Functional Analysis of CaWRKY39 Under Phytophthora capsici Infection in Pepper

doi:10.16420/j.issn.0513-353x.2023-1013

Acta Horticulturae Sinica ›› 2024, Vol. 51 ›› Issue (10): 2297-2310.doi: 10.16420/j.issn.0513-353x.2023-1013

• Genetic & Breeding·Germplasm Resources·Molecular Biology • Previous Articles Next Articles

Functional Analysis of CaWRKY39 Under Phytophthora capsici Infection in Pepper

WU Dan¹, LIU Jiaxin¹, ZHUO Linxi¹, LI Yu¹, LUO Ying¹, ZHOU Yong², YANG Youxin¹^,^*(), YU Ting¹^,^*()

¹ Jiangxi Provincial Key Laboratory for Postharvest Storage and Preservation of Fruits & Vegetables，College of Agronomy，Jiangxi Agricultural University，Nanchang 330045，China
² Key Laboratory of Crop Physiology，Ecology and Genetic Breeding，Ministry of Education，College of Bioscience and Bioengineering，Jiangxi Agricultural University，Nanchang 330045，China

Received:2024-04-19 Revised:2024-07-25 Online:2024-10-25 Published:2024-10-21
Contact: YANG Youxin, YU Ting

Abstract

Abstract:

A WRKY transcription factor gene CaWRKY39 was cloned from pepper（Capsicum annuum L.）inbred line 007EA. The open reading frame of CaWRKY39 was 984 bp，encoding a protein of 327 amino acids，which contained a WRKY domain and a C2H2 zinc finger structure. Amino acid sequence alignment and phylogenetic tree analysis showed that CaWRKY39 had the highest homology with SmWRKY7，SlWRKY4 and StWRKY7 and gene structure analysis showed that CaWRKY39 contained three exons and two introns. Promoter analysis showed that CaWRKY39 promoter included W-box，and cis-acting elements responsive to light，ABA，MeJA，SA and auxin. Subcellular localization results showed that CaWRKY39 was located in the nucleus and cytoplasm. qRT-PCR results showed that CaWRKY39 displayed the highest expression in leaf，followed by stem，root，and other tissues，and its expression level was significantly increased after plant leaves infected by Phytophthora capsici. The function of CaWRKY39 was further studied by VIGS technology. TRV:CaWRKY39-silenced pepper plants were much sensitive to P. capsici infection compared with the TRV:00 control plants. These results of this study showed that CaWRKY39 may be involved in the resistance of pepper to phytophthora blight.

Key words: pepper, CaWRKY39, subcellular localization, Phytophthora capsica, functional analysis

WU Dan, LIU Jiaxin, ZHUO Linxi, LI Yu, LUO Ying, ZHOU Yong, YANG Youxin, YU Ting. Functional Analysis of CaWRKY39 Under Phytophthora capsici Infection in Pepper[J]. Acta Horticulturae Sinica, 2024, 51(10): 2297-2310.

Figures/Tables 11

References 39

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用途 Application	引物名称 Primer name	正向引物（5′-3′） Forward	反向引物（5′-3′） Reverse
亚细胞定位 Subcellular localization	CaWRKY39-GFP	CTCGATACACCAAATCGACTCTAGTCTAGAATGGCTGTGGACTTAATGATG	CAGCTCCTCGCCCTTGCTCACCATGGTACCAGAAGATTCTAAAATAAGACTACTTGTTTC
亚细胞定位 Subcellular localization	GFP	GACGCCATTTCGCCTTTTCAG	GAACTTGTGGCCGTTTACGTCG
基因沉默 VIGS	CaWRKY39	TGAGTAAGGTTACCGAATTCTCTAGAAAAGCCTCCGTTGTCTTCTTCTTCAC	TTTAATGTCTTCGGGACATGCCCGGGATATCTGCCAGTTTCATGCTTATTGC
	CaPDS	TGAGTAAGGTTACCGAATTCTCTAGAAGGTCTTCTTTGGGAACTGA	TTTAATGTCTTCGGGACATGCCCGGGAGACAAACCACCCAAACCTG
	TRV1	ATTGAGGCGAAGTACGATGG	CCATCCACAATTATTTTCCGC
实时荧光定量 qRT-PCR	qCaWRKY39	ACTCCAATCCAACAAGTCCCT	TCATCTTCAGCAGCTTTCCAG
	qCaActin	GACGTGACCTAACTGATAACCTGAT	CTCTCAGCACCAATGGTAATAACTT
	qPcActin	TTTAGTTGGGGGTCTTGTACC	CCTCCACAACCAGCAACA

