大丽轮枝菌蛋白激发子PevD1激活本生烟促分裂原活化蛋白激酶MAPK

doi:10.13560/j.cnki.biotech.bull.1985.2020-0230

摘要/Abstract

摘要：

激活植物免疫系统、提高植物自身抗性是植物病害绿色防控的重要途径之一。促分裂原活化蛋白激酶（MAPK）是植物免疫系统的重要防御信号传导途径。为了探讨大丽轮枝菌蛋白激发子PevD1诱导植物广谱抗病性的分子机制,前期利用转录组测序（RNA-Seq）技术获得了本生烟响应PevD1诱导的差异表达基因,其中大量基因显著富集在MAPK通路上。本研究对这些差异表达基因进行了功能分类并进一步分析,发现这些基因涉及的功能十分广泛,包括参与植物识别的蛋白激酶（LRR-RLK）、调控基因表达的转录因子家族（WRKY和ERF）、与抗病相关的几丁质酶基因、参与钙离子信号传递的钙调蛋白（Calmodulin）、调控活性氧（Reactive oxygen species,ROS）产生的呼吸爆发氧化酶（Rboh）和清除ROS的过氧化氢酶（Catalase）等。从富集在MAPK通路中的差异表达基因中选出10个基因进行了qPCR验证,转录表达模式与转录组测序结果一致。用特异性磷酸化位点抗体杂交技术验证了PevD1激活了本生烟的MAPK,即水杨酸诱导的蛋白激酶SIPK和伤诱导的WIPK被激活。

关键词: 蛋白激发子PevD1, 转录组测序, MAPK, SIPK, WIPK

Abstract:

Plant resistance enhancement via activating plant immune system is one of the important approaches in green management system of plant diseases and pests. Mitogen activated protein kinase（MAPK）cascade is a vital signal transduction pathway in plant innate immune system. In order to investigate the molecular mechanism of Verticillium dahliae elicitor PevD1 inducing broad-spectrum disease resistance,transcriptome sequencing（RNA-seq）data from Nicotiana benthamiana response to PevD1 was obtained and a large number of differential gene expression（DGEs）were significantly enriched in the MAPK pathway. In this paper,DGEs enriched in the MAPK pathway were functionally classified and further analyzed. The involved functions by these DGEs were broad,including protein kinases（LRR- RLK）associated with plant recognition,ERF and WRKY transcription factor family for the regulation of gene expression,chitinase gene involved in disease resistance,calmodulin participated in calcium signaling,breathing outbreak oxidase（Rboh）regulating reactive oxygen species（ROS）and catalase（CAT）clearing ROS,etc. Expression patterns of 10 genes from the DGEs enriched in MAPK pathway were verified by qPCR and it was consistent with the RNA-Seq result. The antibody hybridization technique of specific phosphorylation sites was used to confirm that PevD1activated MAPK in N. benthamiana,i.e.,salicylic acid-induced protein kinase SIPK and wound-induced WIPK were activated by PevD1.

Key words: protein elicitor PevD1, RNA-Seq, MAPK, SIPK, WIPK

贾丰莲, 李泽, 梁颖博, 李广悦, 杨秀芬. 大丽轮枝菌蛋白激发子PevD1激活本生烟促分裂原活化蛋白激酶MAPK[J]. 生物技术通报, 2020, 36(10): 15-24.

JIA Feng-lian, LI Ze, LIANG Ying-bo, LI Guang-yue, YANG Xiu-fen. Verticillium dahliae Elicitor PevD1 Activates MAPKs in Nicotiana benthamiana[J]. Biotechnology Bulletin, 2020, 36(10): 15-24.

