Exploring the Intrinsic Disorder of the WRKY Transcription Factor Family in the Cereals

Mouna Choura^1*, Ahmed Rebaï², Faiçal Brini¹

¹Biotechnology and Plant Improvement Laboratory, Center of Biotechnology of Sfax, University of Sfax, Route Sidi Mansour Km 6, P.O.Box 1177, 3018, Sfax, Tunisia

²Laboratory of Molecular and Cellular Screening Processes, Center of Biotechnology of Sfax, University of Sfax, Route Sidi Mansour Km 6, P.O.Box 1177, 3018, Sfax, Tunisia

^*Corresponding Authors: Mouna Choura, Biotechnology and Plant Improvement Laboratory, Center of Biotechnology of Sfax, University of Sfax, Route Sidi Mansour Km 6, P.O.Box 1177, 3018, Sfax, Tunisia

Received: 09 October 2020; Accepted: 05 November 2020; Published: 16 December 2020

1. Introduction

Intrinsicallydisorderedproteins (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], seventy-eight different families of transcription factors including MYB, AP2-EREBP, bHLH, MADS, C2H2, NAC, HB, WRKY, bZIP and C3H contain different numbers of intrinsicallydisorderedregions (IDRs).

The present paper focused on WRKY transcription factors family. WRKY transcription factors are characterized by their highly conserved DNA-binding domain (DBD), called the WRKY domain.

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.

2. Methods

2.1 Data sources

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

2.2 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.

2.3 Protein binding region prediction

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.

2.4 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].

2.5 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].

3. Results

3.1 Overall disorder content

The overall disorder contents of WRKYs in O.sativa, S.bicolor, Z.mays, T.aestivum, H.vulgare and A.thaliana are listed in Table1. These data clearly show similar mean content of disorder and of disordered binding regions between WRKYs analysed in this study. By grouping proteins according to the percentage of predicted disorder of their sequence, we found that the higher disorder content (>30%) of WRKY proteins is within the interval [30-60%] (Figure 1(A)) and (Supplemental file1).

Prediction of disordered binding regions (DBRs) showed that the average number of DBRs per protein was alsosimilar 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 (B)) and (Supplemental file 1).

Table1: Summary of intrinsic disorder metrics for O.sativa, S.bicolor, Z.mays, T.aestivum, H.vulgare and A.thaliana.

^*Disordered binding regions

We found that the mean of disorder is significantly correlated with the DBR content for A.thaliana, O.sativa, S.bicolor,Z.mays, T. aestivum and H.vulgare (Pearson correlation, r = 0.73, 0.6,0.57, 0.65, 0.65,0.43 respectively).

(A)

Figure 1: Fraction of proteins with different degrees of predicted disorder (A) and disordered binding regions (B) in O.sativa, S.bicolor, Z.mays, T.aestivum, H.vulgare and A.thaliana.

(B)

Figure 1: Fraction of proteins with different degrees of predicted disorder (A) and disordered binding regions (B) in O.sativa, S.bicolor, Z.mays, T.aestivum, H.vulgare and A.thaliana.

3.2 Location of DBRs within proteins

The locations of DBRs within proteins are shown in Figure 2 and (Supplemental file1). We found that DBRs are in N-terminal and C-terminal regions flanking the WRKY domain, and inter-WRKY domains. We also noted short disorder in the WRKY domain itself. This suggests that the disorder content increase with the number of WRKY domains and the pattern of the zinc-finger motif. In fact, members of Group 1 typically contain two WRKY domains, while most proteins with one WRKY domain belong to Group 2. Group 3 proteins have a single WRKY domain with a zinc-finger motif.

(a)

(b)

Figure 2: Schematic representation of disorder regions (in pink) flanking WRKY domain (in green) (a) Protein with one WRKY domain, (b) protein with two WRKY domains.

3.3 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.

3.4 WRKY interaction networks

Based on genomic context, high-throughput experiments, co-expression and text mining, protein-protein networks related to WRKYs in O.sativa (Figure 3), S.bicolor (Figure 4), Z.mays (Figure 5), H.vulgare (Figure 6), T.aestivum (Figure 7) and A.thaliana (Figure 8) were constructed. We then analyzed the topological features of these networks. These parameters are listed in table 2 and in supplemental file 3.

