Figures
Abstract
Oryza officinalis Wall ex Watt, a very important and special wild rice species, shows abundant genetic diversity and disease resistance features, especially high resistance to bacterial blight. The molecular mechanisms of bacterial blight resistance in O. officinalis have not yet been elucidated. The WRKY transcription factor family is one of the largest gene families involved in plant growth, development and stress response. However, little is known about the numbers, structure, molecular phylogenetics, and expression of the WRKY genes under Xanthomonas oryzae pv. oryzae (Xoo) stress in O. officinalis due to lacking of O. officinalis genome. Therefore, based on the RNA-sequencing data of O. officinalis, we performed a comprehensive study of WRKY genes in O. officinalis and identified 89 OoWRKY genes. Then 89 OoWRKY genes were classified into three groups based on the WRKY domains and zinc finger motifs. Phylogenetic analysis strongly supported that the evolution of OoWRKY genes were consistent with previous studies of WRKYs, and subgroup IIc OoWRKY genes were the original ancestors of some group II and group III OoWRKYs. Among the 89 OoWRKY genes, eight OoWRKYs displayed significantly different expression (>2-fold, p<0.01) in the O. officinalis transcriptome under Xoo strains PXO99 and C5 stress 48 h, suggesting these genes might play important role in PXO99 and C5 stress responses in O. officinalis. QRT-PCR analysis and confirmation of eight OoWRKYs expression patterns revealed that they responded strongly to PXO99 and C5 stress 24 h, 48 h, and 72 h, and the trends of these genes displaying marked changes were consistent with the 48 h RNA-sequencing data, demonstrated these genes played important roles in response to biotic stress and might even involved in the bacterial blight resistance. Tissue expression profiles of eight OoWRKY genes revealed that they were highly expressed in root, stem, leaf, and flower, especially in leaf (except OoWRKY71), suggesting these genes might be also important for plant growth and organ development. In this study, we analyzed the WRKY family of transcription factors in O.officinalis. Insight was gained into the classification, evolution, and function of the OoWRKY genes, revealing the putative roles of eight significantly different expression OoWRKYs in Xoo strains PXO99 and C5 stress responses in O.officinalis. This study provided a better understanding of the evolution and functions of O. officinalis WRKY genes, and suggested that manipulating eight significantly different expression OoWRKYs would enhance resistance to bacterial blight.
Citation: Jiang C, Shen QJ, Wang B, He B, Xiao S, Chen L, et al. (2017) Transcriptome analysis of WRKY gene family in Oryza officinalis Wall ex Watt and WRKY genes involved in responses to Xanthomonas oryzae pv. oryzae stress. PLoS ONE 12(11): e0188742. https://doi.org/10.1371/journal.pone.0188742
Editor: Raman Meenakshi Sundaram, ICAR-Indian Institute of Rice Research, INDIA
Received: June 23, 2017; Accepted: November 13, 2017; Published: November 30, 2017
Copyright: © 2017 Jiang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: This study was supported by the NSFC-Yunnan Project (U1302265) and NSFC Project (31460054 to XK, 31460478 to WL, 31401707 to SX) and granted from Yunnan Key Project (2012CI) to XQH.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Since the first WRKY gene of sweet potato is reported in 1994 [1], many WRKY genes have been identified from a wide variety of plant genome [2–7]. A large majority of WRKY genes functions as a positive or negative regulator's response to various biotic and abiotic stresses in plant [8–9]. WRKY have been demonstrated to mediate the regulatory network of salicylic acid (SA), jasmonic acid (JA), gibberellins (GA), abscisic acid (ABA) and ethylene (ET) [10–15], and reported to play an important role in resistance against diseases caused by bacteria or fungi [16]. In rice, 35 WRKYs of which OsWRKY3, OsWRKY13, OsWRKY28, OsWRKY30, OsWRKY31, OsWRKY45, OsWRKY53, OsWRKY70 and OsWRKY71 play important regulatory role in disease resistance-related pathways of bacterial blight, rice blast and sheath blight, and are induced by pathogen [17]. Over-expressing OsWRKY53 and OsWRKY71 enhanced the resistance of transgenic rice against fungal blast and bacterial blight, respectively [18–19]. In addition, WRKY genes also play an important role in the control of leaf senescence, seed formation, dormancy and germination [11, 20–21].
WRKY transcription factors, which have a significant character, contain one or two WRKY domains that are composed of a WRKY motif and a zinc finger motif [3]. The core sequence of a WRKY motif is WRKYGQK with some variants including WRKYGKK, WRKYGEK, WRKYGRK, WKKYGQK, WKRYGQK and WSKYEQK. Zinc fingers include two types of C2H2 motif (C-X4-5-C-X22-23-H-X1-H) and C2HC motif (C-X5–7-C-X23-H-X1-C) [3]. Based on the number of WRKY domains and zinc finger motifs, WRKY genes are usually divided into Ⅰ, Ⅱ and Ⅲ group [2]. Group Ⅰ has two WRKY domains, and is divided into two subgroups, Ia containing C2H2 zinc fingers and Ib containing C2HC zinc fingers; group Ⅱ, which containing one WRKY domain with C2H2 zinc finger, are divided into subgroup Ⅱa, Ⅱb, Ⅱc, Ⅱd and Ⅱe based on their phylogenetic relationship; group III has one WRKY domain with C2HC zinc finger. After analysis of WRKY transcription factor family in Arabidopsis thaliana and rice genomes, the classification of WRKY genes group II is further optimized that the subgroup Ⅱa, Ⅱb, Ⅱd and Ⅱe merged into Ⅱa+b and Ⅱd+e [3–4].
