Transcriptomic Profiling Reveals That the Differentially Expressed PtNAC9 Transcription Factor Stimulates the Salicylic Acid Pathway to Enhance the Defense Response against Bursaphelenchus xylophilus in Pines

Abstract

Pinus, a conifer, dominates the world’s forest ecosystems. But it is seriously infected with pine wood nematode (PWN). Transcription factors (TFs) are key regulators in regulating plant resistance. However, the molecular mechanism of TFs remains thus far unresolved in P. thunbergii inoculated with Bursaphelenchus xylophilus. Here, we used RNA-seq technology to identify differentially expressed TFs in resistant and susceptible pines. The results show that a total of 186 differentially expressed transcription factors (DETFs), including 99 upregulated and 87 downregulated genes were identified. Gene ontology (GO) enrichment showed that the highly enriched differentially expressed TFs were responsible for secondary biosynthetic processes. According to KEGG pathway analysis, the differentially expressed TFs were related to chaperones and folding catalysts, phenylpropanoid biosynthesis, and protein processing in the endoplasmic reticulum. Many TFs such as NAC, LBD, MYB, bHLH, and WRKY were determined to be quite abundant in the DETFs. Moreover, the NAC transcription factor PtNAC9 was upregulated in PWN-resistant and susceptible P. thunbergii and especially distinctly upregulated in resistant pines. By purifying recombinant PtNAC9 protein in vitro, we found that overexpression of PtNAC9 at the early stage of B. xylophilus infection could reduce the degree of disease. We also demonstrated the content of salicylic acid (SA) and the related genes were increased in the PtNAC9 protein-treated plants. These results could be helpful in enhancing our understanding of the resistance mechanism underlying different resistant pine.

1. Introduction

Pine wilt disease (PWD) caused by Bursaphelenchus xylophilus is the most serious forest disease in recent decades in China. B. xylophilus is a major global quarantine forest disease that causes irreparable damage to the vascular system of its hosts, leading to their death within a few months [1,2]. Monochamus alternatus are key vectors of PWD, introducing the B. xylophilus to healthy trees during feeding [3]. Other organisms, such as bacteria and fungi, can also pose a threat [4]. For example, B. xylophilus-related bacteria Pseudomonas toxic metabolites could alter the virulence of nematode and promote the development of PWD [5]. Fungi, such as Ophiostoma spp, Leptographium spp., Graphilbum spp., and Sporothrix spp., serve as a supplementary food source for nematodes to enter the mycophagous stage to maintain their population [6]. PWD has caused disastrous economic and ecological losses in the 40 years of invasion in China. This disease has caused hundreds of millions in losses, with billions of pines dying [7].

In the continuous process of evolution, plants have developed a two-tier innate immune system that includes both PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI) [8,9]. During PTI and ETI, plants trigger a cascade of signaling events, including reactive oxygen species (ROS) generation, MAP kinase (MAPK) activation, production of phytohormones, and transcriptional reprogramming [10,11]. Transcriptional reprogramming is a process that requires transcription factors (TFs) to coordinate spatially and temporally, which is a major feature of plant immunity [12,13,14]. TFs can directly or indirectly bind to RNA polymerase to regulate initiation of transcription by recognizing cis-acting elements in DNA sequences. Transcription is the first stage of gene expression and the main stage of gene regulation [13]. TFs combine with specific DNA sequences to inhibit or enhance gene expression levels, and thus play a major role in plant immunity [15].

In the face of B. xylophilus invasion, pines will produce a series of physiological, biochemical, and molecular changes to improve their defense [16]. Pines induce the production of reactive oxygen species (ROS), calcium ions, or other signaling molecules. At the same time, plant resistance hormones, like ethylene, salicylic acid, and jasmonic acid, also induce expression in pines [17]. The secondary metabolites of conifers play an important role in their defense response. More and more secondary metabolism-related genes have been discovered and are differentially expressed during the infection of PWN [18]. The contents of terpenes and flavonoids in infested pines were closely related to PWD resistance [19]. However, the role of TFs in pines’ resistance to PWN has rarely been studied.

