葡萄抗灰霉病相关转录因子VviERF045下游靶基因的筛选

doi:10.3864/j.issn.0578-1752.2025.13.007

Abstract

Abstract:

【Background】 Gray mold is an important fungal disease that seriously endangers the grapevine (Vitis vinifera) industry worldwide. As an important transcription factor family in plants, ethylene-responsive factor (AP2/ERF) family plays a key role in regulating plant growth and development and stress response. 【Objective】 The study aimed to elucidate the molecular mechanisms by which the grapevine ethylene-responsive factor VviERF045 mediates defense against Botrytis cinerea. Through prediction and functional analysis of its downstream target genes, this research provides new insights into the transcriptional regulatory network associated with grapevine resistance to B. cinerea, establishing a foundation for breeding disease-resistant cultivars. 【Method】 Grapevine leaves were inoculated with B. cinerea using both agar disc and spore suspension methods. The expression profiles of VviERF045 and its predicted targets were evaluated via quantitative real-time PCR (qRT-PCR). Promoter cis-acting elements were analyzed using PlantCARE, while phylogenetic relationships and sequence alignments of VviERF045 were assessed using MEGA 7 and DNAMAN. Functional validation was conducted through Agrobacterium-mediated transient overexpression of VviERF045 in ‘Cabernet Sauvignon’ leaves. To further explore its regulatory landscape, DNA affinity purification sequencing (DAP-seq) was performed on ‘Pinot Noir’ leaves at 12 hours post-inoculation (hpi) with B. cinerea. 【Result】 Transient overexpression of VviERF045 significantly enhanced resistance to gray mold in ‘Cabernet Sauvignon’ leaves. DAP-seq analysis was performed on V. vinifera ‘Pinot Noir’ leaves at 12 hpi. Comparative analysis of peaks between two experimental replicates identified 51 806 consensus peaks. Subsequent genomic annotation revealed these peaks were predominantly located within ± 2 kb regions flanking promoter-transcription start sites (TSS). The top 2 000 most statistically significant peaks were selected for functional characterization through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Through integrated analysis of potential target genes and peak calling, three high-confidence candidate target genes (TCP8, SAP5, and bHLH48) were identified, all showing significant binding peaks in their promoter regions. Subsequent qRT-PCR validation confirmed the transcriptional upregulation of these genes, suggesting their cooperative involvement with VviERF045 in the grapevine’s response to B. cinerea infection.【Conclusion】VviERF045 functions as a positive regulator of grapevine defense against B. cinerea, likely by activating key stress-responsive genes such as TCP8, SAP5, and bHLH48. These results provide mechanistic insights into the pathogen-responsive transcriptional network in grapevine and identify potential molecular targets for breeding resistant V. vinifera cultivars.

Key words: Vitis vinifera, VviERF045, Botrytis cinerea, gray mold, target gene, transient transformation, DAP-seq

ZHAO YuLei, XIN JiaLu, LI ChengNan, LI Shan, XIE XuFei, YIN Xiao. Screening of Target Genes Downstream of VviERF045, a Transcription Factor Associated with Gray Mold Resistance in Vitis vinifera[J].Scientia Agricultura Sinica, 2025, 58(13): 2578-2590.

