Time-Series Transcriptome Analysis of the European Plum Response to Pathogen Monilinia fructigena

Abstract

European plum production is affected by mostly harm Monilinia spp., causing full pathogen brown-rot infections. The plums are the susceptible to the Monilinia fructigena pathogen, which is the most common in Europe. This study aims to analyze the gene expression profiles and signaling pathways of the European plum, cv. Victoria, inoculated with the M. fructigena pathogen at 24, 48, and 72 h post inoculation. By transcriptome sequencing, the number of differentially expressed genes (DEGs) increased over time, with the highest number at 72 hpi, showing the tendency to involve more genes in the response to prolonged exposure to the pathogen. Pathogenesis-related (PR) family and mildew resistance locus O (MLO-like) proteins were expressed the most during plum response to the pathogen. The plum initiates complex defense responses by significantly activating 23 pathways according to Kyoto Encyclopedia of Genes and Genomes (KEGG). In this study, expressed genes over the infection were in response to stress, defense, cell death, and disease resistance. The findings of this study could be used as the basis for further research of markers linked to resistance or susceptibility to disease in plum hybrids at an early age, which will improve the plum breeding process.

1. Introduction

Plums are a botanically diverse stone fruit group that includes up to 40 species and originates from Europe, Asia, and America. The most commercially important species are the hexaploid European plum (Prunus domestica) and the diploid Japanese plum (Prunus simonii and Prunus salicina), both belonging to the Prunus genus, Rosaceae family [1,2,3]. Plum fruit production reaches around 12 million tons a year worldwide, the second highest group of stone fruits (after peaches), according to FAO 2022 data [4]. Fruit quality, nutritional value, productivity, and disease tolerance are the main criteria for horticulture and breeding programs, in which the creation of new cultivars of the European plum are initiated [1,3].

The fungal Monilinia spp. pathogens cause the highest losses of stone fruit production worldwide [5,6]. In the genus Monilinia, the three most common species in Europe are M. fructigena, M. laxa, and M. fructicola [5]. M. fructigena is spread all over the world—in Africa, Asia, and Europe, and it is a quarantine pathogen in the United States, Canada, New Zealand, and Australia [7,8]. The most significant yield losses are observed on pome fruits; however, this pathogen also infects stone fruits, from which plums are the most susceptible [5]. The M. fructigena pathogen causes blossom blight, twin canker, branch infections and, most importantly, fruit rot before and during storage [5,9]. Like other Monilinia spp., M. fructigena is a necrotrophic fungal pathogen, which generates typical brown-rot symptoms, i.e., a concentric brown or white pom-pom-shaped conidial layer on the fruit surface [10,11]. During fruit infection, the M. fructigena pathogen invades the wound by forming a light brown rot spot with the wound in the center. The pathogen quickly expands to the surrounding tissue, damaging the fruit [11]. Climate changes encourage novel virulence alleles’ appearance, which lead to the emergence of new pathogenic species and resistance to existing fungicidal agents [10,12].

Fruit resistance to brown rot depends on constitutive and active defense mechanisms. Stone fruit resistance is associated with a higher content of phenolic compounds in the epidermis and the flesh of the fruit [13]. The thickness and density of the cuticula and the epicuticular wax also adds additional resistance to brown rot [14,15]. Possible induced defense mechanisms in fruits, such as hypersensitivity response, phytoalexin production, pathogenesis-related proteins (PR), reactive oxygen species, and systemic acquired resistance, are activated in cells during host–pathogen interactions. However, a better understanding of the active defense strategies at the genetic level would aid in identifying resistant germplasm resources against Monilinia spp. fungal damage [16].

The interaction between fruit and fungi has been studied using sequencing technologies, by analyzing transcriptomic changes in peach fruit infected with the M. fructicola pathogen [11,13,17] and M. laxa [18]. Moreover, stress, induced with the M. laxa pathogen, was already analyzed in nectarine fruit [10]. Transcriptome analysis is used to better understand the host plant response to infection, by analyzing the interaction mechanisms, discovering genes related to disease resistance and understanding the response pathways [19].

