Skip to main content
Download PDF

Transcriptional insights of citrus defense response against Diaporthe citri

BMC Plant Biology volume 23, Article number: 614 (2023) Cite this article

A Correction to this article was published on 27 December 2023

This article has been updated

Abstract

Citrus melanose, caused by Diaporthe citri, is one of the most important and widespread fungal diseases of citrus. Previous studies demonstrated that the citrus host was able to trigger the defense response to restrict the spread of D. citri. However, the molecular mechanism underlying this defense response has yet to be elucidated. Here, we used RNA-Seq to explore the gene expression pattern at the early (3 days post infection, dpi) and late (14 dpi) infection stages of citrus leaves in response to D. citri infection, and outlined the differences in transcriptional regulation associated with defense responses. The functional enrichment analysis indicated that the plant cell wall biogenesis was significantly induced at the early infection stage, while the callose deposition response was more active at the late infection stage. CYP83B1 genes of the cytochrome P450 family were extensively induced in the callus deposition-mediated defense response. Remarkably, the gene encoding pectin methylesterase showed the highest upregulation and was only found to be differentially expressed at the late infection stage. Genes involved in the synthesis and regulation of phytoalexin coumarin were effectively activated. F6’H1 and S8H, encoding key enzymes in the biosynthesis of coumarins and their derivatives, were more strongly expressed at the late infection stage than at the early infection stage. Collectively, our study profiled the response pattern of citrus leaves against D. citri infection and provided the transcriptional evidence to support the defense mechanism.

Peer Review reports

Background

Citrus is one of the most popular fruits in the world, due to their unique and refreshing flavor, as well as their rich nutrients. Nowadays, citrus is widely grown in over 140 countries throughout the tropical and subtropical regions [1]. The global citrus production in 2019 is over 157 million tons accounting for the largest single category of fruit production in the world (FAO statistics, 2019).

Diaporthe citri is one of the most destructive fungal pathogens of citrus [2, 3]. It infects young leaves, shoots and fruits, and induces black-to-reddish brown, raised pustules (called melanose) on the leaves, twigs, and fruits of citrus [4]. Usually, melanose does not reduce yield but impacts the marketability of citrus fruits, leading to heavy economic losses [2, 5]. D. citri also causes stem-end rot, shoot-blight and dieback, trunk or branch gummosis and rot of all citrus species or varieties worldwide [2, 3, 6,7,8]. D. citri can live as endophyte, saprophyte or parasite on citrus [3, 8, 9], however, in most time of the life cycle it grows and produces conidia and ascospores on dead wood of citrus [2, 4]. Under humid conditions, the conidia extrude from pycnidia in slimy masses or tendrils, and are dispersed by water (rain or overhead irrigation) to susceptible tissues in the citrus canopy [2, 4]. The ascospores produced from spherical perithecia are grown in dead wood and are dispersed by wind over longer distance [2, 4].

When spores of D. citri drop on citrus leaves, they can germinate and penetrate through the cuticle and interact with the epidermal cells, which in turn activates the host defense response, by which the hyphae are restricted in plant cells [10]. The phytoalexin 6, 7-dimethoxy coumarin and cell-division inducer gamma-amino-n-butyric acid (GABA) secreted by citrus cells comprise the mechanical barrier to defend against D. citri invasion [11, 12]. The barrier is formed at the damage site, consisting of necrotic cells, callus and periderm [10]. Despite the microscopic evidence of host defense response, the molecular mechanisms on how host genes are organized to counteract fungal invasion are largely elusive.

In the present study, transcriptomes of the mandarin (Citrus reticulata cv. Hongjv) leaves inoculated with D. citri were profiled at 3 and 14 days post inoculation (dpi). This dataset provides a unique opportunity for investigating transcriptional regulation of citrus in response to D. citri invasion.

Results

RNA-Seq analysis of D. citri-inoculated versus mock-inoculated citrus leaves

In the glasshouse, we inoculated citrus (Citrus reticulata cv. Hongjv) leaves with conidia of D. citri (Dc) (Fig. 1A). At 3 dpi, obvious early disease symptoms were observed, with the leaves showing chlorosis spots. At 14 dpi, the leaves developed visible melanose symptoms, with black spot-like lesions (Fig. 1B), and the symptoms stabilized thereafter. Based on the disease symptoms, the infection process of D. citri was tentatively defined as early (3 dpi) and late (14 dpi) infection stages. To characterize gene expression dynamics during D. citri infection, we performed transcriptome sequencing of Dc-inoculated or mocked citrus leaves at both early and late stages, with each of five biological replicates.

