**3. Discussion**

In a fast-paced world, it is important to expand our knowledge and search for the undiscovered. The results of the present work demonstrate changes in the transcriptomic profile of the mold *P. rubens* in association with stress responses caused by the exposure to EO vapors. According to our literature research, there is no investigation studying the gene expression of *P. rubens* under exposure to the vapors of EOs.

Plant derivates, such as EOs, can modulate, inhibit and even kill the microorganism through specific mechanisms [32]. Unfortunately, the exact mechanism of EO effects is still not entirely clear; their different composition of substances makes them unique. The chemical compositions of assayed EOs are guaranteed by the producer based on the performed a GC/MS analysis and can be checked for each flask of EOs through the web page: https://sourcetoyou.com. EOs possess a wide spectrum of biological activities, including antimicrobial, antiviral, insecticidal and antifungal properties [33].

The regulation of genes responsible for protein synthesis is a limiting factor for cell survival. It can be inhibited in a number of ways, such as by altering the expression level of aminoacyl-tRNA synthetases [34]. In our case, treatments with EOs significantly show their power to decrease (for valyl-aaRS) or increase (tryptophanyl-, lysyl-, asparagynil-, methionyl-, prolyl- and histidyl-aaRS) the expression of several *P. rubens* genes. Modifications of these enzymes lead to changes in the mechanism of the translation of cellular proteins, metabolic and signaling pathways [34].

Amino acid metabolisms are an indispensable process in β-lactam production. Three AAs serve as building blocks, namely α-aminoadipate, L-cysteine and L-valine. A previous analysis demonstrated that the gene Pc20g04020 is considered to be unique for coding the threonine synthase in *P. rubens* [35]. It plays a key role in catalyzing the conversion of O-phospho-L-homoserine into L-threonine and phosphate. In the next step, L-threonine can be converted into another AA, such as L-cysteine. Pc13g07730 is annotated as L-threonine deaminase, which removes an amino group from L-threonine, resulting in the production of 2-oxobutanoate and pyruvate. The corresponding enzyme that catalyzes the conversion of glycine and succinal-CoA into 5-aminolevulinate, CoA and CO2 is 5-aminolevulinate synthase (Pc22g13500), also named as the pyridoxal 5'-phosphate-dependent (PLP) enzyme [35]. These findings suggested that the influence of applied EOs may play an important role in modulating these signaling pathways and, thus, may regulate processes involved in cysteine production, which is very important for beta lactam production [35].

According to current knowledge, gene Pc20g08350 is coding a cystathionine gamma synthase, which is a transsulfuration enzyme involved in the catalysis of the PLP-dependent γ-replacement of O-succinyl-L-homoserine and L-cysteine, yielding L-cystathione and acetate [35]. Hypothetically, the alteration of the β-lactam production can bring to the modification a synthesis of diverse compounds that can increase the vulnerability of the fungus against different environmental factors.

Carbohydrates have an irreplaceable role in fungi and represent an important part of the cell wall (glucans and chitin), as well in the role of storage polysaccharide (glycogen), disaccharide and sugar alcohols [36]. An interesting gene is 6-phosphofructokinase (Pc12g13500) which has a strong similarity to 6-phosphofructokinase pfkA to *Aspergillus niger* [37]. The Pc16g08460 gene is annotated as D-arabinitol dehydrogenase (NADP+) and has a low expression level. If compared with other fungi, this enzyme is considered as one of the few enzymes capable of utilizing arabitol as a main substrate [38]. The downregulated Pc20g15580 gene, coding L-glyceraldehyde reductase, takes part in a D-galacturonate degradation pathway and plays an important role in the pentose and glucuronate interconversion pathways [39].

The genus *Penicillium* includes species well-known for lipolytic enzyme productions, especially lipases and esterases, which are able to use lipids as carbon sources. They also participate as biocatalysts, which provide the hydrolysis of water-soluble short acyl chain esters as sure as water-insoluble long-chain triacylglycerols [40]. Acetyl-CoA-acetyltransferase (Pc22g00420) has a key role in the regulation of ergosterol synthesis and is the first main catalytic enzyme in the mevalonate pathway. This enzyme regulates the transformation of acetotyl-CoA from two molecules of acetyl-CoA [41]. If the ergosterol production is broken down, it leads to inhibition of the fungus growth. The biosynthesis of triglyceride is necessary to provide the decisive energy molecules, as well as in the biosynthesis of fatty acids and phospholipids [42]. Triglycerol-3-phosphate O-acetyltransferase (Pc22g05820) is an integral component of the membrane that has transferase activity. This upregulated enzyme is the first enzyme catalyzing the acylation of glycerol 3-phosphate in the glycerolipid metabolism [43].

Over the past decades, scientists have further explored the role of enzymes in biological processes [44], performing a wide genome sequencing of the filamentous fungus *P. chrysogenum* Wisconsin 54-1255. A transcriptomic analysis demonstrated the altered transcript levels in many metabolic pathways. Some of our outcomes coincided to their published results. The downregulated gene associated with the secondary metabolite productions was the glycoside hydrolases (Pc18g01310), also named as β-N-acetylhexosaminidase, which has a strong similarity to the hypothetical β-hexosaminidase A precursor in *Bacillus halodurans* [44]. This enzyme is an important part in the chitinolytic system in the cell wall of the growing fungus. It plays a key role in the controlling of the cell wall chitin lysis and, at the same time, in protecting cells from rupture [45].

