Study of silver nanoparticle effects on some molecular responses and metabolic pathways of Phytophthora parasitica

The genus Phytophthora includes more than 120 species, causing many devastating diseases to plants, natural ecosystems, and crops worldwide [1]. Phytophthora infestans is the most notable species, and was the primary cause of the destruction of potato monoculture in Europe, contributing to a great famine that resulted in the reduction of the total Irish population by approximately 20% in the 1840s [2]. More research has been focused mainly on P. infestans and P. sojae. P. infestans infects potato and tomato and is a foliar pathogen [3], while P. sojae causes stem and root rot of soybean [4]. P. parasitica is a typical root pathogen that infects over 255 plant Abstract

genera in 90 families [5]. The hosts range from fi eld crops [6] to forest trees [7] in natural habitats [8]. P. parasitica is a worldwide problem causing black shank disease of tobacco [9] and root rot and gummosis of citrus species [10].
Oomycetes which include the genus Phytophthora, are coenocytic [11] and have notable differences in cell wall composition compared to fungi. Oomycetes mainly consist of -1,3-glucan and -1,6-glucan polymers and cellulose, whereas fungal cell walls are mainly composed of chitin [12]. This difference makes oomycetes challenging to control as most of the fungicides target sterol and chitin synthesis which is absent in oomycetes (8). The phenylamide fungicide, metalaxyl, used against oomycete pathogens, targets and inhibits RNA polymerase 1 [13]. However, there has been development of resistance reported against metalaxyl in closely related P. infestans populations [14]. Phytophthora species are well known to develop resistance to chemicals rapidly [15][16][17][18][19].
In order to manage pathogens, new strategies are currently being explored and evaluated with the aid of genomics and transcriptomics. Nanoparticles have gained a lot of interest in recent years for use in disease control based on their biological, chemical, and physical properties. They have been used in disease management based on these unique characteristics [20]. Silver nanoparticles (AgNPs) have been used to control fungal pathogens because of their multiple modes of inhibition to microorganisms [21]. There are several studies reporting the effi cacy of AgNPs for controlling diverse pathogens, including bacteria [22] and fungi [23]. Antibacterial and antifungal activities of AgNPs have been shown to have great potential in controlling bacteria and spore-producing fungi, with less toxicity to the ecosystem [24]. AgNPs affect a broad range of metabolic processes and have been shown to have multiple modes of action that make pathogens less prone to developing resistance [25]. AgNPs act in three signifi cant ways to inhibit microorganisms: they interact with the cell membranes or cell walls, bind to DNA [26], and bind to proteins [27]. Silver ions have been reported to interact with thiols, amides, carboxylates, and hydroxyl groups [28][29][30]. Such interactions can result in targeting membrane-bound cytoplasmic enzymes and proteins as well as DNA. These chemical interactions have been reported to cause changes in the structure of bacterial membranes [26] inhibit respiration [31], and increase reactive oxygen species [30].
Silver nanoparticles (AgNPs) have shown promise for managing P. parasitica because of properties attributed to their size and large ratio of surface area to volume [32]. In a study by Ali, et al. [32], they demonstrated that these Artemisia absinthium based AgNPs inhibited the growth of P. parasitica at various developmental stages. AgNPs exhibited a potent inhibitory effect on the growth of mycelia and zoospore germination. The mycelial growth was reported to be inhibited strongly after 12 hours of incubation with AgNPs. Although several studies have outlined mode of action of AgNPs in bacterial [33,34] and fungal pathogens [23], the precise mode of action of AgNPs against P. parasitica is not fully understood. This study aimed to elucidate the changes in the cellular metabolism induced by AgNP toxicity by quantifying and studying genes associated with the pathways of P. parasitica.

