**2. Results**

#### *2.1. Inhibition of Mycelial Growth Using EOs Vapors*

In present study the inhibition concentrations of eight EOs vapors, for which the microscopic filamentous fungus still survived, were investigated. The microarray hybridizations were carried out with the RNA obtained from the mycelia of *P. rubens* isolated 24 h after exposure *to Cymbopogon flexuosus* (CF), *Cinnamomum cassia* (CC), *Origanum vulgare* (OV), *Thymus vulgaris* (TV) and *Thuja plicata* (TP) EO vapors and 48 h after exposure to *Mentha piperita* (MP), *Eugenia caryophyllata* (EC) and *Melaleuca* *alternifolia* (MA) EO vapors. The incubation time was dependent on the inhibition ability of EOs to the mycelial growth. In the end of the exponential phase, the samples were collected. A concentration of selected EOs was in the range of 0.01–0.05 μg/mL. Under these conditions, the conidia formation and germination of spores was suppressed and coupled with the activation of stress-responding processes. The RNA extracted from the fungus without exposure to EOs, after 24 and 48 h of cultivation, served as the control.

#### *2.2. Impact of Essential Oils on Genome-Wide Gene Expression*

The gene expression profile of *P. rubens* was evaluated by Gene Expression 8 × 15K custom Microarray Slide containing 12,675 genes, from which only 551 are annotated, and the other 12,124 genes encode hypothetical proteins. A microarray-based analysis allowed us to reveal differentially expressed genes (fold change >2) by comparing the expression profiles of genes between the control and the fungus exposed to EOs.

We identified several differently expressed genes (Table 1) for targeted EOs, of which 1430 upregulated genes and 833 downregulated genes were detected in at least four assayed EOs (*n* ≥ 4). However, from 2263 genes (1430 upregulated and 833 downregulated), 148 (80 upregulated and 68 downregulated) genes were with mixed regulation, i.e., genes were not just up- or downregulated by at least four EOs, but also, in at least one sample, they were regulated in the other way (e.g., one gene upregulated by four EOs and downregulated by one EO). These genes were discarded from further analysis. The final number of genes, which had the same regulation in at least half of the samples exposed to different EOs (*n* ≥ 4): 1350 upregulated and 765 downregulated genes (Table S1).


**Table 1.** The number of up- or downregulated genes presented in at least half of the samples after exposure to selected essential oils (EOs).

#### *2.3. Gene Ontology Analysis*

To examine the functions of significantly expressed genes (1350 upregulated and 765 downregulated), we performed gene ontology by gene set enrichment analysis (GSEA). In order to classify genes into functional categories, gene ontology-based overrepresentation analyses were executed. We identified our dataset overrepresented in several biological processes (Figures 1A and 2A), cellular components (Figures 1B and 2B) and molecular functions (Figure 1C).

**Figure 1.** Results of a gene ontology (GO) gene set enrichment analysis (GSEA) of *Penicillium rubens* upregulated genes for different GO domains' (**A**) biological processes, (**B**) cellular components and (**C**) molecular functions. The figures describe the rate of involvement of upregulated genes in the processes (black circles) and, also, about the significance (color changes from blue to red).

**Figure 2.** Results of GO GSEA of *P. rubens* downregulated genes for different GO domains' (**A**) biological processes and (**B**) cellular components. No GO molecular functions were detected. The significance, as well as the rate of involvement of downregulated genes, are marked with the same colors and shapes.

#### *2.4. Functional Classes Analysis*

To interpret the data closer, we used overlapped the functional classification from the COG, KOG and KEGG BRITE databases [31]; however, many of the *P. rubens* genes were not well-characterized or with unknown functions. Genes with significantly changed expressions were classified. The occurrence of up- or downregulated genes in appropriate functional classes were compared to the overall gene representation in functional classes from the whole genome of the fungus presented in a microarray. We identified significantly dysregulated functional classes (Figure 3), where the increased occurrence of up- or downregulated genes was revealed. Figure 3 depicts the most significantly altered functional classes with significantly increased occurrences of upregulated genes: translation, ribosomal structure and biogenesis (*p* = 0.0000018); RNA processing and modification (*p* = 0.0001) and replication, recombination and repair (*p* = 0.01). The observed upregulation is moreover supported, with significantly less than the expected occurrence of downregulated genes in these classes.

Other significantly altered functional classes with statistically significant increases of upregulated genes were: energy production and conversion (*p* = 0.0056) and coenzyme transport and metabolism (*p* = 0.047) and, with the increase of downregulated genes, inorganic ion transport and metabolism (*p* = 0.0007) and secondary metabolites biosynthesis, transport and catabolism (*p* = 0.04). Significant increases in the occurrences of downregulated genes in the functions of unknown classes (*p* = 0.001) point to a number of genes that may play important roles in the processes of the response of *P. rubens* to EOs exposure. Unfortunately, their functions still remain unknown.

**Figure 3.** The most significant pathways affected by vapors of eight different EOs. Based on the results of the GO enrichment analysis, data shows changed expression level of genes (**A**) underexpressed and (**B**) overexpressed. \* *p*-value ≤ 0.05, \*\* *p*-value ≤ 0.01 and \*\*\* *p*-value ≤ 0.001.