Oryza officinalis Wall ex Watt is one of the important and special wild rice species in Oryza genus. It is one of the three wild species indigenous in China and shows abundant genetic diversity and disease resistance features, especially high resistance to bacterial blight [22–23]. The molecular mechanisms of bacterial blight resistance in O. officinalis have not yet been elucidated. WRKY genes play an important role in resistance against diseases caused by bacteria or fungi [16], but there is no analysis of O.officinalis WRKYs due to lack of genome information. Therefore, based on the O. officinalis transcriptome under the strains PXO99 and C5 of Xanthomonas oryzae pv. oryzae (Xoo) stress, 89 genes encoding WRKY transcription factors (OoWRKY) were identified, and the analysis of their structure, molecular phylogenetics, conserved motifs, and stimulation in response to Xoo strains PXO99 and C5 were performed. The results provided insights into the evolution of O. officinalis WRKYs and their functions in Xoo stress responses. To our knowledge, this is the first report of O. officinalis WRKY genes.
Materials and methods
Plant materials and treatments
Oryza officinalis Wall ex Watt (CC, 2n = 2x = 24), strains PXO99 and C5 of Xanthomonas oryza pv. oryza (Xoo) were provided by the Biotechnology & Genetic Germplasm Institute, Yunnan Academy of Agricultural Sciences, Yunnan, China. PXO99 was a Philippines hypervirulent Xoo strain, and C5 was the hypervirulent and respresentative strain in China. The O. officinalis had strong resistance to C5, but its resistance to PXO99 was weak (unpulished data). Xoo strains PXO99 and C5 were used to inoculate O. officinalis.
The PXO99 and C5 were cultured on Nutrient Agar medium at 28°C for 48 h. The bacterial strains were suspended in sterile water, adjusted the concentration of bacterial liquid to OD600 = 0.8~1.0 using the NanoDrop2000, and then used to inoculate O. officinalis leaves. O. officinalis were respectively inoculated with strains PXO99 and C5 by leaf-clipping method [24] at 14: 00~15: 00 under 28~30°C room temperature. The control experiment of plant leaves were cut with sterile water (ddH2O) instead of bacterial liquid by the same method. The treated leaves were collected at 48 h and used for RNA-seq. In addition, the treated leaves were collected at 0 h, 24 h, 48 h, 72 h, 96 h, 120 h respectively, and used for analysis of O. officinalis WRKY genes expression in response to Xoo strains PXO99 and C5 by qRT-PCR.
For analysis of WRKY genes expression in different organs using qRT-PCR, the root, stem, leaf and flower of O. officinalis were collected under normal growth conditions and used for extraction of total RNA.
Analysis of O. officinalis WRKY genes expression in response to Xoo using transcriptomic data
Total RNA was extracted from the leaves inoculated respectively with ddH2O, strains PXO99 and C5 for 48 h according to the manufacturer's instructions of RNeasy Plant Mini Kit and the RNase-Free DNase Set (Qiagen, Germany). RNA pools were constructed using 3 μg of RNA per sample according to the manufacturer’s instructions and sequenced on an Illumina HiSeq 4000 (Illumina, Inc., San Diego, CA, USA) next-generation platform technology. Gene expression levels were calculated in FPKM (Fragments Per Kilobase Per million Fragments mapped) and the FPKM values for each gene in all samples were log10 transformed [25]. Finally, a heat map was generated using TreeView software [26].
Identification of the O. officinalis WRKY genes
BLAST analysis with the O. sativa ssp. Japonica WRKY genes were used to check the predicted WRKY genes from the O. officinalis database. A hidden Markov model (HMM) was constructed using the O. sativa WRKY amino acid sequences [27]. All the potential O. officinalis WRKY proteins were used to identify by HMMER3.1 and BLAST if they contained a WRKY domain. And then the CDD and PFAM databases were used to validate all the potential O.officinalis WRKY genes.
Classification of the O. officinalis WRKY genes
The protein sequences of O. officinalis WRKY genes were aligned by Clustal X2.1 and classified into groups based on the numbers of WRKY domains and zinc finger motifs [2–4]. In brief, O. officinalis WRKY genes were divided into groups' Ⅰ, Ⅱ and Ⅲ. Group Ⅰ contained two WRKY domains and was divided into two subgroups based on zinc finger types, including subgroup Ⅰa with C2H2 zinc finger and subgroup Ⅰb with C2HC zinc finger. The group Ⅱ contained one WRKY domain with C2H2 zinc finger. Group Ⅲ contained one WRKY domain with C2HC zinc finger.
The analysis of protein sequence motifs in O. officinalsis WRKY proteins
MEME (http://meme-suite.org/tools/meme) was used to predict and analyze motifs of O. officinalis WRKY proteins [28]. The numbers of motif were chosen 10 motifs; the motif widths were set between 6 and 50. The other parameters were set to default values.
Phylogenetic analysis
The WRKY domain and full-length protein sequence of O. officinalis WRKY genes were used to analyze their phylogenetic relationship. The multiple protein sequences of O.officinalis WRKY genes were aligned by MUSCLE in MEGA6.0 with default parameters. The neighbor-joining (NJ) phylogenetic trees were constructed based on the aligned results of O. officinalis WRKY domain and full-length protein sequences using MEGA6.0 with bootstrap replications of 1000.
