Genome-wide Identification and Structural, Functional and Evolutionary Analysis of WRKY Components of Mulberry

Abstract

Mulberry is known to be sensitive to several biotic and abiotic stresses, which in turn have a direct impact on the yield of silk, because it is the sole food source for the silk worm. WRKYs are a family of transcription factors, which play an important role in combating various biotic and abiotic stresses. In this study, we identified 54 genes with conserved WRKY motifs in the Morus notabilis genome. Motif searches coupled with a phylogenetic analysis revealed seven sub-groups as well as the absence of members of Group Ib in mulberry. Analyses of the 2K upstream region in addition to a gene ontology terms enrichment analysis revealed putative functions of mulberry WRKYs under biotic and abiotic stresses. An RNA-seq-based analysis showed that several of the identified WRKYs have shown preferential expression in the leaf, bark, root, male flower, and winter bud of M. notabilis. Finally, expression analysis by qPCR under different stress and hormone treatments revealed genotype-specific responses. Taken together, our results briefs about the genome-wide identification of WRKYs as well as their differential response to stresses and hormones. Importantly, these data can also be utilized to identify potential molecular targets for conferring tolerance to various stresses in mulberry.

Figure 1. Illustration of the gene structure of 54 mulberry WRKY genes, showing the distribution of exons and introns (to map scale).

Genes are separated into their respective groups. Exons are shown using solid boxes while introns are shown using solid lines. For greater resolution, two genes >5 kb in length are shown on a separate scale.

Figure 2. Phylogenetic tree depicting WRKY evolution in mulberry.

Clades are highlighted using background colors. Proteins are shown with the predicted MEME motifs (to map scale). Motif annotation is shown as color legends.

Figure 3. Word-cloud showing the identified cis elements in the putative promoter regions of WRKY genes in mulberry.

The intensity and size of the motifs indicate their frequency.

Figure 4. Heat map showing the expression pattern of WRKY genes across five developmental stages.

Heat map was generate using TMM-normalized FPKM values obtained for the respective stages. Legend shows the level of up- or down-regulation.

Figure 5. Heat map showing the comparative expression level of WRKY genes in three genotypes i.e., Morus laevigata, Morus serrata, and Morus notabilis.

Genes with comparatively lower expression values are shown using shades of green and high expression values are represented using shades of red. TMM-normalized FPKM values were used to generate the heat map.

Figure 6. Bar graphs showing the relative fold change of selected WRKY genes in different mulberry species.

Error bars depict standard error for two biological replicates. Three technical replicates for each biological replicates were run. Relative fold changes were obtained using the ∂∂Ct method. Asterisks on top of the error bars represent the significance levels (Students t-test; p value ≤ 0.05). See legend for details.