Identification of the group IIa WRKY subfamily and the functional analysis of GhWRKY17 in upland cotton (Gossypium hirsutum L.)

RESEARCH ARTICLE Identification of the group IIa WRKY subfamily and the functional analysis of GhWRKY17 in upland cotton (Gossypium hirsutum L.) Lijiao Gu1, Libei Li1, Hengling Wei1, Hantao Wang1, Junji Su1, Yaning Guo1,2, Shuxun Yu1* 1 State Key Laboratory of Cotton Biology, Institute of Cotton Research of CAAS, Anyang, Henan, China, 2 College of Agronomy, Northwest A&F University, Yangling, Shanxi, China * a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Gu L, Li L, Wei H, Wang H, Su J, Guo Y, et al. (2018) Identification of the group IIa WRKY subfamily and the functional analysis of GhWRKY17 in upland cotton (Gossypium hirsutum L.). PLoS ONE 13(1): e0191681. https://doi.org/ 10.1371/journal.pone.0191681 Editor: Meng-xiang Sun, Wuhan University, CHINA Received: May 14, 2017 Accepted: January 9, 2018 Published: January 25, 2018 Copyright: © 2018 Gu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: The work was supported by the National Key Research and Development Program of China, grant no. 2016YFD0101006 (http://service.most. gov.cn) to SY. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Abstract WRKY transcription factors play important roles in plant defense, stress response, leaf senescence, and plant growth and development. Previous studies have revealed the important roles of the group IIa GhWRKY genes in cotton. To comprehensively analyze the group IIa GhWRKY genes in upland cotton, we identified 15 candidate group IIa GhWRKY genes in the Gossypium hirsutum genome. The phylogenetic tree, intron-exon structure, motif prediction and Ka/Ks analyses indicated that most group IIa GhWRKY genes shared high similarity and conservation and underwent purifying selection during evolution. In addition, we detected the expression patterns of several group IIa GhWRKY genes in individual tissues as well as during leaf senescence using public RNA sequencing data and real-time quantitative PCR. To better understand the functions of group IIa GhWRKYs in cotton, GhWRKY17 (KF669857) was isolated from upland cotton, and its sequence alignment, promoter cisacting elements and subcellular localization were characterized. Moreover, the over-expression of GhWRKY17 in Arabidopsis up-regulated the senescence-associated genes AtWRKY53, AtSAG12 and AtSAG13, enhancing the plant’s susceptibility to leaf senescence. These findings lay the foundation for further analysis and study of the functions of WRKY genes in cotton. Introduction Throughout their life cycle, plants exhibit a set of complex adjustment mechanisms to perceive and respond to various physiological and developmental signals. Among these diverse adjustment mechanisms, transcriptional regulation mechanisms, which are mainly executed by transcription factors (TFs), play important roles [1]. For example, WRKY proteins constitute one of the largest TF families in plants [2]. Since the first WRKY gene, SPF1, was reported in sweet potato [3], increasing numbers of WRKY genes have been reported in various species, including Arabidopsis thaliana, Gossypium hirsutum, Oryza sativa, Ricinus communis, Manihot esculenta and Cucumis sativus [4–9]. WRKY TFs were named for their conserved WRKY domain, which consists of approximately 60 amino acids containing a conserved WRKYGQK core sequence and a zinc finger- PLOS ONE | https://doi.org/10.1371/journal.pone.0191681 January 25, 2018 1 / 21 Group IIa WRKY genes in upland cotton like motif [2]. The WRKY TF family is divided into three main groups according to the number of WRKY domains and the pattern of zinc finger motifs: group I contains two WRKY domains each with a C2H2 zinc finger motif, whereas group II and group III each contain a single WRKY domain with either a C2H2 zinc finger motif or a C2HC zinc finger motif, respectively. However, group II can be further divided into five subgroups (IIa, IIb, IIc, IId, and IIe) according to the amino acid motifs outside the WRKY domain [2,10–13]. The group IIa WRKY domain possesses a conserved VQR intron in the zinc finger motif nearer to the Cterminus [14]. Previous studies have reported 3 group IIa WRKY genes in Arabidopsis thaliana, 4 in Oryza sativa, 6 in Gossypium hirsutum, 5 in Populus trichocarpa and 4 in Cucumis sativus [9,15,5]. Group IIa WRKY genes appear to include a small number of members, but they participate widely in the regulation of diverse physiological processes, such as defense, trichome development and leaf senescence [16,17,13,18]. In Arabidopsis, the genes AtWRKY18, AtWRKY40 and AtWRKY60 represent the group IIa WRKY subfamily [2]. These three homologs exhibit a complex pattern of physical and functional interactions in response to the microbial pathogens Pseudomonas syringae and Botrytis cinerea [19]. In addition, the over-expression of AtWRKY18 in Arabidopsis can delay leaf senescence, but AtWRKY18 T-DNA insertion lines show an early leaf senescence phenotype. An AtWRKY18-AtWRKY53-mediated signaling pathway is involved in the senescence process [20]. However, more work has focused on the role of these three genes in the abscisic acid (ABA) signaling pathway. For example, the three WRKY genes were identified as negative regulators of ABA signaling [21,22] and can bind to W-box elements in the promoter regions of ABI4 and ABI5 to inhibit the expression of these two genes [23]. In rice, OsWRKY28, OsWRKY62, OsWRKY71 and OsWRKY76 are four members of the OsWRKY group IIa subfamily and are involved in modulating plant innate immunity [24]. OsWRKY28 plays a negative regulatory role in the resistance to rice blast fungus Ina86-137, as determined by phenotypic analysis of an oswrky28 mutant [25]. OsWRKY71 was found to be induced by salicylic acid (SA), methyl jasmonic acid (MeJA) and pathogen infection [26]. OsWRKY62 responds negatively to innate immunity in terms of susceptibility to pathogens [27]. Additionally, inducible alternative splicing of the genes OsWRKY62 and OsWRKY76 participates in pathogen defense, as found through the analysis of over-expression and loss-of-function knockout rice plants [28]. Furthermore, the group IIa WRKY genes have also been studied in other species. For example, the over-expression of PtrWRKY40 can enhance resistance to the necrotrophic fungus B. cinerea in Arabidopsis and susceptibility to Dothiorella gregaria in poplar [29], and TaWRKY71-1 presents a hyponastic leaf phenotype by altering IAA levels in transgenic Arabidopsis [30]. Since the release of abundant genome sequences and publicly availab (...truncated)