The evolution of WRKY transcription factors

Abstract

Background: The availability of increasing numbers of sequenced genomes has necessitated a re-evaluation of the evolution of the WRKY transcription factor family. Modern day plants descended from a charophyte green alga that colonized the land between 430 and 470 million years ago. The first charophyte genome sequence from Klebsormidium flaccidum filled a gap in the available genome sequences in the plant kingdom between unicellular green algae that typically have 1-3 WRKY genes and mosses that contain 30-40. WRKY genes have been previously found in non-plant species but their occurrence has been difficult to explain.

Results: Only two WRKY genes are present in the Klebsormidium flaccidum genome and the presence of a Group IIb gene was unexpected because it had previously been thought that Group IIb WRKY genes first appeared in mosses. We found WRKY transcription factor genes outside of the plant lineage in some diplomonads, social amoebae, fungi incertae sedis, and amoebozoa. This patchy distribution suggests that lateral gene transfer is responsible. These lateral gene transfer events appear to pre-date the formation of the WRKY groups in flowering plants. Flowering plants contain proteins with domains typical for both resistance (R) proteins and WRKY transcription factors. R protein-WRKY genes have evolved numerous times in flowering plants, each type being restricted to specific flowering plant lineages. These chimeric proteins contain not only novel combinations of protein domains but also novel combinations and numbers of WRKY domains. Once formed, R protein WRKY genes may combine different components of signalling pathways that may either create new diversity in signalling or accelerate signalling by short circuiting signalling pathways.

Conclusions: We propose that the evolution of WRKY transcription factors includes early lateral gene transfers to non-plant organisms and the occurrence of algal WRKY genes that have no counterparts in flowering plants. We propose two alternative hypotheses of WRKY gene evolution: The "Group I Hypothesis" sees all WRKY genes evolving from Group I C-terminal WRKY domains. The alternative "IIa + b Separate Hypothesis" sees Groups IIa and IIb evolving directly from a single domain algal gene separate from the Group I-derived lineage.

Figure 1

A group IIb WRKY transcription factor from Klebsormidium flaccidum . ClustalW2 multiple sequence alignment and consensus sequence of WRKY domains from Arabidopsis Group IIb genes and the IIb gene (kfl00189) from Klebsormidium flaccidum. The consensus amino acid sequence is shown and also the number of amino acids in the WRKY domains. Amino acid sequence motifs found in Group IIb WRKY transcription factors are shown and underlined in red.

Figure 2

The WRKY gene family. A. Neighbor joining phylogenetic tree derived from a MUSCLE alignment of WRKY domains from the complete WRKY gene families from the following species: Arabidopsis thaliana, Glycine max, Brachypodium distachyon, Selaginella moellendorffii, Physcomitrella patens, Chlamydomonas reinhardtii, Chlorella variabilis, Coccomyxa subellipsoidea, Micromonas pusilla, Ostreococcus lucimarinus, Ostreococcus tauri, Volvox carteri, Klebsormidium flaccidum, Bathycoccus prasinos, Dictyostelium discoideum, Polysphondylium pallidum, Dictyostelium fasciculatum, Fonticula alba, Acanthamoeba castellanii, Giardia lamblia, Giardia intestinalis, Dictyostelium purpureum, Auxenochlorella protothecoides, Spironucleus salmonicida, Mucor circinelloides, Rhizopus delemar, Absidia idahoensis, Lichtheimia corymbifera, Rhizophagus irregularis, and Mortierella verticillata. Fungal genes are marked with a red dot, unicellular green algae green, diplomonads blue, amoebozoa black, social amoebae purple, and Klebsormidium flaccidum orange. The higher plant WRKY groups are marked I-III. I NTD and I CTD denote the N-terminal and C-terminal domains from Group I proteins. The tree was produced using MEGA 6. B. Maximum likelihood phylogenetic tree using the same MUSCLE alignment.

Figure 3

The distribution of WRKY genes in the tree of life. Red boxed names indicate the presence of WRKY genes.

