棉花黄萎病菌β-葡萄糖苷酶基因的鉴定及其在致病中的功能

doi:10.3864/j.issn.0578-1752.2026.07.002

摘要/Abstract

摘要：

【目的】大丽轮枝菌（Verticillium dahliae）是一种重要的土传病原真菌，可侵染200多种植物，引致黄萎病，对全球农业生产造成严重经济损失。细胞壁降解酶（CWDEs）在病原真菌致病过程中发挥关键作用，鉴定棉花黄萎病菌中的β-葡萄糖苷酶基因并探究其功能，为棉花抗病育种提供新的分子靶点。【方法】通过生物信息学分析，从大丽轮枝菌全基因组鉴定β-葡萄糖苷酶基因，并对其进化关系、保守结构域和表达模式进行系统分析。利用寄主诱导的基因沉默（HIGS）技术在棉花中沉默Vdbg4和Vdbg6，评估其对棉花抗病性的影响。设计靶向Vdbg4（asiR1364）和Vdbg6（asiR1444）的asiRNAs分别与V. dahliae共培养，对共培养V. dahliae的生长发育、碳源利用和胁迫响应能力，以及致病力等进行调查。通过酵母信号肽诱捕试验验证Vdbg6的分泌活性，并利用农杆菌介导的烟草瞬时表达试验检测其是否触发植物免疫反应。【结果】从大丽轮枝菌全基因组中共鉴定18个β-葡萄糖苷酶基因，其中，Vdbg4和Vdbg6受感病棉花品种根系分泌物诱导后显著上调表达。利用HIGS技术分别沉默Vdbg4或Vdbg6，以及同时沉默2个基因，均能显著减轻棉花的发病症状，降低病情指数和真菌生物量。靶向Vdbg4和Vdbg6的asiRNAs（asiR1364、asiR1444和asiR1364+1444）能够抑制病原菌的菌落和菌丝生长，降低产孢和孢子萌发率、碳源利用和胁迫响应的能力以及致病力。Vdbg6具有分泌活性，但它不能引起烟草细胞程序性坏死，也不能抑制由BAX诱导的烟草细胞程序性坏死。【结论】Vdbg4和Vdbg6均参与大丽轮枝菌的生长发育、碳源利用、胁迫响应和致病过程。

关键词: 棉花, 大丽轮枝菌, 细胞壁降解酶, β-葡萄糖苷酶, 寄主诱导的基因沉默, 人工小干扰RNA

Abstract:

【Objective】Verticillium dahliae is a major soil-borne pathogenic fungus that can infect more than 200 plant species, causing Verticillium wilt and leading to severe economic losses in global agricultural production. Cell wall-degrading enzymes (CWDEs) play a critical role in fungal pathogenesis. This study aimed to identify β-glucosidase genes in V. dahliae and explore their functions, thereby providing new molecular targets for cotton disease-resistant breeding.【Method】β-glucosidase genes were identified from the whole genome of V. dahliae through bioinformatic analysis, and their evolutionary relationships, conserved domains, and expression patterns were systematically analyzed. Host-induced gene silencing (HIGS) technology was used to silence Vdbg4 and Vdbg6 in cotton to evaluate the effects of this silencing on cotton disease resistance. Artificial small interfering RNAs (asiRNAs) targeting Vdbg4 (asiR1364) and Vdbg6 (asiR1444) were designed and co-cultured with V. dahliae, respectively. The growth and development, carbon source utilization and stress response abilities, and pathogenicity of V. dahliae in co-culture were investigated. The secretory activity of Vdbg6 was verified using a yeast signal peptide trapping assay, and whether the gene could trigger plant immune responses was detected using an Agrobacterium-mediated tobacco transient expression assay.【Result】A total of 18 β-glucosidase genes were identified from V. dahliae. Among them, the expression levels of Vdbg4 and Vdbg6 were significantly up-regulated after induction by root exudates of a susceptible cotton cultivar. Silencing Vdbg4, Vdbg6 alone, or silencing both genes simultaneously via HIGS, significantly alleviated the disease symptoms of cotton, reduced the disease index, and decreased the fungal biomass. The asiRNAs targeting Vdbg4 and Vdbg6 (asiR1364, asiR1444, and asiR1364+1444) could inhibit the fungal colony and mycelial growth, reduce the sporulation and spore germination rates, and impair the carbon source utilization and stress response abilities, and pathogenicity of V. dahliae. Vdbg6 exhibited secretory activity, but it could neither induce programmed cell death (PCD) nor suppress BAX-induced PCD in tobacco cells.【Conclusion】Both Vdbg4 and Vdbg6 are involved in the growth and development, carbon source utilization, stress response, and pathogenic processes of V. dahliae.