QRT-PCR analysis of O. officinalis WRKY genes expression in response to Xoo and in different organs
The total RNA was extracted with the Omega plant RNA kit (Omega Bio-Tek, Georgia, USA) according to the instructions provided by the manufacturer. A total of 1 μg of RNA was reverse-transcribed into cDNA using PrimeScript RT reagent with the gDNA Eraser kit (TaKaRa, Dalian, China). A control amplicon was generated using the β-actin primers for amplification of β-actin (S1 Table). The primers of O. officinalis WRKY genes were designed by the online tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast) (S1 Table). Gene expression levels were determined by perfoming quantitative real-time polymerase chain reaction (qRT-PCR) in Applied Biosystems QuantStudio 6 Flex (ABI, USA) using SYBR Premix Ex Taq II (TaKaRa) according to the manufacturer's instructions. Datas were analyzed by QuantStudio 6 Flex software (ABI, USA) and the 2-△△CT method [29].
Results
Identification of OoWRKY genes in O. officinalis
All the O. sativa ssp. Japonica WRKY protein sequences were used as queries for the BLAST to identify O. officinalis WRKY proteins. HMM search was also performed against the O. officinalis proteins using WRKY domains. In total, 89 WRKY genes were identified after excluding the WRKYs of repeatable sequences and incomplete WRKY domains. These 89 WRKY genes were named OoWRKY1 to OoWRKY125 corresponding to the names of their orthologs in O. sativa. The details of these OoWRKY genes, such as their locus numbers, types of the encoded WRKY domains and sizes of the deduced peptides, were listed in S2 Table. The 89 OoWRKY proteins ranged from 141 (OoWRKY60) to 1,252 (OoWRKY125) amino acids (aa) in length, with an average length of approximately 370 aa. The MSU IDs of the O. sativa orthologs can also be found in S2 Table.
Classification and phylogenetic analysis of the OoWRKY genes
The 89 OoWRKY genes were classified into different groups and subgroups according to published methods [2, 27]. Thirteen OoWRKY genes with two WRKY domains were belong to group I, which further divided into Ia and Ib based on the zinc finger motifs of C2H2-type (C-X4–5-C-X22–23-H-X1-H) and C2HC-type (C-X7-10-C-X23-H-X1-C), respectively; 50 group II OoWRKY genes, containing a C2H2-type (C-X4–5-C-X22–24-H-X1-H) zinc finger motif, were divided into five subgroups, including 11 group IIa+b, 22 group IIc and 17 group IId+e; 25 OoWRKY genes were belong to group III with a C2HC-type (C-X5–7-C-X23–33-H-X1-C) zinc finger motif (Fig 1). In addition, OoWRKY118 was not able to classify into any other groups, although it had a WRKY domain and a zinc finger structure. All the WRKY domains and the zinc finger sequence of OoWRKY genes were showed as S1 File.
In order to analysis of the phylogenetic relationship of the OoWRKYs, a neighbor-joining (NJ) phylogenetic tree was constructed by using MEGA6.0 for the multiple sequence alignment of all OoWRKY domains with 1000 bootstrap analysis (Fig 2, S2 File). The NJ tree of OoWRKY domains was clustered into three groups (I, II, and III) (Fig 2). The N-terminal domains and C-terminal domains of subgroup Ia OoWRKYs were clustered into clade IaN and IaC respectively. Group II was diverged into four clades, of which subgroups IIa and IIb clustered into one clade, subgroups IId and IIe clustered into another, and subgroup IIc clustered into IIc1 and IIc2 clades. Most of the subgroup IIc OoWRKY domains were clustered into IIc1 neighbored the IaC clade, while the subgroup IIc OoWRKY90 and OoWRKY57 were clustered within the IaC and IaN clades respectively, which demonstrated a close evolutionary relationship between the subgroup IIc and subgroup Ia genes. The Group III OoWRKY genes were clustered into one clade including three subgroup Ib OoWRKYs. OoWRKY118 was scattered amongst the different clades, fell outside of IIa+b and IIc1 clades, which suggested it didn't belong to these subgroups.
The WRKY domain sequences were aligned by MUSCLE in the MEGA6 using the default parameters. The consensus NJ tree was shown by the results of 1,000 bootstrap replications. Bootstrap values were displayed with nodes. Group Ia: hollow diamond; Group Ib: filled triangle; Group IIa: circle; Group IIb: filled circle; Group IIc: filled diamond; Group IId: filled square; Group IIe: square; Group III: triangle; OoWRKY118: red filled triangle.
The phylogenetic tree of OoWRKY domains might miss important information on the evolution of OoWRKY genes. Therefore, a NJ phylogenetic tree was constructed using the multiple sequence alignment of the full-length OoWRKY proteins (Fig 3, S3 File). The full-length proteins' phylogenetic tree was similar to the domain tree, but there were two main differences between two NJ trees. In the OoWRKY domains tree, the subgroup Ia OoWRKY4 was clustered in the IaN and IaC clades, while OoWRKY4 and OoWRKY57 (IIc) were clustered into one clade in the NJ tree of full-length OoWRKY proteins, strongly supported by a high bootstrap value (87). In addition, subgroup IIe OoWRKY 109 was clustered into IIe clade in OoWRKY domains tree, but fell out of IIe clade in the NJ tree of full-length OoWRKY proteins as OoWRKY118, supported by low bootstrap values (<20).
The amino acid sequences were aligned by MUSCLE in the MEGA6 using the default parameters. The NJ tree was shown by the results of 1,000 bootstrap replications. Bootstrap values were displayed with nodes. Group Ia: hollow diamond; Group Ib: filled triangle; Group IIa: circle; Group IIb: filled circle; Group IIc: filled diamond; group IId: filled square; Group IIe: square; Group III: triangle; OoWRKY118: red filled triangle.
