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Genome-wide identification and comparative expression profiling of the WRKY transcription factor family in two Citrus species with different Candidatus Liberibacter asiaticus susceptibility



Salicylic Acid (SA) is a pivotal phytohormone in plant innate immunity enhancement of triggered by various pathogens, such as Candidatus Liberibacter asiaticus (CLas), the causal agent of Huanglongbing (HLB). WRKY is a plant specific transcription factor (TF) family, which plays crucial roles in plant response to biotic stresses. So far, the evolutionary history, functions, and expression patterns under SA treatment and CLas infection of WRKY family are poorly understood in Citrus, despite the release of the genome of several Citrus species. A comprehensive genomic and expressional analysis is worth to conduct for this family.


Here, a genome-wide identification of WRKY TFs was performed in two Citrus species: Citrus sinensis (HLB-sensitive) and Poncirus trifoliata (HLB-tolerant). In total, 52 CsWRKYs and 51 PtrWRKYs were identified, whose physical and chemical properties, chromosome locations, phylogenetic relationships and structural characteristics were comparatively analyzed. Especially, expression patterns of these WRKY genes before and after SA treatment and CLas infection were compared. Based on this result, seven pairs of orthologous WRKY genes showing opposite expression patterns in two Citrus species were screened out. Moreover, two pairs of orthologous WRKY genes with significant differences in the number or type of stress-responsive cis-elements in the promoter regions were discovered. Subcellular localization and transcriptional activation activity assays revealed that these two pairs of orthologous genes are classic WRKY TFs localize in the nucleus and could function as transcriptional activators.


In this study, we systematically analyzed the genomic characterization of WRKY family in two Citrus species, together with the analyses of expression patterns under SA signaling and CLas infection. Our study laid a foundation for further study on the function of WRKY TFs in HLB response and SA signaling of Citrus.

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Huanglongbing, generally referred to as citrus greening, is the most catastrophic and unprecedented disease that impedes citrus growth and threatens global citrus industry [1]. Three ‘Candidatus Liberibacter’ species, ‘Candidatus Liberibacter asiaticus’, ‘Candidatus Liberibacter americanus’, and ‘Candidatus Liberibacter africanus’, have been identified as the causal agents of HLB, among which CLas is the most widespread and harmful species [2]. Two phloem-feeding citrus psyllids, Diaphorina citri (Asian citrus psyllid, ACP) and Trioza erytreae, are natural vectors responsible for the transmission of HLB-associated bacteria [3]. Once infected with CLas, citrus trees will exhibit a series of typical symptoms including yellow or green mottled spots in leaves, stunted twigs, lopsided fruits and abortive seeds. Eventually, the trees will die as the disease progresses [4].

To date, there is no radical cure to eliminate HLB disease. Almost all citrus cultivars can be affected by the HLB-associated bacteria and there is no confirmed resistance [5]. However, the preliminary analysis of different citrus cultivars and varieties has indicated that there are differences in host response to CLas [6]. Evidences showed that many trifoliate orange varieties and its hybrids exhibited lighter symptoms of HLB than the rootstock shoots emerging from the trees’ bases did [7,8,9]. Several accessions of P. trifoliata have also been found to be resistant to ACP by influencing oviposition and lifespan of adults [10, 11]. Therefore, trifoliate orange may have a certain tolerance to CLas infection or psyllid colonization.

SA is a ubiquitous phenolic compound distributed extensively in plants and a vital phytohormone that regulates various stress responses and physiological processes, such as immune response, seed germination, growth and development, stomatal closure, senescence, etc. [12]. Among them, the function of SA in plant immune response to pathogens has been extensively studied. SA signaling constitutes a tightly linked regulatory cascade that plays a pivotal role in establishing Systemic Acquired Resistance (SAR), a long-term and broad-spectrum resistance which exists throughout the whole plant after local pathogen infection [13]. SA content in plants elevates rapidly after pathogen infection, which could be transported to various tissues and organs, and finally induce a series of SAR resistance reactions, such as the expression of disease resistance genes, the interaction between SA and disease resistance proteins, and between other related genes through signal transduction. Recent studies have demonstrated that SA resistance signaling pathway is a complex network, in which multiple TFs are involved to regulate gene expression after pathogen infestation [14, 15].

WRKY TFs comprised plenty members in plant genomes and form indispensable parts of regulator networks that modulate numerous plant processes [16,17,18,19]. The name of the WRKY TFs is derived from the most conspicuous characteristic of these proteins, namely the WRKY domain, a highly conserved region containing approximately 60 amino acids with a core sequence WRKYGQK at its N-terminal end, together with a novel zinc-finger-like domain at its C-terminal [20]. The WRKY proteins are generally categorized into three groups and eight subgroups on the basis of the number of WRKY domain and the feature of zinc finger motif. The proteins possessing two WRKY domains at N-terminal (Group Ia) or C-terminal (Group Ib) were classified into Group I, while those with only one WRKY domain belonged to Group II or Group III. WRKY TFs bind to the cis-element W-box [(T/C)TGAC(T/C)] in the promoter region of its target genes and regulate the expression of related genes [21]. Massive studies have shown that WRKY TFs participate in various biological and physiological processes, including disease resistance [22, 23]. A subgroup IIb WRKY family member CaWRKY6 from Capsicum annuum positively function in Ralstonia solanacearum resistance by activating another WRKY gene, CaWRKY40 [24]. In rice, OsWRKY62 functions as a negative regulator of innate immunity by affecting both basal and Xa21-mediated rice-specific defense responses to the bacterial pathogen Xanthomonas oryzae pv. oryzae [25]. In summary, WRKY family has numerous members with diverse quantitative distributions in different plants and variable mechanisms under pathogen attack.

Although several WRKYs functioning in plant disease defense have been characterized, the molecular mechanisms and the regulatory roles of most WRKYs, especially those from the non-model plants, still remain poorly understood. In the current study, a genome-wide analysis of WRKY TFs was performed in two Citrus species, C. sinensis and P. trifoliata, by implementing bioinformatics approach on the publicly available database of sequenced genome. Multiple sequence alignments, phylogenetic relationships, chromosome distributions, gene duplications, syntenic relationships of WRKY genes were performed. Furthermore, the expression profiles of WRKY orthologous pairs from two Citrus species under SA treatment and CLas-infection were analyzed. Promoter analysis was carried out to investigate stress-responsive cis-elements in seven pairs of orthologous WRKY genes which exhibited strong opposite expression patterns. Additionally, subcellular localization and transcriptional activation activity of two pairs of orthologous WRKY genes with different promoter elements were also investigated. This study provides prospective knowledge for the properties and biological significance of the WRKY family in Citrus and functional research involved in HLB response.


Plant materials and growth conditions

Two Citrus varieties, sweet orange (C. sinensis) and trifoliate orange (P. trifoliata), were grown in containers in Gannan Normal University, Ganzhou, China. Three-month-old plants of each variety were kept under 16: 8 h, light: dark conditions at a temperature of 25 °C. Solutions of 2 mM SA were used to irrigate the potting soil and spray the branches and leaves. Each pot applied the same dosage. Leaves were collected at designated time points, instantly frozen in liquid nitrogen and stored at -80 °C for analyses of gene expression. Furthermore, leaves of C. sinensis and P. trifoliata were also collected for the RNA extraction and the preparation of cDNA template, which was further used for gene cloning and vector construction. Tobacco (Nicotiana benthamiana) plants were planted on potted soil under the same growth conditions for subsequent transient transformation experiment. Three leaves, sampled randomly from three different pots (one leaf per pot), were used as repetitions for each condition.