#### *2.5. Metabolic Pathway Analysis*

In the present study, considerable alterations in gene expression levels to stress responses on EOs vapors were found in *P. rubens*. We have been identified 99 altered pathways, containing 351 genes from our dataset (Figure S1). The occurrence of genes from our dataset was, together, 534, caused by the occurrence of some genes in more than one metabolic pathway. Commonly, we observed significant alterations of gene expressions in various metabolic pathways: nucleotide transport and metabolism; translation and protein biosynthesis processes; amino acid metabolism; metabolism of cofactor and vitamins; basic cellular processes; basic metabolism pathway and energy production and others (Table S2).

#### 2.5.1. Genes Involved in Protein Synthesis

In order to deeply understand the metabolic pathways of these di fferentially expressed genes, they have been grouped and systematically studied. Data analysis revealed that around 28% of genes are associated with the biosynthesis of proteins. The representation of genes that encode enzymes involved in amino acid (AA) metabolisms was strongly diverse. Enzymes coding aminoacyl-tRNA synthetases (aaRS) are located in the cytoplasm of the cell. Among these enzymes, tryptophanyl (Pc12g04630), lysyl (Pc12g09250), asparaginyl (Pc12g15910), methionyl (Pc16g02040), prolyl (Pc16g07440) and histidyl (Pc22g02880) were overexpressed compared to valyl (Pc20g09480), which had decreased expression.

In many pathways, like in β-lactam antibiotic production, analysis revealed changes on the transcriptional level in metabolic pathways, where AAs have a key role. The upregulated Pc20g04020 gene encodes a threonine synthase, an enzyme that is involved in threonine biosynthesis. The other gene, which showed a higher expression level, was Pc13g07730. This overexpressed gene is annotated as L-threonine ammonia-lyase. Results showed that 5-aminolevulinate synthase (Pc22g13500) is another upregulated protein involved in these biosynthetic pathways. In contrast, cystathionine gamma-synthase (Pc20g08350) showed a decreased transcriptional level over the aforementioned genes. Taken together, all these data and the other in Table S2, interference with the functioning of the cell is confirmed at the proteomic level.

#### 2.5.2. Transcriptional Modifications of Genes Encoding Carbohydrates

Our results showed that the metabolism of carbohydrates during the exposition of EOs is generally upregulated. From a range of metabolic pathways, encoded by multiple gene clusters, increased transcriptional levels were achieved: at glycolysis (Pc18g01490, Pc12g10630, Pc20g01630, Pc12g13500, Pc20g04410 and Pc12g16040); the pentose phosphate pathway (Pc12g02790, Pc13g14570, Pc12g13500 and Pc20g04410); fructose and mannose metabolisms (Pc16g12970, Pc22g09390, Pc21g05470, Pc20g01550, Pc13g12020, Pc12g09190, Pc12g13500, Pc21g04410, Pc21g04400 and Pc16g10970) and galactose metabolism (Pc12g13500, Pc14g00310, Pc12g07810 and Pc20g04410). The dominant gene in each group is 6-phosphofructokinase (Pc12g13500). It is responsible for transferring phosphorus-containing groups. In contrast, low levels of downregulated genes involved in the carbohydrate metabolism were obtained. As an example, Pc16g08460 the D-arabinitol dehydrogenase (NADP+) and Pc20g15580, the L-glyceraldehyde reductase. The list of all genes with changed transcript levels is displayed in Table S2.

#### 2.5.3. Changed Expression Level of Genes Involved in Fat Metabolism

The functional analysis of the genes revealed numerous altered metabolic pathways involved in lipid metabolism. Most of them remain with unknown functions that cannot be readily assigned. These hypothetical proteins have a certain similarity with conserve proteins that encode genes for fatty acids; steroid biosynthesis and glycerophospholipid, glycerolipid and sphingolipid metabolisms. Among them, Pc22g00420 is annotated as acetyl-CoA-acetyltransferase, also known as acetoacetyl-CoA thiolase, and demonstrated low levels of expression; it has a key role in the regulation of ergosterol synthesis. The other important protein is glycerol-3-phosphate O-acetyltransferase (Pc22g05820). An analysis revealed the overexpression of this gene. In addition to the above information, all alterations are summarized in Table S2.

#### 2.5.4. Gene Related to Secondary Metabolites

A gene-by-gene analysis showed that among the downregulated genes with hypothetical functions are just a few of them that are included in β-lactam resistance, aflatoxin biosynthesis or ABC transporters. The most interesting gene is pc18g01310, which codes for proteins regulating enzymatic reactions at the level of the cell wall and cellular organelles. It belongs to the group of chitin hydrolases -β-N-acetylhexosaminidase, which is released during autolysis into the medium. In total, two genes encoding enzymes involved in the biosynthesis of aflatoxin have shown reduced expression levels. The Pc12g16460 gene is linked by norsolorinic acid ketoreductase and pc22g19340-coding versiconal hemiacetal acetate esterase. In our investigation, EO treatments increased the expression level of the Pc21g18900 gene. It encodes a hypothetical protein included in monobactam biosynthesis and is named as the 4-hydroxy-tetrahydrodipicolinate synthase.