For breeding purposes of the European plum, the main objective is to create cultivars with genetically determined resistance to Monilinia spp. [20]. The identified and evaluated genes open possibilities for modern plant breeding with integrated genetic engineering, gene editing or marker selection. These technologies allow for faster, more precise and targeted crop improvement compared to traditional breeding. Transcriptomic profiles of the European plum with an induced infection of M. fructigena have not been analyzed yet and the genetic response to host–pathogen interactions requires elucidation. The aim of this study is to analyze the gene expression profiles and signaling pathways of the European plum, inoculated with the M. fructigena pathogen over the infection time period.

2. Materials and Methods

2.1. Inoculation of Plum Fruits with M. fructigena

According to long-term evaluations in Lithuania genetic resources collection of plum in the Lithuanian Research Centre for Agriculture and Forestry Institute of Horticulture (LRCAF IH), European plum cv. Victoria was evaluated as moderately susceptible to brown rot, with a resistance score of 5 (according to Postman et al. [21] scale), showing 25–50% Monilinia spp. infection. Visually intact fruits were collected at the commercial maturity stage from the same orchard in August of 2024.

Fungi mycelium was collected from the European plum from the LRCAF orchards in 2023 and grown on potato dextrose agar (PDA) for 10 days at 22 °C for 16 h in the light and 8 h in the dark. Monilinia fructigena was identified by multiplex PCR using four primers and PCR conditions as described by Cotê et al. [22] and by sequencing internal transcribed spacer (ITS) regions (ITS1 and ITS2) with ITS4 and ITS5 primers (Macrogene, Europe, The Netherlands). The isolate used in this study is identical to M. fructigena isolate DM1082 (Genbank accession No. MT644896.1).

The surface of the plum fruit was disinfected using commercial bleach, according to Tsalgatidou et al. [17]. For more effective fruit inoculation, the surfaces of all plum fruits were wounded with a sterile wooden stick to a depth and width of 2 mm. Fruits, except control (C treatment), were inoculated with M. fructigena mycelium (F treatment) by putting a 0.5 mm diameter plug of M. fructigena mycelium, with the mycelial side down on to the wounded fruit site from the edges of plate 10 days old culture. The inoculated fruits were placed into sealed transparent containers on a sterile wet paper towel. The fruits were incubated for 24, 48, and 72 h at 24 °C with high humidity and under standard photoperiod conditions [17]. The peel and pulp of the plum were collected by cutting a 3 × 3 cm section, 4 mm deep, around the inoculation sites at three time points: 24, 48, and 72 h post inoculation (hpi). Samples were frozen and stored at −20 °C for RNA extraction.

2.2. RNA Isolation and Transcriptome Analysis

Total RNA was extracted with the GeneJET Plant RNA Purification Mini Kit (Thermo Scientific, Waltham, MA, USA), according to the manufacturer’s protocol. RNA quality was evaluated using a Nanodrop Implen GmbH spectrophotometer (Implen, Munich, Germany) and Qubit 1X dsDNA HS (High Sensitivity) assay kit (Thermo Fisher Scientific, Waltham, MA, USA).

In total, 18 samples (3 controls and 3 treatments, including 3 biological replicates) of the European plum ‘Victoria’ tissues were used for transcriptome analysis (see above). Sample preparation and mRNA library construction (poly-A enrichment) was performed by Novogene (Cambridge, UK). The RNA sequencing was conducted with the Illumina NovaSeq x Plus Series (PE150) sequencing system. The accession number of Bioproject in NCBI data base is PRJNA1222764.

2.3. Data Analysis

Raw reads were processed using fastp software [23], processing included cleaning from low-quality reads, removal of adapters and poly-N containing reads. Reads were mapped to the Prunus persica genome (Genbank accession No. GCA_000346465.2), which is a model plant of Rosaceae family, using Hisat2 v2.0.5 software [24]. Gene expression levels were calculated by the expected number of fragments per kilobase of transcript sequence per millions base pairs (FPKM) for each gene, based on the gene length and mapped reads.

For the differential gene expression analysis, DESeq2 [25] software was used. The threshold of significant differential expression values was p < 0.05 and |log2 foldchange| ≥ 1.