Two-year-old plants of each citrus varieties were graft-inoculated with axillary bud of HLB-symptomatic sweet orange plants, which were grafted on the plant’s primary stem. At least three axillary buds were grafted for each plant. Axillary buds from healthy plants were utilized to graft two citrus varieties as control. All plants were maintained in a greenhouse with an average temperature of 25 °C. Leaves and petioles were randomly collected at three months after inoculation for DNA isolation and RNA extraction, with three biological replicates for each experiment.

Screening and identification of the WRKY genes in C. sinensis and P. trifoliata

The WRKY protein sequences of C. sinensis and P. trifoliata were obtained from Citrus Pan-genome to Breeding Database (, respectively. The Hidden Markov Model (HMM) of the WRKY domain (PF03106) was downloaded from the Pfam protein family database ( [26] and used to identify putative WRKY proteins using the HMMER search program (, Version 3.0) with an E-value of less than 1e− 5. All non-redundant protein sequences were selected as putative WRKY proteins and confirmed the presence of the complete WRKY domains using the SMART software program ( [27] and the CDD software program ( [28]. Genes without the conserved WRKY domains were excluded, and only the longest transcript could be reserved. The relative molecular weight (MW) and isoelectric point (pI) of the WRKY proteins from both varieties were calculated using the Compute pI/MW tool ExPASy ( [29].

Phylogenetic, gene structure and motif analysis

Multiple sequence alignment of WRKY proteins was performed through the ClustalW ( for each species individually. Phylogenetic analysis of WRKY peptides based on amino acid sequences was performed separately for three species (C. sinensis, P. trifoliata and A. thaliana) following Neighbor-joining method in MEGA (, Version 11.0), with the maximum likelihood method (bootstrap: 1,000 replicates) [30]. The protein sequences of AtWRKYs were obtained from The Arabidopsis Information Resource (TAIR) database ( The Gene Structure Display Server program (GSDS,, Version 2.0) was used to illustrate the exon-intron structures through matching the genomic sequences and coding sequences (CDS) of the obtained WRKY genes [31]. MEME program (, Version 5.4.1) was employed to predict the conserved motifs in different WRKY proteins [32]. The analysis was performed by keeping number of repetitions, any; maximum number of motifs, 10; and optimum width of the motif ≥ 20.

Chromosomal location and gene duplication analysis

Precise position information on chromosomes for the genes encoding these CsWRKY and PtrWRKY proteins were obtained from the Citrus Pan-genome to Breeding Database. Chromosome location map of the WRKY genes was generated by the software MapInspect ( The genes were plotted separately onto the nine chromosomes according to their ascending order of physical position (bp), from the short-arm telomere to the long-arm telomere. To detect segmental and tandem duplication events, every WRKY sequence was respectively aligned against the other WRKY protein sequences in C. sinensis or P. trifoliata respectively. BLASTp program was used to identify potential homologous gene pairs (E-value < 1e− 5, top three matches) and output format as tabular. Subsequently, the destination tabular file and the GFF file of C. sinensis or P. trifoliata genome were inputted into software MCScanX to analyze duplication types with the flowing parameters: match score (> 20); gap penalty (-1); match size (5); E-value: 1e− 5; max gaps (25) and visualized using software program CIRCOS ( [33].

DNA isolation and CLas quantification

Young leaves of graft-inoculated C. sinensis and P. trifoliata plants with similar age and position were collected. Petioles of three leaves from each plant were combined and grind into powder. DNA extraction was carried out using the CTAB (cetyltrimethylammonium bromide) method according to prior method [34]. DNA concentration was determined using a NanoDrop 2000 (NanoDrop Technologies, Wilmington, DE, USA) and adjusted to 20 ng/µL. Three biological replicates were randomly sampled for each variety sample.

The presence of the CLas pathogen in the isolated DNA samples were confirmed by PCR using the CLas 16 S F/CLas 16 S R primers, and the degree of CLas infection was determined by quantitative PCR (qPCR) according to the protocol of prior research [35]. The CLas 16 S gene was amplified using qCLas 16 S F/qCLas 16 S R primers and citrus 18 S gene was detected as the internal reference using qCt 18 S F /qCt 18 S R primers, respectively (Supplementary Table S1). Each sample was analyzed in three biological replicates and three technical replications. Referring to the Ct values of the CLas 16 S gene, the tested samples with Ct < 31.3 were identified as positive for CLas, and samples with Ct > 36 were CLas negative. The CLas bacterial populations (CLas cells µg− 1 of citrus DNA) were calculated using the formula described by prior study [36]. Samples with similar CLas bacterial populations were selected for further gene expression analysis.

RNA extraction and quantitative real-time (qRT)-PCR analysis

Total RNA was extracted from leaves in accordance with the manufacturer’s instructions of the RNeasy® Plant Mini kit (Qiagen, Germany). The quality and integrity of the total RNA were examined with agarose gel electrophoresis and the NanoDrop 2000. The cDNA was synthesized using PrimeScript™ RT reagent Kit with gDNA Eraser (Takara, Japan). The specific WRKY gene primers were designed using Primer Premier 5.0 software (Supplementary Table S1). Realtime qRT-PCR analysis was done using SYBR GREEN PCR Master Mix (TaKaRa, Japan) on a QuantStudio 5 Applied BioSystem (ThermoFisher Scientific, USA). To normalize the relative expression level of the target genes, Actin was used as the internal reference gene. The qRT-PCR reactions were repeated in three biological and three technical replications, and the 2−ΔΔCt method was applied to calculate the relative expression levels. The heatmaps were constructed using the TBtools software (Version 1.098696) [37], based on the transformed data of log2 values. If an expression level of WRKY gene was more than 2 folds or less than 0.5 fold, we considered the WRKY gene as a significantly expressed gene. The qRT-PCR assays were performed according to prior method [38]. Venn diagram were generated using TBtools software (Version 1.098696) [37], to depicts number of differentially expressed genes commonly found among the two treatments (SA or CLas infection) in C. sinensis or P. trifoliata, separately.

Promoter analysis of putative stress-responsive CsWRKYs and PtrWRKYs

The 2.0 kb upstream promoter sequences from the transcription start site of the selected WRKY genes were retrieved using the ‘Sequence Fetch’ tool of Citrus Pan-genome to Breeding Database in order to perform promoter analysis. Two plant cis-elements database, PlantCARE ( and New PLACE (, were used to analyze the stress-response elements in promoters [39, 40]. The identified cis-elements were then visualized by TBtools (Version 1.098696) [37].

Subcellular localization assay

The full length (FL) of coding sequences without stop codon of CsWRKY7, PtrWRKY39, CsWRKY33 and PtrWRKY32, were separately cloned and inserted into the pBI121-EGFP vector at Xba I and Xma I restriction sites, under the control of the Cauliflower mosaic virus 35 S (CaMV 35 S) promoter. The fusion plasmids and the empty vector were then transformed into Agrobacterium tumefaciens (GV3101), and the suspensions of Agrobacteria were co-transformed with a plasmid coding for a nuclear marker gene VirD2NLS fused to mCherry into tobacco (Nicotiana benthamiana) leaves by Agrobacterium tumefaciens infiltration. The green fluorescence (for GFP) and red fluorescence (for mCherry) were observed through a confocal laser scanning microscope (Leica TCS SP8, Germany) after infected for three days. Transformation and infiltration of tobacco were carried out according to prior study [41].

