Scientia Agricultura Sinica ›› 2019, Vol. 52 ›› Issue (21): 3794-3805.doi: 10.3864/j.issn.0578-1752.2019.21.008

• PLANT PROTECTION • Previous Articles     Next Articles

Identification and Analysis of Differentially Expressed Genes Induced by Protein Elicitor PevD1 in Nicotiana benthamiana

LIANG YingBo,LI Ze,QIU DeWen,ZENG HongMei,LI GuangYue,YANG XiuFen()   

  1. State Key Laboratory for Biology of Plant Disease and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100081
  • Received:2019-05-22 Accepted:2019-06-28 Online:2019-11-01 Published:2019-11-12
  • Contact: XiuFen YANG E-mail:yangxiufen@caas.cn

Abstract:

【Objective】 The objective of this study is to screen the differentially expressed genes (DEGs) induced by protein elicitor PevD1 of Verticillium dahliae in Nicotiana benthamiana by RNA-Seq, and to analyze the potential mechanism of PevD1-induced disease resistance. 【Method】 Leaves of 4-week-old N. benthamiana were infiltrated with 10 μmol·L -1 PevD1 solution, and samples were taken at 6, 12 and 24 h after PevD1 treatment, then RNA was extracted. The mRNA libraries were constructed and sequenced by BGISEQ-500 platform. The DEGs at each time point were screened for GO and KEGG analysis. The leucine-rich repeat receptor-like kinases (LRR-RLKs), transcription factors (TFs) and pathogenesis-related (PR) proteins family in DEGs were analyzed, and qRT-PCR was used to verify the expression levels of relevant DEGs.【Result】 GO functional enrichment and KEGG pathway enrichment analysis indicated that the DEGs at 6 h post infiltration (hpi) were mainly related to cell recognition, photosynthesis, light-harvesting, and were significantly enriched in photosynthesis-antenna protein pathways, terpenoid synthesis pathway, flavonoid and flavonol, and other secondary metabolite synthesis pathways. The DEGs at 12 hpi and 24 hpi were mainly associated with cell recognition and biological functions such as intracellular kinase activity, and were significantly enriched in plant-pathogen interaction pathway, sesquiterpene and triterpenoid biosynthetic pathway, flavonoid and flavonol biosynthetic pathway, linolenic acid metabolism. The unigenes in photosynthesis-antenna proteins, photosynthesis and porphyrin, chlorophyll metabolism pathway were mainly down-regulated, and the unigenes in sesquiterpenoid and triterpenoid biosynthesis, flavone and flavonol biosynthesis, plant-pathogen interaction, phenylpropanoid biosynthesis and terpenoid backbone biosynthesis pathway were mainly up-regulated. After PevD1 induction, a large number of LRR-RLKs, TFs and PR proteins family genes were significantly up-regulated, which were related to elicitor recognition, gene transcriptional regulation and disease resistance. qRT-PCR analysis showed that the expression pattern of the detected DEGs was consistent with the RNA-Seq results.【Conclusion】 PevD1 induced a large number of gene transcriptional rearrangements in N. benthamiana. Lots of LRR-RLKs, TFs and PR proteins family genes were up-regulated, which activated the plant immune system and conferred to plants disease resistant. These results can provide a basis for further study on the mechanism of PevD1-induced immunity in the future.

Key words: Nicotiana benthamiana, Verticillium dahliae, protein elicitor PevD1, RNA-Seq, LRR-RLKs, transcription factor, PR protein

Table 1

Primers for quantitative validation of differentially expressed genes (DEGs)"