Key words: cotton, Verticillium dahliae, cell wall-degrading enzymes, β-glucosidase, host-induced gene silencing (HIGS), artificial small interfering RNA (asiRNA)

李元晶, 袁瑞祥, 李永泰, 孙天歌, 刘峰, 李艳军, 张新宇. 棉花黄萎病菌β-葡萄糖苷酶基因的鉴定及其在致病中的功能[J]. 中国农业科学, 2026, 59(7): 1380-1399.

LI YuanJing, YUAN RuiXiang, LI YongTai, SUN TianGe, LIU Feng, LI YanJun, ZHANG XinYu. Identification and Functional Characterization of β-Glucosidase Genes in Verticillium dahliae for Pathogenicity on Cotton[J]. Scientia Agricultura Sinica, 2026, 59(7): 1380-1399.

图/表 10

表1

表2

大丽轮枝菌中β-葡萄糖苷酶基因的鉴定"

基因ID ID gene	基因名称 Gene name	CDS长度 CDS length (bp)	蛋白长度 Protein length (aa)	染色体Chromosome	分子量 Molecular weight (kDa)	理论等电点Isoelectric point	跨膜结构域数量Transmembrane domain number	亚细胞定位预测Subcellular localization prediction	基因描述 Gene description
VDAG_00134	Vdbg1	2878	813	2	87.54	6.34	1	质膜 Plasma membrane	β-葡萄糖苷酶Beta-glucosidase
VDAG_01062	Vdbg2	3076	897	1	98.58	5.41	0	细胞质 Cytosol	耐热β-葡萄糖苷酶Thermostable Beta-glucosidase
VDAG_01263	Vdbg3	1886	611	1	66.56	6.28	0	细胞质 Cytosol	β-葡萄糖苷酶Beta-glucosidase
VDAG_02542	Vdbg4	2796	798	3	86.19	5.67	0	线粒体Mitochondrion	耐热β-葡萄糖苷酶Thermostable Beta-glucosidase
VDAG_05580	Vdbg5	2489	774	2	85.27	4.98	0	细胞质 Cytosol	β-葡萄糖苷酶Beta-glucosidase
VDAG_06279	Vdbg6	2758	683	8	74.29	5.54	0	细胞外 Extracellular	β-葡萄糖苷酶Beta-glucosidase
VDAG_08139	Vdbg7	2500	816	6	87.91	4.69	0	细胞外 Extracellular	β-葡萄糖苷酶Beta-glucosidase
VDAG_09280	Vdbg8	2605	823	4	89.29	5.93	0	细胞质 Cytosol	耐热β-葡萄糖苷酶Thermostable Beta-glucosidase
VDAG_09750	Vdbg9	3169	876	3	94.85	4.78	0	细胞外 Extracellular	β-葡萄糖苷酶Beta-glucosidase
VDAG_09393	Vdbg10	1943	609	4	67.13	4.88	0	细胞外 Extracellular	周质β-葡萄糖苷酶Periplasmic Beta-glucosidase
VDAG_05611	Vdbg11	2676	833	2	91.40	5.61	0	细胞质 Cytosol	β-葡萄糖苷酶Beta-glucosidase
VDAG_03973	Vdbg12	2598	803	3	104.64	5.51	0	细胞外 Extracellular	β-葡萄糖苷酶Beta-glucosidase
VDAG_02717	Vdbg13	2942	955	3	86.67	5.24	1	细胞质 Cytosol	β-葡萄糖苷酶Beta-glucosidase
VDAG_08263	Vdbg14	2313	746	6	78.45	4.77	0	细胞外 Extracellular	耐热β-葡萄糖苷酶Thermostable Beta-glucosidase
VDAG_08240	Vdbg15	2632	836	6	89.78	5.41	0	细胞质 Cytosol	耐热β-葡萄糖苷酶Thermostable Beta-glucosidase
VDAG_06333	Vdbg16	1995	476	8	53.78	4.87	0	细胞质 Cytosol	β-葡萄糖苷酶Beta-glucosidase
VDAG_02175	Vdbg17	2420	781	7	84.91	5.39	0	细胞外 Extracellular	β-葡萄糖苷酶Beta-glucosidase
VDAG_03143	Vdbg18	648	215	6	23.10	4.88	0	细胞质 Cytosol	β-葡萄糖苷酶Beta-glucosidase

