转录因子NbMYB1R1通过促进活性氧积累抑制病毒侵染

doi:10.3864/j.issn.0578-1752.2024.08.006

Abstract

Abstract:

【Background】Cucumber green mottle mosaic virus (CGMMV) is an important quarantine plant virus in China, which has seriously reduced the production of vegetables and melons worldwide. The MYB protein family is large, multifunctional and present in all eukaryotes. Most MYB proteins act as transcription factors that control development, and metabolism in plants, and regulate biotic and abiotic stress responses. Previous studies have shown that during CGMMV infection, the expression of transcription factor gene NbMYB1R1 was significantly up-regulated. 【Objective】The objective of this study is to clarify the mechanism of NbMYB1R1 involved in CGMMV infection, and to provide a theoretical basis for controlling CGMMV infection. 【Method】MEGA 7.0 was used to construct a phylogenetic tree to analyze the amino acid sequence of NbMYB1R1 protein. The expression vector NbMYB1R1-GFP was constructed, transformed into Agrobacterium GV3101 and infiltrated the tobacco leaves to observe the subcellular localization of NbMYB1R1 by confocal microscopy. The transcriptional levels of NbMYB1R1 at different stages of CGMMV infection and ROS related genes in silenced NbMYB1R1 plants were analyzed using qRT-PCR technology. TRV-mediated gene silencing (VIGS) was utilized to analyze the function of NbMYB1R1, NbAOX1a, and NbAOX1b during CGMMV infection. Transient overexpression of NbMYB1R1 and NbMYB1R1 mutant was conducted to analyze the effect of NbMYB1R1 on CGMMV infection. Trypan blue staining and DAB staining were used to observe whether the cell death caused by the transient overexpression of NbMYB1R1 was related to the programmed cell death (PCD) and the accumulation of ROS. Yeast two-hybrid system was used to verify whether NbMYB1R1 interacted with CGMMV related proteins. 【Result】Phylogenetic tree analysis showed that NbMYB1R1 belonged to the 1R MYB category and had high homology with MYB transcription factors in a variety of tobaccos. The results of subcellular localization showed that NbMYB1R1 was localized in the nucleus. In CGMMV-infected tobacco plants, the transcript level of NbMYB1R1 was significantly changed compared with healthy plants, which was significantly up-regulated at 8 and 12 d of CGMMV infection. CGMMV was inoculated into TRV:NbMYB1R1 and TRV:00 plants. The NbMYB1R1-silenced plants showed the mottled and curled symptoms at 3 dpi, while the control plant appeared the symptoms at 3.5 dpi. Silencing NbMYB1R1 could effectively promote the accumulation of CGMMV at mRNA and protein levels. The CGMMV accumulation in NbMYB1R1 and NbMYB1R1 mutant transiently overexpression leaves was detected, and the results showed that NbMYB1R1 overexpression could effectively inhibit CGMMV infection and DNA binding domain deletion mutant reduced the inhibition of CGMMV by NbMYB1R1. The results of trypan blue and DAB staining showed that NbMYB1R1 overexpression could induce the ROS accumulation and cell death. The transcription level of ROS-related genes in TRV:NbMYB1R1 and TRV:00 plant was also detected, and the results showed that silencing NbMYB1R1 could specifically up-regulate the transcription of AOX1a and AOX1b. After CGMMV inoculation on silencing endogenous genes NbAOX1a and NbAOX1b, the systemic leaves of silenced NbAOX1a and NbAOX1b plants showed mottled and curled symptoms at 4 dpi, while the control plants appeared symptoms at 3.5 d. Meanwhile, the results of CGMMV CP mRNA and protein levels also indicated that silencing NbAOX1a and NbAOX1b could effectively inhibit the accumulation of CGMMV. The yeast two-hybrid results showed that NbMYB1R1 did not directly interact with the CGMMV proteins. 【Conclusion】During the CGMMV infection, the defense-related gene NbMYB1R1 is up-regulated. It speculates that the upregulation of NbMYB1R1 inhibits the transcription of the downstream genes AOX1a and AOX1b and activates the production of ROS in cells, thereby inhibiting viral infection. However, NbMYB1R1 does not produce this effect through a direct interaction with the CGMMV viral protein. NbMYB1R1 plays an important role in CGMMV infection.

