## 3 Results and Discussion
### Plate Inhibition Zone Assay  
The antifungal effect of selenium compounds can be investigated by monitoring the growth of wt and mutants. Different concentrations of test compound are employed to evaluate whether there is concentration dependent decrease in the growth of wt and mutants. In this procedure, yeast-seeded agar was employed to assess the sensitivity of yeast strains. A suspension of yeast culture was mixed in agar medium at 50 \({}^{\circ}\)C and poured with the agar immediately into petri plate. After solidification of agar, holes were cut and 20 \(\upmu\)L of respective concentration of the test substance was added to each well. The plates were incubated at 28 \({}^{\circ}\)C for 20 h which enabled the yeast cells to grow widely from the wells. Figure 5 represents the yeast mutants inhibited by the selenocyanates. These yeast mutants are deficient in genes playing a pivotal role in the glutathione metabolism. The compounds inhibit yap1 and glr1, thus, blocking the synthesis of GSH from GSSG. Similarly, selenocyanates cause a decrease in the production of NADPH required for GSH synthesis by inhibiting the mutants \(\Delta zw\gamma\)I, \(\Delta gnd\)I and \(\Delta trx\)2. This explains the antifungal action of selenocyanates. Figure 6 shows the effect of 20 \(\upmu\)L of selenocyanates at concentrations of 2, 4 and 8 mM, respectively. Both of the compounds diffused well into the agar and exhibited antifungal activity with clear zones of inhibition in concentration dependent manner. Compound **2**, however, exhibited higher antifungal activity as compared to compound **1**, as observed in other assays. Moreover, mutants also exhibited greater zones of inhibition in comparison to wt. These findings are in accordance with those of chemogenetic profiling, which also demonstrated that mutants are more sensitive to selenocyanates compared to wt. This assay provides insights into the higher sensitivity/toxicity of mutants inhibited in chemogenetic screening. Each mutant was compared with the wt at each concentration. Compound **1** inhibited each mutant to a higher degree as compared to wt. Higher zones of inhibition were observed for all the  
Figure 4: Drop test of _Saccharomyces cerevisiae_ cells on CSM medium containing 5 \(\upmu\)M of test compound. The wt BY4742 was compared with the mutants. Drops (10 \(\upmu\)L) of serial dilutions up to 10\({}^{-5}\) were plated out. In the control, both wt and mutant cells behaved similarly and grew down to the 10\({}^{-5}\) dilution. In the drop test, 10 \(\upmu\)L of 10\({}^{-5}\) fold serial dilutions were spotted onto medium containing 5 \(\upmu\)M of the test compound. The control plate comprised of medium only. The ability of wildtype (wt) BY4742 cells to grow in the presence of selenocyanates was compared with that of \(\Delta yap1\), \(\Delta grd\)I. \(\Delta gnd\)I, \(\Delta trx\)2 and \(\Delta zw\gamma\)I yeast mutants. mutants. Compound **2** exhibited higher toxicity to mutants and wt in comparison to compound **1**. However, wt was inhibited to a lesser extent with reference to mutant strains. Figure 5: Schematic representation of the mutants inhibited by the selenocyanates. It also shows the activities of yeast mutants; \(\Delta\)_varp1_ (GSH synthesis and GSSG reduction), either directly (\(\Delta\)_glr1_), or by blocking the production of \(\Delta\)_zzv61_ and \(\Delta\)_gnd1_ (NADPH reducing equivalents) or by suppressing the NADPH-dependent reduction of protein disulfides (\(\Delta\)_trx2_, indirectly \(\Delta\)_zzv61_ and \(\Delta\)_gnd1_). The encircled mutants are the targets of selenocyanates showing hypersensitivity. Dotted lines show the metabolic lesions caused by the deletion mutants (modified from [30]). Figure 6: Plate inhibition zone assay showing the antifungal activity of two selenocyanates against yeast wt and mutants. (**a**) The representative pictures of inhibition zones of yeast mutants and wt (BY4742). Cells were incorporated into 50 \({}^{\circ}\)C warm agar and poured into a Petri plate.