The NAC proteins constitute a large plant-specific TFs family [20,21]. NAC proteins have a highly homologous region at their N-terminus, known as NAC domain, which binds to DNA [22,23]. A large number of the NAC TFs play critical roles in plant growth, development, abiotic stress responses, and plant immunity [24]. The functional analyses of NAC TFs in plant immunity are usually verified by using knockout/knockdown mutants and overexpression lines [2]. For example, the tomato transcription factor NAC29 can enhance tomato resistance to Pseudomonas syringae pv. tomato DC3000 [20]. In general, NAC TFs drive the transcriptional expression of downstream genes by binding to key regions of downstream target genes, thereby achieving its biological functions [25,26]. They typically regulate plant immunity by binding downstream target genes to activate some downstream pathways.

In this study, we mainly focused on the changes in TFs in resistant and susceptible pines inoculated with B. xylophilus and the key signaling pathways involved. Among them, PtNAC9 could effectively improve the resistance of pines and affect the expression of downstream resistance hormone pathways in pines. This result enriched the research of TFs in pines infected with B. xylophilus and explored the resistant components of pines.

2. Results

2.1. Identification of Differentially Expressed Transcription Factors (TFs)

We analyze the expression of differentially expressed transcription factors (DETFs) in resistant and susceptible pine trees to B. xylophilus invasion. The negative values indicate downregulation while the positive values represent the upregulation. The significantly up- and downregulated DETFs are shown in green dots according to the criteria of |log2FC| > 1 and p-values (p < 0.05). The heatmap displayed the expression patterns of DETFs. Many TFs exhibited different upregulation or downregulation trends in resistant and susceptible pines (Figure 1A). A total of 186 differentially expressed TFs were differentially expressed in P. thunbergii inoculation with B. xylophilus (Figure 1B). The RNA-seq dataset was analyzed using DESeq2 and Venn diagrams. The results indicated that there were 108 differentially expressed TFs between HCK and Hin; a total of 99 differentially expressed TFs occurred between H40CK and H40in (Figure 1C). A volcano plot was also generated to show the down- and upregulated expression of differentially expressed TFs (Figure 1D).

2.2. Functional Enrichment and Annotation of TFs in Response to B. xylophilus

We next annotated all differentially expressed transcription factors (DETFs) using the public databases GO and KEGG. GO term enrichment analysis was conducted to determine differentially expressed TFs involved in pines inoculated with B. xylophilus. Genes were classified into categories with three independent sub-parts: biological process (BP), molecular function (MF), and cellular component (CC). The results show that in pines susceptible to B. xylophilus invasion, the majority of the TFs involved in the biological processes were concerned with response to stimulus (GO: 0065007) and signaling (GO: 0023052). Within the molecular functions category, binding (GO: 0005488) was mostly overrepresented. In the cellular component category, the differentially expressed TFs were mostly associated with organelles (GO: 0043226) and cells (GO: 0005623) (Figure 2A). In addition, differentially expressed TFs in pines resistant to B. xylophilus invasion were more involved in the biological processes, including reproduction (GO: 0000003), developmental process (GO: 0032502), and immune system process (GO: 0002376). There was no difference in the cellular component and molecular functions category in resistant pines compared to susceptible pines (Figure 2B).

A search of the KEGG database was performed to determine the pathways with which the differentially expressed TFs were likely to be associated. Especially in susceptible pines, differentially expressed TFs were concentrated in immune-related pathways. For example, the most significantly enriched TFs were involved in plant hormone signal transduction (PATH: 04075), the MAPK signaling pathway (PATH: 04013), and plant–pathogen interaction (PATH: 04626) (Figure 2C).

2.3. Validation of TFs by Real Time-Quantitative PCR

In order to better understand the expression of TFs in susceptible and resistant pines, we performed RT-qPCR to analyze the transcriptional abundance of 10 selected TFs to verify the accuracy of the RNA-seq data. We measured the expression levels of different TFs, including ERF, MYB, bHLH, and NAC. The results show that an NAC TF was significantly upregulated in pines susceptible and resistant to B. xylophilus invasion, especially in resistant pines where its expression level is higher. After sequence alignment, we named it PtNAC9 (Figure 3).

