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Article

Comparative Transcriptome Analysis of Susceptible and Resistant Rutaceae Plants to Huanglongbing

by
Huihong Liao
*,†,
Fuping Liu
†,
Xi Wang
,
Hongming Huang
,
Qichun Huang
,
Nina Wang
and
Chizhang Wei
Horticulture Research Institute, Guangxi Academy of Agricultural Sciences, Nanning 530007, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2025, 15(5), 1218; https://doi.org/10.3390/agronomy15051218 (registering DOI)
Submission received: 29 March 2025 / Revised: 6 May 2025 / Accepted: 13 May 2025 / Published: 17 May 2025
(This article belongs to the Special Issue Resistance-Related Gene Mining and Genetic Improvement in Crops)
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Abstract

:
Huanglongbing (HLB), also known as citrus greening, is a devastating disease affecting the citrus industry worldwide. This study aimed to investigate the transcriptional responses of two Rutaceae species, Ponkan Mandarin (susceptible) and Punctate Wampee (resistant), to HLB infection. Comparative transcriptome analysis was conducted to identify differentially expressed genes (DEGs) and pathways involved in defense mechanisms. The transcriptome data showed that in the susceptible Ponkan Mandarin, there were 1519 upregulated genes and 700 downregulated genes, while in the resistant Punctate Wampee variety, there were 1611 upregulated genes and 1727 downregulated genes. Upon infection, 297 genes were upregulated in both varieties, while 211 genes were downregulated in both. These genes included transcription factors from different families such as WRKY, ERF, and MYB. Ponkan Mandarin primarily relies on pathways like lignin synthesis and cell wall modification to defend against HLB, whereas Punctate Wampee mainly resists HLB by regulating cellular homeostasis and metabolism. Weighted Gene Co-expression Network Analysis (WGCNA) identified ten potential key resistance genes in the resistant Punctate Wampee variety, including genes involved in lignin biosynthesis and genes related to cellular signaling pathways. These findings not only enhance our understanding of the distinct defense mechanisms employed by citrus species against HLB infection but also offer novel perspectives for developing effective prevention and management strategies against this disease.

1. Introduction

Huanglongbing (HLB), also known as citrus greening disease, is one of the most destructive diseases affecting citrus production worldwide. The disease is caused by the bacteria Candidatus Liberibacter spp., which is transmitted by the Asian citrus psyllid (Diaphorina citri) [1]. HLB poses a significant threat to the global citrus industry, leading to severe economic losses due to reduced fruit yield, poor fruit quality, and ultimately tree death [2]. The symptoms of HLB include yellowing of the leaves, shoot dieback, and misshapen, bitter fruits, which render the produce unmarketable [3]. Despite ongoing efforts, there is currently no effective cure for HLB, and management strategies primarily focus on controlling the psyllid vector, removing infected trees, and implementing best horticultural practices to reduce disease spread [4,5].
The Rutaceae family includes many economically important citrus species that are vital for global fruit production, including oranges, mandarins, and grapefruits [6]. Understanding the response of different Rutaceae species to HLB infection is crucial for developing effective and sustainable disease management strategies [2]. Among the Rutaceae, certain species exhibit varying degrees of susceptibility or resistance to HLB, which offers a unique opportunity to explore the genetic basis of disease resistance [7]. In this study, we selected two Rutaceae species with contrasting responses to HLB: Ponkan (Citrus reticulata), a commonly cultivated susceptible variety, and Punctate Wampee (Clausena lansium), a lesser-known but HLB-resistant species [8]. By comparing their transcriptomic responses following HLB inoculation, we aim to uncover the molecular mechanisms underlying susceptibility and resistance to HLB, which could pave the way for improved breeding strategies and disease management.
Previous research on the resistance mechanisms of Rutaceae plants to HLB has identified several key pathways and genes involved in the defense response. Studies have shown that resistant varieties often exhibit enhanced activation of defense-related genes, including those involved in pathogen recognition, signaling pathways, and the production of antimicrobial compounds [7]. Specifically, genes related to salicylic acid (SA) and jasmonic acid (JA) signaling pathways have been found to play an essential role in HLB resistance [9]. These phytohormones are crucial in mediating plant defense responses against biotic stresses, with SA generally associated with systemic acquired resistance (SAR) and JA primarily involved in defense against herbivores and necrotrophic pathogens [10]. Additionally, resistant varieties tend to have upregulated expression of genes involved in reactive oxygen species (ROS) scavenging, which helps mitigate the oxidative stress caused by HLB infection [11,12]. Secondary metabolites, such as flavonoids and terpenoids, have also been implicated in providing resistance by inhibiting pathogen proliferation or strengthening plant cell walls, thereby limiting pathogen entry and spread [13]. The accumulation of these secondary metabolites serves as an important biochemical defense, enhancing the plant’s ability to resist HLB infection.
Several genes have been reported to be involved in HLB resistance, including NPR1 (Nonexpressor of Pathogenesis-Related Genes 1), WRKY transcription factors, and PR (Pathogenesis-Related) genes, which are known to be upregulated in resistant citrus varieties [14,15]. NPR1 is a key regulator of systemic acquired resistance (SAR) and plays a pivotal role in modulating SA-mediated defense responses [16]. In HLB-resistant citrus, the upregulation of NPR1 has been linked to an enhanced ability to activate SAR, resulting in increased resistance to the pathogen [14]. WRKY transcription factors are involved in regulating gene expression in response to biotic stresses, and their upregulation has been correlated with increased HLB resistance [17]. For instance, WRKY70 is known to be a positive regulator of SA signaling and is often induced in response to pathogen attack, thereby enhancing resistance [18]. PR genes, which encode proteins with antimicrobial properties, have been shown to be significantly induced in resistant varieties, contributing to the suppression of pathogen growth [19]. Studies have demonstrated that PR1, PR2, and PR5 proteins are crucial for establishing an effective defense against HLB by inhibiting pathogen proliferation and providing a chemical barrier [20,21]. Furthermore, previous transcriptome studies have identified the role of LRR-RLK (Leucine-Rich Repeat Receptor-Like Kinase) genes in recognizing pathogen-associated molecular patterns (PAMPs) and activating downstream immune responses [22]. LRR-RLKs are important for initiating pattern-triggered immunity (PTI), which is the first line of defense against pathogens. In resistant citrus species, these genes are often found to be more highly expressed, suggesting their involvement in early pathogen recognition and defense activation [23].
In addition to individual genes, several transcriptome studies have provided insights into the broader molecular networks involved in HLB resistance. For instance, a transcriptome analysis of HLB-tolerant “US-897” rootstock revealed the upregulation of genes associated with hormone signaling, secondary metabolite biosynthesis, and ROS detoxification [24]. Specifically, genes related to salicylic acid (SA) and jasmonic acid (JA) signaling were found to be more active, indicating an enhanced hormonal response, while secondary metabolites such as flavonoids played a role in chemical defense. Another study on HLB-resistant pummelo (Citrus grandis) identified differentially expressed genes involved in cell wall modification and lignin biosynthesis, which are thought to enhance structural barriers against pathogen invasion [21]. These cell wall-modifying genes contribute to the strengthening of plant cell walls, thus providing a physical blockade that hinders pathogen spread [25]. Collectively, these findings indicate that a multifaceted defense strategy, involving both immune signaling and physical reinforcement, is crucial for HLB resistance in Rutaceae species.
The objective of this study is to investigate the transcriptome-level differences between Ponkan and Punctate Wampee in response to HLB infection. Specifically, we aim to identify differentially expressed genes (DEGs) associated with defense responses, immune signaling, and other biological pathways that play a role in resistance or susceptibility. By conducting comprehensive analyses of gene expression profiles, gene ontology (GO) enrichment, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, we hope to gain insights into the key factors contributing to resistance against HLB. Understanding these molecular differences will provide a foundation for developing new disease resistance strategies and may facilitate the breeding of HLB-resistant citrus varieties. Moreover, this study will contribute to the broader understanding of plant–pathogen interactions in citrus and could provide novel insights into the development of biotechnological solutions for combating HLB. Additionally, through Weighted Gene Co-expression Network Analysis (WGCNA), we identified ten genes that are potentially associated with HLB resistance in Rutaceae plants.

