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Article

The TIR-Type NLR Protein Is Involved in the Regulation of Phelipanche aegyptiaca Resistance in Cucumis melo

by
Lifeng Xiao
,
Qiuyue Zhao
,
Xiaolei Cao
,
Zhaoqun Yao
* and
Sifeng Zhao
*
Key Laboratory at the Universities of Xinjiang Uygur Autonomous Region for Oasis Agricultural Pest Management and Plant Protection Resource Utilization, Agriculture College of Shihezi University, Shihezi 832003, China
*
Authors to whom correspondence should be addressed.
Agronomy 2023, 13(3), 644; https://doi.org/10.3390/agronomy13030644
Submission received: 3 January 2023 / Revised: 17 February 2023 / Accepted: 18 February 2023 / Published: 23 February 2023
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Abstract

:
Phelipanche aegyptiaca is an obligate holoparasite that causes serious negative effects on crop growth and productivity, effective control of which is difficult due to its unique biological characteristics. In this study, we performed a comparative transcriptome analysis of resistant and susceptible Cucumis melo cultivars (KR1326 and K1237) inoculated with P. aegyptiaca. CmNLR (encodes a TIR-type NLR protein, consistently highly expressed in KR1326 roots) and CmNLRh (homologous gene of CmNLR) were cloned and overexpressed in K1237 roots to verify whether the TIR-type NLR protein can enhance C. melo resistance to P. aegyptiaca. The variations in enzymes related to active oxygen metabolism were further detected in transformed roots. The results showed that (1) some differentially expressed genes (DEGs) were enriched in pathways associated with active oxygen scavenging; (2) several DEGs encoded transcription factors, calcium channel proteins, and receptor-like proteins were upregulated and expressed in KR1326 roots; (3) the complete CmNLR and CmNLRh proteins prevented P. aegyptiaca from connecting to the vascular system of C. melo roots; and (4) stronger active oxygen burst and scavenging capacity were detected in transformed roots. We herein demonstrated that the TIR-type NLR protein confers C. melo resistance to P. aegyptiaca. The results provided clues for finding a new direction for host resistance against parasitic plants and shed new light on the cultivation of resistant varieties to control P. aegyptiaca.

1. Introduction

Symbiotic interactions between species are common in the plant kingdom and can be mutualistic, commensalistic, or parasitic [1]. Parasitic plants have long been a major threat to world agricultural production, causing serious negative effects on crop growth and productivity. Accordingly, parasitic plants can be classified as root and stem parasites according to their parasitic sites on host plants and can also be classified as photosynthesizing hemiparasites and non-photosynthesizing holoparasites, depending on whether they can perform photosynthesis themselves. Parasitic plants are widely distributed, and about 4500 flowering species of plants have adopted a parasitic lifestyle [2,3].
Broomrapes (Orobanche/Phelipanche spp.), the class with the most economically influential parasitic plants in agriculture, are holoparasites that live on the roots of a variety of agricultural crops. Broomrapes have a huge seed bank in the soil; seeds (0.2–0.4 mm) germinate underground in response to specific plant-derived germination stimuli, such as unicolactone, released by the root of the host plant [4,5,6,7,8]. Similar to other parasitic plants, broomrapes form a multicellular organ, called a haustorium, which is necessary for the parasite to invade host tissues and establish vascular connections. The initiation and development of haustoria (special intrusive organs) is a key step in the growth of parasitic plants [2,9,10]. The initiation of a haustorium is largely dependent on haustorium-inducing factors secreted from host roots. The host lignin composition also affects the haustorium induction of the parasite [9]. Thereafter, the haustorium of the parasite penetrates the root cortex of the host plant and establishes a direct connection with root vascular bundles [11]. Host–parasite vascular connections allow the movement of water and nutrients, such as sugars, carbohydrates, minerals, and proteins, between the host plant and the parasite [12,13]. Phelipanche aegyptiaca is an obligate holoparasite that lacks photosynthetic capacity and relies entirely on host plant parasitism for nutrition and life cycle completion [3,14,15].
Majority of the damage caused by these parasites occurs underground, making control extremely challenging. Effective control is also difficult due to their unique biological characteristics, such as massive seed production, ease of dispersal, germination only under specific conditions, and long seed life [15,16,17]. Therefore, the best long-term strategy for controlling parasitic plants should be the identification, breeding, and cultivation of resistant crop varieties. However, there are only a few resistant crop varieties available and few known cases of genetic resistance [18]. To develop crop varieties with durable resistance against P. aegyptiaca, it is essential to fully understand the mechanisms of parasite and host defense. In recent years, the molecular systems of plant parasitism have been extensively studied [3,19,20].
Host resistance to parasitic plants can roughly be classified into three stages: pre-attachment (including seed germination and haustorium induction of parasitic plants), establishment, and post-establishment. The specific recognition of parasitic plants is the host’s resistance to plant parasitism. The ability to sense the presence of invading parasitic plants and induce appropriate defense responses can demonstrate the difference between resistant and susceptible hosts [20]. Some host plants have developed an immune system that detects parasite-derived components and intervenes actively in parasite attack mechanisms [9]. The host response is also shown in the lignification at the infection site. For instance, several metabolites accumulate preferentially in resistant rice cv ‘Nipponbare’ upon Striga. hermonthica infection as compared with the susceptible cv ‘Koshihikari’, with the most apparent difference being lignin-related molecules [21]. The host response is also reflected in the regulation of the jasmonic acid (JA) pathway. For example, JA-biosynthesis genes and the production of JA and JA-isoleucine, the active amino acid conjugate, are upregulated in rice upon infection by S. hermonthica [22], and Arabidopsis JA-related mutants are less susceptible to P. aegyptiaca [23]. However, once elucidated, non-host resistance is a powerful tool for fighting against noxious parasites [24].
Genetic, genomic, and transcriptomic studies have shown that the major host resistance components are R genes encoding NLR domain proteins that play an important role in host immunity by recognizing parasite virulence factors. The induction of host defense responses by the NLR proteins’ model is proposed in three stages: recognition, activation, and defense response [25].
To defend against invading pathogens, plants have developed two layers of immunity: pathogen-associated molecular-pattern (PAMP)-triggered immunity (PTI) conferred by cell surface pattern recognition receptors (PRRs) and effector-triggered immunity (ETI) mediated by resistance proteins (R) [26]. NLRs and PRRs mediate different immune responses but also share similar immune mechanisms [27,28]. Plant nucleotide-binding leucine-rich repeat receptors (NLRs) sense the virulent effects of pathogens and activate defense responses. NLRs are classified as containing coiled-coil (CC) or Toll/interleukin-1 receptors (TIR) [29]. Gene-to-gene mechanisms similar to ETI described in other host–pathogen interactions may be applicable in these host–parasite associations. The successful cloning and functional characterization of R genes have opened the door for further exploration of host resistance mechanisms and provided a focal point for studies to reveal the molecular and genetic factors underlying parasite virulence and host selection [30].
Genetic resistance is one of the main goals of melon breeding programs. In this study, the expression of genes involved in melon resistance against P. aegyptiaca was captured at the attachment stage in resistant/susceptible interactions between C. melo cultivars and P. aegyptiaca based on comparative transcriptome analysis. Furthermore, we cloned and functionally verified one of the NLR genes and its homologous gene. This may provide possible directions for future work.

