1. Introduction
Nematodes are multicellular animals and belong to the superphylum Ecdysozoa. Nematodes are parasitic and free-living worms that can shed their cuticle to grow [
1]. Plant-parasitic nematodes are recognized as major agricultural pathogens [
2]. One of the most destructive plant-parasitic nematodes that can result in significant yield losses and economic damage is soybean cyst nematode (SCN), caused by
Heterodera glycines (HG) Ichinohe [
3]. SCN poses a serious threat to world soybean production, causing annual yield losses in excess of USD 1.5 billion in North America [
4].
Soybeans, as with other plants, fight off pathogens through the initiation of an array of defense mechanisms. A plant defense system is a complex system that consists of several lines of defense. The first layer is the plant’s passive defense mechanism. These include structural barriers, such as the cell wall that can physically block the entry of pathogens into the plant tissues [
5,
6]. If pathogens are successful in passing a plant’s passive defense mechanism, they have the chance to access the nutrients from the plant. However, after the passive defense mechanism, plants still possess two layers of actively induced immune systems. The first layer of the immune response, which is activated by a pathogen-associated molecular pattern (PAMP), is PAMP-triggered immunity (PTI). PAMPs are a diverse set of microbial molecules that have conserved structures perceived by plant surface-exposed receptors called pattern recognition receptors (PRRs) [
7,
8]. These membrane-bound PRRs are receptor-like kinases (RLK) or receptor-like proteins (RLP) with a high variety of intracellular domains. After the perception of PAMPs through PRRs, PTI will be activated as the first layer of the plant immunity system or surface immunity, which restricts pathogen proliferation. PTI signaling components are often targeted by various pathogen virulence effector proteins, resulting in diminished plant defenses and increased pathogen virulence. While PAMPs are conserved molecules that are shared among many different pathogens, effectors are typically species-, race-, or strain-specific molecules that contribute to pathogen virulence by targeting specific host plant processes [
9]. Some plant resistance (R) proteins have evolved to recognize pathogen effectors directly or indirectly by associating with cytoplasmic immune receptors [
10]. These receptors, which often contain a nucleotide-binding leucine-rich repeat domain, activate the second layer of immunity known as effector-triggered immunity (ETI) [
8]. In contrast to PTI, ETI is a highly specific defense response that is triggered by the recognition of pathogen effectors that have been specifically adapted to interact with and manipulate host proteins. It can trigger programmed cell death, called the hypersensitive response (HR), in response to a pathogen attack, which helps to contain the infection and limit the damage caused to the plant [
11].
The most effective management strategy to control SCN populations is using resistant cultivars rather than any other strategies, such as crop rotation or nematicides. Three popular Plant Introductions (PIs) resistant to SCN are PI 437654, PI 548402 (also known as Peking), and PI 88788, which carry resistant loci effective against multiple nematode races [
12,
13]. However, among all these resistant lines, plant breeders have heavily overused PI 88788 in the past three decades as the resistant parent in breeding programs. This has led to the selection of virulent biotypes of SCN and population shifting such as HG type 1.2.5.7, which are able to overcome the PI 88788-type resistance.