2. Materials and Methods

2.1. Plant Material

Two-year-old seedlings of Ponkan (Citrus reticulata) and Punctate Wampee (Clausena lansium) were grown under greenhouse conditions (No. 174, East University Road, Nanning, China). HLB inoculation was performed using grafting carrying Candidatus Liberibacter asiaticus (CLas) scions grafted onto Ponkan and Punctate Wampee. A total of 20 Ponkan and 20 Wampee plants per species (10 inoculated, 10 non-inoculated as controls) were used. All 10 inoculated Ponkan plants (susceptible) showed qPCR-positive results and developed typical HLB symptoms, while 9 out of 10 inoculated Wampee plants (resistant) were qPCR-positive but remained asymptomatic. Leaf samples were collected at 1.5 months post-inoculation to capture the transcriptional response in May 2023. Three biological replicates per variety were collected, with each replicate consisting of leaves from groups of 3 plants (using 9 plants total per treatment group), which allowed us to maintain consistency while accounting for biological variation. Samples were frozen in liquid nitrogen and stored at −80 °C until RNA extraction.

2.2. RNA Sequencing and Transcriptomic Analysis

Total RNA was extracted from leaf samples using the Qiagen RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. To ensure high-quality RNA, an on-column DNase I digestion was performed to remove any residual genomic DNA. RNA quality and concentration were assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Samples with a RIN (RNA Integrity Number) greater than 7.0 and OD260/OD280 ratios between 1.8 and 2.1 were selected for downstream analysis. In addition, the integrity of the RNA was confirmed by running samples on a 1% agarose gel. cDNA libraries were constructed using the Illumina TruSeq RNA Sample Preparation Kit (Illumina, San Diego, CA, USA) following the manufacturer’s protocol. Briefly, poly(A)+ mRNA was isolated from 2 µg of total RNA using oligo-dT magnetic beads. The purified mRNA was fragmented at 94 °C for 8 min, and the fragmented mRNA was reverse transcribed to synthesize first-strand cDNA using random hexamer primers. Second-strand cDNA synthesis was performed using DNA Polymerase I and RNase H. The resulting double-stranded cDNA was end-repaired, A-tailed, and ligated to Illumina sequencing adapters. The cDNA fragments were then purified using AMPure XP beads (Beckman Coulter, Brea, CA, USA) and enriched by PCR amplification to create the final cDNA library. The quality and size distribution of the cDNA libraries were assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Paired-end sequencing (2 × 150 bp) was performed on an Illumina HiSeq platform (Illumina, San Diego, CA, USA), targeting a sequencing depth of at least 30 million reads per sample. Sequencing was performed at a commercial sequencing facility, and quality control measures were implemented to minimize technical variability between samples.