2. Materials and Methods

2.1. Plant Materials and Growth Conditions

P. aegyptiaca seeds were collected from plants growing on tomato cultivars located in the 163rd regiment of the Xinjiang production and construction corps in 2019. Melon seeds (KR1326, resistant Cucumis melo cultivar; K1237, susceptible Cucumis melo cultivar) were obtained from the Xinjiang Academy of Agricultural Sciences. The development of the host–P. aegyptiaca interaction was performed in the Oasis Agriculture Ecology Laboratory in the College of Agriculture at Shihezi University.
The root chamber method was used to prepare RNA-seq samples and assay P. aegyptiaca–melon root interactions. Until the second true leaf appeared, the C. melo seedlings were transferred to a Petri dish (15 cm) that contained a sterile sponge with its surface covered by two layers of sterile filter paper, with the melon root flat on the filter paper and the leaves outside the dish and medical cotton holding the melon seedling joint of the root and stem. After the melon root developed stably, about 200 P. aegyptiaca seeds were sterilized with 70% alcohol for 2 min and 2% sodium hypochlorite for 20 min and then washed with sterile water at least three times. The seeds were gently spread on the seedling roots to parasitize the melon roots. The plants were grown under a 16 h light/8 h dark cycle at 28 °C. After P. aegyptiaca seed inoculation, the P. aegyptiaca–melon root interaction types (such as attachment, haustorium swelling, and tubercle swelling) were observed by stereomicroscope and recorded daily. The number of days post-inoculation (dpi) was used to record the date of feature occurrence.
Planting melon seeds in pots with peat and vermiculite (1:1, v/v) constituted the substrate for pot growth. Seeds of P. aegyptiaca (50 mg) were mixed with 0.5 kg of the substrate before being inoculated, and all plants were grown in growth chambers at 28 °C with a photoperiod of 16 h.

2.2. RNA Sequencing

Melon seedlings were cultivated using the root chamber method mentioned above. After the melon roots developed stably for about 1 week, the seedlings (about 25 days old) had grown their fourth true leaf. Melon seedlings with comparable growth were selected for inoculation and follow-up tests. RNA-seq samples were collected at 9 and 16 dpi. To harvest the interaction site between the melon root and P. aegyptiaca, less than 1 cm adjacent to the haustorium cuts were made at the left and right ends of the melon roots. One biological replicate (100 mg) was obtained from the interaction sites of the same melon plant, with three replicates for each sample. Samples R1, R2, S1, and S2 were from two development stages of the melon roots inoculated with P. aegyptiaca, where “R” refers to resistant melon cultivar KR1326 and “S” refers to susceptible melon cultivar K1237. Stage 1 refers to 9 dpi, the early attachment stage, and stage 2 refers to 16 dpi, the late attachment stage. After harvesting, the samples were quickly frozen in liquid nitrogen and stored at −80 °C for RNA extraction and sequencing.
Total RNA was extracted from samples with TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA degradation and contamination were assessed by 1% agarose gel electrophoresis (Figure S1A). A Nanodrop ND-2000 microspectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) was used to determine the concentration and purity of total RNA (OD260/280 ratio was between 1.9 and 2.1, OD260/230 > 2.0).
Transcriptome sequencing was performed by Novegene Bioinformatics Technology Co. Ltd. (Beijing, China) using the Illumina HiSeq 2500 platform with paired-end sequencing technology.

2.3. Analysis of Gene Expression and Differentially Expressed Genes (DEGs)

Low-quality reads were removed from raw reads in fastq format by FASTQC, and those containing ploy-N were removed by FASTP [31]. The result was high-quality clean data (clean reads). Clean reads were sequenced with transcripts from the melon genome (CM3.6.1). The number of read counts on each gene for each sample comparison was further obtained, and FPKM (fragments per kilobase per million) transformation was performed to analyze the gene expression level [32].
The identification of DEGs between different samples was performed using the DESeq R package. Genes with a false discovery rate (FDR) < 0.05 and |log2fold change| > 1 were considered differentially expressed [33]. Functional annotation of the unigenes was performed by a BLAST search against public databases, including the Swiss-Prot, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Functional enrichment analysis of GO annotation results of significantly differentially expressed genes (DEGs) was performed using the clusterProfiler 4.0R package [34]. KEGG path enrichment analysis of DEGs was performed using KOBAS 3.0 (KEGG Orthology-Based Annotation System) [35].

