HIGS-SsCCS转基因拟南芥的菌核病抗性鉴定

doi:10.3864/j.issn.0578-1752.2020.04.008

Abstract

Abstract:

【Objective】Sclerotinia stem rot is a kind of fungal disease caused by Sclerotinia sclerotiorum. The host range of S. sclerotiorum is wide, which seriously endangers the quality of many crops. The objective of this study is to enhance the resistance to stem rot by silencing the virulence genes of S. sclerotiorum in host via the host-induced gene silencing (HIGS) technology, and to provide new ideas for breeding of sclerotinia stem rot resistance.【Method】The gene encoding a copper chaperone for copper/zinc superoxide dismutase of S. sclerotiorum (SsCCS) was selected as the target gene, the sequences were analyzed by bioinformatics tools, and the phylogenetic tree was constructed using MEGA6.0 software. The specific interference fragment was selected for amplification after comparing the genome of Arabidopsis thaliana and S. sclerotiorum, respectively. HIGS vector containing the RNAi structure of SsCCS was transferred into wild type A. thaliana Col-0 mediated by Agrobacterium, and the stable HIGS-CCS transgenic A. thaliana lines were screened by DNA identification and labeling. The leaves of HIGS-CCS transgenic plants grown for 4-5 weeks were selected to analyze the resistance to sclerotinia stem rot according to the lesion area at 24 h after inoculated with S. sclerotiorum. The relative expression level of SsCCS during infecting was analyzed by qRT-PCR. The accumulation of H₂O₂ during the period of interaction between transgenic plants and S. sclerotiorum was detected by DAB staining at 6, 12 and 24 hpi.【Result】Bioinformatics analysis showed that the length of genome sequence of SsCCS (SS1G_00102) is 1 010 bp, while the length of its coding sequence (CDS) is 759 bp, encoding a protein with 253 amino acids, the molecular weight is 2 7176.96 Da, the isoelectric point (PI) is 5.04. SsCCS has 87% amino acid homology to BcCCS (EDN25358) while far to AtCCS (AT1G12520.1). By aligning with the genome of S. sclerotiorum and A. thaliana, a 314 bp specific interference fragment was selected and constructed the HIGS vector successfully and transformed into A. thaliana. The lesion of T₁ and T₂ generation transgenic lines was smaller than that of wild type A. thaliana 24 h after inoculated with strain 1980. From the T₂ generation, three stably expressed T₃ generation HIGS-CCS transgenic A. thaliana lines (HIGS-CCS-5, HIGS-CCS-8, HIGS-CCS-13) were obtained. Compared with wild type A. thaliana, the lesion area on the HIGS-CCS transgenic plants was reduced by 46% to 61% 24 h after inoculated with strain 1980. The expression of SsCCS was significantly reduced by 98% in the HIGS-CCS transgenic plants compared to that in wild type A. thaliana at 6 h after inoculated with S. sclerotiorum strain 1980. Furthermore, the accumulation of H₂O₂was decreased in transgenic plants as revealed by DAB staining, indicating the ROS production was reduced in transgenic plants.【Conclusion】The resistance to stem rot can be significantly enhanced by silencing the SsCCS of S. sclerotiorum in A. thaliana via the HIGS. This study provides a reference for the resistance improvement to sclerotinia stem rot of host crops, such as rapeseed.

Key words: Arabidopsis thaliana, host-induced gene silencing (HIGS), Sclerotinia sclerotiorum, SsCCS, disease resistance, sclerotinia stem rot

YaRu CHAI,YiJuan DING,SiYu ZHOU,WenJing YANG,BaoQin YAN,JunHu YUAN,Wei QIAN. Identification of the Resistance to Sclerotinia Stem Rot in HIGS-SsCCS Transgenic Arabidopsis thaliana[J].Scientia Agricultura Sinica, 2020, 53(4): 761-770.