The expressed DEGs were annotated by Gene Ontology (GO) [26] and Kyoto Encyclopedia of Genes and Genomes (KEGG) [27] enrichment analysis using the cluster Profiler (4.0) software [28]. The differential genes related to fungal infection at three time points (24, 48, and 72 hpi) with 3 experimental replicates were analyzed. The relative expression profiles of DEGs, determined by qRT–PCR analysis, were visualized using SAS (Statistical Analysis System) (2011) SAS Version 9.3 [29].

Data of principal component analysis, heatmap, GO categorization, and significantly enriched DEGs were visualized using Science and Research Online Plot, n.d. (SRplot) software [30]. R software (4.4.3) [31] was used for upset plot and KEGG pathway enrichment analysis visualizations.

2.4. Quantitative Real-Time PCR Validation

To validate the RNA-seq data, quantitative real-time PCR (qRT-PCR) was used. The total RNA samples from the European plum controls and infected samples as described above were used. The first-strand cDNA was synthesized from previously extracted RNA samples using the Maxima H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. The qRT-PCR was performed using the Applied Biosystems QuantStudio 5 PCR system (Thermo Fisher Scientific, Waltham, MA, USA). The qRT-PCR reaction was performed using the PowerUp™ SYBR™ Green Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) with standard thermo cycling mode (60 °C for annealing and extension) according to the manufacturer’s protocol. The qRT-PCR reactions were performed with three biological replicates. Specific primers for 12 randomly selected DEGs were designed, and their expression profiles were analyzed (Table S1). The normalization of gene expression was performed with NADH dehydrogenase subunit 5 of the NADH ubiquinone oxidoreductase complex (nad5), coding mitochondrial gene of higher plants [32]. NAD5 gene is used for internal control and due to the constant expression of this gene in transcriptome data, for transcript validation through RT-qPCR [33]. The 2^-ΔΔCT method [34] was used to calculate the gene expression relative to the selected reference gene (nad5), using three technical replicates for each DEG evaluation. The statistical analysis was performed using Duncan’s multiple range test.

3. Results

3.1. Transcriptome Sequencing Results

To identify transcriptomic changes in time dynamics of the European plum (Prunus domestica) fruits in response to Monilinia fructigena infection, RNA-seq for 18 sequencing libraries (including three biological replicates) was performed. In total, 777,575,418 G high-quality reads and 116.62 G raw bases were generated from the European plum pulp (

Table A1. Summary data of quality control (QC) of the European plum transcriptome sequencing throughout 24, 48, and 72 hpi with M. fructigena infection. C—control plum, F—plum, inoculated with M. fructigena pathogen.

Sample Library	Raw Reads	Raw Bases, G	Clean Reads	Clean Bases, G	Error Rate, %	Q20	GC pct, %
C_24	43,077,636.00	6,46	42,109,142.00	6.32	0.01	98.49	45.33
	46,969,746.00	7.05	45,559,624.00	6.83	0.01	98.50	44.49
	44,588,494.00	6.69	42,679,676.00	6.40	0.01	98.70	45.80
Average	44,878,625.33	6.73	43,449,480.67	6.52	0.01	98.56	45.21
C_48	40,840,226.00	6.13	40,081,022.00	6.01	0.01	98.63	45.93
	41,759,202.00	6.26	40,218,422.00	6.03	0.01	98.53	46.08
	41,459,352.00	6.22	39,908,394.00	5.99	0.01	98.75	45.38
Average	41,352,926.67	6.20	40,069,279.33	6.01	0.01	98.64	45.80
C_72	48,350,406.00	7.25	47,224,864.00	7.08	0.01	98.64	44.86
	41,882,526.00	6.28	40,893,806.00	6.13	0.01	98.69	45.83
	42,733,784.00	6.41	41,984,820.00	6.30	0.01	98.54	44.97
Average	44,322,238.67	6.65	43,367,830.00	6.50	0.01	98.62	45.22
F_24	42,799,956.00	6.42	41,868,796.00	6.28	0.01	98.46	45.61
	42,027,560.00	6.30	40,638,812.00	6.10	0.01	98.72	45.86
	48,588,966.00	7.29	47,264,042.00	7.09	0.01	98.67	45.10
Average	44,472,160.67	6.67	43,257,216.67	6.49	0.01	98.62	45.52
F_48	41,425,328.00	6.21	40,386,776.00	6.06	0.01	98.76	45.24
	40,753,924.00	6.11	39,872,974.00	5.98	0.01	98.52	46.01
	41,746,662.00	6.26	40,747,272.00	6.11	0.01	98.49	45.49
Average	41,308,638.00	6.19	40,335,674,00	6.05	0.01	98.59	45.58
F_72	42,496,534.00	6.37	41,609,950.00	6.24	0.01	98.49	46.26
	41,317,856.00	6.20	40,546,588.00	6.08	0.01	98.60	46.21
	44,757,260.00	6.71	43,730,384.00	6.56	0.01	98.77	46.39
Average	42,857,216.67	6.43	41,962,307.33	6.29	0.01	98.62	46.29
Total	777,575,418.00	116.62	757,325,364.00	113.59	N/A	N/A	N/A