Transactivation assay

The FL or three truncated fragments (N, W, and C) of four WRKY genes (CsWRKY7, PtrWRKY39, CsWRKY33 and PtrWRKY32) were amplified and inserted into pGBKT7 vector at EcoR I and BamH I restriction sites to generate sixteen constructs. The recombinant plasmids and the negative control, pGBKT7 plasmids, were further separately transformed into Y2HGold yeast cells according to the manufacturer’s protocol (Matchmaker® Gold Yeast Two-Hybrid Library Screening System, Takara). The SD/-Trp medium were used to cultivate the yeast transformants. Transcriptional activation activity of the transformed yeast cells was monitored after incubation at 30℃ for three to five days on SD/-Trp or SD/-Trp/-His/-Ade medium supplemented with the chromogenic substrate X-α-gal (Sigma-Aldrich, USA).


Identification of WRKY TFs in C. sinensis and P. trifoliata

To identify WRKY family genes in C. sinensis and P. trifoliata, Hidden Markov Model (HMM) profile from the Pfam database and BLASTp search were performed against reference genomes using the consensus sequence of WRKY domain. In the genome assemblies of C. sinensis and P. trifoliata, 94 CsWRKYs and 87 PtrWRKYs were identified. After removing the redundant sequences and the sequences with only partial WRKY domains, 52 WRKY members were identified in C. sinensis and 51 were identified in P. trifoliata. Detailed information of CrWRKYs and PtrWRKYs were presented in Supplementary Table S2 and S3 respectively, including gene ID, chromosome No., strand information, start and end position on chromosome, exon and intron number, length of Open Reading Frame (ORF) sequence, amino acid (aa) number, relative molecular weight, isoelectric point and classification of individual gene members. The identified WRKYs were named according to their ascending orders on the nine chromosomes.

In C. sinensis, the lengths of the CsWRKY proteins ranged from 116 (CsWRKY2) to 724 aa (CsWRKY14), with an average of approximately 381 aa. The molecular weights of the CsWRKY proteins were between 13.35 kDa (CsWRKY2) and 78.03 kDa (CsWRKY14). The predicted isoelectric point values of CsWRKYs varied from 5.06 (CsWRKY41) to 9.83 (CsWRKY37). Out of 52 members, 22 were identified on the positive strand and 30 were on the negative strand (Supplementary Table S2).

The identified PtrWRKY encode peptides, which ranged from 162 (PtrWRKY8/9) to 1390 aa (PtrWRKY21) with an average length of 428 aa. The molecular weight and isoelectric point of PtrWRKY peptides were ranging from 18.22 (PtrWRKY34) to 159.05 kDa (PtrWRKY21) and 4.94 (PtrWRKY50) to 9.80 (PtrWRKY51) respectively. Out of 51 members, 26 were identified on the negative strand and 25 on the positive strand (Supplementary Table S3).

Multiple sequence alignment and phylogenetic analysis

In order to further classify the WRKY proteins, we analyzed multiple sequence alignment of domain sequences, and constructed a NJ phylogenetic tree of CsWRKY (52 members) and PtrWRKY proteins (51 members) using WRKY sequences of model plants Arabidopsis thaliana (72 members) [42] as reference to represent each group and subgroup filled with different colors (Fig. 1).

Fig. 1
figure 1

Phylogenetic analysis of WRKYs in Citrus sinensis (Cs), Poncirus trifoliata (Ptr) and Arabidopsis thaliana (At). The phylogenetic tree was created using MEGA by the Neighbor-Joining (NJ) method with 1,000 bootstrap replicates. The CsWRKYs (red), PtrWRKYs (yellow) and AtWRKYs (blue) were clustered into groups I, II, and III including eight subgroups (Ia; Ib, IIa, IIb, IIc, IId, IIIa, IIIb)

Generally, the WRKY proteins are classified in three groups and five subgroups, depending on the number of the WRKY domain and the feature of zinc finger motif they possess. The members having two WRKY domains at N-terminal (Group Ia) and C-terminal (Group Ib), along with a C2H2 type zinc-finger motif, were grouped into Group I, while the members possessing only one WRKY domain and a C2H2 type zinc-finger motif belonged to Group II, and Group III members have only one WRKY domain and a C2H type zinc-finger motifs. The WRKY domain contains one or two highly conserved short peptide WRKYGQK, which considered to be important for recognizing and binding to W-box elements. A multiple sequence alignment of the core WRKY domain of CsWRKYs and PtrWRKYs was shown in Supplementary Figure S1. WRKYGQK sequences represented the major variant in 52 WRKY proteins from C. sinensis and 51 WRKY proteins from P. trifoliata. The detailed study showed that the variation arose from amino acid substitutions of Q amino acid to K amino acid, and the mutated WRKYGKK sequence was observed only in five WRKY proteins (CsWRKY2, CsWRKY13, PtrWRKY8, PtrWRKY9 and PtrWRKY21). Interestingly, the two CsWRKY proteins and three PtrWRKY proteins carrying these mutations occur in the same Group Ib.

As per our research findings, the CsWRKY protein family have 9 members in Group Ia, 15 in Group Ib; 3 in Group IIa, 8 in Group IIb, 5 in Group IIc, 6 in Group IId; and 6 in Group IIIa. While in P. trifoliata, we found 9 genes in Group Ia, 13 in Group Ib, 3 in Group IIa, 8 in Group IIb, 5 in Group IIc, 7 in Group IId and 6 encoding PtrWRKY proteins in Group IIIa. Noteworthily, neither C. sinensis nor P. trifoliata has any Group IIIb members (Fig. 2A). The distribution of WRKY genes in C. sinensis was comparable to that of P. trifoliata (Fig. 2B). The number of WRKY members of Group Ia, Group IIa, Group IIb, Group IIc and Group IIIa in C. sinensis and P. trifoliata were exactly the same, and their distributions in these two Citrus species were nearly same, namely 17.3% (Ia in C. sinensis) and 17.4% (Ia in P. trifoliata), 5.8% and 5.9% (IIa), 15.4% and 15.7% (IIb), 9.6% and 9.8% (IIc) and 11.5% and 11.8% (IIIa), respectively. Although the numbers of WRKY members differ in most subgroups between Citrus and Arabidopsis, the distribution of WRKYs was similar among these crops.

Fig. 2
figure 2

Distribution of WRKY genes in different species and groups. (A) Number of WRKY members in each subgroup of Citrus sinensis, Poncirus trifoliata and Arabidopsis thaliana. (B) Percentage distribution of WRKY members in C. sinensis, P. trifoliata and A. thaliana

Gene structure analysis of WRKY genes

The similarity and diversity of gene structures play prominent role in the evolution of gene family, and the positions of introns/exons are commonly well-conserved in orthologous genes. To investigate the structural diversity of the CsWRKY and PtrWRKY genes, we analyzed the distribution of introns/exons by comparing genomic and CDS sequences. The Group I genes have 2–6 exons in C. sinensis and 2–12 exons in P. trifoliata. All members from both citrus varieties of the Group IIa genes possess 4 or 5 exons. Variable numbers of exons were also found in Group IIb genes, 4–6 exons in CsWRKY genes, and 4–8 exons in PtrWRKY genes. In Group IIc members, 3 exons were found mostly in case of CsWRKY and PtrWRKY genes with Cs5g30250.4 (CsWRKY20) possessing 2 exons and Pt5g004720.1 (PtrWRKY37) possessing 4 exons, exceptionally. Overall, there was considerable diversity in the number of exons (2–12) and the length of exons in both citrus varieties. However, at the subfamily level, members exhibited similar gene structures in terms of exon number and length. For instance, all members of Group IId and Group IIIa contain 3 exons in both C. sinensis and P. trifoliata (Fig. 3A-B).