Fig. 1
figure 1

Disease development in citrus leaves infected by Diaporthe citri. A The symptom of Citrus reticulata cv. Hongjv infected by D. citri in the glasshouse at 14 dpi. B Pictures of D. citri infection progress in citrus leaves up to 14 dpi (days post inoculation)

Full size image

The percentage of reads of all the samples that aligned to the citrus reference genome was 97.0–97.7% (Table S1). A total of 16,896 genes (representing 60.1% of the total number of annotated genes) were detected under the threshold of 1 CPM (count per million) in at least half of the samples. Concerning the pathogen transcriptome, the number of reads mapped to the D. citri genome [13] from the harvested samples was very low (< 0.01%), prohibiting further analysis of fungal transcriptomes. Principal component analysis separated samples into four groups, reflecting the difference of development stages and fungal invasion (Fig. 2A). Differentially expressed genes between Dc-inoculated and mocked samples at each time point were identified (adjusted P < 0.05 and fold change > 2) and listed in Table S2. In general, more upregulated genes than downregulated genes were observed at both time points. Specifically, 1,994 of the 3,458 DEGs were upregulated at 3 dpi, whereas 1,666 of the 3,031 DEGs were upregulated at 14 dpi (Fig. 2B,C). Among all DEGs, up to 1,768 were commonly found in both time points, including 948 co-upregulated genes and 773 co-downregulated genes (Fig. 2D).

Fig. 2
figure 2

Overview of differential gene expression of citrus leaves infected by D. citri. A Principal component analysis (PCA) of citrus leaves at different time points with or without Dc infection. B, C Volcano plots summarizing differentially expressed genes at 3 and 14 dpi, respectively. Each dot represents an upregulated or downregulated gene. D Venn diagrams representing common and distinct differentially expressed genes between 3 and 14 dpi

Full size image

Overview of citrus transcriptional regulation in response to D. citri infection

Functional enrichment analysis was performed for all DEGs using clusterProfiler [14] (adjusted P < 0.05), which identified 154 and 210 enriched GO (gene ontology) terms (Fig. 3A, B and Table S3), and 10 and 12 enriched KEGG (kyoto encyclopedia of genes and genomes) pathways, at 3 and 14 dpi, respectively (Fig. 3C, D and Table S4).

There were 97 GO terms enriched at both 3 and 14 dpi, which are mainly involved in response-related biological processes (33 GOs), such as “response to reactive oxygen species”, “immune response”, “response to fungus”, “response to chitin”, “response to wounding”, and “induced systemic resistance”. Several phytohormone response related processes were also found at both 3 and 14 dpi, including “response to ethylene”, “response to abscisic acid”, “response to salicylic acid”, and “response to jasmonic acid”. Moreover, seven KEGG pathways were enriched at both 3 and 14 dpi. Prominent among these was “plant–pathogen interactions” pathway, which comprised the largest number of DEGs over other pathways.

Moreover, 57 GO terms were enriched specifically at 3 dpi, among these the most significantly enriched GO terms were mainly associated with the biosynthesis of plant cell wall, i.e., “plant-type secondary cell wall biogenesis”. In contrast, 113 GO terms were enriched only at 14 dpi, and the significantly enriched GO terms were mainly involved in the plant hormone metabolism and regulation, such as “regulation of hormone levels”, “hormone metabolic process”, and “salicylic acid metabolic process”. In addition, we found that three and five KEGG pathways were enriched only at 3 and 14 dpi, respectively.

Fig. 3
figure 3

Overview of functional enrichment of citrus leaves in response to D. citri infection. Functional enrichment analysis was performed using ClusterProfiler. A, B Significantly induced GO (gene ontology) categories (top 10 GOs belong to “biological process”) at 3 and 14 dpi, respectively. C, D Significantly induced KEGG pathways at 3 and 14 dpi, respectively

Full size image

Plant perception and signal transduction upon D. citri infection

Recognition is one of the earliest events in plant-pathogen interaction [15]. Plants are able to recognize potential pathogens through host sensors (PRRs, pattern-recognition receptors), and to trigger a series of defense responses called PAMP-triggered immunity (PTI) [16].

MAPK (Mitogen-activated protein kinase) cascades play central roles in the PTI signaling pathway, transducing signals from PRRs to downstream components for regulation [17]. In this study, the MAPK pathway was significantly induced at both 3 and 14 dpi, which consisted of 51 and 37 DEGs, respectively (Fig. 3C). CERK1 (chitin elicitor receptor kinase 1) is a LysM receptor kinase that belongs to PRRs and is essential for chitin elicitor signaling upon fungal invasion [18]. We observed three significantly upregulated genes encoding CERK1, and two of them were upregulated at both 3 and 14 dpi (Cr_hj_2g001380, Cr_hj_2g001390), while the other one was only upregulated at 14 dpi (Cr_hj_2g001370) (Fig. 4A).

In addition to the front line of innate immune system (i.e., PTI), plants have evolved diverse R proteins, most of which belong to the NB-LRR class, to recognize effector proteins secreted by the pathogens, and subsequently trigger the effector-triggered immunity (ETI) [17]. We found that 25 NB-LRR genes were differentially expressed, most of which (N = 24) were upregulated (Fig. 4B). Among these NB-LRR genes, DSC1 (disease resistance-like protein 1), which is known to be able to trigger hypersensitive response in plant defense, showed the highest level of upregulation specifically at 14 dpi.

Fig. 4
figure 4

Differential expression of PRRs (pattern recognition receptors), NB-LRR (nucleotide-binding site–leucine-rich repeat protein) and phytohormone genes. Heatmap depicting differential expressed genes of PRRs (A), NB-LRR (B), and phytohormone (C) at 3 and 14 dpi. Differential expressed genes are annotated by eggNOG database. Expression values are presented as log2(fold change)

Full size image

Differential expression of phytohormone associated genes

Phytohormones are small molecules produced by plants to regulate diverse physiological processes and plant–pathogen interactions, including plant defense [19]. Genes involved in the biosynthesis and signaling of SA (salicylic acid), JA (jasmonic acid), ET (ethylene), and ABA (abscisic acid) were differentially expressed following D. citri inoculation. We identified 74 SA-, 72 JA-, 64 ET-, 115 ABA-related DEGs, and most of them were upregulated (Fig. 4C).

