Transcriptome analysis of WRKY gene family in Oryza officinalis Wall ex Watt and WRKY genes involved in responses to Xanthomonas oryzae pv. oryzae stress

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.

Fig 1. The distribution of the OoWRKY genes based on groups or subgroups.

Fig 2. Phylogenetic analysis of the OoWRKY domains.

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.

Fig 3. Phylogenetic analysis of full-length OoWRKY proteins.

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.

Fig 4. The conserved motifs arrangement of OoWRKY proteins based on their phylogentic relationships.

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.

Fig 5. Expression profile analysis of OoWRKY genes by RNA-seq data.

(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.

Fig 6. Expression of eight differentially expressed OoWRKY genes in response to PXO99 and C5.

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.

Fig 7. The organ-specific expression levels of eight differentially expressed OoWRKY genes in root, stem, leaf, and flower.

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.