图/表 5

参考文献 43

[1]	Xu J, Zhang S. Mitogen-activated protein kinase cascades in signaling plant growth and development[J]. Trends in Plant Science, 2015,20:56-64. doi: 10.1016/j.tplants.2014.10.001 URL pmid: 25457109
[2]	Xu J, Yang K, Yoo SJ, et al. Reactive oxygen species in signalling the transcriptional activation of WIPK expression in tobacco[J]. Plant Cell & Environment, 2014,37(7):1614-1625.
[3]	Asai T, Tena G, Plotnikova J, et al. MAP kinase signalling cascade in Arabidopsis innate immunity[J]. Nature, 2002,415:977-983. URL pmid: 11875555
[4]	Suarez-Rodriguez MC, Adams-Phillips L, et al. MEKK1 is required for flg22 -induced MPK4 activation in Arabidopsis plants[J]. Plant Physiology, 2007,143:661-669. URL pmid: 17142480
[5]	Qiu JL, Fiil BK, Petersen K, et al. Arabidopsis MAP kinase 4 regulates gene expression through transcription factor release in the nucleus[J]. EMBO Journal, 2008,27:2214-2221. doi: 10.1038/emboj.2008.147 URL pmid: 18650934
[6]	Brader G, Djamei A, Teige M, et al. The MAP kinase kinase MKK2 affects disease resistance in Arabidopsis[J]. Molecular Plant-Microbe Interactions, 2007,20:589-596. URL pmid: 17506336
[7]	Xiong L, Yang Y. Disease resistance and abiotic stress tolerance in rice are inversely modulated by an abscisic acid-inducible mitogen- activated protein kinase[J]. Plant Cell, 2003,15:745-759. doi: 10.1105/tpc.008714 URL pmid: 12615946
[8]	Samuel MA, Ellis BE. Double jeopardy:both overexpression and suppression of a redox- activated plant mitogen-activated protein kinase render tobacco plants ozone sensitive[J]. Plant Cell, 2002,14:2059-2069. doi: 10.1105/tpc.002337 URL pmid: 12215505
[9]	Wang B, Yang X, Zeng H, et al. The Purification and characterization of a novel hypersensitive-like response-inducing elicitor from Verticillium dahliae that induces resistance responses in tobacco[J]. Applied Microbiology and Biotechnology, 2012,93:191-201. doi: 10.1007/s00253-011-3405-1 URL pmid: 21691787
[10]	Bu B, Qiu D, Zeng H, et al. A fungal protein elicitor PevD1 induces Verticillium wilt resistance in cotton[J]. Plant Cell Reports, 2014,14:461-470.
[11]	Zhang Y, Gao Y, Liang Y, et al. Verticillium dahliae PevD1, an Alt a 1-like protein, targets cotton PR5-like protein and promotes fungal infection[J]. Journal of Experimental Botany, 2019,70(2):613-626. URL pmid: 30295911
[12]	Gomez-Gomez L, Boller T. FLS2:an LRR receptor -like kinase involved in the perception of the bacterial elicitor flagellin in Arabidopsis[J]. Molecular Cell, 2000,5:1003-1011. doi: 10.1016/s1097-2765(00)80265-8 URL pmid: 10911994
[13]	Zipfel C, Kunze G, Chinchilla D, et al. Perception of the bacterial PAMP EF -Tu by the receptor EFR restricts agrobacterium -mediated transformation[J]. Cell, 2006,125:749-760. URL pmid: 16713565
[14]	Miya A, Albert P, Shinya T, et al. CERK1, a LysM receptor kinase, is essential for chitin elicitor signaling in Arabidopsis[J]. Proceedings of the National Academy of Sciences of the United States of America, 2007,104:19613-19618. URL pmid: 18042724
[15]	Wan J, Zhang XC, Neece D, et al. A LysM receptor -like kinase plays a critical role in chitin signaling and fungal resistance in Arabidopsis[J]. Plant Cell, 2008,20:471-481. URL pmid: 18263776
