG followed by30 μL of 1:1 suspension ofProtein A beads. Supernatant was collected and 10 μL of this lysate was taken as input

Chromatin immunoprecipitation(ChIP)

The desired S.cerevisiae strain was grown overnight in YPD liquid medium and yeast cells were harvested by centrifugation at 2,500x gin 15 mL polypropylene tubes. Yeast cells were washed with PBS, suspended in 500 μL lysis buffer (Section 2.1.6.2) and were transferred to a 1.5 mL microcentrifuge tube. Yeast cells were incubated for 15 min on a thermomixer set at 65 ̊C and 750 rpm.After incubation, a volume equivalent to 500 μLof glass beads (0.5 mm) were added and cells were lysedon a beating apparatus by mixing three times for 45 seconds each with intermittent cooling on ice to prevent overheating.Cell lysates were centrifuged at 12,000 x gfor 5 min and upper aqueous phase was transferred carefully to a new 1.5 mLmicrocentrifuge tube, to which 275 μL of 3M sodium acetate was added. To this solution, 500 μL of chloroform was added, mixed well, and centrifuged at 12,000 x gfor 5 min at 4°C (this step was repeated twice). Supernatant was transferred to a new 1.5 mLmicrocentrifuge tubeand500 μL of isopropanolwas added and mixed well by inverting the tube 3-4 times. To precipitate genomic DNA, the suspension was centrifuged at 12000 x gfor 15minat 4°C. Precipitated genomic DNA was washed with 70% ethanol and dried at room temperature. The genomic DNA pellet was dissolved either in 100μLof Sigma molecular biology grade water or TE buffer supplied with Qiagen plasmid mini prep kit, andadd 1 μL of RNase solution (30 mg/mL) was added to this and incubated at 37oC for 1 h. The extracted genomic DNA was checked for integrityon a 0.8%agarose gel by electrophoresis and stored at -20oC

Genomic DNA isolation by glass bead lysis method

After 14-16 h incubation, hybridization buffer was decanted to a radioactive liquid waste container. Membranes were washed twice 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 1X SSC buffer at room temperature exposed to a phosphorimager screen for 1 h and scanned using a phosphorimager (Fuji Film FLA-9000). The data were analysed by densitometry using Fuji Film Multigauge software V3.11 and graphs were plotted using GraphPad Prism5 software.Note:Depending on signal saturation or non-specificity, high stringency washes were performed starting from 0.5X SSC followed by 0.2X SSC or 0.1X SSC wash buffers containing 0.1% SDSat room temperature

Post-hybridization washes

Yeast were grown in appropriate medium while the logarithmicphase and 1 OD600cells wereharvested and chilled on an ice(Elion and Warner, 1986). Allsubsequent steps,unless specified, werecarried out at4°C or on ice. Cells were collected by centrifugation at 2500 gfor 6min and washed with2 mLof TMN (Section 2.1.6.4). Cells were suspended in 1 mL of ice cold permeabilization buffer (Section 2.1.6.4) and incubated for 15 min.Cells were pelleted and incubated with 100μLof transcription assay buffer (Section 2.1.6.4) containingradiolabelled [α-32P]UTP. After incubation for 10 min at30°C and 300 rpm in a shaking drybath (Eppendorf),1mLof cold TMN containing1 mM nonradioactive UTP was added, the cells were collected by centrifugation, and RNA was prepared by the hot phenol methodas described in Section 2.2.8. Equal counts of labelled RNA were used for hybridization. The membrane was pre incubated with 50 mL of hybridization buffer for 20-30 min, and hybridization was performed with labelled RNA at 65°C for 15 h in 20 mL of hybridization buffer

Transcription run on analysis

apparatus.Membranes were neutralised in 2X SSC, and denatured plasmids were cross-linked to Hybond-N+membranes usingaUV cross linker at 2000 J/inch square energy for 2 min

+610),end 5’ETS (+611 to +952),25S (+5270 to +5630) and NTS2(gifted byDr. Susan J Baserga) (Gallagheret al., 2004); ACT1 cDNA (+175 to +701) cloned into TOPO vector; pUC12 plasmid containing 5S rDNA construct (giftedby Dr. Purnima Bhargava). Empty TOPO plasmid and genomic DNA extracted from wild type yeast were used as controls. Plasmids and gDNA were extracted, quantitated and denatured in alkaline denaturing solution. 10 μg of each plasmid and gDNAin replicates weredenaturedin alkali, blotted on a Hybond N+membrane using a 96-well Dot Blot

The following plasmids (Table 2.2)were used as probes to detect the transcribed RNA (Fig. 2.1). The TOPOplasmids containing rDNA start (+1 to +177), 5’ETS (+351 to

Dot blot membrane preparation

Cellswere grown in YPD to an OD600of 0.5-0.7. Cells equivalent to 1OD600were washed with synthetic complete medium without uacil twice and suspended in SC-Ura containing 3 μCi/mLof [14C]uracil for 5 min. Cells were pelleted and washed with SC-Ura medium twice and suspended in 0.5 mLof AE solution(Section 2.1.6.2). 1OD600of this cell suspension was counted in a liquid scintillation counter (Perkin Elmer-Tricarb 2900). The cpm values obtained were and converted into moles based on thespecific activity of [14C] uracil and plotted using GraphPad Prism5

Uracil uptake assay

labelled RNAs to N+Hybond membrane (GE Life Science), the radiolabelled rRNA was detected using a phosphorimager (Fuji Film FLA-9000)

Overnight grown yeast were sub-cultured at 0.2 OD600and growntill 0.8 OD600.Cells equivalent to 1 OD600were harvested and were washed in SC-Ura medium, suspended in 1 mL of SC-Ura medium containing 3μCi/mL of [14C]uracil and pulselabelledfor 15 min at30°C.After incubation the cells were pelleted and a chase was performed by adding SC medium containing 240 mg/L unlabelled uracil. Samples were taken at 0, 1, 5, 10and 20min after the chase, centrifuged at 12000 x gfor 1 min at 4°C, and total RNA was isolated from cells by the hot-phenol techniquedescribed in Section 2.2.8.Equal total RNA was loaded on a 1.2% formaldehyde-agarose gel. After transfer of

Pulse-chase analysis of rRNA synthesis

Overnight grown yeast were sub-cultured at 0.2 OD600and growntill 0.8 OD600. Cells equivalent to 1 OD600were harvested and washed twice with SC-Uramedium to remove any residual uracil from the cells. Cells were incubated in SC-Ura medium containing 3 μCi/mL [14C]uracil for 1, 5, 10 and 20 min, and RNA was extracted as described in Section 2.2.8. Equal total RNA was resolved on a formaldehyde agarosegel and transferred to an N+Hybond membrane (GE Life Sciences). Radiolabeled rRNA was detected using a phosphorimager scanner (Fujifilm FLA-9000)

