Pathogen-free PTI induction with a plant conditioner

: The effects of ELICE16INDURES, a well-known plant conditioner developed by the Research Institute for Medicinal Plants and Herbs Ltd. Budakalasz, Hungary, were studied in a soybean population. The active ingredients of the compound have been selected to help elicit general immunity in plants without pathogenic damage, thereby roborizing the healthy plant population and preparing it for possible future biotic stressors. Here we have analyzed changes in the expression levels of genes encoding enzymes involved in the catalysis of metabolic pathways that induce and regulate PAMP-triggered immunity (PTI) at two different time points and treatments. Twenty-three different enzymes were analyzed that catalyze different metabolic pathways, such as the biosyntheses of jasmonic acid, salicylic acid, ethylene, phenylpropanoid, flavonoid, and phytoalexin biosynthesis and cellular detoxification processes. Bioinformatical softwares werw used to analyze the results. It has been found that some of the primary defense mechanisms (e.g., Mitogen-Activated-Protein Kinase (MAPK) cascade, jasmonic acid biosynthesis, flavonoid and phytoalexin biosynthesis, etc.) that intensify following the attack of pathogens can be activated without the intrusion of the actual pathogen by an immunochemical. Thus, we proved that plant resistance can be artificially conditioned.

In the population treated by lower dose of plant conditioner after the first treatment 966,199 of 9,919,253 reads were assembled to 23 reference sequences and produced 23 contigs.
A clear increase in the expression level of mitogen-activated protein kinase cascade (MAPK cascade) enzymes was observed at the first sampling time in this study, as the result of the lower dose treatment (Fig. 2). This elevated levels of the cascades enzymes are the first indication of the immunostimulatory effect of ELICE16INDURES. Plants protect themselves against stresses by recognizing and transmitting signals inside the cell via signaling pathways such as the MAPK pathway [29]. Cell surface receptors immediately send a message to the nucleus about the presence of a virulent microorganism. This intracellular messaging occurs through MAPK cascades. MAPK is activated by various biotic and abiotic stresses and is regulated by the upstream regulator MAPK kinase (MAPKK), which in turn is regulated by MAPKK kinase (MAPKKK). This modulation is conserved in all eukaryotes.
As a result of the attack of some infectious agents, the thickening of the plant cell wall is primarily observed as a typical stress response [42]. This thickening is caused by the deposition of silica and callose in the cell wall. When microbes attack the plant, a polymer of callose is formed and deposited at a high rate in the space between the cell wall and the cell membrane. This helps to concentrate antimicrobial compounds (e.g., phytoalexins, ROS, etc.) in the plant at the site of the pathogen entry and also acts as a physical barrier. In the part of the plant cell in contact with the pathogen, locally, vesicles, small spheres and granules form from the cytoplasm. From the material released from these granules, papillae rich in phenols and callose are formed, which are located in the space between the cell membrane and the solid cell wall. Simultaneously, cell wall thickening and a gradual membrane surrounding the pathogen develop, leading to immobilization and death of the infected cell [43]. An important component of the granules is the enzyme NADPH oxidase (NOX), which produces superoxide anions (O2 •-) using oxygen. The resulting superoxide anion is then converted to hydrogen peroxide (H2O2) by the enzyme superoxide dismutase (SOD), and H2O2 induces the formation of crosslinks between cell wall proteins. This makes the cell wall proteins insoluble, which strengthens the cell wall. This rapid reaction precedes the transcription of genes involved in defense [44,45]. In our experiment, the level of the enzyme amine oxidase (AO), which catalyzes the synthesis of locally produced H2O2 in different cellular organs, was also increased. Recent research has supported the involvement of enzymes of the AO protein family in the synthesis of H2O2, which is formed separately in different cellular organs, and H2O2 is probably play a key role in preventing biotic stressor-induced infection [46]. This is accompanied by an increase in the expression of the genes of ascorbate peroxidase (APX) enzyme, which indirectly indicates an increase in the amount of H2O2, a compound that plays a primary role in stabilizing cell walls. Egbichi et al. (2014) [47] measured increased level of H2O2 followed by an increase in APX levels in soybeans induced by nitric oxide applied externally. As a result, increased antioxidant synthesis and tolerance to abiotic stress were demonstrated. Accumulated and excess H2O2 is neutralized and converted to water by the enzyme APXs or catalase (CAT). One of the key enzymes in the Halliwell-Asada cycle is APX, and since there is no CAT production in chloroplasts, the H2O2 generated here is neutralized by this cycle [48].
