多组学技术揭示葡萄叶片响应灰葡萄孢菌侵染的抗性机制

doi:10.3785/j.issn.1008-9209.2018.11.121

浙江大学学报（农业与生命科学版）

2019, Vol. 45

Issue (3): 306-316 DOI: 10.3785/j.issn.1008-9209.2018.11.121

植物保护

多组学技术揭示葡萄叶片响应灰葡萄孢菌侵染的抗性机制

方献平(

),和雅妮,奚晓军,查倩,张丽勍,蒋爱丽(

)

上海市农业科学院林木果树研究所, 上海 201403

Multi-omics reveals the resistance mechanism of grape leaves in response to Botrytis cinerea

Xianping FANG(

),Yani HE,Xiaojun XI,Qian ZHA,Liqing ZHANG,Aili JIANG(

)

Institute of Forestry and Pomology, Shanghai Academy of Agricultural Sciences, Shanghai 201403, China

全文: PDF(2992 KB) HTML

摘要：

以葡萄灰霉病高抗品种‘申丰’叶片为试验材料，采用基于液相色谱质谱联用的非标记定量蛋白质组学和非靶向定量代谢组学技术，比较了叶片在灰葡萄孢菌侵染胁迫3 d后体内蛋白质和代谢物的差异变化水平。试验结果表明，葡萄叶片中有1 374个蛋白质和33种小分子代谢物在病菌侵染后发生了1.5倍以上的差异表达（P＜0.05）。功能注释和代谢通路富集等生物信息学分析发现，灰葡萄孢菌侵染对叶绿体蛋白表达影响最大，且主要集中在植病互作、植物激素与生物碱合成3条信号路径上。基于多组学数据的联合分析进一步表明，水杨酸合成与信号转导通路中的分支酸、水杨酸、异分支酸丙酮酸裂解酶pchB、转录因子TGA和病程相关蛋白PR-1表达水平显著上调。水杨酸介导的抗病信号通路的全面激活是葡萄叶片抵御灰葡萄孢菌侵染的有效手段。本研究发现为后续深入揭示葡萄灰霉病菌互作分子机制及葡萄抗病新品种选育奠定了理论研究基础。

关键词： 葡萄; 灰葡萄孢菌; 蛋白质组学; 代谢组; 水杨酸

Abstract:

Liquid chromatography and mass spectrometry based label-free proteomics and non-target metabolomics technology were used to study the proteome and metabolome change of disease-resistant grape cultivar ‘Shenfeng’ infected with Botrytis cinerea. There were 1 374 proteins and 33 metabolites showing more than 1.5-fold changes in ‘Shenfeng’ leaves infected with B.cinerea, respectively. The differentially expressed proteins and metabolites were analyzed by gene ontology annotation and bioinformatics. The results showed that B. cinerea infection changed the expression level of chloroplast proteins, and mainly focused on plant and pathogen interaction, synthesis pathways of plant hormones and alkaloids. Multi-omics analysis further showed that there was a consistent increase in the expression levels of chorismic acid, salicylic acid, isochorismic pyruvate lyase pchB, transcription factor TGA and pathogenesis-related protein PR-1 in the salicylic acid-mediated disease-resistant signal transduction pathway. The full activation of salicylic acid-mediated disease resistance signaling pathway is an effective means for grape leaves to resist B. cinerea infection. The result is of great benefit to further deeply reveal the molecular mechanisms of plant-pathogen interactions and the breeding of pathogen-resistant grape varieties.

Key words: grape Botrytis cinerea proteomics metabolomics salicylic acid

收稿日期: 2018-11-12 出版日期: 2019-06-25

CLC:

S 663.1

基金资助: 国家葡萄产业技术体系项目(CARS-29-10)

通讯作者: 蒋爱丽 E-mail: fxpbio@163.com;putaojal@163.com

作者简介: 方献平（https://orcid.org/0000-0001-7461-9170），E-mail：fxpbio@163.com

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引用本文:

方献平,和雅妮,奚晓军,查倩,张丽勍,蒋爱丽. 多组学技术揭示葡萄叶片响应灰葡萄孢菌侵染的抗性机制[J]. 浙江大学学报（农业与生命科学版）, 2019, 45(3): 306-316.