). A quality score of 20 (Q20) was in the range of 98.46 to 98.77% with an average of 98.61%. The proportion of guanine (G) and cytosine (C) bases (GC content) was in the range of 44.49 to 46.39%, with an average of 45.60%.

In total, 185 million transcripts were identified in all analyzed samples with an average of more than 10 million per sample (Table S2). For alignment of reads to the reference, the model plant P. persica for Rosaceae family was used [35]. On average, 64.25% of reads were mapped to the P. persica reference genome. The highest number of transcripts was observed in the F_24 sample with an average of more than 11 million, and the lowest number (about 8 million) was observed in the F_72 sample.

3.2. Evaluation of Expressed Genes Across Sampling Points

To visualize the gene expression data of 61,765,758.33 genes in a total of 18 samples (including three biological replicates) across controls (C_24, C_48, and C_72) and inoculated plum (F_24, F_48, and F_72), principal component analysis (PCA) was carried out (Figure S1). Replicates within each group cluster closely together, indicating consistency in the data for each condition. Samples of all controls and inoculated fruits appear in distinct regions of the plot, showing that infection alters gene expression significantly. According to both principal components, all control samples shifted over time; however, inoculated fruit samples shifted only according to PC2.

The number of uniquely expressed genes in the plum cv. Victoria inoculated with the M. fructigena pathogen (F_24, F_48, and F_72) and its controls (C_24, C_48, and C_72) is shown in Figure 1. In the control samples, the highest number of unigenes (264 genes) was observed at the first sampling point. After 48 hpi, the number of unigenes decreased to 40 genes, and then increased again to 59 genes at 72 hpi. Similar tendencies were observed among transcripts of inoculated fruits. After 24 hpi, the number of unigenes was 96; after 48 hpi, it decreased to 44 genes; and then increased to 189 genes at 72 hpi. In the inoculated samples, the highest number of uniquely expressed genes was observed after 72 hpi.

In total, 11,611 genes were commonly found between all samples. The commonly found genes in all samples were excluded from the upset plot diagram (Figure 1). All controls (C_24, C_48, and C_72) and two inoculated samples (F_24 and F_48) shared 700 commonly expressed genes. In inoculated samples at three sampling points (F_24, F_48, and F_72), more than 300 commonly expressed genes were observed. In inoculated samples at all analyzed time points and the control sample at 24 hpi, over 200 genes were commonly expressed. These results indicate that the expression of unigenes constantly changes during fungal infection in plum fruit.

3.3. Comparative Analysis of Differentially Expressed Genes (DEGs)

To analyze the number of differentially expressed genes (DEGs) during the infection of plum, the data were compared to the gene expression data from control samples, using p < 0.05 as a screening criterion. To visualize gene expression distribution, Volcano plots were generated (Figure 2). The number of differentially expressed genes in the inoculated plum samples increased from 13% (2 522) in the F_24 sample to 48.9% (9 208) in the F_72 sample. The number of up-regulated DEGs rose from 7.2% (1384) in F_24 to 25.4% (4789) in F_72 samples. Similarly, the down-regulated DEGs increased from 5.8% (1138 genes) in F_24 to 23.5% (9639 genes) in the F_72 sample. During biotic stress, the highest number of activated and suppressed genes was observed after 72 hpi (F_72). Over the time period from 24 to 72 h, the fruit of the European plum cv. Victoria involved an increasing number of genes in its response to the pathogen.