Treatment with ELICE16INDURES also increases the levels of the enzyme cytochrome P450 monooxygenase (CYP), which is known to be a catalyst for phytoalexin production after cell wall thickening. The gene expression changes of the CYP enzyme family and their effects were examined in soybean [49]. The cytochrome P450 (CYP; P450s) superfamily is the largest family of enzymatic proteins in plants and other organisms. P450s protect the plants from various biotic and abiotic stressors. Cytochrome P450 monooxygenases (P450s) catalyze the oxidation of various substrates using oxygen and NAD(P)H. Plant P450s are involved in the biosynthesis of primary and secondary metabolites with diverse biological functions. Among others, they are involved in phytoalexin biosynthesis and hormone metabolism [50,51].
Phytoalexins are low molecular weight antimicrobial compounds produced by plants during the attack of a wide variety of plant pathogens. The phytoalexins produced are host-specific, such as camalexin in Arabidopsis [52], some diterpenoids in rice [53], gliceollins in soybeans [54], and serotonin in wheat [55]. Chamalexin is derived from tryptophan and the enzyme CYP is involved in the biosynthesis of chamelexin. In the absence of phytoalexin production plants become vulnerable to attack by a number of pathogens [56]. In Arabidopsis, several phytoalexin-deficient (pad = phytoalexin-deficient) mutants have been identified that were unable to accumulate the phytoalexin chamalexin after infection with the pathogen. The pad3 mutant impaired resistance to Alternaria brassicicola infection [57], while the pad4 mutant is characterized by increased susceptibility to a number of pathogens [58].
As a result of our plant conditioning treatment, an increase in the expression levels of EDS1-2 and PAD4 (Enhanced Disease Susceptibility1 and its co-regulator Phytoalexin Deficient4) enzymes were observed, which promote increased phytoalexin production. According to EDS1 and PAD4 [59] are well-known regulators of both basal and resistance (R) gene-mediated resistance. Two EDS1-like (GmEDS1a / GmEDS1b) and one PAD4-like (GmPAD4) proteins have been identified in soybeans that are required for signal transduction of resistance. Although the expressions of EDS1 and PAD4 genes were increased by biotic stressors, this was not resulted in an increase in SA level.
At the same time, soybean plants showed increased resistance to Pseudomonas syringae pv glycinea. Studies in Arabidopsis plants have revealed associations between the SA pathway and the EDS1-2/PAD4 genes involved in the functioning of this pathway. Until recently, it has been hypothesized that these two enzymes are involved in the SA pathway, but a new research suggests that they operate independently but in parallel pathways [60]. Genetic and transcriptomic data also revealed an EDS1/PAD4-regulated branch being independent of SA in basal and TNL immunity [58,[61][62][63][64]. The EDS1-2/PAD4 pathway contributes to plant protection as a kind of helper mechanism when functioning of the SA pathway is insufficient. When gene expression in the SA pathway following the pathogen attack is inadequate, the EDS1-2/PAD4 pathway is also activated. In the putative system, EDS1-2 and PAD4, in addition to associated SA biosynthesis, maintain important SA-related resistance function, thereby increasing the strength of inherent immunity [60].
The Enhanced Disease Susceptibility5 (EDS5) gene is also closely related to the plant protection system, resulting in increased disease resistance. In healthy plants, the level of EDS5 gene expression is markedly low, but it significantly increases in parallel with the EDS1-2/PAD4 pathway upon infection [65]. Although the level of EDS5 enzyme in healthy plants is extremely low, the biostimulant ELICE16INDURES successfully induced the higher expression of that gene in our experiment, thus strengthening the disease resistance of the plants. The EDS5 protein also elicits immunity induced by basal or pathogen-associated molecular patterns [59,65].
The initial steps of the JA biosynthetic pathway include sequential conversion of α-linolenic acid (LnA) to OPDA by lipoxygenase (LOX), allene-oxide-synthase (AOS), and allene-oxide-cyclase (AOC), which operate in the chloroplasts [66][67][68]. LOX enzymes and oxylipins-enzymatic formation derived from lipid hydroperoxides-produced by them also play a significant role in the defense responses of pathogen-infected plants. Oxylipines such as jasmonates and octadecanoids are oxygen-saturated compounds derived from polyunsaturated fatty acids. While SA regulates protection against biotrophic pathogens, JA and methyl-JA (MeJA) primarily regulate immunity to necrotrophic pathogens and pests [69]. Jasmonates include JA which can be metabolized to MeJA and to its biologically active form JA-isoleucine (JA-Ile) [12,70].
Octadecanoids include 12-oxophytodienolic acid (OPDA) and its derivatives. These compounds are synthesized by one of the seven different branches of the LOX pathway, the initial compound of which is α-linolenic acid (α-LnA) [71]. The primary mediator of the damage response is JA, and the synthesis of OPDA is a crucial step in its formation. OPDA synthesis is catalyzed by the AOC enzyme [72]. The immunostimulator used in this study increased the activity of the LOX enzyme, which plays an important role in catalyzing the biosynthesis of JA. The expression level of AOCwhich is another key enzyme in the synthesis of JA-was also increased. This enzyme catalyzes the formation of the OPDA precursor. SA is critical to SAR, but ISR is less dependent on SA and mainly regulated by JA and ET [12,73]. One possible explanation for this is that MAPK kinases suppress the SA pathway and induce the JA pathway, activating defensins coded by a group of defensive genes.