Exploratory transcriptome analysis of P. parasitica transcriptome in response to AgNPs
The exploratory expression studies we used provided a general overview of the impact of AgNPs on P. parasitica and provided targets for qRT-PCR. Clean reads from all fourteen libraries were mapped to the P. parasitica genome INRA 310.3 (AGFV00000000.2) ("(dataset) Broad Institute [35] Phytophthora parasitica INRA-310 Genome sequencing and assembly. National Center for Biotechnology Information,"). All the samples showed overall mapping coverage of 74-85% to the P. parasitica genome. Details of the mapping and sequences are given in Table S1. Dramatic differences in P. parasitica transcriptome were observed in the samples treated with AgNPs compared to the control samples.
To check the variation among all the samples and the quality of our data, we conducted principal component analysis (PCA) ( Figure 1A) and hierarchical clustering (HC) ( Figure S1). The normalized differences in expression patterns were used to compute a distance matrix. Distances among samples and replicates can be displayed in two dimensions. PCA discriminated between treatments and replicates based on 76% of the variance (PC1 accounted for 53% of the variance and PC2 for 23% of the variance). Clustering results indicated low variation among the treatment replicates, indicating reproducibility of the biological replicates. P. parasitica treated with AgNPs showed considerable separation compared to the mock-treated control samples at all time points, indicating transcriptome changes in response to the application of AgNPs ( Figure 1A). Furthermore, the biological reps for each time point following AgNPs exposure clustered together but were separated from those at the other time points.
Genes that were altered signifi cantly in their expression in treated samples were identifi ed for each time point at 15 min, 1 hr, and 15 hr (Table S2). The highest number of differentially expressed genes (DEGs), 3818, was found in AgNP-treated samples at 1 hr vs mock-treated control samples at 0 hr. We also compared the DEGs among AgNP-treated samples at different time points and found the highest number of DEGs among AgNP-treated samples at 15 hr vs. AgNP-treated samples at 15 min. Venn diagram analysis indicated a total of 4,028 genes were under expressed collectively in all treated samples at time points 15 min, 1 hr, and 15 hr compared to mock-treated control at 0 hr, while 3,170 genes were over expressed at these time points ( Figure 1B). Gene expression comparisons with adjacent time points showed a total of 4,531 genes under expressed and 4,108 genes over expressed in samples treated with AgNPs ( Figure S2). The genes at each time point in these Venn diagram analyses are provided in Table S3. We identifi ed several processes that were downregulated, such as ribosome biogenesis, DNA replication, and respiration. The results of RNA-Seq give a general picture, and the results are broadly in line with previous studies [34,36] that have reported the same process to be signifi cantly affected in response to AgNPs. Gene ontology (GO) enrichment analysis is frequently used to determine the biological activity of genes. The differentially expressed genes were mapped in terms of their GO analysis to obtain the possible effect of AgNPs. It was seen that GO categories such as metabolic processes and cellular processes were predominantly affected. Unlike fungicides and antibiotics, AgNPs appear to target several processes. Based on the preliminary RNA-Seq data, we identifi ed genes associated with oxidative stress, which were altered in gene expression following exposure to AgNPs.