基因ID Gene ID 基因名称Gene name 正向引物Forward primer 反向引物Reverse primer
Niben101Scf00245g03004 LHCA4 CTCCTACTTATCTTGATGGAA CACTGGTGAGAACCTCAG
Niben101Scf01094g03014 PTI5 TTCCCAGAAATGTCCTTC GCTTCTTCTGCTGTTTCA
Niben101Scf01297g04006 WRKY7 GGTCTCCTATCAGTAAGTC GCTTGTGGTTTGAACTAC
Niben101Scf01426g00001 ERF3 CTTCGAGCTGGATCTCAC GTCGCCACAAAGAATCAA
Niben101Scf02430g03006 WRKY8 CCTACTGAACTCTTGGAC CTGCTCTTCCAGTTAAAAG
Niben101Scf03160g01004 NAC90 TGTCGAGTTTACGTCATA TCAGCTGCTAATTCTTGA
Niben101Scf03816g01001 SOBIR1 CACCAGAATACCATCAGA GATCCTCAGAAGTCATTACA
Niben101Scf04504g03008 LHCB3 CCAAGTAGTGCTAATGGG AGTGGGTCAAAGTACTGG
Niben101Scf06017g02002 MYB4 CACCCACTTGAAGAAGAG CTTGGATGTTGTTAAAATTGTG
Niben101Scf06509g02006 RLK5 GCTGTTAAGAGGATTTGG GCAACAAAGCAGTTTTAC
Niben101Scf10735g00018 PR10 CTGGAAATGGAGGATGTA AACGGTCTTGAAAAGTTC
Niben101Scf18667g01002 MKS1 GCTTCTCTTCCACCTATA GGGCTAGATAAATAACTGTG

Fig. 1

GO enrichment analysis of DEGs at each time point after PevD1 inducement A, B, and C represent the GO enrichment results of DEGs at 6, 12 and 24 h, respectively. The results only show the top 20 terms with the smallest Q-value. The terms labeled with red circles are significantly enriched under the threshold of Rich factor>0.2 and Q-value<0.05"

Fig. 2

KEGG enrichment analysis of DEGs at each time point after PevD1 inducement"

Fig. 3

The number of DEGs in gene annotation of PRGs at each time point after PevD1 treatment"

Table 2

Screened DEGs of LRR-RLKs"

Unigene ID log2FC 蛋白特性
Protein property
6 hpi 12 hpi 24 hpi
Niben101Scf00160g06027 2.80 3.42 2.12 Putative receptor-like protein kinase At3g47111
Niben101Scf00182g06001 5.47 Pollen receptor-like kinase 2
Niben101Scf00245g00006 10.00 6.88 4.19 Probable LRR receptor-like serine/threonine-protein kinase At1g74361
Niben101Scf00742g01037 2.35 Protein STRUBBELIG-RECEPTOR FAMILY 3-like isoform X4
Niben101Scf00744g08015 2.10 1.82 Receptor-like protein kinase HAIKU3
Niben101Scf00887g04011 1.80 2.26 Putative receptor-like protein kinase At3g47111
Niben101Scf00953g00004 2.65 3.06 1.66 Receptor-like protein kinase HAIKU3
Niben101Scf01203g01002 2.12 4.39 2.65 Probable LRR receptor-like serine/threonine-protein kinase At3g47570 isoform X2
Niben101Scf01205g02008 -2.00 -2.10 Probable LRR receptor-like serine/threonine-protein kinase At4g36181
Niben101Scf01225g04031 2.51 2.85 2.08 Probably inactive leucine-rich repeat receptor-like protein kinase At5g48381
Niben101Scf01278g09008 2.04 1.54 Probable LRR receptor-like serine/threonine-protein kinase At1g56130 isoform X3
Niben101Scf01673g02002 6.26 Probable LRR receptor-like serine/threonine-protein kinase RLK
Niben101Scf01819g01001 1.31 2.21 Phytosulfokine receptor 2
Niben101Scf02217g05010 1.65 2.29 Receptor-like protein kinase HSL2
Niben101Scf02531g00007 2.88 LRR receptor-like serine/threonine-protein kinase EFR
Niben101Scf02862g00007 -1.90 -2.80 Probable LRR receptor-like serine/threonine-protein kinase At4g36181
Niben101Scf03374g08019 3.80 3.71 1.10 Probable LRR receptor-like serine/threonine-protein kinase At3g47571
Niben101Scf03816g01001 2.03 2.07 1.39 AHW85126.2//leucine-rich repeat receptor-like kinase
Niben101Scf04053g03008 2.06 2.50 1.79 Receptor-like protein kinase HAIKU3
Niben101Scf04099g05004 2.38 2.35 1.72 Leucine-rich repeat receptor-like serine/threonine/tyrosine-protein kinase SOBIR2
Niben101Scf05349g01040 1.30 2.32 2.20 Protein STRUBBELIG-RECEPTOR FAMILY 5 isoform X2
Niben101Scf05928g03007 5.32 6.75 3.32 Probable LRR receptor-like serine/threonine-protein kinase At1g74361
Niben101Scf06509g02006 5.34 5.87 2.71 Receptor-like protein kinase 6
Niben101Scf08137g01034 2.82 3.29 Putative receptor-like protein kinase
Niben101Scf08564g00001 1.15 3.10 Probable LRR receptor-like serine/threonine-protein kinase At1g56141
Niben101Scf10381g03006 3.10 2.42 Probable LRR receptor-like serine/threonine-protein kinase At4g08851
Niben101Scf20124g00014 2.27 2.20 1.73 Leucine-rich repeat receptor-like tyrosine-protein kinase PXC4

