Abstract
Background
Akebia trifoliata, belonging to the Lardizabalaceae family, is a well-known Chinese traditional medicinal plant, susceptible to many diseases, such as anthracnose and powdery mildew. WRKY is one of the largest plant-specific transcription factor families and plays important roles in plant growth, development and stress response, especially in disease resistance. However, little was known about the numbers, characters, evolutionary relationship and expression of WRKY genes in A. trifoliata in response to plant disease due to lacking of A. trifoliata genome.
Results
A total of 42 putative AktWRKY genes were identified based on the full-length transcriptome-sequencing data of A. trifoliata. Then 42 AktWRKY genes were divided into three major groups (Group I-III) based on the WRKY domains. Motif analysis showed members within same group shared a similar motif composition, implying a functional conservation. Tissue-specific expression analysis showed that AktWRKY genes could be detected in all tissues, while few AktWRKY genes were tissue specific. We further evaluated the expression of AktWRKY genes in three varieties in response to Colletotrichum acutatum by qRT-PCR. The expression patterns of AktWRKY genes were similar between C01 and susceptible variety I02, but distinctly different in resistant variety H05. In addition, it showed that more than 64 percentages of AktWRKY genes were differentially expressed during fungal infection in I02 and H05. Furthermore, Gene ontology (GO) analysis showed that AktWRKY genes were categorized into 26 functional groups under cellular components, molecular functions and biological processes, and a predicted protein interaction network was also constructed.
Conclusions
Results of bioinformation analysis and expression patterns implied that AktWRKYs might play multiple function in response to biotic stresses. Our study could facilitate to further investigate the function and regulatory mechanism of the WRKY in A. trifoliata during pathogen response.
Similar content being viewed by others
Introduction
The WRKY transcription factor family is one of the largest transcriptional regulatory gene families in plants, which can regulate downstream transcription through specifically recognizing and binding with the cognate cis-element W-box (TTGACT/C), which is usually located in the promoter region of genes related to growth, development and stress response [1,2,3].
Structurally, WRKY transcription factors contain one or two conserved WRKY domains, which consist of a signature sequence (WRKYGQK) along with a C2H2 or C2HC zinc-binding motif. Generally, WRKY transcription factors are classified into three major groups based on their structure. Group I WRKYs contain two WRKY domains at the N- and C-terminal, each of which is followed by a C2H2 zinc-finger, while Group II WRKYs have only one WRKY domain. Similarly, Group III also contains only one WRKY domain, but there is a C2HC zinc-finger motif at the C-terminal of the WRKY domain instead of C2H2 zinc-finger [4,5,6,7]. By inferring the results of a nuclear magnetic resonance solution structure of the C-terminal WRKY domain of AtWRKY4, the conserved WRKY domain is composed of a four-stranded beta-sheet with a zinc binding pocket, which is directly involved in DNA binding [8, 9]. Previous studies have shown that WRKY domain sequences can directly bind to W-box (C/T)TGAC(C/T) cis-regulatory element, which were found in the promoter region of the target genes [3, 10]. These (C/T)TGAC(C/T) sequence elements contain the invariant TGAC core that mediate transcriptional responses to biotic stresses. Thus, plant genes that contain TGAC core in the promoter regions are considered as defense-associated genes [5, 11]. Interactions between the WRKY domain and the W box have been demonstrated by numerous binding experiments, both in vitro and in vivo, and these interactions can also be regulated post-translationally, since these bindings can be inhibited by phosphatase and protein-kinase inhibitors [12, 13]. Previous studies have proven that WRKYs play essential roles in plant growth, development, and abiotic or biotic stresses responses [14,15,16]. For instance, AtWRKY12 could partly mediate the effect of GA3 in controlling flowering time and regulating the formation of pith secondary wall in Arabidopsis [17,18,19]. Wintersweet WRKY71 was proven to be involved in the regulation of flowering and leaf senescence in Arabidopsis [20]. Interestingly, WRKY transcription factors, although are transcription factors themselves, are also regulated by other WRKYs [21]. For example, WRKY18 was able to bind directly to different W-boxes in the WRKY53 promoter region, thereby repressing the expression of WRKY53. Thus, WRKY18 could act as a positive senescence regulator due to its repressing function on WRKY53 [22]. Furthermore, WRKYs have been found to be involved in various biotic and abiotic stress defense responses, such as viruses, bacterial pathogens, fungi, heat, drought [23,24,25,26,27,28]. For instance, PlWRKY65, as a disease resistance-related transcriptional activator, could exert a regulatory effect on JA and SA signals to enhances the resistance of Paeonia lactiflora to Alternaria tenuissima [29]. Additionally, it has been reported that VvWRKY30 overexpression lines had higher antioxidant activities and lower reactive oxygen species contents under salinity stress, thus enhancing their resistance to salt stress [30].
Since WRKY transcription factors play critical roles in plant development and stresses resistance, WRKYs have been identified genome-wide from various plant species with the development of high-throughput sequencing, including 74 WRKYs in Arabidopsis, 103 in rice, 86 in Brachypodium distachyon, 197 in soybean, and 54 in pineapple [5, 21, 31,32,33,34]. However, the numbers, characters, evolutionary relationships and expressions of WRKY genes in Akebia trifoliata (Thunb.) Koidz. were completely unknown. A. trifoliata belonging to the Lardizabalaceae family, was mainly distributed in the eastern part of Asia, which was a well-known Chinese traditional medicinal plant, as its antiphlogistic, antineoplastic and diuretic characters [35, 36]. The wild resources of A. trifoliata were on the verge of exhaustion because of overexploitation. Due to its medicinal and edible value, A. trifoliata has been developing as an artificial cultivation commercial crop in Hunan and Jiangxi province in China. However, the cultivated A. trifoliata seedlings were susceptible to disease, thus it is very important to protect them from pathogen. In this study, a total of 42 putative AktWRKY genes were identified based on the full-length transcriptome-sequencing data of A. trifoliata. Subsequently, the characters of AktWRKYs and their expression patterns in response to Colletotrichum acutatum have been further analyzed. Our results could provide the novel insight into protein structures, evolutionary relationships, and expression pattern of WRKYs in A. trifoliata, which could also facilitate to further investigate the biological functions of AktWRKYs under biotic stresses.