表2

图1

图2

图3

图4

图5

图6

图7

图8

参考文献 59

[1]	SUNILKUMAR G, CAMPBELL L M, PUCKHABER L, STIPANOVIC R D, RATHORE K S. Engineering cottonseed for use in human nutrition by tissue-specific reduction of toxic gossypol. Proceedings of the National Academy of Sciences of the United States of America, 2006, 103(48): 18054-18059.
[2]	BILLAH M, LI F G, YANG Z E. Regulatory network of cotton genes in response to salt, drought and wilt diseases (Verticillium and Fusarium): Progress and perspective. Frontiers in Plant Science, 2021, 12: 759245. doi: 10.3389/fpls.2021.759245
[3]	LI H Y, DAI J C, QIN J, SHANG W J, CHEN J Y, ZHANG L, DAI X F, KLOSTERMAN S J, XU X M, SUBBARAO K V, et al. Genome sequences of Verticillium dahliae defoliating strain XJ592 and nondefoliating strain XJ511. Molecular Plant-Microbe Interactions, 2020, 33(4): 565-568. doi: 10.1094/MPMI-11-19-0320-A
[4]	SHORT D P G, SANDOYA G, VALLAD G E, KOIKE S T, XIAO C L, WU B M, GURUNG S, HAYES R J, SUBBARAO K V. Dynamics of Verticillium species microsclerotia in field soils in response to fumigation, cropping patterns, and flooding. Phytopathology, 2015, 105(5): 638-645. doi: 10.1094/PHYTO-09-14-0259-R
[5]	TIAN J, KONG Z S. Live-cell imaging elaborating epidermal invasion and vascular proliferation/colonization strategy of Verticillium dahliae in host plants. Molecular Plant Pathology, 2022, 23(6): 895-900. doi: 10.1111/mpp.v23.6
[6]	FRADIN E F, THOMMA B P H J. Physiology and molecular aspects of Verticillium wilt diseases caused by V. dahliae and V. albo-atrum. Molecular Plant Pathology, 2006, 7(2): 71-86. doi: 10.1111/mpp.2006.7.issue-2
[7]	康立茹, 刘燕, 杨永峰, 王永, 王黎胜, 刘湘萍, 付崇毅, 狄洁增, 廉勇, 高婧. 大丽轮枝菌及其致病机制的研究进展. 现代农业, 2022, 47(3): 49-51.
	KANG L R, LIU Y, YANG Y F, WANG Y, WANG L S, LIU X P, FU C Y, DI J Z, LIAN Y, GAO J. Research progress of Verticillium dahliae and its pathogenic mechanism. Modern Agriculture, 2022, 47(3): 49-51. (in Chinese)
[8]	TANG C, XIONG D G, FANG Y L, TIAN C M, WANG Y L. The two-component response regulator VdSkn7 plays key roles in microsclerotial development, stress resistance and virulence of Verticillium dahliae. Fungal Genetics and Biology, 2017, 108: 26-35. doi: 10.1016/j.fgb.2017.09.002
[9]	CANTU D, VICENTE A R, LABAVITCH J M, BENNETT A B, POWELL A L T. Strangers in the matrix: Plant cell walls and pathogen susceptibility. Trends in Plant Science, 2008, 13(11): 610-617. doi: 10.1016/j.tplants.2008.09.002 pmid: 18824396
[10]	KUBICEK C P, STARR T L, GLASS N L. Plant cell wall-degrading enzymes and their secretion in plant-pathogenic fungi. Annual Review of Phytopathology, 2014, 52: 427-451. doi: 10.1146/annurev-phyto-102313-045831 pmid: 25001456
[11]	YANG Y K, ZHANG Y, LI B B, YANG X F, DONG Y J, QIU D W. A Verticillium dahliae pectate lyase induces plant immune responses and contributes to virulence. Frontiers in Plant Science, 2018, 9: 1271. doi: 10.3389/fpls.2018.01271
[12]	CHEN J Y, XIAO H L, GUI Y J, ZHANG D D, LI L, BAO Y M, DAI X F. Characterization of the Verticillium dahliae exoproteome involves in pathogenicity from cotton-containing medium. Frontiers in Microbiology, 2016, 7: 1709.