WRKY motifs analysis of OoWRKY proteins
Most of OoWRKY proteins had a conserved WRKY motif, but some of them had a variant motif such as WKKY, WVKY, WRMC and WIKY (S2 Table). The sequence W(R/K) (K/R) Y is also recommended as the consensus sequence for the WRKY motif [27]. For example, the OoWRKY proteins had variant WRKY motifs of WKKY (OoWRKY60), WIKY (OoWRKY63 and OoWRKY125), while OoWRKY61 had two variant motifs of WVKY and WNKY. In addition, OoWRKY118 had a variant motif of WRMC, which was different from W(R/K) (K/R) Y motifs. Although these OoWRKY proteins had a variant WRKY motif, they also contained the conserved zinc finger motifs.
Similarly, the WRKYGQK heptamer was present in the WRKY domain about 83% of OoWRKY proteins. WRKYGQK variants such as WVKYGQK (OoWRKY61), WNKYGQK (OoWRKY61) and WIKYDQK (OoWRKY63, -125) were found in subgroupⅠb OoWRKY proteins. The WRKYGKK (OoWRKY7, -10, -26, -67 and -77) and WKKYGQK (OoWRKY60) motifs were found in subgroup IIc OoWRKY proteins. OoWRKY46, -52, -55, -84, -97 and -114 with WRKYGEK were found in group III. In addition, OoWRKY118, falling outside of group Ⅰ, II, and III, had a variant WRMCGQN that different from other WRKYGQK heptamer. The variant WRKYGQK of OoWRKYs was exhibited in S1 Fig with red box and S2 Table.
Analysis of conserved motifs in OoWRKY proteins
MEME4.11.4 online software was used to analyze OoWRKY protein motifs, and Fig 4 and S2 Fig showed the ten conservative motifs in OoWRKY proteins. The same group of OoWRKYs had substantially consistent conserved motifs, which indicated there might be similar genetic functions. In subgroup Ⅰa, the C-terminal WRKY domains were consisted of motif 1, motif 2 and motif 3, while N-terminal domain only had motif 1. The WRKY domains of subgroup Ⅰb and group Ⅲ were composed of motif 1 and motif 4. The WRKY domains consisting of different motifs in subgroupⅠa and subgroup Ⅰb suggested that functional differentiation might occur in the groupⅠOoWRKY genes. The WRKY domains of subgroups' Ⅱa, Ⅱb, Ⅱc, and Ⅱe were consisted of similar motifs (mainly motif 1 + motif 3 ± motif 2), while the WRKY domains of Ⅱd were mainly consitented of motif 1 and motif 5, indicating subgroups Ⅱd might have specific function.
The NJ tree was constructed from the amino acid sequences of OoWRKYs using MUSCLE and MEGA6 with 1,000 bootstrap replications. The conserved motifs in the OoWRKY proteins were identified by MEME. In total, ten conserved motifs were identified and showed in different colors.
The expressed analysis of OoWRKY genes based on RNA-seq data
Based on the RNA-seq data analysis of O. officinalis, the expression level of 89 OoWRKY genes changed in the O. officinalis under Xoo stress at 48 h (Fig 5A), however, only eight OoWRKY genes were >2-fold up-regulated or down-regulated (p<0.01) (Fig 5B). Among these eight OoWRKY genes, six OoWRKY genes, including OoWRKY3, OoWRKY13, OoWRKY26, OoWRKY30, OoWRKY53 and OoWRKY70, displayed increases in expression by >2-fold under PXO99 and C5 stress 48 h. In contrast, OoWRKY111 was significantly down-regulated (>2-fold, p<0.01) under PXO99 and C5 stress 48 h. In addition, OoWRKY71 displayed increases in expression by <2-fold (p<0.01) under PXO99 stress, while increases in expression by >2-fold (p<0.01) were observed under C5 stress. These eight genes displayed significant changes in expression might play important roles in PXO99 and C5 stress responses.
(A) The digital gene expression profiling of 89 OoWRKY genes in the CK, PX and CF transcript. The CK, PX and CF were dealt with ddH2O, Xoo strains PXO99 and C5 for 48 h respectively. Transcriptome data (Fragments Per Kilobase per Million fragments mapped, FPKM) was used to measure the expression levels of OoWRKY genes. (B) The selected significantly differential expression OoWRKY genes under PXO99 and C5 stress 48 h. One and two asterisks respectively indicated significant difference (< 2-fold, p<0.01) and extremely significant difference (> 2-fold, p<0.01) in gene expression in O. officinalis treated with PXO99 and C5 compared with the treated with ddH2O.
Expression patterns of selected eight OoWRKY genes in response to Xoo strains PXO99 and C5
In order to confirm some of the OoWRKY genes important for bacterial blight resistance, eight significantly differential expression OoWRKY genes were selected and their expression patterns were quantified by qRT-PCR at different time points under Xoo strains PXO99 and C5 stress. As shown in the Fig 6, seven OoWRKY genes were significantly un-regulated, while one OoWRKY displayed marked decreases in expression under Xoo stress (p<0.05). The expression levels of OoWRKY13, OoWRKY53, OoWRKY70 and OoWRKY71 were strongly increased under Xoo stress, especially at 72 h (Fig 6, red square frame).The expression of OoWRKY71 under PXO99 stress 72 h was 20 times higher than that of 0 h. In contrast, the expression of OoWRKY111 kept decreasing during PXO99 and C5 treatment, while displayed increases during the ddH2O treatment (CK). Interestingly, the expression patterns of OoWRKY3 reversed in response to PXO99 and C5, which was significantly up-regulated under PXO99 stress 24 h and 48 h, while significantly down-regulated under C5 stress 72 h, 96 h and 120 h. OoWRKY26 was significantly up-regulated under PXO99 and C5 stress, but the expression level was lower than that of treated with ddH2O (CK) from 72 h to 120 h. Except OoWRKY13 and OoWRKY111, overall expressed tendency of other six OoWRKYs was significantly up-regulated under Xoo stress. The OoWRKYs, except OoWRKY111, were not significantly change at the different treatment time points in CK. The qRT-PCR results of the eight OoWRKYs under Xoo stress 48 h were consistent with the RNA-seq data (Fig 6, black square frame), which further demonstrated the reliability of our RNA-seq data. The expressed tendency of the same OoWRKY gene was almost unanimous under PXO99 and C5 stress, whether it was up-regulated or down-regulated.