[14C]uracillabelling of total RNA

After the RNA samples were resolved, the gel was placed in a tray and incubated for 5-10 min in DEPC treated water followed by 10X SSC buffer. A nylon membrane (N+Hybond, GE Life Sciences) was cut to the size of the gel and it was rinsed in 10X SSC buffer for 5 min. In the same tray, 10X SSC buffer was filled and a gel running boat (15 cm length) was placed in an inverted position. Whatmannumber 3 filter paper (wick) was cut to an appropriate size and placed on the inverted boat in such a way that the longer edges of the paper should touch the buffer. The gel was placed upside down on the wick and care was taken not to allow any air bubbles between the gel and wick. On top of the gel, three Whatman number 3 papers that were cut to the size of gel were placed by avoiding the air bubbles. A bundle of blotting sheets was placed on top of this, on which appropriate weightwas placed and was allowed the transfer to take place for 15 –16 h

Capillary transfer of RNA on to the membrane

A mixture of 40 mL of DEPC treated water and 0.6 gof agarose was meltedby boiling. After cooling down the temperature to 55°C, 8.4 mL of formaldehyde (final concentration 2.2 M)and 5 mL of 10X MOPS were added, mixed well and poured into a boat to cast a gel

Preparation of formaldehyde agarose gel(50 mL) and RNA sample

Yeast were grown till mid-log phase 0.6-0.8 OD600in an appropriate medium and 10 mL of culture was pelleted at 2500 x g. Cells were suspended in 350μLof AEbuffer,mixed with 50μLof 10% sodium dodecyl sulphate and 400μLacid phenol(pH4.3) and immediately shaken vigorously on a dry bath (Eppendorf)at 65°C for 15 min.The tube was then quickly chilled on iceand centrifuged at 12000 x gfor 15 min to separate the aqueous phase from the phenol. After centrifugation, the aqueous phase was transferred to a new tube and extracted with an equal volume of chloroform.RNA was precipitated by adding 50 μLof 3 M sodium acetate (pH 5.3) and equal volume of 100% ethanol followed by incubation at -20°C for 2 h, andcentrifugation at maximum speed for 30 min at 4°C. The pellet obtained was washed in 70% ethanol, dried at room temperature anddissolved in an appropriate volume of DEPC-treated water. The concentration of RNA was estimated by measuring A260using a Nano Drop Spectrophotometer (ND1000). To monitor different classes of rRNA levels, 10 μg of total RNA from each strain was resolved on a 1.2% formaldehyde-agarose gel

RNA extraction by hot-phenol method

placed on ice.The samples were centrifuged at 10,000x gfor 10 min at 4°C and the supernatant was extracted by avoiding the glass beads. Absorbance of thelysatewas measuredat 260 nm, and it was divided into 200 μL aliquots and frozen at -80°C. No difference in the ribosome profiles were detected between thesesamples and fresh samples analysed immediately.Polysomes were analysed by centrifugation through a 10-50% sucrose continuous gradient. The gradient was prepared by pipetting, gradient buffers (Section 2.1.6.3), 5 mLof each layer onto the bottom of an 11 mLopen top centrifuge tube, covering the tube with paraffin filmand by placing it horizontally on a flat surface at 40C for 2 h. The ribosome sample (cell lysate) was loaded on top, and gradient was centrifuged at 100,000x gat4°C for 6 h in an SW41 rotor (Beckman). Anamount of lysate equivalent to 10 A260units was loaded on the gradient. Ribosome levels were measured by the gradient analysis with an ISCO UV-6gradient collector with continuous monitoring atA254.Polysomepurification was performed by layering the cell lysate on 37% sucrose solution in an 11 mLopen top centrifuge tube, and centrifuged at 100,000x gfor 14 h in an SW41 rotor(Beckman). The pellet was suspended in 50 to 100 μL of lysis buffer. An amount oflysate equivalent to 0.7A260unitswas loaded on the gradient. Ribosome levels were measured by gradient analysis with an ISCO UV-6gradient collector with continuous monitoring atA254.Ribosome subunits were analysedby centrifugation through a 10-30% sucrose continuous gradient. The gradient was prepared by pipetting 2.2 mLof each layer onto the bottom of a 5 mLopen top centrifuge tube, sealing it with paraffin filmand placing it horizontally on a flat surface at 4°C for 2 h.The ribosome sample wasloaded on top, and gradient was centrifuged at 100,000x gand 40C for 4.5 hin an SW55rotor (Beckman). Ribosome subunit levels were measured by gradient analysis with an ISCO UV-6gradient collector with continuous monitoring atA254.Ribosome profiles with yeast cell lysates and purification of ribosomes from yeast were performed with the help of Mr. Aluri Srinivasand Dr. Umesh Varshney

The method to analyse ribosome profiles was adapted from (Leeet al., 1992). Yeast cells were grown to 0.2 to 0.8 OD600at 30°C in 200mLof YPD,and cycloheximide was added to this media at 50μg/mLfinal concentration. The culture was placedand mixed continuously onanice and salt mixture for 2-5 min and centrifuged immediately at 4,000 x g. The culture was not allowed to stay for a longer time on the ice and salt mixture to avoid freezing. The cell pellet wassuspended in 1mLof lysisbuffer (Section 2.1.6.3),andtransferred to a2 mL microfuge tube, to which1mLglass beads (0.45-0.6mm diameter)were added, and lysed by bead beating for 10 min with intervals (30 sec on time and 1min off time).During the 1 min off time, tubes were

Ribosome profiles

Yeast weregrown in YPD (Difco) overnight,and sub-cultured at 0.2 OD600. Cells were harvested at 0.6-0.8 OD600. 1 OD600of each culture was used for the labelling. Cells were washed in SC-Metmedium twice, suspended in SC-Metmedium containing 25μCi/mLof 35S Met-Cys and pulsed for 15 min. Cells were washed twice in methionine-free medium and suspended in 300Lof Tris-saline. Cell suspension was counted in a liquid scintillation counter (Perkin Elmer-Tricarb 2900). The cpm values obtained were plotted using GraphPad Prism5

35S-Met uptake assay

growntill the OD600reached 0.8-1.0. Cells equivalent to 1OD600of each culture was taken for the labellingof total protein. Cells were washed in methionine-free synthetic complete medium(SC-Met) twice, suspended in SC-Met mediumcontaining 25μCi/mLof 35S Met-Cys twin label mixand incubatedfor 1min, 5 min and 15min. Cells were washed twice in ice-cold SC-Met medium twice and suspended in 500 μL of Tris-salinecontaining protease inhibitor cocktail.To this,300 μL ofglass beads (0.45-0.6mm diameter)were added and cells were lysed for 10 min by bead beating (with intervals of 1 min on time and 30 sec off time).The lysate was centrifuged at high speed for 15 minat 4°C. To the supernatant,sodium deoxycholate was added to a final concentration of 0.1 mg/mLand incubated on ice for 30 min. To this solution, 20% trichloroacetic acid was addedto a final concentration of 6%, incubated for 1 h on ice, and centrifugedat high speedfor 20minat 4°C. The pellet was suspended in 300 μL of Tris-saline and counted inaliquid scintillation counter (Perkin Elmer-Tricarb 2900). The cpm values obtained were plotted using GraphPad Prism5