Materials and methods
Database search and identification of WRKY transcription factors
All 42 putative WRKY proteins were retrieved from full-length transcriptome sequencing data of A. trifoliata (unpublished data). Arabidopsis WRKY protein sequences were downloaded from the database of The Arabidopsis Information Resource (TAIR, https://www.arabidopsis.org/). The AktWRKY proteins were identified by blastp method using Geneious software with Arabidopsis WRKY proteins as query sequences. The identified AktWRKY proteins in A. trifoliata were rechecked and confirmed to avoid repetition and verify the reliability of our results: a) short AktWRKY sequences with an incomplete WRKY domain have been removed, b) all putative non-redundant sequences were assessed with UniProt and SMART (http://smart.embl-heidelberg.de/) analyses, respectively. The neighbor-joining (NJ) phylogenetic tree based on 42 putative WRKY proteins were constructed to classify the AktWRKYs. All putative WRKY gene family members in A. trifoliata were designed their names base on the homologs in Arabidopsis.
Protein Motifs, Structure Analysis and Phylogenetic Analysis
The conserved motifs in the WRKY proteins were predicted using MEME suite (http://meme.sdsc.edu/meme/cgi-bin/meme.cgi). The parameters were set as follows: maximum number, 6; site distribution, any number of repetitions; minimum width, 10; and maximum width, 80. The graph was generated by TBtools v1.068. Subsequently, multiple alignment analyses of the WRKY domains sequence were performed by ClustalW (www.ebi.ac.uk/clustalw/). The phylogenetic tree based on the alignment of WRKY domains in rice, Arabidopsis, and A. trifoliata was constructed using MEGA X software with NJ method and following parameters: p-distance and pairwise deletion. Bootstrap analysis was performed with 1000 replicates.
Plant materials and expression analysis
A. trifoliata, which is not a protected plant, was identified by Prof. Liao Liang according to Flora of China. The original samples (C01, I02, and H05) were obtained from Yuncheng City, Shanxi Province, Lu’an City, Anhui Province, and Xiangyang City, Hubei Province, respectively. The voucher specimens deposited in Jiujiang University (accession number JJU130C01, JJU130I02, and JJU130H05, respectively). The seedlings were planted in Mutong yard in Jiujiang University, Jiujiang, Jiangxi province, China. A. trifoliata tissues of buds, young leaves, mature leaves, stems, female flowers, and male flowers were collected from the Mutong yard. For pathogen infection analysis, A. trifoliata seedlings were sprayed with spores of C. acutatum. After infection for 6 h, the leaves were collected for genes expression analysis. Total RNA was extracted from plant tissues by using Trizol reagent (Invitrogen, USA) according to the manufacturer’s protocol. A RNase-free DNaseI (TaKaRa, Japan) was used to remove genomic DNA contamination. First-strand cDNA was synthesized using PrimeScript™ 1st Strand cDNA Synthesis Kit (TaKaRa, Japan) according to the manufacturer’s protocol. The expression of the all AktWRKY genes was assessed upon the qPCR result analysis. For tissue-specific analysis, the average of total ΔCTvalue (ΔCT. average) was subtracted from all other ΔCT values to obtain second normal standardization, according to the previous method [37]. For phytopathogen infection analysis, the expression level of test genes was calculated with the 2 −ΔΔCT method. The data were statistically analyzed using an OriginPro 7.5 software. Gene specific primers for quantitative real-time PCR are listed in Additional file 4. Public Arabidopsis expression datasets were obtained from the Gene Expression Omnibus (GEO) and the AtGenExpress Consortium (Arabidopsis eFP Browser).
Gene ontology annotation and protein interaction analysis
To perform the functional classification, all putative AktWRKY genes were analyzed using Blast2GO basic software. And then, the results were performed by using an online tool WEGO (http://wego.genomics.org.cn/) to compare and plot Gene Ontology annotation results. Protein-protein interactions were predicted by the STRING 11.0 program (https://string-db.org/) based on an Arabidopsis association model with the confidence parameter set at a threshold of 0.35.
Results and discussion
Identification of WKRY transcription factors in A. trifoliata
As one of the largest gene families of transcriptional regulators in plant, WRKY transcription factor family plays critical roles in regulating plant growth and development as well as abiotic or biotic stress responses [38,39,40]. Although the functions and evolutionary relationships of WRKYs in several model plants have been investigated, little is known about this family genes in the Chinese traditional medicinal plant, A. trifoliata. To systematically explore the organizational structure, evolutionary relationship and function of WRKYs in A. trifoliata, the full-length transcriptome-sequencing data of this species were applied to identify WRKY genes in silico. A total of 42 AktWRKY genes were identified by searching the transcriptome-sequencing datasets using total Arabidopsis WRKY genes as queries (Table 1 and Additional file 1). All 42 deduced AktWRKY proteins contained at least one highly conserved WRKY domains, while 11 of them had two WRKY domains. As shown in Table 1, the deduced AktWRKY proteins contained amino acid residues between 145 (AktWRKY51) to 747 (AktWRKY34), the range of which was similar to that of other dicotyledons, such as Glycyrrhiza glabra and Santalum album [41, 42]. Their molecular weight (MW) varied between 16.77 kDa (AktWRKY51) to 81.41 kDa (AktWRKY25), while the isoelectric point (pI) of 21 AktWRKYs were acidic and the other 21 were basic proteins. A neighbor-joining phylogenetic tree was preformed to investigate the phylogenetic classification of the WRKY proteins in A. trifoliata according to previous report [5]. As shown in phylogenetic tree, all 42 deduced AktWRKY proteins were clustered into three main groups, namely, Group I, II, and III (Fig. 1). Seven AktWRKYs containing one WRKY domain and a C2CH zinc-binding motif at the C-terminal were classified as Group III, while twenty-three main clustering proteins with one WRKY domain and a C2H2 motif were clustered into Group II, which was further divided into five subgroups, named Group IIa-IIe. The twelve remaining proteins were included in Group I.
