Active site prediction and docking study

For clonogenic assays, 1x103 (A549) or 2x103 (E-10) cells were seeded per well of a six well tissue culture plate and grown for 15 days. For identification of signaling pathways various inhibitors were used viz, PI3K inhibitor LY294002 (10μM), MEK inhibitor PD98059 (10μM) or p38 inhibitor SB203580 (10μM). Cells were grown in the presence of inhibitor for seven days following which fresh medium was added. For staining, cells were washed twice with PBS and fixed in 10% formalin for 10 minutes, washed extensively with water and stained with 0.25% crystal violet prepared in 25% methanol for 4hrs at 4°C. Plates were then washed with milli Q water and dried before scanning

Clonogenic Assay

Quantitative measurement of periplasmic acid phosphatase activity in phosphate-starved C. glabratacells was performedas mentioned previously (Orkwis et al., 2010). A total of 0.5 OD600YNB-grown and phosphate-starved cells were collected, washed thrice with cold water and oncewith cold 0.1 M sodium acetatebuffer (pH 4.2). Washed cells were resuspendedin 500 μl sodium acetate (0.1 M)and incubated at 30 ̊C with constant stirring. After 10 min incubation, 500 μl freshly-prepared solution of 20 mM p-nitrophenyl phosphate in 0.1 M sodium acetate(pH 4.2) was added to the cell suspension. Enzymatic activity was stopped after incubation at 25 ̊C for 20 min by addition of 250 μl sodium carbonate (1 M)tothe reaction mix. Resultant colour change was measured by monitoring absorbance at 400 nm. Acid phosphatase activity was expressed as a ratio of OD400to OD600 to normalize against cell density

Determination of acid phosphatase activity

C. glabratacells grown either in RPMI medium or harvested from THP-1 macrophages were collected, washed with DEPC treated water and were disrupted with glass beads in trizol. Total RNA was isolated using acid phenol extraction method and frozen at -80C. Quality of RNA was examined by determiningtheRNAintegrity number (RIN) before microarrayanalysis.Microarray experiments wereperformed atOcimum Biosolutions Ltd., Hyderabad (http://www.ocimumbio.com). Briefly, a4x44K GE Agilent array comprised of 10,408 probes representing 5,205 ORFs of C. glabratawas used wherein average number of replicates for each probe was four to five. Feature Extraction software version 10.7.3.1. (Agilent) and Quantile normalization was used for data analysis. Hierarchical clustering was performed using Complete Linkage methodwith Euclidean Distance as distance measure. Data arethe average of two hybridizations from biological replicates ofeach sample and raw data sets for this study areavailable at the Gene Expression Omnibus database(Accession number -GSE38953)

Microarray 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

using the GENESPRING GX (Version 12.0) software,normalized to 75 percentile shift and represent the average of two hybridizations from biological replicates for each sample. Functional annotation of differentially regulated gene set(≥1.5 Fold change with p≤0.05)was performed using the GENESPRING GX (Version 12.0) softwareand GO terms with p<0.05 were considered as statistically significant. Using the REVIGO tool(http://revigo.irb.hr), redundant and significantly overlapping GO terms were removed and summarized. In REVIGO analysis, S. cerevisiaedatabase was chosen for GOterm sizes andtheallowed similarity value was set to 0.5(small).Additionally, to identify the overlap among differentially expressed genes, functional category analysis was performed usingthefungal specific annotation tool FUNGIFUN (https://sbi.hki-jena.de/FungiFun/FungiFun.cgi). Significantly enriched FunCat (Functional Catalogue) associated pathways were extracted usingthewhole C. glabratagenome as background and compared across differentially regulated gene sets. The parameters used for FUNGIFUN analysis were cut-off p=0.05; Fisher’s exact test; FunCatlevel 3. Raw data sets for this study are available attheGene Expression Omnibus database (http://www.ncbi.nlm. nih.gov/geo; accession no. GSE60741

Log-phase C. glabratacells were grown either in YNB or YNB medium supplemented witheither50 μMBPS(iron limiting) or 500 μMferric chloride (iron excess) for 2 h. Cells were spun down at 4,000 rpm for 5 min and washed twice with ice-cold DEPC-treated water. Total RNA was extracted usingtheacid phenolisolationmethod, resuspended in nuclease-free water and stored at -80°C. The frozen RNA samples were sent to Genotypic Technology Ltd., Bangalore (http://www.genotypic.co.in) wherein quality of RNA samples wasdetermined by examining the RNA integrity number (RIN) before performing microarray analysis. Next, the 8x15 GE Agilent array,comprised of 60mer oligonucleotides representing a total of 5,503 C. glabrataORFs (three replicates of each probe on average),was used for single colour microarray experiments.Datawereextracted

Microarray analysis