2.3. Data Processing

Raw reads were quality-checked using FastQC (v0.12.1) and Trimmomatic was used to remove low-quality bases and adapters. Cleaned reads were aligned to the reference citrus genome using HISAT2 (v2.2.1), and read counts for each gene were generated using featureCounts (v2.0.3) [26,27]. Quality metrics such as mapping efficiency and gene body coverage were also calculated.

2.4. Differential Expression and Functional Enrichment Analysis

The WGCNA was performed with WGCNA-shinyApp (https://github.com/ShawnWx2019/WGCNA-shinyApp, accessed on 15 May 2024) [28]. The raw count values of all the genes were normalized using the variance-stabilizing transformation method, and then the gene sets were filtered twice. First, genes with 90% of samples having a count value less than 10 were removed. Then, genes were further filtered using the “median absolute deviation” method [29]. The normalized count values of the remaining genes were utilized to calculate the suggested power value. Then, the module net was constructed with parameters of “min Module size = 30” and “module cuttree height = 0.25”. All CLas infected samples were designated as “HLB”, while CLas free samples were designated as “MOCK”. The correlation between module and trait data (such as “HLB”) was computed, and significant “module-trait” pairs were utilized to identify hub genes. Differential expression analysis was conducted using DESeq2. Genes with an adjusted p-value (FDR) < 0.05 and a log2 fold change >1 or <−1 were considered significantly differentially expressed. Comparisons were made between HLB-infected and mock-inoculated plants within each variety, as well as between Ponkan and Punctate Wampee. Volcano plots and heatmaps were generated to visualize DEGs. Functional annotation was performed using Blast2GO. GO enrichment analysis categorized DEGs into biological processes, molecular functions, and cellular components. KEGG pathway enrichment analysis identified significant metabolic and signaling pathways. Weighted Gene Co-expression Network Analysis (WGCNA) identified gene modules correlated with HLB resistance, and ten hub genes potentially associated with resistance were identified.

3. Results

3.1. Transcriptome Analysis of the Effects of Huanglongbing on Ponkan Mandarin and Punctate Wampee

Principal component analysis (PCA) and correlation analysis demonstrated high reproducibility of the transcriptome data, confirming their reliability for subsequent analyses (Figure 1A,B and Figure 2A,B). Additionally, the clustering heatmap of differentially expressed genes indicates high reproducibility of the transcriptome data, supporting further analysis (Figure S1). Upon treatment, 1519 genes in Ponkan Mandarin were upregulated, while 700 genes were downregulated after HLB infection (Figure 1C, Table S1). KEGG enrichment analysis showed that the upregulated genes were primarily associated with the “Amino sugar and nucleotide sugar metabolism” and “Phenylpropanoid biosynthesis” pathways; also, the brassinosteroid biosynthesis pathway was enriched after HLB infection, whereas the downregulated genes were enriched in the “Photosynthesis” and “Glycolysis/Gluconeogenesis” pathways (Figure 1D,E). These findings indicate a significant inhibition of photosynthesis in Ponkan Mandarin following HLB infection. Conversely, activating the lignin biosynthesis pathway suggests a potentially crucial role for lignin in defense against HLB.
Gene ontology (GO) enrichment analysis further revealed that, after HLB infection, the upregulated genes were significantly enriched in pathways related to the “external encapsulating structure” and “cell wall”, while the downregulated genes were primarily enriched in the “plastid” and “chloroplast” pathways (Figure 1F,G, Figures S2 and S3). This provides additional evidence that HLB infection strongly impacts photosynthesis and respiration in susceptible Ponkan Mandarin. Moreover, the activation of lignin biosynthesis and cell wall formation pathways underscores their importance in the defense response to HLB infection.
In Huanglongbing-infected Punctate Wampee, 1611 genes were upregulated and 1727 genes were downregulated (Figure 2C, Table S2). KEGG enrichment analysis revealed that the upregulated genes were primarily enriched in the “Starch and sucrose metabolism” pathway, while the downregulated genes were significantly enriched in the “Ribosome” and “Photosynthesis” pathways, indicating a substantial effect of HLB infection on leaf photosynthesis (Figure 2D,E). GO enrichment analysis indicated that the upregulated genes were mainly enriched in the “ribonucleoside monophosphate catabolic process” and “ATP metabolic process”, whereas the downregulated genes were enriched in pathways related to the “thylakoid”, “chloroplast thylakoid”, and “organelle subcompartment” (Figure 2F,G, Figures S4 and S5), These results suggest that in Huanglongbing-resistant Citrus reticulata, the plant triggers a series of intracellular metabolic responses to mitigate HLB infection.