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References 42

[1]	BOLAND G J, HALL R . Index of plant hosts of Sclerotinia sclerotiorum. Canadian Journal of Plant Pathology, 1994,16(2):93-108.
[2]	BOLTON M D, THOMMA B P, NELSON B D . Sclerotinia sclerotiorum(Lib.) de Bary: Biology and molecular traits of a cosmopolitan pathogen. Molecular Plant Pathology, 2006,7(1):1-16.
[3]	ZHANG Y, WANG X, CHANG X, SUN M, ZHANG Y, LI W, LI Y . Overexpression of germin-like protein GmGLP10 enhances resistance to Sclerotinia sclerotiorum in transgenic tobacco. Biochemical and Biophysical Research Communications, 2018,497(1):160-166.
[4]	张河山, 胡亚亚, 张娜, 魏学军, 杨文香, 刘大群 . 寄主诱导的基因沉默(HIGS)技术研究进展. 农业生物技术学报, 2013,21(5):603-611.
	ZHANG H S, HU Y Y, ZHANG N, WEI X J, YANG W X, LIU D Q . Progress of host-induced gene silencing (HIGS) technology. Journal of Agricultural Biotechnology, 2013,21(5):603-611. (in Chinese)
[5]	张晓娟, 张羽, 胡胜武 . 油菜菌核病抗性机理及抗病遗传育种研究进展. 分子植物育种, 2016,14(3):704-711.
	ZHANG X J, ZHANG Y, HU S W . Progress on resistance mechanism of Sclerotinia sclerotiorum and genetic breeding program on disease resistant rapeseed. Molecular Plant Breeding, 2016,14(3):704-711. (in Chinese)
[6]	FIRE A, XU S Q, MONTGOMERY M K, KOSTAS S A, DRIVER S E, MELLO C C . Potent and specific genetic interference by double- stranded RNA in Caenorhabditis elegans. Nature, 1998,391(6669):806-811.
[7]	WIANNY F, ZERNICKA-GOETZ M . Specific interference with gene function by double-stranded RNA in early mouse development. Nature Cell Biology, 2000,2(2):70-75.
[8]	SVOBODA P, STEIN P, HAYASHI H, SCHULTZ R M . Selective reduction of dormant maternal mRNAs in mouse oocytes by RNA interference. Development, 2000,127(19):4147-4156.
[9]	NUNES C C, DEAN R A . Host-induced gene silencing: A tool for understanding fungal host interaction and for developing novel disease control strategies. Molecular Plant Pathology, 2012,13(5):519-529.
[10]	WANG M, WEIBERG A, LIN F M, THOMMA B P, HUANG H D, JIN H . Bidirectional cross-kingdom RNAi and fungal uptake of external RNAs confer plant protection. Nature Plants, 2016,2(10):16151.
[11]	NOWARA D, GAY A, LACOMME C, SHAW J, RIDOUT C, DOUCHKOV D, HENSEL G, KUMLEHN J, SCHWEIZER P . HIGS: Host-induced gene silencing in the obligate biotrophic fungal pathogen Blumeria graminis. The Plant Cell, 2010,22(9):3130-3141.
[12]	PANWAR V, MCCALLUM B, BAKKEREN G . Endogenous silencing of Puccinia triticina pathogenicity genes through in planta-expressed sequences leads to suppression of rust diseases on wheat. The Plant Journal, 2013,73(3):521-532.
[13]	KOCH A, KUMAR N, WEBER L, KELLER H, IMANI J, KOGEL K H . Host-induced gene silencing of cytochrome P450 lanosterol C14 α-demethylase-encoding genes confers strong resistance to Fusarium species. Proceedings of the National Academy of Sciences of the United States of America, 2013,110(48):19324-19329.
[14]	GHAG S B, SHEKHAWAT U K, GANAPATHI T R . Host-induced post-transcriptional hairpin RNA-mediated gene silencing of vital fungal genes confers efficient resistance against fusarium wilt in banana. Plant Biotechnology Journal, 2014,12(5):541-553.