[13]	GUI Y J, ZHANG W Q, ZHANG D D, ZHOU L, SHORT D P G, WANG J, MA X F, LI T G, KONG Z Q, WANG B L, et al. A Verticillium dahliae extracellular cutinase modulates plant immune responses. Molecular Plant-Microbe Interactions, 2018, 31(2): 260-273. doi: 10.1094/MPMI-06-17-0136-R
[14]	WANG D, CHEN J Y, SONG J, LI J J, KLOSTERMAN S J, LI R, KONG Z Q, SUBBARAO K V, DAI X F, ZHANG D D. Cytotoxic function of xylanase VdXyn4 in the plant vascular wilt pathogen Verticillium dahliae. Plant Physiology, 2021, 187(1): 409-429. doi: 10.1093/plphys/kiab274
[15]	MARUTHACHALAM K, KLOSTERMAN S J, KANG S, HAYES R J, SUBBARAO K V. Identification of pathogenicity-related genes in the vascular wilt fungus Verticillium dahliae by Agrobacterium tumefaciens-mediated T-DNA insertional mutagenesis. Molecular Biotechnology, 2011, 49(3): 209-221. doi: 10.1007/s12033-011-9392-8
[16]	GUI Y J, CHEN J Y, ZHANG D D, LI N Y, LI T G, ZHANG W Q, WANG X Y, SHORT D P G, LI L, GUO W, et al. Verticillium dahliae manipulates plant immunity by glycoside hydrolase 12 proteins in conjunction with carbohydrate-binding module 1. Environmental Microbiology, 2017, 19(5): 1914-1932. doi: 10.1111/emi.2017.19.issue-5
[17]	王艺潼, 李海博, 王雨晴, 吴志宏, 高贻宙. 层出镰刀菌分泌蛋白阿魏酸酯酶(FpESD)的鉴定及功能研究. 农业生物技术学报, 2025, 33(2): 386-399.
	WANG Y T, LI H B, WANG Y Q, WU Z H, GAO Y Z. Identification and functional analysis of the secretory protein feruloyl esterase (FpESD) in Fusarium proliferatum. Journal of Agricultural Biotechnology, 2025, 33(2): 386-399. (in Chinese)
[18]	邢启凯, 王欣芳, 彭军波, 张玮, 燕继晔, 李永华. 可可毛色二孢菌全基因组非经典分泌蛋白的预测及致病相关性分析. 植物病理学报, 2024, 54(1): 102-115. doi: 10.13926/j.cnki.apps.001625
	XING Q K, WANG X F, PENG J B, ZHANG W, YAN J Y, LI Y H. Genome-wide prediction and pathogenic analysis of non-classical secreted proteins of Lasiodiplodia theobromae. Acta Phytopathologica Sinica, 2024, 54(1): 102-115. (in Chinese)
[19]	郑菲. 真菌第五家族纤维素酶的基因挖掘与分子改良研究[D]. 北京: 北京林业大学, 2019.
	ZHENG F. Gene excavation and molecular engineering of glycoside hydrolyase amily 5 cellulase from fungi[D]. Beijing: Beijing Forestry University, 2019. (in Chinese)
[20]	徐金涛. 瑞氏木霉β-葡萄糖苷酶在纤维素酶诱导表达过程中作用机制的研究[D]. 济南: 山东大学, 2014.
	XU J T. The study on the roles of β-glucosidases in induced expression of cellulase genes in Trichoderma reesei[D]. Jinan: Shandong University, 2014. (in Chinese)
[21]	XU M W, LI H W, LUO H Y, LIU J Y, LI K Q, LI Q Q, YANG N, XU D L. Unveiling the role of β-glucosidase genes in Bletilla striata’s secondary metabolism: A genome-wide analysis. International Journal of Molecular Sciences, 2024, 25(23): 13191. doi: 10.3390/ijms252313191
[22]	林文细. 内生真菌Microsphaeropsis sp. S59 β-葡萄糖苷酶Bgl812酶学性质及其生物转化人参皂苷[D]. 南昌: 江西科技师范大学, 2024.
	LIN W X. Endophytic fungi Microsphaeropsis sp. S59 enzymatic properties of β-glucosidase Bgl812 and its biotransformation of ginsenosides[D]. Nanchang: Jiangxi Science and Technology Normal University, 2024. (in Chinese)
[23]	邱萍. 基于多组学解析棉花与大丽轮枝菌的互作机制[D]. 武汉: 华中农业大学, 2023.