The relative expression levels of eight OoWRKY genes were measured by PXO99 and C5 stress 24 h, 48 h, 72 h, 96 h and 120 h compared with 0 h, respectively. Three independent replicates were used to generate each expression value. The error bars represented standard deviations. One and two asterisks respectively represented significantly different (p<0.05, n = 3) and extremely significant difference (p<0.01, n = 3) when assessed by Duncan's multiple range test.
The organ specific expression of eight differentially expressed OoWRKY genes
To confirm the functions of eight differentially expressed OoWRKY genes under PXO99 and C5 stress, we analyzed the expression of these OoWRKY genes in root, stem, leaf and flower of O. officinalis using qRT-PCR. The results showed that the eight OoWRKY genes were constitutive expression genes in root, stem, leaf and flower of O. officinalis (Fig 7). The expression levels of OoWRKY71 and OoWRKY111 were lower in leaf than that of in root, especially OoWRKY71, while the expression level of OoWRKY111 was similar in stem, leaf and flower. The expression levels of other six OoWRKY genes were the highest in leaf, which was 3~400 times more than that of in root. The results suggested that OoWRKY111 mainly functioned in root, while the other six OoWRKYs mainly functioned in leaf in the normal growth.
The relative expression levels of eight OoWRKY genes were measured by the expression of stem, leaf and flower compared with root respectively. Three independent replicates were used to generate each expression value. The error bars represented standard deviations.
Discussion
The important of identification of O. officinalis WRKY genes
The WRKY transcription factors are one of the largest gene families, which involving in a wild variety of functions including plant development and stress response, and WRKY genes have been identified in many species, including kinds of plants, green algae, protozoa and slime mold [11]. Although WRKY genes of cultured rice and wild rice species O. nivara have many research reported [30], the WRKY genes in wild rice species of O. officinalis have not been studied. O. officinalis shows abundant genetic diversity and high resistance to bacterial blight [22–23, 31]. Thus, research on the WRKY genes of O. officinalis could not only provide a reference and theoretical basis for revealing the role of O. officinalis WRKY genes in the response mechanism of high resistance to bacterial blight, but also enrich the plant WRKY families.
In this study, a total of 89 OoWRKY genes were firstly identified from the transcriptome of O. officinalis by using an HMM model. The 89 OoWRKY genes were classified into groups' Ⅰ, Ⅱ and Ⅲ based on the number of WRKY domains and zinc finger motifs of WRKY domain sequences. Although OoWRKY118 had a WRKY domain and a zinc finger motif, OoWRKY118 could not be classified into group Ⅱ or Ⅲ for its WRKY domain and zinc finger type different from other OoWKRY genes. Comparison of the sequences of WRKY domains between OoWRKY118 and OsWRKY118 (LOC_Os08g09900.1), there were same variant WRMC and zinc finger motifs (S3 Fig), suggesting the difference sequence of OoWRKY118 was not due to transcriptome assembling of O. officinalis. However, the OoWRKY numbers of identified from O. officinalis transcriptome database (670 Mb, 89 WRKY genes) was small compared with the O. sativa (440 Mb, 103 WRKY genes) and Arabidopsis (107 Mb, 72 WRKY genes), but our results provided a solid foundation for the study of the evolution and functions of O. officinalis WRKY genes.
The evolution analysis of O. officinalis WRKY genes
The diversification of WRKYs classified into groups' Ⅰ, Ⅱ, Ⅲ and Ⅳ results from a long evolutionary history. There have been proposed several hypotheses to explain the evolution of WRKY genes. Hypothesis 1 proposes that subgroup Ia genes are the ancestor of other WRKY genes [4]. Hypothesis 2 propose that IIc-like genes are the common ancestor of subgroups Ia and IIc genes, and the other WRKYs evolve from subgroup Ia genes [32]. Hypothesis 3 proposes subgroup Ia genes are the most primitive ancestor and all other WRKYs evolve from the C-terminal domain of the subgroup Ia genes. Meanwhile, hypothesis 3 also suggests subgroups Ⅱ WRKY genes evolve directly from WRKYs that containing one WRKY domain [33]. Xu et al [30] propose that subgroup IIc genes are the original ancestor of WRKYs, and group I, Ⅲ and the other subgroup II WRKYs evolve from IIc genes.