3.2. Comparative Transcriptomics Reveals Key Genes Involved in the Response to Huanglongbing in Citrus

After identifying differentially expressed genes in Ponkan Mandarin and Punctate Wampee following Huanglongbing inoculation, we conducted a comparative analysis of these genes. The results showed that 297 genes were upregulated in both Ponkan Mandarin and Punctate Wampee (Table S3), while 211 genes were downregulated in both. Additionally (Table S4), 31 genes were upregulated in Punctate Wampee but downregulated in Ponkan Mandarin, whereas 68 genes were upregulated in Ponkan Mandarin but downregulated in Punctate Wampee (Figure 3C) (Table S5). Notably, among the genes specifically upregulated in Punctate Wampee were several transporter genes, including Cs3g26380 and Cs7g12620 (ATP-binding cassette transporters), Cs6g05990 (cation/H antiporter), Cs7g22940 (aluminum-activated malate transporter), and three ABC transporters (Cs6g13326, Cs4g17100, and Cs4g09150). These genes may contribute to Punctate Wampee’s resistance to HLB. Additionally, analysis of commonly upregulated and downregulated transcription factors in both Ponkan Mandarin and Punctate Wampee revealed that numerous transcription factors, including those from the zinc finger, MYB, bZIP, ERF, NAC, and MADS gene families, may play essential roles in HLB resistance (Figure 3C,D). These transcription factors likely act as critical upstream regulatory signals governing resistance in Rutaceae species against HLB.

3.3. Identification of Core Genes Conferring Huanglongbing Resistance in Rutaceae Species Through WGCNA

Using Weighted Gene Co-Expression Network Analysis (WGCNA), we systematically analyzed the transcriptome data from Punctate Wampee and Ponkan citrus infected with Huanglongbing. The data were normalized, and an optimal soft-thresholding power of 16 was selected to construct a scale-free gene co-expression network (Figure S6). This analysis clustered highly co-expressed genes into seven distinct modules, each labeled with a unique color (Figure 4A). Correlation analysis revealed that the blue module was significantly positively correlated with Huanglongbing resistance (R = 0.89, p < 0.01) (Figure 4B), with genes only upregulated in the Punctate Wampee varieties, suggesting that genes within the blue module may play a key role in the resistance mechanism (Figure 4C,D). Functional annotation further indicated that genes in the blue module were highly enriched in defense-related signaling pathways and secondary metabolic processes, highlighting the potential importance of these pathways in disease resistance (Figure 4E, Table 1). Core gene identification within the blue module identified Cs7g30890 (Clathrin heavy chain 1), Cs1g20380 (auxin-independent growth promoter), and Cs6g05890 (heat shock protein) as key genes with high module membership (MM) and gene significance (GS) scores, suggesting that these genes may serve as crucial regulatory factors in the defense response. Additionally, there are two cellulose synthase-related proteins (Cs07g05150, Cs07g07870), two serine/threonine-protein genes (Cs8g14490, Cs1g12370), and the casein kinase protein (Cs5g26620). In the resistance of citrus to HLB, Clathrin heavy chain 1 restricts pathogen spread through endocytosis and promotes the transport of defense molecules. Serine/threonine-protein phosphatase regulates defense signaling pathways to enhance disease resistance gene expression. Casein kinase I isoform delta-like modulates the timing and stages of defense responses to strengthen immunity, while cellulose synthase-like protein enhances disease resistance by reinforcing the physical barrier of the cell wall and activating defense signals. These genes work synergistically to enhance citrus resistance to HLB. Future studies will employ gene knockout and overexpression experiments to further elucidate the specific mechanisms by which these core genes contribute to HLB resistance, providing potential molecular targets for breeding disease resistant citrus varieties.

3.4. Proposed Working Model of Huanglongbing Resistance in Ponkan Mandarin and Punctate Wampee

Based on the study’s results, a potential working model of resistance in Ponkan and Wampee under Huanglongbing infection is proposed (Figure 5), describing interactions among key genes and signaling pathways.
In Ponkan Mandarin, upon Huanglongbing infection, pathways related to cell wall metabolism were significantly upregulated, including phenylpropanoids, flavonoids, and amino sugar and nucleotide metabolism (Figure 5). Conversely, pathways associated with photosynthesis were notably downregulated. The upregulation of cell wall-related pathways suggests an important role in enhancing structural defense mechanisms against the pathogen. The phenylpropanoid pathway is known to be involved in the synthesis of lignin, which strengthens cell walls and acts as a barrier to pathogen invasion. Flavonoids, which are secondary metabolites, may also contribute to antimicrobial activity, providing an additional chemical defense layer. The increased activity of amino sugar and nucleotide metabolism could be linked to the synthesis of structural components essential for cell wall reinforcement. Meanwhile, the downregulation of photosynthesis-related pathways might indicate a reallocation of energy and resources from growth processes to defense responses. Overall, these pathways, especially those involved in cell wall synthesis, appear to play crucial roles in Ponkan’s defense against Huanglongbing by enhancing the physical and chemical barriers that limit pathogen spread.
In Punctate Wampee, upon Huanglongbing infection, genes related to starch and sucrose metabolism and ribonucleoside monophosphate catabolism were significantly upregulated, while the ribosome metabolic pathway was notably suppressed (Figure 5). The upregulation of starch and sucrose metabolism suggests that the plant may be mobilizing energy reserves to mount an effective defense response. This could enhance the availability of energy and carbon skeletons required for various defense processes, including the production of defense-related metabolites. The increase in ribonucleoside monophosphate catabolism might contribute to nucleic acid turnover, which could be crucial for regulating stress-responsive gene expression and adapting to pathogen-induced cellular changes. On the other hand, the downregulation of the ribosome pathway implies a reduction in protein synthesis, possibly as a strategy to conserve energy and resources during stress conditions. This reallocation of resources might help prioritize the activation of defense mechanisms over normal cellular functions. Overall, these metabolic changes suggest that Punctate Wampee adopts a strategy of energy reallocation and stress adaptation to effectively combat HLB infection.
In both Ponkan and Punctate Wampee, numerous transcription factors showed differential expression, along with several genes related to transporter proteins, indicating that these genes might also play significant roles in the resistance of Rutaceae plants to HLB (Figure 5). Transcription factors are known to regulate gene expression in response to biotic stress, potentially activating defense-related genes. Transporter proteins may be involved in the movement of defense compounds or signaling molecules, thus contributing to the overall defense strategy of the plants.