Key words: cucumber green mottle mosaic virus (CGMMV), NbMYB1R1, transcription factor, pathogenic mechanism, reactive oxygen species (ROS)

JIANG XingLin, YU LianWei, FU Han, AI Niu, CUI YingJun, LI HaoHai, XIA ZiHao, YUAN HongXia, LI HongLian, YANG Xue, SHI Yan. The Transcription Factor NbMYB1R1 Inhibits Viral Infection by Promoting ROS Accumulation[J].Scientia Agricultura Sinica, 2024, 57(8): 1490-1505.

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Figures/Tables 11

Table 1

Primers used in this study"

用途Use	引物名称Primer name	引物序列Primer sequence (5′-3′)
酵母双杂交系统 Yeast two-hybrid	pGADT7- NbMYB1R1-F	ATATGGCCATGGAGGCCAGTGAATTCATGTCTAACGAATGCGGAAACAAAGA
	pGADT7- NbMYB1R1-R	ATCTGCAGCTCGAGCTCGATGGATCCAGCCACACTAATTATGCTATCCCC
	pGBKT7-CP-F	TGCATATGGCCATGGAGGCCGAATTCATGGCTTACAATCCGATCACACCTA
	pGBKT7-CP-R	TGCGGCCGCTGCAGGTCGACGGATCCAGCTTTCGAGGTGGTAGCC
	pGBKT7-MP-F	TGCATATGGCCATGGAGGCCGAATTCATGTCTCTAAGTAAGGTGTCAGTCG
	pGBKT7-MP-R	TGCGGCCGCTGCAGGTCGACGGATCCGGTGTGATCGGATTGTAAGCC
	pGBKT7-129K-F	TGCATATGGCCATGGAGGCCGAATTCATGGCAAACATTAATGAACAAATCA
	pGBKT7-129K-R	TGCGGCCGCTGCAGGTCGACGGATCCTTTGGTAGGCACAGTGGTAGC
	pGBKT7-MET-F	TGCATATGGCCATGGAGGCCGAATTCATGGCAAACATTAATGAACAAATCA
	pGBKT7-MET-R	TGCGGCCGCTGCAGGTCGACGGATCCCTCGATCTGTACCTTCCTAACCTTCA
	pGBKT7-HEL-F	TGCATATGGCCATGGAGGCCGAATTCACATTAGTTGACGGAGTGCC
	pGBKT7-HEL-R	TGCGGCCGCTGCAGGTCGACGGATCCTTTGGTAGGCACAGTGGTAGC
	pGBKT7-P2-F	TGCATATGGCCATGGAGGCCGAATTCCAATTAATGCAGAACTCGCTGTATG