[15]	ANDRADE C M, TINOCO M L P, RIETH A F, MAIA F C O, ARAGÃO F J L, . Host-induced gene silencing in the necrotrophic fungal pathogen Sclerotinia sclerotiorum. Plant Pathology, 2016,65(4):626-632.
[16]	焦春香 . 活性氧与植物防卫反应. 大理学院学报(自然科学), 2006,5(10):73-76, 79.
	JIAO C X . Reactive oxygen species in plant stress and defense. Journal of Dali University (Natural Science), 2006,5(10):73-76, 79. (in Chinese)
[17]	IMLAY J A, LINN S . DNA damage and oxygen radical toxicity. Science, 1988,240(4857):1302-1309.
[18]	BYRD S, REINS D, DOETSCH P W . Effects of oxidative DNA damage on transcription by RNA polymerases. Free Radical Biology and Medicine, 1990,9(1):47.
[19]	XU L, CHEN W . Random T-DNA mutagenesis identifies a Cu/Zn superoxide dismutase gene as a virulence factor of Sclerotinia sclerotiorum. Molecular Plant-Microbe Interactions, 2013,26(4):431-441.
[20]	BOWLER C, VAN CAMP W, VAN MONTAGU M, INZÉ D . Superoxide dismutase in plant. Critical Reviews in Plant Science, 1994,13(3):199-218.
[21]	BARONDEAU D P, KASSMANN C J, BRUNS C K, TAINE J A, GETZOFF E D . Nickel superoxide dismutase structure and mechanism. Biochemistry, 2004,43(25):8038-8047.
[22]	邹国林, 裘名宜, 朱彤 . 超氧化物歧化酶研究的历史、现状及应用前景. 氨基酸杂志, 1991(3):28-32.
	ZOU G L, QIU M Y, ZHU T . The history, current status and application prospects of superoxide dismutase research. Journal of Amino Acids, 1991(3):28-32. (in Chinese)
[23]	VELUCHAMY S, WILLIAMS B, KIM K, DICKMAN M B . The CuZn superoxide dismutase from Sclerotinia sclerotiorum is involved with oxidative stress tolerance, virulence, and oxalate production. Physiological and Molecular Plant Pathology, 2012,78:14-23.
[24]	HIMELBLAU E, MIRA H, LIN S J, CULOTTA V C, PEÑARRUBIA L, AMASINO R M . Identification of a functional homolog of the yeast copper homeostasis gene ATX1 from Arabidopsis. Plant Physiology, 1998,117(4):1227-1234.
[25]	SCHMIDT P J, RAE T D, PUFAHL R A, HAMMA T, STRAIN J, O’HALLORAN T V, CULOTTV V C . Multiple protein domains contribute to the action of the copper chaperone for superoxide dismutase. The Journal of Biological Chemistry, 1999,274(34):23719-23725.
[26]	LI Y, CAO X L, ZHU Y, YANG X M, ZHANG K N, XIAO Z Y, WANG H, ZHAO J H, ZHANG L L, LI G B, ZHENG Y P, FAN J, WANG J, CHEN X Q, WU X J, ZHAO J Q, DONG O X, CHEN X W, CHERN M, WANG W M . Osa-miR398b boosts H₂O₂ production and rice blast disease-resistance via multiple superoxide dismutases. New Phytologist, 2019,222(3):1507-1522.
[27]	周元委 . 叶绿素合成酶基因在甘蓝型油菜及拟南芥种子生育酚合成过程中的作用研究[D]. 武汉: 华中农业大学, 2017
	ZHOU Y W . The role of chlorophyll synthetase for tocopherol synthesis in canola and Arabidopsis seeds[D]. Wuhan: Huazhong Agricultural University, 2017. (in Chinese)
[28]	张莉芳, 杨正修, 张学文, 龙炎杏, 赵燕 . 荠菜雌蕊调控转录因子SPT基因的克隆与拟南芥遗传转化. 作物研究, 2018,32(3):202-207.
	ZHANG L F, YANG Z X, ZHANG X W, LONG Y X, ZHAO Y . Cloning of the gene of pistil-regulated transcription factor in Capsella bursa-pastoris and its genetic transformation in Arabidopsis thaliana. Crop Research, 2018,32(3):202-207. (in Chinese)
[29]	远俊虎, 丁一娟, 杨文静, 闫宝琴, 柴亚茹, 梅家琴, 钱伟 . 利用TRV-HIGS技术鉴定核盘菌致病相关的分泌蛋白基因. 中国农业科学, 2019,52(23):4274-4284.