用途 Application	引物名称 Primer name	正向引物（5′-3′） Forward	反向引物（5′-3′） Reverse
亚细胞定位 Subcellular localization	CaWRKY39-GFP	CTCGATACACCAAATCGACTCTAGTCTAGAATGGCTGTGGACTTAATGATG	CAGCTCCTCGCCCTTGCTCACCATGGTACCAGAAGATTCTAAAATAAGACTACTTGTTTC
亚细胞定位 Subcellular localization	GFP	GACGCCATTTCGCCTTTTCAG	GAACTTGTGGCCGTTTACGTCG
基因沉默 VIGS	CaWRKY39	TGAGTAAGGTTACCGAATTCTCTAGAAAAGCCTCCGTTGTCTTCTTCTTCAC	TTTAATGTCTTCGGGACATGCCCGGGATATCTGCCAGTTTCATGCTTATTGC
	CaPDS	TGAGTAAGGTTACCGAATTCTCTAGAAGGTCTTCTTTGGGAACTGA	TTTAATGTCTTCGGGACATGCCCGGGAGACAAACCACCCAAACCTG
	TRV1	ATTGAGGCGAAGTACGATGG	CCATCCACAATTATTTTCCGC
实时荧光定量 qRT-PCR	qCaWRKY39	ACTCCAATCCAACAAGTCCCT	TCATCTTCAGCAGCTTTCCAG
	qCaActin	GACGTGACCTAACTGATAACCTGAT	CTCTCAGCACCAATGGTAATAACTT
	qPcActin	TTTAGTTGGGGGTCTTGTACC	CCTCCACAACCAGCAACA

元件 Element	序列 Sequence	位置/bp Location	数量 Number	位点功能 Function of site
TC-rich repeats	GTTTTCTTAC	-813	1	参与防御和胁迫反应的顺式作用元件 cis-Acting element involved in defense and stress responsiveness
LAMP-element	CTTTATCA	-168/-92	2	光响应元件的一部分 Part of a light responsive element
Box 4	ATTAAT	-1 583/-939/ -348	3	参与光响应的保守DNA模块的一部分 Part of a conserved DNA module involved in light responsiveness
ATCT-motif	AATCTAATCC	-1 456/-384	2	参与光响应的保守DNA模块的一部分 Part of a conserved DNA module involved in light responsiveness
circadian	CAAAGATATC	-1 520	1	参与昼夜节律调控的顺式作用元件 cis-Acting regulatory element involved in circadian control
3-AF1 binding site	TAAGAGAGGAA	-1 643	1	光响应元件 Light responsive element
AE-box	AGAAACTT	-1 474	1	光响应模块的一部分 Part of a module for light response
ABRE	ACGTG	-1 257	1	参与脱落酸反应的顺式作用元件 cis-Acting element involved in the abscisic acid responsiveness
ARE	AAACCA	-1 960/ -1 886	2	厌氧诱导所必需的顺式作用元件 cis-Acting regulatory element essential for the anaerobic induction
TGA-element	AACGAC	-653	1	生长素响应元件 Auxin-responsive element
AT-rich sequence	TAAAATACT	-1 861	1	最大诱导子介导的激活元件 Element for maximal elicitor-mediated activation
ATC-motif	AGTAATCT	-387	1	参与光响应的保守DNA模块的一部分 Part of a conserved DNA module involved in light responsiveness
AT1-motif	AATTATTTTTTATT	-359	1	光响应模块的一部分 Part of a light responsive module
AuxRR-core	GGTCCAT	-996	1	参与生长素响应的顺式作用元件 cis-Acting regulatory element involved in auxin responsiveness
TCA-element	CCATCTTTTT	-1 983/-140	2	参与水杨酸反应的顺式作用元件 cis-Acting element involved in salicylic acid responsiveness
G-Box	CACGTT	-1 257	1	参与光响应的顺式作用元件 cis-Acting regulatory element involved in light responsiveness
CGTCA-motif	CGTCA	-218	1	参与MeJA响应的顺式作用元件 cis-Acting regulatory element involved in the MeJA-responsiveness
I-box	GTATAAGGCC	-271	1	光响应元件的一部分 Part of a light responsive element