2.4. Subcellular Localization and Functional Verification of PtNAC9

To better describe the function of PtNAC9 in plants, we determined its subcellular localization and transiently expressed a red fluorescent protein (RFP)-tagged PtNAC9 in Nicotiana benthamiana by agroinfiltration. The results show that PtNAC9 is mainly expressed in the nucleus (Figure 4A). We also counted the fluorescent intensity, indicating that PtNAC9 as a TF functioned in the nucleus of plants. To further determine whether PtNAC9 affects immune-related components, the PtNAC9 protein was first produced in the yeast Pichia pastoris and confirmed by Western blot (Figure 4B). We infiltrated purified PtNAC9 protein into N. benthamiana leaves. The purified GFP was used as a control. RT-qPCR analysis revealed that the SA-related genes (NtPR1 and NtPR2) were upregulated in N. benthamiana (Figure 4C), suggesting that PtNAC9 might be involved in the regulation of plant immunity.

2.5. PtNAC9 Positively Regulates the Resistance of Pines to B. xylophilus

In order to better understand the role of PtNAC9 in susceptible and resistant pines during B. xylophilus infection, we tested PtNAC9’s relative expression level at different infection times. The results show that PtNAC9 was upregulated in susceptible pines with peak expression at 12 h after B. xylophilus infection, while PtNAC9 always a high expression level in resistant pines (Figure 5A,B). To further estimate the contribution of PtNAC9 to the resistance of pines, the pines were treated with purified PtNAC9 protein and then inoculated with B. xylophilus. Notably, symptoms did not appear in the PtNAC9 protein-treated pines until 15 dpi (Figure 5C). At 15 dpi, the morbidity of GFP protein-treated pines was obviously higher than PtNAC9 protein-treated pines (Figure 5D). Moreover, the degree of morbidity also proved that the PtNAC9 protein could delay the occurrence of disease (Figure 5E). These data indicate that PtNAC9 is positively required for the resistance of pines to B. xylophilus infection.

2.6. PtNAC9 Influences the Accumulation of Defense-Related Plant Hormones and Genes

Transcription factors (TFs) play an important role in regulating the expression of downstream genes and endogenous hormones. To investigate whether PtNAC9 influenced pine defense responses, susceptible and resistant pine seedlings were treated with PtNAC9 protein and then inoculated with B. xylophilus. The results show that PtNAC9 induced the expression of salicylic acid (SA), which was increased more in resistant pines (Figure 6A). RT-qPCR analysis confirmed that PR genes (PR-1b and PR-5) are distinctly upregulated in pines inoculated with B. xylophilus and treated with the PtNAC9 protein. It is worth noting that the expression level of PtPR-5 in susceptible pines after PtNAC9 treatment reached the level of resistant pines (Figure 6B,C). The enzyme activity of SOD, POD, and PAL were also higher than that in the control, especially after PtNAC9 treatment, and the content of POD and SOD in susceptible pines was consistent with the expression of resistant pines, while there was a significant difference in the levels between the two before treatment (Figure 6D–F). These results indicate that PtNAC9 is indeed a defense-related TF in pines.

3. Discussion

Pine wilt disease (PWD) has caused widespread destruction to forests in our country [27]. Resistance breeding is the most effective measure to control PWD [28,29]. To reduce the PWD damage, our team introduced resistant Pinus thunbergii from Japan many years ago [30,31]. Field inoculation experiments confirmed that P. thunbergii 40# was the resistant pine. In addition, we performed transcriptome difference analysis between P. thunbergii 40# and susceptible P. thunbergii to better reveal the resistance mechanism [19]. Reactive oxygen species (ROS) and lignin synthesis metabolism pathways were demonstrated to play a key role in pine resistance to Bursaphelenchus xylophilus. In addition to them, recognition of the binding sites in DNA sequences is of great significance for understanding gene transcriptional activity and gene expression. So, the function of transcription factors (TFs) in different resistant pines is also worth studying.