4. Discussions

The results of this study provide significant insights into the distinct responses of Ponkan Mandarin and Punctate Wampee to HLB infection, highlighting key pathways and regulatory mechanisms involved in their defense.

4.1. Differential Defense Strategies

The transcriptome analysis revealed distinct and species-specific defense mechanisms between Ponkan Mandarin and Punctate Wampee when exposed to HLB infection (Figure 5). In Ponkan, the significant upregulation of cell wall-related pathways, including phenylpropanoids, flavonoids, and amino sugar metabolism, suggests that the reinforcement of cell wall integrity is a primary strategy to combat HLB infection. The phenylpropanoid pathway is known to play a pivotal role in the biosynthesis of lignin, which strengthens cell walls and provides a physical barrier against pathogen invasion [7,30]. As secondary metabolites, flavonoids exhibit limited antimicrobial activity that contributes to plant chemical defense by partially suppressing pathogen growth, though they cannot fully prevent infection [31]. Previous studies have indicated that phenylpropanoid and flavonoid biosynthesis pathways are critical in enhancing the structural rigidity of plant cell walls and producing compounds that can limit pathogen spread and reduce disease severity [32,33].
The upregulation of amino sugar and nucleotide metabolism observed in Ponkan suggests that cell wall component synthesis is also being enhanced to fortify physical barriers [34]. Such changes are crucial for reinforcing the structural integrity of the cell wall, making it difficult for the pathogen to infiltrate host tissues. Although soft tissues lack constitutive rigidity, pathogen-induced deposition of cell wall components (e.g., callose) may locally reinforce structural integrity at infection sites, potentially slowing pathogen progress. Moreover, it has been documented that the activation of these pathways helps create a resistant environment by synthesizing compounds like chitin and glucosamine, which are key components in strengthening the plant’s defensive structure [35]. However, although structural reinforcement of cell walls (e.g., callose and lignin deposition) limits pathogen spread, these modifications do not deter insect vectors from feeding on young, non-lignified tissues. Insect feeding preferences and the developmental stage of plant tissues remain critical factors in pathogen acquisition and transmission.
Conversely, Punctate Wampee showed a distinct defense strategy characterized by the upregulation of starch and sucrose metabolism and ribonucleoside monophosphate catabolism, while the ribosome metabolic pathway was notably downregulated. The increased activity of starch and sucrose metabolism suggests an adaptive mechanism in Punctate Wampee to mobilize energy reserves in response to pathogen infection. Mobilizing these energy reserves is likely crucial for supporting defense-related biosynthetic processes and producing secondary metabolites that can inhibit pathogen growth and spread. Previous research has highlighted the importance of carbohydrate metabolism in plant immunity, as it helps provide the necessary energy and carbon skeletons needed for synthesizing defense compounds [36,37]. Sucrose, in particular, acts as a signaling molecule that modulates the activation of defense genes, which may further contribute to a coordinated immune response [38].
Furthermore, the upregulation of ribonucleoside monophosphate catabolism might indicate a process of nucleic acid turnover, enabling the plant to rapidly adapt to the stress caused by the pathogen [39]. This metabolic change could facilitate the production of stress-responsive molecules and modulate gene expression to better prepare for biotic stress conditions [40]. The suppression of the ribosome metabolic pathway in Punctate Wampee suggests a reduction in protein synthesis, potentially conserving energy and rerouting it toward essential defense processes [41]. This strategy of reducing non-essential cellular functions is common among plants undergoing pathogenic stress as it allows for the reallocation of resources to activate stronger defense responses, thus enhancing overall resistance [42].
Together, these findings illustrate that while Ponkan relies heavily on physical and chemical barriers by reinforcing its cell wall integrity, Punctate Wampee appears to adopt a metabolic reprogramming strategy, reallocating its resources to generate energy and defense-related compounds. These contrasting strategies highlight the species-specific responses and emphasize the complexity of plant immune mechanisms against HLB infection. Understanding these pathways in more detail provides valuable insight into the molecular basis of resistance in Rutaceae plants and may offer new avenues for breeding HLB-resistant citrus varieties.