	pGBKT7-P2-R	TGCGGCCGCTGCAGGTCGACGGATCCCTTAGAGACATCTATGTAAAGACTAC
瞬时过表达 Over-expression	NbMYB1R1-GFP-F	ACGAGCTGTACAAGGGTACCATGTCTAACGAATGCGGAAACAAAGA
	NbMYB1R1-GFP-R	CGGACTCTAGTTCATCTAGAAGCCACACTAATTATGCTATCCCC
	NbMYB1R1^121-127-GFP-F	CCTACTCAGGTGGCCCTCCGCCGAAATAACATCAA
	NbMYB1R1^121-127-GFP-R	GTTATTTCGGCGGAGGGCCACCTGAGTAGGGGTC
基因沉默 Virus induced gene silencing (VIGS)	NbMYB1R1 VIGS-F	AGTGGTCTCTGTCCAGTCCTCAAGACAAGGACCCCTACTC
	NbMYB1R1 VIGS-R	GGTCTCAGCAGACCACAAGTGGTATTGGCCTAATGGGCT
	NbAOX1a/b VIGS-F	AGTGGTCTCTGTCCAGTCCT GTAGGAGGAATGTTGTTGCACT
	NbAOX1a/b VIGS-R	GGTCTCAGCAGACCACAAGT ATTACCCTTGTCTAATTCCTTGAGG
实时荧光定量PCR Quantitative real-time PCR	NbUBC-qPCR-F	TTTCGGTCCTGATGATACTCCC
	NbUBC-qPCR-R	CACAGAGCAAAGACTGGATTGA
	NbMYB1R1-qPCR-F	GGGTTCATGCTCTTTGGTGT
	NbMYB1R1-qPCR-R	ACGGGTCCTAGCAGAAGGAT
	NbAOX1a-qPCR-F	TTGAGAACGTTCCTGCTCCT
	NbAOX1a-qPCR-R	TGGAGAGTCCCTTGAGCTGT
	NbAOX1b-qPCR-F	TTGAGAACGTTCCTGCTCCT
	NbAOX1b-qPCR-R	GGTGCTGGAGAGTCCTTGAG
	NbGST-qPCR-F	TGAAGGAAAGCGAAGCACAAT
	NbGST-qPCR-R	ACGACGGCGCGAATCAAACA
	NbSOD-qPCR-F	GCCGTCCTTAGCAGCAGTGAA
	NbSOD-qPCR-R	CCGGGTTTTAGGCCAGAGACAT
	NbAPX1-qPCR-F	GGAGTGGTTGCTGTTGAAGTC
	NbAPX1-qPCR-R	GGAGAGCCTTGTCTGATGG
	NbCAT3-qPCR-F	TGCGCATCACAATAATCACCAT
	NbCAT3-qPCR-R	TGTCGCGCTTTCCTGTCAA
	NbGPX2-qPCR-F	TTGTTTTGCCACTACCTTGTTCAG
	NbGPX2-qPCR-R	TTCATTTGGGGGTGTCAGATTAG
	CP-qPCR-F	ACAAGGTACCGCTTTCCAGA
	CP-qPCR-R	TACGACAGACGAGGGTAACG