Wild type and knock out yeast strains were grown in YPD (Difco) whereas synthetic complete medium without uracil was used for the KCS1 complementedstrains. Overnight grown yeast were subcultured in appropriate medium at 0.2 OD600and

Protein synthesis analysis

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

To monitor yeast cell death 1 OD600equivalent cellsfrom mid-log and overnight growncultures were pelleted, washed in PBS and the cell pellet was suspended in 100 μL of PBS. 20 μLof 0.4% trypan bluesolution was added to 20 μLof cell suspension and incubated for 10 min. 20 μL of this suspension was placed on a slide, covered with a cover slip, and cell death was measured by scoring dead cells that take up the dye.To monitor cell viability, cells equivalent to 10-5OD600 from mid-log and stationary phase cultures were plated on YPD-agar, incubated at 30°C for 48 h, and colonies were counted to extrapolate viable cell count per OD600

To determine yeast cell mass, cells equivalent to 5 OD600were harvested from mid-log and overnight growncultures, and washed twice with PBS. Cell pellets were dried at 50°C for 20 minand the dry weight of yeast was measured. To assess the cell number, cells in mid-log or stationary phase were counted using a Neubauer chamber and the number of cells present in 1 OD600was calculated

Cell mass, cell numberand viability assessment

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

S. cerevisiae strains were routinely grown either in rich YPD mediumorsyntheticcomplete medium (SC)(Section 2.1.5.1) at 30°C with continuous shaking at 200 rpm unless otherwise stated. In general, S. cerevisiae frozen glycerol stocks were revived on 2% YPD medium by streaking and allowed to grow for 1-2 days. S. cerevisiae strains harbouringaplasmid containingthe URA3geneas the auxotrophy selectionmarker were revived on synthetic complete medium lacking uracil (SC-Ura).To prepare liquid cell culture, a single colony of each S. cerevisiae strain was inoculated either in YPD or SC-Ura medium and grown for 14-16 h. S. cerevisiae strains streaked on plates were sealed with paraffin film (parafilm M) and stored at 4°C for a maximum period of 2 weeks.Protein over expression in yeast was carried in presence of galactose instead of glucose as the carbon source, as the plasmid pYesGex 6p2 carries the GALpromoter under which yeast proteins were expressed

Strains and culture conditions

Luciferase assay was performed using luciferase assay systems (Promega #1500). Cells were transfected with shRNA. Following 24 hrs.of shRNA transfection, cells were transfected separately with FOP-Flash and TOP-Flashvectors in the absence and presence of Wnt3a plasmids. After 24 hrs, media was removed, and the cells were rinsed twice with 1X PBS. After removing the final wash, the cells were incubated with lysis buffer (1X lysis reagent: CCLR; 20μl/well for a96-wellplate, or 400 μl/60mm culture dish, or 900μl/100mm culture dish). Cells were collected in a microcentrifuge tube and were centrifuged at 14000 rpm for 10 minutes. The cell lysate (supernatant) was transferred to a new tube. 20μl of cell lysate was mixed with 100μl of Luciferase Assay Reagent(LAR), and the amount of light produced was measuredin luminometer by usinga delay time of 2 sec and a read time of 10 sec.

Luciferase reporter assay

44KDa to 670 KDa; Ovalbumin (44KDa), Conalbumin (75KDa), Aldolase (158KDa), Ferritin (440KDa), Thyroglobulin (669KDa)] purchased fromGE Healthcare Life Science. Blue dextran was used for the void volume of the column. The molecular weight of the protein complex fractions was calculated from the plot. The different fractions that were collected as eluates from the column were concentrated,and the presence of WWP2 or WWP1 as a monomer or multimer was identified by western blotting using specific antibodies for WWP2 and WWP1

The WWP2-WWP1 heterodimeric complex formation under normal conditions and upon cisplatin-induced stress conditions was studied by gel exclusion chromatography. HEK293T cells untreated or treated with cisplatin were harvested,and cell lysate was prepared by using the standard protocol. Sephacryl S-200 (GE Healthcare) columns were equilibrated with 1X NETN (without Triton X-100) at a flow rate of 1ml/min. 0.8ml of cell lysate (1mg/ml) was passed through the Sephacryl S-200 column,and different fractions (fraction size; 500μl) were collected using Bio-Rad 2110 fraction collectorat the same flowrate. To determine the molecular weight of the fractions, column was calibrated with high molecular weight markers [range

Gel filtration

The reactions were carried out at 30 °C for 15 min in 25μlof ubiquitylation reaction buffer (40mM Tris-HCl at pH 7.6, 2mM DTT, 5mM MgCl2, 0.1M NaCl, 2mM ATP) containing the following components: 100μM ubiquitin, 20nM E1 (UBE1), 100nM UbcH5b (all from Boston Biochem). The bacterially purified MBP-WWP2 and MBP-WWP1 E3 ligases were added to the reaction mixture. The bacterially purified and GST bound GST-protein, GST-p73, and GST-ΔNp73 were used as the substrate in the reaction. After the ubiquitylation reaction, the GST beads werewashed five times with 1X NETN buffer and boiled withan equal volume ofSDS-PAGE loading buffer. The ubiquitination of the substrates was determined by western blotting with the substrate-specific antibody

In vitroubiquitylation assay

immunoprecipitation by using substrate specific antibody or pull-down by affinity trapping the substrate tag. The IP/pull-down complexes wereanalyzed by detecting the ubiquitination of substrate protein by using either substrate specificantibody or ubiquitin antibody through western blotting

HeLa cells were transfected with various combinations of plasmids. At 24 h post-transfection, cells were treated with MG132 (10μM) for 6 h and the whole-cell extracts were prepared by NETN lysis. The cell lysatewas subjected to

In vivoubiquitylation assay

Cells were transfectedwith various combinations of plasmids and treated with cycloheximide (50ug/ml) 24hrs.post-transfection. Cells were harvested at different time points, and the protein levels were determined by using the standard protocol for western blotting/immunoblotting

Cycloheximide-chase assay

Cells were grown overnight on coverslips and transfected with various combinations of plasmids. Post 24 hrs. of transfection, cells were washed with PBS and then fixed in 3% w/v paraformaldehyde in 1X PBS containing 50 mM sucrose for 15 minutes at room temperature. Cells were permeabilized with permeabilization buffer i.e. 0.5% Triton X-100 buffer containing20mM HEPES at pH 7.4, 50mM NaCl, 3mM MgCl2 and 300mM sucrose and were incubated for 5 min.at room temperature.Cells were washed twice with 1X PBS and blocked with 3% BSA/PBS for 30 minutes. Cells were incubated with specificprimary antibody diluted in the blocking buffer. After 2 hours of incubation, cells were washed thrice with 1X PBS (each washfor 5 minutes). The 1X PBS was removed,and the cellswere incubatedwith specific FITC or Rhodamine-conjugated secondary antibodyat 37°C for 30 min.To visualize nuclei, cells were co-stained with DAPI (10 μg/ml). Cellswere washed thrice with 1X PBS and after final wash, coverslips containing cell weremounted on the slidesusing glycerine containing paraphenylenediamine. The cells were analyzed using confocal microscopy facility at CDFD