The former finding is also supported by a spectacular increase in the expression level of the enzyme methyl ketone synthase (MKS-1,2) in our experiment. MKS enzymes are induced by MAPK/ERK (MEKK) enzymes and MEKK enzymes are involved in MeJa synthesis. Thus, we indirectly demonstrated that our immunostimulator stimulated MeJa synthesis, resulting in the activation of some defensin genes. The significant role of defensins in the development of immune responses has also been demonstrated recently in soybean [74].
An additional cellular response is a sudden increase in the levels of plant glutathione S-transferases (GST). GSTs are ubiquitous multifunctional enzymes encoded by large gene families. The complexing and detoxifying effects of glutathione have long been known [75,76], but new research has also revealed novel features [77]. A characteristic feature of GST genes is that their expression can be triggered by a wide range of stress condition. Numerous transcriptomic studies have demonstrated that distinct groups of GSTs are specifically induced in the early stages of bacterial, viral, and fungal infections. Several studies demonstrated that the up-or downregulation of GSTs greatly influences the rate of pathogen proliferation and thus the severity of the course of infections [77]. The expression level of the GST enzyme, which is involved in general cell detoxification processes was also increased in our experiment. It is well documented that GST enzymes in higher plants may be induced by a variety of fungal pathogens, xenobiotics, and elicitors, wounding, and phytohormones [78][79][80][81][82]. increased. NOX uses oxygen to produce superoxide anions (O2 • -), and SOD catalyzes the conversion of the superoxide radical to H2O2 in the organisms, so the enhanced defense function of the plant cell wall is also manifested here. Plasma membrane NOXs are key producers of ROS, and play important roles in the regulation of plant-pathogen interactions. Zhang et al. (2019) [83] performed a comprehensive analysis of the NOX gene family in the soybean genome (Glycine max) and 17 NOX (GmNOX) genes were identified. It is already proved that the stresses produced in plants by high temperatures, drought, toxic metals, salinity, and biotic pathogenes can cause molecular damage directly or indirectly through the formation of ROS. To scavenge ROS and to avoid oxidative damage, efficient enzymatic and non-enzymatic antioxidant systems are present in the cells. One of them in soybean is SOD, an enzyme that alternately catalyzes the dismutation (or partitioning) of the superoxide (O2 • -) radical -produced as a by-product of oxygen metabolism -into ordinary molecular oxygen (O2) and hydrogen peroxide (H2O2) [84,85].
Besides NOXs, oxalate oxidases (OxO) and apoplast AOs are enzymatic sources of ROS production [86-89]. These H2O2-generated enzymes have been shown to mediate lignification and cross-linking, and programmed cell death in response to both normal growth and biotic and abiotic stresses [89][90][91][92][93][94]. The level of AO enzyme, which catalyzes the synthesis locally in different cellular organs produced H2O2, also increased in our experiment. The level of APXs responsible for neutralizing the already redundant H2O2 also increased. Similar to the results of the first sampling time, gene expressions of CYP450 monooxygenase, EDS1-2, EDS5, and IFS enzymes were increased, enhancing the induction of phytoalexin production. Lozovaya et al. (2007) [95] transformed soybean roots with genes derived from the enzymes CHS and IFS, leading to increased phytoalexin (gliceollin) production and resistance to pathogenic fungi.
At the cellular level, malonyl-CoA is formed from acetyl-CoA using the enzyme acetyl-CoA-carboxylase (ACC). The compound is then converted to flavonoids by chalcone synthase (CHS) and coumaroyl-CoA, which are cyclized by chalcone isomerase (CHI). Cyclization results in the formation of flavanone derivatives. These undergo further transformation in processes catalyzed by particular enzymes. Although the central pathway for flavonoid biosynthesis is conserved in plants, depending on the species, a group of enzymes, such as hydroxylases, reductases, isomerases, and several dioxygenases modify the basic flavonoid skeleton, leading to the different flavonoid subclasses [96]. Flavone synthase (FS) produces flavones, isoflavone synthase (IFS) synthesizes isoflavones, while isoflavone hydroxylase (IH), pterocarpane synthase (PTS), and pterocarpane hydroxylase (PTH) together form pterocarpans. Glycinol, a precursor compound of phytoalexins (gliceollidine, gliceollin), is derived from pterocarpane by further transformation [97]. Lygnin et al. (2013) [98] demonstrated that rapid accumulation of gliceollin during infection contributes to the inherent protective response in soybeans. In addition, according to these authors, the level of the enzyme LOX, which plays a role in the production of oxylipins (e.g., JA), was also increased.  In the higher dose-treated population, the expression levels of the MAP cascade, MEKK and MKS 1,2 enzymes increased at the first treatment time (Fig. 4), proving that all of them are responsible for defensin production. The expression levels of SOD and APX enzymes involved in primary cell wall thickening were significantly increased; while CHS, IFS and EDS1-2/PAD4-enzymes that support phytoalexin synthesis-were elevated. Ultimately, the expression level of the GST enzyme responsible for cell detoxification processes was also increased.

