Verticillium dahliae Elicitor PevD1 Activates MAPKs in Nicotiana benthamiana

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

Abstract

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

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.

Figures/Tables 5

References 43

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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