Table 1

Table 2

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 5

Fig. 6

Fig. 7

Fig. 8

Fig. 9

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[2]	LI YiPu, TONG LiXiu, LIN YaNan, SU ZhiJun, BAO HaiZhu, WANG FuGui, LIU Jian, QU JiaWei, HU ShuPing, SUN JiYing, WANG ZhiGang, YU XiaoFang, XU MingLiang, GAO JuLin. Investigation of Low Nitrogen Tolerance of ZmCCT10 in Maize [J]. Scientia Agricultura Sinica, 2023, 56(6): 1035-1044.
[3]	YAN PeiHan, LUO JianMing, HAO ChenXing, SUN ZiQing, YE RongChun, LI Yi, LIU Lian, SHENG Ling, MA XianFeng, DENG ZiNiu. Identification of the Transcription Factor WRKY75 of CmPR4A in Citron C-05 and Its Function Analysis in Resistance to Citrus Canker Disease [J]. Scientia Agricultura Sinica, 2023, 56(21): 4304-4317.
[4]	HAN XiaoWen, HAN Shuo, HU YiFeng, WANG MengRu, CHEN ZhongYi, ZHU YongXing, YIN JunLiang. Genome-Wide Identification of AP2/ERF Gene Family in Alternanthera philoxeroides and Its Expression Patterns Under Herbicide Stresses [J]. Scientia Agricultura Sinica, 2023, 56(20): 4021-4034.
[5]	DING GuoHua, XIAO GuangHui, ZHU LiPing. Genome-Wide Identification and Expression Analysis of NLP (NIN- Like Protein) Transcription Factor Gene Family in Cotton [J]. Scientia Agricultura Sinica, 2023, 56(19): 3723-3746.
[6]	HA DanDan, ZHENG HongXia, ZHANG ZhenHao, ZHU LiHong, LIU Hao, WANG JiaoYu, ZHOU Lei. Fluorescent Labeling and Observation of Infection Structure of Fusarium verticillioides [J]. Scientia Agricultura Sinica, 2023, 56(18): 3556-3573.
[7]	YU LianWei, JIANG XingLin, YANG LingLing, WANG He, ZHANG YuYang, XIE LiNa, XIA ZiHao, LI HongLian, YANG Xue, SHI Yan. Function of Transcription Factor NbERF RAP2-1 in Cucumber Green Mottle Mosaic Virus Infection [J]. Scientia Agricultura Sinica, 2023, 56(15): 2919-2928.
[8]	ZHANG Nan, HAN GuangJie, LIU Qin, LI ChuanMing, QI JianHang, LU YuRong, XIA Yang, XU Jian. Response and Function of the Transcription Factor CncC in Cnaphalocrocis medinalis Infected with Baculovirus CnmeGV [J]. Scientia Agricultura Sinica, 2023, 56(13): 2491-2503.
[9]	YOU YuWan,ZHANG Yu,SUN JiaYi,ZHANG Wei. Genome-Wide Identification of NAC Family and Screening of Its Members Related to Prickle Development in Rosa chinensis Old Blush [J]. Scientia Agricultura Sinica, 2022, 55(24): 4895-4911.
[10]	ZHANG Jie, JIANG ChangYue, WANG YueJin. Functional Analysis of the Interaction Between Transcription Factors VqWRKY6 and VqbZIP1 in Regulating the Resistance to Powdery Mildew in Chinese Wild Vitis quinquangularis [J]. Scientia Agricultura Sinica, 2022, 55(23): 4626-4639.
[11]	PANG HaoWan,FU QianKun,YANG QingQing,ZHANG YuanYuan,FU FengLing,YU HaoQiang. Maize Transcription Factor ZmEREB93 Negatively Regulates Kernel Development [J]. Scientia Agricultura Sinica, 2022, 55(19): 3685-3696.
[12]	YANG ShengDi,MENG XiangXuan,GUO DaLong,PEI MaoSong,LIU HaiNan,WEI TongLu,YU YiHe. Co-Expression Network and Transcriptional Regulation Analysis of Sulfur Dioxide-Induced Postharvest Abscission of Kyoho Grape [J]. Scientia Agricultura Sinica, 2022, 55(11): 2214-2226.
[13]	LIU RuiDa, GE ChangWei, WANG MinXuan, SHEN YanHui, LI PengZhen, CUI ZiQian, LIU RuiHua, SHEN Qian, ZHANG SiPing, LIU ShaoDong, MA HuiJuan, CHEN Jing, ZHANG GuiYin, PANG ChaoYou. Cloning and Drought Resistance Analysis of Transcription Factor GhMYB108 in Gossypium hirsutum [J]. Scientia Agricultura Sinica, 2022, 55(10): 1877-1890.
[14]	MA ShuanHong, WAN Jiong, LIANG RuiQing, ZHANG XueHai, QIU XiaoQian, MENG ShuJun, XU NingKun, LIN Yuan, DANG KunTai, WANG QiYue, ZHAO JiaWen, DING Dong, TANG JiHua. Candidate Gene Association Analysis of Maize Transcription Factors in Flowering Time [J]. Scientia Agricultura Sinica, 2022, 55(1): 12-25.
[15]	SHA RenHe,LAN LiMing,WANG SanHong,LUO ChangGuo. The Resistance Mechanism of Apple Transcription Factor MdWRKY40b to Powdery Mildew [J]. Scientia Agricultura Sinica, 2021, 54(24): 5220-5229.