Characterization of the WRKY gene family in Akebia trifoliata and their response to Colletotrichum acutatum

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.

Keywords: Akebia trifoliata; Colletotrichum acutatum; WRKY transcription factors; biotic stress.

Fig. 1

The phylogenetic tree of the AktWRKY proteins. The NJ tree was constructed from the amino acid sequences of AktWRKYs using MEGA10.0 with 1000 bootstrap replicates

Fig. 2

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

Fig. 3

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

Fig. 4

NJ analyses of 252 conserved WRKY domains from O. sativa, Arabidopsis, and A. trifoliata. The domains clustered into eight major subgroups, IN, IC, IIa, IIb, IIc, IId, IIe, and III

Fig. 5

ClustalW amino acid sequence alignment of typical A. trifoliata WRKY domains. Gaps (dots) have been inserted for optimal alignment. Black and gray shading indicate the presence of identical and conserved amino acid residues, respectively. Consensus amino acid residues are shown below the alignment

Fig. 6

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

Fig. 7

The expression level of 42 AktWRKY genes in three different varieties of A. trifoliata (C01, I02 and H05) after C. acutatum infection for 6 h. The red columns represented up-regulated genes, while the green columns represented down-regulated genes

Fig. 8

Gene ontology analysis of identified AktWRKYs. Three main categories, including cellular component, molecular function, and biological process were defined by GO classification. Left and right y-axis represented the percentage and number of genes, respectively

Fig. 9

Protein-Protein interaction of AktWRKYs based on AtWRKYs orthologs as predicted by STRING search tool. The thickness of the lines represents the level of interaction between proteins