Fig. 3
figure 3

Gene structure and conserved motif analysis of WRKY genes in Citrus sinensis and Poncirus trifoliata. (A) Phylogenetic relationship of WRKY proteins from C. sinensis and P. trifoliata. (B) Exon/intron structures of CsWRKYs and PtrWRKYs. The exons and introns were represented by yellow boxes and black lines, respectively. The dark blue boxes indicated the upstream and/or downstream untranslated region. (C) The distribution of 10 conserved motifs in WRKY proteins, identified by MEME program, was shown by different colored blocks. The numbers 1–10 in the center of the blocks represent different motifs. The sequences of these conserved motifs were listed in Supplementary Table S4

Conserved domain analysis

WRKY proteins usually possess additional conserved motifs that might be involved in activating the functions of WRKY proteins [43]. Therefore, other motifs may also serve unknown functional or structural roles along with the conserved residue of 60 amino acids of WRKY domain. The conserved motifs of CsWRKYs and PtrWRKYs were studied using the MEME program for structural diversity analysis and functional prediction. As a result, 10 conserved motifs were identified and the length of these motifs ranged from 21 to 50 amino acids (Supplementary Table S4). Amongst them, motif 1 and 3 were annotated as the WRKY domain. Out of the 8 remaining non-redundant motifs in CsWRKYs and PtrWRKYs, the function of the majority of motifs could not be predicted. Only two functional motifs of bZIP motif (motif 5) and plant-zinc cluster domain (motif 8) could be identified by MEME in both CsWRKY and PtrWRKY proteins. The 50 amino acid-long bZIP like motif denoted as motif 5, were distributed predominantly in Group IIa and Group IIb. The plant-zinc cluster domains of length 24 amino acids were explicitly presented in Group IIc. Except motif 1 and 2 being present in all the sequences, motif 5 and motif 6 are specifically and commonly shared by closely related Group IIa and Group IIb members. Similarly, motif 7 and motif 9 are unique to Group IIb members, as well as motif 3 and motif 10 only presented in Group Ia. The result suggesting the potential functions of these conserved motifs in their respective subgroups (Fig. 3C).

Chromosomal locations and gene duplication

In total, 52 and 51 members of WRKY genes were identified in the genome of C. sinensis and P. trifoliata, respectively. Out of 52, only 41 of CsWRKY genes could be mapped on the chromosome. The precise location of 11 genes could not be determined, which were orange1.1t00419.1 (CsWRKY42), orange1.1t00425.1 (CsWRKY43), orange1.1t00472.1 (CsWRKY44), orange1.1t01175.1 (CsWRKY45), orange1.1t01686.2 (CsWRKY46), orange1.1t01713.1 (CsWRKY47), orange1.1t01779.1 (CsWRKY48), orange1.1t02600.1 (CsWRKY49), orange1.1t02759.1 (CsWRKY50), orange1.1t04068.1 (CsWRKY51), and orange1.1t05133.1 (CsWRKY52). As shown in Fig. 4, distribution of CsWRKY genes was scattered throughout all nine chromosomes of C. sinensis. Most of the genes (10 genes, 19.2%) were located in chromosome 7, followed by chromosome 2 (7 genes, 13.5%), chromosome 6 (6 genes, 11.5%), chromosome 4 (5 genes, 9.6%), chromosome 5 (4 genes, 7.7%), chromosome 9 (4 genes, 7.7%) and chromosome 1 (3 genes, 5.8%). Chromosome 3 and 8 each has one gene.

In P. trifoliata, chromosome 3 had the largest number of PtrWRKY genes (11 genes, 21.6%), while chromosome 5, 7 and 9 had the smallest number (3 genes, 5.9%). Nine PtrWRKY genes were found on chromosomes 1 (17.6%). Eight PtrWRKY genes were distributed on both chromosomes 2 and 4 (15.7%) followed by five on chromosomes 6 (9.8%). Only one gene, PtUn004120.1 (PtrWRKY51), could not be located in any definite chromosome. Interestingly, no PtrWRKY was detected in chromosome 8 (Fig. 4).

Fig. 4
figure 4

Chromosomal mapping and collinearity of WRKY genes among Citrus sinensis and Poncirus trifoliata. The horizontal columns represent chromosomes with the chromosome numbers placed in the middle and the gene ID shown outside the plot. The gray lines indicated the collinear blocks within these two species genomes, and the syntenic WRKY gene pairs were highlighted with the blue lines. Tandemly duplicated genes are represented by green blocks and the genes located on the duplicated segmental regions have been highlighted by red lines. The exact position of these WRKY genes was documented in Supplementary Table S2 and S3

It is well established that gene duplication plays a crucial role in the expansion of gene families in plant evolution. To further examine the evolution of WRKY genes in both varieties of citrus, genome duplication events were investigated for segmental and tandem duplications. Totally, only one pair of tandem duplication, Cs7g29570.1 (CsWRKY35) and Cs7g29580.1 (CsWRKY36) could be detected on chromosome 7 in C. sinensis (represented by green block in Fig. 4). In addition, two CsWRKY genes located on the duplicated segmental regions of chromosomes made up to two segmental duplication events (Cs6g10120.1/CsWRKY24 and Cs7g29570.1/CsWRKY35) (represented by red line in Fig. 4). Similar result was found in the P. trifoliata genomes, the PtrWRKY genes, Pt4g019960.1 (PtrWRKY36) and Pt4g019950.1 (PtrWRKY35), were observed to be tandem duplicated on chromosome 4 (represented by green block in Fig. 4). Four PtrWRKY genes located on the duplicated segmental regions of chromosomes made up to two pairs of segmental duplications (Pt3g002870.1/PtrWRKY20 and Pt3g037470.1/PtrWRKY26; Pt4g019960.1/PtrWRKY36 and Pt6g011450.1/PtrWRKY42 represented by red line in Fig. 4). In both C. sinensis and P. trifoliata, the tandem and segmental gene duplication events are not that significant, suggesting that these phenomena may play much insignificant role in the evolution of CsWRKY and PtrWRKY genes. Moreover, orthologous relationships of WRKYs between C. sinensis and P. trifoliata were analyzed, 48 orthologous gene pairs were identified (represented by blue line in Fig. 4). A total of seven WRKY genes could not found orthologous genes between C. sinensis and P. trifoliata (four genes from C. sinensis and three genes from P. trifoliata). It is worth noting that except one gene (Pt2g024060.1/PtrWRKY13) in P. trifoliata, the other six WRKY genes (Cs1g03100.1/CsWRKY1, Cs1g03870.1/CsWRKY2, Cs6g06940.1/CsWRKY22, Pt1g023510.4/PtrWRKY9, Pt3g023910.2/PtrWRKY21) were all belonged to Group Ib.