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References 41

[1]	SUN X Y, XIA X, GUAN X. Genome-wide identification and characterisation of stress-associated protein gene family to biotic and abiotic stresses of grapevine. Pathogens, 2022, 11(12): 1426.
[2]	WILCOX W F, GUBLER W D, UYEMOTO J K. Compendium of Grape Diseases, Disorders, and Pests, 2nd ed. The American Phytopathological Society (APS), 2016.
[3]	FEDORINA J, TIKHONOVA N, UKHATOVA Y, IVANOV R, KHLESTKINA E. Grapevine gene systems for resistance to gray mold Botrytis cinerea and powdery mildew Erysiphe necator. Agronomy, 2022, 12(2): 499.
[4]	FENG K, HOU X L, XING G M, LIU J X, DUAN A Q, XU Z S, LI M Y, ZHUANG J, XIONG A S. Advances in AP2/ERF super-family transcription factors in plant. Critical Reviews in Biotechnology, 2020, 40(6): 750-776. doi: 10.1080/07388551.2020.1768509 pmid: 32522044
[5]	LICAUSI F, OHME-TAKAGI M, PERATA P. APETALA2/ethylene responsive factor (AP2/ERF) transcription factors: Mediators of stress responses and developmental programs. New Phytologist, 2013, 199(3): 639-649. doi: 10.1111/nph.12291 pmid: 24010138
[6]	LI M Y, XU Z S, HUANG Y, TIAN C, WANG F, XIONG A S. Genome-wide analysis of AP2/ERF transcription factors in carrot (Daucus carota L.) reveals evolution and expression profiles under abiotic stress. Molecular Genetics and Genomics, 2015, 290(6): 2049-2061.
[7]	NAKANO T, SUZUKI K, FUJIMURA T, SHINSHI H. Genome-wide analysis of the ERF gene family in Arabidopsis and rice. Plant Physiology, 2006, 140(2): 411-432.
[8]	MENG X Z, XU J, HE Y X, YANG K Y, MORDORSKI B, LIU Y D, ZHANG S Q. Phosphorylation of an ERF transcription factor by Arabidopsis MPK3/MPK6 regulates plant defense gene induction and fungal resistance. The Plant Cell, 2013, 25(3): 1126-1142.
[9]	SUZUKI H, ITO T, OGATA T, TSUKAHARA Y, NELSON R S, SASAKI N, MATSUSHITA Y. Overexpression of NtERF5, belonging to the ethylene response factor gene family, inhibits potato virus X infection and enhances expression of jasmonic acid/ethylene signaling marker genes in tobacco. Journal of General Plant Pathology, 2024, 90(3): 125-133.
[10]	OUYANG Z G, LIU S X, HUANG L H, HONG Y B, LI X H, HUANG L, ZHANG Y F, ZHANG H J, LI D Y, SONG F M. Tomato SlERF. A1, SlERF.B4, SlERF.C3 and SlERF.A3, members of B3 group of ERF family, are required for resistance to Botrytis cinerea. Frontiers in Plant Science, 2016, 7: 1964.
[11]	GAO H, JIANG L Y, DU B H, NING B, DING X D, ZHANG C Z, SONG B, LIU S S, ZHAO M, ZHAO Y X, RONG T Y, LIU D X, WU J J, XU P F, ZHANG S Z. GmMKK4-activated GmMPK6 stimulates GmERF113 to trigger resistance to Phytophthora sojae in soybean. The Plant Journal, 2022, 111(2): 473-495.
[12]	TEZUKA D, KAWAMATA A, KATO H, SABURI W, MORI H, IMAI R. The rice ethylene response factor OsERF83 positively regulates disease resistance to Magnaporthe oryzae. Plant Physiology and Biochemistry, 2019, 135: 263-271.
[13]	JIN J H, WANG M, ZHANG H X, KHAN A, WEI A M, LUO D X, GONG Z H. Genome-wide identification of the AP2/ERF transcription factor family in pepper (Capsicum annuum L.). Genome, 2018, 61(9): 663-674.
[14]	XU Z S, CHEN M, LI L C, MA Y Z. Functions of the ERF transcription factor family in plants. Botany, 2008, 86(9): 969-977.
[15]	MARUYAMA Y, YAMOTO N, SUZUKI Y, CHIBA Y, YAMAZAKI K, SATO T, YAMAGUCHI J. The Arabidopsis transcriptional repressor ERF9 participates in resistance against necrotrophic fungi. Plant Science, 2013, 213: 79-87.
[16]	ZHU Y X, ZHANG X M, ZHANG Q H, CHAI S Y, YIN W C, GAO M, LI Z, WANG X P. The transcription factors VaERF16 and VaMYB306 interact to enhance resistance of grapevine to Botrytis cinerea infection. Molecular Plant Pathology, 2022, 23(10): 1415-1432.
[17]	王梦楠. 中国野生山葡萄转录因子VaERF20克隆与功能研究[D]. 杨凌: 西北农林科技大学, 2018.
	WANG M N. Cloning and functional analysis of a VaERF20 transcription factor in Chinese wild Vitis amurensis[D]. Yangling: Northwest A&F University, 2018. (in Chinese)
[18]	柴生樾. 中国野生山葡萄抗灰霉病转录因子VaERF99功能及其作用机理[D]. 杨凌: 西北农林科技大学, 2021.
	CHAI S Y. Study on the function and mechanism of the transcription factor VaERF99 for Botrytis cinerea resistance in Vitis amurensis[D]. Yangling: Northwest A&F University, 2021. (in Chinese)
[19]	O’MALLEY R C, HUANG S C, SONG L, LEWSEY M G, BARTLETT A, NERY J R, GALLI M, GALLAVOTTI A, ECKER J R. Cistrome and epicistrome features shape the regulatory DNA landscape. Cell, 2016, 165(5): 1280-1292. doi: S0092-8674(16)30481-0 pmid: 27203113
[20]	BI Y, WANG H, YUAN X, YAN Y Q, LI D Y, SONG F M. The NAC transcription factor ONAC083 negatively regulates rice immunity against Magnaporthe oryzae by directly activating transcription of the RING-H2 gene OsRFPH2-6. Journal of Integrative Plant Biology, 2023, 65(3): 854-875.
[21]	LIU Y S, LIU Q W, LI X W, ZHANG Z J, AI S K, LIU C, MA F W, LI C. MdERF 114 enhances the resistance of apple roots to Fusarium solani by regulating the transcription of MdPRX63. Plant Physiology, 2023, 192(3): 2015-2029.