The next a ffected upregulated gene is Pc21g18900, which belongs to the DapA family proteins, catalyzing the condensation of (S)-aspartate-beta-semialdehyde ((S)-ASA) and pyruvate to 4-hydroxy-tetrahydrodipicolinate (HTPA) [46]. It is located in the cytoplasm of the cell and has a predicted role in the biosynthesis of the secondary metabolites (monobactams) and lysine [47]. To date, the function of this enzyme is still not well-understood [48].

The study of the gene expression of *P. rubens* exposed to eight di fferent EO vapors was never done before using a microarray system; moreover, we were able to ge<sup>t</sup> a big amount of data at once, which is currently almost unknown. This experiment can be considered as pioneering in understanding the effect of several EOs on various *P. rubens* biochemical pathways. The information obtained by this study allowed us to begin to comprehend the antifungal mechanisms of EOs in a more complete way.

#### **4. Materials and Methods**

#### *4.1. Essential Oils*

The study was performed using the following commercial EOs: arborvitae (TP) from *T. plicata* Donn., cassia (CC) from *C. cassia* L., clove (EC) from *E. caryophyllata* Thunb., lemongrass (CF) from *C. flexuosus* Nees ex Steud., melaleuca (MA) from *M. alternifolia* Maiden, Betche. oregano (OV) from *O. vulgare* L., peppermint (MP) from *M. piperita* L. and thyme (TV) from *T. vulgaris* L. (doTERRA, Pleasant Grove, UT, USA). In order to avoid photo-oxidation, the EOs were preserved in dark glass vials.

#### *4.2. Fungal Strain and Fungicide Activity of the Vapor Phase of EOs*

Antifungal activity of eight EOs were investigated against airborne mold *Penicillium rubens Wisconsin* 54-1255 (American Type Culture Collection-ATCC 28089). The fungus was cultivated on Malt Extract Agar (MEA; Sigma-Aldrich, Saint Louis, MO, USA) at 28 ◦C. In order to determine the fungicide activity of volatized EOs, a 5-mm square of growing fungal mycelia from MEA was placed into 5 mL of malt extract broth (MEB; Sigma-Aldrich) inside small (60-mm diameter) Petri dishes and incubated at room temperature (24–26 ◦C). Different quantity of EOs (Table 2) was applied on the inner surface of the Petri dish lid at dose levels of 1 μL/1 mL air space. As controls, served fungus *P. rubens* were placed into MEB medium without the application of EOs. To prevent vapor leakage, the Petri dishes were sealed with parafilm and incubated in the dark for 24 h for TP, CC, CF, OV, and TV; MP; EC and MA for 48 h at 22 ◦C. When the fungus reached the exponential phase, it was harvested. After cultivation, 50–100 mg of samples were collected by inoculation loop under aseptic conditions into sterile RNase-free plastic tubes, quickly frozen in liquid nitrogen and lysed in 1 mL of commercially available TRIzolTM Reagent (Thermo Fisher Scientific, Waltham, MA, USA). Before storage at −80 ◦C or directly used to perform RNA isolation, the samples needed to be incubated for 30 min at room temperature.


**Table 2.** Used essential oils their highest noncytotoxic concentrations.

#### *4.3. RNA Isolation and Quality Control*

Total RNA was isolated from fungal mycelia using a Direct-zolTM RNA MiniPrep Plus (Zymo Research, Irvine, CA, USA) kit according to the protocol of the manufacturer. RNA quality was evaluated using the Experion Automated Electrophoresis System for RNA analysis (Bio-Rad Laboratories, Inc., Hercules, CA, USA), and the RNA concentration was measured using Nanodrop ND-2000 (Thermo Fisher Scientific). Total RNA degradation/quality was determined based on the 18S and 28S rRNA ratio. RNA samples with the RQI number (calculated based on the 18S/28S rRNA ratio) above the value 7.5 were selected for subsequent gene expression analysis.

#### *4.4. Microarray Analysis of Gene Expression*

Microarray analysis was performed using the total RNA and comparing the gene expression of the control sample with the samples treated with EOs. One-hundred nanograms of total RNA were amplified and labeled with a Low Input Quick Amp Labeling Kit (Agilent, Santa Clara, CA, USA) according to the manufacturer's instructions. After, labeling samples were purified using a GeneJET RNA Purification Kit (Thermo Fisher Scientific) to remove unincorporated nucleotides. Three-hundred nanograms of labeled test samples vs. control samples were mixed and applied onto a *Penicillium rubens* Gene Expression 8x15K custom microarray slide (Agilent Technologies) and hybridized 17 h at 65 ◦C by rotating the slide at the speed 10 rpm in a hybridization oven (Agilent Technologies). After hybridization, two wash steps were performed (Gene Expression Wash Buffer Kit; Agilent Technologies), and the slide was scanned at a resolution of 2 μm using an Agilent Microarray Scanner.

#### *4.5. Image and Data Analysis*

Tagged Image File Format (TIFF) multiscan image was converted and processed using Feature Extraction Software 12.1 (Agilent Technologies) to acquire spot intensities. Acquired data were analyzed in GeneSpring 12.6 GX software to obtain differences in the gene expression. Significant differences in the fold of the gene expression change between the control and treated samples were ≥2.0. Finally, a pathway analysis was performed to reveal the molecular pathways significantly modified in our experiment (*p* ≤ 0.05). The R programming language version 3.3.3 [49] and the openxlsx library (github) were used. To perform a KEGG enrichment analysis, the Bioconductor library clusterProfiler [50] was applied to genes with a *p*-value cutoff 0.05 and BH *p*-value adjustment method (Benjamini – Hochberg method) [51]. Heatmaps from the KEGG enrichment analysis were created by Bioconductor libraries complexHeatmap [52] and circlize [53]. Biochemical pathways' graphical representations were obtained using the pathview [54] library from Bioconductor.