Expression profiles of WRKY genes under SA treatment

To gain insights into the potential functions of the WRKY genes in plant disease resistance, we analyzed the expression patterns of all CsWRKYs and PtrWRKYs under SA treatment by qRT-PCR. Of the 52 WRKY genes in C. sinensis, 17 genes (CsWRKY1, 3, 5, 9, 10, 12, 14, 15, 25, 26, 27, 36, 37, 38, 40, 43, and 49) have no remarkable differences in transcript abundance, while the other 35 CsWRKY genes showed alteration of transcript levels in response to the SA treatment. The expression patterns of the genes varied among different members. Thirty-one CsWRKY genes (CsWRKY2, 4, 6, 7, 8, 11, 13, 16, 17, 20, 21, 23, 24, 28, 29, 30, 31, 32, 33, 34, 35, 39, 41, 42, 44, 46, 47, 48, 50, 51, and 52) showed increased expression levels during the SA treatment, while four CsWRKY genes (CsWRKY18, 19, 22, and 45) were down-regulated by SA treatment (Fig. 5A, Supplementary Table S5).

The WRKY genes in P. trifoliata exhibited a similar expression pattern to that in C. sinensis. Most of the PtrWRKY genes (PtrWRKY1, 2, 3, 8, 9, 11, 13, 14, 15, 17, 20, 21, 22, 23, 24, 26, 27, 28, 29, 32, 36, 37, 39, 41, 42, 43, 46, 47, and 49) were significantly induced by SA treatment. Only PtrWRKY18, 19, and 38 showed markedly decreased expression levels under SA treatment, and the remaining 10 PtrWRKY genes showed no conspicuous changes in their expression levels (Fig. 5B, Supplementary Table S5).

Fig. 5
figure 5

Expression profiles of WRKY genes of Citrus sinensis (A) and Poncirus trifoliata  (B) under SA treatment. Expression analysis was carried out in leaves of C. sinensis and P. trifoliata at different time points (0 h, 3 and 6 h after irrigation of 2 mM SA). The qPCR results of WRKY genes were normalized by log2 transform. The heatmap constructed by TBtools software. Color scale erected vertically at the right side of the diagram. Higher expression levels are shown in red, bigger shape and lower expression levels are shown in green, smaller shape

Expression profiles of WRKY genes under CLas-infection

To further determine the function of the WRKY genes in response to HLB disease, efforts were made to analyze expression patterns of all WRKY genes from C. sinensis and P. trifoliata in health and CLas-infected plants. After three months of CLas-inoculation via grafting, seven of the eight C. sinensis seedlings were identified as positive plants by PCR amplification, while only three of the P. trifoliata seedlings were detected with the presence of the CLas pathogen (Fig. 6A-B, Full-length gels are presented in Supplementary Figure S2). Further qPCR analysis showed that the CLas 16 S Ct values of positive C. sinensis seedlings were significantly lower than those of positive P. trifoliata seedlings. Meanwhile, the bacterial populations of CLas of the grafted C. sinensis seedlings were also considerably higher than those of P. trifoliata seedlings (Fig. 6C-E). These results verified previous researches that P. trifoliata has a relative tolerance to HLB compared with C. sinensis. Materials from CLas-inoculated plants of C. sinensis (CLas-2, 4 and 5) and P. trifoliata (CLas-2, 3 and 6) with similar bacterial populations of CLas were selected for gene expression analysis. A considerable amount of WRKY genes (CsWRKY2, 5, 6, 7, 8, 9, 12, 13, 14, 17, 20, 21, 22, 23, 25, 26, 27, 28, 29, 30, 33, 35, 36, 40, 44, 47, 50, 51, and 52) in C. sinensis displayed notably decreased expression levels in CLas-infected plants than that in healthy plants. Eight CsWRKY genes (CsWRKY4, 16, 18, 19, 24, 32, 41, and 45) were remarkably induced in CLas-infected plants, while 15 CsWRKY genes (CsWRKY1, 3, 10, 11, 15, 31, 34, 37, 38, 39, 42, 43, 46, 48, and 49) showed no alteration of transcript levels between healthy and CLas-infected plants (Fig. 6F, Supplementary Table S6).

In P. trifoliata, 20 PtrWRKY genes (PtrWRKY5, 8, 9, 16, 22, 23, 25, 26, 27, 28, 30, 31, 34, 35, 37, 41, 44, 45, 48, and 51) showed no response to CLas-infection, 15 genes (PtrWRKY3, 4, 11, 13, 15, 17, 19, 21, 24, 32, 38, 39, 42, 43, and 50) were up-regulated and 16 genes (PtrWRKY1, 2, 6, 7, 10, 12, 14, 18, 20, 29, 33, 36, 40, 46, 47, and 49) were down-regulated after CLas-infection (Fig. 6G, Supplementary Table S6).

Fig. 6
figure 6

The detection of the C Las 16S gene and the expression profiles of WRKY genes before and after C Las-inoculation. (A-B) The PCR determination results of the CLas pathogens at three months after grafting in Citrus sinensis seedlings (A) and Poncirus trifoliata seedlings (B). Full-length gels are presented in Supplementary Figure S2. (C-D) The mean threshold cycle values (Ct) at three months after grafting. Different letters on top of the bars indicate significant differences between the highest Ct value of CLas 16 S gene (#1 for the P. trifoliata) and the others based on a Tukey’s test (p < 0.05). (E) The bacterial populations (Log10CLas cells µg− 1 of citrus DNA) at three months after grafting. Different letters on the top of the column indicate significant differences between the highest bacterial population (#6 for the C. sinensis) and the others based on a Tukey’s test (p < 0.05). H, Healthy plant; CLas, CLas-inoculated plant. (F-G) Expression analysis was carried out in leaves of C. sinensis (F) and P. trifoliata  (G) three months after inoculation. The qPCR results of WRKY genes were normalized by log2 transform. The heatmap constructed by TBtools software. Color scale erected vertically at the right side of the diagram, Higher expression levels are shown in red, bigger shape and lower expression levels are shown in green, smaller shape

Differential WRKY genes expression analysis

The similar expression patterns of WRKY gene under SA treatment and CLas-infection were likely related to the response of HLB in SA-dependent pathway. Firstly, a venn diagram was used to reveal the common expression changes among the two treatments in C. sinensis or P. trifoliata, separately. Six categories of SA↑ (up-regulated by SA treatment), SA↓ (down-regulated by SA treatment), SA (no significant change by SA treatment), HLB↑ (up-regulated by CLas-infection), HLB↓ (down-regulated by CLas-infection), and HLB (no significant change by CLas-infection) were performed. As shown in Fig. 7A, five up-regulated CsWRKY genes overlapped in the SA↑ and HLB↑ common comparisons, and one down-regulated CsWRKY gene was detected between SA↓ and HLB↓ common comparisons. Similarly, eleven common PtrWRKY genes were found to be up-regulated in the SA↑ and HLB↑ comparisons and one PtrWRKY gene was down-regulated in both SA↓ and HLB↓ categories (Fig. 7B). Further alignment of these WRKY genes revealed that three pairs of WRKY genes (CsWRKY4/PtrWRKY15, CsWRKY24/PtrWRKY42, and CsWRKY32/PtrWRKY3) from SA↑ and HLB↑ comparisons were orthologous genes, suggesting that these genes may be involved in the SA-dependent resistance to HLB.