Fig. 4

Analysis of transcription factors (TFs)"

Table 3

Screened PR protein family DEGs after PevD1 inducement"

PR 家族基因
PR family gene
Unigene ID log2 Ratio (WT_PevD1/WT_CK) 蛋白特性
Protein property
6 hpi 12 hpi 24 hpi
PR1 Niben101Scf00107g03008 -2.10 2.38 4.32 Pathogenesis-related protein 1A
Niben101Scf00953g03008 5.98 3.86 Basic form of pathogenesis-related protein 1
Niben101Scf00953g03009 4.60 7.73 5.58 Basic form of pathogenesis-related protein 1-like
Niben101Scf01999g07002 3.17 2.58 Pathogenesis-related protein 1C-like
Niben101Scf03376g03004 -6.50 2.87 6.24 Pathogenesis-related protein 1A
Niben101Scf04053g02006 1.64 5.47 3.81 Basic form of pathogenesis-related protein 1-like
Niben101Scf04053g02007 5.23 8.30 6.87 Basic form of pathogenesis-related protein 1
PR2 Niben101Ctg13736g00004 3.09 4.15 Glucan endo-1,3-beta-glucosidase
Niben101Scf01001g00003 2.86 3.91 Glucan endo-1,3-beta-glucosidase
Niben101Scf01001g00004 2.86 3.91 Glucan endo-1,3-beta-glucosidase
Niben101Scf01001g00005 2.86 3.91 Glucan endo-1,3-beta-glucosidase
Niben101Scf01001g00006 3.70 Glucan endo-1,3-beta-glucosidase
Niben101Scf01934g02004 5.40 3.21 Glucan endo-1,3-beta-glucosidase
Niben101Scf03905g02017 1.32 5.13 2.91 Glucan endo-1,3-beta-glucosidase
Niben101Scf04869g03002 3.25 3.39 Glucan endo-1,3-beta-glucosidase
PR3 Niben101Scf02041g00002 1.57 3.85 Acidic endochitinase Q
Niben101Scf03200g01014 2.22 2.15 Endochitinase A
Niben101Scf03949g00023 4.49 1.38 Endochitinase 3
Niben101Scf07491g00003 4.87 3.48 Endochitinase A
PR4 Niben101Scf01015g01002 1.84 4.45 Wound-induced protein WIN1-like
Niben101Scf02171g00007 2.01 5.84 3.06 Endochitinase PR4-like
Niben101Scf12045g06025 1.41 4.73 6.29 Pathogenesis-related protein PR-4B
PR5 Niben101Scf03937g02019 1.69 Pathogenesis-related protein 5-like
Niben101Scf05732g04031 4.88 Pathogenesis-related protein 5-like
PR6 Niben101Scf04053g01004 6.66 Pathogenesis-related leaf protein 6-like
PR10 Niben101Scf10735g00018 1.36 2.10 2.50 Pathogenesis-related protein PR-10
PR11 Niben101Scf01789g04010 2.80 3.62 2.77 Chitotriosidase-1-like
Niben101Scf06295g04023 1.91 2.85 2.19 Chitotriosidase-1-like
PR17 Niben101Scf03385g02011 2.02 3.33 Uncharacterized protein LOC104232799

Fig. 5

Validation of DEGs by qRT-PCR"