Figure 4: Gene expression levels of control and population treated by higher dose of plant conditioner after the first treatment
In the population treated by higher dose of plant conditioner after the second treatment 566,317 of 8,795,753 reads were assembled to 23 reference sequences and produced 23 contigs.
A higher dose of the plant conditioner at a later time (Fig. 5) induced the MAPK cascade and increased NOX activity, and the latter promotes the production of reactive oxygen species involved in cell wall thickening. In addition, the level of the enzyme ACC, the first enzyme in the pathway that initiates the production of phytoalexins [99] has been increased. The ACC enzyme converts acetyl-CoA to malonyl-CoA. CHS, which induces the second step of the pathway, promotes the formation of the flavonoid structure and also increases the gene expression levels of IFS enzymes, which catalyze the biosynthesis of isoflavones. The change in the expression levels of the additional enzymes studied in our work did not show a significant difference as a result of the treatments.  Our examinations were carried out on the soybean tables of PlantArt Research Ltd. in the outskirts of the town of Tata, Hungary, in 2020. Samples were taken from both untreated plots and plots treated with ELICE16INDURES plant conditioner at doses of 20 g . ha -1 and 240 g . ha -1 , applied two times (25 May, 2020 and 10 July, 2020), and sampled two days after treatments. Fresh leaf samples were collected in RNA-shield (Zymo Research, Irvine, US) preservative, then sequenced by Xenovea Ltd, Szeged, Hungary. Data were validated, followed by a full bioinformatics analysis of the sequencing results.