Xianping FANG,Yani HE,Xiaojun XI,Qian ZHA,Liqing ZHANG,Aili JIANG. Multi-omics reveals the resistance mechanism of grape leaves in response to Botrytis cinerea. Journal of Zhejiang University (Agriculture and Life Sciences), 2019, 45(3): 306-316.

链接本文:

http://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2018.11.121 或 http://www.zjujournals.com/agr/CN/Y2019/V45/I3/306

图1 模拟和真实接种灰葡萄孢菌3 d后‘申丰’‘巨峰’葡萄叶片的病症变化

图2 葡萄叶片病菌处理前后的差异蛋白质组鉴定

基因名 Gene name	蛋白质信息 Protein description	序列覆盖率 Sequence coverage/%	分子质量 Molecular mass/kDa	比值(接菌组/模拟组) Ratio (pathogen-infected/mock-infected)	P值 P value
VIT_08s0007g03490	酰基载体蛋白 Acyl carrier protein	26.2	14.800	0.16±0.02	0.011 3
VIT_14s0068g01280	V型ATP酶亚基C V-type proton ATPase subunit C	27.4	42.700	0.17±0.02	0.011 5
VIT_12s0035g01140	Ras相关蛋白 Ras-related protein	57.3	23.900	0.22±0.03	0.007 7
VIT_08s0040g01910	V型ATP酶亚基G V-type proton ATPase subunit G	71.0	12.200	0.24±0.01	0.008 1
VIT_06s0004g05210	转录起始因子ⅡA亚基1 Transcription initiation factor ⅡA subunit 1	9.7	43.000	0.25±0.01	0.000 5
VIT_17s0053g01010	果糖-1,6-二磷酸醛缩酶 Fructose-1, 6-bisphosphate aldolase	19.9	34.300	0.26±0.02	0.007 2
VIT_06s0061g00810	细胞周期蛋白依赖激酶 Cyclin-dependent kinases	53.4	10.607	0.26±0.03	0.001 1
VIT_07s0191g00090	14-3-3样蛋白 14-3-3-like protein	67.5	28.781	0.26±0.03	0.002 4
VIT_18s0001g10520	脂质转移蛋白 Lipid-transfer protein	50.8	13.599	0.26±0.02	0.021 4
VIT_00s0317g00050	脱氢抗坏血酸还原酶 Dehydroascorbate reductase	78.3	23.689	0.29±0.01	0.004 9
VIT_06s0004g05920	增殖细胞核抗原 Proliferating cell nuclear antigen	69.8	29.600	0.32±0.02	0.000 6
VIT_13s0147g00100	复制蛋白A Replication protein A	17.1	18.200	0.32±0.03	0.001 9
VIT_19s0014g01570	UBP1相关蛋白2C UBP1-associated protein 2C	40.8	45.420	0.32±0.01	0.003 3
VIT_08s0040g00470	钙调素-7亚型X1 Calmodulin-7 isoform X1	71.5	16.800	0.33±0.08	0.009 7
VIT_02s0025g01300	枯草杆菌蛋白酶SBT 2.5 Subtilisin-like protease SBT 2.5	28.4	10.765	3.03±0.35	0.013 8
VIT_05s0020g03180	光系统Ⅰ反应中心亚单位Ⅱ Photosystem Ⅰ reaction center subunit Ⅱ	59.8	22.481	3.11±0.21	0.003 8
VIT_14s0060g00820	茎环结合蛋白 Stem-loop binding protein	45.2	42.330	3.12±0.22	0.004 9
VIT_14s0066g02380	蛋白质转运蛋白SEC13同系物B Protein transport protein SEC13 homolog B	58.1	32.720	3.12±0.33	0.005 7
VIT_14s0006g01400	钙结合蛋白CML23 Calcium-binding protein CML23	31.7	16.500	3.13±0.26	0.006 5
VIT_17s0000g02480	钙结合过敏原Ole8 Calcium-binding allergen Ole8	80.6	10.400	3.27±0.28	0.006 5
VIT_03s0097g00700	病程相关蛋白1 Pathogenesis-related protein 1	24.0	17.600	3.33±0.24	0.004 4
VIT_05s0077g00810	钙调蛋白4 Calmodulin-4	25.4	21.200	3.35±0.32	0.009 9
VIT_03s0088g00680	病程相关蛋白B1-2 Pathogenesis-related protein B1-2	20.0	17.900	3.76±0.12	0.000 2
VIT_18s0001g14500	内质同系物 Endoplasmin homolog	52.8	93.200	3.86±0.29	0.004 2
VIT_17s0000g00580	钙调蛋白 Calmodulin	71.5	16.900	3.89±0.19	0.000 8
VIT_15s0046g02110	晚期冗余蛋白 Late abundant protein	47.7	16.567	4.08±0.12	0.000 8
VIT_14s0030g02220	转位蛋白TATA Translocase protein TATA	38.5	14.700	4.13±0.36	0.013 6
VIT_02s0025g02140	管腔结合蛋白5 Luminal-binding protein 5	66.8	73.600	4.16±0.25	0.002 5
VIT_16s0098g01580	管腔结合蛋白亚型X2 Luminal-binding protein 5 isoform X2	63.8	73.500	4.52±0.11	0.000 1
VIT_19s0090g01570	40S核糖体蛋白S8 40S ribosomal protein S8	37.5	24.800	4.56±0.36	0.024 4
VIT_08s0007g03620	60S酸性核糖体蛋白P2 60S acidic ribosomal protein P2	79.1	11.500	4.87±0.34	0.003 2
VIT_08s0091g00240	异分支酸合成酶 Isochorismate pyruvate lyase	34.1	13.700	5.12±0.41	0.002 6
VIT_06s0061g00770	50S 核糖体蛋白L9 50S ribosomal protein L9	41.5	22.200	7.13±0.35	0.001 1