3.4. Functional Annotation and Classification of DEGs by GO Enrichment Analysis

To compare the expression of genes between the European plum controls and inoculated plums, Gene ontology analysis with padj < 0.05 was performed. DEGs were grouped into three functional categories (GO terms): biological process (BP); cellular component (CC); and molecular function (MF) (Figure 3). In total, 80 different GO terms were expressed during biotic stress, compared to the control.

Overall, the number of genes over the inoculation period increased in all three functional categories. The highest impact of the fungi is shown in the BP category, where the total number of genes increased 4.5 times (from 287 genes in F_24 to 1297 genes in F_72). In the biological processes category, 37 GO terms were obtained. Three GO terms, found in all sampling points, were defense response, protein ubiquitination, and protein modification by small protein conjugation.

Over the time period, the impact of the pathogen in the CC group increased significantly (1.9 times), with the number of genes rising from 93 in F_24 to 172 in F_72. In total, 12 GO terms were annotated in the cellular components group. Only uniquely expressed GO terms across all sampling points were found. The most prominent GO terms, with the highest number of associated genes, were an integral component of the membrane, identified in the F_48 sample with 134 genes. This was followed by terms ribosome, with 129 genes and cell periphery with 43 genes, both observed in the F_72 sample.

In the MF category, the total number of genes increased from 663 (F_24) to 1318 (F_72). Genes were distributed into 31 GO terms. Seven GO terms were common throughout all sampling points with the highest number of genes associated with transferase activity, transferring glycosyl groups (84 genes in F_24, 110 in F_48, and 201 genes in F_72).

According to the GO enrichment analysis, DEGs which were responsible for defense response, were involved in the biological processes group. During the inoculation, the highest number of these genes was observed in the F_72 sample (38 genes), followed by the F_48 (34 genes) and F_24 (21 genes) samples. During the impact of the pathogen, the number of defense response genes increased throughout time. The infection of plum fruit tissue triggers a defense response, which may enhance resistance to pathogen infection by regulating intracellular metabolism.

3.5. Functional Annotation and Classification of DEGs by KEGG Enrichment Analysis

During the fungal interaction on plum fruit over the time period, 23 significant (padj < 0.05) pathways of three KEGG categories were activated (Figure 4A). The highest number of pathways (20) was activated in the metabolism (M) category, followed by the environmental information processing (EIP) category with two pathways in the F_24 and F_48 samples. Additionally, one pathway was activated in the genetic information processing (GIP) category (F_72).

Six significantly enriched pathways were found commonly, at the infection recognition stage (F_24) and at the response to pathogen stage (F_48) (Figure 4B). In the metabolites category, four commonly expressed pathways between the F_24 and F_48 samples were found with the most abundant appearance of DEGs in the metabolism of pyruvate, phenylpropanoid, biosynthesis of various plants secondary metabolites, and 2- Oxocarboxylic acid. In the environmental information processing category, plant hormone signal transduction and MAPK signaling pathways, were highly expressed in the F_24 and F_48 samples. The highest enrichment of DEGs was found in plant hormone signal transduction, followed by pyruvate metabolism and MAPK signaling pathways (Figure 4B).

The alpha-linolenic acid metabolism pathway was highly significantly expressed and common to inoculated plum fruits in the F_48 and F_72 samples. This pathway was more enriched in the F_72 sample, showing that the alpha-linolenic acid metabolism pathway was important in longer exposure to the pathogen.

At the infection recognition stage (F_24), four pathways were activated uniquely in the M group: carbon and nitrogen metabolism; terpenoid backbone biosynthesis; and photosynthesis antenna proteins. Higher number of uniquely expressed pathways were found in the response to pathogen (F_48), with 11 uniquely expressed pathways. The highest gene count was found in the biosynthesis of cofactors (71) and the biosynthesis of amino acids (67 genes). In the F_72 sample, only the ribosome pathway was expressed uniquely (GIP group), which is important for host plant long-term response.