SA is best known as a defense-related hormone. Plant perception of pathogen attack can induce SA synthesis, leading to activation of plant immunity. NPR1-like gene has been identified as a key component of the SA signaling pathway and is required for plant disease resistance. We observed differential expression of three NPR1-like genes, among which Cr_hj_7g013060 and Cr_hj_7g013090 were only upregulated and downregulated at 3 dpi, respectively, while the other gene (Cr_hj_7g013220) was upregulated at both 3 and 14 dpi. NPR1-like proteins interact with TGACG-binding (TGA) transcription factors and connect with defense response. Here, two TGA2 transcription factors (Cr_hj_5g031660 and Cr_hj_1g005970) were found only upregulated at 3 dpi, which mediated pathogenesis-related (PR) gene expression and disease resistance. Five PR genes were highly expressed, mainly showing upregulation.

ET is a major component of the blend of defense signals that is produced in many plant-pathogen interactions, and functions as an important regulator of plant immunity. Unlike other plant hormones, ET-related DEGs mainly showed upregulation at 3 dpi only (Fig. 4C), suggesting that the ET pathway is possibly involved in the early response of citrus defense. Several genes involved ET-signaling pathway were upregulated (Table S2). The ethylene receptor ETR2, which localized to the ER membrane [20], was upregulated at 3 dpi only (2.1-fold) (Table S2). Concordantly, some regulatory elements downstream of ETR2 were also induced at 3 dpi (Table S2), including the transcription factor EIN3 and defense-related gene CHiB [21]. ET- and JA-signaling often regulate synergistically to activate defense-related genes upon pathogen infection [22] Ethylene response factors (ERFs) act downstream of the intersection between the ET and JA pathways, which are key elements regulating defense response genes [23]. Noteworthy, our data showed that ERFs were upregulated at 3 dpi but downregulated at 14 dpi.

Cell wall modification of citrus leaves following D. citri infection

The germinated D. citri conidia penetrate the epidermal cell of citrus leaves, which triggers the self-defense reaction of citrus host, leading to formation of a mechanical barricade tissue to inhibit further development of the fungus. The plant cell wall forms a dynamic physical barrier that protects the host against pathogen invasion. Alterations of the cell wall structure may facilitate the restriction of disease development [24, 25].

Accordingly, we observed that 12 enriched GO terms were associated with the modification of plant cell wall. Remarkably, six of these GO terms were enriched only at 3 dpi and all of them were associated with cell wall biogenesis. Prominent among these was “plant-type secondary cell wall biogenesis” category, including 19 enriched genes (Fig. 5A). These upregulated genes were mainly involved in plant secondary cell wall synthesis, such as FLA11 involved in the initiation of secondary cell wall development, CESA required for cellulose synthesis in secondary cell wall, and IRX10 that is essential for glucuronoxylan biosynthesis.

As a comparison, five of 12 enriched cell wall-related GO terms were specific to 14 dpi, which included the biological process of callose deposition (2 GOs), cell wall thickening (2 GOs) and cell wall modification (1 GOs). Among them, the “Defense response by callose deposition in cell wall” category was the most enriched one, containing 20 DEGs (Fig. 5B). Within the “Defense response by callose deposition in cell wall” category, 11 out of 20 DEGs belonged to the cytochrome P450 family, and 9 were annotated as CYP83B1.

We noted that five DEGs showed highly upregulated expression at 14 dpi (Fig. 2C), while no significant changes at 3 dpi. The pectin methylesterase (Cr_hj_5g001890) was the most highly expressed gene (log2FC > 25), whose function is associated with demethylesterification of pectin.

Fig. 5
figure 5

Genes involved in the plant cell wall modification and the coumarin synthesis were effectively activated. Heatmap depicting differentially expressed genes of plant-type secondary cell wall biogenesis (A) and defense response by callose deposition in cell wall (B). Coumarin biosynthesis pathway (C) and the corresponding differentially expressed genes at 3 and 14 dpi (D)

Full size image

Coumarin biosynthesis pathways are triggered in infected leaves

In addition to forming a mechanical barrier through the change of cell structure, previous studies have indicated that citrus can produce a phytoalexin (scoparone, 6, 7-dimethoxy coumarin) to inhibit D. citri infection [12]. Coumarins are ubiquitously found in higher plants where they synthesized in the phenylpropanoid pathway (Fig. 5C). Accordingly, KEGG enrichment analysis demonstrated that “phenylpropanoid biosynthesis” pathway was significantly induced in citrus upon D. citri infection both at 3 and 14 dpi. In this study, we identified seven candidate DEGs associated with coumarin synthesis and regulation (Fig. 5D). All of the genes were upregulated at 3 and/or 14 dpi except for one that was downregulated at 3 dpi. Among them, F6’H1 (Feruloyl-CoA 6’-Hydroxylase1) and S8H (scopoletin 8-hydroxylase), which belong to a large enzyme family of the 2-oxoglutarate and Fe2+-dependent dioxygenases, were found to be more strongly upregulated at 14 dpi than at 3 dpi. Cr_hj_2g032020, ortholog of F6’H1, was upregulated by 176.1-fold at 3 dpi and by 377.4-fold at 14 dpi. Cr_hj_2g031990, another F6’H1 gene, was upregulated at 14 dpi only (1050.0-fold). Similarly, S8H (Cr_hj_9g028260) was also significantly differentially expressed (243.1-fold upregulated) only at 14 dpi.