[16]	Hayafune M, Berisio R, Marchetti R, et al. Chitin-induced activation of immune signaling by the rice receptor CEBiP relies on a unique sandwich-type dimerization[J]. Proceedings of the National Academy of Sciences of the United States of America, 2014,111:E404-E413. URL pmid: 24395781
[17]	Chinchilla D, Zipfel C, Robatzek S, et al. A flagellin-induced complex of the receptor FLS2 and BAK1 initiates plant defence[J]. Nature, 2007,448(7152):497-500. URL pmid: 17625569
[18]	Heese A, Hann DR, Gimenez-Ibanez S, et al. The receptor-like kinase SERK3/BAK1 is a central regulator of innate immunity in plants[J]. Proceedings of the National Academy of Sciences of the United States of America, 2007,104(29):12217-12222. URL pmid: 17626179
[19]	Yamaguchi K, Yamada K, Ishikawa K, et al. A receptor-like cytoplasmic kinase targeted by a plant pathogen effector is directly phosphorylated by the chitin receptor and mediates rice immunity[J]. Cell Host Microbe, 2013,13:347-357. doi: 10.1016/j.chom.2013.02.007 URL pmid: 23498959
[20]	Shinya T, Yamaguchi K, Desaki Y, et al. Selective regulation of the chitin-induced defense response by the Arabidopsis receptor-like cytoplasmic kinase PBL27[J]. Plant Journal, 2014,79:56-66. doi: 10.1111/tpj.12535 URL pmid: 24750441
[21]	Zhang C, Chen H, Zhuang RR, et al. Overexpression of the peanut CLAVATA1-like leucine-rich repeat receptor-like kinase AhRLK1 confers increased resistance to bacterial wilt in tobacco[J]. Journal of Experimental Botany, 2019,70(19):5407-5421. URL pmid: 31173088
[22]	Phukan UJ, Jeena GS, Shukla RK. WRKY transcription factors:molecular regulation and stress responses in plants[J]. Frontiers in Plant Science, 2016,7:760. doi: 10.3389/fpls.2016.00760 URL pmid: 27375634
[23]	Eulgem T, Somssich IE. Networks of WRKY transcription factors in defense signaling[J]. Curr Opin Plant Biology, 2007,10:366-371.
[24]	Rushton PJ, Somssich IE, Ringler P, et al. WRKY transcription factors[J]. Trends in Plant Science, 2010,15:247-258. doi: 10.1016/j.tplants.2010.02.006 URL pmid: 20304701
[25]	Mao G, Meng X, Liu Y, et al. Phosphorylation of a WRKY transcription factor by two pathogen-responsive MAPKs drives phytoalexin biosynjournal in Arabidopsis[J]. Plant Cell, 2011,23:1639-1653. doi: 10.1105/tpc.111.084996 URL pmid: 21498677
[26]	Chen H, Lai Z, Shi J, et al. Roles of Arabidopsis WRKY18, WRKY40 and WRKY60 transcription factors in plant responses to abscisic acid and abiotic stress[J]. BMC Plant Biology, 2010,10:281. URL pmid: 21167067
[27]	Yan L, Liu ZQ, Xu YH, et al. Auto and cross-repression of three Arabidopsis WRKY transcription factors WRKY18, WRKY40, and WRKY60 negatively involved in ABA signaling[J]. Journal of Plant Growth Regulation, 2013,32:399-416.
[28]	Yang KY, Liu Y, Zhang S. Activation of a mitogen-activated protein kinase pathway is involved in disease resistance in tobacco[J]. Proceedings of the National Academy of Sciences of the United States of America, 2001,98:741-746. doi: 10.1073/pnas.98.2.741 URL pmid: 11209069
[29]	Yoshioka H, Numata N, Nakajima K, et al. Nicotiana benthamiana homologs NbrbohA and NbrbohB participate in H₂O₂ accumulation and resistance to phytophthora infestans[J]. Plant Cell, 2003,15(3):706-718. doi: 10.1105/tpc.008680 URL pmid: 12615943