3.6. Genes Involved in Infection Response

During the interaction with the pathogen, 41 DEGs were involved in the defense response over the time period, according to functional GO enrichment analysis (Table S3). The expression levels of DEGs are shown in the heatmap (Figure 5). DEGs are involved in response to M. fructigena encode pathogenesis-related (PR) proteins, mildew resistance locus O (MLO-like) proteins, single-stranded DNA-binding protein, nematode resistance protein-like, and phytohormone-binding protein.

In total, 22 pathogenesis-related family proteins PR-10 and PR-4 were significantly up-regulated. The highest number of significantly activated PRs was observed in F_72 sample (22 DEGs); however, the expression of these genes was highest after 48 hpi (F_48) (72% of all expressed PRs). After 24 hpi (F_24), 15 PRs were expressed; however, their expression level was the lowest compared to samples in other time points.

Moreover, 13 DEGs of mildew resistance locus O protein of MLO1, MLO3, MLO4, MLO8, MLO11, MLO12, and MLO13 genes were activated or suppressed across all time points. The highest number of up-regulated MLOs was observed in the F_48 sample (8), followed by the F_72 (7) and F_24 (5) samples. Five down-regulated MLOs were identified in F_72 and one was identified in F_48. The highest expression of up-regulated MLO-like DEGs was observed in the F_72 (5) sample.

One nematode resistance protein-like HSPRO2 gene was activated in the F_48 sample and the expression level of this DEG was the highest in F_72 sample. Single-stranded DNA-binding protein, encoded by the WHY1 gene, was suppressed at all time points with the highest suppression level in the F_72 sample. The phytohormone-binding protein gene MTR_3g055120 was suppressed only in F_72 hpi.

According to the NCBI database [36], all gene sequences matched the genome of Prunus persica and are involved in defense responses. These genes were also observed in five more pathways. PR-10 and MTR_3g055120 DEGs were involved in the abscisic acid signaling pathway, enabling abscisic acid binding, protein phosphatase inhibitor activity, and signaling receptor activity. These genes are expressed at all sampling points, with one DEG being suppressed in the F_72 sample. Additionally, the HSPRO2 gene was activated in the F_48 and F_72 samples, involving the tryptophan catabolic process to kynurenine, which enables heme binding and metal ion binding. Regulation of the WHY1 gene was suppressed in the F_72 sample, which enables single-stranded DNA binding. Three PR genes activate RNA nuclease activity and are involved in defense responses to bacteria and fungi pathways, with the highest expression observed in the F_48 sample.

According to the KEGG pathways annotation, the highest impact of DEGs from plum defense response to infection were found in the MAPK signaling pathway in the F_24 and F_48 samples, with 31 significantly expressed DEGs (Table S4, Figure 6A). Higher expression of DEGs was observed in the F_48 sample (64.5%), comparing with F_24, showing that the expression of DEGs increased over time. Significantly expressed DEGs were classified as protein kinases, ATPase, endochitinase, transcription factors, aminotransferases, ethylene receptor, and response sensor, as well as the BTB/POZ domain, regulatory, F-box, ethylene-insensitive, and pathogenesis-related proteins. The MAPK signaling pathway activates pathogen infection, pathogen attack, and phytohormones pathways.

In the MAPK signaling pathway, 12 DEGs responsible for the response to pathogen infection were significantly activated (Figure S2). Up-regulated DEGs belonged to pathways which activate early defense response (F_24), camalexin and ethylene synthesis (F_24 and F_48), and late defense response to pathogen (F_48). In the pathogen attack pathway, four DEGs were common in both time points: three were up-regulated, and one showed a change in expression level from down-regulated (F_24) to up-regulated (F_48) (Figure S2). The pathogen attack pathway promotes cell death, H₂O₂ production, pathogen defense, and the accumulation of reactive oxygen species as part of the plant’s defense mechanism. The PR1 DEG, involved in pathogen defense and pathogen infection pathways, was significantly up-regulated only in the F_48 sample. In the phytohormones pathway, related to ethylene biosynthesis, in the early stage of pathogen interaction (F_24), 8 DEGs were significantly up-regulated (Figure S2). Later (F_48), the number of significantly up-regulated DEGs increased to 14. In the jasmonic acid biosynthesis pathway, two DEGs (transcription factors) were significantly up-regulated (F_24) and reduced to one (F_48).