Soybean cyst resistance is a complex trait with polygenic inheritance. The first quantitative trait loci (QTL) underlying the resistance to
H. glycines (i.e., rhg) were reported in the early 1960s [
14,
15]. Among several reported QTL, the QTL on chromosomes 18 (
rhg1) and 8 (
Rhg4) are the two major resistance ones that have been consistently mapped and reported in a variety of soybean germplasm [
13,
16,
17]. In some SCN-resistant lines, such as PI 88788,
rhg1 with recessive action is sufficient to provide resistance to certain races of SCN and display an incompatible interaction with the nematode [
13]. In other resistant sources, such as PI 548402, resistance to SCN requires both
rhg1 and
Rhg4, while
Rhg4 exhibits a dominant gene action [
18]. Brucker et al. 2005 [
12] classified
rhg1 into two types:
rhg1-a in PI 548402 (also known as Peking-type) with low copy number (three or fewer repeats), which reacts with
Rhg4 to provide greater resistance to SCN, and
rhg1-b in PI 88788-type soybeans, which poses high copy number (four or more repeats) and provides the resistance without interacting with
Rhg4 [
12,
19]. SCN-susceptible lines such as Williams 82 and Lee 74 have only a single copy of
rhg1 [
19]. Previous studies discovered that the SCN resistance governed by
rhg1 is mediated through a 31-kb segment that is tandemly repeated and carries three genes, including a predicted amino acid transporter (
Glyma18g02580), an SNAP protein predicted to participate in the disassembly of SNARE membrane trafficking complexes (
Glyma18g02590), and a protein with aWI12 (wound-inducible protein 12) region without functionally characterized domains (
Glyma18g02610) [
19,
20]. On the other hand, map-based cloning of the
Rhg4 locus revealed that a single gene encoding a serine hydroxymethyltransferase (SHMT,
Glyma08g11490) is responsible for the resistance [
21].
By deploying defense mechanisms, SCN-resistant soybean genotypes are able to mount effective immune responses against SCN and minimize the damage caused to their production. However, pathogens have also evolved sophisticated strategies to evade or overcome these defenses, which has led to an ongoing evolutionary arms race between plants and pathogens. Therefore, studying soybean–SCN interactions can be challenging due to the complex nature of these interactions and the fact that the molecular mechanisms involved can be highly dynamic and context-dependent. It is important to use sensitive and high-throughput methods that can capture the complexity of these interactions and provide a comprehensive view of the molecular changes that occur during infection.
RNA sequencing enables high throughput analysis of the transcriptome landscape of cells. In host cells infected with pathogens, two organisms interact with markedly distinct transcriptomes. Dual RNA sequencing is a powerful in silico analysis method that enables the simultaneous study of the gene expression responses of both pathogens and host cells from the same samples, thereby deepening our understanding of their interaction [
22,
23]. In this study, we aimed to understand how SCN invasion modulates soybean gene expression, while simultaneously examining pathogen reactions across multiple hosts (e.g., compatible, semi-compatible, semi-incompatible, incompatible soybean). By utilizing dual RNA sequencing, we provide novel insights into the intricate interplay between host and parasite, revealing a diversity of defense mechanisms in soybean and virulence genes in the soybean cyst nematode. This study marks the first time that such a method has been employed to investigate soybean–SCN interactions, offering new insights into this crucial area of research.
3. Discussion
The main objective of this study was to investigate the defense mechanism of PI 437654, a highly resistant soybean line (FI = 0%), against SCN HG type 1.2.5.7. PI 548402, and PI 88788, which exhibit lower levels of resistance (FI = 10% and 63%, respectively), were also included to validate resistant genes and identify the resistance genes unique to PI 437654. Furthermore, the roots of these three resistant lines, as well as the susceptible line Lee 74 (FI = 100%), were analyzed to study changes in soybean root transcriptome in response to SCN. To identify candidate genes responsible for resistance in PI 437654, GO enrichment analyses were employed, which allowed for the extraction of gene sets involved in the enriched biological process. These findings provide insight into the mechanisms underlying resistance to SCN, particularly in PI 437654, and can aid in the development of more resistant soybean cultivars. Simultaneously, dual RNA sequencing allows us to examine SCN reactions across multiple soybean hosts (e.g., compatible, semi-compatible, semi-incompatible, incompatible) to study the expression of SCN pathogenicity genes during the infection process.