Immunofluorescence

Cells (HEK293T) were transfected with various combinations of plasmids/siRNAsor treated with cisplatin. Cells were washed first with 1X PBSand then with Met-/Cys-1X DMEM supplemented with dialyzed FBS(10%). Cells were then incubated with Met-/Cys-DMEM supplemented with dialyzed FBS in the incubatorfor 1h (met/cysstarvation). Cells were taken out from the incubator,and the culture media was removed. Cells were placed behind the radioactivity protective shield and DMEM supplemented with 35S met/cys (200μCi) was added to the cells. Plates containing radioactive media were then put into the acrylic box and incubated for 1h at 37°C in a CO2 incubator. Plates were taken out and kept behind the radioactivity protective shield;the radioactive media was disposedofin the radioactive liquid waste. One set of cells washarvested for 0 time point, other sets of cells were washed twice with 1X PBS and were incubated with normal medium containing 2mM each of cysteine and methionine. Cell plates were put in the acrylic box and incubated at 37°C in a CO2 incubator. Cells were harvested at different time points. Cells were collected in ice-cold PBS and were lysed using the standard cell lysis protocol. Cell lysate were subjected to immunoprecipitation(IP). IP complex is separated on SDS-gel using standard protocol. The gel was transferred onto PVDF membrane, and the membrane was dried. Dried membrane was exposed in a cassette and the signal was detected using phosphorimager. Later the same blots were probed with specific antibodies

35S met/cys pulse-chase assay

Triton X-100, 0.5 mg/ml RNase A, 40 μg/ml propidium iodide). Cell nuclei were then incubated for 30 min. at 30°C and were subsequently analyzed by FACS. The hypodiploid nuclei in the histograms were considered and represented aspercentage of apoptotic cells

Apoptosis was measured by Nicoletti method (Nicoletti et al., 1991). Cells treated with appropriate apoptotic stimuli for the indicated times were harvested by centrifugation at 800g for 5 minutes at room temperature. Cells were washed once with PBS, and then resuspended in hypotonic PI lysis buffer (1% sodium citrate, 0.1%

Apoptosis

A bacterial clone expressing the recombinant protein was grown overnight in 5ml of LB medium with 50ug/ml ampicillinand the next day culture was transferred into 500ml of LB medium with 50 ug/ml ampicillin. Bacterial culture was allowed to grow at 37°C until reached an OD600 of 0.6-0.8 and then induced with IPTG (1 mM) for 4 h at 37°Cor overnight at 16°C to induce fusion protein expression. Subsequently, bacterial culture was centrifuged at 4000 RPM for 10 minutes (At this point bacterial cell pellets can be stored at -20° C for later use). The bacterial cell pellet was washed once with PBS and resuspended in 10ml of lysis buffer (1X NETN and protease inhibitors) followed by sonication (45 sec. pulse was given three times). The cell lysate was centrifuged for 10 minutes at 14000 RPM. The supernatant containing the GST fusion proteins was added to the 200μl of 50% GST/MBP beads. After 2 hours of incubation at 4°C, beads were washed three times with 1 ml lysis buffer. Purified proteins were eluted by using GST/MBP elution buffer. The recombinant proteins were analyzed by SDS-PAGE followed by either Coomassie staining orwestern blotting

GST/MBP recombinant protein purification from bacteria

protein agarose beads for 1 hour at 4 c and then washed three times with 1X NETN. The proteins bound to S-protein agarose beads were resolved by SDS-PAGE and visualized by Coomassie staining. Proteinspresent in the gels were analyzed by Mass Spectroscopy

HEK293T cells were transfected with S-protein/FLAG/SBP (streptavidin binding protein)-triple tagged WWP2/Dvl2 and then three weeks later puromycin-resistant colonies were selected and screened for WWP2/Dvl2 expression. The positive stable cells were then maintained in RPMI1640 supplemented with 10% FBS and 2 g/ml puromycin. The stable cells(harvested cells from ~30 tissue culture plates of 10cm size)were collected in 1X PBS by scraping them off the plates, and were lysedwith NETN buffer (20 mM Tris-HCl,pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 % Nonidet P-40) containing 50 mM -Glycerophosphate, 10 mm NaF, 0.5 mM PMSF, 1 g/ml of each Pepstatin and Aprotinin on ice for 20 minutes. After removal of cell debris by spinning, cell lysates were incubated with streptavidin sepharose beads for 1 hour at 4C. The bound proteins were washed three times with 1X NETN and then eluted twice with 2 mg/ml Biotin for 60 minutes at 4 C. The eluateswere incubated with S-

Tandem affinity purification

final supernatant, beads were boiled in the equal volume of 2X SDS loading buffer (Lamelli Buffer) for 5 min. at 95°C. Bound protein complexes were collected by brief centrifugation (1 min.) at 14000 RPM and the supernatant containing the eluted protein fraction was resolved in SDS gel and analyzed by western blotting technique. For denaturing immunoprecipitation, cells were harvested in 1X PBS andpelleted. Cell pellet was resuspended in denaturing lysis buffer (50mM Tris-HCL, 100mM β-mercaptoethanol, 1% SDS, 5mM EDTA,0.5mM PMSF, 1 mg/ml aprotinin and 1 mg/ml pepstatin) (200μl for 100mm culture dish) and boiled for 10 min at 99°C. Cells suspensionwas briefly sonicated. Ice-cold 1X NETN (800μl) was added to the suspension after sonication and incubated on ice for 20min.,cells were centrifuged at 14000 rpm for 10 min. The supernatant was collected and used for immunoprecipiation by following the standard protocol

Protein-A or protein G beads (50 μl of 50% agarose beads) were washed twice with NETN lysis buffer. 2-5 μg of specific antibody was added to beads (in 1ml NETN) and were incubated with beads for 1 h at 4°C on a rotary shaker. Beads were collected by spinning at 500 g for 2 min.and supernatant was removed. 200-600 μg of totalprotein (mammalian cell lysate or bacterial cell lysate) was added to the beads and incubated for 2 hrs at 4°C on a rotary shaker. Beads were washed four times with the lysis buffer, each time by centrifuging at 500 g for 2 min at 4°C. After discarding the