4.2. Role of Transcription Factors and Transporter Proteins

The differential expression of numerous transcription factors (TFs) and transporter proteins observed in both Ponkan Mandarin and Punctate Wampee highlights the significant role these components play in orchestrating complex defense responses against HLB infection. Transcription factors, such as WRKY, MYB, and NAC, are known to be key regulators of plant immunity, where they facilitate the transcriptional reprogramming of defense-associated genes. These TFs are often implicated in various signaling pathways, including those that activate pathogen-responsive genes and regulate hormone-mediated immune signaling, such as salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) pathways [43,44]. WRKY TFs, in particular, have been extensively studied and are known to regulate both basal defense and systemic acquired resistance (SAR) by modulating SA-responsive gene expression [45,46]. The WRKY family’s ability to bind to W-box elements in the promoters of target genes makes them central players in fine-tuning the plant immune response. MYB transcription factors, on the other hand, are also crucial for defense regulation, particularly in secondary metabolite biosynthesis and lignin production, both of which enhance physical and chemical barriers against pathogen invasion [47]. These MYB proteins are often activated in response to biotic stress and contribute to the accumulation of phenolic compounds, such as flavonoids, which possess antimicrobial properties that deter pathogen growth. For instance, studies have shown that MYB75, a member of the R2R3-MYB family, regulates anthocyanin biosynthesis, enhancing pathogen resistance by accumulating these phenolic metabolites [48]. Similarly, NAC TFs play multifaceted roles, including cell wall modification, hypersensitive response (HR) regulation, and modulation of reactive oxygen species (ROS) production, all of which are critical components of plant immunity [49,50]. NAC transcription factors, such as ANAC019 and ANAC055, have been found to mediate JA-responsive defense, linking hormonal signaling to transcriptional regulation during pathogen attack [51].
In addition to transcription factors, transporter proteins are integral to the plant’s defense strategy. Transporters are involved in the movement of defense-related compounds, such as phytoalexins, hormones, and other secondary metabolites, which are crucial for the plant’s chemical defense mechanisms [52,53]. For example, ATP-binding cassette (ABC) transporters have been shown to play a role in transporting antimicrobial compounds across cell membranes, thereby contributing to localized and systemic resistance against pathogens [54]. Moreover, studies have shown that metal ion transporters, such as Nramp and ZIP families, contribute to the homeostasis of essential micronutrients like iron and zinc, which can influence pathogen virulence and plant immune responses [55]. The ability of these transporter proteins to maintain ion homeostasis under pathogenic stress conditions ensures that critical cellular functions, such as ROS detoxification and signal transduction, remain intact, thereby supporting overall plant resilience to HLB.
Furthermore, the interplay between these transcription factors and transporter proteins adds an additional layer of complexity to the plant’s immune system. For example, the upregulation of transcription factors may directly enhance the expression of specific transporter proteins involved in detoxification processes, secondary metabolite secretion, or nutrient allocation. This integrated regulatory mechanism suggests that both transcription factors and transporters work in tandem to ensure a rapid and efficient defense response to HLB infection. Such coordination allows the plant to activate localized responses, such as cell wall reinforcement, while simultaneously managing systemic signals that prepare distal tissues for potential pathogen invasion.

4.3. Comparative Insights and Resistance Mechanisms

Compared to previous studies on HLB, our findings align with the general observation that plants adopt both structural and biochemical defense mechanisms. For example, overexpression of certain defense-related genes, including those involved in shikimate pathway and pathogen response, is crucial for tolerance to HLB [7]. Similarly, studies by Hu et al. identified significant roles for genes involved in secondary metabolite biosynthesis, including phenylpropanoids and flavonoids, which contribute to resistance against the pathogen [56]. The distinct pathways activated in Ponkan and Punctate Wampee provide new insights into species-specific responses. While Ponkan primarily focuses on enhancing its physical barriers, Punctate Wampee appears to optimize its metabolic resources to fuel defense-related processes. This suggests that a combination of both strategies could be crucial for developing effective HLB-resistant cultivars. Notably, the shikimate pathway contributes to citrus defense against HLB by producing aromatic amino acids, which feed into phenylpropanoid metabolism to generate lignin and flavonoids. These compounds enhance physical barriers (cell wall reinforcement) and directly suppress Candidatus Liberibacter asiaticus proliferation. However, the pathway’s role is context-dependent: while its activation in susceptible cultivars like Ponkan Mandarin may reflect a compensatory defense response, the systemic nature of HLB likely undermines its efficacy. CLas may exploit shikimate-derived metabolites (e.g., chorismate) for survival, creating a paradox where host metabolic efforts inadvertently support pathogen persistence. Additionally, the delayed or spatially restricted activation of the pathway in phloem—CLas’s primary niche—could limit its protective impact. This highlights the need to evaluate shikimate flux dynamics in infected tissues and explore strategies to decouple defense metabolite production from pathogen resource exploitation.

4.4. Implications for Breeding Programs and Future Directions

The identification of key pathways and regulatory genes, such as those involved energy metabolism and hub genes associated with resistance to HLB, provides valuable targets for breeding programs aimed at enhancing HLB resistance. Marker-assisted selection or genetic engineering approaches could be employed to introduce or enhance these traits in susceptible citrus varieties, potentially improving their resilience to HLB infection. Future research should focus on functional validation of the identified candidate genes, particularly the transcription factors and transporter proteins, to establish their specific roles in HLB resistance. Additionally, exploring the interactions between different signaling pathways, such as hormone signaling and secondary metabolite production, could provide a more comprehensive understanding of the defense response.