In this study, we systematically analyzed the expression of TFs, which were able to govern the transcriptional reprogramming associated with plant immunity, in susceptible and resistant pines infected with B. xylophilus. We measured the relative expression of 10 differentially TFs. It was found that one of the TFs, PtNAC9, was upregulated in both susceptible pines and resistant pines, especially obviously in resistant pines. More importantly, PtNAC9 was consistently upregulated throughout the B. xylophilus infection stage in resistant pines. Meanwhile, we successfully cloned PtNAC9 and continue to study its role in pines’ resistance to PWD. The NAC protein plays a key role in plants’ stress response networks [32]. However, there has been relatively limited research on NAC transcription factors in pines. We found that PtNAC9 was mainly concentrated in the nucleus. The nucleus is the control center of cells and plays an important role in their metabolism, growth, and signaling pathways, and is the main site of genetic material [33], indicating that PtNAC9 subcellular localization was consistent with transcription factor characteristics. Since comparable genetic tools are still not available in pines. We attempted to express and purify PtNAC9 by Pichia pastoris to better understand the function of PtNAC9. The results show that PtNAC9 played a positive role in resistance to B. xylophilus. Consistent with this notion, the tomato NAC gene SlSRN1 is a positive regulator of defense responses against Botrytis cinerea and Pseudomonas syringae pv. tomato (PstDC3000) [34]. SlNAC35 overexpression improved resistance to bacterial pathogens in transgenic tobacco [35].

In order to clarify the role of PtNAC9 in plant immunity, we expressed the PtNAC9 protein in tobacco leaves. PtNAC9rec induced the expression of pathogenesis-related (PR) genes in N. benthamiana and pines. PR proteins are considered encoded by the host plant but induced only in pathological or related situations, and phytohormones serve as secondary signals to activate PR gene expression [36]. For example, PR genes are normally activated during systemic acquired resistance (SAR), which is related to salicylic acid (SA) [37]. We also confirmed that PR gene expression was induced by PtNAC9 in the salicylic acid-dependent pathway, suggesting that PtNAC9 might regulate the level of SA in pines to affect PWD resistance.

In addition, PtNAC9rec also improved the activity of several enzymes in pines. The activity PAL can be induced to increase the synthesis of secondary metabolites and improve plant resistance [38,39]. PAL is involved in the synthesis of lignin, which is an important component of the cell wall and plays an important role in maintaining plant morphological structure. PAL is also involved in the synthesis of flavonoids, which have important functions in plant resistance and plant–microbial interactions. Antioxidant enzymes (POD and SOD) were induced to improve the resistance of PWD. Their activity level could reflect the degree to which plants are affected by a pathogen [40]. To solidify PtNAC9’s role in resistance, we analyzed the changes of pathogenesis-related genes and enzymes in susceptible and resistant pines. The level and content of the genes and enzymes in resistant pines were originally higher than in susceptible pines. When pines were treated with PtNAC9rec, these indicators in the susceptible pines could reach the initial level of resistant pines or even higher, indicating that PtNAC9 might be a vital TF in pines resistant to P. thunbergii. Unfortunately, there is no mature and stable genetic transformation system of pines, and we were currently unable to clarify the regulatory role of PtNAC9 and its pathway in immune response in pines. Our study suggested a transcription factor, PtNAC9, from the pines that plays a key function during B. xylophilus invasion.

4. Materials and Methods

4.1. Identification of Differentially Expressed Transcription Factors (DETFs)

According to the previous transcriptome data in our lab [19], different libraries containing differentially expressed transcription factors (DETFs) were analyzed using the FPKM method. The number of mapping reads for each gene was determined by FPKM, and the gene expression levels were assessed. With a log-fold expression change of |log2FoldChange| > 1, significant p-value < 0.05, DEGs were filtered using a DEGseq algorithm. High-quality genes were used in further analysis. High-quality reads were used to map back to the assembled transcripts.

4.2. Functional Annotation and Pathway Analysis

TopGO (v2.50.0) was used to describe GO functional classification of biological processes, molecular functions, and cellular components. KEGG mapping was carried out using ClusterProfiler (v4.6.0) software for analysis of biochemical pathways of annotated genes.

