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Journal of Zhejiang University (Agriculture and Life Sciences)  2019, Vol. 45 Issue (3): 306-316    DOI: 10.3785/j.issn.1008-9209.2018.11.121
Plant protection     
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
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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 wordsgrape      Botrytis cinerea      proteomics      metabolomics      salicylic acid     
Received: 12 November 2018      Published: 25 June 2019
CLC:  S 663.1  
Corresponding Authors: Aili JIANG     E-mail: fxpbio@163.com;putaojal@163.com
Cite this article:

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.

URL:

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


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

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 
Fig. 1 Symptom of Vitis vinifera cultivar ‘Shenfeng’ and ‘Jufeng’ leaves induced by mock and B. cinerea-infection for three days
Fig. 2 Proteomic identification of V. vinifera before and after B. cinerea infection

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

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
Table 1 Quantitation information of differentially expressed proteins for more than three folds
Fig. 3 GO functional category analysis of differentially expressed proteins
Fig. 4 GO function (A) and KEGG pathway enrichment (B) analysis of differentially expressed proteins

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
Table 2 Thirty-three differentially expressed metabolites (more than 1.5-fold)
Fig. 5 KEGG enrichment analysis of differentially expressed metabolites
Fig. 6 Disease resistance signaling pathways of salicylic acid with the participation of up-regulated proteins and metabolites
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