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)

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

SiRNAandshRNA

All the primers (sequences)used for cloning the above-mentioned genes are providedin AppendixI

using gateway cloning method (Invitrogen). P73domain deletions were cloned in SFB destination vector. WWP2, WWP1, HACE1, E6AP, and PPM1G were cloned into SFB (S-protein/Flag/streptavidin binding protein (SBP) triple tag), GFP,and Myc mammalian destination vectors using the Gateway cloning technology (Invitrogen). WWP2 domain deletions were cloned into Myc-destination vector. WWP1 domain deletions were cloned into SFB-destination vector. PPM1G domain deletions were cloned into SFB mammalian destination vector using Gateway cloning. Bacterially expressing GST-p73, GST-∆Np73, GST-PPM1G, MBP-WWP1, MBP-WWP2, GST-WWP2, GST-WWP1 and GST-HACE1 were generated by using gateway technology. Ubiquitin WT and all the mutants were cloned into hemagglutinin (HA) mammalian destination vector. Flag-tagged Dvl2was purchased from Addgene. Dvl2 domain deletions were cloned into SFB-destination vectors. All the plasmid constructs generated in the present study are mentioned in table 2.Table 2: Plasmids used in the study

TAp73α and ∆Np73α were kindly gifted by Alex Zaika, Vanderbilt University. Full-length p73 and ∆Np73 were cloned into Myc and HA mammalian destination vectors

Expression plasmids

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

Antibodies

The following chemicalswere used in the present study: Ampicillin, EDTA (USB), dNTPs, Taq DNA polymerase(Fermentas), Pfu DNA polymerase (Stratagene), DpnI (New England Biolabs), Plasmid miniprep, midiprep, and maxiprepkits(Qiagen, and Invitrogen), glycine, EGTA, NaCl, Tris (Fisher Scientific), NH4Cl, acrylamide(SRL), Cisplatin, Doxorubicin, MG132,Cadmium chloride,Nonident P-40, propidium iodide (PI),bis-acrylamide, SDS, TEMED, Ammonium persulphate (APS), CoomassieBrilliant Blue, DAPI, IPTG, kanamycin, Aprotinin, pepstatin, PMSF, -Glycerophosphate, Sodium Fluoride (NaF),Biotin, and DMSO(Sigma), Luciferase assay kit (Promega#1500), Gateway cloning kit, DMEM, FBS, RPMI, Opti-MEM medium,Met-/Cys-DMEM, dialyzed FBS,trypsin-EDTA, L-glutamine, PBS, Lipofectamine 2000, Oligofectamine, (Invitrogen), PEI(Polysciences), milkpowder (Warana), protein G agarose beads, Streptavidin sepharose beads, Glutathionesepharose beads, MBP beads (GE Healthcare), S-protein beads (Novagen/Calbiochem), HA beads(Covance), LB media(Himedia)

Chemicals and reagents

Laboratory of Cell Death & Cell SurvivalCentre for DNA Fingerprinting and DiagnosticsHyderabad-500 001, India

Neelam Chaudhary

Studies on functional interactome of WWP2: AnHECT-typeE3 ubiquitin ligase

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

Xanthomonasand E.colicolony PCR

and finally resuspended in 100 μl sterile water. Bacterial cell suspension was aliquoted in 20 μl volume. The above procedure was followed for all the three strains and cell suspension of three different strains were mixed together in 1:1:1 ratio. For conjugation to occur, 20 μl of the above mixture was spottedon the LB agar plate and incubated at 37°C for 12-16 h. Next, the conjugation drops were streaked on LB agar plate containing appropriate antibiotics to select the S17-1 recipient containing recombinant plasmid.S17-1 was directly conjugated with Xanthomonasstrain. S17-1 strain containing recombinant plasmid (3 ml) and recipient Xanthomonasstrain (100 ml) was grown overnight with appropriate antibiotics. Cells were harvested and washed thrice as mentioned earlier. Xanthomonasstrain was finally dissolvedin 600-700 μl sterile water and S17-1 strain was dissolved in 3 ml sterile water. 50 μl Xanthomonascell suspension and 10 μl S17-1 cell suspension were mixed together and 20 μl was spotted on PS agar plate. After 40 h of incubation at 28°C, each conjugation drop was dissolved in 400 μl water separately and plated on PS agar medium with rifampicin (counter-selectable marker) and plasmid specific antibiotics for specific selection of Xanthomonascolony with recombinant plasmid