qRT-PCR indicates alteration of oxidative stress-related genes in exposure to AgNPs
Glutathione S-transferase (GST), which is associated with stress in plants [37] and fungi [38] and which was identifi ed in the exploratory transcriptome analyses, was targeted in qRT-PCR given that the transcript levels of GST were differentially expressed in samples that were treated with AgNPs compared to the mock-treated control. Based on qRT-PCR, GST was signifi cantly upregulated at 15 min and 1 hr in AgNP-treated samples, but then the expression level decreased signifi cantly at 15 hr (Figure 2 A). GST has been implicated in the detoxifi cation of xenobiotic substances, and it was also indicated to be differentially regulated in our pathway analysis.
The signifi cant increase in GST gene expression in response to AgNPs, reveals the activation of this protein as part of the cellular antioxidant defense system. (Figure 2 A). Expression of glutathione peroxidase (EC 1.11.1.9), which is known to remove hydrogen peroxide, was also upregulated [33,39].
Glutathione activity is an important process involved in removing reactive oxygen species (ROS) generated endogenously during xenobiotic metabolism [40]. Glutathione redox is the marker for oxidative stress that plays a crucial role in resisting oxidative stress and maintaining cellular oxidationreduction homeostasis [41]. Upregulation of GST in response to AgNPs and depletion of GSH suggests the imbalance caused in the cellular system and resulting ROS. The ROS can ultimately oxidize DNA and protein. Interestingly, AgNPs have been reported to act by generating ROS and depleting glutathione [39]. Oxidative stress refers to a state where GSH is depleted while oxidized glutathione accumulates [42]. In A. brassicicola, GST transcription was found to be elevated by heavy metals [43]. Bacterial GST genes are suggested to play an essential role in the degradation of xenobiotics and protect from oxidative stress. In a recent study on multidrug resistant P. aeruginosa, AgNPs had a strong bactericidal effect. Transmission electron microscopy showed AgNPs entering P. aeruginosa cells and impairing their structure and morphology by creating a disequilibrium of oxidation and reduction reactions and failing to eliminate excess reactive oxygen species [33,44]. Our analysis substantiates the previous reports where depletion of glutathione signifi es oxidative stress [42]. An increased level of reduced glutathione is considered as a natural defense system in cells to overcome the metal generated oxidative stress [45,46]. The initial upregulation of the genes might be involved in defending the cells from the stress induced by AgNPs. With recent advances, the importance of GST and its role in the diverse cellular process was explored, confi rming it to be a critical target for many inhibitors and drugs [47]. Flavin adenine dinucleotide (FAD)-binding domain, which was also identifi ed in exploratory transcriptome analyses as differentially expressed, has previously been associated with stress in fungi [48]. We used qRT-PCR to measure the expression level of the FAD-binding domain in P. parasitica following exposure to AgNPs. FAD-binding protein was expressed at 15 min post exposure and then was under expressed in all treated samples with AgNP-treated at 1 hr and 15 hr ( Figure 2B). FADbinding proteins are well known for their vital function in chromatin remodeling [49], DNA repair, protein folding [50], and dehydrogenation [51]. Exposure to AgNPs has enhanced their expression, likely to counteract the damage caused by the AgNPs. The qRT-PCR expression data support the gene expression data indicated by RNA-Seq analysis. FAD is involved in the dehydrogenation of many metabolites and has a role in oxidation [52].
Stress at the cellular level is also induced in response to nanoparticles. Stress in cells is manifested by the production of stress related proteins, including heat shock proteins (Hsp), which are produced in cells that are exposed to extreme temperature, pH, nanoparticles, or heavy metals. Research indicated that heat shock protein expression increased fourfold in response to AgNPs [53]. Heat shock factors (HSFs) are activated in response to acute stress that binds the Hsp genes and mediates transcription [54]. Our qRT-PCR analysis confi rmed that HSF-type DNA binding proteins were also over expressed at the initial exposure to AgNPs. ( Figure 2C).
Initial high expression of ATP-binding cassette (ABC) transporter is also indicative of self-defense. ABC transporters exude a variety of substrates, including extracellular toxins from the cell [55,56]. However, research has indicated that AgNPs induce cell death and inhibit the effl ux mechanism of the emergence of multidrug resistant (MDR) cancer cells [57]. ABC transporters have also been reported to be differentially regulated in response to AgNPs in P. aeruginosa [36]. In exploratory transcriptome analysis, expression of ABC transporter was increased at 15 min and decreased slightly later  Figure   2D). ABC transporters have also been reported to be involved in toxicant effl ux and virulence [58][59][60]. These transporters serve as an apparatus for cell defense in many organisms, extruding various substrates [55]. The high expression in treated samples in response to AgNPs would indicate a stress response.
Alkyl hydroperoxide reductase subunit (AhpC) a subunit of thioredoxin, which has been reported to be an essential antioxidant and that has been associated with oxidative stress response as it directly reduces hyperoxides [61]. In this study it was found in exploratory transcriptome analysis to be downregulated two fold at 1 hr (Table S4) (Table S5).

qRT-PCR reveals alteration of protein kinases in response to AgNPs
Apart from stress related genes, genes that contain protein kinase domains were also highly altered based on exploratory transcriptomic analysis and qRT-PCR data. Protein kinases play an important role in many cellular processes. The exploratory RNA-Seq analysis revealed that the expression of protein kinase domains signifi cantly increased in treated samples on exposure to AgNPs (Table S2). We selected two kinases, a serine-threonine protein kinase catalytic domain (IPR000719) and a protein tyrosine kinase (IPR001245) from our transcriptomic analysis and observed their expression using qRT-PCR. We observed that protein kinase domain expression was high in all the treated samples while no change was observed in control samples ( Figure 3A). qRT-PCR  proteins and act as an on/off switch for many vital cellular processes including cell division, cell cycle, and metabolism [64]. Protein kinases play an essential role in intracellular signal communication and transcription as protein kinases regulate protein phosphorylation. Protein kinases regulate the protein phosphorylation by catalyzing the phosphorylation reaction where the gamma phosphate of ATP is transferred [65]. The ability of cells to perceive, recognize, and respond to physical and chemical stimuli is the key to their survival.
Signal transduction is therefore important to react quickly and effi ciently to these stimuli. A study on epidermal growth factor signal transduction indicated reduction of phosphorylation on exposure to silver nanoparticles and caused interference in signaling [66]. Perception of signals leads to certain modifi cations in the cell such as posttranslational modifi cations including phosphorylation and plays crucial roles in regulation of key cellular activities including protein binding affi nity, stability, and activity [67]. Protein phosphorylation is regulated by protein kinases and kinases play an important role in signaling, cell division, and gene transcription [68]. Protein kinases have also been involved in non-catalytic process such as survival [69]. As the role of protein kinases is involved in several cellular processes, the differential expression on exposure to AgNPs results in changes in many protein activities which could be detrimental to the entire cellular machinery.