[1] BARBARA D J, CLEWES E . Plant pathogenic Verticillium species: How many of them are there? Molecular Plant Pathology, 2003,4(4):297-305.
[2] DURESSA D, ANCHIETA A, CHEN D, KLIMES A ,GARCIA-PEDRAJAS M D,DOBINSON K F,KLOSTERMAN S J. RNA-seq analyses of gene expression in the microsclerotia of Verticillium dahliae. BMC Genomics, 2013,14:607.
[3] CARROLL C L, CARTER C A, GOODHUE R E ,LAWELL C C L, SUBBARAO K V. A review of control options and externalities for Verticillium wilts. Phytopathology, 2018,108(2):160-171.
[4] 邱德文 . 我国植物病害生物防治的现状及发展策略. 植物保护, 2010,36(4):15-18.
QIU D W . Current status and development strategy for biological control of plant diseases in China. Plant Protection, 2010,36(4):15-18. (in Chinese)
[5] WANG B, YANG X, ZENG H, LIU H, ZHOU T, TAN B, YUAN J, GUO L, QIU D . The purification and characterization of a novel hypersensitive-like response-inducing elicitor from Verticillium dahliae that induces resistance responses in tobacco. Applied Microbiology and Biotechnology, 2012,93(1):191-201.
[6] 王炳楠, 杨秀芬, 曾洪梅, 邱德文 . 大丽轮枝菌分泌蛋白激发子的分离纯化及生物功能研究. 生物技术通报, 2011(11):166-171.
WANG B N, YANG X F, ZENG H M, QIU D W . Purification and its bioassay of secreted elicitor protein fromVerticillium dahliae. Biotechnology Bulletin, 2011(11):166-171 (in Chinese)
[7] 卜冰武, 邱德文, 曾洪梅, 郭立华, 袁京京, 杨秀芬 . 大丽轮枝菌蛋白激发子PevD1诱导棉花抗病性及作用机理. 植物病理学报, 2014,44(3):254-264.
BU B W, QIU D W, ZENG H M, GUO L H, YUAN J J, YANG X F . Induced resistance and mechanism of protein elicitor PevD1 against Verticillium dahliae in cotton. Acta Phytopathologica Sinica, 2014,44(3):254-264. (in Chinese)
[8] ZHANG Y, GAO Y, LIANG Y, DONG Y, YANG X, QIU D . Verticillium dahliae PevD1, an Alt a 1-like protein, targets cotton PR5-like protein and promotes fungal infection. Journal of Experimental Botany, 2019,70(2):613-626.
[9] WANG L K, FENG Z X, WANG X, WANG X W, ZHANG X G . DEGseq: An R package for identifying differentially expressed genes from RNA-seq data. Bioinformatics, 2010,26(1):136-138.
[10] SCHMITTGEN T D, LEE E J, JIANG J . High-throughput real-time PCR. Methods in Molecular Biology, 2008,429:89-98.
[11] SAIJO Y, LOO E P, YASUDA S . Pattern recognition receptors and signaling in plant-microbe interactions. The Plant Journal, 2018,93(4):592-613.
[12] LIANG X, ZHOU J M . Receptor-like cytoplasmic kinases: Central players in plant receptor kinase-mediated signaling. Annual Review of Plant Biology, 2018,69:267-299.
[13] SU J, YANG L, ZHU Q, WU H, HE Y, LIU Y, XU J, JIANG D, ZHANG S . Active photosynthetic inhibition mediated by MPK3/MPK6 is critical to effector-triggered immunity. PLoS Biology, 2018,16(5):e2004122.
[14] CHINCHILLA D, ZIPFEL C, ROBATZEK S, KEMMERLING B , NÜRNBERGER T, JONES J D, FELIX G, BOLLER T. A flagellin- induced complex of the receptor FLS2 and BAK1 initiates plant defence. Nature, 2007,448(7152):497-500.
[15] SCHULZE B, MENTZEL T, JEHLE A K, MUELLER K, BEELER S, BOLLER T, FELIX G, CHINCHILLA D . Rapid heteromerization and phosphorylation of ligand-activated plant transmembrane receptors and their associated kinase BAK1. The Journal of Biological Chemistry, 2010,285(13):9444-9451.
[16] LIEBRAND T W ,VAN DEN BURG H A, JOOSTEN M H. Two for all: Receptor-associated kinases SOBIR1 and BAK1. Trends in Plant Science, 2014,19(2):123-132.
doi: 10.1016/j.tplants.2013.10.003