Since compatible conjugation does not exist between Xanthomonasand E.coliDH5α strain.Therefore, upon getting the appropriate clones in DH5α, conjugation was performed with S17-1 (recipient strain) and PRK600 (helper strain). All the three strains (DH5α with clone, S17-1 and PRK600 strain of E.coli) were grown overnight at 37°C with constant shaking at 200 rpm in 3 ml LB broth. Cells from 1 ml overnight grown cultures were harvested by centrifugation followed by three washes with s

Xanthomonas conjugation

shaking at 200 rpm. 1% of overnight grown culture was inoculated in 100 ml fresh PS medium and grown to obtain log-phase culture. Log phase Xanthomonas culture was kept on ice for 10-15 min, aliquoted in 50 ml pre-chilled centrifuge tubes and centrifuged at 4000-5000 g at 4°C for 10 min. Supernatant was discarded and pellet from each tube was gently resuspended in 10-20 ml sterile chilled water. Next, cells were harvested by centrifugation at 4000 g at 4°C for 10 min and supernatant was discarded. Harvested cells were washed twice and finally resuspended in adequate amount of prechilled sterile water. 100 μl of cell suspension was aliquoted in sterile 1.5 ml microcentrifuge tubes and kept on ice. For transformation, Xanthomonaselectrocompetent cells and appropriate amount of plasmid DNA was mixed, and kept on ice in laminar hood. This mixture was added to 1 mm electroporation cuvettes (Biorad) and tapped gently to allow the cells to settle properly in order to avoid air bubbles. Competent cells were electroporated (1800 V, 25 μF, 200 Ω, 1mm cuvette) followed by immediate addition of fresh PS broth in the cuvette, mixed properly and taken in the microcentrifuge tubes. Microcentrifuge tubes containing transformed cells were incubated at 28°C for 2 hours with continuous shaking for recovery. After recovery, cells were plated on specific medium with appropriate antibiotics and incubated in 28°C plate incubator

For electrocompetent cell preparation, single colony of desired Xanthomonasstrain was inoculated in 5 ml PS medium and grown overnight at 28°C

Xanthomonastransformation

E.coliDH5α strain was transformed with plasmids carrying appropriate inserts to generate clones, and Xanthomonas deletion strains. Ultracompetent cells stored at -80°C were thawed on ice for 5-10 min. 5 μl ligated plasmid was added to 100 μl ultracompetent cells and incubated on ice for 30 min. Next, competent cells were subjected to heat shock at 42°C for 90 seconds. Cells were immediately transferred on ice for 2-3 min. Next, 1 ml LB medium was added and cells were allowed to recover for 1 h on a shaker incubator set at 37°C. After the recovery, cells were centrifuged at 3000 g for 3 min. Medium supernatant was discarded and cells were resuspended in 100 μl fresh sterile medium. Cells were plated on LB agar containing appropriate antibiotics. Plates were incubated at 37°C for 12-16 h

E.colitransformation

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

Preparation of E.coliultracompetent cells

Xanthomonas strains were grown in PS medium for 14-16 h at 28°C with continuous shaking at 200 rpm. 1 ml of bacterial cultures were ten-fold serially diluted in water and 100 μl volume of each dilution was plated on PS agar plates to get the colony forming units (CFUs). Similarly, 5 μl volume of each dilution was spotted on PS agar plates containing different concentration of streptonigrin and different detergents for intracellular iron and membrane sensitivity assay, respectively. Plates were incubated at 28°C and images were captured after 2-8 days of incubation depending upon m

Serial dilution plating and spotting assay

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

Growth analysis and determination of generation time

Xanthomonasstrains were routinely grown in rich PS medium, at 28°C with continuous shaking at 200 rpm unless otherwise stated (New Brunswick Scientific, Innova 43, U.S.A.). In general, Xanthomonas frozen glycerol stocks were revived on PSA medium by streaking,and allowed to grow for 3-4 days. To prepare liquid culture, a loopful of each Xanthomonasstrain was inoculated in PS medium and grown for 24-30 h. Xanthomonasstrains on plates were stored at 4°C for a maximum period of 1 week. For growth of X. oryzaepv. oryzaein Minimal (MM9); (Kelemu and Leach, 1990)and XOM2 media (minimal media which induces hrp genes in X. oryzaepv. oryzae),(Tsuge et al., 2002), first the strains were grown in PS medium to a cell density of 109cells/ml and then centrifuged at 5000 g to concentrate the cells and washed twice with sterile water to remove media components sticking to the cells. Washed cells were inoculated in MM9 and XOM2 medium and grown for overnight.Escherichia coliDH5α,used for cloning purposes, was revived on LB medium containing nalidixic acid and grown at 37°C with continuous shaking at 200 rpm. LB medium was supplemented with appropriate antibiotics to grow the bacterial strains carrying plasmids. For plasmid purification, bacterial strains were grown overnight in LB broth medium containing suitable antibiotics.Antibiotics were used at a final concentration of 50 μg/ml rifampicin, kanamycin, streptomycin and trimethoprim; 100 μg/ml ampicillin; 25 μg/ml nalidixic acid; 10 μg/ml cephalexin, chloramphenicol and gentamicin; 20 μg/ml cyclohexamide and 5 μg/ml tetracyclin