2.4. Construction of Transgene Expression Plasmids and Generation of Transgenic Plants

The full-length CmNLR protein was predicted based on the first ATG and longest open reading frame (ORF) to an in-frame stop codon, and the full-length PCR amplicon of this coding region was mobilized into the pCAMBIA3301 vector by homologous recombination at the SalI restriction site using a one-step cloning kit (Vazyme-Biotech, Nanjing, China). The homologous gene CmNLRh was constructed in the same way (Figure S1B). The products were then transformed into Escherichia coli competent cells, and colony PCR was performed (Figure S1C). Positive clones were selected according to the colony PCR results, and the recombinant plasmid was confirmed by its sequence. All pCAMBIA3301 plasmids (pCAMBIA3301—CmNLR, pCAMBIA3301—CmNLRh, and pCAMBIA3301) were transformed into Agrobacterium rhizogenes strain K599 for heterogeneous expression in susceptible C. melo cultivar K1237. The positive strains were selected, and verification was performed according to colony PCR verification (Figure S1D).
K1237 seeds were germinated in sterilized water for 2 days, and then germinated seeds were planted in a plastic cave dish containing vermiculite. When the cotyledons spread out after seedling emergence, the seedlings were inoculated with A. rhizogenes strains carrying pCAMBIA3301 plasmids below the cotyledons perpendicular to the stem using the needle of a syringe. The seedlings were covered with a disposable cup soon after the inoculation and grown overnight at 22 °C in the dark. The disposable plastic cup was removed, and the bottom was cut off. The cup was placed directly over the seedlings and filled with vermiculite until the vermiculite covered the inoculation site. Seedlings were cultured at 25 °C with a photoperiod of 16 h light and 8 h dark; vermiculite was added daily to cover the inoculation wound and sprayed with water for moisture. New roots emerged from the inoculation site after about 2 weeks. The original roots of the melon seedlings were removed after the new root system had developed stably, and transgenic plants were then generated. The transformed melon seedlings were cultured at 28 °C with a photoperiod of 16 h light and 8 h dark. About 20 d after transformation, seedlings with regenerated roots were moved to rhizotrons and grown at 28 °C for 14 d before P. aegyptiaca inoculation. At 14, 21, and 28 dpi, parasite–melon root interaction events were scored, and the percentage of each interaction type was determined for transgenic roots. The seedlings with regenerated roots were also moved to a pot culture with P. aegyptiaca seeds and grown at 28 °C for 28 d. After root washing, the number of P. aegyptiaca on the transgenic melon roots was counted, and plant height and fresh weight were measured.

2.5. GUS Assay

The transgenic roots were soaked in a GUS staining solution medium after cleaning with distilled water and incubated overnight at 37 °C. After overnight incubation, the blue location on the white background was the expression site of the GUS marker gene. Specific operation steps were performed according to the instructions (Solarbio, Beijing, China).

2.6. qRT-PCR

Total RNA was extracted from independent samples using the EASYspin Plus Plant RNA Kit (Aidlab, Beijing, China) following the manufacturer’s instructions. cDNA was synthesized using the EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen, Beijing, China) used for qRT-PCR analysis on the 7500 Real Time PCR System using the ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech Co., Nanjing, China). The gene-specific primers used for qRT-PCR amplification are shown in Supplemental Table S2. The PCR conditions consisted of pre-denaturation at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 10 s and annealing and extension at 60 °C for 30 s. Each measurement had three biological replicates, and each biological replicate included three technical replicates. The 2−ΔΔCT method was used to calculate the relative expression level of each gene to the endogenous control GAPDH.

2.7. Determination of Enzyme Activity

Transgenic K1237 roots were collected and quickly frozen in liquid nitrogen and then stored at −80 °C for reserve. The content of H2O2 and O2− and the activity of peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD) were determined using micromethods at OD415, OD530, OD470, OD240, and OD560, respectively. Three sample replicates were set for each treatment group, with three technical replicates. Of the tissue samples, 0.1 g was used in each test. Specific operation steps were performed according to the instructions of the kits provided by Solarbio, Beijing, China. The calculation formulas were as follows:
H2O2(μmol/g) = ∆Ameasure/(∆Anorm/Cstandard-solution) × Vsample/(Vsample/Vextract × W) = 2 × ∆Ameasure/∆Anorm/W
https://www.solarbio.com/goods-9127.html (accessed on 2 February 2023)
O2−(μmol/g) = 2x × Vsample/(Vsample/Vextract × W) = 2x/W
https://www.solarbio.com/goods-9214.html (accessed on 2 February 2023)
POD(U/g) = ΔA/0.005 × Vtotal-reaction-volume/(W/Vtotal-extract-volume × Vsample-volume)/T = 9800 × ΔA/W
https://www.solarbio.com/goods-9116.html (accessed on 2 February 2023)
CAT(U/g) = [ΔA × Vtotal-reaction-volume/(ε × d) × 106]/(Vsample-volume/Vtotal-extract-volume × W)/T = 764.5 × ΔA/W
https://www.solarbio.com/goods-12544.html (accessed on 2 February 2023)
SOD(U/g) = [Inhibitory-percentage/(1−Inhibitory-percentage) × Vtotal-reaction-volume]/(500 × Vsample-volume/Vtotal-extract-volume) × F = 0.02 × Inhibitory-percentage/(1−Inhibitory-percentage) × F
https://www.solarbio.com/goods-9124.html (accessed on 2 February 2023).