Almost all of hypotheses could explain the evolution of OoWRKYs. Subgroup IIc WRKYs are ancestor of all WRKYs and display most diverse [4, 32]. The subgroup IIc OoWRKY genes were clustered into IIc1 and IIc2 clades in the phylogenetic analysis, indicating subgroup IIc OoWRKY genes were diverse. It was generally suggested that subgroup IIc WRKY genes evolved from C-terminal WRKY domain of subgroup Ia genes by losing the N-terminal WRKY domain. The subgroup IIc OoWRKY domain sequences were very similar to C-terminal OoWRKY domains of subgroup Ia, and they were many amino acids align identically (S4 Fig). In addition, subgroup IIc1 OoWRKY genes were clustered nearby the subgroup Ia. Subgroup IIc OoWRKY90 was clustered into IaC clade (Fig 2), which indicated that OoWRKY90 might evolve from the C-terminal domains of subgroup Ia. However, our results were also approved the hypothesis 2 that subgroup IIc genes were the ancestor of other WRKY genes. Most of subgroup IIc OoWRKY genes in IIc1 clade contained an RVE sequence in their zinc finger motifs, while most of group I OoWRKYs contained an HVE sequence in their zinc finger motifs (S4 Fig). OoWRKY90 contained an HVE sequence in its zinc finger motif, which might be the reason OoWRKY90 (IIc1) clustered into IaC in the phylogenetic tree. In addition, the subgroup IIc2 of OoWRKY56 and OoWRKY119 contained the same HVE sequence as IaC in their zinc finger motifs, indicating the subgroup Ia genes might evolve from subgroup IIc2 WRKYs by double WRKY domain. The close relationship between IIc and IaC indicated that the subgroup IIc (OoWRKY90) evolved from the C-terminal WRKY domain of the subgroup Ia, or subgroup Ia genes evolved from subgroup IIc WRKYs by double WRKY domain.
Hypotheses suggest the group Ⅲ WRKY genes evolve from the subgroup Ia and IId genes [32]. However, based on the zinc finger motifs of group Ⅲ and subgroup Ia WRKYs, Xu et al [30] propose group Ⅲ WRKYs diverge first from ancient subgroup IIc genes. They also propose if the group Ⅲ WRKYs evolve from subgroup Ia, then the zinc finger type of subgroup Ia WRKYs would be a C2HC [30]. However, all of the subgroup Ia WRKYs contain C2H2 zinc finger type, and all of group Ⅲ WRKYs contain C2HC zinc finger type [16, 30, 34]. Thus, based on the phylogenetic and zinc finger motif analysis of group Ⅲ OoWRKY genes in O. officinalis, we were more inclined to approve the hypothesis of Xu et al [30] that group Ⅲ OoWRKY genes diverged from subgroup IIc genes.
The function of conserved motifs in O. officinalis WRKY genes
Although the WRKYGQK is highly conserved in most WRKY domains, frequently occurring variants of the core sequence are WRKYGKK and WRKYGEK [35]. Most of OoWRKY genes contained the WRKYGQK heptamer, while five subgroup IIc OoWRKY proteins (OoWRKY7, -10, -26, -67 and -77) and six group Ⅲ OoWRKY proteins (OoWRKY46, -52, -55, -84, -97 and -114) contained WRKYGKK and WRKYGEK, respectively. The variant WRKYGKK and its functions have also been reported in most plant species [35–37]. The WRKY domain-DNA interactions can activate the plant development and defense [38], but the WRKYGKK variant reduces DNA-binding to the W-box, which reported losing the ability of binding to W-box domain in soybean [39]. In our study, the expression level of OoWRKY26 was significantly up-regulated before 72 h under PXO99 and C5 stress, but the OoWRKY26 expression level under Xoo stress after 72 h was lower than that in CK (ddH2O treatment), suggesting that the regulated expression of OoWRKY26 under Xoo stress after 72 h might be mainly due to mechanical injury during inoculation treatment. PXO99 and C5 induced the expression of OoWRKY26 in O. officinalis, while WRKY26 enhances the tolerance of plants to high temperature stress regulated in Arabidopsis thaliana [40]. Therefore, OoWRKY26, containing WRKYGKK heptamer, might be simultaneously involved in biotic and abiotic stress responses in O. officinalis.
Identification of conserved domains on OoWRKY genes could help elucidate their functions in O. officinalis. Extra domains were found in OoWRKY proteins by using the MOTIF search program (S3 Table). Interestingly, subgroup Ib OoWRKY125 contained not only two WRKY domains but also an NB-ARC domain. NB-ARC domain, sharing by plant resistance gene products and regulators, functioned as a signaling motif in plant resistance [41]. The genes that contain both WRKY and NB-ARC domains are classified as RWRKY genes, which are also found in OsRWRKY1 and OsRWRKY2 [41]. NB-ARC domain has also been identified in Arabidopsis thaliana, Fragaria vesca, Glycine max, Sorghum bicolor, Setaria italica, and Theobroma cacao [33, 41]. NACHT domain, an evolutionary conserved protein domain associated with apoptosis and MHC transcriptional activation [42], was also found in OoWRKY125. Therefore, we conjectured OoWRKY125 might play an important role in the immune response of O. officinalis. Plant_zn_clust only occured in subgroup IId OoWRKYs, indicating these genes possessed special function. FLYWCH zinc finger domains were found in subgroup IId OoWRKY6, subgroup IIe OoWRKY39 and group III OoWRKY40, which suggested they might play a role in DNA-RNA binding or protein-protein interactions [43]. FAR1 domain, involving in the phyA-signaling pathway [44], was found in subgroup Ⅰa OoWRKY24 and subgroup IIc OoWRKY7, -26, -67. It suggested that these OoWRKYs with FAR1 domain might function in the phytochrome signaling pathway in O. officinalis. Mito_fiss_reg domain, relating to mitochondrial fission [45], was presented in subgroup IIa OoWRKY71. OsWRKY71, involving in elicitor-induced defense responses in rice [46], also contains the Mito_fiss_reg, and the subgroup IIa OsWRKY transcription factors are reported to mediate rice innate immunity [47]. Therefore, the Mito_fiss_reg domain could help to explore the subgroup IIa OoWRKYs' functional mechanism in O. officinalis. These extra domains could help elucidate the function and evolutionary relationships of OoWRKY genes.