表1 葡萄叶片差异表达3.0倍以上的蛋白质定量信息

图3 差异表达蛋白质的GO功能归类分析

图4 差异表达蛋白质的GO功能（A）和KEGG代谢通路富集（B）分析

代谢物 Metabolites	质荷比 m/z	保留时间 Retention time/min	离子模式 Ion mode	比值 Ratio
L-脲基丙氨酸 L-Oxalylalbizziine	202.0	3.41	阴性	0.15±0.01
3-乙酰氧基-2-羟丙基十八酸盐 3-(Acetyloxy)-2-hydroxypropyl octadecenoate	423.3	12.11	阴性	0.36±0.01
色氨酸 Tryptophane	200.0	3.41	阴性	0.36±0.01
N1-反式阿魏拉美汀 N1-trans-feruloylagmatine	289.2	6.41	阴性	0.46±0.00
3-乙酰氧基-2-羟丙基二十烷酸盐 3-(Acetyloxy)-2-hydroxypropyl icosanoate	451.3	13.23	阴性	0.48±0.01
C19-鞘氨醇-1-磷酸 C19-Sphingosine-1-phosphate	376.3	6.14	阴性	0.50±0.02
3-（2,3-二羟基-3-甲基丁基）-4-羟基苯甲酸甲酯 3-(2,3-dihydroxy-3-methylbutyl)-4-hydroxybenzoat	277.1	0.95	阴性	0.50±0.02
3-己二酸 3-Hexenedioic acid	143.0	7.93	阳性	0.50±0.01
2-（1-乙氧基乙氧基）丙酸 2-(1-Ethoxyethoxy)propanoic acid	347.2	3.87	阴性	0.51±0.01
邻苯二甲酸 Phthalic acid	149.0	8.22	阴性	0.51±0.01
八乙二醇 Octaethylene glycol	388.3	3.63	阴性	0.52±0.02
扎波汀 Zapotinin	329.1	7.35	阴性	0.53±0.00
苦杏仁碱H Acrimarine H	536.2	16.07	阴性	0.54±0.05
三氟乙酸 Trifluoroacetic acid	113.0	17.11	阳性	0.55±0.01
2-苯基丁酸 2-Phenylbutyric acid	147.1	0.19	阴性	0.55±0.02
氧化芳樟醇D 3-[己糖基-（1->6）-葡萄糖苷] Linalool oxide D 3-[apiosyl-(1->6)-glucoside]	509.2	6.97	阳性	0.55±0.00
二十烷酰乙醚 Eicosanoyl-EA	338.3	12.70	阴性	0.58±0.05
肾上腺素 Epinephrine	166.1	2.02	阴性	0.59±0.05
12S-羟基-16-十七烷酸 12S-Hydroxy-16-heptadecynoic acid	327.2	4.68	阳性	1.54±0.09
MGDG[20∶5(5Z,8Z,11Z,14Z,17Z)/16∶3(7Z,10Z,13Z)]	815.5	16.84	阳性	1.64±0.21
DG[14∶0/18∶3(6Z,9Z,12Z)/0∶0]	585.5	12.63	阴性	1.67±0.15
MGDG[18∶3(9Z,12Z,15Z)/16∶3(7Z,10Z,13Z)]	769.5	12.63	阴性	1.67±0.09
13-氧肟 13-OxoODE	293.2	7.07	阳性	1.69±0.18
MGDG[18∶3(9Z,12Z,15Z)/18∶3(9Z,12Z,15Z)]	797.5	13.48	阴性	1.74±0.14
13S-羟基十八二烯酸 13S-Hydroxyoctadecadienoic acid	341.2	5.32	阳性	1.85±0.12
2-正戊基呋喃 2-Pentylfuran	277.2	7.08	阴性	1.94±0.09
PI[13∶0/18∶2(9Z,12Z)]	837.5	14.88	阳性	2.01±0.14
氧分子 Oxygen	82.0	0.79	阴性	2.02±0.15
鸟嘌呤 Guanine	152.1	1.23	阴性	2.04±0.19
鳄梨碱 Avocadyne	329.2	4.90	阳性	2.06±0.15
分支酸 Chorismic acid	226.0	8.32	阴性	2.92±0.13
（E）-2-辛烯醛 (E)-2-octenal	109.1	6.97	阴性	3.82±0.21
水杨酸 Salicylic acid	138.0	10.65	阳性	3.84±0.24

