## 3 Results and Discussion
### Yeast Genome Haploinusfficiency Screening  
Yeast chemogenetic profiling was carried out to elucidate the possible mechanisms of the fungicidal properties of allicin. The haplinsufficiency screening was performed by employing wildtype BY4743 and 1152 yeast deletion strains independently by employing 50 uM alicin. Figure 4: Allicin shows antifungal activity via the gas phase. A 20 uL drop of an 80 mM solution of allicin was placed at the lid of a Petri dish and covered by the base with bacteria-seeded agar inverted over it. Thus, the agar did not come into contact with the droplet. Inhibition of growth was visible as an inhibition zone with reduced fungal growth. Control plates without allicin exhibit complete fungal growth. However, allicin-containing plates show growth inhibition. The mutants and wildtype were tested by measuring the growth curves overnight with and without allicin. This screening resulted in the hypersensitivity of three yeast mutant strains as compared to the wildtype. Quantitative Identification of Hypersensitive Strains  
According to previous studies, it is required to make a comparison of treated and untreated growth profiles as many heterozygous mutants grow slowly in rich culture medium ([PERSON] et al., 1999). In the present investigation, mutants such as \(\Delta\)_npa3_, \(\Delta\)_mcm5_ and \(\Delta\)_hts1_ were recognized as hypersensitive in comparison to the untreated strains. Moreover, these mutants exhibited lower growth compared to wildtype in the allicin-treated samples (Figure 5). Figure 6 represents the endpoint values of treated wildtype and hypersensitive mutants after 15 h of treatment. It shows that there was greater inhibition of growth of mutants related to the wildtype after treatment with 50 \(\upmu\)M of allicin. Figure 5: Effects of allicin (50 \(\upmu\)M) on the growth of wildtype BY4743 and \(\Delta\)_npa3_, \(\Delta\)_mcm5 and \(\Delta\)_hts1_ mutants in CSM (complete supplement medium). The red line in each graph represents the untreated (culture in CSM only) and blue line shows the treatment with 50 \(\upmu\)M of allicin. There is greater difference between treated and untreated in case of hypersensitive yeast mutants as compared to BY4743. The experiments were repeated twice with similar results. Error bars show standard deviation about the mean. The hypersensitivity of yeast to allicin was explored by screening wildtype and yeast mutants against allicin. Drug targets can be uncovered by hypersensitivity to a drug of a heterozygous deletion strain containing one copy of a gene as compared to a wildtype strain containing two copies. HIP probes heterozygous strains, while most of the genetic screens examine the phenotypic results of a complete gene deletion in the homozygous strains. Thus, HIP is advantageous as compared to HOP because, with complete deletions, it is difficult to determine the primary effect of the compound and impossible to detect the effect of any essential gene [47]. Yeast haploinsufficiency screening has been widely employed to decipher the mechanisms and modes of action of compounds exhibiting antifungal activities, as for example, the determination of MOA of saponin, _a-hederin_, from ivy leaves (_Hedera helix_) by haploinsufficiency profiling (HIP) [55]. This saponin exhibits activity against _Candida albicans_ and its antifungal MOA was similar to that of the known calcineurin inhibitor FK506 [56]. During chemogenetic screening, various yeast deletion strains lacking specific genes were employed and toxicity was observed in the presence of the drug with reference to the wildtype. The mutants that are hypersensitive to drugs compared to the wildtype may give clue about the possible relationship between the drug and the gene for a specific protein/enzyme absent in the mutant and its role in metabolism or defense. This technique is simple, easy and presents a robust avenue for the cost-effective and prompt screening of numerous compounds in order to identify the possible targets and modes of action of cytotoxic compounds [57].