Fig. 7
figure 7

Venn diagram of WRKY genes of Citrus sinensis and Poncirus trifoliata under SA treatment and/or HLB infection. (A) Diagram of overlapping CsWRKYs under SA treatment and HLB infection. (B) Diagram of overlapping PtrWRKYs under SA treatment and HLB infection. (C) Diagram of overlapping CsWRKYs and PtrWRKYs under SA treatment. (D) Diagram of overlapping CsWRKYs and PtrWRKYs under HLB infection. Lines with different colors and traits represent different categories. Cs, C. sinensis; Ptr, P. trifoliata; SA↑, up-regulated by SA treatment; SA↓, down-regulated by SA treatment; SA, no significant change by SA treatment; HLB↑, up-regulated by HLB infection; HLB↓, down-regulated by HLB infection; HLB, no significant change by HLB infection

The opposite expression patterns of WRKY genes across varieties were probably related to differences in tolerance to HLB. Furthermore, we identified the WRKY genes with inverse expression patterns between two varieties under SA treatment or CLas-infection, respectively. No common genes represented opposite expression pattern was observed between C. sinensis and P. trifoliata under SA treatment (Fig. 7C). However, six pairs of orthologous WRKY genes (CsWRKY6/PtrWRKY17, CsWRKY7/PtrWRKY39, CsWRKY23/PtrWRKY43, CsWRKY33/PtrWRKY32, CsWRKY47/PtrWRKY24, and CsWRKY52/PtrWRKY11) overlapped in the Cs HLB↓ and Ptr HLB↑ comparisons, and one pair of orthologous WRKY gene (CsWRKY19/PtrWRKY18) was detected between Cs HLB↑ and Ptr HLB↓ comparisons (Fig. 7D). These results suggest that the opposite expression WRKY genes under CLas-infection may contribute to the variation of HLB resistance between different citrus varieties.

Prediction of putative stress-responsive WRKYs and analysis of their promoters

The interaction between TF and the stress-inducible cis-elements present in the promoter modulates the expression of gene regulatory networks response to the respective physical, environmental and biological stress [44]. As the orthologous WRKY genes exposed opposite expression patterns between C. sinensis and P. trifoliata under CLas-infection, we speculate that these alterations may be related to their upstream regulatory factors. To verify this assumption, we investigated the cis-elements in the promoter of seven pairs of orthologous WRKY genes with opposite expression patterns (six pairs from Cs HLB↓/Ptr HLB↑ comparisons, one pair from Cs HLB↑/Ptr HLB↓ comparisons). Subsequently, the − 2.0 kb promoter regions of these candidate WRKY genes were analyzed to identify stress-responsive cis-elements (Fig. 8, Supplementary Table S7). Among them, the promoter elements of three pairs of orthologous WRKY genes (CsWRKY23/PtrWRKY43, CsWRKY47/PtrWRKY24, and CsWRKY6/PtrWRKY17) were not significantly different in both type or quantity. There was little difference among two pairs of orthologous WRKY genes. For instance, CsWRKY19 has a MYB-TF-binding site in its promoter region, while PtrWRKY18 hasn’t. Similarly, an ABF-TF-binding site was captured in the promoter of CsWRKY52, but its’ orthologous gene, PtrWRKY11, contains no such cis-element in the promoter. Noteworthily, there exist five MYC-TF-binding sites in the promoter of CsWRKY7, while no existence of MYC-TF-binding site could be detected in the promoter of the orthologous gene PtrWRKY39. Conversely, PtrWRKY32 contains four ABF-TF-binding sites in the promoter region whereas no ABF-TF-binding site was detected in the promoter of CsWRKY33.

Fig. 8
figure 8

Cis-elements analysis in the promoters of candidate WRKY genes. Stress-responsive cis-elements were identified in the 2.0 kb upstream promoter region of WRKY genes candidates. Different colored rectangles represent different elements. Detailed information of sequence and position of these elements was described in Supplementary Tables S7

Subcellular localization and transcriptional activation activity

Typical TFs usually have two basic characteristics: located in the nucleus and have transcriptional activation activity. For deeper study, we cloned two pairs of orthologous WRKY genes (CsWRKY7/PtrWRKY39, and CsWRKY33/PtrWRKY32) with opposite expression patterns under CLas-infection and significant differences in either type or quantity of cis-elements in promoter regions. Meanwhile, their subcellular localizations were analyzed by fusing the full length cDNAs with green fluorescent protein (GFP) under the drive of the CaMV 35 S promoter. Transient expression experiment was performed by Agrobacterium-mediated infiltration of Nicotiana benthamiana leaves. Co-transformation of a nucleus marker gene fused to mCherry was conducted to confirm the localization of nucleus. Control imaging of the epidermis of tobacco showed that the green fluorescence filled throughout the entire cell, including the membrane, the cytoplasm and the cell nucleus. The green fluorescence signals of four WRKY proteins, CsWRKY7, PtrWRKY39, CsWRKY33 and PtrWRKY32, were exclusively distributed in the nucleus (Fig. 9A).

Fig. 9
figure 9

Subcellular localization and transcriptional activation assay of selected WRKY proteins. (A) The 35 S::GFP and 35 S::GFP-WRKY plasmids were transiently infiltrated into Nicotiana benthamiana leaves by Agrobacterium tumefaciens-mediated transformation and observed under a confocal laser scanning microscope. (B) Schematic structure of the four WRKY proteins. (C) The empty vector (pGBKT7) constructs and fusion constructs (FL or truncated fragments) were transformed into yeast Y2HGold strain and incubated on SD/-Trp (SDO), SD/-Trp/-His/-Ade (TDO) and TDO added with X-α-gal

To determine the transcriptional activation activity of these four nuclear localized WRKY proteins in gene expression regulation, the GAL4-responsive reporter system in yeast was carried out. Four tested WRKY proteins were truncated into N-terminal (N), W-terminal (containing the conserved WRKY domain, W) and C-terminal (C) according to the amino acid structure (Fig. 9B). The full length (FL) and three truncated fragments (N, W, and C) of WRKYs were fused to pGBKT7 to generate effectors, respectively. All the fusion plasmids were independently transformed into the Y2HGold yeast strain and exhibited visible white colonies on the SD medium lacking tryptophan (SD/-Trp, SDO). All yeast transformants containing FL fragments of CsWRKY7, PtrWRKY39, CsWRKY33, and PtrWRKY32 survived on the SD/-Trp/-Ade/-His medium (TDO) and exhibited blue pigment on the SD/-Trp/-Ade/-His medium added with X-α-Gal (TDO + X-α-Gal). Among all truncated fragments, only yeast cells transformed with the effectors containing CsWRKY7-N, PtrWRKY39-N, CsWRKY33-C and PtrWRKY32-C grew and displayed GAL4 activity on the medium supplemented with X-α-gal (Fig. 9C). These results demonstrate that the four WRKY proteins have transcriptional activation activity, among which CsWRKY7 and PtrWRKY39 exhibit strong transcriptional activation ability at its N-terminal respectively, and the C-terminal is necessary for the transactivation of CsWRKY33 and PtrWRKY32.


In this study, gene architectures, protein motifs, gene duplications and syntenic relationships of the WRKY family were globally analyzed in two Citrus species. Furthermore, the expression pattern and functional analysis of the WRKY genes were further investigated.