Immunoprecipitation

lysed with ice-cold NETN lysis buffer (20mM Tris-HCl, pH 8.0, 100mM NaCl, 1mM EDTA, 0.5% Nonidet P-40) containing protease inhibitors (0.5 mM PMSF, 1 mg/ml aprotinin and 1 mg/ml pepstatin) for 30 min.on ice, and then centrifuged at 14000 RPM for 10 min. at 4°C. Protein concentration in the supernatant (cell lysate) was estimated by Bradford assay. 20ug of the protein lysate was mixed with6X SDS loading dye (100 mMTris pH6.8, 4% SDS, 0.2% bromophenol blue, 20% glycerol and 200 mMβ-mercaptoethanol), boiled for 5 minutes at 99°C. Protein samples were resolved electrophoreticallyon SDS-polyacrylamide gels and transferred onto PVDF-membrane (Amersham Biosciences) using a semi-dry transfer apparatus (Biorad) for 1h at a constant current of 500 mA. Membranes were blocked for 30 min.with 5% non-fat dry milk powderdissolvedin 1X PBS and then immunoblotted overnight with primary antibody diluted in blocking buffer. Membranes were washed four times (each 5 min.) with 1X TBST followed by incubation with secondary antibody conjugated with horseradish peroxidase (HRP) for one hour at room temperature. The membrane was washed four times with 1X TBST (each wash for 5 min.), and the specific proteins on the membrane were detected by using enhanced chemiluminescent (ECL) reagents in 1:1 ratio (Amersham)

At 24 or 48 h of transfection, cell culture media was removed, cells were collected in 1X PBS by scraping them off the plate. Cells were harvested by centrifugation at 4000 RPM for 5 min.at 4° C. Pellet was washed twice with ice-cold 1X PBS and

Western blotting or immunoblotting

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

Cells were plated in a manner that they were 30-50% confluent on the day of transfection.Cells were washed with serum-free medium,and the serum-free medium was added to the cells as per plate size. SiRNA was diluted in the serum-free medium, and oligofectamine was diluted in serum-free media, separately (Table 10). Both the complexes were incubated at room temperature for 5 min. Diluted siRNA wasmixed gently with diluted oligofectamine and incubated at room temperature for 15 min. The final transfection mixture was added dropwise to the cells and mixed properly by gentle rocking. Cells were incubated for 4 hrs.,and the growth medium containing 10% FBS was added to the plates without removing the previous medium. Cells were incubated overnight at 37°C in a CO2 incubator. After overnight incubation, the siRNA transfection was repeated using the same protocol. Cells were harvested after 24-48 hours of second round siRNA transfection. The knockdown was detected bychecking the protein levels throughwestern blotting. (Note: SiRNA transfection is carried out in antibiotic free medium)Table 10: SiRNA transfection methodology

SiRNA

RNA interference

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

Table 8: Lipofectamine plasmid-transfection methodology

For transfection with Lipofectamine, cells were plated in antibiotic-free medium 24 h before transfection and were transfected at a confluency of 70-80% as per the manufacturer’s protocol. The plasmid of interest was incubated in serum free media,and Lipofectaminewas incubated in serum free media forseparately5minutes. The plasmid and the Lipofectamine mixtures(Table 8)were mixedgentlyand incubated at room temperature for 20 min.;thetransfection mixture was added dropwise to the cells. Transfection media was replaced with the fresh complete medium after 6 hrs.of transfection and cell are harvested after 24 hours

Plasmid transfection using Lipofectamine 2000

293T cells or HeLa cells were transfected with various plasmids as per the designed experiments using Lipofectamine 2000 (Invitrogen) reagent or PEI

PlasmidTransfectionof mammalian cells

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

PCR product was incubated with 1ul of Dpnl restriction enzyme for 2-3 hours at 37ºC, following which Dpn1 treated PCR product was transformed into DH5α competent bacterialcells. Mutant colonies were screened andconfirmed using DNA sequencing

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

PCR product was incubated with 1ul of Dpnl restriction enzyme for 2-3 hours at 37ºC, following which Dpn1 treated PCR product was transformed into DH5α competent bacterialcells. Mutant colonies were screened andconfirmed using DNA sequencing

The various mutant plasmids were generatedbyusing PCR-based site-directed mutagenesis protocol (Stratagene).Briefly, primers carrying the desired nucleotide changes were used in a PCR reaction to amplify the nascent mutantplasmids from the wild type parent plasmid. The PCR reaction was set up according to manufacturer’s protocol(Table 5)using a high-fidelity Pfu DNApolymerase and donor-plasmids of the desired geneas the template. Following reactionmixture and the cycling conditions were used for site-directed mutagenesis

Site-directed mutagenesis

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

Amplified PCR products were run on Agarose gel to check for the amplification ofgene of interests

PCR amplification of the gene of interests was carried out by following the method mentioned in table 4.Table 4: PCR methodology

Polymerase chain reaction (PCR)

Four week old tomato S-22 cultivar (acts as non-host for Xanthomonas oryzae pv. oryzicola) were syringe-infiltrated with a suspension of Xocstrains and water control. Plants were incubated in green house for 24 h with minimum and maxium temperature of 26 and 28°C, respectively and relative humidity of 65%. Callose deposition assay was performed as a marker for hypersensitvity response in non host plant as described previously (Hauck et al., 2003). Leaf picture was captured at this stage to observe the HR browning of leaf. For assaying callose deposition by aniline blue staining, infilterated leaves were removed from plant,dipped in lactophenol solution and incubated at 65°C in water bath until the cholorohyll is completely removed. Leaves were rehydrated by washing with 50% ethanol, and finally rinsed with water. For aniline blue staining, leaves were incubated in 0.01% aniline blue solution, prepared in 100 mM K2HPO4(pH 9.5), for 15-20 min in dark. Subsequently, leaves were washed with water and observed for callose deposition in epifluorescence microscope (Stereo, Lumar V7, Zeiss) under UV illumination

In plantahypersensitive response (HR) and callose deposition assay

flash (Thermoscientific). β-Glucuronidase activity for GUS was expressed as nanomoles of MU produced/minute/108 cells

In planta siderophore gene expression was studied by measuring β-glucuronidase activity. GUS marked BXOR1 strain and wild-typeBXOR1 (control) were inoculated in the leaves of 14 day old susceptible Taichung Native 1 (TN-1) variety of rice. After 10 days of inoculation, leaves were crushed and dissolved in 1 ml of MUG extraction buffer without adding MUG substrate (4-methylumbelliferyl β-D-glucuronide). Subsequently, 250 μl extraction buffer containing MUG was added, and incubated at 37°C for appropriate time (Jefferson et al., 1987). Next, 75-μl aliquots were taken from each reaction mixture, and the reaction was terminated by the addition of 675 μl Na2CO3 (0.2 M). Fluorescence was measured against 4-methyl-umbelliferone (MU; Sigma) as standard at excitation/emission wavelength of 365/455 nm, respectively in

