Assay for reduced glutathione(GSH)

Acridine orange/ethidium bromide (AO –EB) staining

After PCR, 1 μl of Dpn1 enzyme (10U/μl) was added to the amplification mix and incubated at 37°C for 6hours. After that, 10ml of the amplification mix was taken to transform Dh5a cells. Positive clones were selected after confirming the sequence of plasmid DNA

The PCR parameters were as follows

The reaction mix included 2ml of PSKll(39+) (50ng) containing wild type K-Ras cDNA , 5ml 10x buffer, 20pmoles of primers , 1ml of 10mM dNTP mix and 1ml of deep vent polymerase (NEB).

The following K-Ras-ras mutants were generated by site directed mutagenesis according to the protocol described in QuickChange site directed mutagenesis kit (Stratagene). The primers used are shown in the following table

Site Directed Mutagenesis

were maintained in log-phase by continuous passaging in fresh YNB medium every 4 h

For phosphate starvation,yeastcells grown to log-phase in YNB medium were harvested,washed with water,transferred to either regular YNBor YNB medium lackingphosphate and were grown for 16 h at 30 ̊C. Cells cultured in YNB medium

Phosphate starvationof yeast cells

Nucleosomal-associated DNA was extracted from RPMI-grownand macrophage-internalized C. glabratacells using EZ NucleosomalDNA prep kit (ZYMO Research),treated withmicrococcal nuclease digestionfor 2.5, 5, 7.5 and 10 min at 25ºC andwasresolved on 2% agarose gel

Micrococcal nuclease digestion assay

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

For Yeast colony PCR, yeast cells were subjected to zymolyase (MP Biomedicals, 0832092) digestion to obtain thespheroplast. To perform zymolyase digestion, a digestion cocktail was prepared in 1XPBS consisting of zymolyase (2.5mg/ml) and sorbitol (1.2 M). The cocktail was dispensed in 0.2ml PCR tubes in 10 μl aliquots anda tip-full of yeast cells wasadded to these tubes. Tubes were incubated at 37°C for 2-3 h and 1 μl of digested mixture was used as a template in a PCR reaction

Yeast colony PCR

For Yeast colony PCR, yeast cells were subjected to zymolyase (MP Biomedicals, 0832092) digestion to obtain thespheroplast. To perform zymolyase digestion, a digestion cocktail was prepared in 1XPBS consisting of zymolyase (2.5mg/ml) and sorbitol (1.2 M). The cocktail was dispensed in 0.2ml PCR tubes in 10 μl aliquots anda tip-full of yeast cells wasadded to these tubes. Tubes were incubated at 37°C for 2-3 h and 1 μl of digested mixture was used as a template in a PCR reaction

Yeast colony PCR