Figure 4

Fungal and moss Group III WRKY proteins. A. Part of the neighbor joining phylogenetic tree shown in Figure 2 derived from a MUSCLE alignment of WRKY domains. The WRKY domains from Mucor circinelloides, Rhizopus delemar, Absidia idahoensis, Lichtheimia corymbifera, Rhizophagus irregularis, and Mortierella verticillata are indicated by red dots and form a distinct clade. Numbers indicate bootstrap values from 1,000 replicates. B. ClustalW2 multiple sequence alignment and consensus sequence of WRKY domains from fungi incertae sedis. The conserved WKNNGNT amino acid sequence is shown C. ClustalW multiple sequence alignment and consensus sequence of WRKY domains from Physcomitrella patens Group III proteins (PpWRKY35-38). The conserved WKKYGNK amino acid sequence is shown.

Figure 5

WRKY transcription factors that are not from higher plants. A. Bootstrap consensus tree (1,000 replicates) of a Neighbor Joining phylogenetic tree derived from a MUSCLE alignment of WRKY domains from the species described in Figure 2. Shown is a non-higher plant clade that contains algae (green), diplomonad (blue), and amoebozoa (black) WRKY transcription factors. B. Phylogenetic tree of Group I-like WRKY proteins from social amoebae and other amoebozoa. Groups I CTD, I NTD, IIc and an intermediate clade are shown. Unicellular green algae WRKY domains are marked with a green dot, amoebozoa black, social amoebae purple and Klebsormidium flaccidum in orange.

Figure 6

Consensus positions of the PR, VQR, and algal I CTD introns. A. The consensus amino acid sequences of WRKY domains from Arabidopsis Groups I, IIc, IId, IIe, and III, derived by ClustalW together with the position of the conserved PR intron. B. The consensus amino acid sequences of WRKY domains from Arabidopsis Groups IIa and IIb together with the position of the conserved VQR intron. C. The carboxy terminal domains from four algal Group I WRKY proteins together with the position of the conserved intron. The species and name of each gene are shown.

Figure 7

A comparison of WRKY domains with GCM1 and FLYWCH domains. ClustalW multiple sequence alignment of all WRKY domains from Arabidopsis, together with representative GCM1 and FLYWCH domains. The WRKY signature sequence is marked, as is the zinc finger domain with the conserved zinc binding residues.

Figure 8

Distribution of the eight R protein-WRKY families. A phylogenetic tree of sequenced plant genomes is presented. The distribution of the eight R protein-WRKY families (RW1-RW8) in shown by red arrows. Based partly on phylogenetic analysis at http://phytozome.jgi.doe.gov.

Figure 9

Phylogenetic analyses of the R protein-WRKY (RW) families. A. Neighbor Joining phylogenetic tree derived from a MUSCLE alignment of full length R protein-WRKY proteins. Numbers indicate bootstrap values from 1,000 replicates. B. Non-rooted version of the same tree as presented in A.

Figure 10

HMMER analyses of the R protein-WRKY families. Next to each predicted protein in the phylogenetic tree is the HMMER-derived overview of protein architecture with protein domains shown. WRKY domains are shown in reddish purple, TIR domains in green, leucine rich repeat domains in blue or black, NB-ARC domains in lilac, calmodulin-binding domains in yellowish green, NAC domains in dark purple, and B3 domains in green. The number of WRKY domains and their groups are shown to the right of the proteins.

Figure 11

Overview of the evolution of WRKY transcription factors. Boxes represent WRKY domains. Red boxes indicate postulated progenitor domains. Blue boxes indicate WRKY domains from present day species. Green boxes indicate FLYWCH, GCM1 and BED zinc finger domains. Conserved introns are shown in red lettering. The four major flowering plant WRKY lineages are shown in large light blue boxes. Currently existing groups of WRKY transcription factors not found in multicellular plants are shown in large light green boxes. The direction of evolution is shown by arrows.