[17] ZIPFEL C, KUNZE G, CHINCHILLA D, CANIARD A, JONES J D, BOLLER T, FELIX G . Perception of the bacterial PAMP EF-Tu by the receptor EFR restricts Agrobacterium-mediated transformation. Cell, 2006,125(4):749-760.
[18] NG D W K, ABEYSINGHE J K, KAMALI M . Regulating the regulators: The control of transcription factors in plant defense signaling. International Journal of Molecular Sciences, 2018,19(12):3737.
[19] DIETZ K J, VOGEL M O, VIEHHAUSER A . AP2/EREBP transcription factors are part of gene regulatory networks and integrate metabolic, hormonal and environmental signals in stress acclimation and retrograde signalling. Protoplasma, 2010,245(1/4):3-14.
[20] GAUTAM J K, NANDI A K . APD1, the unique member of Arabidopsis AP2 family influences systemic acquired resistance and ethylene-jasmonic acid signaling. Plant Physiology and Biochemistry, 2018,133:92-99.
[21] JIN Y, PAN W, ZHENG X, CHENG X, LIU M, MA H, GE X . OsERF101, an ERF family transcription factor, regulates drought stress response in reproductive tissues. Plant Molecular Biology, 2018,98(1/2):51-65.
[22] PANDEY S P, SOMSSICH I E . The role of WRKY transcription factors in plant immunity. Plant Physiology, 2009,150(4):1648-1655.
[23] ZHENG Z, QAMAR S A, CHEN Z, MENGISTE T . Arabidopsis WRKY33 transcription factor is required for resistance to necrotrophic fungal pathogens. The Plant Journal, 2006,48(4):592-605.
[24] CHUJO T, TAKAI R, AKIMOTO-TOMIYAMA C, ANDO S, MINAMI E, NAGAMURA Y, KAKU H, SHIBUYA N, YASUDA M, NAKASHITA H, UMEMURA K, OKADA A, OKADA K, NOJIRI H, YAMANE H . Involvement of the elicitor-induced gene OsWRKY53 in the expression of defense-related genes in rice. Biochimica et Biophysica Acta (BBA)-Gene Structure and Expression, 2007,1769(7/8):497-505.
[25] DUBOS C, STRACKE R, GROTEWOLD E, WEISSHAAR B, MARTIN C, LEPINIEC L . MYB transcription factors in Arabidopsis. Trends in Plant Science, 2010,15(10):573-581.
[26] LIU J, OSBOURN A, MA P . MYB transcription factors as regulators of phenylpropanoid metabolism in plants. Molecular Plant, 2015,8(5):689-708.
[27] MA Q H, WANG C, ZHU H H . TaMYB4 cloned from wheat regulates lignin biosynthesis through negatively controlling the transcripts of both cinnamyl alcohol dehydrogenase and cinnamoyl-CoA reductase genes.Biochimie, 2011,93(7):1179-1186.
[28] HUANG Y, LI T, XU Z S, WANG F, XIONG A S, HUANG Y, LI T, XU Z S, WANG F, XIONG A S . Six NAC transcription factors involved in response to TYLCV infection in resistant and susceptible tomato cultivars. Plant Physiology and Biochemistry, 2017,120:61-74.
[29] YAN J, TONG T, LI X, CHEN Q, DAI M, NIU F, YANG M, DEYHOLOS M K, YANG B, JIANG Y Q . A novel NAC-type transcription factor, NAC87, from oilseed rape modulates reactive oxygen species accumulation and cell death. Plant and Cell Physiology, 2018,59(2):290-303.
[30] WANG G, ZHANG S, MA X, WANG Y, KONG F, MENG Q . A stress-associated NAC transcription factor (SlNAC35) from tomato plays a positive role in biotic and abiotic stresses. Physiologia Plantarum, 2016,158(1):45-64.
[31] VAN LOON L C, REP M, PIETERSE C M J . Significance of inducible defense-related proteins in infected plants. Annual Review of Phytopathology, 2006,44:135-162.