3. Results

3.1. The Analysis of RNA-Seq Data

We performed transcriptome profiling of the melon roots inoculated with P. aegyptiaca, and 12 libraries were generated from the resistant and susceptible C. melo cultivars at 9 and 16 dpi. After data filtering, approximately 121.22 Gb of clean reads were obtained, and at least 6.45 Gb of clean reads were generated for each library. The Q30 percentage (the percentage of bases with a quality score of 30 or higher) of each sample was approximately 92%, and the GC (guanine and cytosine) percentage of each sample was 42.70–49.31% (Table S1), suggesting the accuracy of the sequence data. According to the Pearson correlation analysis between samples, the reproducibility and reliability of transcriptome data met the requirements for further analysis (Figure S2).

3.2. Functional Annotation of DEGs between KR1326 and KR1327

Between KR1326 and K1237, a total of 744 (315 upregulated and 429 downregulated) differentially expressed genes (DEGs) were identified at the early attachment stage (stage 1, 9 dpi) (Figure 1A), and 590 (283 upregulated and 307 downregulated) DEGs were identified at the late attachment stage (stage 2, 16 dpi) (Figure 1B).
KEGG functional annotation and classification of DEGs at the early attachment stage and the late attachment stage were both assigned 10 significant shared terms. From the bubble map of the DEG pathway enrichment analysis, we found that alpha-linolenic acid metabolism, thiamine metabolism, peroxisome, biosynthesis of amino acids, brassinosteroid biosynthesis, cutin, suberine and wax biosynthesis, carbon fixation in photosynthetic organisms, pentose phosphate pathway, arginine and proline metabolism, and homologous recombination were significant enrichment pathways at the early attachment stage (Figure 1C). Carbon fixation in photosynthetic organisms; photosynthesis-antenna proteins; carbon metabolism; alanine, aspartate, and glutamate metabolism; diterpenoid biosynthesis; glycosaminoglycan degradation; brassinosteroid biosynthesis; linoleic acid metabolism; phenylpropanoid biosynthesis; and arachidonic acid metabolism were marked enrichment pathways at the late attachment stage (Figure 1D).
Some DEGs were involved in two pathways related to active oxygen scavenging: peroxisome and phenylpropanoid biosynthesis. At 9 dpi, 7 genes (2 upregulated genes and 5 downregulated genes) were differentially expressed in peroxisome, including 1 gene that encodes catalase (CAT) that was upregulated in KR1326 roots (Figure 2). At 16 dpi, 10 genes (2 upregulated genes and 8 downregulated genes) were differentially expressed in phenylpropanoid biosynthesis, including 1 gene encoding peroxidase (POD) that was upregulated in KR1326 roots (Figure 3).
The GO pathway analysis of the DEGs revealed that diverse pathways were represented in the transcriptome dataset. The multi-type bubble plots of the DEG pathway enrichment analysis (only the top 30 pathways are shown) showed that, at the early attachment stage, the oxidoreductase activity, catalytic activity, nucleotide binding, kinase activity, binding, phosphotransferase activity, lyase activity, and strictosidine synthase activity were significantly shared GO terms in the molecular function protein category; two translocon complexes were markedly shared terms in the cellular component category; chromosome organization, phosphorylation, oxidation–reduction process, DNA conformation change, protein–DNA complex assembly or subunit organization, phosphorus metabolic process, phosphate−containing compound metabolic process, phage shock, cellular response to virus, and kinetochore assembly or organization were the shared terms in the biological process category (Figure 1E). At the late attachment stage, the oxidation–reduction process, transmembrane transport, single−organism process, metabolic process, lipid modification or glycosylation, and biosynthetic process were markedly shared terms in the biological process category; binding, oxidoreductase activity, catalytic activity, transporter activity, ATPase activity, synthase activity, and hydrolase activity were significantly shared GO terms in the molecular function category (Figure 1F).
The similarity was that differentially expressed genes at both attachment stages were enriched in oxidation−reduction related pathways, such as the oxidation−reduction process and oxidoreductase activity pathway.

3.3. Identification of Upregulated DEGs in C. melo KR1326

A comparison of the DEGs in KR1326 and K1237 at two attachment stages showed that 192 DEGs were significantly differentially expressed at both stages (Figure 4A), with 70 of these DEGs (Figure 4B) upregulated in response to P. aegyptiaca across melon genotypes, of which 245 and 213 were detected at the early attachment stage and the late attachment stage, respectively, presumably reflecting the general progression of infection within the plant tissue.
According to the annotation of these 70 upregulated DEGs in C. melo KR1326 (Figure 4C), some genes were most likely involved in host immunity directly, among which, one was related to a transcription factor, one to a calcium channel protein, one to a glutathione peroxidase, one to an aspartic protease in guard cells, five to transferase-like proteins, ten to receptor-like proteins (six to serine/threonine-protein kinase, one to a proline-rich receptor-like protein, one to a putative LRR receptor-like protein, and two to TIR-NBS-LRRs class disease resistance protein), and so on. Among the rest, nearly 16% (11 of 70) of these upregulated DEGs were annotated as having an unknown function (Figure 4C). Novel00280, one of the 70 upregulated genes, encoded a TIR-NBS-LRR class protein and was annotated as a disease-resistance protein. Based on its uniquely high level of expression in C. melo KR1326 roots, we selected this candidate for further detailed analysis.