In plantaGUS expression assay for siderophore cluster

grown culture was inoculated in fresh PS medium with or without 50 μM 2, 2’-dipyridyl and grown at 28°C. At regulartime intervals, 1 ml culture was removed to determine OD at 600 nm. Furthermore, for GUS assay, 1 ml culture was centrifuged to obtain the pellet, which was washed once in sterile miliQ water, and resuspended in 250 μl volume of 1 mM MUG (4-methylumbelliferyl β-D-glucuronide) extraction buffer (50 mM sodium dihydrogen phosphate [pH 7.0], 10 mM EDTA, 0.1% Triton X-100, 0.1% sodium lauryl sarcosine, and 10 mM β-mercaptoethanol),and incubated at 37°C (Jefferson et al., 1987). After appropriate time intervals, 75 μl aliquotes were taken from each reaction mixture, and reaction was terminated by adding 675 μl Na2CO3 (0.2 M). Fluorescence was measured against 4-methyl-umbelliferone as the standard at excitation/emission wavelength of 365/455 nm, respectively. Likewise, GFP activity was measured in Varioscan flash (Thermoscientific) at exitation/emission wavelength of 472/512 nm, respectively by taking 200 μl of culture directly

For reporter assay, GUS and GFP marked Xanthomonas oryzaepv. oryzicolastrains and control strains were grown overnight in PS medium. 0.2

Reporter assays with β-Glucuronidase (GUS) and green fluorescent protein (GFP)

2 bed volumes of methanol, and equilibrated with 5 bed volumes of distilled water. In order to reduce the water solubility of siderophore in the supernatant, it was acidified to pH 2 using concentarted HCl. This acidified supernatant was passsed through the column, and finally eluted with 160 ml methanol by collecting approximately 60 fractions (2 ml each) of the flow through. Siderophore assay was done on CAS plate with each collected fraction. Fraction that gave orangish-yellow halo for the siderophore on CAS plate, was combined together, dried in rotary evaporator and finally reconstituted in 1 ml methanol for further quantification using HPLC as described previously (Amin et al., 2009).For HPLC analysis, siderophore samples were filtered through filter membrane (porosity, 0.45 μ). Next, 10 μl sample was injected into Agilent C18 (4.6mm×250mm×5μm) column (gradient:(A=H2O/0.1%TFA), (B= CH3CN/0.1%TFA) 0-30% B in 10 min, 30-45% B in 15 min,45-0%B in 20 min at a flow rate of 1 ml/min). Similarly, standard vibrioferrin (siderophore produced by Xanthomonas) was also estimated through HPLC for comparison. Fe(III) bound vibrioferrin complex was prepared by incubating FeCl3.6H2O and apo-vibrioferrin for overnight. This complex was detected at300 nm (RT 10.998 min), whereas apo-vibrioferrin was detected at 220 nm at RT 10.988 min. The siderophore concentration in the samples were determined by peak area and calculated against the standard curves obtained from standard vibrioferrin. The siderophore from the test samples were detected at 300 nm, which confirms that majority of the vibrioferrin isolated from the culture was present in bound form

Different Xanthomonas oryzaepv. oryzicola strains were grown overnight in PS medium at 28 °C and 200 rpm. 0.2% of the overnight grown culture was inoculated in the the fresh PS medium supplemented with 50 μM 2, 2’-dipyridyl, and grown till OD600 reached to 1. Cultures were centrifuged at 12,000 g for 50 min to get the cell free culture supernantant, which was collected into acid treated bottles. Excess exoplysaccharide was removed by centrifugation for longer time. Siderophore was initially isolated by column chromatography as described previously (Wright, 2010). Briefly, 220 g of XAD-16 resin was soaked overnight and packed into the column (2.4×30 cm), column was wa

HPLC based siderophore estimation from culture supernatant of different strains of X. oryzaepv. oryzicola

concentarion in the samples were determined based on their peak area against standard oxalic acid plot.For GC-MS analysis, N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) derivatization was performed with the dried HPLC fraction of samples as well as standards as described previously (Šťávová et al., 2011). Briefly, 200 μl BSTFA, and 100 μl hexane were added to the sample, and incubated at 50 °C for 70 min. GC analyses were performed using a Shimazdu GP 2010 plus instrument equipped with an autosampler, and a split injector.Separations were accomplished using a 30-m long DB-5 capillary column, 0.25 mm internal diameter (I.D.) at a constant helium flow rate of 1.5 mL/min. Samples (10 μL) were injected with a split ratio of 10 into the column at 100 °C. The final column temperature program started at 100 °C and attained final temperature 280°C with a gradient increase of 5 °C/min. The MS data (total ion chromatogram,TIC) was acquired in the full scan mode (m/z of 50–500) at a scan rate of 1000 amu using the electron ionization (EI) with an electron energy of 70 eV. The acquired spectrum was searched against standard NIST-05 library

Xanthomonas oryzaepv. oryzicola strains were grown overnight in PS medium supplemented with appropriate antibiotics. 0.2 % of the overnight grown culture was reinoculated in 250 ml of fresh PS medium supplemented with 50 μM 2,2’-dipyridyl, and allowed to grow till OD600reached 1. Cultures were centrifuged to obtain cell free culture supernatant, concentrated on vaccum evaporator, and freeze dried at regular time intervals to remove the water completely. Oxalic acid was estimated from the dried supernatant by using Agilent 1100 series HPLC system as described previously with slight modifications (Ding et al., 2006). In brief, dried supernatant fractions of different cultures were dissolved in mobile phase of pH 2.7, and allowed to stand for 3 h for the precipitation of humic substances. These samples were filtered through membrane filter (porosity, 0.45 μm), and 20 μl volume of the filtrate was injected into the Agilent C18 (4.6 mm× 250 mm× 5 μm) column. The mobile phase used was 10 mM KH2PO4-CH3OH (95:5, pH 2.7), and the samples were separated by isocratic elution at 0.8 mL/min at 26°C temperature. Standard oxalic acid was detected in similar way in mobile phase (pH 2.7 at 210 nm) with retention time (RT) of 6.7 min. Likewise, oxalic acid in the test samples were also detected at 210 nm with RT 6.7

Oxalic acidestimation from culture supernatant of different strains of X. oryzaepv. oryzicola by HPLC and GCMS analysis

CFUs/ml) onto fully expanded leaf, and pricking with sterile needle to facilitate the entry of bacteria inside the leaves through wound. To detrmine the growth of bacteria inside leaves, 1 cm2 leaf area surrounding the inoculation site was cut at regular time intervals, surface sterilized by dipping in 2% (vol/vol) sodium hypochlorite for 2 min, and washed twice in sterile water. For getting the CFUs, leaves were crushed using mortar and pestle, serially diluted, and plated on PSA medium containing appropriate antibiotics