Reactive oxygen species

Cellular H2O2 is an important reactive oxygen species (ROS) that acts as a signaling molecule regulating various physiological and defense processes [26, 27]. The increasing ROS in plants leads to an oxidative burst that induces cell death and limits further pathogen infection [28]. In this study, the GO term “reactive oxygen species metabolic process” was significantly induced both at 3 and 14 dpi, with 33 and 25 upregulated genes, respectively (Table S3). RBOHD (Respiratory burst oxidase homolog protein D), a calcium-dependent NADPH oxidase that generated superoxide, involves in the ROS generation upon pathogen invasion [29]. We showed that RBOHD was upregulated by 24.8-fold at 3 dpi and by 70.8-fold at 14 dpi (Table S2). Glycolate oxidase (GLO) was able to generate peroxisomal H2O2 to modulate redox state and induce programmed cell death (PCD) [30]. Four GLOs gene were differentially expressed, with one upregulated at both 3 and 14 dpi (2.6- and 3.0-fold, respectively), and the other three upregulated only at 14 dpi (range from 2.8- to 8.3-fold) (Table S2).

Discussion

D. citri is one of the most destructive fungal pathogens of citrus, which has a worldwide distribution [2]. Previous studies reported that citrus leaves can initiate a series of defense responses to limit D. citri invasion and the development of the lesion [10, 12, 31]. However, this finding is based on microscopic examination and HPLC detection, while robust evidence at the molecular level is lacking. In this study, we performed an RNA-seq to profile gene expression in citrus leaves at the early (3 dpi) and late (14 dpi) infection stages upon D. citri challenge, and provided molecular insight into the defense response triggered by the fungus (Fig. 6).

Fig. 6
figure 6

Overview of defense response model against D. citri in citrus leaves. The early (3 days post inoculation) and late (14 days post inoculation) infection stages show different regulation patterns in response to D. citri infection. The number of graphics represents the quantity of differentially expressed genes for each category

Full size image

Plants have been intricately linked with pathogens, and also have evolved several strategies to recognize them [32]. A large number of perception-related genes were induced in citrus leaves following D. citri infection, including MAPKs, PRRs, and NB-LRRs. Remarkably, the MAPK cascade pathway is intensively triggered, both at the early and late infection stages. MAPK cascades constitute a network of signaling cascades responsible for multiple defense responses, including defense gene activation, cell wall modification, phytohormone biosynthesis, ROS production and Hypersensitive response (HR) [17]. This suggests that the MAPK cascades is likely to play a central role in the defense response of citrus leaves against D. citri infection. More DEGs were found at the early infection stage than at the late infection stage, while more GO terms and KEGG categories were enriched at the late infection stage. This implies that citrus leaves induce more genes of different functional categories participating in the defense response at the late infection stage.

Activation of MAPKs is one of the earliest signaling events for plants to percept PAMPs and pathogen effectors [17]. PTI and ETI were effectively triggered when citrus leaves were attacked by D. citri. ROS and HR are downstream components in PTI or ETI, and are associated with pathogen inhibition [33]. ROS have been proposed to act as antimicrobial molecules involved in the reinforcement of plant cell wall and callose deposition to limit the spread of pathogens [34]. We found that ROS biogenesis in citrus leaves was significantly induced at both early and late infection stages. Recognition of effectors by R proteins leads to the activation of ETI, which is often associated with HR at the place of infection. Early responses of the plant defense include pathogen-induced HR, which reduces pathogen penetration and transmission through local plant cell death at the site of infection [32]. In this study, the late infection stage exhibited more stronger expression of R proteins than does the early infection stage. The HR process of citrus leaves against D. citri also mainly involved at the late infection stage, possibly mediated by the two highly upregulated genes.

The previous evidence of citrus leaves defending against D. citri through the self-defense response was from microscopic and scanning electron microscopic (SEM) examinations. This evidence indicated that citrus leaves can limit the expansion of D. citri mainly through mechanical barriers formed by cell division and coumarin secretion [10, 12, 31]. In the RNA-Seq dataset, we focused on the modification of plant cell wall and the biosynthesis of coumarins. Dissecting the expression of these genes could provide more insight into the self-defense response of citrus leaves against D. citri.

