## 3 Results and Discussion
### Activity of Selenocyanates in Drop Test  
The drop test is an efficient standard technique for quantifying the activities of test substances on yeast. In this method, the test compound at a given concentration is incorporated into molten agar medium and poured immediately into plates. A total of 10 \(\mu\)L of a 10 th dilution series of a freshly growing yeast culture is spotted onto medium containing the test compound. After incubation for 48 h at 28 \({}^{\circ}\)C, individual colonies can be counted and compared in the plates with or without the compound. A fixed concentration of test substance is employed in drop test and serial dilutions of microbial culture are tested. Figure 4 shows the comparison of the yeast mutants and wildtype on plates in the presence and absence of compound.  
In the absence of compound in the medium, the wt and yeast mutants grew equally well down to the 10\({}^{-5}\) fold dilution. Compound **1** at a concentration of 5 \(\mu\)M inhibited the wildtype and mutants demonstrating a greater inhibition in \(\Delta zw\Omega\), \(\Delta yap1\), \(\Delta glr1\) and slightly in \(\Delta gnd1\) and \(\Delta trx2\) cells. Compound **2** inhibited the growth of wt to greater extent as compared to compound **1**. The mutants \(\Delta zw\Omega\) and \(\Delta glr1\) were found to be the most sensitive to compound 2. These findings confirm the results of chemogenetic profiling and reveal considerable toxicity of selenocyanates particularly of compound **2** towards yeast. The Yap1 is a member of AP-1 family of transcription factors and in response to oxidative stress, Yap1 activates antioxidant genes [45]. Yap1 regulates the antioxidant genes, such as Glr1, Gsh1, Trx1 and Trx2 [39]. The transcriptional activity of Yap1 is governed by redox dependent control at subcellular level [46]. The C-terminal domain contains a nuclear export signal (NES) while N-terminal has nuclear localization signal NLS [47]. Yap1 is exported via Crm1p from nucleus in the absence of oxidative stress [48]. However, during oxidative stress, Yap1 nuclear export is hampered due to conformational change. It results in the increased transcriptional activity of Yap1 [49].  
The hypersensitivity of Yap1, towards the selenocyanates under investigation, explains the increase in oxidative stress. The similar mode of action has been proposed for resveratrol which is a polyphenol and produces oxidative stress at lower concentration [50]. Yap1 cells are sensitive to  
Figure 3: Effects of selenocyanates on the growth in shake culture of wt BY4742 and \(\Delta yap1\), \(\Delta glr1\), \(\Delta zw\Omega\), \(\Delta gnd1\) and \(\Delta trx2\) mutant yeast cells in complete supplement medium (CSM). (**a**,**c**) CSM alone (control), (**b**) 15 \(\mu\)M of Compound **1**, and (**d**) 15 \(\mu\)M of Compound **2**. The experiments were repeated twice (growth curves were measured in triplicates) with similar results.  
resveratrol which activates the transcriptional activity of Yap1 [51]. Allicin also modifies thiols by oxidation and has also been shown to activate Yap1 [38].  
These results also highlight the pivotal role of oxidative stress and GSH metabolism as the underlying mode(s) of action of selenocyanates. Further studies are needed to explore the additional mechanisms responsible for the antimicrobial activity of selenocyanates, probably beyond their redox-activity.