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ISSN : 1598-5504(Print)
ISSN : 2383-8272(Online)
Journal of Agriculture & Life Science Vol.54 No.4 pp.1-5
DOI : https://doi.org/10.14397/jals.2020.54.4.1

Isolation and Expression Analysis of a WRKY Transcription Factor in Sweet potato

Dong-Hwan Shim1, Yun-Hee Kim2,*
1Department of Forest Genetic Resources, National Institute of Forest Science, Suwon 16631, Republic of Korea
2Department of Biology Education, IALS, Gyeongsang National University, Jinju, Republic of Korea
*Corresponding author: Yun-Hee Kim Tel:
+82-55-772-2237 Fax: +82-55-772-2239 E-mail:
cefle@gnu.ac.kr
June 19, 2020 July 23, 2020 August 3, 2020

Abstract

A new WRKY transcription factor gene was isolated by ESTs screening from a cDNA library of suspension cultured cells of Sweet potato (Ipomoea batatas). The 2,285 bp cDNA fragment, IbWRKY, was sequenced, from which a 505 amino acid residue protein was deduced. A search of the protein BLAST database identified significant similarity to other plant WRKY31 protein sequences. RT-PCR analysis showed expression patterns of IbWRKY31 in various intact tissues and suspension cultured cells of Sweet potato, and in leaves exposed to different stresses. The IbWRKY31 gene was highly expressed in suspension cultured cells. In leaf tissues, IbWRKY31 showed strong expression during salicylic acid and methyl jasmonate treatments. Expression of IbWRKY31 was also induced under various abiotic stress and pathogen infection conditions, such as wounding, H2O2, MV, PEG, NaCl, and bacterial pathogen infection. These results suggest that IbWRKY31 is involved in plant responses to various stress conditions, such as abiotic stresses and pathogen infection through a defense signaling pathway.


초록


    Ministry of Science, ICT and Future Planning
    (2018R1A1A1A05018446

    Introduction

    Plants have a variety of active defense mechanisms to protect themselves from various environmental conditions. A common feature of plant defense responses is the transcriptional activation of a large number of genes upon abiotic and biotic stress conditions. Transcriptional regulation of gene expression is largely mediated by the specific recognition of cis-acting promoter elements by trans-acting sequence specific DNA-binding transcription factors. Among the several classes of transcription factors associated with plant defense responses are the recently identified DNAbinding proteins containing WRKY domains that appear to be unique to plants (Eulgem et al., 2000). The WRKY domains contain a conserved WRKYGQ sequence followed by a Cys2His2 or Cys2HisCys zinc-binding motif (Eulgem et al., 2000). A number of studies have shown that WRKY proteins have regulatory functions in plant defense responses to SA-dependent signaling during pathogen infection (Eulgem et al., 1999;Chen & Chen, 2000;Dellagi et al., 2000;Kim et al., 2000). Interestingly, WRKY proteins are encoded by a multigene family (Eulgem et al., 2000). However, although there is considerable corroborative evidence that expressions of WRKYs is involved in responses and resistance to pathogen infection, the regulation of the different WRKYs during exposure to pathogen infection and stress response is not yet well understood in different plant species.

    Sweet potato (Ipomoea batatas) is a relatively drought-tolerant root crop, however Sweet potato is recognized as a comparatively stress-tolerant plant, molecular mechanisms underlying stress tolerance are not well defined. Previously expressed sequence tags (ESTs) from a full-length enriched cDNA library prepared from suspension cultured cells of Sweet potato were isolated and characterized (Kim et al., 2006). Expression analysis showed that some Sweet potato genes isolated from the EST library were responded to various environmental stress conditions (Kim et al., 2006). Therefore, the investigation of Sweet potato EST pools might yield valuable genetic information about the molecular genetic regulatory networks involved in stress-response pathway of Sweet potato.

    In this study, we isolated and characterized the IbWRKY gene from an EST library of suspension cultured cells in Sweet potato. The results suggest that the IbWRKY gene plays a role in responses to various stress conditions, such as abiotic stress and pathogen infection in Sweet potato.

    Materials and Methods

    1. Plant materials

    In the previously study, to construct the cDNA library, total RNA was extracted from suspension cultures of 15 DAS cells (Kim et al., 2006). Poly (A) RNA was prepared with the polyA Track mRNA isolation system (Promega, USA). After in vivo mass-excision of the library, plasmid DNA from 1700 randomly selected colonies was prepared and subjected to fluorescence cycle sequencing, using the ABI Big Dye Cycle Sequencing kit (PE Applied Biosystems, USA). Sweet potato (Ipomoea batatas L. Lam. cv. White Star) was obtained from Bioenergy Crop Research Center, National Crop Research Institute, RDA, Muan, Jeonnam, Korea. Plants were cultivated in a growth chamber in soil at 25°C with a photoperiod of 16 h light/8 h dark for 50 days. For gene expression analysis during plant growth, Sweet potato plants were grown in a greenhouse for 3 months. Suspension cultured cells (1 g fresh weight), subcultured at 14 days intervals, were inoculated into 50 mL of MS (Murashige & Skoog, 1962) basal medium supplemented with 2,4-dichlorophenoxyacetic acid (1 mg/L) and sucrose (30 g/ L).