[30]	Adachi H, Ishihama N, Nakano T, et al. Nicotiana benthamiana MAPK-WRKY pathway confers resistance to a necrotrophic pathogen Botrytis cinerea[J]. Plant Signaling & Behavior, 2016,11(6):e1183085. URL pmid: 27191816
[31]	Ogata T, Okada H, Kawaide H, et al. Involvement of NtERF3 in the cell death signaling pathway mediated by SIPK/WIPK and WRKY1 in tobacco plants[J]. Plant Biology, 2015,17:962-972. URL pmid: 25996234
[32]	Zuo KJ, Qin J, Zhao JY, et al. Over-expression GbERF2 transcription factor in tobacco enhances brown spots disease resistance by activating expression of downstream genes[J]. Gene, 2007,391:80-90. URL pmid: 17321073
[33]	Shin R, Park JM, An JM, et al. Ectopic expression of Tsi1 in transgenic hot pepper plants enhances host resistance to viral, bacterial, and oomycete pathogens[J]. Molecular Plant -Microbe Interactions, 2002,15:983-989. URL pmid: 12437295
[34]	Dong L, Cheng Y, Wu J, et al. Overexpression of GmERF5, a new member of the soybean EAR motif-containing ERF transcription factor, enhances resistance to Phytophthora sojae in soybean[J]. Journal of Experimental Botany, 2015,66:2635-2647. URL pmid: 25779701
[35]	Zhang G, Chen M, Li L, et al. Overexpression of the soybean GmERF3 gene, an AP2/ERF type transcription factor for increased tolerances to salt, drought and diseases in transgenic tobacco[J]. Journal of Experimental Botany, 2009,60(13):3781-3796. URL pmid: 19602544
[36]	李业, 陈银华, 吴家和, 等. OsRboh 基因家族在水稻免疫应答中的表达及功能分析[J]. 生物工程学报, 2011,27(11):1574-1585. pmid: 22393712
	Li Y. Chen YH, Wu JH, et al. Expression and functional analysis of OsRboh gene family in rice immune response[J]. Chinese Journal of Biotechnology, 2011,27(11):1574-1585. URL pmid: 22393712
[37]	刘秋圆, 贺浩华, 胡丽芳. 植物Rboh基因功能及其活性调节机制的研究进展[J]. 生物技术通报, 2013,11:8-13.
	Liu QY, He HH, Hu LF. Research progress in plant Rboh gene function and activity regulation mechanism[J]. Biotechnology Bulletin, 2013,11:8-13.
[38]	Boudsocq M, Willmann MR, McCormack M, et al. Differential innate immune signalling via Ca²+ sensor protein kinases[J]. Nature, 2010,464:418-422. URL pmid: 20164835
[39]	Yuasa T, Ichimura K, Mizoguchi T, et al. Oxidative stress activates ATMPK6, an Arabidopsis homologue of MAP kinase[J]. Plant Cell Physiology, 2001,42:1012-1016. URL pmid: 11577197
[40]	Zhang S, Klessig DF. Salicylic acid activates a 48-kD MAP kinase in tobacco[J]. Plant Cell 1997,9:809-824.
[41]	Boller T, Felix G. A renaissance of elicitors:perception of microbe -associated molecular patterns and danger signals by pattern-recognition receptors[J]. Annual Review of Plant Biology, 2009,60:379-406. URL pmid: 19400727
[42]	Liang X, Zhou J. Receptor-like cytoplasmic kinases:central players in plant receptor kinase-mediated signaling[J]. Annual Review of Plant Biology, 2018,69:267-299. doi: 10.1146/annurev-arplant-042817-040540 URL pmid: 29719165
[43]	Nishiuchi T, Shinshi H, Suzuki K. Rapid and transient activation of transcription of the ERF3 gene by wounding in tobacco leaves:possible involvement of NtWRKYs and autorepression[J]. The Journal of Biological Chemistry, 2004,279:55355-55361. URL pmid: 15509567

