Exploring the Intrinsic Disorder of the WRKY Transcription Factor

The WRKY transcription factors superfamily are involved in diverse biological processes in plants including responses to biotic and abiotic stresses. It is also known that WRKYs exert many of its biological functions via interaction with other proteins of the signalling pathway. To understand the potential roles of intrinsic disorder in the functioning of WRKYs, we investigated the intrinsic disorder distribution of WRKYs in O.sativa, S.bicolor, Z.mays, T.aestivum, H.vulgare and A.thaliana and their protein-protein interactions. The analysis revealed that their intrinsic disorder distributions are similar. The data show that hub proteins have a higher disorder content in all species. These findings indicate that the peculiarities of the intrinsic disorder of WRKYs are evolutionary conserved, and therefore could be of crucial for their biological activities. The multifunctionality of WRKYs suggests that these proteins might utilize intrinsic disorder for their various functions.


Introduction
Intrinsically disordered proteins (IDPs) lack a defined tertiary structure, yet they play key biological roles. The IDPs are over represented in signalling and transcription regulatory networks which involve hubs having several protein partners [1,2].
Transcription factors are modular proteins that contain at least one DNA-binding domain, recognize and bind to specific DNA sequences, involved in transcription regulation. The analysis reported that the AT-hooks and basic regions of DNA-binding domains of transcription factors exhibit high level of disorder [3].
According to [4] The WRKY domain is characterised by the conserved amino acid sequence WRKYGQK at its N-terminal end, with a zinc-finger-like motif. The WRKY domain is found in one or two copies in a superfamily of plant transcription factors involved in the regulation of various physiological processes, including pathogen defence, response to salt stress, response to cold, response to wounding, salicylic response and the biosynthesis of secondary metabolites. The WRKY domain is required to DNA binding. WRKY TFs are structurally classified into three main groups (I, II and   III), and also multiple subgroups (e.g. IIa, IIb and IIc, etc.) based on the number of WRKY domains [5].
Here, we aim to investigate the long disorder in the WRKY transcription factors in O.sativa, S.bicolor, Z.mays, T.aestivum, H.vulgare and A.thaliana.

Data sources
The dataset was retrieved from the plant transcription factor database Plant TFDB 5.0 [6].

Protein disorder prediction
The prediction of intrinsic protein disorder was carried out by IUPred2A web server (http://iupred.enzim.hu/) [7]. This server disordered region from amino acid sequence based on pairwise energy content. There are three different prediction types: long disorder, short disorder, and structured domains. Here, we used long disorder prediction type in which at least 30 consecutive disordered residues are considered.

Protein binding region prediction 94
The binding regions prediction was used by ANCHOR tool (http://anchor.enzim.hu/) [8], which is based on the IUPred program mentioned above. Anchor predicts binding regions located in disordered proteins from the amino acid sequence.
The software IUPred and ANCHOR were requested from the authors and were compiled and executed locally.

Protein-protein interaction networks and topological analysis
The protein-protein interactions of WRKYs in O.sativa, S.bicolor, Z.mays, T.aestivum, H.vulgare and A.thaliana were obtained from STRING version 11.0 database [9].
The topological and statistical parameters of networks have been analysed using Cytoscape (version 3.5.1) [10].

Gene Ontology analysis
The gene ontology (GO) analyses were performed by agriGOv2.0 tools. It is specifically focused on gene ontology (GO) enrichment analyses of plant and agricultural species [11].

Overall disorder content
The Prediction of disordered binding regions (DBRs) showed that the average number of DBRs per protein was also similar among studied species (Table1). When proteins were grouped according to intervals of DBR residues content, we found higher number of WRKYs in the [5-10) interval excepting T.aestivum (Figure 1

Location of DBRs within proteins
The locations of DBRs within proteins are shown in Figure 2

Gene ontology (GO) analysis
The enrichment analysis of WRKY proteins revealed similar GO categories among the studied species. The cellular components encompassed the nucleus, intracellular membrane-bounded organelle, organelle and cell. Their molecular functions are mainly related to sequence-specific DNA binding, transcription factor activity and binding.
The biological processes mediated by the WRKYs included regulation of transcription, regulation of cellular metabolic process and biological process, regulation of defense response and gene expression. The Gene Ontology categories are listed by organism in supplemental file 2.

WRKY interaction networks
Based on genomic context, high-throughput experiments, co-expression and text mining, protein-protein networks  The highly ranked nodes, namely hubs, are identified by combining degree distribution and betweenness centrality measures. In all studied species, we have noticed that these hubs have high disorder content and disorder binding

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We noticed that in spite of the strong evolutionary conservation of the WRKY DNA-binding domain, the flanking sequences of proteins are highly divergent, which might reflect their different functions.   In agreement with previous report, we found that AtWRKY33 acts as a one of the hubs in the protein-protein interaction in the WRKYs of A.thaliana [14]. AtWRKY33 (disorder content=0.65). Moreover, AtWRKY33 is involved in the regulation of the defense pathways mediating responses to P. syringae and necrotrophic fungal pathogens [15]. It is also involved in response to salt stress and abscisic acid (ABA) signalling [16].
Previous study suggests that OsWRKY6 (disorder content=0.5) positively regulates defense responses through activation of OsICS1 expression and OsWRKY6 stabilization [17]. Furthermore, HvWRKY46 (disorder content=0.68), denoted also SUSIBA2 has been demonstrated as a regulatory transcription factor in starch synthesis and involved in carbohydrate anabolism [18].

Conclusion
In