Time dynamics have an impact on the MAPK signaling pathway in response to pathogen infection, attack and the biosynthesis of ethylene phytohormone during the infection, with a higher number of DEGs in F_48 than in the F_24 samples. Only the jasmonic acid biosynthesis pathway showed a higher number of DEGs in F_24 than in F_48 samples.

In the plant hormone signal transduction pathway, 15 DEGs were significantly expressed in the alpha-linolenic acid and phenylalanine metabolism pathways (Table S5, Figure S3, Figure 6B). In total, 80% of these DEGs were up-regulated and 20% were down-regulated. The alpha-linolenic acid metabolism pathway is responsible for senescence and stress response. A total of 8 DEGs involved in this pathway were significantly expressed and classified as transcription factors, synthetase, TIFY, and coronatine-insensitive proteins. In the phenylalanine metabolism pathway, eight DEGs were significantly expressed over the time period, in response to disease resistance. In the F_24 sample, two DEGs were activated and two were suppressed, whereas in the F_48 sample, the number of activated DEGs increased to five, and the number of suppressed DEGs decreased to one. The PR-1 gene was significantly activated in the F_48 sample. In the plant hormone signal transduction pathway, a higher proportion of significantly expressed up- and down-regulated DEGs was observed in F_24 sample (53.3%).

The alpha-linolic acid metabolism pathway was activated in the F_48 and F_72 samples (Figure S4 and Figure 6C). In total, 45 DEGs were significantly expressed, and 53.3% of them were common for both time points (Table S6). Commonly found DEGs were classified as lipoxygenase, CoA ligase, oxidoreductase, allene oxide synthase and cyclase, alcohol dehydrogenase, oxophytodienoate reductase, acyl-coenzyme A oxidase, CoA thiolase, multifunctional protein, and jasmonic acid carboxyl methyltransferase. Most of the DEGs were significantly up-regulated (75.6%) and 24.4% of DEGs were down-regulated. Higher expression levels of both activated and suppressed DEGs were observed in the F_72 sample in 91.1% of the DEGs.

3.7. Validation of RNA-Seq Data

In order to validate RNA-seq results, the expression of 12 randomly selected DEGs was evaluated with quantitative real-time PCR (qRT-PCR). In qRT-PCR data, 11 genes showed similar expression direction with RNA-seq results (up-regulated). One DEG (LOC18773995) in qRT-PCR showed positive regulation; however, in RNA-seq data of the F_48 and F_72 samples, it was down-regulated, and the expression level at 48 hpi was insignificant (Figure S5).

4. Discussion

The highest damage to the European plum worldwide is caused by fungal pathogens of the genus Monilinia spp. The plum’s response to fungal Monilinia spp. pathogens has not yet been analyzed at the molecular level [37]. The ability to sequence transcriptomic changes during plum fungal interaction sites enables the analysis of genes involved in infection response. This study provides a comprehensive transcriptomic time-series analysis of the European plum cv. Victoria response to M. fructigena inoculation and highlights the key genes and signaling pathways activated during pathogen progression.

In this study, the M. fuctigena pathogen changes gene expression of the plum fruit by activating more DEGs over time, with the highest number of DEGs in the F_72 sample (Figure 2). The increasing number of DEGs was also reported throughout the infection in peach fruits, which were infected with the M. fructicola pathogen, with the highest number of reported DEGs in the F_48 sample [13,17]. Our results also agree with the research performed by Balsells-Llauradó et al. [10], which found that the number of up-regulated DEGs in mature peach fruit infected with M. laxa increased over time, while in immature fruit, it increased progressively until 24 hpi and then decreased at 48 hpi.

The main effects of biotic stress of M. fructigena were in the biological processes category of Gene Ontology classification in the F_24 and F_48 samples, with cell wall modifications, organic substances’ metabolic and catabolic processes, and protein localization. These functions were also observed in peach fruit infected with the M. fructicola pathogen [11]. Moreover, in this study, the highest gene count was observed in peptide and amide biosynthesis in the F_72 sample. Peptides and amides are secondary metabolites that have shown remarkable long-term effectiveness against fungal infections in previous research [38,39].