Strains and culture conditions

Microbiological methods

Liquid scintillation cocktail5 g PPO (2,5-diphenyloxazol)0.3 g POPOP (1,4-bis (5 phenyl 1,2-oxazole) Benzene Volume was adjusted to 1L with toluene.MUG (4-methylumbelliferyl β-d-glucuronide)extraction buffer1 mM MUG substrate50 mM Sodium dihydrogen phosphate (pH-7.0)10 mM EDTA0.1% Triton X-1000.1% Sodium lauryl sarcosine10 mM β-MercaptoethanolLactophenol solution (100 g)25 g Lactic acid (20.66 ml)25 g Phenol 50 g Glycerol (39.77 ml)These three components were mixed together and 1 volume of lactophenol was added to 2 volumes of ethanol

CAS solutiona) 0.06 g Chrome Azurol S dye in 50 mlb) Fe (III) solution: 10 ml1 mM FeCl310 mM HClc) 0.072 g HDTMA in 40 mlAll the above three solutions were mixed together and autoclaved prior to use

Other solutions

Transformation buffer I (Tfb-I) 30 mM Potassium acetate100 mM Rubidium chloride (RbCl2)10 mM Calcium chloride dihydrate (CaCl2.2H2O)50 mM Manganese chloride tetrahydrate (MnCl2.4H2O)15% (v/v) GlycerolpH was adjusted to 5.8 with 10% acetic acid and volume was adjusted to 500 ml with H2O. Transformation buffer II (Tfb-II) 10 mM MOPS75 mM CaCl2.2H2O10 mM RbCl2.2H2O15% GlycerolpH was adjusted to 6.5 with KOH (Potassium hydroxide) and volume was adjusted to 100 ml with H2O.

Buffers for E. colielectrocompetent cell preparation

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

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

Buffers for enzyme assaysCellulase assay

10% APS -30 μlTEMED -3 μlSDS loading buffer (2X)100 mM Tris-HCl (pH-6.8)20% (v/v) Glycerol4% (W/V) SDS0.02% Bromophenol Blue10% β-MercaptoethanolSDS-loading buffer was prepared as 2X stock solution in H2O and used at 1X concentration.SDS-PAGE running buffer14.4 g Glycine3.03 g Tris methylamine1 g SDSDissolved in H2O and volume was adjusted to 1L with H2O.Buffers for western blot analysisTransfer buffer (1 litre)14.4 g Glycine3.03 g Tris methylamine800 ml H2O 200 ml methanolBlocking and wash buffers (PBS-T)5% Fat-free milk0.05% Tween-20Volume was adjusted to 100 ml with1XPBS

Whole cell lysis buffer50 mM Sodium acetate 410 mg Sodium acetate anhydrous was dissolved in 80 ml H2O. pH was adjusted to 5.4 with glacial acetic acid and finally volume was adjusted to 100 ml with H2O.1 mM PMSF (phenylmethylsulfonyl fluoride) in isopropanol.Dialysis buffer50 mM Trizma basepH was adjusted to 7.5 by using concentrated HCl.Silver stainingFixing solution50% ethanol10% glacial acetic acid0.05% formaldehydeFinal volume was adjusted with sterile H2O.0.2% Silver nitrate solution (AgNO3)0.2 g AgNO3

0.075% formaldehyde (37% stock) Dissolved in 100 ml of H2O. Stored at 4°C for 1 hour in brown colored bottle.Developing solution 6% Sodium carbonate (Na2CO3)0.05% Formaldehyde (37% stock)0.02% Sodium thiosulphateStorage buffer50% EthanolSDS-PAGE30% Acrylamide solution29 g Acrylamide1 g Bis-acrylamideAcrylamide solution was prepared in H2O.Resolving gel mix (12%) (10 ml)H2O -3.3 ml30% Acrylamide:Bisacrylamide mix (29:1) -4 ml1.5 M Tris-HCl (pH-8.8) -2.5 ml10% SDS -100 μl10% Ammonium persulphate (APS) -100 μlN, N, N’,N’,-Tetramethylethylenediamine (TEMED) -4 μlStacking gel mix (5%, 3 ml)H2O -2.1 ml30% acrylamide:bisacrylamide mix (29:1) -500 μl1.5 M Tris-HCl (pH-6.8) -380 μl 10% SDS -30 μl