表2 33种变化1.5倍以上的差异代谢物

图5 差异代谢物参与的KEGG代谢通路富集分析

图6 差异蛋白质与代谢物共同参与的水杨酸抗病信号通路

1	MLIKOTA G F , SMILANICK J L . Postharvest control of table grape gray mold on detached berries with carbonate and bicarbonate salts and disinfectants. American Journal of Enology Viticulture, 2001,52(1):12-20.
2	WILLIAMSON B , TUDZYNSKI B , TUDZYNSKI P , et al . Botrytis cinerea: the cause of grey mould disease. Molecuar Plant Pathology, 2007,8(5):561-580.
3	陈宇飞,文景芝,李立军 .葡萄灰霉病研究进展.东北农业大学学报,2006,37(5):693-699． CHEN Y F , WEN J Z , LI L J . Research progress of grape grey mildew. Journal of Northeast Agricultural University, 2006,37(5):693-699. (in Chinese with English abstract)
4	FANG X P , CHEN J P , DAI L Y , et al . Proteomic dissection of plant responses to various pathogens. Proteomics, 2015,15(9):1525-1543.
5	MUKHERJEE A K , CARP M J , ZUCHMAN R , et al . Proteomics of the response of Arabidopsis thaliana to infection with Alternaria brassicicola . Journal of Proteomics, 2010,73(4):709-720.
6	YAO Y A , WANG J , MA X, et al . Proteomic analysis of Mn-induced resistance to powdery mildew in grapevine. Journal of Experimental Botany, 2012,63(14):5155-5170.
7	FIEHN O . Metabolomics: the link between genotypes and phenotypes. Plant Molecular Biology, 2002,48:155-171.
8	BATOVSKA D I , TODOROVA I T , NEDELCHEVA D V , et al . Preliminary study on biomarkers for the fungal resistance in Vitis vinifera leaves. Journal of Plant Physiology, 2008,165(8):791-795.
9	VON S V, ZHANG W , KANAWATI B , et al . The Arabidopsis glucosyltransferase UGT76B1 conjugates isoleucic acid and modulates plant defense and senescence. The Plant Cell, 2011,23(11):4124-4145.
10	WINTER D , SEIDLER J , ZIV-LEHRMAN S , et al . Simultaneous identification and quantification of proteins by differential 16O/18O labeling and UPLC-MS/MS applied to mouse cerebellar phosphoproteome following irradiation. Anticancer Research, 2009,29(12):4949-4958.
11	NIEHL A , ZHANG Z J , KUIPER M , et al . Label-free quantitative proteomic analysis of systemic responses to local wounding and virus infection in Arabidopsis thaliana . Journal of Proteome Research, 2013,12(6):2491-2503.
12	CAMARIES G , SCALSCHI L , VICEDO B , et al . An untargeted global metabolomic analysis reveals the biochemical changes underlying basal resistance and priming in Solanum lycopersicum, and identifies 1-methyltryptophan as a metabolite involved in plant responses to Botrytis cinerea and Pseudomonas syringae . The Plant Journal, 2015,84(1):125-139.
13	SADE D , SHRIKI O , CUADROS-INOSTROZA A , et al . Comparative metabolomics and transcriptomics of plant response to tomato yellow leaf curl virus infection in resistant and susceptible tomato cultivars. Metabolomics, 2015,11(1):81-97.