In this research, a long-term SCN stress at 5 and 10 dpi was chosen for RNA sequencing analysis to investigate the dual transcriptome reaction between soybean and SCN. Hammond et al. (2004) [
27] classified genes that respond to stress into two categories: “early” and “late” genes. The “early” genes exhibit a rapid response and are generally non-specific to target stresses, while the “late” genes have a delayed expression but can have a significant impact on the morphology, physiology, and/or metabolism of plants. Furthermore, these “late” genes are often specific to target stresses. According to Matsye et al. (2011) [
28], Peking-type resistance is characterized by a rapid and potent resistant reaction that results in the formation of a necrotic region around the syncytium by 5 dpi. On the other hand, PI 88788-type resistance is a prolonged but potent resistant reaction, which does not show any cytological evidence of a reaction at 5 dpi. Previous studies on cell fate against SCN have found that selecting “late” genes increases the likelihood of identifying specific genes. Consequently, 5 and 10 dpi were used for dual transcriptome reaction between soybean and SCN. The results of our dual transcriptome analysis provide valuable insight into the complex reaction of soybean gene expression in response to SCN parasitism. Furthermore, the coordinated expression of SCN HG type 1.2.5.7 genes potentially involved in parasitism against different soybean lines can now be better understood.
According to the total number of reads mapped to H. glycines, results indicated that SCN HG type 1.2.5.7 had the lowest penetration in PI 437654, possibly due to a strong passive defense mechanism, while it had the highest penetration in Lee 74, likely due to a physical barrier weakness. A significant decrease in the frequency of reads in Lee 74 at 10 dpi suggested that SCN HG type 1.2.5.7 had overcome the defense mechanism of Lee 74, leading to successful development into adult females and males. In PI 548402, the observed reduction in read frequency may be attributed to the strong two-layered immunity system, which overcomes SCN HG type 1.2.5.7. It is possible that the second-stage juveniles (J2) nematode cannot form specialized feeding sites, syncytia, and, therefore, are unable to copulate, leading to their eventual starvation and death. At 10 dpi, while the total number of reads mapped to H. glycines in PI 88788 was significantly reduced, it still remained relatively large. This observation may suggest that there was a battle between the SCN and PI 88788, where neither was able to fully overcome the other.
Comparing differentially expressed genes (DEGs) within each genotype under infested and non-infested conditions at two time points, 5 and 10 dpi, demonstrated that the number of DEGs significantly decreased in PI 437654, PI 548402, and PI 88788 at 10 dpi. In contrast, the number of DEGs remarkably increase in Lee 74, which may be due to the ability of SCN effector proteins to hijack and alter the gene expression patterns of Lee 74, leading to the induction of a large number of DEGs.
Host transcriptional pattern reprogramming is often triggered by pathogen invasion. Plasma membrane Ca channels are among the early sensors that respond to pathogen attacks by increasing Ca influx into the cell cytoplasm, as reported by Sun et al. (2015) [
29]. Ca2+ sensor proteins, such as calmodulin, EF-hand domain, and Ca2+-dependent protein kinases (CDPKs), detect the transient increase in Ca
2+ signatures, as observed by Houqing Zeng et al. (2017) [
30] and Sun et al. (2015) [
29]. Calmodulin, despite lacking enzymatic activity, binds to calmodulin-binding proteins, thereby stimulating the synthesis and accumulation of SA during immunity, as highlighted by Choudhury et al. (2017) [
31] and Gilroy et al. (2016) [
32]. SA, in turn, activates systemic acquired resistance (SAR), providing broad-spectrum and long-lasting resistance against pathogens [
29]. The study identified calmodulin-binding proteins
(Glyma.19G229500,
Glyma.03G232400, and
Glyma.19G229400) at 5 dpi and calmodulin-binding proteins (
Glyma.19G229500,
Glyma.05G237200,
Glyma.07G093900, and
Glyma.09G182400) at 10 dpi in PI 437654, but not in Lee 74.
Glyma.03G232400 and
Glyma.19G229400 were unique to PI 437654, and
Glyma.05G237200 and
Glyma.07G093900 were also observed in PI 88788 (
Figure 7).