3.4. Characteristics of Novel00280, a Predicted Disease Resistance Protein

GO enrichment results showed that Novel00280 was significantly involved in these GO terms, including oxidoreductase activity, catalytic activity, oxidation–reduction process and binding at the early attachment stage (Figure 5A) and oxidoreductase activity, oxidation–reduction process, single–organism process or metabolic process, catalytic activity, and ion binding at the late attachment stage (Figure 5B), which were related to oxidation–reduction pathways.
The full-length cDNA sequence of Novel00280 contained a 2325 bp long open reading frame encoding 774 amino acids. The encoded full-length protein had a typical TIR domain at the N-terminus, an NB-ARC domain after the TIR, and the predicted LRRs arranged in tandem near the C-terminus of the protein, as shown in the structural composition displayed in Figure S3A. The secondary structure showed that the protein contained an alpha helix (51.81%), extended strand (13.57%), beta turn (5.04%), and random coil (29.59%) (Figure S3B). The predicted 3-D structure of the protein is displayed in Figure S3C. The results of phylogenetic tree analysis revealed that Novel00280 was most similar to XM008467437.2 (Figure S3D), which contains a 2463 bp cDNA sequence (gene ID: 103503300) encoding a 821 amino acids (protein ID: XP_008465659.2). Differences in their encoded protein sequences between Novel00280 and XM008467437.2 were found in the LRR domain (Figure S3E).
As the gene was screened in C. melo cultivars, and based on its unique structural characteristics, we designated the gene Novel00280 and its homologous genes XM008467437.2 as CmNLR and CmNLRh.

3.5. Overexpression of CmNLR and CmNLRh Could Enhance the Immune Response to P. aegyptiaca

To confirm the presence of the CmNLR gene in the C. melo cultivar, primers (Supplementary Table S2) of CmNLR and CmNLRh were designed across the full-length coding region and used in genomic PCR amplification.
By overexpressing CmNLR and CmNLRh genes in susceptible C. melo cultivar K1237 using Agrobacterium rhizogenes plant transformation (Figure S4A), we verified whether CmNLR affects melon root innate immunity. Transgenic roots were detected with GUS staining and qRT-PCR (Figure S4B,C).
In the interaction between the host roots and P. aegyptiaca simulated by the root chamber method, transgenic K1237 roots expressing CmNLR and CmNLRh showed a significantly higher frequency of browning events than control transgenic roots with P. aegyptiaca starting at 14 dpi and significantly less tubercle swelling events at 21 dpi, indicating that the overexpression of CmNLR and CmNLRh in host roots stimulated the host resistance response (Figure 6). Tubercle expansion is a well-characterized phenotype, which indicates a successful vascular connection between P. aegyptiaca and its host. We did not observe any tubercle expansion events on the transgenic K1237 roots expressing CmNLR and CmNLRh inoculated with P. aegyptiaca at 21 dpi. Tubercle swelling and expansion events occurred with increasing frequency at 28 dpi, most likely because their expression levels were already low (Figure S4C).
Transcriptome analysis showed that the immune response of the C. melo cultivar roots included active oxygen scavenging, and the CmNLR gene was involved in several oxidation–reduction-related pathways. We further examined the enzyme activity of the transgenic K1237 root system with CmNLR- or CmNLRh-overexpression transformation. The results of the enzymatic activity assays showed that oxidative burst significantly in transgenic K1237 roots expressing the CmNLR and CmNLRh proteins compared with the control transgenic K1237 roots (Figure 7). H2O2 burst, especially, occurred at the early attachment stage (7 dpi) in the CmNLRh roots. Meanwhile, the H2O2 burst occurred continuously and progressively in the CmNLR roots. The content of O2− increased significantly at the late attachment stage (21 dpi) in all transgenic roots. Transgenic K1237 roots expressing the CmNLR or CmNLRh proteins displayed higher antioxidant amounts and activity than control transgenic roots, including peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD), which were related to ROS scavenging. POD remained highly active in transgenic roots. In particular, CAT activity was significantly higher than in control transgenic roots starting at 7 dpi. However, SOD activity remained high only in transgenic K1237 roots expressing CmNLRh throughout the attachment stage (Figure 7).
Furthermore, transgenic K1237 roots expressing the CmNLR and CmNLRh proteins and control transgenic roots were then challenged with P. aegyptiaca in pots. At 28 dpi, transgenic K1237 roots showed significantly less P. aegyptiaca parasitism, especially in CmNLRh roots (Figure 8A,B). There was no significant difference in the length of stems between the transformed plants inoculated with P. aegyptiaca (Figure 8C). However, transgenic K1237 roots expressing CmNLRh showed a significant difference in the fresh weight of the stem (Figure 8C).