As the first physical and defensive barrier against pathogens, the plant cell wall usually undergoes dynamic remodeling as an immune response to prevent infection by pathogens [35]. We found that gene expression associated with the plant cell wall exhibited a pattern of temporal regulation. Specifically, plant cell wall biogenesis was mainly induced at the early infection stage, while plant callose deposition response was more active at the late infection stage. This suggests that dynamic cell wall response processes in citrus employ different defense strategies to adapt to D. citri challenge. In this study, a considerable number of CYP83B1 genes were involved in defense response by callose deposition in cell wall. In Arabidopsis, mutant cyp83B1 was impaired in the induction of the callose response [36]. This suggests that CYP83B1 may play an important role in the defense response of cell wall callose deposition and may contribute to the restricted growth of D. citri. Remarkably, we observed that a highly upregulated gene at the late infection stage, annotated as PME17, which involves in regulation of pectin demethylesterification. We speculated that citrus leaves were likely to block the further expansion of D. citri by regulating the demethylesterification of pectin at the late infection stage. This was supported by a previous study, which demonstrated that PME17 triggers pectin methylesterases activity and involves in defense against Botrytis cinerea in Arabidopsis [37]. Specifically, a greater development of B. cinerea mycelium was observed around the inoculation site of pme17 mutant compared to the wild type plant [37].

Coumarins are phytoalexins derived from the phenylpropanoid pathway and are widely found in higher plants [38]. In general, coumarins can be classified into two types, simple (e.g., scopolin, scopoletin and fraxetin) and complex coumarins [39]. Numerous studies have shown that coumarins exhibited antimicrobial activity against plant pathogens, including bacteria, fungi, viruses and oomycetes [39]. Indeed, the extent and timing of coumarin accumulation has often been associated with the level of disease resistance [39]. For instance, the Hevea rubber tree variety resistant to the fungus Microcyclus ulei and the oomycete Phytophthora palmivora accumulated scopoletin faster and more persistently upon pathogen infection than the susceptible variety [40]. Scopoletin in tobacco is accumulated during a hypersensitive response [41]and is considered to be involved in virus resistance [42]. In citrus, coumarins, such as xanthyletin and scoparone, can act as phytoalexins in resistance to pathogens [43, 44]. Scoparone is the most intensively studied phytoalexin in citrus. Many studies have reported the antagonistic effect of scoparone against pathogens in citrus, such as Penicillium digitatum, P. italicum, Phytophthora citrophthora, as well as D. citri [44]. Citrus leaves were able to produce scoparone upon D. citri attacking, and restrict the pathogen development [12]. Our RNA-Seq dataset supports the coumarin biosynthesis in citrus leaves against D. citri and indicates that the coumarin response is stronger at the late infection stage than at the early infection stage. In addition, the S8H gene is also induced (at the late infection stage only) among the coumarin biosynthesis pathway in response to D. citri infection. However, S8H gene is not directly involved in scoparone synthesis, which implies the production of other coumarins, possibly fraxetin, a coumarin derived from scopoletin [39].

Conclusions

The differentially expressed genes were analyzed by RNA-Seq analysis between D. citri- versus mocked samples at 3- and 14-days post inoculation, representing the early and late infection stages, respectively. 1994 out of the 3458 differentially expressed genes were upregulated at the early infection stage, whereas 1666 out of the 3031 differentially expressed genes were upregulated at the late infection stage. Collectively, our study profiled the defense response of citrus leaves against D. citri infection, involving the coumarin synthesis and plant cell wall modification.

Methods

Plant material, D. citri inoculation and harvesting

Eight-year-old “Hongjv” cultivar citrus plants were grown in a greenhouse with a 28/20°C day/night temperature regime and natural sunlight at Zhejiang University, located in Hangzhou, Zhejiang Province, China. D. citri was cultured on potato dextrose agar at 26 °C. To inoculate citrus leaves, conidial suspension of D. citri was prepared from a 45-day-old culture. The concentration of the conidial suspension was adjusted to 1 × 106 mL−1 using a hemacytometer. The young leaves were sprayed with the conidial suspensions. After inoculation, the entire plant was placed in a clear plastic bag sprayed with water for three days to ensure high humidity. The same procedure was followed for the mock inoculation, where distilled water was used instead. Leaf samples were collected at 3- and 14-days post inoculation (dpi) from the pathogen-inoculation and the mock-inoculation treatments. The samples were frozen in liquid nitrogen immediately after collection and kept at -80 °C until use. Five biological replicates were prepared for each time point.

RNA extraction, library construction and sequencing

RNA was extracted from 100 mg leaf material using the RNeasy Plant Mini kit according to the manufacturer’s instructions (Qiagen, Germany). The quantity and quality of extracted RNA were assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, USA). RNA-Seq libraries were prepared from 3 µg of each purified RNA with NEBNext UltraTM RNA Library Prep Kit of Illumina (NEB, USA) according to the manufacturer’s instructions. Briefly, the fragmentation of mRNA was enriched from total RNA by Oligo (dT) beads, and double-stranded cDNAs were synthesized. Libraries were purified using the AMPure XP system (Beckman Coulter, USA), and quantified using the Agilent 2100 Bioanalyzer. The pooled libraries were subjected to cluster generation and sequencing was carried out on the Illumina NovaSeq 6000 platform (250-bp, paired-end reads).

Data processing and analysis

The raw reads were filtered using Trimmomatic [45] to remove adapters and low quality bases. Hisat2 was used for the clean reads to align with the C. reticulata reference genome assembly [46]. The read numbers mapped to each gene were counted using featureCounts [47]. Gene expression was calculated using TPM (transcripts per million) normalization. DESeq2 [48] was used to identify differentially expressed genes (DEGs) (adjusted P value < 0.05, fold change > 2). DEGs were mapped to the KEGG (Kyoto Encyclopedia of Genes and Genomes) [49] pathways and GO (gene ontology) [50] functional categories using eggNOG-mapper [51]. ClusterProfiler [14] was used to perform GO and KEGG functional enrichment analysis of the DEGs (Benjamini-Hochberg adjusted P value < 0.05).