5. Conclusions

This study elucidated species-specific defense mechanisms in Ponkan Mandarin and Punctate Wampee against HLB. Early responses to CLas infection, including callose deposition and reactive oxygen species (ROS) bursts, triggered cell wall reinforcement in Ponkan Mandarin through phenylpropanoid and flavonoid pathways. In contrast, Punctate Wampee prioritized metabolic reprogramming to redirect energy resources toward defense-related biosynthesis. Transcription factors (WRKY, MYB, NAC) and transporters played pivotal roles in coordinating these responses.
Our findings highlight the complexity of citrus immunity: while physical barriers like cell wall thickening may delay pathogen spread, their efficacy is constrained by the systemic nature of HLB. Notably, the current study focused on early-to-mid infection stages (1.5 months post-inoculation). Future studies should incorporate longer-term sampling points (e.g., 6–12 months) to evaluate the durability of these defense strategies and their interaction with chronic pathogen pressure.
These insights provide a foundation for breeding HLB-resistant cultivars through targeted manipulation of defense pathways, coupled with strategies to address both pathogen invasion and insect vector behavior.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/agronomy15051218/s1. Figure S1. Heatmap of differentially expressed genes in Ponkan Mandarin and Punctate Wampee following Huanglongbing infection. Figure S2. Cnet plot: GO enrichment of the upregulated DEGs in Ponkan Mandarin. Figure S3. Cnet plot: GO enrichment of the downregulated DEGs in Ponkan Mandarin. Figure S4. Cnet plot: GO enrichment of the upregulated DEGs in Punctate Wampee. Figure S5. Cnet plot: GO enrichment of the downregulated DEGs in Punctate Wampee. Figure S6. Sample clustering dendrogram (A) and soft thresholding power selection (B). Table S1. Differentially expressed genes in Ponkan Mandarin following Huanglongbing (HLB) infection. Table S2. Differentially expressed genes in Punctate Wampee following Huanglongbing (HLB) infection. Table S3. Genes downregulated in Ponkan Mandarin but upregulated in Punctate Wampee following Huanglongbing (HLB) infection. Table S4. Genes upregulated in both Ponkan Mandarin and Punctate Wampee. Table S5. Genes downregulated in both Ponkan Mandarin and Punctate Wampee.

Author Contributions

H.L.: Supervision, Conceptualization, Methodology, Funding acquisition, Writing—review and editing. F.L.: Formal analysis, Methodology, Writing—original draft. X.W., H.H., Q.H., N.W. and C.W.: Data curation, Formal analysis, Methodology. All authors have read and agreed to the published version of the manuscript.

Funding

National Natural Science Foundation of China (32,060,658); Guangxi Characteristic Crop Experimental Station, China (TS202122), Guangxi Academy of Agricultural Sciences Basic Scientific Research Business Special Project (Guinongke 2021YT051), China.

Data Availability Statement

The data sets supporting the results of this article are included within the article and its additional files. All the other data are available in the main text or the Supplementary Data.

Conflicts of Interest

The authors declare that they have no competing interests.