Names	Forward primers（5'- 3'）	Reverse primers（5'- 3'）
Actin	GCAAGGAAATCACCGCTTTGG	TGCCTGCTGGAATGTGCTAAG
BAK1	GGTCAATAAGGAAGCGTATA	GGTCAATAAGGAAGCGTATA
SERK2	TTGGGATTAGAGAATAACAAG	TTGGGATTAGAGAATAACAAG
ACS6	AGGGTTTGCTTATAAACAATC	AGGGTTTGCTTATAAACAATC
ACS1	GACAGAGACACACTGAAA	GACAGAGACACACTGAAA
WRKY22	AGGACAGACCCAAATATG	AGGACAGACCCAAATATG
MKS1	GCTTCTCTTCCACCTATA	GCTTCTCTTCCACCTATA
MYC2	GGAGCTGAATTGTTTAATTTC	GGAGCTGAATTGTTTAATTTC
ERF1	GTCCTTGGTCTCTTCTTA	GTCCTTGGTCTCTTCTTA
WRKY7	GGTCTCCTATCAGTAAGTC	GGTCTCCTATCAGTAAGTC
WRKY8	CCTACTGAACTCTTGGAC	CCTACTGAACTCTTGGAC

Names	Forward primers（5'- 3'）	Reverse primers（5'- 3'）
Actin	GCAAGGAAATCACCGCTTTGG	TGCCTGCTGGAATGTGCTAAG
BAK1	GGTCAATAAGGAAGCGTATA	GGTCAATAAGGAAGCGTATA
SERK2	TTGGGATTAGAGAATAACAAG	TTGGGATTAGAGAATAACAAG
ACS6	AGGGTTTGCTTATAAACAATC	AGGGTTTGCTTATAAACAATC
ACS1	GACAGAGACACACTGAAA	GACAGAGACACACTGAAA
WRKY22	AGGACAGACCCAAATATG	AGGACAGACCCAAATATG
MKS1	GCTTCTCTTCCACCTATA	GCTTCTCTTCCACCTATA
MYC2	GGAGCTGAATTGTTTAATTTC	GGAGCTGAATTGTTTAATTTC
ERF1	GTCCTTGGTCTCTTCTTA	GTCCTTGGTCTCTTCTTA
WRKY7	GGTCTCCTATCAGTAAGTC	GGTCTCCTATCAGTAAGTC
WRKY8	CCTACTGAACTCTTGGAC	CCTACTGAACTCTTGGAC

Gene ID	log2 Fold Change			Protein properties
Gene ID	6 hpi	12 hpi	24 hpi	Protein properties
Niben101Scf09644g00009	/	/	3.90	ERKY33,WRKY transcription factor 33
Niben101Scf03374g08019	3.80	3.71	1.10	LRR-RLK,probable LRR receptor-like serine/threonine-protein kinase At3g47571
Niben101Scf07608g00007	/	8.30	/	CALM,calmodulin
Niben101Scf05928g03007	5.32	6.75	3.32	probable LRR receptor-like serine/threonine-protein kinase At1g74361
Niben101Scf04406g07003	/	/	5.6	SERK2,PREDICTED：somatic embryogenesis receptor kinase 2-like isoformX1,
Niben101Scf18667g01002	/	/	2.6	MKS1,MAP kinase substrate 1
Niben101Scf00821g16001	3.67	7.77	3.44	BAK1,brassinosteroid insensitive 1-associated receptor kinase 1,RecName：Full=Leucine-rich repeat protein 1
Niben101Scf01090g02001	4.83	/	5.0	ERF1,ethylene-responsive transcription factor 1,PREDICTED：ethylene-responsive transcription factor 1B
Niben101Scf00219g00006	4.27	/	/	CALM,calmodulin
Niben101Scf37108g00003	4.27	6.46	/	PP2C,protein phosphatase 2C,PREDICTED：putative late blight resistance protein homolog R1B-12
Niben101Scf04133g02009	5.18	6.37	/	CALM,calmodulin,PREDICTED：probable calcium-binding protein CML45
Niben101Scf01297g04006	5.85	6.26	/	WRKY33,WRKY transcription factor 33
Niben101Scf01942g04001	5.89	5.76	3.07	WRKY33,WRKY transcription factor 33
Niben101Scf04053g02006	/	5.64	3.48	PR1,pathogenesis-related protein 1
Niben101Scf09512g03008	/	5.38	/	ACS1_2_6,1-aminocyclopropane-1-carboxylate synthase 1/2/6
Niben101Scf07491g00003	/	4.89	3.24	CHIB,basic endochitinase B,PREDICTED：endochitinase A,
Niben101Scf01398g00003	/	4.76	2.10	PRKAA,AMPK,PREDICTED：putative late blight resistance protein homolog R1B-16 isoform X1
Niben101Scf01015g01002	1.84	4.45	1.265	CHIB,basic endochitinase B,PREDICTED：wound-induced protein WIN1-like
Niben101Scf07345g00016	2.38	3.46	/	RBOH,respiratory burst oxidase
Niben101Scf04403g02004	2.06	5.85	1.72	PRF,disease resistance protein
Niben101Scf02171g00008	3.3	2.82	1.24	MPK3,mitogen-activated protein kinase 3,wound-inuduced protein kinase,WIPK
Niben101Scf14996g00009	1.0	1.6	1.69	CAT,catalase
Niben101Scf02041g00002	1.0	1.57	3.51	CHIB,basic endochitinase B
Niben101Scf03461g05022	1.0	1.45	1.23	PRKAA,AMPK,5'-AMP-activated protein kinase,catalytic alpha subunit,PREDICTED：putative late blight resistance protein homolog R1B-23