The number of AktWRKY genes belonging to each subgroup was compared to the number of WRKYs in other plant species, in which the WRKY gene family has been fully identified and characterized, including Arabidopsis, Brachypodium distachyon, rice, kiwifruit (Actinidia chinensis), grape and tomato (Fig. 2 and Additional file 2) [21, 43, 44]. Comparing the number of WRKY genes in each subgroup, Group I and Group IIc experienced a significant expansion by having majority members in dicot species. In addition, the number of Group I WRKY genes was similar in A. trifoliata, Arabidopsis, B. distachyon, rice, grape and tomato, but it showed a significant expansion in kiwifruit, which might attribute to the two whole-genome duplications of kiwifruit genome [45]. On the contrary, it was apparent that variations in the number of WRKY genes in Group III were the primary cause of the diversity of WRKY gene family size in monocot species, i. e. B. distachyon and rice [21, 31]. These results suggested that numerous duplications and diversifications of Group III WRKY genes might be occurred after the divergence of the monocots and dicots. Previous studies have reported that the Group III of WRKY genes, as a newly defined group, was the most dynamic group with respect to gene family evolution [46]. In this study, the number of WRKY genes in Group III was relatively less in A. trifoliata, suggesting that this group had experienced less gene duplications during the evolutionary course, which could attribute to the fact that A. trifoliata located at the basal clade of the phylogenetic tree.
The distribution of WRKY transcription factors from Arabidopsis, B. distachyon, rice, A. trifoliata, kiwifruit (A. chinensis), grape and tomato. The width of the band represents the percentage of WRKYs in each group. Green represents group I, blue represents group IIa, orange represents group IIb, cyan represents group IIc, yellow represents group IId, purple represents group IIe, and red represents group III
Conserved motifs of AktWRKYs
To better understand the conservation and diversity of AktWRKYs, the conserved motifs of all the putative AktWRKY proteins were predicted by MEME online program. The distribution of motifs in each group and the diversity of conserved motifs were shown in Fig. 3. Generally, most members within the same group or subgroup shared a similar motif composition, which implied that AktWRKYs homologs located under the same group might have similar functions. However, AktWRKY members of different groups had no common conserved motifs except for the WRKY domain at the C-terminal. The motif 1 and 2 of group I contained conserved heptapeptides WRKYGQK sequence, representing the C-terminal and N-terminal WRKY domains, respectively. Obviously, the C-terminal and N-terminal WRKY domains were differentiated, suggesting that these two WRKY domains might be different in origin or functional differentiation. It was consistent with the fact that the specific binding to W-box was mediated mainly by the C-terminal WRKY domain, whereas N-terminal WRKY domain showed weak binding activity [9]. Further, insight into motif analysis of group II indicated that most of members of group IIa and IIb shared similar motif composition, while group IId and IIe shared motif 1, 2 and 4. The members of group IIc were relatively different with other subgroup members, even within subgroup itself. Most members of group III contained motif 1, which shared a tripeptide HTC residue at the C-terminal of the motif. Overall, these results present group-based motif analysis of AktWRKYs, while the functions of most of these motifs need to be further investigated.
Diagram showed information of different motifs and their sequence logos for all AktWRKY proteins. A-C Distribution of conserved motifs in the 42 AktWRKYs. A, B, and C for Group I, Group II and Group III, respectively. Each motif was represented by a colored box. Blue hollow boxes represented the WRKY domains. D-F The logo of each motif (Color figure online). D, E, and F for Group I, Group II and Group III, respectively
Comparative analysis of WRKY domains
To understand the diversity and evolutionary relationships of the AktWRKY domains, we compared the WRKY domains from the two other sequenced plant genomes (O. sativa and Arabidopsis). An unrooted neighbor-joining comparative phylogenetic tree was constructed by using MEGA software version 7 from the 252 conserved WRKY domains among these three plant species. As shown in Fig. 4, the complete WRKY domains were divided into three major groups (I, II and III), and all groups were present in monocots and eudicots. Of the three major groups, group II was the largest major group in the phylogenetic tree, with 118 WRKY domains distributed in five subgroups, including 9 in IIa, 19 in IIb, 49 in IIc, 18 in IId and 23 in IIe. Group I contained 77 WRKY domains, which were divided into subgroup IN and IC, containing 41 and 36 WRKY domains, respectively. Apparently, the subgroup IN and IC were clustered into different clades, suggesting that these domains originated from different ancestors and maintained their own differentiation after the lineage divergence. In addition, group I was considered to be the oldest group, located on the basal clade of the phylogenetic tree [47]. Subgroup IC and IIc were closely clustered, which was consistent with the previous results [48]. Meanwhile, no species-specific WRKY domain subgroups were observed in these three species, and WRKY domains belonging to the same group had similar conserved domain compositions, implying that WRKY family genes were conserved during plant evolution. Furthermore, multiple sequence alignment of the 42 AktWRKY protein domains was performed based on the conserved WRKY domain using clustal W software, containing approximately 60 amino acids for each AktWRKY (Fig. 5). The highly conserved heptapeptide sequence WRKYGQK was found within a total of 37 AktWRKYs, while five proteins (AktWRKY20, AktWRKY30, AktWRKY46, AktWRKY50 and AktWRKY51) were different due to one or two amino acid substitution. The protein AktWRKY50 and AktWRKY51 were found to contain a WRKYGKK sequence, while AktWRKY20, AktWRKY30 and AktWRKY46 contained a WCKYGRK, WMKYGQK and WEKYGQQ sequence, respectively. In addition, CX4CX22-23HXH and CX4CX23HXH zinc finger motifs were found in the N-terminal and C-terminal of group I AktWRKYs, respectively. CX5CX23HXH motifs were found in subgroup IIa, IIb, IId and IIe AktWRKYs, and CX7CX23HTC motifs were found in group III AktWRKYs, while the zinc finger motifs in subgroup IIc were same with C-terminal of group I AktWRKYs.