4.3. Real-Time Quantitative PCR

The total RNA of pines was extracted with an RNA Isolation Kit (TIANGEN, Beijing, China) according to the manufacturer’s protocol. Then, RNA was used for cDNA synthesis using HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme Biotech Co., Ltd., Nanjing, China). Gene expression levels were calculated on the basis of four technical replications. The RT-qPCRs were performed on an ABI Prism 7500 PCR instrument under the following conditions: 95 °C for 2 min, 40 cycles at 95 °C for 30 s, and 60 °C for 30 s to calculate cycle threshold values, followed by a dissociation program of 95 °C for 15 s, 60 °C for 1 min, and 95 °C for 15 s to obtain melt curves.

4.4. Agrobacterium Tumefaciens Infiltration Assays

The A. tumefaciens transformation was performed as described [41]. A. tumefaciens carrying recombinant strains were grown in Luria–Bertani media plus 50 mg/mL of kanamycin and 50 mg/mL of rifampin for 48 h, harvested, and washed with 10 mM MgCl₂ three times, resuspended in 10 mM MgCl₂ to a final OD₆₀₀ of 0.4, and then incubated at room temperature for 1 to 3 h before infiltration.

4.5. Subcellular Localization

The full coding sequence (CDS) of PtNAC9 was inserted into the pBINRFP vector. Transformants with PtNAC9 in the Agrobacterium tumefaciens strain GV3101 were used for the infiltration of the leaves of 4-week-old N. benthamiana plants. A. tumefaciens cells were infiltrated into N. benthamiana leaves with needleless syringes. For fluorescence observations, patches of N. benthamiana leaves were cut after 36 hpi and used for confocal imaging on an LSM710 microscope (Zeiss, Oberkochen, Germany).

4.6. Eukaryotic Expression of the Recombinant PtNAC9 Protein

PtNAC9 was linked to the eukaryotic expression vector, pPICZαA by homologous recombination. We had the GFP protein as a control. The recombinant vectors were transformed into Pichia pastoris KM71H (Muts) (Invitrogen, Waltham, MA, USA) by electroporation. PtNAC9 clones were grown in yeast extract–peptone–dextrose (YPD) medium containing 100 µg/mL zeocin at 30 °C for 2–3 days. Buffered glycerol complex medium (BMGY) and buffered methanol–complex medium (BMMY) were used to induce protein expression. Purification of recombinant PtNAC9 from the culture supernatant was performed by affinity chromatography using Ni-NTA Superflow resin (Qiagen, Nanjing, China).

4.7. Inoculation Assay

A sterile blade was used to cut a small wound deep into the xylem on pine stems, and a cotton ball was inserted. The incision and cotton ball were then covered with a funnel-shaped parafilm. The suspension containing 2000 nematodes was injected into the xylem in each pine. According to the color of the needles, the symptoms were evaluated and categorized using five levels. The morbidity and morbidity degrees of the pines were calculated according to PWD symptoms as described previously.

4.8. Determination of Salicylic Acid and Enzyme Activity in Pines

The content of salicylic acid (SA) and activities of superoxide dismutase (SOD), phenylalanineammonialyase (PAL) and peroxidase (POD) were determined in the stem treated with purified PtNAC9 protein and nematode inoculation was performed 2 h later. The stems were frozen in liquid nitrogen and ground to a fine powder using a mortar and pestle. The contents of SA, SOD, PAL, and POD were calculated based on previous studies [42].

4.9. Statistical Analysis

All data are presented as means ± standard deviation (SD) of three replicates. Statistical analyses were performed by the analysis of variance (ANOVA) using data processing system (DPS) version 9.50. The significant differences were analyzed by the Duncan’s multiple range test. Differences at p < 0.05 were considered to be significant.

5. Conclusions

We identified a transcription factor, PtNAC9, that played a key role during the B. xylophilus invasion. It could effectively improve the resistance of pines, and induce the expression of pathogenesis-related genes, resistance hormones, and antioxidant enzymes. Our results provide a reference for further study of transcription factors in different resistances of P. thunbergii infected with B. xylophilus to better reveal the resistance mechanisms of PWD.