    2. Analysis of nucleotide and protein sequences

    Sequence identities were determined using the multiple sequence alignments were performed using the Clustal X (www. clustal.org/clustal2/), National Center for Biotechnology Information (NCBI) BLAST search tool (https://blast.ncbi.nlm.nih.gov/), and GeneDoc programs. The deduced proteins were predicted using the ExPasy (http://www.expasy.org/tools) programs.

    3. Stress treatment

    Sweet potato plants grown at 25°C for 50 days were used for stress treatments. For treatments with phyto-hormones, the third leaf from the top was detached from each plant, placed into a conical tube containing 30 mL of sterile water (control) or 0.1 mM each solution such as SA, MeJA, ET and ABA, and then incubated at 25°C for 48 h under light conditions. For treatments with hydrogen peroxide (H2O2, 400 mM), methyl viologen (MV, 0.05 mM), PEG (30%) and NaCl (0.1 mM), Sweet potato leaves were incubated in conical tubes containing 30 mL of each chemical solution at 25 ◦C for 48 h under light conditions. Sterile water was used as a control for chemical stress treatments. For wounding treatment, the third leaves from the top were wounded by exerting pressure with a needle puncher according to methods of Huh et al., (1997). For bacterial treatment, Pectobacterium chrysanthemi (Erwinia chrysanthemi, KCTC 2569) was used following the methods of Jang et al., (2004).

    4. Gene expression analysis

    Total RNA was extracted from Sweet potato using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and, first strand cDNA was generated from total RNA of 2 μg using MMLV reverse transcriptase (Promega, Madison, WI, USA) in according to the manufacturer’s instructions. Gene-specific primers used for PCR reactions were as follows: the IbWRKY31 primer set (5’- GTCGAGAGTGGGAGGAATGAA-3’, 5’-TTACCGATATTG TGCCCCCG-3’) used to amplify a 186 bp product from cDNA coding for IbWRKY31, and alpha-tubulin (GenBank accession number, BM878762.1) gene-specific primers (5’-CAACTAC CAGCCACCAACTGT-3’, 5’- CAAGATCCTCACGAGCTTC AC-3’) to amplify the tubulin gene as an internal standard..

    Results

    1. Isolation and sequence analysis of the IbWRKY31

    To analyze the ESTs from the cDNA library of suspension cultured cells of Sweet potato (Kim et al., 2006), we searched the sequences using the NCBI database. The classification was based on the best homology match of BLASTX searches against Arabidopsis and other plant protein sequences. The novel WRKY gene was found among the most abundant ESTs. Its cDNA is 2,285 bp in length and encodes a deduced 505 amino acid residue protein (Fig. 1A). Database searches revealed that the amino acid sequence of the IbWRKY31 protein contains a conserved, WRKY domain that is present in a large family of plant WRKY proteins (Fig. 1B). Alignment and comparison of its amino acid sequence with those of other WRKY31 genes isolated from various plant species showed that it shares 91% homology with Ipomoea triloba WRKY31 and 71% homology with I. nill WRKY31 (Fig. 1B). Therefore, we suggest that IbWRKY31 can be classified as a member of the plant WRKY31 proteins, and is likely to have a similarly important role in pathogen infection-related stress responsive gene expression.

    2. Differential expression of IbWRKY31 in intact Sweet potato tissues and suspension cultured cells

    The expression patterns of the IbWRKY31 gene were investigated in various intact tissue in whole plants and suspension cultured cells of Sweet potato (L, leaf; S, stem; FR, fibrous root; TR, thick pigmented root; SR, storage root; SUS, suspension cultured cells) by RT-PCR analysis (Fig. 2). The results demonstrate that there is considerable variation in the levels of IbWRKY31 expression in different Sweet potato tissues. The IbWRKY31 gene was strongly expressed in suspension cultured cell conditions, whereas it was weakly expressed in tact tissues. During suspension cultured cell growth, IbWRKY31 showed high expression levels at 0.5 days after subculture.

    3. Differential responses of Sweet potato IbWRKY31 to phyto-hormone and chemical stresses

    To investigate the phyto-hormone-related responses of Sweet potato IbWRKY31, gene expression changes in response to salicylic acid (SA), methyl jasmonate (MeJA), ethephone (ET) and abscisic acid (ABA) treatment during time course for 48 h in leaves of Sweet potato were analyzed by RT-PCR (Fig. 3A). The IbWRKY31 gene exhibited increasing expression patterns in response to SA and MeJA treatments, whereas IbWRKY31 was not expressed during ET and ABA treatments. The IbWRKY31 expression was also induced by various chemical stressors such as H2O2, MV, PEG and NaCl in Sweet potato leaves for 48 h treatments. (Fig. 3B).