Pathways affected in response to AgNPs
Differential expression of 28 ribosomal biogenesis genes in response to AgNPs was an interesting observation, which suggests that ribosomes in P. parasitica could be a major target for AgNPs. Ribosome biogenesis involves maturation of ribosomal rRNAs and assembly into ribosomal subunits [70].
Ribosomes function at the heart of translation machinery for converting mRNA into protein and plays a vital role in cell growth in eukaryotes [71]. Ribosome biogenesis is highly conserved among eukaryotes and initial steps include rRNA precursors and transcription and assembly of factors and ribosomal proteins. In the eukaryotic cell, ribosome biogenesis requires more than 250 non-ribosomal assembly factors, of which many are vital. Despite many potential targets, only a few chemical inhibitors have been reported so far [72]. From pathway analysis, we observed that the genes that have a role in methylation and pseudouridylation were downregulated in AgNPs treated samples. These play important roles in modifi cation of RNA ( Figure S3). Our results indicated that AgNPs work as an inhibitory agent by interfering with ribosome biogenesis pathways by altering the expression of the genes essential for assembly of 90s pre-ribosome and affecting maturation of 40S and 60S ( Figure S3).
Many other major pathways were altered including glutathione metabolism (Figure 4), glycolysis ( Figure S4), and TCA cycle ( Figure S5). DNA replication ( Figure S6) was also signifi cantly altered. These pathways are essential to maintain an energy source for the cell. They play a central role in providing energy and precursors for various metabolic reactions of the cell [73]. Apart from these, purine ( Figure S7) and pyrimidine biosynthesis ( Figure S8) was altered in our analysis after exposure to AgNPs. Both these pathways play a major role in many cellular processes.

Preparation of silver nanoparticles
AgNPs were synthesized using an aqueous extract of Artemisia absinthium [32]. The plant of A. absinthium was raised in a greenhouse and dried at room temperature. 1g of leaf tissue powder was boiled in 10 ml of deionized water for 5 min. The plant extract was cooled to room temperature and fi ltered through a 0.45 um fi lter. This aqueous extract and AgNO3 (2 mM) were then mixed in equal volumes. After 24 hr, the mixture was centrifuged at 14,000 × g for 10 min. This was repeated fi ve times and the resulting AgNPs were used for in vitro analysis ( [32]. The preparation and characteristics of these AgNPs has been reported previously [32].

Inhibition of P. parasitica in conditions in vitro
The data on the inhibitory potential of AgNPs on P. parasitica have been previously reported by Ali, et al. [32], where the study confi rmed that AgNPs were highly potent in inhibiting mycelial growth, production and germination of zoospores, and germ tube elongation [32]. The AgNPs used in the study were synthesized previously by Ali, et al. [32] and the synthesis was confi rmed by recording UV-vis spectra [32]. AgNPs were also characterized physically using transmission electron microscopy, dynamic light scattering and zeta potential [32].

RNA-Seq data analyses
The raw reads were fi ltered through to eliminate the low-quality reads and maintain only the Phred score >20 depending on wrapper script Trim galore [74]. Trim galore was also used to remove the adapters and checked for quality of reads per sample using FastQC [75]. TopHat2 software [76] was used to map high quality reads to the reference genome  [79] was used to calculate differential gene expression.
We used cutoff value of corrected p -values < 0.05 to select the deferentially expressed genes ( Figure S9).

KEGG analysis
To visualize the involvement of the differentially expressed genes in biological pathways and look for the response of P.
parasitica to AgNPs at 15 min, 1hr and 15hr, Kyoto Encyclopedia of Genes and Genomes (KEGG) ontology of differentially expressed genes was done by using KEGG Automatic Annotation Server (KAAS) and assigned KO terms for each group of genes were mapped on KEGG pathways using Pathview [80].

qRT-PCR analysis
Based on our exploratory RNA-Seq data, seven genes that were differentially expressed and had importance in many essential cellular processes were selected. The selected seven genes were subjected to quantitative real-time PCR using a previously published method [81]. Gene-specifi c primers ( control for the study of gene expression in P. parasitica [82].
Melting curve analysis was carried to verify the amplifi cation of a specifi c single gene by each primer pair.

Conclusion
AgNPs have robust inhibitory effects on P. parasitica.
AgNPs are known to inhibit multiple pathways, and can be utilized as an alternative way of controlling plant pathogens [83,84]. To the best of our knowledge, this is the fi rst report

Supplementary materials
Supplementary materials can be found in attachment and RNA-Seq data is deposited in NCBI.  Tables-S1-S6)