	QIU P. Interaction mechanism analysis between cotton and Verticillium dahliae based on mμlti-omics[D]. Wuhan: Huazhong Agricultural University, 2023. (in Chinese)
[24]	PÉREZ-HERNÁNDEZ A, GONZÁLEZ M, GONZÁLEZ C, VAN KAN J A L, BRITO N. BcSUN1, a B. cinerea SUN-family protein, is involved in virulence. Frontiers in Microbiology, 2017, 8: 35.
[25]	JOURDAN S, FRANCIS I M, DEFLANDRE B, TENCONI E, RILEY J, PLANCKAERT S, TOCQUIN P, MARTINET L, DEVREESE B, LORIA R, et al. Contribution of the β-glucosidase BglC to the onset of the pathogenic lifestyle of Streptomyces Scabies. Molecular Plant Pathology, 2018, 19(6): 1480-1490. doi: 10.1111/mpp.2018.19.issue-6
[26]	ZHANG X Y, CHENG W H, FENG Z D, ZHU Q H, SUN Y Q, LI Y J, SUN J. Transcriptomic analysis of gene expression of Verticillium dahliae upon treatment of the cotton root exudates. BMC Genomics, 2020, 21(1): 155. doi: 10.1186/s12864-020-6448-9
[27]	张小雪. 黄萎病菌VdxyL3和VdITP1基因对棉花的抗病性影响[D]. 石河子: 石河子大学, 2021.
	ZHANG X X. Effects of VdxyL3 and VdITP1 genes of Verticillium wilt on disease resistance of cotton[D]. Shihezi: Shihezi University, 2021. (in Chinese)
[28]	XIONG X P, SUN S C, ZHANG X Y, LI Y J, LIU F, ZHU Q H, XUE F, SUN J. Corrigendum: GhWRKY70D13 regulates resistance to Verticillium dahliae in cotton through the ethylene and jasmonic acid signaling pathways. Frontiers in Plant Science, 2020, 11: 1045. doi: 10.3389/fpls.2020.01045
[29]	WANG H, CHEN B, TIAN J, KONG Z S. Verticillium dahliae VdBre1 is required for cotton infection by modulating lipid metabolism and secondary metabolites. Environmental Microbiology, 2021, 23(4): 1991-2003. doi: 10.1111/emi.v23.4
[30]	CHEN L H, MA X H, SUN T G, ZHU Q H, FENG H J, LI Y T, LIU F, ZHANG X Y, SUN J, LI Y J. VdPT 1 encoding a neutral trehalase of Verticillium dahliae is required for growth and virulence of the pathogen. International Journal of Molecular Sciences, 2023, 25(1): 294. doi: 10.3390/ijms25010294
[31]	LIVAK K J, SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2 (-Delta Delta C(T)) Method. Methods, 2001, 25(4): 402-408. doi: 10.1006/meth.2001.1262
[32]	陈丽华. 棉花黄萎病菌糖代谢相关基因VdST和VdPT1的功能及RNAi研究[D]. 石河子: 石河子大学, 2023.
	CHEN L H. Functional and RNAi studies of sugar metabolism related genes VdST and VdPT1 in Verticillium dahliae from cotton[D]. Shihezi: Shihezi University, 2023. (in Chinese)
[33]	李聪, 王允. 棘孢木霉糖苷水解酶3基因家族的生物信息学及表达模式分析. 微生物学通报, 2023, 50(1): 1-12.
	LI C, WANG Y. Bioinformatics and expression analyses of glycoside hydrolase 3 genes in Trichoderma asperellum. Microbiology China, 2023, 50(1): 1-12. (in Chinese)
[34]	吕峻元. 大丽轮枝菌M35家族金属蛋白酶VdM35-1和VdASPF2的功能研究[D]. 北京: 中国农业科学院, 2023.
	LÜ J Y. Functional analysis of VdM35-1 and VdASPF2 in Verticillium dahliae M35 family[D]. Beijing: Chinese Academy of Agricultural Sciences, 2023. (in Chinese)
[35]	DOU D L, KALE S D, WANG X, JIANG R H Y, BRUCE N A, ARREDONDO F D, ZHANG X M, TYLER B M. RXLR-mediated entry of Phytophthora sojae effector Avr1b into soybean cells does not require pathogen-encoded machinery. The Plant Cell, 2008, 20(7): 1930-1947. doi: 10.1105/tpc.107.056093