During the M. fructigena impact on plum fruit, the highest number of DEGs involved in defense response (according to GO classification) was observed in the F_72 sample. The expression of these DEGs was the highest in the F_48 sample. Gene expression analysis showed that the ones of the most important defense response proteins were the PR-10 family proteins, which displays ribonuclease activity after stress stimulus of fungal elicitors [40]. This study agrees with other research, where the higher expression of PR-10 family proteins after M. fructicola infection was identified in plum [38] and in peach [14]. In our study, the expression levels of PR-10 genes of moderately susceptible to brown-rot cv. Victoria plum do not agree with the expression levels of susceptible cv. ‘Veeblue’; however, they coincide with resistant cv. Violette PR-10 gene expression [41]. The up-regulation of PR-10 in response to fungal infection was also shown in other plants like wheat [42], parsley [43], rubber trees [44], apples [45], and strawberry [46]. All these studies agree that the PR-10 expression depends on biotic stress, and this gene group is involved in fungal disease resistance. The knowledge about PR-10 protein family expression over the infection period is important for crop and fruits improvement.

The second highest group of defense response proteins found in cv. Victoria is encoded by Mildew resistance locus O family genes. The MLO transmembrane protein plays a key role in plant immunity by accumulating on the plasma membrane at the site of pathogen penetration [47]. These genes were significantly expressed in peach, infected with M. fructicola and M. laxa pathogens [17,47].

The host plant resistance mechanism to fungal infection is determined by significantly enriched pathways. At the infection recognition (F_24) and disease progression (F_48) stages, primary plant metabolism was modified by activating metabolites, which encourage host plant protection and defense response [10,11,17]. Environmental information processing pathways were activated after stress stimulus of the fungi. The MAPK signaling pathway is essential for the transduction of developmental and environmental signals. Mitogen-activated protein kinases are found in the nucleus and cytoplasm and play a key role in different cellular processes [48]. This pathway encourages pathogen infection and attack, as well as phytohormones synthesis in the host plant. Jasmonic acid and ethylene phytohormones were significantly activated in the MAPK signaling pathway, which are responsible for defense response in the host plant against necrotrophic pathogens [49,50]. Jasmonic acid produces antioxidant protection in host plants [11]. In this study, the MAPK signaling pathway was significantly activated, as well as in M. fructicola-infected peaches [11,17].

In host plant defense response against biotrophic pathogens, salicylic acid was synthetized in the plant hormone signal transduction pathway, which is important for the detection of the pathogen at the site of infection and encourage broad-spectrum long-lasting resistance [50]. In this study, the analyzed enrichment of the plant’s hormone signal transduction pathway was also observed in other stone fruits (peach and nectarine), infected with M. fructicola and M. laxa pathogens, respectively [10,11,17].

The alpha-linolic acid metabolism pathway was activated during the inoculation (F_48) and in long-lasting resistance (F_72), initiating plant oxyphospholipids and jasmonic acid biosynthesis [51]. The alpha-linolic acid pathway modifies the flexibility of cell membranes, acts as a regulator for defense genes’ activation in the host plant, and promotes fungal spore colonization [52]. This pathway was also observed in peach, immature and mature nectarine infected with M. fructicola and M. laxa pathogen [10].

5. Conclusions

In this study, the induced defenses in plum fruits of cv. Victoria during the interaction with the M. fructigena pathogen was determined. This is the first comparative study to analyze the European plum genetic response to M. fructigena at transcriptomic level. The differentially expressed genes involved in resistance to the M. fructigena pathogen were observed right after 24 h post infection, with the peak of differentially expressed genes being at 48 h. PR family and MLO-like proteins were expressed the most during plum response to the pathogen. During the M. fructigena interaction MAPK signaling pathway, plant hormone signal transduction and alpha-linolic acid metabolism pathways were expressed in response to stress, defense, cell death, and disease resistance over the host plant–fungi interaction. DEGs were involved in pathogen infection and attack, synthesis of phytohormones and metabolism of alpha-linolenic acid and phenylalanine. The findings of this study could be used as the basis for further research of markers linked to resistance or susceptibility to disease in the plum hybrids at an early age, which will improve the plum breeding process.