	YUAN J H, DING Y J, YANG W J, YAN B Q, CHAI Y R, MEI J Q, QIAN W . Identification of genes encoding secretory proteins related to the pathogenicity of Sclerotinia sclerotiorum using TRV-HIGS. Scientia Agricultura Sinica, 2019,52(23):4274-4284. (in Chinese)
[30]	GUAN L M, SCANDALIOS J G . Hydrogen peroxide-mediated catalase gene expression in response to wounding. Free Radical Biology and Medicine, 2000,28(8):1182-1190.
[31]	陈秀秀, 张彤, 余倩文, 周薇, 安逸民, 杜秉昊, 郭长虹 . 紫花苜蓿F-box蛋白基因MsFTL的克隆及功能分析. 植物遗传资源学报, 2019,20(3):750-759.
	CHEN X X, ZHANG T, YU Q W, ZHOU W, AN Y M, DU B H, GUO C H . Cloning and functional analysis of F-box protein gene MsFTL in alfalfa(Medicago sativa L.). Journal of Plant Genetic Resources, 2019,20(3):750-759. (in Chinese)
[32]	ZHU X, QI T, YANG Q, HE F, TAN C, MA W, VOEGELE R T, KANG Z, GUO J . Host-induced gene silencing of the MAPKK gene PsFUZ7 confers stable resistance to wheat stripe rust. Plant Physiology, 2017,175(4):1853-1863.
[33]	ZHANG T, JIN Y, ZHAO J H, GAO F, ZHOU B J, FANG Y Y, GUO H S . Host-induced gene silencing of the target gene in fungal cells confers effective resistance to the cotton wilt disease pathogen Verticillium dahlia. Molecular Plant, 2016,9(6):939-942.
[34]	WEIBERG A, WANG M, LIN F M, ZHAO H, ZHANG Z, KALOSHIAN I, HUANG H D, JIN H . Fungal small RNAs suppress plant immunity by hijacking host RNA interference pathways. Science, 2013,342(6154):118-123.
[35]	HUA C, ZHAO J H, GUO H S . Trans-Kingdom RNA silencing in plant-fungal pathogen interactions. Molecular Plant, 2018,11(2):235-244.
[36]	WILLIAMS B, KABBAGE M, KIM H J, BRITT R, DICKMAN M B . Tipping the balance:Sclerotinia sclerotiorum secreted oxalic acid suppresses host defenses by manipulating the host redox environment. PLoS Pathogens, 2011,7(6):e1002107.
[37]	KABBAGE M, YARDEN O, DICKMAN M B . Pathogenic attributes of Sclerotinia sclerotiorum: switching from a biotrophic to necrotrophic life style. Plant Science, 2015,233:53-60.
[38]	ZHANG M X, WANG Q H, XU K, MENG Y L, QUAN J L, SHAN W X . Production of dsRNA sequences in the host plant is not sufficient to initiate gene silencing in the colonizing oomycete pathogen Phytophthora parasitica. PLoS ONE, 2011,6(11):e28114.
[39]	GOVINDARAJULU M, EPSTEIN L, WROBLEWSKI T, MICHELMORE R W . Host-induced gene silencing inhibits the biotrophic pathogen causing downy mildew of lettuce. Plant Biotechnology Journal, 2015,13(7):875-883.
[40]	WANG L, GE Y, KANG Y J . Effect of copper on nuclear translocation of copper chaperone for superoxide dismutase-1. Experimental Biology and Medicine, 2016,241(14):1483-1488.
[41]	ROLKE Y, LIU S, QUIDDE T, WILLIAMSON B, SCHOUTEN A, WELTRING K M, SIEWERS V, TENBERGE K B, TUDZYNSKI B, TUDZYNSKI P . Functional analysis of H₂O₂-generating systems in Botrytis cinerea: The major Cu-Zn-superoxide dismutase (BCSOD1) contributes to virulence on French bean, whereas a glucose oxidase (BCGOD1) is dispensable. Molecular Plant Pathology, 2004,5(1):17-27.
[42]	张金平 . “植物中的果蝇”—拟南芥. 农药市场信息, 2016(10):70-71.
	ZHANG J P . “Drosophila in Plants”- Arabidopsis. Pesticide Market News, 2016(10):70-71. (in Chinese)