Availability of data and materials

Raw RNA-Seq reads have been deposited in the NBCI SRA under the BioProject accession numbers PRJNA734968. The used genome and the gene model information can be found from the following website: http://115.159.76.36/index.html?data=data/hj.

Change history

References

  1. Chen Y, Barzee TJ, Zhang R, Pan Z. Chapter 9 – Citrus. In: Pan Z, Zhang R, Zicari S, editors. Integrated Processing Technologies for Food and Agricultural By-Products. Academic Press; 2019. p. 217–42.

  2. Jiang L, Xu F, Huang Z, Huang F, Chen G, Li H. Occurrence and control of citrus melanose caused by Diaporthe Citri. Acta Agriculturae Zhejiangensis. 2012;24(4):647–53.

    Google Scholar 

  3. Guarnaccia V, Crous PW. Emerging citrus diseases in Europe caused by species of Diaporthe. IMA Fungus. 2017;8(2):317–34.

    Article  PubMed  PubMed Central  Google Scholar 

  4. Nelson, S. Citrus Melanose. Plant Disease. 2008;59:1–5.

  5. ur Rehman F, Kalsoom M, Sultan A, Adnan M, Junaid S, Akram H, Tariq MA, Shafique T, Zafar MI. Citrus Melanose and Quality Degradation of Fruit by this Disease: a review. Open Access J Biog Sci Res. 2020;3(5):1–4.

  6. Chen G, Jiang L, Xu F, Li H. In vitro and in vivo screening of fungicides for controlling citrus melanose caused by Diaporthe citri. J Zhejiang Univ Agric Life Sci. 2010;36(4):440–4.

  7. Fawcett HS. The Cause of Stem-end Rot of Citrus Fruits: (Phomopsis citri N. sp.). Phytopathology. 1912;2(3):109–13.

  8. Huang F, Hou X, Dewdney MM, Fu Y, Chen G, Hyde KD, Li H. Diaporthe species occurring on citrus in China. Fungal Divers. 2013;61(1):237–50.

    Article  Google Scholar 

  9. Mondal S, Vicent A, Reis R, Timmer L. Saprophytic colonization of citrus twigs by Diaporthe citri and factors affecting pycnidial production and conidial survival. Plant Dis. 2007;91(4):387–92.

  10. Arimoto Y, Homma Y, Misato T. Studies on citrus melanose and citrus stem-end rot by Diaporthe citri (Faw.) Wolf. Part 3. Mode of reaction in citrus fruit and leaf against Infection of D. citri. Jpn J Phytopathol. 1982;48(5):559–69.

    Article  Google Scholar 

  11. Arimoto Y, Ohsawa T, Homma Y. Studies on citrus melanose and citrus stem-end rot by Diaporthe citri (Faw.) Wolf. Part 12. Identification of a wound hormone-like substance in Melanose spot. Jpn J Phytopathol. 1991;57(4):568–72.

  12. Arimoto Y, Homma Y, Ohsawa T. Studies on citrus melanose and citrus stem-end rot by Diaporthe citri (Faw.) Wolf. Part 5. Identification of a phytoalexin in melanose spot. Jpn J Phytopathol. 1986;52(4):620–5.

  13. Gai Y, Xiong T, Xiao X, Li P, Zeng Y, Li L, Riely BK, Li H. The genome sequence of the citrus melanose pathogen Diaporthe citri and two Citrus-related Diaporthe species. Phytopathology. 2021;111:779–83.

  14. Yu G, Wang L-G, Han Y, He Q-Y. clusterProfiler: an R package for comparing biological themes among gene clusters. OMICS. 2012;16(5):284–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Maffei ME, Mithöfer A, Boland W. Before gene expression: early events in plant–insect interaction. Trends Plant Sci. 2007;12(7):310–6.

    Article  CAS  PubMed  Google Scholar 

  16. Yuan M, Jiang Z, Bi G, Nomura K, Liu M, Wang Y, Cai B, Zhou J-M, He SY, Xin X-F. Pattern-recognition receptors are required for NLR-mediated plant immunity. Nature. 2021;592(7852):105–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Meng X, Zhang S. MAPK cascades in plant Disease resistance signaling. Annu Rev Phytopathol. 2013;51(1):245–66.

  18. Miya A, Albert P, Shinya T, Desaki Y, Ichimura K, Shirasu K, Narusaka Y, Kawakami N, Kaku H, Shibuya N. CERK1, a LysM receptor kinase, is essential for chitin elicitor signaling in Arabidopsis. Proc Natl Acad Sci USA. 2007;104(49):19613–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Berens ML, Berry HM, Mine A, Argueso CT, Tsuda K. Evolution of hormone signaling networks in plant defense. Annu Rev Phytopathol. 2017;55:401–25.

    Article  CAS  PubMed  Google Scholar 

  20. Chen Y-F, Shakeel SN, Bowers J, Zhao X-C, Etheridge N, Schaller GE. Ligand-induced degradation of the ethylene receptor ETR2 through a proteasome-dependent pathway in Arabidopsis. J Biol Chem. 2007;282(34):24752–8.