Exogenous iron supplementation was performed as described previously (Chatterjee and Sonti, 2002). Briefly, leaves of 40-day-old greenhouse-grown rice plants of the susceptible rice cultivar Taichung Native-1 (TN-1) were cut with scissors 2 cm above the junction of the leaf blade and leaf sheath. These cut leaves (25 leaves per flasks) were dipped in 250 ml conical flasks containing 200 ml 1μg/mlof Benzyl amino purine (BAP) in double distilled water. BAP (a cytokine hormone) maintain the detached rice leaves in fresh condition for longer period. For iron supplementation, FeCl3 was added to a final concentration of 50 μM (stock-10 mM). Prior to inoculation with different strains of Xanthomonas oryzaepv. oryzicola, the leaves were maintained overnight on a laboartory bench top. Strains were inoculated into the leaves by needle pricking method by dropping 20μl of bacterial suspension (approx. 1 × 108bacterial

Exogenous iron supplementation and bacterial growth assay in rice leaves

Ferric-iron-reduction activity of Xanthomonas oryzaepv. oryzicolawas measured using ferrozine, a chromogenic ferrous iron chelator, as described previously (Velayudhan etal., 2000; Worst et al., 1998). For estimating the ferric reductase activity, Xanthomonas oryzaepv. oryzicolastrains were grown in 20 ml PS medium carrying appropriate antibiotics for 24 h to OD600 of 1. Cell free PS media was incubated under similarcondition to be used as control. Chromogenic ferrous iron chelator, ferrozine was added to a final concentration of 1 mM, and FeCl3was added as ferric iron source to a final concentration of 50 μM, and incubated at 28ºC. At regular time intervals, 1 ml aliquotes were taken from the test culture and control, centrifuged to remove the cells, and absorbance of the magenta coloured Fe2+-ferrozine complex in the cell free culture supernantant was measured at 535 nm by using control supernatant as reference. The Fe2+reduction activity was quantified as micromoles of Fe2+-Ferrozine complex formed

Assay for ferric reductase activity

media containing 50 μM 2’2’-dipyridyland grown for 24 h at 28°C with continuous shaking at 200 rpm. Cells were harvested by centrifugation at 7000 g for 10 min at 4 °C, washed twice with 50 mM phosphate buffer (pH-7.4), and finally resuspended in phosphate buffer. The bacterial suspension was then diluted with chelex-100 treated PS to get a final OD600of 1.0 and incubated at 28°C for 5 min. Iron transport assay was initated by adding 55FeCl3(American radiolabeled chemicals, Inc., St. Louis, USA,specific activity 10.18 mci/mg) to a final concentration of 0.4 μM into the bacterial suspension. The radiolabelled stock solution was diluted with water and 1M sodium ascorbate for 55Fe3+uptake and 55Fe2+uptake studies, respectively. For uptake of FeCl3bound vibrioferrin, both vibrioferrin (7.6 mM stock) and 55FeCl3were incubated in 1:1 ratio by diluting it appropriately with water and uptake was initiated with a final concentartion of 0.4 μM. To stop the uptake, 200 μl of bacterial cell suspension was layered and immediately centifuged (13000 g; 1 min) through 300 μl of di-butylphthalate and di-octyl phthalate (1:1) mixture. The upper aqueous layer and organic solvent was aspirated, and pellet was resuspended in 100 μl Triton-X-100. The suspension was added to 5 ml scintillation cocktail, and radioactivity count was determined in the 3H channel of scintillation counter (Perkin Elmer, Liquid Scintillation analyzer, Tri-Carb 2910 TR, USA). As control, both Fe2+and Fe3+uptake assays were performed in presence of proton motive force uncoupler carbonylcyanidep-trifluoromethoxyphenylhydrazone (FCCP; 50 μM), to distinguish between non-specific uptake of readiolabelled Fe by the bacterial cells. However, no significant increase in the incorporation of Fe2+and Fe3+ was observed in presence of FCCP, which indicated that iron uptake by these strains is energy-dependent process

In vitro transport assay was performed by using radiolabelled iron to measure the capacity of Xanthomonas oryzaepv. oryzicola strains to transport 55Fe(II) and 55Fe(III) forms of iron as described previously with slight modifications (Ardon et al., 1997; Velayudhan et al., 2000). For iron uptake asssay, Xocwild-type BXOR1 strain, ∆rpfF mutant and the complemented strain harboring full length rpfFgenewere grown overnight in PS medium. 0.2% of the overnight grown culture was inoculated in fresh P

55Fe uptake assay

Intracellular iron content in different Xanthomonas oryzaepv. oryzicolastrains was measured by using atomic absorption spectroscopy as described previously with few modifications (Velayudhan et al., 2000). For estimation of intracellular iron, different strains of Xanthomonas oryzaepv. oryzicolawere grown overnight in 3 ml PS media with appropriate antibiotics for differentially marked strains. 0.2% of the overnight grown culture was inoculated in 250 ml PS medium alone or PS plus 2, 2’-dipyridyl for iron stravation, and grown to an OD600 of 1.2. Cells were then pelleted down by centrifuging at 7000 g for 10 min, and washed twice with phosphate buffer saline (PBS). After washing, cells were lyophilized, and their dry weights were determined. Lyophilized cells were then dissolved in 30% nitric acid at 80ºC for 12 h and diluted 10-fold with miliQ water. Iron content was determined by atomicabsorption spectroscopy using ICP-OES (JY 2000 sequential Inductively Coupled Plasma Optical Emisson spectrometer,Jobin Yvon, Horiba, France). Iron level was quantified against aqueous standard of iron traceable to NIST (National institute of standards and technology, India)

Estimation of intracellular iron content

culture dishes and dishes were incubated at 28ºC. OD600 was measured after 16 and 42 h of incubation, and percentage inhibition of growth was determined with respect to the growth in the corresponding control cultures containing PS media without streptonigrin

For streptonigrin sensitivity assay, different strains of Xanthomonas oryzaepv. oryzicolawere grown overnight with appropriate antibiotics as described earlier. 0.2% of primary inoculum was added into fresh PS medium and grown for 24 h till the OD600reached 0.6. Serial dilution of bacterial cultures were performed as mentioned earlier, and 5μl diluted cultures were spotted on PSA plates containing different concentration of streptonigrin (0.05 μg/ml, 0.1 μg/ml and 0.15 μg/ml). Plates were incubated at 28ºC for 72 h and plate images were captured and analyzed for comparative growth inhibitionin different strains caused by streptonigrin. Further, streptonigrin sensitivity assay in liquid broth was performed by growing different strains as described previously (Wilson et al., 1998).Briefly, Xanthomonas oryzae pv. oryzicolastrains were grown to an OD of 1 in PS medium with appropriate antibiotics. Cells were pelleted down, and resuspended in fresh PS medium at an OD600of 0.6. Next, 100 μl culture was inoculated in 4 ml PS medium with or without streptonigrin. Streptonigrin was added to a final concentration of 0.1μg/ml into

