ROS assay

Tests for Tannins

Transformation of ligated product in chemically competent E. coli host cells (DH5αααα strain)

Ligation reactions

. Statistical analysis

Lactic dehydrogenase

For adherent cells from which lysates have to be prepared , culture medium was removed and cells were washed with ice cold 1X PBS twice and then scraped with cell scraper in Cell Lysis buffer. Cells were rotated at 4°C for 30min at cold room and centrifuged at 13000 rpm for 10min at 4°C. The supernatant was collected and protein concentration was estimated using BCA assay. For standard western, 50-70μg of protein was loaded on to the gel

Lysate Preparation for Immuno- blotting

Log-phasecells grown in YPD medium containing or lacking CaCl2and FK506 were collected, PBS-washed and loaded with ratiometric, high affinity, membrane-permeable calcium indicator, Fura-2 AM (10 M; Sigma #47989). After 30 min incubation at 30◦C, labelled cells were washed thrice with cold PBS, suspended in PBS and fluorescence was recorded at 505 nm with dual excitation at 340 and 380 nm. The ratio of fluorescence intensities between 340 and 380 nm, representing Ca-bound and Ca-free Fura-2 molecules, respectively, reflected free intracellular calcium concentrations

Measurement of intracellular calciumlevels

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

PI-3 kinase reaction set up and phopsholipid extraction

C. glabrata cells were grown overnight in 10 ml YPD liquid medium. Cells were harvested at 2,500g for 5 min, resuspended in 400 μl Buffer A (50 mM Tris-HCl, 10 mM EDTA, 150 mM NaCl, 1% Triton X-100 and 1% SDS) and were transferred to a 2 ml microcentrifuge tube. Equal volume of phenol-chloroform-isoamyl alcohol (PCI) solution was added tocell suspension and tubes were vortexed for 2-3 min. After incubation at 42 ̊C for 30 minon a thermomixer set at 800 rpm (Eppendorf), cell debris was removed by centrifugation at 7,500g for 10 minand aqueous phase (300-350 μl) was carefully transferred to a new 2 ml microcentrifuge tube. Genomic DNA was precipitated with800 μl chilled absolute ethanol and 35 μl sodium acetate (3 M, pH 5.2). DNA pellet was washed with chilled 70% ethanol and dried at room temperature for 5-10 min. Genomic DNA pellet was dissolved either in 50 μl 0.1X TE or molecular biology grade water containing 0.3 μl Ambion RNase cocktail and incubated at 37 ̊C for 30 minfor digestionof RNA. After RNA degradation, 100 μl of 0.1X TE or nuclease-free water was added to the tube and stored at -20ºC. Quality of extracted genomic DNA was checkedon 0.6% agarosegel by electrophoresis

Genomic DNA isolationby quick genomic DNA extraction method

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

To extract DNA from agarose gels,the QIAquick® gel extraction kit (QIAGEN, 28706) was used. For purification of PCR amplified DNA products,the QIAquick® PCR purification kit (QIAGEN, 28106) was used. Clean-up of enzymatic reactions was performedusing the MinElute® Reaction Cleanup kit (QIAGEN, 28204). Allprotocolswere followed as per manufacturer’s instruction

Gel extraction, PCR purification and Reaction clean-up

To extract DNA from agarose gels,the QIAquick® gel extraction kit (QIAGEN, 28706) was used. For purification of PCR amplified DNA products,the QIAquick® PCR purification kit (QIAGEN, 28106) was used. Clean-up of enzymatic reactions was performedusing the MinElute® Reaction Cleanup kit (QIAGEN, 28204). Allprotocolswere followed as per manufacturer’s instructions

Gel extraction, PCR purification and Reaction clean-up