[22]	LEIDA C, DAL RÌ A, DALLA COSTA L, GÓMEZ M D, POMPILI V, SONEGO P, ENGELEN K, MASUERO D, RÍOS G, MOSER C. Insights into the role of the berry-specific ethylene responsive factor VviERF045. Frontiers in Plant Science, 2016, 7: 1793.
[23]	DE MICCOLIS ANGELINI R M, ROTOLO C, MASIELLO M, GERIN D, POLLASTRO S, FARETRA F. Occurrence of fungicide resistance in populations of Botryotinia fuckeliana (Botrytis cinerea) on table grape and strawberry in southern Italy. Pest Management Science, 2014, 70(12): 1785-1796.
[24]	MA N, SUN P, LI Z Y, ZHANG F J, WANG X F, YOU C X, ZHANG C L, ZHANG Z L. Plant disease resistance outputs regulated by AP2/ERF transcription factor family. Stress Biology, 2024, 4(1): 2. doi: 10.1007/s44154-023-00140-y pmid: 38163824
[25]	LI Z, ZHANG Y, REN J, JIA F L, ZENG H M, LI G Y, YANG X F. Ethylene-responsive factor ERF114 mediates fungal pathogen effector PevD1-induced disease resistance in Arabidopsis thaliana. Molecular Plant Pathology, 2022, 23(6): 819-831.
[26]	KHERADPOUR P, KELLIS M. Systematic discovery and characterization of regulatory motifs in ENCODE TF binding experiments. Nucleic Acids Research, 2014, 42(5): 2976-2987. doi: 10.1093/nar/gkt1249 pmid: 24335146
[27]	唐玉瑾. 葡萄VviBSs转录因子功能和机理研究[D]. 杨凌: 西北农林科技大学, 2022.
	TANG Y J. Stugy on the function and mechanism of VviBSs transcription factor[D]. Yangling: Northwest A&F University, 2022. (in Chinese)
[28]	ZHANG S L, WANG L, YAO J, WU N, AHMAD B, VAN NOCKER S, WU J Y, ABUDUREHEMAN R, LI Z, WANG X P. Control of ovule development in Vitis vinifera by VvMADS28 and interacting genes. Horticulture Research, 2023, 10(6): uhad070.
[29]	TANG Q, WEI S S, ZHENG X D, TU P C, TAO F. APETALA2/ ethylene-responsive factors in higher plant and their roles in regulation of plant stress response. Critical Reviews in Biotechnology, 2024, 44(8): 1533-1551.
[30]	LU X, ZHANG L, ZHANG F Y, JIANG W M, SHEN Q, ZHANG L D, LV Z Y, WANG G F, TANG K X. AaORA, a trichome-specific AP2/ERF transcription factor of Artemisia annua, is a positive regulator in the artemisinin biosynthetic pathway and in disease resistance to Botrytis cinerea. New Phytologist, 2013, 198(4): 1191-1202.
[31]	PENG Y, LIANG M R, ZHANG X, YU M, LIU H, CHENG Z M, XIONG J S. FaERF 2 activates two β-1,3-glucanase genes to enhance strawberry resistance to Botrytis cinerea. Plant Science, 2024, 347: 112179.
[32]	WANG L, LIU W D, WANG Y J. Heterologous expression of Chinese wild grapevine VqERFs in Arabidopsis thaliana enhance resistance to Pseudomonas syringae pv. tomato DC3000 and to Botrytis cinerea. Plant Science, 2020, 293: 110421.
[33]	SPEARS B J, MCINTURF S A, COLLINS C, CHLEBOWSKI M, CSEKE L J, SU J B, MENDOZA-CÓZATL D G, GASSMANN W. Class I TCP transcription factor AtTCP8 modulates key brassinosteroid-responsive genes. Plant Physiology, 2022, 190(2): 1457-1473. doi: 10.1093/plphys/kiac332 pmid: 35866682
[34]	AGUILAR-MARTÍNEZ J A, SINHA N. Analysis of the role of Arabidopsis class I TCP genes AtTCP7, AtTCP8, AtTCP22, and AtTCP23 in leaf development. Frontiers in Plant Science, 2013, 4: 406.
[35]	GUO S Y, XU Y Y, LIU H H, MAO Z W, ZHANG C, MA Y, ZHANG Q R, MENG Z, CHONG K. The interaction between OsMADS57 and OsTB1 modulates rice tillering via DWARF14. Nature Communications, 2013, 4: 1566.
[36]	SPEARS B J, HOWTON T C, GAO F, GARNER C M, MUKHTAR M S, GASSMANN W. Direct regulation of the EFR-dependent immune response by Arabidopsis TCP transcription factors. Molecular Plant-Microbe Interactions, 2019, 32(5): 540-549.
[37]	GIRI J, DANSANA P K, KOTHARI K S, SHARMA G, VIJ S, TYAGI A K. SAPs as novel regulators of abiotic stress response in plants. Bioessays, 2013, 35(7): 639-648. doi: 10.1002/bies.201200181 pmid: 23640876
[38]	SHU X, DING L, GU B, ZHANG H J, GUAN P Y, ZHANG J X. A stress associated protein from Chinese wild Vitis amurensis, VaSAP15, enhances the cold tolerance of transgenic grapes. Scientia Horticulturae, 2021, 285: 110147.
[39]	FELLER A, MACHEMER K, BRAUN E L, GROTEWOLD E. Evolutionary and comparative analysis of MYB and bHLH plant transcription factors. The Plant Journal, 2011, 66(1): 94-116. doi: 10.1111/j.1365-313X.2010.04459.x pmid: 21443626
[40]	LI M, SUN L, GU H, CHENG D W, GUO X Z, CHEN R, WU Z Y, JIANG J F, FAN X C, CHEN J Y. Genome-wide characterization and analysis of bHLH transcription factors related to anthocyanin biosynthesis in spine grapes (Vitis davidii). Scientific Reports, 2021, 11(1): 6863. doi: 10.1038/s41598-021-85754-w pmid: 33767241
[41]	ZHANG Y P, ZHANG L, MA H, ZHANG Y C, ZHANG X M, JI M M, VAN NOCKER S, AHMAD B, ZHAO Z Y, WANG X P, GAO H. Overexpression of the apple (Malus×domestica) MdERF100 in Arabidopsis increases resistance to powdery mildew. International Journal of Molecular Sciences, 2021, 22(11): 5713.