元件 Element	序列 Sequence	位置/bp Location	数量 Number	位点功能 Function of site
TC-rich repeats	GTTTTCTTAC	-813	1	参与防御和胁迫反应的顺式作用元件 cis-Acting element involved in defense and stress responsiveness
LAMP-element	CTTTATCA	-168/-92	2	光响应元件的一部分 Part of a light responsive element
Box 4	ATTAAT	-1 583/-939/ -348	3	参与光响应的保守DNA模块的一部分 Part of a conserved DNA module involved in light responsiveness
ATCT-motif	AATCTAATCC	-1 456/-384	2	参与光响应的保守DNA模块的一部分 Part of a conserved DNA module involved in light responsiveness
circadian	CAAAGATATC	-1 520	1	参与昼夜节律调控的顺式作用元件 cis-Acting regulatory element involved in circadian control
3-AF1 binding site	TAAGAGAGGAA	-1 643	1	光响应元件 Light responsive element
AE-box	AGAAACTT	-1 474	1	光响应模块的一部分 Part of a module for light response
ABRE	ACGTG	-1 257	1	参与脱落酸反应的顺式作用元件 cis-Acting element involved in the abscisic acid responsiveness
ARE	AAACCA	-1 960/ -1 886	2	厌氧诱导所必需的顺式作用元件 cis-Acting regulatory element essential for the anaerobic induction
TGA-element	AACGAC	-653	1	生长素响应元件 Auxin-responsive element
AT-rich sequence	TAAAATACT	-1 861	1	最大诱导子介导的激活元件 Element for maximal elicitor-mediated activation
ATC-motif	AGTAATCT	-387	1	参与光响应的保守DNA模块的一部分 Part of a conserved DNA module involved in light responsiveness
AT1-motif	AATTATTTTTTATT	-359	1	光响应模块的一部分 Part of a light responsive module
AuxRR-core	GGTCCAT	-996	1	参与生长素响应的顺式作用元件 cis-Acting regulatory element involved in auxin responsiveness
TCA-element	CCATCTTTTT	-1 983/-140	2	参与水杨酸反应的顺式作用元件 cis-Acting element involved in salicylic acid responsiveness
G-Box	CACGTT	-1 257	1	参与光响应的顺式作用元件 cis-Acting regulatory element involved in light responsiveness
CGTCA-motif	CGTCA	-218	1	参与MeJA响应的顺式作用元件 cis-Acting regulatory element involved in the MeJA-responsiveness
I-box	GTATAAGGCC	-271	1	光响应元件的一部分 Part of a light responsive element