4. Discussion

Plants are constantly threatened by numerous pathogens and have evolved sophisticated defense mechanisms to recognize and respond to such attacks [26]. The primary task of plant immunity is the successful recognition of pathogens that guide the activation of defense responses [36]. Along with microbial pathogens, parasitic plants represent another category of threat to crops and have become a constraint on agriculture around the world. The way host plants recognize parasitic plants is similar to the perception of microbial pathogens [37]. It is necessary to understand the resistance mechanism at the molecular level, which is expected to provide the possible targets for effective control strategies [38]. However, relatively little is known about the immune response of host plants [3,23,39].
In host–parasite plant interactions, haustorial formation and a vascular connection with the host plant attempted by the parasite plant occur at the attachment stage, leading to a compatible or incompatible interaction. We investigated the mechanistic basis of compatible and incompatible interactions between P. aegyptiaca and K1237 and KR1326 based on a comparison of the transcriptomic method (Figure 1). Some DEGs involved in peroxisome and phenylpropanoid biosynthesis were related to active oxygen scavenging, which corresponds to oxidoreductase activity from the GO enrichment pathway (Figure 2 and Figure 3). This is consistent with the conclusion that plants may undergo physiological and biochemical changes, such as enhanced activity of antioxidant enzymes in response to the stress of parasitic plants to enhance their anti-parasitic ability [40,41], which is basically consistent with the mechanism of plant resistance to the invasion of pathogenic microorganisms.
The available advances in transcriptome information, in many cases [42,43,44,45,46], allow for comparative analyses to give us a deeper understanding. The challenge now is to translate the information from these resources into molecular details about host–parasite interactions. Quite a few genes among the 70 upregulated genes in KR1326 at both attachment stages were most likely involved in host immunity, such as some genes encoding a the transcription factor, calcium channel protein, and receptor-like protein (including two TIR-NBS-LRR class disease resistance proteins) (Figure 4), which indicated that KR1326 initiated a series of signal transduction responses against the invasion of P. aegyptiaca and resulted in an incompatible interaction. Some LRR-containing receptors from parasite-resistant crop varieties have been discovered and characterized. Sunflower recognizes O. cumana by LRR-RLK HAOR7, a well-studied homolog of the immune receptor Xa21 that makes rice resistant to bacterial blight [47]. It is likely that HAOR7 detects an avirulence factor of O. cumana. LRR receptor-based recognition has also been found in tomato, where LRR-rlp CuRe1 recognizes a small peptide from Cuscuta reflexa, the stem parasitic plant, and interacts with an adaptor kinase S1SOBIK1 to effectively resist pathogens [37]. Another case is found in the interaction between S. gesnerioides and cowpea; the resistance is mediated by RSG3-301 (resistance to S. gesnerioides race 3 in cowpea cultivar B301), encoding an R protein with an NLR domain [48].
As one of the most important plant disease-resistant proteins, NLRs have attracted extensive attention from researchers since their discovery. Here, CmNLR, encoding a TIR-NBS-LRR protein, was consistently highly expressed in KR1326 at two attachment stages, and significantly participated in oxidoreductase activity, catalytic activity, and oxidation-reduction process at the early attachment stage (Figure 5A) and oxidoreductase activity, oxidation–reduction process, single-organism process or metabolic process, and catalytic activity at the late attachment stage (Figure 5B). The results showed that CmNLR was involved in physiological and biochemical changes related to immunity, such as the enhancement of antioxidant enzyme activity in melon in response to P. aegyptiaca. In the present study, the overexpression of CmNLR and its homolog gene CmNLRh in K1237 roots could improved the resistance of melon to P. aegyptiaca, especially in the root compartment study; the results were remarkable (Figure 7). Previous studies have confirmed that when RSG3-301 expression is knocked down by virus-induced gene silencing (VIGS) in the multirace-resistant cowpea cultivar B301, S. gesnerioides can invade the endodermis and establish xylem–xylem connections with the host vascular system [48]. These results show that NLR proteins can enhance host resistance to parasites. However, there was no significant difference in the number of P. aegyptiaca in the transgenic K1237 root system expressing CmNLR, probably because the expression of CmNLR always remained relatively low, especially in the later stage (Figure 8).
Meanwhile, we detected that their overexpression resulted in a stronger ROS burst and scavenging capacity in transformed roots compared with control plants (Figure 6), which further demonstrated that the host defense responses to parasitic plants include a localized hypersensitive response that serves to prevent the spread of infection by triggering cell death.
Furthermore, NLR resistance genes are significantly enriched in parasites, such as S. hermonthica, suggesting an underlying important function in parasitic plants. Moreover, haustorium genes are also enriched in LRR domains [44]. A novel decoy effector SHR4z (suppressor of host resistance 4z) was identified from the haustorium of S. gesnerioides, which can suppress the hypersensitive response in host cowpea plants. SHR4z has significant homology to the short LRR domain in somatic embryogenesis receptor kinase (SERK) [49]. We can speculate that to overcome host resistance, parasitic plants target components of signal transduction pathways activated by R genes containing LRR domains to disrupt defense response cascades directly or indirectly. Therefore, in the foreseeable future, the investigation of NLRs remains a hot topic in the field of plant resistance research.
Functional characterization supplies insight into further understanding of the resistance mechanism of melon to P. aegyptiaca infection and provides new ideas for crop resistance improvement. Nevertheless, our current study is far from complete, and there are still many open questions that need to be further explored. For example, in the fight against the invasion of parasitic plants, the signal transduction pathways involved in the defense response are not clear, and the effector proteins and the specific recognition process are unknown; therefore, further research on how the plant immune system senses and regulates parasitic plants is imperative.

5. Conclusions

An important conclusion from this study was that the TIR-type NLR protein confers C. melo resistance to P. aegyptiaca. The overexpression of CmNLR (encoding a TIR-type NLR protein, consistently highly expressed in KR1326) and CmNLRh (homologous gene of CmNLR) in K1237 roots prevented P. aegyptiaca from connecting to the vascular system of C. melo roots. A stronger ROS burst and scavenging capacity were detected in the transformed roots. Moreover, several genes involved in the regulation of host immunity that were upregulated in the resistant C. melo cultivar KR1326 have been studied and reported in previous studies. This is consistent with the mechanism of plant resistance to the invasion of pathogenic microorganisms. These results can provide valuable resources for the breeding of melon against parasitism.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy13030644/s1, Figure S1: Validation results of each step in the construction process of recombinant plasmid; Figure S2: Pearson correlation analysis between transcriptome samples from melon roots; Figure S3: Gene and protein structural features of Novel00280; Figure S4: Detection of transformed roots; Table S1: Evaluation of sample sequencing data melon transcriptome samples; Table S2: Primers used in this study.