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NCBI登录号 NCBI accession number	注释 Description	引物序列 Primer sequence
XP_002272228.1	转录因子TCP8 Transcription factor TCP8	F: GCTTCACTCAACCTCGTCACTCC
XP_002272228.1	转录因子TCP8 Transcription factor TCP8	R: AGGACGCAGGCTTAGAGGATGTAG
XP_003633555.1	A20/AN1型锌指胁迫相关蛋白5 Zinc finger A20 and AN1 domain-containing stress-associated protein 5	F: GGTGAATCGGTGCTCTGGATGC
XP_003633555.1		R: CGGCGGTCTTGTAATCGTAGCTG
XP_002282897.1	转录因子bHLH48 Transcription factor bHLH48	F: GCGAAAGGAGCGAGAGAAGAAGG
XP_002282897.1	转录因子bHLH48 Transcription factor bHLH48	R: GCTATGGCTATCAGTGGCTTGGC
XP_002276899.1	含LOB结构域蛋白41 LOB domain-containing protein 41	F: TGCGGATGAGTTGTAATGGCTGTC
XP_002276899.1	含LOB结构域蛋白41 LOB domain-containing protein 41	R: GCGAGGAAGACGGTGGCATTG
KX179904.1	乙烯转录因子ERF045，ENTAV115 Ethylene transcription factor (ERF045)	F: TCTGAAATTCGCCACCCACTTCTG
KX179904.1		R: TTCTCGCCCTCGGACCACAC
VIT_12s0178g00200	内参 Actin	F: AACCCCAAGGCCAACAGAGAAAA
VIT_12s0178g00200	内参 Actin	R: CACCATCACCAGAATCCAGCACA