The eight significantly differential expression O. officinalis WRKYs in response to bacterial blight resistance
WRKY transcription factor plays an important role in the defense mechanism of plant disease resistance as a positive regulator or negative regulator [8]. There have been identified more than 100 WRKY genes from O. sativa, of which 35 WRKY genes play an important role in disease resistance, such as OsWRKY3, OsWRKY13, OsWRKY28, OsWRKY30, OsWRKY31, OsWRKY45, OsWRKY53, OsWRKY70, OsWRKY71, they play an important regulatory role in disease resistance-related pathways of bacterial blight, rice blast and sheath blight [17]. Although many plant WRKY genes associated with disease resistance have been reported, there is no any research report about resistance to disease-related WRKY genes in O. officinalis due to lacking of its genomic information. Based on the expression levels of 89 OoWRKY genes of O. officinalis under PXO99 and C5 stress, we selected eight OoWRKY genes of marker changed in expression under Xoo stress.
Liu et al [48] reveal that OsWRKY3, as a transcriptional activator in SA-dependent or JA-dependent on disease-resistant signal cascades, is down-regulated at 12 h and up-regulated at 48 h under Xoo stress. We found the expression patterns of OoWRKY3 were different in response to PXO99 and C5. PXO99 induced the expression of OoWRKY3, whereas C5 inhibited its expression. The expression level of OoWRKY3 was significantly up-regulated under PXO99 stress 24 h and 48 h, while significantly down-regulated under C5 stress from 72 h to 120 h. Although the OoWRKY3 expression was significantly up-regulated under PXO99 as reported by Liu et al [48], Liu et al do not clear the strains of Xoo. OoWRKY3 had different expression patterns in response to different Xoo strains, and the expression of OoWRKY3 was the highest in leaf, which indicated it mainly functioned in leaf. Therefore, we suggested OoWRKY3 may regulate different defense genes expression in response to different Xoo strains by self expression level. OsWRKY13 directly or indirectly mediates disease resistance to bacterial blight and fungal blast through activation of SA-dependent pathways, suppression of JA-dependent pathways [49]. In addition, OsWRKY13 also activates the flavonoid biosynthesis pathway, which enhances the biosynthesis of antimicrobial flavonoid phytoalexins [50]. The expression level of OoWRKY13 was 2~3 times higher under PXO99 and C5 stress than that of ddH2O treated (CK), indicating that the increased expression of OoWRKY13 was induced by PXO99 and C5. The up-regulated expression of OoWRKY13 also activated the flavonoid biosynthesis pathway and increased the accumulation of flavonoid. Flavonoid can accumulate as phytoalexins in plant to enhance plants resistance to pathogen [51]. There is report that the flavonoid phytoalexin in resistant rice contribute to blast resistance [52]. Therefore, OoWRKY13 might enhance the resistance to PXO99 and C5 in O. officinalis by activating the flavonoid biosynthesis pathway and increasing the accumulation of flavonoid, indicating OoWRKY13 might be a new bacterial blight resistance gene of O. officinalis.
OsWRKY30 is constitutive expression gene in the root and leaf of O. sativa, and the expression of OsWRKY30 induces rapidly by Magnaporthe grisea, SA and JA [53]. Ramamoorthy et al [54] suggest the expression level of OoWRKY30 cannot be observed in the root and leaf of normal growth. Over-expression of OsWRKY30 enhances the resistance of transgenic rice plants to fungal blast [55]. Han et al [56] prove that OsWRKY30 mediates disease resistance to bacterial blight as a positive regulator through the SA signaling pathway. The expression level of OoWRKY30 displayed increases only under PXO99 and C5 stress 48 h, indicating the affection of PXO99 and C5 on OoWRKY30 was transient. The expression level of OoWRKY30 was very high in stem and leaf of normal growth in O. officinalis, which was 300~500 times more than that in root. As a high and constitutive expression gene in leaf, we speculated OoWRKY30 would play an important role in maintaining the normal growth of leaf, and its high expression level was enough to regulate the resistance-related pathway in O. officinalis without increasing its expression under PXO99 and C5 stress.
OsWRKY53 is a transcriptional activator in plant defense, which triggers the expression of other transcription factors of the same or different family of genes that involved in disease resistance-related metabolic pathways [18]. Over-expression of OsWRKY53 enhances the resistance of transgenic rice plants to bacterial blight and fungal blast [18]. The OoWRKY53 expression level was the highest in the normal growth leaf of O. officinalis among eight OoWRKY genes, and its expression significantly increased under PXO99 and C5 stress, indicating that OoWRKY53 not only played a role in the normal growth of leaf, but also played an important role in disease resistance to PXO99 and C5 in O. officinalis.
OsWRKY71 plays an important role in rice defense response. Over-expression of OsWRKY71 enhances the resistance of transgenic rice to the Xoo strain 13571 [19]. The sequences of WRKY function domains between OsWRKY71 and OoWRKY71 were completely consistent (S5 Fig showed with black box). Therefore, OoWRKY71 also had the resistance to Xoo strain 13571 as OsWRKY71. In O. officinalis, the expression of OoWRKY71 was extremely low in the normal growth of leaf, but the expression level significantly increased under PXO99 and C5 stress. In view of the analysis of OoWRKY71 expression trends under PXO99 and C5 stress, we suggested the up-regulated expression of OoWRKY71 would enhance the resistance of O. officinalis to the PXO99 and C5. So, we speculated OoWRKY71 not only had resistance to Xoo strain 13571 as OsWRKY71 but also resistance to strains PXO99 and C5, and the bacterial blight resistance of OoWRKY71 might be stronger than OsWRKY71 due to OoWRKY71's strong response to the PXO99 and C5. OoWRKY71 should have an important effect on bacterial blight resistance in O. officinalis.