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

Buffers and solutions for protein extraction, analysis by SDS-PAGE (sodium dodecyl sulphate-polyacrylamaide gel electrophoresis) and silver staining

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

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

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

PBS was prepared as a 10X stock solution and used as a 1X concentration.Tris-HCl buffer0.5 M Trizma BasepH was adjusted to 7.6 using concentrated HCl.Tris-Cl buffer was prepared as a 10X stock solution and used as 1X concentartion.Tris-EDTA (TE) buffer10 mM Tris-HCl (pH 8.0)1mM EDTATris Acetic acid-EDTA (TAE) buffer40 mM Tris base0.5 M EDTApH was adjusted to 8.5 with glacial acetic acidTAE buffer was prepared as a 50 X stock solution and used at 1 X concentartion.Potassium Phosphate buffer (0.1 M)1 M Potassium phosphate dibasic (K2HPO4)1 M Potassium phosphate monobasic (KH2PO4)61.5 ml of 1 M K2HPO4was mixed with 38.5 ml of 1 M KH2PO4, pH was adjusted to 7.0 and volume was adjusted to 1 L with H2O

Phosphate-Buffered Saline (PBS)137 mM NaCl2.7 mM KCl10 mM Na2HPO42 mM KH2PO4pH was adjusted to 7.3 before autoclaving

Common buffers

Buffers and solutions

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

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

Bacterial media

Media

Rifr, Apr, Kmr, Gmr, Tetrand Spcrindicate resistant to rifampicin, ampicillin, kanamycin, gentamicin and spectinomycin, respectively.Table 2.2: List of oligonucleotides used in the study

hydrochloric acid, sulphuric acid, methanol, acetic acid, acetone and nitric acid were purchased from Fischer Scientific. Protease inhibitor tablets were procured from Roche. Hybond-P membranes for protein transfer were purchased from Amersham Biosciences. Taq DNA polymerase and Hi-fidelity Taq DNA polymerase were purchased from Thermoscientific and Larova, respectively. SYBR-green kit for real-time PCRwas procured from Qiagen and Thermoscientific. Superscript SS-III RT kit was obtained from Invitrogen. Random hexamers were obtained from Qiagen. Different restriction enzymes used for cloning and mutation generation were purchased from New EnglandBiolabs (NEB). Plasmid DNA purification, PCR purification, gel extraction and reaction clean up kits were procured from Qiagen. Medium components for bacterial culture viz., sucrose, agar, Luria Bertani (LB), Nutrient Agar (NA), peptone, yeast extract, beef extract, magnesium chloride hexahydrate (MgCl2.6H2O) and potassium sulphate (K2SO4) were purchased from Himedia. Table 2.1: List of strains and plasmids used in the study

Agarose, phenol, dimethyl sulphoxide (DMSO), sodium acetate, sodium carbonate, sodium bicarbonate, mangenese sulphate, tris methylamine, trizma base, sodium dodecyl sulphate (SDS), formamide, ethylenediaminetetraacetic acid (EDTA), glycerol, polyethylene glycol, tributyrin, ammonium persulphate, TEMED, acrylamide, bis-acrylamide, coomassie brilliant blue (CBB), β-mercaptoethanol, chloroform, formaldehyde, nuclease free water, diethylpyrocarbonate (DEPC), isopropanol, ferrozine, glycine, sodium lauryl sarcosine, carbonylcyanidep-trifluoromethoxyphenylhydrazone (FCCP), benzyl amino purine (BAP), ferrozine, tween-20, triton-X-100, aniline blue, trisodium citrate dehydrate, remazol brilliant blue-xylan (RBB-xylan), lactic acid, nicotinic acid, hexadecyltrimethyl ammonium bromide (HDTMA), p-nitrophenol, carboxymethyl cellulose (CMC cellulose), sodium phosphate dibasic, sodium phosphate monobasic, rubidium chloride, ferrous sulphate, ferric chloride, ammonium sulphate, 2,5-diphenyloxazol (PPO), 1,4-bis (5 phenyl 1,2-oxazole) Benzene (POPOP) and 2, 2-dipyridyl were purchased from Sigma Chemicals. Sodium hypochloride, disodium hydrogen orthophosphate dehydrate, sodium chloride, sodium hydroxid

Chemicals, kits and culture medium components

Allbacterial strains and plasmids used in this study are listed in Table 2.1

Oligonucleotides

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