    Article  CAS  PubMed  Google Scholar 

  21. Meng X, Xu J, He Y, Yang K-Y, Mordorski B, Liu Y, Zhang S. Phosphorylation of an ERF transcription factor by Arabidopsis MPK3/MPK6 regulates plant defense gene induction and fungal resistance. Plant Cell. 2013;25(3):1126–42.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Verma V, Ravindran P, Kumar PP. Plant hormone-mediated regulation of stress responses. BMC Plant Biol. 2016;16(1):1–10.

    Article  Google Scholar 

  23. Lorenzo O, Piqueras R, Sánchez-Serrano JJ, Solano R. Ethylene response factor1 integrates signals from ethylene and jasmonate pathways in plant defense. Plant Cell. 2003;15(1):165–78.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Hückelhoven R. Cell wall–associated mechanisms of disease resistance and susceptibility. Annu Rev Phytopathol. 2007;45:101–27.

    Article  PubMed  Google Scholar 

  25. Malinovsky FG, Fangel JU, Willats WG. The role of the cell wall in plant immunity. Front Plant Sci. 2014;5:178.

    Article  PubMed  PubMed Central  Google Scholar 

  26. Quan LJ, Zhang B, Shi WW, Li HY. Hydrogen peroxide in plants: a versatile molecule of the reactive oxygen species network. J Integr Plant Biol. 2008;50(1):2–18.

    Article  CAS  PubMed  Google Scholar 

  27. Torres MA, Jones JD, Dangl JL. Reactive oxygen species signaling in response to pathogens. Plant Physiol. 2006;141(2):373–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Apel K, Hirt H. Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu Rev Plant Biol. 2004;55:373–99.

    Article  CAS  PubMed  Google Scholar 

  29. Torres MA, Dangl JL, Jones JD. Arabidopsis gp91phox homologues AtrbohD and AtrbohF are required for accumulation of reactive oxygen intermediates in the plant defense response. Proc Natl Acad Sci USA. 2002;99(1):517–22.

    Article  CAS  PubMed  Google Scholar 

  30. Foyer CH, Bloom AJ, Queval G, Noctor G. Photorespiratory metabolism: genes, mutants, energetics, and redox signaling. Annu Rev Plant Biol. 2009;60:455–84.

    Article  CAS  PubMed  Google Scholar 

  31. Arimoto Y, Homma Y, Misato T. Studies on citrus melanose and citrus stem-end rot by Diaporthe citri (Faw.) Wolf Part 4. Antifungal substance in Melanose Spot. Jpn J Phytopathol. 1986;52(1):39–46.

  32. Jones JD, Dangl JL. The plant immune system. Nature. 2006;444(7117):323–9.

    Article  CAS  PubMed  Google Scholar 

  33. Komis G, Šamajová O, Ovečka M, Šamaj J. Cell and developmental biology of plant mitogen-activated protein kinases. Annu Rev Plant Biol. 2018;69:237–65.

    Article  CAS  PubMed  Google Scholar 

  34. Camejo D, Guzmán-Cedeño Á, Moreno A. Reactive oxygen species, essential molecules, during plant–pathogen interactions. Plant Physiol Biochem. 2016;103:10–23.

    Article  CAS  PubMed  Google Scholar 

  35. Wan J, He M, Hou Q, Zou L, Yang Y, Wei Y, Chen X. Cell wall associated immunity in plants. Stress Biol. 2021;1(1):1–15.

    Article  Google Scholar 

  36. Clay NK, Adio AM, Denoux C, Jander G, Ausubel FM. Glucosinolate metabolites required for an Arabidopsis innate immune response. Science. 2009;323(5910):95–101.

    Article  CAS  PubMed  Google Scholar 

  37. Del Corpo D, Fullone MR, Miele R, Lafond M, Pontiggia D, Grisel S, Kieffer-Jaquinod S, Giardina T, Bellincampi D, Lionetti V. AtPME17 is a functional Arabidopsis thaliana pectin methylesterase regulated by its PRO region that triggers PME activity in the resistance to Botrytis Cinerea. Mol Plant Pathol. 2020;21(12):1620–33.

  38. Bourgaud F, Hehn A, Larbat R, Doerper S, Gontier E, Kellner S, Matern U. Biosynthesis of coumarins in plants: a major pathway still to be unravelled for cytochrome P450 enzymes. Phytochem Rev. 2006;5(2):293–308.

    Article  CAS  Google Scholar 

  39. Stringlis IA, De Jonge R, Pieterse CM. The age of coumarins in plant–microbe interactions. Plant Cell Physiol. 2019;60(7):1405–19.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Giesemann A, Biehl B, Lieberei R. Identification of scopoletin as a phytoalexin of the rubber tree Hevea brasiliensis. J Phytopathol. 1986;117(4):373–6.

    Article  CAS  Google Scholar 

  41. Gachon C, Baltz R, Saindrenan P. Over-expression of a scopoletin glucosyltransferase in Nicotiana tabacum leads to precocious lesion formation during the hypersensitive response to Tobacco mosaic virus but does not affect virus resistance. Plant Mol Biol. 2004;54:137–46.