14	HAO L , WANG J X , PAGE D , et al . Comparative evaluation of MS-based metabolomics software and its application to preclinical Alzheimer’s disease. Scientific Reports, 2018,8(1):9291-9300.
15	DURSO G , PIZZA C , PIACENTE S , et al . Combination of LC-MS based metabolomics and antioxidant activity for evaluation of bioactive compounds in Fragaria vesca leaves from Italy. Journal of Pharmaceutical and Biomedical Analysis, 2018,150:233-240.
16	MAHESH H B , SUBBA P , ADVANI J , et al . Multi-omics driven assembly and annotation of the sandalwood (Santalum album) genome. Plant Physiology, 2018,176:2772-2788.
17	BLANCO-MIGUEZ A , FDEZ-RIVEROLA F , SANCHEZ B , et al . Resources and tools for the high-throughput, multi-omic study of intestinal microbiota. Briefings in Bioinformatics, 2017,18(6):156-157.
18	LEI R , JIANG H S , HU F , et al . Chlorophyll fluorescence lifetime imaging provides new insight into the chlorosis induced by plant virus infection. Plant Cell Reports, 2017,36(2):327-341.
19	LIU J , YANG J , BI H P , et al . Why mosaic? Gene expression profiling of African cassava mosaic virus-infected cassava reveals the effect of chlorophyll degradation on symptom development. Journal of Integrative Plant Biology, 2014,56(2):122-132.
20	CLEMENTE-MORENO M J , DIAZ-VIVANCOS P , RUBIO M , et al . Chloroplast protection in plum pox virus-infected peach plants by L-2-oxo-4-thiazolidine-carboxylic acid treatments: effect in the proteome. Plant Cell Environment, 2013,36(3):640-654.
21	RAGHVENDRA A S . Photosynthesis: a Aomprehensive Treatise. London, UK: Cambridge University Press, 1998:72-86.
22	SARRY J E , KUHN L , DUCRUIX C , et al . The early responses of Arabidopsis thaliana cells to cadmium exposure explored by protein and metabolite profiling analyses. Proteomics, 2006,6(7):2180-2198.
23	NAKABAYASHI R , SAITO K . Integrated metabolomics for abiotic stress responses in plants. Current Opinion in Plant Biology, 2015,24:10-16.
24	FEUSSNER I , POLLE A . What the transcriptome does not tell: proteomics and metabolomics are closer to the plants’ patho-phenotype. Current Opinion in Plant Biology, 2015,26:26-31.
25	BEDNAREK P , OSBOURN A . Plant-microbe interactions: chemical diversity in plant defense. Science, 2009,324:746-748.
26	HUFFAKER A , KAPLAN F , VAUGHAN M M , et al . Novel acidic sesquiterpenoids constitute a dominant class of pathogen-induced phytoalexins in maize. Plant Physiology, 2011,156:2082-2097.
27	WILDERMUTH M C , DEWDNEY J , WU G , et al . Isochorismate synthase is required to synthesize salicylic acid for plant defence. Nature, 2001,414(6863):562-565.

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