Structural and Functional Insights into WRKY3 and WRKY4 Transcription Factors to Unravel the WRKY–DNA (W-Box) Complex Interaction in Tomato (Solanum lycopersicum L.). A Computational Approach

Abstract

The WRKY transcription factors (TFs), play crucial role in plant defense response against various abiotic and biotic stresses. The role of WRKY3 and WRKY4 genes in plant defense response against necrotrophic pathogens is well-reported. However, their functional annotation in tomato is largely unknown. In the present work, we have characterized the structural and functional attributes of the two identified tomato WRKY transcription factors, WRKY3 (SlWRKY3), and WRKY4 (SlWRKY4) using computational approaches. Arabidopsis WRKY3 (AtWRKY3: NP_178433) and WRKY4 (AtWRKY4: NP_172849) protein sequences were retrieved from TAIR database and protein BLAST was done for finding their sequential homologs in tomato. Sequence alignment, phylogenetic classification, and motif composition analysis revealed the remarkable sequential variation between, these two WRKYs. The tomato WRKY3 and WRKY4 clusters with Solanum pennellii showing the monophyletic origin and evolution from their wild homolog. The functional domain region responsible for sequence specific DNA-binding occupied in both proteins were modeled [using AtWRKY4 (PDB ID:1WJ2) and AtWRKY1 (PDBID:2AYD) as template protein structures] through homology modeling using Discovery Studio 3.0. The generated models were further evaluated for their accuracy and reliability based on qualitative and quantitative parameters. The modeled proteins were found to satisfy all the crucial energy parameters and showed acceptable Ramachandran statistics when compared to the experimentally resolved NMR solution structures and/or X-Ray diffracted crystal structures (templates). The superimposition of the functional WRKY domains from SlWRKY3 and SlWRKY4 revealed remarkable structural similarity. The sequence specific DNA binding for two WRKYs was explored through DNA-protein interaction using Hex Docking server. The interaction studies found that SlWRKY4 binds with the W-box DNA through WRKYGQK with Tyr408, Arg409, and Lys419 with the initial flanking sequences also get involved in binding. In contrast, the SlWRKY3 made interaction with RKYGQK along with the residues from zinc finger motifs. Protein-protein interactions studies were done using STRING version 10.0 to explore all the possible protein partners involved in associative functional interaction networks. The Gene ontology enrichment analysis revealed the functional dimension and characterized the identified WRKYs based on their functional annotation.

Structural and functional dissection of differentially expressed tomato WRKY transcripts in host defense response against the vascular wilt pathogen (Fusarium oxysporum f. sp. lycopersici)

“…Of these three upregulated WRKY members only two members SolyWRKY33 and SolyWRKY37 were used for further studies. The functional characterization of the SolyWRKY4 (XP_004235494.1) has been already reported in our previous publication [ 61 ].The Blast-p program research in the NCBI protein database ( http://blast.ncbi.nlm.nih.gov/blast.cgi [ 62 ] was used to find sequential homologs for both SolyWRKY33 and SolyWRKY37. Blast annotations were filtered using subject to query coverage of >96% and with sequence identity > 62%.…”

In silico Prediction, Characterization, Molecular Docking, and Dynamic Studies on Fungal SDRs as Novel Targets for Searching Potential Fungicides Against Fusarium Wilt in Tomato

“…It was found that all the replaced residues in the FOXG_04696 were present in either major or another major (first two) cavities predicted by the metapocket server. In our previous studies, the structure of the functional domain of the proteins that belong to the WRKY gene superfamily members has been modeled for its qualitative and quantitative evaluation, to unravel the DNA–protein interaction studies, in a stimulus-specific manner in tomato ( Aamir et al, 2017 , 2018 ). In our results, the computational screening revealed the docking site and energy score values for all the 37 fungicides, to evaluate their efficacy against the FOL pathogen.…”

The Role of Tomato WRKY Genes in Plant Responses to Combined Abiotic and Biotic Stresses

“…However, it should be stressed that slight changes in the DNA-binding domain may have an important effect on the binding specificity, and sequence homologs may be highly similar yet have different functions ( Tao et al, 2009 , 2011 ; Du et al, 2014 ). For example, the close tomato homologs SlWKRY3 and SlWRKY4 are predicted to interact with the W-box DNA through a different motif, RKYGQK, and WRKYGQK, respectively ( Lai et al, 2008 ; Aamir et al, 2017 ). There is evidence that motifs outside the WRKY domain may provide binding specificity to WRKYs ( Phukan et al, 2016 ).…”