[32] ANTONIW J F, RITTER C E, PIERPONT W S ,VAN LOON L C. Comparison of three pathogenesis-related proteins from plants of two cultivars of tobacco infected with TMV. Journal of General Virology, 1980,47(1):79-87.
[33] VAN LOON L C . Regulation of changes in proteins and enzymes associated with active defence against virus infection//Active Defense Mechanisms in Plants, 1982,37:247-273.
[34] LAGRIMINI L M, BURKHART W, MOYER M, ROTHSTEIN S . Molecular cloning of complementary DNA encoding the lignin- forming peroxidase from tobacco: Molecular analysis and tissue- specific expression. Proceedings of the National Academy of Sciences of the United States of America, 1987,84(21):7542-7546.
[35] 李瑞博, 崔秀明, 刘玉忠, 吴志刚, 林淑芳, 申业, 黄璐琦 . 三七病程相关蛋白1基因的克隆与表达分析. 药学学报, 2014,49(1):124-130.
LI R B, CUI X M, LIU Y Z, WU Z G, LIN S F, SHEN Y, HUANG L Q . Cloning and expression analysis of pathogenesis-related protein 1 gene of Panax notoginseng. Acta Pharmaceutica Sinica, 2014,49(1):124-130. (in Chinese)
[36] MELCHERS L S, APOTHEKER-DE GROOT M,VAN DER KNAAP J A, PONSTEIN A S, SELA-BUURLAGE M B, BOL J F, CORNELISSEN B J, VAN DEN ELZEN P J, LINTHORST H J. , A new class of tobacco chitinases homologous to bacterial exo-chitinases displays antifungal activity. The Plant Journal, 1994,5(4):469-480.
[37] OKUSHIMA Y, KOIZUMI N, KUSANO T, SANO H . Secreted proteins of tobacco cultured BY2 cells: Identification of a new member of pathogenesis-related proteins. Plant Molecular Biology, 2000,42(3):479-488.
doi: 10.1023/A:1006393326985
[1] PEI YueHong,LI FengWei,LIU WeiNa,WEN YuXia,ZHU Xin,TIAN ShaoRui,FAN GuangJin,MA XiaoZhou,SUN XianChao. Characteristics of Cysteine Proteinase Gene Family in Nicotiana benthamiana and Its Function During TMV Infection [J]. Scientia Agricultura Sinica, 2022, 55(21): 4196-4210.
[2] YuXia WEN,Jian ZHANG,Qin WANG,Jing WANG,YueHong PEI,ShaoRui TIAN,GuangJin FAN,XiaoZhou MA,XianChao SUN. Cloning, Expression and Anti-TMV Function Analysis of Nicotiana benthamiana NbMBF1c [J]. Scientia Agricultura Sinica, 2022, 55(18): 3543-3555.
[3] MingYue GONG,XiaoTian DUAN,TingTing YU,Jie WANG,LiLi SHEN,Ying LI,MingHong LIU,YongLiang LI,HongKun LÜ,SongBai ZHANG,JinGuang YANG. Cloning of Hsc70-2 and Its Promoting Effect on Potato virus Y Infection in Nicotiana benthamiana [J]. Scientia Agricultura Sinica, 2020, 53(4): 771-781.
[4] LIU HaiYang, WANG Wei, ZHANG RenFu, RAXIDA ·ABDURAHMAN, YAO Ju. Fungal Community Structure of Cotton-Field Soil Under Different Incidences of Cotton Verticillium Wilt [J]. Scientia Agricultura Sinica, 2019, 52(3): 455-465.
[5] YUAN JunHu,DING YiJuan,YANG WenJing,YAN BaoQin,CHAI YaRu,MEI JiaQin,QIAN Wei. Identification of Genes Encoding Secretory Proteins Related to the Pathogenicity of Sclerotinia sclerotiorum Using TRV-HIGS [J]. Scientia Agricultura Sinica, 2019, 52(23): 4274-4284.
[6] LIU RuiChi,CHENG YouPu,CHAI ALi,SHI YanXia,XIE XueWen, PATIGULI,LI BaoJu. Establishment and Application of a Triplex PCR Detection System for Vegetable Soil-Borne Pathogens [J]. Scientia Agricultura Sinica, 2019, 52(12): 2069-2078.
[7] WU GenTu, CHEN GuangXiang, ZHANG JiaYuan, HU Qiao, MA MingGe, DOU YanXia, LI MingJun, QING Ling. Disease Resistance of Rice stripe virus NS3-Transgenic Nicotiana benthamiana [J]. Scientia Agricultura Sinica, 2019, 52(10): 1710-1720.
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed   
No Suggested Reading articles found!