Author Contributions

L.X., Q.Z. and S.Z. designed the research. L.X., Q.Z. and X.C. performed the experiments. L.X., Q.Z. and Z.Y. performed the data analysis and interpretation. L.X., Q.Z. and S.Z. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China [grant numbers 32160649, 31460467]; and XPCC fund [grant numbers 2018CB022].

Data Availability Statement

The transcriptome data of Cucumis melo in this study have been submitted to the SRA database in NCBI (https://www.ncbi.nlm.nih.gov/sra/PRJNA929515 (accessed on 1 February 2023).

Acknowledgments

We are grateful to Xinli Ma for providing seeds of melon cultivars, including the KR1326 and K1237. We thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Volcano plots for GO and KEGG enrichment of differentially expressed genes (DEGs) of melon cultivar roots. (A) Differential expression patterns of 744 melon root DEGs selected as significant at 9 days post-inoculation (dpi) between KR1326 and K1237 samples. (B) Differential expression patterns of 590 melon root DEGs selected as significant at 16 dpi between KR1326 and K1237 samples. Red represents upregulated DEGs, green represents downregulated DEGs, and blue represents stably regulated genes. (C) GO analysis of DEGs between KR1326 and K1237 at 9 dpi (top 30 terms). (D) GO analysis of DEGs between KR1326 and K1237 at 16 dpi (top 30 terms). (E) KEGG pathway enrichment of DEGs between KR1326 and K1237 at 9 dpi. (F) KEGG pathway enrichment of DEGs between KR1326 and K1237 at 16 dpi. R1—KR1326 (resistant melon cultivar) samples at 9 dpi; S1—K1237 (susceptible melon cultivar) samples at 9 dpi; R2—KR1326 samples at 16 dpi; S2—K1237 samples at 16 dpi.
Figure 1. Volcano plots for GO and KEGG enrichment of differentially expressed genes (DEGs) of melon cultivar roots. (A) Differential expression patterns of 744 melon root DEGs selected as significant at 9 days post-inoculation (dpi) between KR1326 and K1237 samples. (B) Differential expression patterns of 590 melon root DEGs selected as significant at 16 dpi between KR1326 and K1237 samples. Red represents upregulated DEGs, green represents downregulated DEGs, and blue represents stably regulated genes. (C) GO analysis of DEGs between KR1326 and K1237 at 9 dpi (top 30 terms). (D) GO analysis of DEGs between KR1326 and K1237 at 16 dpi (top 30 terms). (E) KEGG pathway enrichment of DEGs between KR1326 and K1237 at 9 dpi. (F) KEGG pathway enrichment of DEGs between KR1326 and K1237 at 16 dpi. R1—KR1326 (resistant melon cultivar) samples at 9 dpi; S1—K1237 (susceptible melon cultivar) samples at 9 dpi; R2—KR1326 samples at 16 dpi; S2—K1237 samples at 16 dpi.
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Figure 2. Expression patterns of differentially expressed genes (DEGs) in the peroxisome pathway. The heat map shows DEG expression levels that enriched the peroxisome pathway in KR1326 and K1237 root inoculation with P. aegyptiaca at 9 and 16 dpi. Blue to orange indicates the gene expression level from low to high. Expression levels were estimated using log2 (fold change) for each gene, with color intensity correlating to the fold change level. The group of transcriptome samples of melon is shown as the footer denotes. ACOX, acyl-CoA oxidase; ACSL, acyl-CoA synthetase; FAR, fatty acyl-CoA reductase; IDH, isocitrate dehydrogenase; HAO, hydroxy-acid oxidase; CAT, catalase.
Figure 2. Expression patterns of differentially expressed genes (DEGs) in the peroxisome pathway. The heat map shows DEG expression levels that enriched the peroxisome pathway in KR1326 and K1237 root inoculation with P. aegyptiaca at 9 and 16 dpi. Blue to orange indicates the gene expression level from low to high. Expression levels were estimated using log2 (fold change) for each gene, with color intensity correlating to the fold change level. The group of transcriptome samples of melon is shown as the footer denotes. ACOX, acyl-CoA oxidase; ACSL, acyl-CoA synthetase; FAR, fatty acyl-CoA reductase; IDH, isocitrate dehydrogenase; HAO, hydroxy-acid oxidase; CAT, catalase.
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Figure 3. Expression patterns of differentially expressed genes (DEGs) in the phenylpropanoid biosynthesis pathway. The heat map shows DEG expression levels that enriched in the phenylpropanoid biosynthesis pathway in KR1326 and K1237 root inoculation with P. aegyptiaca at 9 and 16 dpi. Blue to orange indicates the gene expression level from low to high. Expression levels were estimated using log2 (fold change) for each gene, with color intensity correlating to the level of fold change. The group of transcriptome samples of melon is shown as the footer denotes. BGLU, beta-glucosidase; 4CL1, 4-coumarate-CoA ligase 1; HCT, hydroxycinnamoyl transferase; POD, peroxidase; SCPL19, serine carboxypeptidase-like 19.
Figure 3. Expression patterns of differentially expressed genes (DEGs) in the phenylpropanoid biosynthesis pathway. The heat map shows DEG expression levels that enriched in the phenylpropanoid biosynthesis pathway in KR1326 and K1237 root inoculation with P. aegyptiaca at 9 and 16 dpi. Blue to orange indicates the gene expression level from low to high. Expression levels were estimated using log2 (fold change) for each gene, with color intensity correlating to the level of fold change. The group of transcriptome samples of melon is shown as the footer denotes. BGLU, beta-glucosidase; 4CL1, 4-coumarate-CoA ligase 1; HCT, hydroxycinnamoyl transferase; POD, peroxidase; SCPL19, serine carboxypeptidase-like 19.