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Figure 1. Transcriptome analysis of Ponkan Mandarin infected with Huanglongbing. (A) PCA of Ponkan Mandarin control samples (CK) and after treatment (T). PT denotes samples inoculated with HLB, whereas PCK represents samples not inoculated with HLB. (B) Correlation analysis between treatment and control samples. (C) Volcano plot analysis of differentially expressed genes in the transcriptome. (D,E) represent KEGG enrichment pathways among upregulated and downregulated genes, respectively. (F,G) represent the GO-enriched pathways among upregulated and downregulated genes, respectively.
Figure 1. Transcriptome analysis of Ponkan Mandarin infected with Huanglongbing. (A) PCA of Ponkan Mandarin control samples (CK) and after treatment (T). PT denotes samples inoculated with HLB, whereas PCK represents samples not inoculated with HLB. (B) Correlation analysis between treatment and control samples. (C) Volcano plot analysis of differentially expressed genes in the transcriptome. (D,E) represent KEGG enrichment pathways among upregulated and downregulated genes, respectively. (F,G) represent the GO-enriched pathways among upregulated and downregulated genes, respectively.
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Figure 2. Transcriptome analysis of Punctate Wampee infected with Huanglongbing. (A) PCA of Punctate Wampee control samples (CK) and after treatment (T). H denotes samples inoculated with HLB, whereas CK represents samples not inoculated with HLB. (B) Correlation analysis between treatment and control samples. (C) Volcano plot analysis of differentially expressed genes in the transcriptome. (D,E) represent KEGG enrichment pathways among upregulated and downregulated genes, respectively. (F,G) represent the GO-enriched pathways among upregulated and downregulated genes, respectively.
Figure 2. Transcriptome analysis of Punctate Wampee infected with Huanglongbing. (A) PCA of Punctate Wampee control samples (CK) and after treatment (T). H denotes samples inoculated with HLB, whereas CK represents samples not inoculated with HLB. (B) Correlation analysis between treatment and control samples. (C) Volcano plot analysis of differentially expressed genes in the transcriptome. (D,E) represent KEGG enrichment pathways among upregulated and downregulated genes, respectively. (F,G) represent the GO-enriched pathways among upregulated and downregulated genes, respectively.
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Figure 3. Combined analysis of differentially expressed genes in Ponkan Mandarin and Punctate Wampee following Huanglongbing inoculation. (A) Venn diagram analysis of differential genes in Punctate Wampee and Ponkan Mandarin after HLB inoculation. PT denotes Ponkan Mandarin samples inoculated with HLB, whereas PCK represents samples not inoculated with HLB. H denotes Punctate Wampee samples inoculated with HLB, whereas CK represents samples not inoculated with HLB. (B) Heatmap analysis of differentially expressed genes upregulated in Punctate Wampee but downregulated in Ponkan Mandarin. (C,D) Transcription factors that are simultaneously upregulated or downregulated in Punctate Wampee and Ponkan Mandarin, respectively.
Figure 3. Combined analysis of differentially expressed genes in Ponkan Mandarin and Punctate Wampee following Huanglongbing inoculation. (A) Venn diagram analysis of differential genes in Punctate Wampee and Ponkan Mandarin after HLB inoculation. PT denotes Ponkan Mandarin samples inoculated with HLB, whereas PCK represents samples not inoculated with HLB. H denotes Punctate Wampee samples inoculated with HLB, whereas CK represents samples not inoculated with HLB. (B) Heatmap analysis of differentially expressed genes upregulated in Punctate Wampee but downregulated in Ponkan Mandarin. (C,D) Transcription factors that are simultaneously upregulated or downregulated in Punctate Wampee and Ponkan Mandarin, respectively.
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Figure 4. WGCNA of hub genes associated with resistance to Huanglongbing. (A) Dendrogram of gene clustering. (B) Heatmap of module–Trait Relationships. (C) The module membership and gene significance in MEbule, Module membership vs. gene significance, cor = 0.89, p < 1 × 10−200. (D) Expression levels of genes from MEbule module. (E) Identification of the hub genes of DEGs in the MEbule modules.
Figure 4. WGCNA of hub genes associated with resistance to Huanglongbing. (A) Dendrogram of gene clustering. (B) Heatmap of module–Trait Relationships. (C) The module membership and gene significance in MEbule, Module membership vs. gene significance, cor = 0.89, p < 1 × 10−200. (D) Expression levels of genes from MEbule module. (E) Identification of the hub genes of DEGs in the MEbule modules.
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Figure 5. Proposed model of differential resistance mechanisms to Huanglongbing in Punctate Wampee and Ponkan Mandarin. A blue star with orange circles represents the Candidatus Liberibacter spp. injection. TFs: transcription factors. MYBs, MADSs, NACs, and ERFs represent MYB, MADS, NAC, and ERF transcription factors, respectively.
Figure 5. Proposed model of differential resistance mechanisms to Huanglongbing in Punctate Wampee and Ponkan Mandarin. A blue star with orange circles represents the Candidatus Liberibacter spp. injection. TFs: transcription factors. MYBs, MADSs, NACs, and ERFs represent MYB, MADS, NAC, and ERF transcription factors, respectively.
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Table 1. Ten hub genes related to Huanglongbing resistance.
Table 1. Ten hub genes related to Huanglongbing resistance.
Gene IDPT vs. PCK (log2FC)HT vs. HCK (log2FC)Description
Cs7g308900.180.96Clathrin heavy chain 1
Cs1g229000.221.02Putative uncharacterized protein
Cs1g203800.551.11Similarity to auxin-independent growth promoter
Cs6g058900.381.55Heat shock protein
Cs7g051500.131.10Cellulose synthase A catalytic subunit
Cs7g07870−0.030.75Cellulose synthase-like protein
Cs3g273100.490.86Similar to patched sphingolipid transporter
Cs8g144900.001.19Serine/threonine-protein phosphatase
Cs1g123700.040.93Serine/threonine-protein phosphatase
Cs5g266200.311.06Casein kinase I isoform delta-like
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Liao, H.; Liu, F.; Wang, X.; Huang, H.; Huang, Q.; Wang, N.; Wei, C. Comparative Transcriptome Analysis of Susceptible and Resistant Rutaceae Plants to Huanglongbing. Agronomy 2025, 15, 1218. https://doi.org/10.3390/agronomy15051218

AMA Style

Liao H, Liu F, Wang X, Huang H, Huang Q, Wang N, Wei C. Comparative Transcriptome Analysis of Susceptible and Resistant Rutaceae Plants to Huanglongbing. Agronomy. 2025; 15(5):1218. https://doi.org/10.3390/agronomy15051218

Chicago/Turabian Style

Liao, Huihong, Fuping Liu, Xi Wang, Hongming Huang, Qichun Huang, Nina Wang, and Chizhang Wei. 2025. "Comparative Transcriptome Analysis of Susceptible and Resistant Rutaceae Plants to Huanglongbing" Agronomy 15, no. 5: 1218. https://doi.org/10.3390/agronomy15051218

APA Style

Liao, H., Liu, F., Wang, X., Huang, H., Huang, Q., Wang, N., & Wei, C. (2025). Comparative Transcriptome Analysis of Susceptible and Resistant Rutaceae Plants to Huanglongbing. Agronomy, 15(5), 1218. https://doi.org/10.3390/agronomy15051218

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