Tissue-specific expression patterns of AktWRKY genes
Numerous studies have demonstrated that the abundance of transcription factor genes varied greatly in different tissues and at different developmental stages, and played critical roles in regulating plant growth and development. For instance, WRKY12 was specifically expressed in pith and cortex cells of stem and hypocotyls, playing a critical role in pith secondary wall formation, while WRKY13 transcripts were highly abundant in the juvenile phase but decreased over time, implying that WRKY13 was involved in the control of age-mediated pathway [17,18,19]. Thus, tissue- and developmental stage-specific gene expression patterns might provide clues to gene functional divergence during evolution [49]. To investigate the patterns and expression levels of putative AktWRKY genes, the expression levels of 42 A. trifoliata WRKY genes in six tissues (buds, young leaves, mature leaves, stems, female flowers and male flowers) were determined by qRT-PCR (Fig. 6). Our results showed that AktWRKY genes could be detected in all test tissues, and exhibited distinct expression patterns. Generally, most AktWRKY genes were relatively highly abundant in leaves, especially in young leaves, while most AktWRKY genes were expressed at low levels in stem and flowers. However, tissue-specific expression of WRKY genes was also observed in A. trifoliata. For instance, AktWRKY21 was particularly highly expressed in stems but less expressed in female flowers. AktWRKY17 exhibited extremely high levels in young leaves, but low in the other tissues, implying this gene might be involved in leaf development. AktWRKY28 showed high levels of expression in buds and female flowers, while AktWRKY19 were highly expressed in young leaves and male flowers. Previous reports demonstrated that WRKY genes with high expression in plant tissues were often found to be able to regulate target genes involved in the relevant processes of plant growth and organs development [50, 51]. Thus, tissue-specific AktWRKY genes in this study might provide some useful clues for further investigation of their biological functions in the growth and organs development of A. trifoliata. In addition, some clustered gene pairs showed the same expression pattern, such as AktWRKY03/20, AktWRKY26/33, AktWRKY07/11, AktWRKY27/65, etc., implying that they might be functionally redundant. On the other hand, AktWRKY gene pairs with different expression patterns might executed different biological functions in plant growth and development.
Expression patterns of WRKY genes in A. trifoliata in different tissues. YL for young leaves, ML for mature leaves, FF for female flowers, MF for male flowers. The expression values of the 42 AktWRKY genes were assessed upon the qPCR result analysis. Red represents a higher level of abundance while green signify lower expression levels
Expression analysis of AktWRKY genes in response to phytopathogen
As crucial components of plant defense signaling networks, WRKY genes were known to exhibit complex response patterns [16, 52]. The function of WRKY genes in the regulation of plant response to biotic stresses has been studied in many plant species, especially in Arabidopsis, rice and tomato [7]. It is well known that the functions of AtWRKY genes response to phytopathogens have been well studied. Expression profiles of AtWRKY genes under various phytopathogen stresses indicated that most of AtWRKYs were involved in various biotic stresses response (Additional file 5) [53,54,55,56,57,58,59,60]. For example, AtWRKY3, AtWRKY4, AtWRKY53 and AtWRKY70 played positive roles in disease defense against the necrotrophic fungal pathogen and the biotrophic pathogen Pseudomonas syringae [61,62,63]. AtWRKY33, which was activated by two sigma factor binding proteins, SIB1 and SIB2, was essential for defense toward the necrotrophic fungus Botrytis cinerea [62, 64, 65], while AtWRKY18 and PtrWRKY18 could activate pathogenesis-related genes, and increase resistance to the biotrophic pathogens [66, 67]. Moreover, some WRKY genes, such as AtWRKY7 and AtWRKY48, had direct negative effects on plant defense responses [68, 69]. In regards to AktWRKY expression in response to biotic stresses, three different varieties of A. trifoliata (C01, susceptible variety I02 and resistant variety H05) were infected with a polyphagous fungal plant pathogen, C. acutatum. Then, qRT-PCR was performed on C. acutatum infected leaves in order to investigate the expression profiles of A. trifoliata in response to C. acutatum (Additional file 3). As shown in Fig. 7, a large number of AktWRKY genes were induced by C. acutatum infection. It has been shown that four WRKY genes (AktWRKY03, 12, 28 and 33) were highly expressed in all three tested A. trifoliata varieties, which were consistent with the previous reports that their homologs (AtWRKY3, BrWRKY12, BnWRKY15 and AtWRKY33) enhanced resistance to phytopathogens through transcriptional activation of defense-related genes [61, 65, 70, 71]. These results suggested that, at least, these four genes might be involved in plant disease resistance. Furthermore, AktWRKY28 showed an expression pattern different from AktWRKY03 and AktWRKY33, with high abundance 6 hpi in susceptible variety, but low abundance in the resistant one, although the expression level of which were almost similar in the control samples (Additional file 6). Such divergent behaviors were previously found in rice and cacao [72, 73]. For instance, knock-out of OsWRKY28 led to a two-fold increase in resistance to Magnaporthe oryzae, while overexpression of OsWRKY28 resulted in enhanced susceptiblility to M. oryzae [72, 74]. Therefore, it was speculated that AktWRKY28, like OsWRKY28 and TcWRKY28, might act as negative regulator of basal defense responses to pathogens infection. Generally, the expression patterns of AktWRKY genes in C01 and susceptible variety I02 were similar, but distinctly different in resistant variety H05, implying a functional divergence of AktWRKYs in response to phytopathogen infection between susceptible and resistant varieties. In total, a number of nine AktWRKY genes (AktWRKY11, 18, 21, 31, 47–2, 51, 65, 70, and 74) increased after C. acutatum infection in H05, but decreased in C01 and I02. On the other hand, thirteen AktWRKY genes, including AktWRKY04, 13, 14, 19, 23, 25, 32, 34, 39, 40, 47–1, 57–1, and 68, were up-regulated after C. acutatum infection in C01 and I02, but down-regulated in H05. For example, AktWRKY18 and AktWRKY70 were induced by C. acutatum in resistant variety (Additional file 6), which was consistent with the previous results that WRKY18 and WRKY70 could positively modulate defense-related gene expression and disease resistance in Arabidopsis [63, 67]. Although analysis of WRKY expression was helpful in discriminating the role and function of these proteins at the tissue and organism levels, further molecular and biological experiments are needed to investigate their biological function.
Gene ontology annotation and interaction analysis of AktWRKY proteins
Functional annotation of proteins and prediction of protein-protein interaction could help us predict their possible regulatory functions, and provide great support for further investigation of gene families. Based on the similarity of peptide sequences, the Gene Ontology (GO) annotations of one protein could be coordinately transferred to another [75, 76]. Here, GO annotations of 42 AktWRKY proteins were analyzed using the Blast2GO basic tool (Fig. 8). Among the protein sequences annotated in the GO database, AktWRKY proteins were categorized into three main categories (i. e. biological processes, molecular functions, and cellular components) and 26 subcategories. In the category of biological process, most AktWRKYs were identified to be involved in the ‘regulation of cellular process’ (GO:0009987), ‘regulation of biological process’ (GO:0050789), biological regulation (GO:0065007), metabolic process (GO:0008152), and so on. However, three AktWRKYs (AktWRKY08, 27, and 28) were predicted to be involved in developmental process, including cell differentiation and leaf senescence, while four AktWRKYs (AktWRKY26, 33, 50, and 51) appeared to be involved in defense response to biotic stimulus. The molecular functions of AktWRKYs were associated mostly with ‘binding’ (GO:0005488) and ‘transcription regulator activity’ (GO:0140110). The cellular component of this protein family included organelle (GO:0043226), cell part (GO:0044464) and cell (GO:0005623), besides, all AktWRKYs were predicted to be localized in the nucleus (Table 1).