    Article  CAS  PubMed  Google Scholar 

  42. Chong J, Baltz R, Schmitt C, Beffa R, Fritig B, Saindrenan P. Downregulation of a pathogen-responsive Tobacco UDP-Glc: phenylpropanoid glucosyltransferase reduces scopoletin glucoside accumulation, enhances oxidative stress, and weakens virus resistance. Plant Cell. 2002;14(5):1093–107.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Yu J, Wang L, Walzem RL, Miller EG, Pike LM, Patil BS. Antioxidant activity of citrus limonoids, flavonoids, and coumarins. J Agric Food Chem. 2005;53(6):2009–14.

    Article  CAS  PubMed  Google Scholar 

  44. Ortuño A, Botia J, Fuster M, Porras I, García-Lidón A, Del Río J. Effect of scoparone (6, 7-dimethoxycoumarin) biosynthesis on the resistance of tangelo Nova, Citrus paradisi, and Citrus aurantium fruits against Phytophthora Parasitica. J Agric Food Chem. 1997;45(7):2740–3.

    Article  Google Scholar 

  45. Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30(15):2114–20.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Wang L, He F, Huang Y, He J, Yang S, Zeng J, Deng C, Jiang X, Fang Y, Wen S. Genome of wild mandarin and domestication history of mandarin. Mol Plant. 2018;11(8):1024–37.

    Article  CAS  PubMed  Google Scholar 

  47. Liao Y, Smyth GK, Shi W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics. 2014;30(7):923–30.

    Article  CAS  PubMed  Google Scholar 

  48. Love MI, Huber W, Anders S. Moderated estimation of Fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15(12):1–21.

    Article  Google Scholar 

  49. Kanehisa M, Furumichi M, Tanabe M, Sato Y, Morishima K. KEGG: new perspectives on genomes, pathways, Diseases and Drugs. Nucleic Acids Res. 2017;45:D353–61.

    Article  CAS  PubMed  Google Scholar 

  50. Consortium GO. The Gene Ontology (GO) database and informatics resource. Nucleic Acids Res. 2004;32:D258-261.

    Article  Google Scholar 

  51. Huerta-Cepas J, Forslund K, Coelho LP, Szklarczyk D, Jensen LJ, Von Mering C, Bork P. Fast genome-wide functional annotation through orthology assignment by eggNOG-mapper. Mol Biol Evol. 2017;34(8):2115–22.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We would like to thank Xiao-xiao Feng from the agricultural experiment station of Zhejiang University for her assistance during the experiment.

Funding

This research was funded by the National Natural Science Foundation of China (32102148), the Key Research and Development Program of Zhejiang Province (2019C02022) and the Earmarked Fund for China Agriculture Research System (CARS-26).

Author information

Authors and Affiliations

  1. The Key Laboratory of Molecular Biology of Crop Pathogens and Insects of Ministry of Agriculture, The Key Laboratory of Biology of Crop Pathogens and Insects of Zhejiang Province, Institute of Biotechnology, Zhejiang University, Hangzhou, 310058, Zhejiang, China

    Pudong Li, Xiaoe Xiao, Jingrui Wang, Fan Niu, Jiangnan Huang, Bianyue Xie, Lu Ye, Chaofan Zhang, Hongye Li & Chen Jiao

  2. Quzhou Academy of agricultural and Forestry Sciences, Quzhou, 323000, Zhejiang, China

    Dengliang Wang & Qun Wu

  3. Agricultural Characteristic Industry Development Center of Quzhou City, Quzhou, Zhejiang, 323000, China

    Xueliang Zheng

  4. School of Grassland Science, Beijing Forestry University, Beijing, 100083, China

    Yunpeng Gai

Authors
  1. Pudong Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xiaoe Xiao
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jingrui Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Fan Niu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jiangnan Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Bianyue Xie
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Lu Ye
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Chaofan Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Dengliang Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Qun Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Xueliang Zheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Yunpeng Gai
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Hongye Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Chen Jiao
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

C.J. and H.L. conceived and supervised the project. P.L. and X.X. performed experiments. P.L., J.W., F.N., JH., B.X., L.Y., Y.G. and C.Z. analyzed the data. D.W., Q.W., X.Z., contributed to sample preparation. P.L. and C.J. wrote the manuscript. C.J. revised the paper.

Corresponding authors

Correspondence to Hongye Li or Chen Jiao.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

The original online version of this article was revised: an error has been spotted in the affiliations of the first two authors, Pudlong Li and Xiaoe Xiao. These authors are affiliated to Aff1 only.

Supplementary Information

Additional file 1: Table S1.

Summary statistics of the RNA-Seq data.

Additional file 2: Table S2.

Differentially expressed genes calculated by DESeq2.

Additional file 3: Table S3.

Significantly induced GO (gene ontology) categories.

Additional file 4: Table S4.

Significantly induced KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Li, P., Xiao, X., Wang, J. et al. Transcriptional insights of citrus defense response against Diaporthe citri. BMC Plant Biol 23, 614 (2023). https://0-doi-org.brum.beds.ac.uk/10.1186/s12870-023-04624-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://0-doi-org.brum.beds.ac.uk/10.1186/s12870-023-04624-x

Keywords

BMC Plant Biology

ISSN: 1471-2229

Contact us