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引物名称 Primer name	引物序列 Primer sequence
SsCCS-SF	CCC AAGCTT GAATTC TATCTACGGCTGGTTTATGG
SsCCS-SR	TAA GATATC GCATACCTTTGCTAATCTCATCC
SsCCS-AF	CGC GGATCC CTCGAG TATCTACGGCTGGTTTATGG
SsCCS-AR	TAA CTGCAG GCATACCTTTGCTAATCTCATCC
SsTubF	GTGAGGCTGAGGGCTGTGA
SsTubR	CCTTTGGCGATGGGACG
RT-SsCCSF	TCCGTGTTCCCCTTTATCCT
RT-SsCCSR	CCTCAATCGCCCTTACAATCT
PIF	GCCCTTCCTCCCTTTATTTC
PIR	GAGCTGACATCGACACCAAC
M13F	TTGTAAAACGACGGCCAG
M13R	ACACAGGAAACAGCTATGAC
RVF	CGCACAATCCCACTATCCTT
RVR	AAAAGACAAAAGTGGGGTAG

编号 Code	T₁代 T₁generation				T₂代病斑面积 T₂ generation lesion area (cm²)
编号 Code	阳性种子数 Positive	非阳性种子数 Negative	分离比 Segregation ratio	病斑面积 Lesion area (cm²)	T₂代病斑面积 T₂ generation lesion area (cm²)
WT	-	-	-	1.46±0.26a	1.02±0.36a
HIGS-CCS-13	73	27	3﹕1	0.69±0.2b	0.79±0.27b
HIGS-CCS-8	69	31	3﹕1	0.71±0.17b	0.48±0.21b
HIGS-CCS-5	78	22	3﹕1	0.89±0.13b	0.55±0.23b
HIGS-CCS-14	79	21	3﹕1	0.82±0.27b	0.93±0.27a
HIGS-CCS-7	92	8	15﹕1	0.77±0.18b
HIGS-CCS-18	69	31	3﹕1	0.91±0.53a
HIGS-CCS-22	85	25	3﹕1	1.11±0.08a
HIGS-CCS-3	71	29	3﹕1	1.11±0.25a
HIGS-CCS-19	44	56	1﹕1	1.12±0.11a
HIGS-CCS-4	56	44	1﹕1	1.15±0.13a
HIGS-CCS-17	100	0	-	1.27±0.28a
HIGS-CCS-9	78	22	3﹕1	1.37±0.18a
HIGS-CCS-11	95	5	15﹕1	1.21±0.24a
HIGS-CCS-2	70	30	3﹕1	0.91
HIGS-CCS-20	76	34	3﹕1	1.02
HIGS-CCS-1	89	11	15﹕1	1.13
HIGS-CCS-12	74	26	3﹕1	1.26

Identification of the Resistance to Sclerotinia Stem Rot in HIGS-SsCCS Transgenic Arabidopsis thaliana

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