Subsequently, to systematically analyze the interaction of AktWRKY proteins, a predicted protein interaction network containing 16 AktWRKY proteins was constructed based on Arabidopsis homologous genes using STRING 11.0 software with the confidence parameter set at a threshold of 0.35 (Fig. 9). Among these proteins, the interaction between AtWRKY33 (AktWRKY33 and 58) and AtWRKY22 (AktWRKY74) were related to the Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway of plant MAPK signaling pathway (ath04016) and plant-pathogen interaction (ath04626) [77,78,79,80,81]. The interaction network among AtWRKY18 (AktWRKY18), AtWRKY33 (AktWRKY33 and 58), AtWRKY40 (AktWRKY40), AtWRKY53 (AktWRKY46 and 53), and AtWRKY70 (AktWRKY54 and 70) enriched significantly GO term in defense response, including bacterium (GO: 0042742), fungus (GO:0050832), chitin (GO:0010200) and salicylic acid (GO:0009751) [22, 65, 67, 82]. In addition, these five AtWRKYs and corresponding ortholog AktWRKYs were also involved in a stronger interaction network with other proteins. Overall, the results showed that there were multiple interactions among AktWRKY proteins, implying that AktWRKY proteins were involved in multiple stress responses.
Conclusion
In conclusion, a total of 42 WRKY genes were identified based on transcriptome sequences, which was the first study on the organizational structure and abundance of WRKY in A. trifoliata. The classification, protein structure, conserved motif composition, and phylogenetic relationship of AktWRKYs were systematically analyzed and compared, which provide a basis for further study of the molecular and functional structure of AktWRKYs. Furthermore, based on qPCR results, the expression patterns of tissues-specific and phytopathogen-responsive AktWRKYs were obtained, providing useful information for further investigation of the function of AktWRKYs in response to biotic stresses. Our study could help researchers better understand the function and regulatory mechanism of WRKYs in A. trifoliata during pathogen response.
Availability of data and materials
Data generated or analyzed during this study were are available in the NCBI Short Read Archive with accession number PRJNA795256 and the Additional files. Public Arabidopsis expression datasets were obtained from the AtGenExpress Consortium (Arabidopsis eFP Browser) and the Gene Expression Omnibus (Accession No. GSE61418, GSE106811, GSE83478, GSE107786, GSE129011, GSE146189, GSE168015 and GSE179628).
References
Chen L, Song Y, Li S, Zhang L, Zou C, Yu D. The role of WRKY transcription factors in plant abiotic stresses. Biochim Biophys Acta. 2012;1819(2):120–8.
Rushton DL, Tripathi P, Rabara RC, Lin J, Ringler P, Boken AK, et al. WRKY transcription factors: key components in abscisic acid signalling. Plant Biotechnol J. 2012;10(1):2–11.
Ciolkowski I, Wanke D, Birkenbihl RP, Somssich IE. Studies on DNA-binding selectivity of WRKY transcription factors lend structural clues into WRKY-domain function. Plant Mol Biol. 2008;68(1–2):81–92.
Bakshi M, Oelmuller R. WRKY transcription factors: Jack of many trades in plants. Plant Signal Behav. 2014;9(2):e27700.
Eulgem T, Rushton PJ, Robatzek S, Somssich IE. The WRKY superfamily of plant transcription factors. Trends Plant Sci. 2000;5(5):199–206.
Rinerson CI, Rabara RC, Tripathi P, Shen QJ, Rushton PJ. The evolution of WRKY transcription factors. BMC Plant Biol. 2015;15:66.
Rushton PJ, Somssich IE, Ringler P, Shen QJ. WRKY transcription factors. Trends Plant Sci. 2010;15(5):247–58.
Yamasaki K, Kigawa T, Inoue M, Tateno M, Yamasaki T, Yabuki T, et al. Solution structure of an Arabidopsis WRKY DNA binding domain. Plant Cell. 2005;17(3):944–56.
Maeo K, Hayashi S, Kojima-Suzuki H, Morikami A, Nakamura K. Role of conserved residues of the WRKY domain in the DNA-binding of tobacco WRKY family proteins. Biosci Biotechnol Biochem. 2001;65(11):2428–36.
Eulgem T, Somssich IE. Networks of WRKY transcription factors in defense signaling. Curr Opin Plant Biol. 2007;10(4):366–71.
Rushton PJ, Somssich IE. Transcriptional control of plant genes responsive to pathogens. Curr Opin Plant Biol. 1998;1(4):311–5.
Wang Z, Yang P, Fan B, Chen Z. An oligo selection procedure for identification of sequence-specific DNA-binding activities associated with the plant defence response. Plant J. 1998;16(4):515–22.
Yang P, Chen C, Wang Z, Fan B, Chen Z. A pathogen- and salicylic acid-induced WRKY DNA-binding activity recognizes the elicitor response element of the tobacco class I chitinase gene promoter. Plant J. 1999;18(2):141–9.
Baillo EH, Hanif MS, Guo Y, Zhang Z, Xu P, Algam SA. Genome-wide Identification of WRKY transcription factor family members in sorghum (Sorghum bicolor (L.) moench). PLoS One. 2020;15(8):e236651.
Wu ZJ, Li XH, Liu ZW, Li H, Wang YX, Zhuang J. Transcriptome-wide identification of Camellia sinensis WRKY transcription factors in response to temperature stress. Mol Gen Genomics. 2016;291(1):255–69.
Jiang J, Ma S, Ye N, Jiang M, Cao J, Zhang J. WRKY transcription factors in plant responses to stresses. J Integr Plant Biol. 2017;59(2):86–101.
Li W, Wang H, Yu D. Arabidopsis WRKY Transcription Factors WRKY12 and WRKY13 Oppositely Regulate Flowering under Short-Day Conditions. Mol Plant. 2016;9(11):1492–503.