Genome-wide exploration and evolutionary analysis of WRKY genes among two Citrus species

A BLASTp search and HMMER analysis revealed a total of 52 WRKY proteins in C. sinensis genome and 51 WRKY proteins in P. trifoliata genome. Previous research excavated 50 non-redundant WRKY genes in sweet orange by using “WRKY & Citrus” as keywords to search within ten databases [45]. Detailed comparison exhibited that all of the 50 CsWRKYs were included in the WRKY genes identified in our study, and two more CsWRKYs (orange1.1t01175.1 and orange1.1t02759.1) were discovered in our results compared with the previous article, suggesting that our approach of BLASTp search after constructing the Hidden Markov Model could identify potential WRKY genes more comprehensively and completely. In both C. sinensis and P. trifoliata, most genes were discovered to be closely related to each other due to similarities in their intron-exon structures, indicating their close phylogenetic relationship. This remarkable resemblance in intron-exon structure also supports the concept of a common ancestry [46, 47]. It was also observed in both species that the intron number of the overwhelming majority of WRKY genes was higher in Group IIb than in other Groups. Similar findings in Arabidopsis, sesame, sorghum, and sugar beet were reported [20, 48,49,50]. A recent study demonstrated a higher frequency of intron loss than the frequency of intron gain after segmental duplication in rice [51]. These results suggesting that Group Ia subfamily might have the original genes, from which other clusters were derived. Furthermore, the lowest number of introns were present in Group IIc, indicating that WRKY genes from Group IIc in Citrus might be in a posterior position in evolution. Other species i.e. Chenopodium quinoa and Isatis indigotica also exhibited the same feature [52, 53].

The conserved motif analysis revealed interesting facts regarding the gene evolution. The motif 4 of Group I proteins, occurring just before the motif 1 containing WRKY domain, were also found in Group IIb proteins (Fig. 3), indicating that the Group IIb genes have been originated from the loss of the N-terminal WRKY domain of Group I genes. Additionally, the phylogenetic closeness and the conserved motifs of Group Ib, Group IId and Group IIIa indicate their evolution from a common origin. Notably, collinearity analysis exhibited that seven WRKY genes from C. sinensis and P. trifoliata had no orthologous genes between each other, among which six WRKY genes belonged to Group Ib (Fig. 4). Furthermore, five WRKY proteins containing a mutated WRKY domain of WRKYGKK with Q amino acid mutation to K amino acid all belonged to Group Ib (Supplementary Figure S1). Combined with the analysis of collinearity, we speculated that WRKY genes from Group Ib in Citrus might be in a relatively anterior position in evolution. Whereas, most members of other subgroups may have not evolved from the subgroup. Thus, our data support the theory of evolution of WRKY genes that the Group I is the oldest group, and Group II along with Group III have been evolved from Group I. In addition, our study further investigated that WRKY members of Group II and Group III may have been evolved from Group Ia in Citrus.

According to the research on the evolution of WRKY family between Arabidopsis and rice, some of the rice WRKY genes in Group III are evolutionarily more active than those in Arabidopsis, suggesting that evolution is more active in the Group III and these members of the group may have more functions in the monocotyledonous plants [42]. This variation in the distribution of WRKY genes among monocotyledons and dicotyledons suggests that members of the Group III have been evolved independently after the dissection of the monocotyledons and dicotyledons. It’s worth mentioning that neither C. sinensis nor P. trifoliata has any members classified into Group IIIb. On the basis of the number of amino acid residues between the inner amino acids of the C2H zinc finger motifs, Group III was divided into two subgroups as Groups IIIa (n ≤ 23) and IIIb (n ≥ 24) respectively. Therefore, the deletion of Group IIIb WRKY genes in the genomes of two Citrus species might be due to the shortening of amino acids of these genes during evolution, which showed diversity in the length of WRKY domains.

Distribution of genes suggests unlike gene structure diversity of WRKYs in different species. Thus, the uneven distribution of genes is evidence of genetic variations resulting as an evolutionary process of these two species after divergence from each other [54]. The recent gene duplication events including segmental duplication and tandem duplication were thought to be key driving forces in the expansion and evolution of gene families, as well as the raw materials for new biological functions. Because of their considerable contribution to various physiological processes, the WRKY family in angiosperms is likely to expand rapidly during evolution. Recent gene duplication events have been reported to be more extensive in the expansion of WRKY genes in many crops, such as rice and Arabidopsis [42]. Nonetheless, in some cases, recent duplications seemed to play no substantial role in WRKY gene expansion [55, 56]. In our case also, we detected few tandem and segmental gene duplications in two Citrus species, C. sinensis and P. trifoliata, but not that conspicuous as compared to rice and Arabidopsis. Notably, we observed that all pairs of tandem-duplicated genes in both C. sinensis and P. trifoliata genome belonged to Group IIIa, which is consistent with a previous study on Arabidopsis [42]. Our study revealed that tandem duplication events of Group III WRKY gene family were considered as main contributors to the rapid expansion of Citrus species WRKY gene family.

The expression and promoter analysis of WRKY genes provide important clues for their function under CLas-infection

Previous studies have shown that WRKYs involved in pathogenic mechanism in plants. SA is a vital hormone in the immunological responses to pathogens. Several WRKY genes have been reported to be induced by SA and play a crucial role in plant disease resistance. CaWRKY6 transcriptionally activated CaWRKY40 and positively regulated Ralstonia solanacearum resistance in pepper [24]. VqWRKY31 was confirmed to fight against powdery mildew pathogen attack in grapevine [57]. The mechanism of their resistance to pathogen infection was all related to SA signaling. In our current study, expression pattern analysis of WRKY family genes from C. sinensis and P. trifoliata showed that most of the genes displayed different up- and down-regulation patterns in response to SA treatment and CLas-infection. Several genes showed a significant induction multiple. For example, the expression levels of CsWRKY24 reached 259.17 and 92.20 times at 3 and 6 h after SA treatment, respectively (Fig. 5, Supplementary Table S5). CsWRKY4 and PtrWRKY43 separately exhibited an enhanced expression level of 17.99-fold and 48.56-fold compared to healthy plants (Fig. 6, Supplementary Table S6), suggesting that some WRKY genes could be stimulated by SA treatment or CLas-infection. Further investigation revealed a high percentage of overlap between WRKY genes which expressed dramatically after CLas-infection and SA treatment. Previous studies have shown that homologous genes of these WRKY genes function in the SA-mediated plant disease resistance. For instance, AtWRKY6 (a homologous gene of CsWRKY4/PtrWRKY15) positively influenced the pathogen defense-associated PR1 promoter activity, most likely involving NPR1 function due to the elevated transcription levels of NPR1 in Arabidopsis thaliana [58]. AtWRKY70 (a homologous gene of CsWRKY24/PtrWRKY42) have been identified as a node of convergence for integrating SA- and JA-mediated signaling events during plant response to bacterial pathogens. Gain or loss function of WRKY70 led to opposite effects on JA-mediated resistance to A. brassicicola and the SA-mediated resistance to E. cichoracearum, suggesting that it had a vital role in balancing SA-dependent and JA-dependent defense pathways [59]. Given that orthologous genes among different plants are generally considered to retain similar functions and to share other key properties, we hypothesize that these homologous WRKY genes in Citrus may likewise play a critical role in the SA-induced resistance pathway to HLB. Notably, AtWRKY11, the homologous gene of last Citrus WRKY orthologous gene pairs, has been discovered to be a negative regulator of the basal resistance to Pseudomonas syringae pv tomato (Pst). Loss function of AtWRKY11 enhanced resistance to both avirulent and virulent Pst strains [60]. Another study showed that two Arabidopsis TFs, AtWRKY11 and AtWRKY70, were identified as essential regulators in Induced Systemic Resistance (ISR) triggered by Bacillus cereus AR156. AR156 treatment dramatically induced the transcription of AtWRKY70, but inhibited that of AtWRKY11 in Arabidopsis leaves. AtWRKY11 regulated AR156 triggering ISR by activating JA signaling pathway, while AtWRKY70 regulated ISR by activating SA signaling pathway [61]. These researches indicate that there are multiple regulatory pathways in the mechanisms of WRKY genes in disease resistance signaling pathway.