Glyma.19G229500 and
Glyma.09G182400 were validated in both PI 548402 and PI 88788, with
Glyma.19G229500 being differentially expressed at both time points and validated by other resistant lines. These findings align with previous studies by Kofsky et al. (2021) [
33] and Zhang et al. (2017) [
34] that reported the presence of calmodulin-binding proteins in transcriptome comparisons of different genotypes under SCN HG type 2.5.7-treated conditions. Additionally, the study detected two differentially expressed EF-hand domain proteins (
Glyma.19G160100 and
Glyma.16G214800) in PI 437654 at 5 dpi, but not in other resistant lines (
Table 2 and
Figure 7).
Protein kinase is a critical component of intracellular signal transduction and plays an essential role in stress response [
35]. In current research, three proteins with protein kinase activity, namely,
Glyma.07G184000,
Glyma.17G173000, and
Glyma.18G219600, were identified at 5 dpi. Among them,
Glyma.07G184000 and
Glyma.17G173000 were validated using both PI 548402 and PI 88788, while
Glyma.18G219600 was only observed as differentially expressed in PI 437654. These protein kinases identified as SCN-responsive genes in the current study are aligned with the observations made by Ithal et al., 2007 [
36], and Han et al., 2015 [
37], which also detected these genes in response to SCN HG type 0 and HG type 1.2.3.5.7 [
36,
37].
Transcription factors play crucial roles in signal transduction by activating or suppressing the expression of defense genes and regulating the crosstalk between different signaling pathways. As they bind to specific cis-acting elements in gene promoters, transcription factors are positioned at the penultimate step of the signal cascade to directly control the downstream target gene expression. In the current study, several proteins with AP2/ERF domains, such as
Glyma.03G162400,
Glyma.03G162700,
Glyma.05G186700,
Glyma.10G186800,
Glyma.13G122500,
Glyma.13G123100,
Glyma.19G163700, and
Glyma.19G163900, which have transcription regulator activity, were found to be differentially upregulated in PI 437654 at 5 dpi. These genes were confirmed to play an important role against SCN HG type 1.2.5.7 through their expression in other resistant lines, including PI 548402 and PI 88788. The presence of AP2 is aligned with the observations made by Kofsky et al. [
33], who studied resistant and susceptible resources from
G. soja against SCN HG type 2.5.7. Another upregulated transcription factor in all three resistant lines at 5 dpi is the WRKY protein (e.g.,
Glyma.08G018300). At 10 dpi, six WRKY genes, including
Glyma.04G223300,
Glyma.13G267600,
Glyma.17G222300,
Glyma.13G267500,
Glyma.14G103100, and
Glyma.18G213200, were detected in PI 437654. Of these, the first three were also observed in PI 88788, and the last three were validated in both PI 548402 and PI 88788. The upregulation of WRKY genes as SCN-responsive genes in this study is supported by Zhang et al., 2017 [
34], as well as the observations made by Ithal et al., 2007 [
36], Han et al., 2015 [
37], and Kofsky et al., 2021 [
33].
Plants encounter various stresses and ROS such as hydrogen peroxide (H
2O
2), superoxide anions (O
2•−), hydroxyl radical (
•OH), and singlet oxygen (
1O
2) play important roles in signal transduction. However, excessive ROS accumulation can be harmful and even lead to cell death. Thus, a delicate balance between ROS production and ROS-scavenging pathways must be maintained [
38]. In the present study, at 5 dpi, several peroxidase genes including
Glyma.19G011800,
Glyma.01G130500,
Glyma.03G038300,
Glyma.03G038500,
Glyma.09G023000,
Glyma.09G057100,
Glyma.15G052700, and
Glyma.20G001400 were found to be differentially expressed in PI 437654, but not in Lee 74. Among these peroxidase genes,
Glyma.01G130500,
Glyma.03G038500,
Glyma.09G023000,
Glyma.09G057100, and
Glyma.20G001400 were observed in both PI 548402 and PI 88788, while
Glyma.19G011800 and
Glyma.03G038300 were only validated in PI 548402.