Functional Analysis of CaWRKY39 Under Phytophthora capsici Infection in Pepper

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[2]	LI Yifei, YANG Xiaomiao, WANG Chunping, DUAN Minjie, HUANG Qizhong, HUANG Renzhong, ZHANG Shicai. A New Processing Pepper Cultivar‘Yanjiao 465’ [J]. Acta Horticulturae Sinica, 2024, 51(9): 2223-2224.
[3]	QIU Hui, ZHU Dejuan, ZHANG Yongle, GAO Yujie, LI Liu, WANG Guoping, HONG Ni. Interaction Between the Coat Protein of Apple Chlorotic Leaf Spot Virus and Two E3 Ubiquitin Ligases of Pear and Their Subcellular Localization [J]. Acta Horticulturae Sinica, 2024, 51(8): 1715-1727.
[4]	DUAN Minjie, LI Yifei, WANG Chunping, YANG Xiaomiao, HUANG Renzhong, HUANG Qizhong, ZHANG Shicai. Integrated Transcriptomic and Targeted Metabolomic Analysis Reveals Regulation of Carotenoid Accumulation During Pepper Fruit Development [J]. Acta Horticulturae Sinica, 2024, 51(8): 1773-1791.
[5]	SHI Fengyan, WANG Zhidan, ZHANG Xi, WANG Xiuxue, ZOU Chunlei. Research Progress on the Mechanism of Pepper Resistance to Phytophthora Blight [J]. Acta Horticulturae Sinica, 2024, 51(7): 1665-1682.
[6]	ZHANG Ru, CHEN Lingzhi, WANG Lanlan. A New Pepper F₁ Hybrid Cultivar‘Longjiao 13’ [J]. Acta Horticulturae Sinica, 2024, 51(7): 1709-1710.
[7]	GUO Rui, CHEN Gao, ZHAO Huixai, QIAN Yike, LAN Hong, WAN Heping, CHEN Chanyou. A New Purple Pepper Cultivar‘Jiangda Zijiao 1’ [J]. Acta Horticulturae Sinica, 2024, 51(5): 1175-1176.
[8]	FU Wen, ZHU Chenghong, LAN Jiayi, LI Shi, ZHANG Zheng, LIU Feng, DAI Xiongze. Study on the Quality Characteristics of Fresh and Immature Pepper [J]. Acta Horticulturae Sinica, 2024, 51(3): 616-630.
[9]	ZHANG Shaoping, LI Zhou, JU Yudong, LIAN Dongmei, LIN Bizhen, YAO Yunfa, WU Songhai, HONG Jianji, LAI Zhengfeng. Advances in Research on Membrane Lipid Peroxidation Reaction During the Quality Deterioration Period of Fresh Pepper [J]. Acta Horticulturae Sinica, 2024, 51(12): 2945-2961.
[10]	LUO Yin, LUO Xirong, WU Shiqi, LI Tangyan, LI Jing, QIU Huarong, QIN Cheng. Karyotype Analysis of Four Cultivars of Peppers Based on Fluorescence in situ Hybridization [J]. Acta Horticulturae Sinica, 2024, 51(10): 2311-2319.
[11]	ZOU Xuexiao, YANG Sha, ZHU Fan, YUAN Fang. Progress and Trends in Development of Pepper Industry: High Quality，Good Taste of Fresh Pepper [J]. Acta Horticulturae Sinica, 2024, 51(1): 27-38.
[12]	LIU Shida, QIU Zelan, LI Xiaogang, ZHONG Jie, ZHU Junzi. Identification and Mechanism Characterization of Antagonistic Actinomyces SD-64 Against Pepper Southern Blight [J]. Acta Horticulturae Sinica, 2024, 51(1): 175-189.
[13]	LIAO Fangfang , HU Mingwen, ZHU Wenchao , SU Dan , BAI Liwei , and CHEN Lin. A New Fresh-eating Type Pepper Cultivar‘Layan 201’ [J]. Acta Horticulturae Sinica, 2023, 50(S1): 67-68.
[14]	YANG Jing , ZHANG Li , ZHAO Xiayun , QU Fei , WEN Linhong , WANG Tianwen , JIANG Fangfang , YANG Jinyan , and LI Zhengli, . A New Pepper Cultivar‘Qianjiao 12’ [J]. Acta Horticulturae Sinica, 2023, 50(S1): 69-70.
[15]	YAN Xi, HE Lei, LAI Wei, LIU Chongzheng, YANG Hong , and JIANG Hong. A New Pepper Cultivar‘Qianla 16’ [J]. Acta Horticulturae Sinica, 2023, 50(S1): 71-72.