A Comprehensive Study of the WRKY Transcription Factor Family in Strawberry

Abstract

WRKY transcription factors play critical roles in plant growth and development or stress responses. Using up-to-date genomic data, a total of 64 and 257 WRKY genes have been identified in the diploid woodland strawberry, Fragaria vesca, and the more complex allo-octoploid commercial strawberry, Fragaria × ananassa cv. Camarosa, respectively. The completeness of the new genomes and annotations has enabled us to perform a more detailed evolutionary and functional study of the strawberry WRKY family members, particularly in the case of the cultivated hybrid, in which homoeologous and paralogous FaWRKY genes have been characterized. Analysis of the available expression profiles has revealed that many strawberry WRKY genes show preferential or tissue-specific expression. Furthermore, significant differential expression of several FaWRKY genes has been clearly detected in fruit receptacles and achenes during the ripening process and pathogen challenged, supporting a precise functional role of these strawberry genes in such processes. Further, an extensive analysis of predicted development, stress and hormone-responsive cis-acting elements in the strawberry WRKY family is shown. Our results provide a deeper and more comprehensive knowledge of the WRKY gene family in strawberry.

Keywords: WRKY family; differential expression; homeolog; paralog; pathogens; ripening; strawberry.

Figure 1

Protein domains (PFAM v32.0) found in FvWRKY proteins. Only the largest splice forms are represented. See Table S2 for more details.

Figure 2

Chromosome mapping of the Fragaria vesca WRKY gene family. Segmental duplicated gene pairs (syntenic paralogs) share the same colors. Tandemly duplicated gene clusters are colored in pink, and underlined genes are collinear with genes outside that syntenic block. Ruler size is in Megabases.

Figure 3

GEvo analyses comparing microsynteny between genomic regions from A. thaliana and F. vesca. (A) Genomic regions containing the genes FvWRKY60, FvWRKY61 and AtWRKY70 with red blocks and connectors show high-scoring sequence pairs between both sequences. The region shown in B is framed. (B) Homology details among FvWRKY60, FvWRKY61 (black arrows) and AtWRKY70. A short, non-WRKY gene (FvH4_7g26100, red arrow) was identified as part of the FvWRKY59-60-61 tandem (see text for details).

Figure 4

Chromosome mapping of the Fragaria × ananassa WRKY gene family within each subgenome. Segmental duplicated gene pairs (syntenic paralogs) share the same colors. Non-syntenic gene duplicates are underlined. Tandemly duplicated gene clusters are colored in orange. Ruler size is in Megabases.

Figure 5

Phylogenetic analysis of Fragaria vesca (Fv), Arabidopsis thaliana (At) and Vitis vinifera (Vv) WRKY proteins. WRKY proteins are clustered into Groups I + IIc, IIa + IIb, IId + IIe and III. R protein-WRKY from Fv and At are clustered within their respective groups. The tree was inferred using the Neighbor-Joining method (1000 bootstrap replicates) and drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the p-distance method and are in the units of the number of amino acid differences per site. All positions with less than 95% site coverage were eliminated. Connecting lines represent shared synteny between Fv-Vv (red) and Fv-At (blue).

Figure 6

GO functional annotation plot comparing the number and percentage of FvWRKY and FaWRKY proteins sharing the same functions. The y-axis represents the number (right) and the percentage (left) of WRKY genes in Fragaria × ananassa (green) and in Fragaria vesca (blue) for each GO category (x-axis).

Figure 7

Phylogenetic tree of the AtWRKY55 and AtWRKY54/70 orthologous genes (red and blue branches, respectively) in strawberry (Fv), soybean (Glyma), walnut (WALNUT), apple (MD), grapevine (Vv) and Amborella (AmTr). Strawberry orthologs to AtWRKY54/70 paralogs underwent tandem duplications after the gamma hexaploidization and independently to the more recent WGD events in other species. The evolutionary history was inferred using the Maximum Likelihood method (100 bootstraps) with optimized parameters (TN + G + I). The tree is drawn to scale, with lengths of branches representing the number of substitutions per site. Analyses were conducted in MEGA7.

Figure 8

Phylogenetic tree of the FvWRKY family showing expression values during fruit ripening (Ruegen receptacle tissue at 15 and at 22 DPA and Yellow Wonder receptacle tissue at 15 and at 22 DPA: Yw-15D, Yw-22D, Rg-15D and Rg-22D, respectively) and roots (Root, collected from 7-week-old plants grown in aerated hydroponic culture, and Root_P, after 2 days of inoculation with Phytophthora cactorum). FvWRKY paralogs are shown in red (see Table 1. Color scale represents the expression level as log-transformed TPM (Transcripts per Million) values.

Figure 9

(A) Heatmap of differentially expressed FaWRKY genes during strawberry fruit ripening stages in receptacle (White_R, Turning_R, and Red_R; left side) and achene (White_A, Turning_A, and Red_A; right side) tissues. Changes in gene expression, with respect to green fruit tissues, are represented as log2 fold change if absolute values were >1 (padj < 0.01), otherwise, they were colored in grey. (B) Venn diagram showing those genes which were differentially expressed in receptacle and achene only, or in both.

Figure 10

Heatmap of differentially expressed FaWRKY genes in response to C. fructicola infection in leaves. Changes of gene expression in infected strawberry (Inf_24h, Inf_72h, Inf_96h; 24, 72 and 96 h after spore inoculation, respectively) vs. mock-inoculated strawberry are represented as log2 fold change if absolute values were >1 (padj < 0.01), in a color scale from lowest (blue) to highest (red). Otherwise, they were colored grey.