[36]	MA A F, ZHANG D P, WANG G X, WANG K, LI Z, GAO Y H, LI H C, BIAN C, CHENG J K, HAN Y N, et al. Verticillium dahliae effector VDAL protects MYB 6 from degradation by interacting with PUB25 and PUB26 E3 ligases to enhance Verticillium wilt resistance. The Plant Cell, 2021, 33(12): 3675-3699. doi: 10.1093/plcell/koab221
[37]	DEGLI ESPOSTI C, GUERRISI L, PERUZZI G, GIULIETTI S, PONTIGGIA D. Cell wall bricks of defence: the case study of oligogalacturonides. Frontiers in Plant Science, 2025, 16: 1552926. doi: 10.3389/fpls.2025.1552926
[38]	NÜHSE T S. Cell wall integrity signaling and innate immunity in plants. Frontiers in Plant Science, 2012, 3: 280. doi: 10.3389/fpls.2012.00280 pmid: 23248636
[39]	FONG M, BERRIN J G, PAËS G. Investigation of the binding properties of a multi-modular GH45 cellulase using bioinspired model assemblies. Biotechnology for Biofuels, 2016, 9(1): 12. doi: 10.1186/s13068-016-0428-y
[40]	宁淑杰. 大丽轮枝菌内切葡聚糖酶基因沉默与分析[D]. 重庆: 西南大学, 2014.
	NING S J. Silence and analysis of the endoglucanase genes in Verticillium dahliae[D]. Chongqing: Southwest University, 2014. (in Chinese)
[41]	BOUTROT F, ZIPFEL C. Function, discovery, and exploitation of plant pattern recognition receptors for broad-spectrum disease resistance. Annual Review of Phytopathology, 2017, 55: 257-286. doi: 10.1146/annurev-phyto-080614-120106 pmid: 28617654
[42]	AHMAD S, MA H, AKHTAR M W, ZHANG Y P, ZHANG X Z. Directed evolution of Clostridium phytofermentans glycoside hydrolase family 9 endoglucanase for enhanced specific activity on solid cellulosic substrate. BioEnergy Research, 2014, 7(1): 381-388. doi: 10.1007/s12155-013-9382-8
[43]	HENRISSAT B, DAVIES G J. Glycoside hydrolases and glycosyltransferases. Families, modules, and implications for genomics. Plant Physiology, 2000, 124(4): 1515-1519. doi: 10.1104/pp.124.4.1515 pmid: 11115868
[44]	GUO B Y, SATO N, BIELY P, AMANO Y, NOZAKI K. Comparison of catalytic properties of multiple β-glucosidases of Trichoderma reesei. Applied Microbiology and Biotechnology, 2016, 100(11): 4959-4968. doi: 10.1007/s00253-016-7342-x
[45]	MA Z C, SONG T Q, ZHU L, YE W W, WANG Y, SHAO Y Y, DONG S M, ZHANG Z G, DOU D L, ZHENG X B, et al. A Phytophthora sojae glycoside hydrolase 12 protein is a major virulence factor during soybean infection and is recognized as a PAMP. The Plant Cell, 2015, 27(7): 2057-2072. doi: 10.1105/tpc.15.00390
[46]	JUNG S, SONG Y, KIM H M, BAE H J. Enhanced lignocellulosic biomass hydrolysis by oxidative lytic polysaccharide monooxygenases (LPMOs) GH61 from Gloeophyllum trabeum. Enzyme and Microbial Technology, 2015, 77: 38-45. doi: 10.1016/j.enzmictec.2015.05.006
[47]	ZHENG Q. Effects of root exudates and phenolic acids from differently resistant cotton cultivars on. Cotton Science, 2012; 24(4): 363-369.
[48]	WU Y X, FANG W P, ZHU S J, JIN K Y, JI D F. The effects of cotton root exudates on the growth and development of Verticillium dahliae. Frontiers of Agriculture in China, 2008, 2(4): 435-440. doi: 10.1007/s11703-008-0079-2