Glyma.15G052700 was the only unique peroxidase gene that was differentially expressed in PI 437654 at 5 dpi. At 10 dpi,
Glyma.09G277900,
Glyma.20G169200, and
Glyma.11G161600 were identified as peroxidase genes, while
Glyma.05G161300 was differentially expressed and annotated as a glutathione S-transferase (GST). The first two peroxidase genes were confirmed using PI 88788, while the last peroxidase and GST were only identified in PI 437654. These findings are in agreement with the observation by Miraeiz et al., 2020 [
39], who studied RNA-Seq profiling of Peking, Fayette, Williams 82, and a wild relative (
Glycine soja, PI 468916) against SCN HG type 0.
Oxidative enzymes are pivotal to plant metabolism, catalyzing a wide array of reactions involved in hydroxylation, DNA repair, and post-translational modification, as well as the activation and catabolism of plant growth regulators. The 2OG-Fe(II) oxygenase superfamily and cytochrome P450 (CYP) are two important classes of these enzymes [
40]. In this investigation, four genes belonging to the 2OG-Fe(II) oxygenase superfamily (
Glyma.03G096500,
Glyma.07G124400,
Glyma.08G169100, and
Glyma.14G058700) were identified at 5 dpi, and two genes (
Glyma.04G227900 and
Glyma.18G273200) were detected at 10 dpi in PI 437654, but not in Lee 74 (
Figure 7). Additionally, 11 genes annotated as Cytochromes P450 were identified at 10 dpi in PI 437654 (e.g.,
Glyma.01G135200,
Glyma.02G156100,
Glyma.03G143700,
Glyma.05G022100,
Glyma.09G049200,
Glyma.10G114600,
Glyma.11G062500,
Glyma.11G062600,
Glyma.11G062700,
Glyma.15G156100, and
Glyma.16G195600) (
Table 3). This study’s results reveal that only two P450 genes (
Glyma.04G035600 and
Glyma.09G144300) were validated and differentially expressed in PI 548402 but not in Lee 74 (
Figure 7 and
Table 4), implying that these genes play a crucial role in soybean defense against SCN infection. Our findings are in agreement with previous studies by Ithal et al., 2007 [
36], and Han et al., 2015 [
37], further highlighting the importance of oxidative enzymes in the plant’s defense response.
The plant cell wall can serve as a protective and physical barrier for limiting pathogen penetration into the plant cell [
41,
42]. Lignin, a three-dimensional polymer, is a major component of plant cell walls, composed of monomeric units such as syringyl, guaiacyl, and p-hydroxyphenyl, derived from phenylalanine in the cytoplasm and transported to the cell wall. The monomeric units are polymerized by laccase and peroxidase enzymes, which catalyze the random cross-linking necessary for the formation of the lignin polymer [
43,
44,
45]. Polymerized lignin reinforces the strength and rigidity of plant cell walls and is a key component of the plant’s response to environmental factors [
46,
47]. In this study, nine differentially expressed peroxidase genes were over-represented in PI 437654 at 5 dpi (
Table 2). Based on KEGG analyses, these genes are involved in the phenylpropanoid pathway and are directly responsible for producing syringyl lignin, guaiacyl lignin, 5-hydroxy-guaiacyl lignin, and p-hydroxyphenyl lignin. Among these genes,
Glyma.01G130500,
Glyma.03G038500,
Glyma.09G023000,
Glyma.09G057100, and
Glyma.20G001400 were validated through PI 548402 and PI 88788 and were not found to be differentially expressed in Lee 74.
Glyma.03G038300 and
Glyma.19G011800 were only validated in PI 548402 and were not observed in the susceptible line (
Figure 7 and
Table 4). Another peroxidase gene that was differentially expressed at 5 dpi was
Glyma.15G052700, which was only observed in PI 437654 and not in the susceptible line (
Figure 7 and
Table 4). At 10 dpi, one differentially expressed laccase gene and nine peroxidase genes were detected.