Gene ID	log2 Fold Change			Protein properties
Gene ID	6 hpi	12 hpi	24 hpi	Protein properties
Niben101Scf09644g00009	/	/	3.90	ERKY33,WRKY transcription factor 33
Niben101Scf03374g08019	3.80	3.71	1.10	LRR-RLK,probable LRR receptor-like serine/threonine-protein kinase At3g47571
Niben101Scf07608g00007	/	8.30	/	CALM,calmodulin
Niben101Scf05928g03007	5.32	6.75	3.32	probable LRR receptor-like serine/threonine-protein kinase At1g74361
Niben101Scf04406g07003	/	/	5.6	SERK2,PREDICTED：somatic embryogenesis receptor kinase 2-like isoformX1,
Niben101Scf18667g01002	/	/	2.6	MKS1,MAP kinase substrate 1
Niben101Scf00821g16001	3.67	7.77	3.44	BAK1,brassinosteroid insensitive 1-associated receptor kinase 1,RecName：Full=Leucine-rich repeat protein 1
Niben101Scf01090g02001	4.83	/	5.0	ERF1,ethylene-responsive transcription factor 1,PREDICTED：ethylene-responsive transcription factor 1B
Niben101Scf00219g00006	4.27	/	/	CALM,calmodulin
Niben101Scf37108g00003	4.27	6.46	/	PP2C,protein phosphatase 2C,PREDICTED：putative late blight resistance protein homolog R1B-12
Niben101Scf04133g02009	5.18	6.37	/	CALM,calmodulin,PREDICTED：probable calcium-binding protein CML45
Niben101Scf01297g04006	5.85	6.26	/	WRKY33,WRKY transcription factor 33
Niben101Scf01942g04001	5.89	5.76	3.07	WRKY33,WRKY transcription factor 33
Niben101Scf04053g02006	/	5.64	3.48	PR1,pathogenesis-related protein 1
Niben101Scf09512g03008	/	5.38	/	ACS1_2_6,1-aminocyclopropane-1-carboxylate synthase 1/2/6
Niben101Scf07491g00003	/	4.89	3.24	CHIB,basic endochitinase B,PREDICTED：endochitinase A,
Niben101Scf01398g00003	/	4.76	2.10	PRKAA,AMPK,PREDICTED：putative late blight resistance protein homolog R1B-16 isoform X1
Niben101Scf01015g01002	1.84	4.45	1.265	CHIB,basic endochitinase B,PREDICTED：wound-induced protein WIN1-like
Niben101Scf07345g00016	2.38	3.46	/	RBOH,respiratory burst oxidase
Niben101Scf04403g02004	2.06	5.85	1.72	PRF,disease resistance protein
Niben101Scf02171g00008	3.3	2.82	1.24	MPK3,mitogen-activated protein kinase 3,wound-inuduced protein kinase,WIPK
Niben101Scf14996g00009	1.0	1.6	1.69	CAT,catalase
Niben101Scf02041g00002	1.0	1.57	3.51	CHIB,basic endochitinase B
Niben101Scf03461g05022	1.0	1.45	1.23	PRKAA,AMPK,5'-AMP-activated protein kinase,catalytic alpha subunit,PREDICTED：putative late blight resistance protein homolog R1B-23