Table2: Network parameters calculated for each species

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 regions (DBRs) allowing them high flexibility and interaction with multiple partners, of which participate in different functions.

Figure 3: Protein-Protein interaction networks of WRKYs in O.sativa.

Figure 4: Protein-Protein interaction networks of WRKYs in S.bicolor.

Figure 5: Protein-Protein interaction networks of WRKYs in Z.mays.

Figure 6: Protein-Protein interaction networks of WRKYs in H.vulgare.

Figure 7: Protein-Protein interaction networks of WRKYs in T.aestivum.

Figure 8: Protein-Protein interaction networks of WRKYs in A.thaliana.

3.5 Evolutionary conservation of intrinsic disorder of WRKYs in cereals

Comparative analysis of WRKY DNA-binding domain and flanking regions by sequence alignment of some WRKYs in O.sativa, S.bicolor, Z.mays, T.aestivum, H.vulgare and A.thaliana showed some interesting insights (Figure 9).

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.

Figure 9: Multiple alignment of WRKY domain and flanking sequences of some members from A.thaliana, T.aestivum,O.sativa, S.bicolor, H.vulgare and Z.mays performed by clustal O [19]. Domain and DNA-binding sequences are highlighted in yellow and purple respectively.

To understand whether the intrinsic disorder is evolutionary conserved among studied species, we evaluated the evolutionary relationships between few AtWRKYs (from Group I, Group II and Group III) and their orthologs in O.sativa, S.bicolor, Z.mays, T.aestivum, H.vulgare (Figure10).

Figure 10: The phylogenetic tree of some WRKY proteins in the studied species.

Ta, Triticum aestivum: At, Arabidopsis thaliana: Hv, Hordeum vulgare, Os, Oryza sativa, Zm, Zea mays, Sb, Sorghum bicolor. The phylogenetic tree was constructed with MEGA5.2 [20] using a bootstrap test of phylogeny with a minimum evolution test and a parameter of 1000 replications.

The results indicated that WRKY family members within the same group, having similar functions, have also similar disorder content and DBR numbers among the species (Table 3).

Table3: Some WRKYs used in the phylogenetic analysis.

^*WRKY domain position

4. Discussion

This study has aimed to explore the protein disorder in the WRKYs in O.sativa, S.bicolor, Z.mays, T.aestivum, H.vulgare and A.thaliana. We found that these proteins have similar disorder content, DBR numbers and biological processes among these species. Similar conclusions were reached by performing the disorder analysis on the complete proteomes of these species [12,13]. The results have also shown that the WRKY domain has regions of conserved disorder as well as the flanking disordered sequences. This indicates that disorder tendencies are kept in these proteins, suggesting that their function depends on disorder such as in protein-protein/DNA interaction. By examining the protein-protein interaction networks of the WRKYs in these species, we found that these networks are highly connected by hubs, having a higher disorder content, particularly important for flexible binding with many interaction partners allowing thus multiple functions. Hereafter, some examples are discussed.

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].

5. Conclusion

In this study, we have provided a comparative analysis of the protein disorder of WRKY transcription factors in O.sativa, S.bicolor, Z.mays, T.aestivum, H. vulgare, and A. thaliana. Interestingly, we found similar and conserved disorder features such as the disorder content, the disorder binding regions (DBR) numbers and the Gene ontology including biological processes, molecular function and cellular components. The data provided here points towards the important role of the disorder of WRKYs in different biological processes notably in stress tolerance, deserving hence further investigation and experimental validation.

Acknowledgements

This work was supported by the Ministry of Higher Education and Scientific Research, Tunisia.

Conflicts of interest

The authors declare that they have no conflicts of interests.

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Supplementary Data

Supplemental file 1 Intrinsic disordered proteins in of WRKYs in A.thaliana, O.sativa, S.bicolor, Z.mays, T.aestivum and H.vulgare predicted by IUPred. It contains the disorder means, the DBRs their positions and their lengths.

Supplemental file 2 Gene Ontology (GO) analysis of WRKYs in A.thaliana, O.sativa, S.bicolor, Z.mays, T.aestivum and H.vulgare including biological process, molecular functions and cellular components.

Supplemental file 3 Topological features of WRKYs in A.thaliana, O.sativa, S.bicolor, Z.mays, T.aestivum and H.vulgare.