There are no reports about the function of WRKY26 and WRKY111 genes in rice. WRKY26, regulating positively in the signaling pathway of ethylene activation and heat shock protein, enhances the tolerance of plants to high temperature stress [40]. OoWRKY26 displayed increases in expression under PXO99 and C5 stress 24 h, 48 h, 72 h, but the expression level of OoWRKY26 under Xoo stress from 72 h to 120 h was lower than that ddH2O treated (CK), which indicated that the expression chances of OoWRKY26 under Xoo stress starting from 72 h might be due to mechanical injury during inoculation treatment. Xoo infects the rice from the wound of leaf, stem, or root, then propagate and transmit in the vascular bundle. The expression of OoWRKY111 continued to decrease significantly under PXO99 and C5 stress, suggesting OoWRKY111 acted as a negative regulator mediated resistance-related pathways of genes in O. officinalis. The expression of OoWRKY111 was higher in root than that in leaf, stem, and flower. Therefore, we speculated the OoWRKY111 would play an important role in defense of the pathogen infection in root.
It can predict gene function by analyzing the expression of the gene in certain condition and the organism. The eight OoWRKY genes displaying significant change in expression under Xoo strains PXO99 and C5 stress might play important roles in Xoo stress responses in O. officinalis. The expressed tendency of the same OoWRKY gene was almost unanimous under PXO99 and C5 stress, whether it was up-regulated or down-regulated, but the expression levels were different. The O. officinalis had strong resistance to C5, but its resistance to PXO99 was weak (unpulished data). The differentially expressed levels of OoWRKY genes in response to PXO99 and C5 might be one of the reasons for the different resistance of O. officinalis to the PXO99 and C5. In this study, the marked expression changes of eight OoWRKYs under Xoo stress and the specific expression in root, stem, leaf, and flower supplemented the results of existing studies, and analyzed the function of OoWRKY genes in response to PXO99 and C5 in O. officinalis, which provided a theoretical basis of studying the WRKY genes in mechanism of O. officinalis-Xoo.
Conclusion
We identified 89 O. officinalis WRKY genes and focused on those involved in response to Xoo stress. The classification, structure, and evolutionary characteristics of O. officinalis WRKY genes were analyzed. OoWRKY genes expression patterns revealed that eight OoWRKY genes displaying significant changes in expression might play an important role in PXO99 and C5 stress responses in O.officinalis. In addition, tissue expression profiles showed that the eight OoWRKY genes also played different roles in O. officinalis development and exhibit differential expression level in different tissues. OoWRKY13 might enhance the resistance to PXO99 and C5 in O. officinalis by activating the flavonoid biosynthesis pathway and increasing the accumulation of flavonoid, and might be a new bacterial blight resistance gene of O. officinalis. OoWRKY71 had resistance to Xoo strain 13571 as OsWRKY71; even the resistance of OoWRKY71 might be stronger than OsWRKY71 due to OoWRKY71's strong response to the strains PXO99 and C5. In conclusion, our study established a structural and functional framework to investigate O. officinalis WRKY genes by the RNA-seq under the condition of lacking of O. officinalis genome. This study provided a solid foundation for the study of the evolution and the functions of O. officinalis WRKY genes in response to Xoo stress.
Supporting information
S1 Fig. The multiple alignments of OoWRKY domain peptide.
Red square frames were WRKYGQK variants. Black lines represented WRKY motifs; Black solid triangle and black background indicated cysteine and histidine residues of zinc finger motifs. The red square frames represented the WRKYGQK variants.
https://doi.org/10.1371/journal.pone.0188742.s001
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S2 Fig. The motif structure of ten motifs in OoWRKYs.
https://doi.org/10.1371/journal.pone.0188742.s002
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S3 Fig. The amino acid alignment of the WRKY domain and zinc finger motif in OoWRKY118.
Black line represented WRKY motifs; Black solid triangle indicated cysteine and histidine residues of zinc finger motifs.
https://doi.org/10.1371/journal.pone.0188742.s003
(TIF)
S4 Fig. The amino acid alignment of subgroup Ia C-terminal OoWRKY domains and subgroup IIc OoWRKY domains.
https://doi.org/10.1371/journal.pone.0188742.s004
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S5 Fig. The amino acid alignments between OoWRKY71 and OsWRKY71.
The amino acids of black frame were the WRKY domains.
https://doi.org/10.1371/journal.pone.0188742.s005
(TIF)
S1 Table. qRT-PCR primers of significantly different expression OoWRKYs.
https://doi.org/10.1371/journal.pone.0188742.s006
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S2 Table. WRKY genes identified in O. officinalis and their orthologs in O. sativa sbb japonica.
https://doi.org/10.1371/journal.pone.0188742.s007
(XLSX)
S3 Table. Extra domains found in OoWRKY proteins.
https://doi.org/10.1371/journal.pone.0188742.s008
(XLSX)
S1 File. The amino acid sequences of WRKY in O. officinalis.
The red backgrounds were the amino acid sequences of conserved WRKY and zinc finger motif.
https://doi.org/10.1371/journal.pone.0188742.s009
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S2 File. Aligment of OoWRKY domain amino acid sequences.
The alignment was performed by MUSCLE in the MEGA6.
https://doi.org/10.1371/journal.pone.0188742.s010
(MEG)
S3 File. Aligment of OoWRKY full length amino acid.
The alignment was performed by MUSCLE in the MEGA6.
https://doi.org/10.1371/journal.pone.0188742.s011
(MEG)
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