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Figure 4. Differentially expressed genes (DEGs) and functional annotation in melon roots. (A) Venn plot of melon DEGs at 9 and 16 dpi between the KR1326 and K1237 samples. (B) Venn plot of melon upregulated DEGs at 9 and 16 dpi between KR1326 and K1237 samples. (C) Clustering analysis of 70 co-upregulated DEGs in KR1326 samples at 9 and 16 dpi. The heat map part indicates the level of log2 fold change of gene expression, with color intensity correlating to the level of the fold change. The footer denotes the group of transcriptome samples of the melon root.
Figure 4. Differentially expressed genes (DEGs) and functional annotation in melon roots. (A) Venn plot of melon DEGs at 9 and 16 dpi between the KR1326 and K1237 samples. (B) Venn plot of melon upregulated DEGs at 9 and 16 dpi between KR1326 and K1237 samples. (C) Clustering analysis of 70 co-upregulated DEGs in KR1326 samples at 9 and 16 dpi. The heat map part indicates the level of log2 fold change of gene expression, with color intensity correlating to the level of the fold change. The footer denotes the group of transcriptome samples of the melon root.
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Figure 5. GO enrichment showing the pathways in which the upregulated gene Novel00280 was significantly involved at 9 dpi (A) and 16 dpi (B) in C. melo KR1326.
Figure 5. GO enrichment showing the pathways in which the upregulated gene Novel00280 was significantly involved at 9 dpi (A) and 16 dpi (B) in C. melo KR1326.
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Figure 6. Differential response of transgenic K1237 roots to parasitism by P. aegyptiaca. (A) Representative photos illustrating the phenotypic response of transgenic K1237 roots. The appearance of P. aegyptiaca contacting the roots of transgenic K1237 at 14, 21, and 28 dpi are shown. Bars, 1 mm. (B) Occurrence frequency of phenotypic event categories during the interaction of P. aegyptiaca with transgenic K1237 roots at 14, 21, and 28 dpi. The abbreviations of the phenotypic event categories are as follows: BR, browning reaction; TS, tubercle swelling; and TE, tubercle expansion. The interaction event ratio of each category was obtained by dividing the number of events in each category by the total number of phenotypic events occurring in the transgenic K1237 root system. Statistical analysis was done with 5 independent transgenic plants.
Figure 6. Differential response of transgenic K1237 roots to parasitism by P. aegyptiaca. (A) Representative photos illustrating the phenotypic response of transgenic K1237 roots. The appearance of P. aegyptiaca contacting the roots of transgenic K1237 at 14, 21, and 28 dpi are shown. Bars, 1 mm. (B) Occurrence frequency of phenotypic event categories during the interaction of P. aegyptiaca with transgenic K1237 roots at 14, 21, and 28 dpi. The abbreviations of the phenotypic event categories are as follows: BR, browning reaction; TS, tubercle swelling; and TE, tubercle expansion. The interaction event ratio of each category was obtained by dividing the number of events in each category by the total number of phenotypic events occurring in the transgenic K1237 root system. Statistical analysis was done with 5 independent transgenic plants.
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Figure 7. Enzyme activity of transgenic K1237 roots: the H2O2 and O2− contents and the activities of POD, CAT, and SOD. H2O2, hydrogen peroxide; O2−, superoxide anion; POD, peroxidase; CAT, catalase; SOD, superoxide dismutase.
Figure 7. Enzyme activity of transgenic K1237 roots: the H2O2 and O2− contents and the activities of POD, CAT, and SOD. H2O2, hydrogen peroxide; O2−, superoxide anion; POD, peroxidase; CAT, catalase; SOD, superoxide dismutase.
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Figure 8. Overexpression of CmNLR and CmNLRh in K1237 roots enhanced the melon immune response to P. aegyptiaca. Plant phenotype (A), number of P. aegyptiaca (B), length of stem ((C) left), and fresh weight of stem ((D) right) of potted transgenic K1237 cultivar 28 days after inoculation with P. aegyptiaca. Each measurement had ten replicates and the experiment was repeated three times. Bar, 1 cm. * indicate significant differences.
Figure 8. Overexpression of CmNLR and CmNLRh in K1237 roots enhanced the melon immune response to P. aegyptiaca. Plant phenotype (A), number of P. aegyptiaca (B), length of stem ((C) left), and fresh weight of stem ((D) right) of potted transgenic K1237 cultivar 28 days after inoculation with P. aegyptiaca. Each measurement had ten replicates and the experiment was repeated three times. Bar, 1 cm. * indicate significant differences.
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Xiao, L.; Zhao, Q.; Cao, X.; Yao, Z.; Zhao, S. The TIR-Type NLR Protein Is Involved in the Regulation of Phelipanche aegyptiaca Resistance in Cucumis melo. Agronomy 2023, 13, 644. https://doi.org/10.3390/agronomy13030644

AMA Style

Xiao L, Zhao Q, Cao X, Yao Z, Zhao S. The TIR-Type NLR Protein Is Involved in the Regulation of Phelipanche aegyptiaca Resistance in Cucumis melo. Agronomy. 2023; 13(3):644. https://doi.org/10.3390/agronomy13030644

Chicago/Turabian Style

Xiao, Lifeng, Qiuyue Zhao, Xiaolei Cao, Zhaoqun Yao, and Sifeng Zhao. 2023. "The TIR-Type NLR Protein Is Involved in the Regulation of Phelipanche aegyptiaca Resistance in Cucumis melo" Agronomy 13, no. 3: 644. https://doi.org/10.3390/agronomy13030644

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