Glyma.01G108200 was annotated as a laccase gene and was validated by both PI 548402 and PI 88788. Out of the nine peroxidase genes, only
Glyma.09G277900 and
Glyma.20G169200 were not found to be differentially expressed in Lee 74 and were validated only by PI 88788 (
Figure 7 and
Table 5). Another peroxidase gene that was differentially expressed at 10 dpi was
Glyma.11G161600, which was only observed in PI 437654 and did not significantly upregulate in Lee 74 (
Figure 7 and
Table 3). In addition, dirigent is another cell-wall-related gene that modulates cell wall metabolism [
48]. At 10 dpi, Glyma.19G151200, which was annotated as a dirigent gene, was not detected in the susceptible line and was validated in PI 88788 (
Figure 7). Our finding about the role of peroxidase, laccase, and dirigent in cell wall rigidity against SCN invasion is supported by Afzal et al., 2009 [
49], which compared two NILs including rhg1rhg1/Rhg4Rhg4 and Rhg1Rhg1/Rhg4Rhg4 against SCN HG type 0 and Miraeiz et al., 2020 [
39] who studied different soybean lines against SCN HG type 0. Glycoside hydrolase is another gene involved in cell wall polysaccharide metabolism [
50]. Among the DEGs observed during SCN invasion in PI 437654,
Glyma.12G053900 at 5 dpi and
Glyma.11G129300,
Glyma.12G054200, and
Glyma.13G346700 at 10 dpi were upregulated. All of these genes are annotated as glycoside hydrolases.
Glyma.12G053900 was observed in both PI 548402 and PI 88788, and
Glyma.11G129300 was validated through PI 548402. Lipoxygenase is another gene that induces cell wall modification to limit pathogen invasion [
51], and at 10 dpi, the lipoxygenase gene
Glyma.19G263300 was detected in PI 437654 and validated in PI 88788.
Interestingly, the rhg1 genes (e.g.,
Glyma18g02580,
Glyma18g02590, and
Glyma18g02610) and Rhg4 gene (
Glyma.08G108900), which are promising genes known to confer SCN resistance to HG type 0, were not identified as significantly different in the current study, consistent with the findings of Zhang et al., 2017 [
34]. Additionally, the pathways of plant–pathogen interaction, carbon fixation in the photosynthetic organism, and carbon metabolism, which were the top three enriched pathways for upregulated DEGs in PI 437654, were not observed in other resistant lines. This finding is consistent with the observation by Shi et al., 2021 [
52], who studied PI 437654 against SCN HG type 1.2.3.5.7. However, this result is in contrast to the reports of Zhang et al., 2017 [
34], who studied
Glycine soja interaction with SCN HG type 2.5.7 and identified genes involved in carbon fixation and photosynthesis pathways as remarkably downregulated. According to Shi et al., 2021 [
52], a large number of DEGs that were upregulated in the incompatible soybean variety PI 437654 were involved in the plant hormone pathway, MAPK signaling, and phenylpropanoid biosynthesis pathway, which is in agreement with the results of the current study on PI 437654.
Dual RNA sequencing has provided a significant advantage in investigating the interaction between pathogen and host simultaneously. Through transcriptome analysis of SCN HG type 1.2.5.7, novel secreted effectors with potential roles in the SCN–host interaction have been identified. The establishment and maintenance of the syncytium, a crucial step for the long-term parasitic success of SCN, involves multiple stages such as hatching stimuli, host attraction, root penetration, tissue modification, feeding site formation, and immune system suppression. The pathogenicity and severity of SCN are dependent on the successful completion of these stages, as well as the pathogen’s classification as pathogenic or non-pathogenic. This study represents the first investigation of the SCN transcriptome in different hosts with varying levels of resistance, shedding light on the virulence strategies employed by SCN to overcome resistant hosts. Notably, 51 putative effectors showing differential expression patterns in both resistant and susceptible lines were identified, with 39 of them being newly discovered. Furthermore, only a limited overlap was observed between the effectors identified in PI 437654 and those identified in other resistant and susceptible lines, highlighting the adaptive ability of SCN to modulate its virulence genes to overcome resistant hosts.