Ma Z, Li W, Wang H, Yu D. WRKY transcription factors WRKY12 and WRKY13 interact with SPL10 to modulate age-mediated flowering. J Integr Plant Biol. 2020;62(11):1659–73.
Wang H, Avci U, Nakashima J, Hahn MG, Chen F, Dixon RA. Mutation of WRKY transcription factors initiates pith secondary wall formation and increases stem biomass in dicotyledonous plants. Proc Natl Acad Sci U S A. 2010;107(51):22338–43.
Huang R, Liu D, Huang M, Ma J, Li Z, Li M, et al. CpWRKY71, a WRKY Transcription Factor Gene of Wintersweet (Chimonanthus praecox), Promotes Flowering and Leaf Senescence in Arabidopsis. Int J Mol Sci. 2019;20(21):5325.
Wen F, Zhu H, Li P, Jiang M, Mao W, Ong C, et al. Genome-Wide Evolutionary Characterization and Expression Analyses of WRKY Family Genes in Brachypodium distachyon. DNA Res. 2014;21(3):327–39.
Potschin M, Schlienger S, Bieker S, Zentgraf U. Senescence Networking: WRKY18 is an Upstream Regulator, a Downstream Target Gene, and a Protein Interaction Partner of WRKY53. J Plant Growth Regul. 2014;33(1):106–18.
Dang FF, Wang YN, Yu L, Eulgem T, Lai Y, Liu ZQ, et al. CaWRKY40, a WRKY protein of pepper, plays an important role in the regulation of tolerance to heat stress and resistance to Ralstonia solanacearum infection. Plant Cell Environ. 2013;36(4):757–74.
Mao G, Meng X, Liu Y, Zheng Z, Chen Z, Zhang S. Phosphorylation of a WRKY transcription factor by two pathogen-responsive MAPKs drives phytoalexin biosynthesis in Arabidopsis. Plant Cell. 2011;23(4):1639–53.
Oh SK, Baek KH, Park JM, Yi SY, Yu SH, Kamoun S, et al. Capsicum annuum WRKY protein CaWRKY1 is a negative regulator of pathogen defense. New Phytol. 2008;177(4):977–89.
Park CJ, Shin YC, Lee BJ, Kim KJ, Kim JK, Paek KH. A hot pepper gene encoding WRKY transcription factor is induced during hypersensitive response to Tobacco mosaic virus and Xanthomonas campestris. PLANTA. 2006;223(2):168–79.
Sarris PF, Duxbury Z, Huh SU, Ma Y, Segonzac C, Sklenar J, Derbyshire P, Cevik V, Rallapalli G, Saucet SB, et al. A Plant Immune Receptor Detects Pathogen Effectors that Target WRKY Transcription Factors. Cell. 2015;161(5):1089–100.
Gao YF, Liu JK, Yang FM, Zhang GY, Wang D, Zhang L, et al. The WRKY transcription factor WRKY8 promotes resistance to pathogen infection and mediates drought and salt stress tolerance in Solanum lycopersicum. Physiol Plant. 2020;168(1):98–117.
Wang X, Li J, Guo J, Qiao Q, Guo X, Ma Y. The WRKY transcription factor PlWRKY65 enhances the resistance of Paeonia lactiflora (herbaceous peony) to Alternaria tenuissima. Hortic Res. 2020;7:57.
Zhu D, Hou L, Xiao P, Guo Y, Deyholos MK, Liu X. VvWRKY30, a grape WRKY transcription factor, plays a positive regulatory role under salinity stress. Plant Sci. 2019;280:132–42.
Ross CA, Liu Y, Shen QJ. The WRKY Gene Family in Rice (Oryza sativa). J Integr Plant Biol. 2007;49(6):827–42.
Schmutz J, Cannon SB, Schlueter J, Ma J, Mitros T, Nelson W, Hyten DL, Song Q, Thelen JJ, Cheng J, et al. Genome sequence of the palaeopolyploid soybean. Nature. 2010;463(7278):178–83.
Tripathi P, Rabara RC, Langum TJ, Boken AK, Rushton DL, Boomsma DD, et al. The WRKY transcription factor family in Brachypodium distachyon. BMC Genomics. 2012;13:270.
Xie T, Chen C, Li C, Liu J, Liu C, He Y. Genome-wide investigation of WRKY gene family in pineapple: evolution and expression profiles during development and stress. BMC Genomics. 2018;19(1):490.
Li L, Yao X, Zhong C, Chen X, Huang H. Akebia: A Potential New Fruit Crop in China. Hortscience. 2010;45(1):4–10.
Li L, Xu-Zhong C, Xiao-Hong Y. Geographic Distribution and Resource Status of Three Important Akebia Species. J Wuhan Botanical Res. 2010;30:497–506.
Sun R, Wang K, Guo T, Jones DC, Cobb J, Zhang B, et al. Genome-wide identification of auxin response factor (ARF) genes and its tissue-specific prominent expression in Gossypium raimondii. Funct Integr Genomics. 2015;15(4):481–93.
Robatzek S, Somssich IE. A new member of the Arabidopsis WRKY transcription factor family, AtWRKY6, is associated with both senescence- and defence-related processes. Plant J. 2001;28(2):123–33.
Sun W, Ma Z, Chen H, Liu M. Genome-wide investigation of WRKY transcription factors in Tartary buckwheat (Fagopyrum tataricum) and their potential roles in regulating growth and development. Peer J. 2020;8:e8727
Chen C, Chen X, Han J, Lu W, Ren Z. Genome-wide analysis of the WRKY gene family in the cucumber genome and transcriptome-wide identification of WRKY transcription factors that respond to biotic and abiotic stresses. BMC Plant Biol. 2020;20(1):443.
Yan H, Li M, Xiong Y, Wu J, Da Silva JAT, Ma G. Genome-Wide Characterization, Expression Profile Analysis of WRKY Family Genes in Santalum album and Functional Identification of Their Role in Abiotic Stress. Int J Mol Sci. 2019;20(22):5676.
Goyal P, Manzoor MM, Vishwakarma RA, Sharma D, Dhar MK, Gupta S. A Comprehensive Transcriptome-Wide Identification and Screening of WRKY Gene Family Engaged in Abiotic Stress in Glycyrrhiza glabra. Sci Rep-UK. 2020;10(1):373.