元件名称 Element name	序列 Sequence	元件数量 Number of elements	功能 Function
ATCT-motif	AATCTAATCC	1	光响应元件Light-responsive element
Gap-box	CAAATGAA（A/G）A	1	光响应元件Light-responsive element
GARE-motif	TCTGTTG	1	赤霉素响应元件Gibberellin-responsive element
GATT-motif	CTCCTGATTGGA	1	光响应元件Light-responsive element
GT1-motif	GGTTAA	1	光响应元件Light-responsive element
I-box	TGATAATGT	1	光响应元件Light-responsive element
TCA-element	TCAGAAGAGG	2	水杨酸响应元件Salicylic acid-responsive element
TC-rich repeats	ATTCTCTAAC	1	防御和胁迫响应元件Defence and stress-responsive element
TCT-motif	TCTTAC	1	光响应元件Light-responsive element

基因名称 Gene name	基因编号 Gene ID	P值 P value	FDR	蛋白名称 Uniprot_ID	注释 Description
LOC100266605	XP_002272228.1	4.42E-133	2.33E-128	F6GYS5	转录因子TCP8 Transcription factor TCP8
LOC100852428	XP_003633555.1	2.01E-125	7.16E-121	F6HBB0	A20/AN1型锌指胁迫相关蛋白5 Zinc finger A20 and AN1 domain-containing stress-associated protein 5
LOC100241856	XP_002282897.1	7.67E-121	2.14E-116	D7TI19	转录因子bHLH48 Transcription factor bHLH48

Screening of Target Genes Downstream of VviERF045, a Transcription Factor Associated with Gray Mold Resistance in Vitis vinifera

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