To analyze the difference of WRKY gene in SA signaling and HLB response between disease tolerant and susceptible varieties, we screened WRKY genes with opposite expression patterns after two treatments in sweet orange and trifoliate orange, resulting in the identification of seven pairs of homologous WRKY genes (Fig. 7C-D), suggesting that these genes may play a pivotal role in the differences in susceptibility of citrus varieties to HLB disease. The cis-elements present in the promoter regions are important molecular switches that control a wide range of gene regulatory networks [62]. Our analysis of these seven pairs of homologous WRKY genes revealed significant differences in the promoter cis-elements of two homologous WRKY gene pairs (CsWRKY7/PtrWRKY39, CsWRKY33/PtrWRKY32). Previous studies showed that AtWRKY33 (a homologous gene of CsWRKY7/PtrWRKY39) participates in the SA signal pathway and plays a negative regulatory role in resisting Botrytis cinerea concomitant with increased expression of SA-regulated PR-1 gene, and reduced expression of JA-regulated genes such as PDF1.2 in wrky33 mutant plants [63]. However, the specific upstream regulatory factors of WRKY33 are still not clear. WRKY45 (a homologous gene of CsWRKY33/PtrWRKY32 in rice) is an important TF in the SA signaling pathway, which mediates chemically induced resistance to a variety of pathogens [64]. One research revealed that the TF Ideal Plant Architecture 1 (IPA1) was phosphorylated at Ser163 within its DNA binding domain along with the alteration of DNA binding specificity after the infection of Magnaporthe oryzae. Phosphorylated IPA1 binds to the promoter of WRKY45 and activates its expression, thereby enhancing disease resistance in rice [65]. These findings imply that the disease resistance conferred by WRKY45 might be closely related to its promoter activity and upstream regulators. In conclusion, the above findings suggest that the opposite expression patterns of two pairs of homologous WRKY genes (CsWRKY7/PtrWRKY39, CsWRKY33/PtrWRKY32) may also be related to their upstream regulatory factors between disease-tolerant and disease-susceptible citrus varieties.

The candidate WRKYs are typical TFs

A close relationship between the function of TFs and their localization has been demonstrated by numerous studies [66]. In the present study, the two screened pairs of homologous WRKY genes, CsWRKY7, PtrWRKY39, CsWRKY33 and PtrWRKY32 were all localized in the nucleus (Fig. 9A). Transcriptional activation analysis showed that all the four WRKY proteins exhibited transcriptional activity in yeast cells (Fig. 9C), demonstrating that these WRKY proteins are typical TFs with transcriptional activation regulatory ability and function in the nucleus, thus implying that their functions may be related to the direct regulatory effects on downstream target genes. However, more in-depth studies are needed to elucidate the upstream regulators and potential downstream target genes in SA signaling pathway and CLas-infection. Our results provide comprehensive information for the function of WRKY genes in Citrus and take important significance in the identification of HLB resistance genes as well as the investigation of molecular mechanism and regulatory network in response to HLB and regulated by SA signaling pathway.


A total of 52 and 51 WRKY genes were identified and analyzed in two Citrus species: C. sinensis and P. trifoliata, respectively. qRT-PCR and promoter analysis showed that the expressions of 2 pairs of candidate orthologous WRKY genes were opposite between two Citrus species, and these genes might be involved in the HLB response and SA signaling pathway. Experiments indicated that these WRKY genes (CsWRKY7/PtrWRKY39, CsWRKY33/PtrWRKY32) are classic WRKY TFs with transcriptional regulation function localized in the nucleus.

Data Availability

The sequence information of Citrus and Arabidopsis WRKY family genes were collected from Citrus Pan-genome to Breeding Database ( and The Arabidopsis Information Resource ( respectively. All data used during the current study are included in this published article and its supplementary information files or available from the corresponding author on reasonable request.



Sequence, amino acid


Asian Citrus Psyllid


Candidatus Liberibacter asiaticus


Cetyltrimethylammonium Bromide


Full Length


Green Fluorescent Protein




Induced Systemic Resistance


Open Reading Frame


Quantitative Polymerase Chain Reaction


Quantitative real-time Polymerase Chain Reaction


Salicylic Acid


Systemic Acquired Resistance


Transcription Factor


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This work was financially supported by the National Natural Science Foundation of China (32060670), Natural Science Foundation of Jiangxi Province (20202BABL215011), “Double Thousand Plan” Foundation of Jiangxi Province (461869), Natural Science Foundation of Jiangxi Province (20212ACB205001) and The Major Science and Technology R&D Program of Jiangxi Province (20194ABC28007).

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Authors and Affiliations



WSD and MW conceived and designed the research; WSD performed the experiments and conducted the bioinformatics analysis; WSD wrote the first version of the manuscript while TP and JHL revised the paper. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Min Wang.

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The test Citrus materials were collected from the citrus germplasm orchard of the Center of National Navel Orange Engineering Research situated in Ganzhou under the permission of Center of National Navel Orange Engineering Research. This article did not contain any studies with human participants or animals and did not involve any endangered or protected species. All experimental studies and experimental materials involved in this research are in full compliance with relevant institutional, national and international guidelines and legislation.

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Additional file 1: Table S1.

List of primer sequences used in this study

Additional file 2: Table S2.

Proposed nomenclature and important features of WRKYs from Citrus sinensis

Additional file 3: Table S3.

Proposed nomenclature and important features of WRKYs from Poncirus trifoliata

Additional file 4: Figure S1.

Multiple sequence alignment of WRKY domains in CsWRKYs and PtrWRKYs

Additional file 5: Table S4.

Conserved motifs in CsWRKY and PtrWRKY proteins.

Additional file 6: Table S5.

qRT-PCR values of WRKY genes under SA treatment

Additional file 7: Figure S2.

The uncropped gel of Fig. 6A-B.

Additional file 8: Table S6.

qRT-PCR values of WRKY genes under HLB infection

Additional file 9: Table S7.

List of stress-responsive cis-acting elements present in 2 kb upstream region of WRKY genes

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Dai, WS., Peng, T., Wang, M. et al. Genome-wide identification and comparative expression profiling of the WRKY transcription factor family in two Citrus species with different Candidatus Liberibacter asiaticus susceptibility. BMC Plant Biol 23, 159 (2023).

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  • Citrus
  • WRKY transcription factor
  • Genome-wide identification
  • Salicylic acid
  • Huanglongbing