Guo C, Guo R, Xu X, Gao M, Li X, Song J, et al. Evolution and expression analysis of the grape (Vitis vinifera L.) WRKY gene family. J Exp Bot. 2014;65(6):1513–28.
Jing Z, Liu Z. Genome-wide identification of WRKY transcription factors in kiwifruit (Actinidia spp.) and analysis of WRKY expression in responses to biotic and abiotic stresses. Genes Genom. 2018;40(4):429–46.
Wu H, Ma T, Kang M, Ai F, Zhang J, Dong G, et al. A high-quality Actinidia chinensis (kiwifruit) genome. Hortic Res. 2019;6:117.
Zhang Y, Wang L. The WRKY transcription factor superfamily: its origin in eukaryotes and expansion in plants. BMC Evol Biol. 2005;5:1.
Wu KL, Guo ZJ, Wang HH, Li J. The WRKY family of transcription factors in rice and Arabidopsis and their origins. DNA Res. 2005;12(1):9–26.
Nan H, Gao LZ. Genome-Wide Analysis of WRKY Genes and Their Response to Hormone and Mechanic Stresses in Carrot. Front Genet. 2019;10:363.
Yao Q, Xia E, Liu F, Gao L. Genome-wide identification and comparative expression analysis reveal a rapid expansion and functional divergence of duplicated genes in the WRKY gene family of cabbage, Brassica oleracea var capitata. Gene. 2015;557(1):35–42.
Luo M, Dennis ES, Berger F, Peacock WJ, Chaudhury A. MINISEED3 (MINI3), a WRKY family gene, and HAIKU2 (IKU2), a leucine-rich repeat (LRR) KINASE gene, are regulators of seed size in Arabidopsis. Proc Natl Acad Sci U S A. 2005;102(48):17531–6.
Yu F, Huaxia Y, Lu W, Wu C, Cao X, Guo X. GhWRKY15, a member of the WRKY transcription factor family identified from cotton (Gossypium hirsutum L.), is involved in disease resistance and plant development. BMC Plant Biol. 2012;12:144.
Dong H, Tan J, Li M, Yu Y, Jia S, Zhang C, et al. Transcriptome analysis of soybean WRKY TFs in response to Peronospora manshurica infection. Genomics. 2019;111(6):1412–22.
Mahdi L, Huang M, Zhang X, Nakano RT, Kopp LBAS, Jacob F, et al. Plant mixed lineage kinase domain-like proteins limit biotrophic pathogen growth. bioRxiv. 2019:681015.
Park YS, Kim SK, Kim SY, Kim KM, Ryu CM. The transcriptome analysis of the Arabidopsis thaliana in response to the Vibrio vulnificus by RNA-sequencing. PLoS One. 2019;14(12):e225976.
Badet T, Voisin D, Mbengue M, Barascud M, Sucher J, Sadon P, et al. Parallel evolution of the POQR prolyl oligo peptidase gene conferring plant quantitative disease resistance. PLoS Genet. 2017;13(12):e1007143.
Abulfaraj AA, Mariappan K, Bigeard J, Manickam P, Blilou I, Guo X, et al. The Arabidopsis homolog of human G3BP1 is a key regulator of stomatal and apoplastic immunity. Life Sci Alliance. 2018;1(2):e201800046.
Nasim Z, Fahim M, Gawarecka K, Susila H, Jin S, Youn G, et al. Role of AT1G72910, AT1G72940, and ADR1-LIKE 2 in Plant Immunity under Nonsense-Mediated mRNA Decay-Compromised Conditions at Low Temperatures. Int J Mol Sci. 2020;21(21):7986.
Menna A, Dora S, Sancho-Andres G, Kashyap A, Meena MK, Sklodowski K, et al. A primary cell wall cellulose-dependent defense mechanism against vascular pathogens revealed by time-resolved dual transcriptomics. BMC Biol. 2021;19(1):161.
Liu T, Cao L, Cheng Y, Ji J, Wei Y, Wang C, et al. MKK4/5-MPK3/6 Cascade Regulates Agrobacterium-Mediated Transformation by Modulating Plant Immunity in Arabidopsis. Front Plant Sci. 2021;12:731690.
Rausch S. The function of Arabidopsis microRNAs in defense against the necrotrophic fungal pathogen Alternaria brassicicola. PhD thesis. Tübingen: Eberhard Karls Universität Tübingen; 2018. https://doi.org/10.15496/publikation-16913.
Lai Z, Vinod K, Zheng Z, Fan B, Chen Z. Roles of Arabidopsis WRKY3 and WRKY4 transcription factors in plant responses to pathogens. BMC Plant Biol. 2008;8:68.
Zheng Z, Qamar SA, Chen Z, Mengiste T. Arabidopsis WRKY33 transcription factor is required for resistance to necrotrophic fungal pathogens. Plant J. 2006;48(4):592–605.
Hu Y, Dong Q, Yu D. Arabidopsis WRKY46 coordinates with WRKY70 and WRKY53 in basal resistance against pathogen Pseudomonas syringae. Plant Sci. 2012;185-186:288–97.
Lai Z, Li Y, Wang F, Cheng Y, Fan B, Yu JQ, et al. Arabidopsis sigma factor binding proteins are activators of the WRKY33 transcription factor in plant defense. Plant Cell. 2011;23(10):3824–41.
Birkenbihl RP, Diezel C, Somssich IE. Arabidopsis WRKY33 is a key transcriptional regulator of hormonal and metabolic responses toward Botrytis cinerea infection. Plant Physiol. 2012;159(1):266–85.
Jiang Y, Guo L, Ma X, Zhao X, Jiao B, Li C, et al. The WRKY transcription factors PtrWRKY18 and PtrWRKY35 promote Melampsora resistance in Populus. Tree Physiol. 2017;37(5):665–75.
Chen C, Chen Z. Potentiation of developmentally regulated plant defense response by AtWRKY18, a pathogen-induced Arabidopsis transcription factor. Plant Physiol. 2002;129(2):706–16.
Kim KC, Fan B, Chen Z. Pathogen-induced Arabidopsis WRKY7 is a transcriptional repressor and enhances plant susceptibility to Pseudomonas syringae. Plant Physiol. 2006;142(3):1180–92.