Streptonigrin sensitivtity assay

ForEPS isolation,X. oryzaepv. oryzicolastrains were plated on PS agar plateand incubated at 28°C. Bacterial lawn was dissolved in 15 ml 1X PBS and 100 μl formamide, and centrifuged at 12,000 g for 6-8 min at RT. Before centrifugation, 1 ml cell suspension was diluted, and plated to get the CFUs. For EPS precipitation, 250 ml chilled acetone was added to the supernatant, and kept at 4°C for overnight (Dharmapuri and Sonti, 1999). EPS was pelleted down at 7000 g for 10 min at 4°C, washed with 10 ml acetone, and kept for drying. After drying, it was dissolved in appropriate volume of water, and quantitated by colorimetric method for estimation of pentoses and hexoses by phenol-sulphuric acid method (Dharmapuri and Sonti, 1999)

EPS isolation and estimation

For biofilm and attachment assays, Xanthomonas oryzaecells were grown in PS media with appropriate antibiotics at 28°C with constant shaking at 200 rpm. 0.2% of the overnight grown culture was inoculated into the fresh PS media and grown till the OD reached 0.6-0.7 at 600 nm. 4 ml culture was inoculated into 12 well polystyrene culture plates, and incubated for 24 h and 48 h at 28°C without shaking. After 24 h, cultures were discarded, and wells were washed with 4 ml of water to remove loosely attached cells. The adherence was examined by staining the cells with 1% crystal violet solution for 30 min at room temperature. After incubation, excess crystal violet stain was removed by washing the wells with 3 ml water. Images were captured for visualizing the stained biofilm on polystyrene plate. Finally, crystal violet stained biofilm was dissolved in 80% ethanol, and quantified by taking OD at 560 nm. Similar procedures were repeated for the polystyrene plate with culture incubated for 48 h. For attachment, cells were grown similarly in 12 well polystyrene culture plates for 24 h, rinsed once with sterile water to remove loosely attached cells then attached cells were collected by vigorous washing with sterile water. Attached cells were diluted, and plated to get the CFUs

Static biofilm and attachment assay

In planta growth assay for different strains of Xanthomonas oryzaepv. oryzicolawas performed by counting CFUs. For getting the CFUs, 1 cm2 leaf area surrounding the site of inoculation was cut and surface sterilized by dipping the leaf in 1% (vol/vol) sodium hypochlorite for 2 min followed by three washes with sterile water. To get the CFUs, sterilized leaves were crushed using mortar and pestle, and diluted appropriately for plating on PSA plate containing suitable antibiotics for differentially marked strains

In plantabacterial growth assay

To study the virulence of Xanthomonas oryzaepv. oryzicolastrains on rice plant two different inoculation methods, syringe infiltration and wound inoculation methods, were implimented. For infiltration method, bacterial suspension comprising of 1 × 108 cells/ml were infiltrated with needleless syringe into leaves of 4 to 6 week-old rice cultivar of susceptible Taichung Native-1 (TN-1) (Hopkins et al., 1992; Wang et al., 2007). Wound inoculation method was carried out by dropping an aliquot of 20 μl bacterial suspension comprised of 1 ×108cells/ml onto fully expanded leaf of 6-8 week green-house grown Taichung Native-1 cultivar of rice, and pricking with sterile needle for facilitating the entry of Xocinside the leaves throgh wound. For inititation of disease symptom, the inoculated plants were incubated in greenhouse with minimum and maximum temperatures of approximately 25 to 30 °C, respectively, and a relative humidity of approximately 60%. Water soaking symptom and lesion development was measured 4 to 10 days after inoculation. Likewise, for infiltration by wound inoculation method, lesion length was measured 14 days after inoculation. In both the cases, no lesions were observed in control experiments in which the leaves were inoculated with sterile wate

Virulence assay on rice plant

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

For determining the motility of Xanthomonas oryzae pv.oryzae strains, swim plate assay was performed as described previously (Robleto et al., 2003; Tremaroli et al., 2010)with slight modifications. Briefly, swim plates were prepared with PSA medium containing 0.1% agar. For motility assay, cells were grown at a density of 109cells, which corresponds to an OD of 0.6. Cells were concentrated by centrifugation at 3000 g for 5 min, washed and resuspended in 1/10 volume of sterile water. 5 μl cell suspension was inoculated at the center of the swim plates and incubated for 36-48 h at 28°C. Toget the quantitative measurement of the motility of each strain, diameter of the motility zone was determined at appropriate time point

Motility assay

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

development. Absorbance was measured at 490 nm, and concentration of glucose production was calculated against glucose standard. Cellulase activity is expressed as micromoles of reducing sugar (glucose) released per minute per 109cells. For plate assay, cell-free culture supernatant of X. oryzaepv. oryzaestrains were inoculated in wells of 0.2% CMC agarose plates. In addition, cellulase assay was also performed by spotting the colony on 0.2% CMC PSA plates. Plates were incubated for 8 to 24 h and stained with congo red to observe the halo formation as described previously (Wood and Bhat, 1988). Extracellular xylanase activity in different X. oryzaepv. oryzae strains was measured using 0.2% 4-O-methyl-D-glucurono-D-xylanremazol Brilliant Blue R (RBB-Xylan) (Sigma-Aldrich) as substrate (Biely et al., 1988)on 1% agarose plates. Xylanase activity is indicated by production of halo around the bacterial colony (Ray et al., 2000). Similarly, for lipase activity p-nitrophenyl butyrate was used as substrate. Lipase activity was calculated by measuring the level of p-nitrophenol released upon hydrolysis of p-nitrophenyl butyrate at 410 nm (Acharya and Rao, 2002). Lipase activity was expressed as micromoles of p-nitrophenol released permin per109cells. For plate assay, colonies were spotted on 1% PSA plates containing 0.5% Tributyrin in 100 mM Tris (pH 8) and 25 mM CaCl2 and halo formation was observed for lipase activity

For extracellular enzyme assays, X. oryzaepv. oryzae strains were grown in PS, MM9 and XOM2 media to an OD of 0.6, and centrifuged at 12,000 g for 10 min to collect the supernatant. The supernatant was taken as an extracellular fraction and cell pellet was plated by dilutionplating to get the CFUs per milliliter of culture. Extracellular cellulase activity was measured using phenol-sulphuric acid (H2SO4) method, which measures pentoses and hexoses (concentration of glucose released) upon cellulase activity (DuBois et al., 1956). Briefly, a specific amount of supernatant was taken and volume was adjusted to 300 μl by adding 50 mM acetate buffer (pH-5.4). To this, 1% carboxy methyl cellulose (CMC) substrate solution was added and mixed well. This mixture was incubated at 28°C for 30 min, and the reaction was stopped by adding 1 ml ice-cold ethanol. Solution was mixed well, kept on ice for 5 min and centrifuged at 12,000 g for 5 min. Supernatant was recovered and 5% phenol was added to it, mixed well followed by adding 1 ml H2SO4. The tube was incubated at RT for 20 min for co

Extracellular enzyme assays

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

A microtipful cells of bacterial strain from appropriate medium was resuspended in 20 μl sterile water and incubated at 98°C for 10 min for cell lysis. 2 μl of heat-lysed cell suspension was used as template in 25 μl PCR reaction