Sample sequencing protocol
Approximately 30

Preprocessing
Bioinformatical softwers were used to data analyses and evaluation. During pre-processing, qualitative analysis was performed and raw sequencing data consisted of 80 bp long single-end sequences were prepared from which the transcriptomes were reconstructed, the reference transcriptome was prepared, and gene expression analyzes were performed. During pre-processing, reads were purified sample-by-sample (filtered for Q30 and 'N').

Quality control and filtering of libraries
The analysis is performed by a nine-module FastQC program [100], which provides a quick overview of whether the data are suitable for further analysis and whether there are any problems or distortions in the read sequences that affect further analysis. Statistics were generated from filtered reads. During purification, reads and bases containing adapters, contaminant sequences, and short or poor quality reads due to possible sequencing errors were filtered out from the raw data. Screening was performed with Trimmomatic and GenoUtils [100,101]. Filtered directories are named clean-prefixed. SRA (unassembled sequence reads) data was deposited in the NCBI under the accession PRJNA778970, SRA submission SUB10637174.

Computer Processing of Samples and Creating a Reference Transcript Database
To create the reference, the sequenced libraries were treated together. The pooled reads were combined with a 3module Trinity program based on the Bruijn graph in order to reconstruct the longest possible mRNA fragments (transcripts) from the short readings (80 bp). The Trinity program allows de novo assembly. When running Trinity, we worked with a value of a 25 K-mer, saving files in fasta extension. Gene-level analyses were performed from generated data [102,103]. TSA (Transcriptome Shotgun Assembly) data was deposited in the NCBI under the accession PRJNA778970, TSA temporary submission SUB10815164 .

Using a reference transcript to search for unique sequences
Gene sequences of enzymes involved in the metabolic processes that may be affected by the immunochemical used in the experiment were collected from known related species (Glycine max and Glycine soybean) using the NCBI public database (https://www.ncbi.nlm.nih. gov/nuccore). In the next step, we retrieved the expressed contigs from our own reference transcriptome database using the Blast+ alignment program [104].

Matching short reads to reference sequences
Using Geneious Prime 9.0 bioinformatics software [105], short reads of transcripts obtained from the samples of the control and those from two different dose-treated strains were aligned to the reference sequences, and these data were further analyzed. First, individual RPM (Reads per million mapped read) values were calculated for each gene (equation is shown below), and these values were then plotted on figures separately for each treatment and time point.

=
(1) In some special RNA-seq protocols, especially in the shallow RNA-seq methods, reads are generated only from one end of the RNA molecule, regardless of the length of the molecule. Since RPM does not take transcript length into account, this unit reflects the actual number of transcripts more accurately. From the RPM values we obtained the individual expression levels of the enzyme sequences operating in the given treatment at a given time, i.e. the degree of their really expression. From the RPM values we obtained the individual expression levels of the enzyme sequences reacting to the treatments applied at a given time, that is, the degree of their expression [106].

Functional annotation
Functional annotation and Gene Ontology (GO) analyses were carried out using OmixBox, Biobam, as follows: 1. Sequences were blasted against NCBI nr (non-redundant) Viridiplantae database (downloaded in 2019) applying blastn configuration locally; 2. To retrieving GO terms associated with the 10 Hits obtained by the Blast search, GO mapping and annotation were performed. GeneBank identifiers (gi) and the primary blast Hit identifiers were used to retrieve UniProt IDs making use of a mapping file from PIR (Non-redundant Reference Protein Database) including PSD, UniProt, Swiss-Prot, TrEMBL, RefSeq, GenPept and PDB. Accessions were searched directly in the db x ref table of the GO database. BLAST results were searched directly in the gene-product table of the GO database; 3. GO annotation were specified according to GO terms of molecular function, cellular component, and biological process.

Conclusions
In our experiments, it has been found that some of the pathogen-induced immune responses of plants can also be elicited using certain immunochemicals. Pathogen-induced SAR (systemic acquired resistance) tends to activate the SA pathway, while priming-induced ISR (induced systemic resistance) triggers other pathways, mainly JA and ET biosynthesis. The results of our experiment are in accordance of these in soybean treated with a plant conditioner, thus proving that ISR can be induced by a natural immunochemical without the presence of a pathogen. General immunity is an inherent, genetically encoded trait of a plant that provides protection to organisms that are not otherwise able to defend themselves through metabolic pathways. Although the plant conditioner used did not increase the expression of all the genes involved in the defense, it activated some of the primary plant responses immediately after infection (e.g., cell wall thickening, accumulation of phenolic compounds, activation of antioxidant enzymes, etc.). Due to its effect, the long-term use of similar immunochemicals has a beneficial immune-boosting effect on the plants. Another advantage of the plant conditioner investigated here (ELICE16INDURES) is that its ingredients can be used in the long run, and so this also can be a sustainable, environmentally safe way of agricultural production. Thus, plant-based antimicrobial agents may also provide an excellent alternative for organic farmers.