Xing DH, Lai ZB, Zheng ZY, Vinod KM, Fan BF, Chen ZX. Stress- and pathogen-induced Arabidopsis WRKY48 is a transcriptional activator that represses plant basal defense. Mol Plant. 2008;1(3):459–70.
Han X, Zhang L, Zhao L, Xue P, Qi T, Zhang C, et al. SnRK1 Phosphorylates and Destabilizes WRKY3 to Enhance Barley Immunity to Powdery Mildew. Plant Commun. 2020;1(4):100083.
Liu F, Li X, Wang M, Wen J, Yi B, Shen J, et al. Interactions of WRKY15 and WRKY33 transcription factors and their roles in the resistance of oilseed rape to Sclerotinia infection. Plant Biotechnol J. 2018;16(4):911–25.
Delteil A, Blein M, Faivre-Rampant O, Guellim A, Estevan J, Hirsch J, et al. Building a mutant resource for the study of disease resistance in rice reveals the pivotal role of several genes involved in defence. Mol Plant Pathol. 2012;13(1):72–82.
De Almeida DSM, Oliveira Jordão Do Amaral D, Del-Bem L, Bronze Dos Santos E, Santana Silva RJ, Peres Gramacho K, et al. Genome-wide identification and characterization of cacao WRKY transcription factors and analysis of their expression in response to witches' broom disease. PLoS One. 2017;12(10):e187346.
Chujo T, Miyamoto K, Shimogawa T, Shimizu T, Otake Y, Yokotani N, et al. OsWRKY28, a PAMP-responsive transrepressor, negatively regulates innate immune responses in rice against rice blast fungus. Plant Mol Biol. 2013;82(1–2):23–37.
Kulmanov M, Schofield PN, Gkoutos GV, Hoehndorf R. Ontology-based validation and identification of regulatory phenotypes. Bioinformatics. 2018;34(17):i857–65.
Cheng L, Lin H, Hu Y, Wang J, Yang Z. Gene function prediction based on the Gene Ontology hierarchical structure. PLoS One. 2014;9(9):e107187.
Hsu FC, Chou MY, Chou SJ, Li YR, Peng HP, Shih MC. Submergence confers immunity mediated by the WRKY22 transcription factor in Arabidopsis. Plant Cell. 2013;25(7):2699–713.
Wang Y, Schuck S, Wu J, Yang P, Doring AC, Zeier J, et al. A MPK3/6-WRKY33-ALD1-Pipecolic Acid Regulatory Loop Contributes to Systemic Acquired Resistance. Plant Cell. 2018;30(10):2480–94.
Kanehisa M, Goto S. KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 2000;28(1):27–30.
Kanehisa M. Toward understanding the origin and evolution of cellular organisms. Protein Sci. 2019;28(11):1947–51.
Kanehisa M, Furumichi M, Sato Y, Ishiguro-Watanabe M, Tanabe M. KEGG: integrating viruses and cellular organisms. Nucleic Acids Res. 2021;49(D1):D545–51.
Chen J, Yin Y. WRKY transcription factors are involved in brassinosteroid signaling and mediate the crosstalk between plant growth and drought tolerance. Plant Signal Behav. 2017;12(11):e1365212.
Acknowledgements
We especially appreciate the conversation with the members of our group in developing some of the ideas presented in this study.
Funding
This work was supported by National Natural Science Foundation of China [31560075, 31760047, 31960041], Natural Science Foundation of Jiangxi Province [20202BABL203045], and Open Project of Key Laboratory of Biopesticides and Chemical Biology, Ministry of Education, Fujian Agriculture and Forestry University [Keylab2020–01].
Author information
Authors and Affiliations
Contributions
FW designed the experiments and organized the manuscript. FW, and XW wrote the manuscript. FW, XW, TL, and MJ performed the analyses. FW and LL edited the manuscript. All the authors discussed the results and contributed to the manuscript. All authors read and approved the final manuscript.
Corresponding author
Ethics declarations
Ethics approval and consent to participate
No specific permits were required for the plant collection. The locations were not privately-owned or protected in any way. The Akebia trifoliata is not a protected plant in the List of Chinese Key Protected Wild Plants (http://www.plant.csdb.cn/protectlist), and our collection and experiment comply with the REGULATIONS OF THE PEOPLE’S REPUBLIC OF CHINA ON WILD PLANTS PROTECTION.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Additional information
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Additional file 1.
The CDs and amino acid sequences of WRKYs in A. trifoliata.
Additional file 2.
The number of WRKY genes belonging to each subgroup in different species.
Additional file 3.
Expression data of the AktWRKY family genes after C. acutatum infection.
Additional file 4.
The list of primer-sets of AktWRKY genes for qRT-PCR.
Additional file 5.
Expression patterns of AtWRKY genes in response to biotic stresses obtained from the GEO (A) and the Arabidopsis eFP Browser (B). (A) Expression profiles of AtWRKY genes in leaves (12, 24, 48 and 72 hpi) under Vibrio vulnificus (GSE61418); mature leaves (24 hpi) under Sclerotinia sclerotiorum (GSE106811); rosette leaves (3dpi) under Alternaria brassicicola (GSE83478); leaves (24 hpi) under Pst DC3000 hrcC (GSE107786); leaves (48 hpi) under Golovinomyces orontii (GSE129011); seedlings (0.5 hpi) under flg22 (GSE146189); roots (1, 2, 3, 4 and 6 dpi) under Fusarium oxysporum (GSE168015); and seedlings (24 hpi) under Agrobacterium (GSE179628). (B) Expression profiles of AtWRKY genes in leaves under phytopathogens. Bc, Botrytis cinerea; Ps, Pseudomonas syringae; Pi, Phytophthora infestans; Eo, Erysiphe orontii. The red represented up-regulated genes, while the blue represented down-regulated genes. The gray block indicated missing data.
Additional file 6.
The expression level of AktWRKY genes provided as -ΔCt values in three different varieties of A. trifoliata after C. acutatum infection.
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.
About this article
Cite this article
Wen, F., Wu, X., Li, T. et al. Characterization of the WRKY gene family in Akebia trifoliata and their response to Colletotrichum acutatum. BMC Plant Biol 22, 115 (2022). https://doi.org/10.1186/s12870-022-03511-1
Received:
Accepted:
Published:
DOI: https://doi.org/10.1186/s12870-022-03511-1