[49]	凌海春. VdNRPSl1在大丽轮枝菌生长发育及致病宿主过程中的功能研究[D]. 重庆: 西南大学, 2022.
	LING H C. Functional study of VdNRPSl1 in the growth and pathogenesis of Verticillium dahliae[D]. Chongqing: Southwest University, 2022. (in Chinese)
[50]	CHEN L H, CHEN B, ZHU Q H, ZHANG X Y, SUN T G, LIU F, YANG Y L, SUN J, LI Y J. Identification of sugar transporter genes and their roles in the pathogenicity of Verticillium dahliae on cotton. Frontiers in Plant Science, 2023, 14: 1123523. doi: 10.3389/fpls.2023.1123523
[51]	WANG Y, KAMAU S, SONG S L, ZHANG Y, ZHANG X Y. Identification of high-affinity nicotinic acid transporter genes from Verticillium dahliae and functional analysis based on HIGS technology. Journal of Cotton Research, 2025, 8(1): 20. doi: 10.1186/s42397-025-00215-3
[52]	LIU S Y, CHEN J Y, WANG J L, LI L, XIAO H L, ADAM S M, DAI X F. Molecular characterization and functional analysis of a specific secreted protein from highly virulent defoliating Verticillium dahliae. Gene, 2013, 529(2): 307-316. doi: 10.1016/j.gene.2013.06.089
[53]	TZIMA A K, PAPLOMATAS E J, RAUYAREE P, OSPINA- GIRALDO M D, KANG S. VdSNF1, the sucrose nonfermenting protein kinase gene of Verticillium dahliae, is required for virulence and expression of genes involved in cell-wall degradation. Molecular Plant-Microbe Interactions, 2011, 24(1): 129-142. doi: 10.1094/MPMI-09-09-0217
[54]	ZHANG W Q, GUI Y J, SHORT D P G, LI T G, ZHANG D D, ZHOU L, LIU C, BAO Y M, SUBBARAO K V, CHEN J Y, et al. Verticillium dahliae transcription factor VdFTF 1 regulates the expression of multiple secreted virulence factors and is required for full virulence in cotton. Molecular Plant Pathology, 2018, 19(4): 841-857. doi: 10.1111/mpp.2018.19.issue-4
[55]	陈星州. 果生刺盘孢效应蛋白CFEP15调控油茶免疫的分子机制研究[D]. 长沙: 中南林业科技大学, 2025.
	CHEN X Z. Molecular mechanisms of Colletotrichum fructicola effector protein CFEP15 regulating immunity in Camellia oleifera anthracnose[D]. Changsha: Central South University of Forestry & Technology, 2025. (in Chinese)
[56]	GUO X Y, LI Y, FAN J, XIONG H, XU F X, SHI J, SHI Y, ZHAO J Q, WANG Y F, CAO X L, et al. Host-induced gene silencing of MoAP 1 confers broad-spectrum resistance to Magnaporthe oryzae. Frontiers in Plant Science, 2019, 10: 433. doi: 10.3389/fpls.2019.00433
[57]	WANG M, WEIBERG A, LIN F M, THOMMA B P H J, HUANG H D, JIN H L. Bidirectional cross-Kingdom RNAi and fungal uptake of external RNAs confer plant protection. Nature Plants, 2016, 2: 16151. doi: 10.1038/nplants.2016.151 pmid: 27643635
[58]	SONG Y, THOMMA B P H J. Host-induced gene silencing compromises Verticillium wilt in tomato and Arabidopsis. Molecular Plant Pathology, 2018, 19(1): 77-89. doi: 10.1111/mpp.2018.19.issue-1
[59]	WANG Y P, CHENG X, SHAN Q W, ZHANG Y, LIU J X, GAO C X, QIU J L. Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. Nature Biotechnology, 2014, 32(9): 947-951. doi: 10.1038/nbt.2969 pmid: 25038773

基因 Gene	asiRNA	正义链 Sense strand (5′-3′)	反义链 Antisense strand (5′-3′)
Vdbg4	asiR1364	GGCUGAAGAUCACAACCAATT	UUGGUUGUGAUCUUCAGCCTT
Vdbg6	asiR1444	CGAUUGUGGUCUUCCAUAATT	UUAUGGAAGACCACAAUCGTT