    4. Differential expression of IbWRKY31 in response to wounding and pathogen infection

    The expression patterns of IbWRKY31 in response to physical stress treatment and pathogenic infection were investigated by RT-PCR. IbWRKY31 expression increased strongly 12 h after wounding treatment, whereas in the control conditions expression was not detected (Fig. 4A). Expression patterns of the IbWRKY31 gene were also investigated in the leaves after infection with a bacterial pathogen (P. chrysanthemi) (Fig. 4B). IbWRKY31 expression was strongly induced by bacterial infection from 20 h, and slightly increased under untreated control conditions after 8 h. These results suggest that IbWRKY31 responds differently to various kinds of abiotic stress and bacterial pathogen infection.

    Discussion

    Among the various plant WRKYs, the studies of WRKY31 genes has not been characterized in detail (Phukan et al., 2016). It is reported that WRKY31 regulated resistance to Botryosphaeria dothidea through the SA signaling pathway by interacting with HIR4 in apple only (Zhao et al., 2019). In this study, IbWRKY31 was isolated from suspension cultured cells of Sweet potato, and the response to abiotic stress and bacterial pathogen infection was characterized. A comparison of stressed and unstressed plants revealed that the expression levels of the IbWRKY31 gene increased in response to wounding, chemical stresses, SA, MeJA treatment, and bacterial pathogen infection (Figs. 3 and 4). However, the expression levels of IbWRKY31 did not change significantly in response to ABA and ET treatments. These results suggest that the IbWRKY31 gene is involved in responses to SA and/or MeJA-related stress-signaling pathway during abiotic stresses and pathogen defense-mediated plant stresses in Sweet potato.

    IbWRKY31 showed the highest expression during growth as cell suspension cultures (Fig. 2). Plant cell suspension culture conditions result in the exposure of cells to higher levels of oxidative stress compared with whole plant culture conditions (Kwak et al., 1995). The results of this study exhibited that the expression of the IbWRKY31 gene also increased following chemical mediated oxidative stress conditions, such as with H2O2 and MV (Fig. 3). These findings indicate that the expression of the IbWRKY31 gene might be regulated in suspension cell cultures and under stress-related chemical treatment-mediated oxidative stress conditions.

    The data presented here suggest that IbWRKY31 might have roles in SA and/or MeJA-related abiotic stress tolerance and pathogenic resistance. Further investigation will be required to elucidate the exact role of the IbWRKY31 gene in the regulation of the defense signal pathway in Sweet potato under stress conditions.

    Acknowledgments

    This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT, and Future Planning (2018R1A1A1A05018446).

    Figure

    JALS-54-4-1_F1.gif

    Sequence analysis of the IbWRKY31 full-length cDNA. (A) Amino acid sequences of IbWRKY31 protein. (B) Multiple alignments of the amino acid sequences of IbWRKY31 proteins isolated from various plants. Conserved WRKYPQ domain was shown in red box. Ipomoea triloba WRKY31 (XP_031124630.1), Ipomoea nil WRKY31 (XP_019168717.1), Nicotiana sylvestris WRKY31 (XP_00 9776376.1), Vitis vinifera WRKY31 (XP_002263115.1), Capsicum annuum WRKY31 (NP_001311693.1), Ipomoea batatas WRKY31 (MN938932).

    JALS-54-4-1_F2.gif

    Expression patterns of IbWRKY31 in various intact tissues and suspension cultured cells of Sweet potato. Total RNAs were extracted from the leaf (L), stem (S), fibrous root (R), storage root (SR), and suspension-cultured cells (SUS). Alpha-tubulin was utilized as a control for equal loading. RT-PCR analyses were repeated at least three times.

    JALS-54-4-1_F3.gif

    Expression patterns of IbWRKY31 under phyto-hormone and chemical stress conditions. (A) Expression patterns of IbWRKY31 leaves in response to SA, MeJA, ET and ABA treatment for 48 h. (B) Expression patterns of IbWRKY31 under stress-related chemical treatments (400 mM H2O2, 0.05 mM MV, 30% PEG and 100 mM NaCl) for 48 h. Alpha-tubulin was utilized as a control for equal loading. RT-PCR analyses were repeated at least three times.

    JALS-54-4-1_F4.gif

    Expression patterns of IbWRKY31 under physical stress and pathogen infection conditions. (A) Expression patterns of IbWRKY31 in leaves under wounded conditions for 72 h. (B) Expression patterns of IbWRKY31 following infection of Sweet potato leaves with a bacterial pathogen (P. chrysanthemi). The mock involved treatment with 10 mM MgCl2. Alpha-tubulin was utilized as a control for equal loading. RT-PCR analyses were repeated at least three times.

    Table

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