Screening and verification of SgMPK6-interacting proteins of Stylosanthes

doi:10.11686/cyxb2023273

Abstract

Abstract:

The mitogen-activated protein kinase （MAPK） cascade plays a key role in regulating plant immunity. Anthracnose is a serious disease affecting the production of Stylosanthes （Stylo）. The SgMPK6 gene of Stylo has been shown to function in resistance against anthracnose， which is caused by Colletotrichum gloeosporioides. To investigate the interacting proteins downstream of SgMPK6 in the response to C. gloeosporioides infection， a yeast two-hybridization technique was used to screen the cDNA library of Stylo using the SgMPK6 kinase domain as the bait. Seventy-four potential interacting proteins were obtained. The interactions between the SgMPK6 kinase domain and the bHLH transcription factors SgbHLH32， SgbHLH33， and SgbHLH44 were verified in yeast two-hybrid experiments. Phosphorylation site analysis revealed that all three transcription factors contained MAPK phosphorylation sites. The results of qRT-PCR analyses showed that SgbHLH32， SgbHLH33， and SgbHLH44 were significantly up-regulated in Stylo by C. gloeosporioides infection. Together， these results suggest that SgbHLH32， SgbHLH33， and SgbHLH44 are substrates of SgMPK6 and may be involved in resistance against anthracnose. The results of this study provide a basis for further analysis of the role of SgMPK6 in resistance to C. gloeosporioides infection.

Key words: Stylosanthes, yeast two-hybrid, MPK6, anthracnose, bHLH transcription factors

Fang WANG, Shi-zi ZHANG, Rong-hui DAI, Li-yun YANG, Li-juan LUO, Ling-yan JIANG. Screening and verification of SgMPK6-interacting proteins of Stylosanthes[J]. Acta Prataculturae Sinica, 2024, 33(7): 84-93.

Figures/Tables 11

References 31

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引物名称 Primer names	序列 Sequences （5′-3′）
pGBKT7-SgMPK6-F	CATGGAGGCCGAATTCATGGAAGGAGGAGCTGC
pGBKT7-SgMPK6-R	GCAGGTCGACGGATCCACTGATATTCCGGGTTAAATGCC
pGBKT7-SgMPK6-KD-F	CATGGAGGCCGAATTCTTCGAAGTCACCGCCAAATAC
pGBKT7-SgMPK6-KD-R	GCAGGTCGACGGATCCACGGGTTAAATGCCAGAGACT
pGADT7-SgbHLH32-F	GGAGGCCAGTGAATTCATGGGGTGCAACAAAGGG
pGADT7-SgbHLH32-R	CGAGCTCGATGGATCCATTATTGGAACTGTACCAACTCACC
pGADT7-SgbHLH33-F	GGAGGCCAGTGAATTCATGGAGAGAGACATGCTTCC
pGADT7-SgbHLH33-R	CGAGCTCGATGGATCCATCACATCATGTCATGGATCCC
pGADT7-SgbHLH44-F	GGAGGCCAGTGAATTCATGCATATGAGAGGTTTGTTTCC
pGADT7-SgbHLH44-R	CGAGCTCGATGGATCCACTATGAATGGGTGTTGGATTCAG
qRT-SgbHLH32-F	AAATCAGGAGCTTGAATCACGT
qRT-SgbHLH32-R	CACATGCAAATCCACCATCCTC
qRT-SgbHLH33-F	CGTACCCACTACTGCTGCTG
qRT-SgbHLH33-R	TGTCAGGTCCGAAATCGAAGG
qRT-SgbHLH44-F	GTCGAGCAGGGTGAGGAAGTA
qRT-SgbHLH44-R	CATGGCTGCTCCAACATCTTC
UBCE1-F	CAGATCAAGCTGCTGACGAA
UBCE1-R	GAACAAGCGATCATCAGGTTT

引物名称 Primer names	序列 Sequences （5′-3′）
pGBKT7-SgMPK6-F	CATGGAGGCCGAATTCATGGAAGGAGGAGCTGC
pGBKT7-SgMPK6-R	GCAGGTCGACGGATCCACTGATATTCCGGGTTAAATGCC
pGBKT7-SgMPK6-KD-F	CATGGAGGCCGAATTCTTCGAAGTCACCGCCAAATAC
pGBKT7-SgMPK6-KD-R	GCAGGTCGACGGATCCACGGGTTAAATGCCAGAGACT
pGADT7-SgbHLH32-F	GGAGGCCAGTGAATTCATGGGGTGCAACAAAGGG
pGADT7-SgbHLH32-R	CGAGCTCGATGGATCCATTATTGGAACTGTACCAACTCACC
pGADT7-SgbHLH33-F	GGAGGCCAGTGAATTCATGGAGAGAGACATGCTTCC
pGADT7-SgbHLH33-R	CGAGCTCGATGGATCCATCACATCATGTCATGGATCCC
pGADT7-SgbHLH44-F	GGAGGCCAGTGAATTCATGCATATGAGAGGTTTGTTTCC
pGADT7-SgbHLH44-R	CGAGCTCGATGGATCCACTATGAATGGGTGTTGGATTCAG
qRT-SgbHLH32-F	AAATCAGGAGCTTGAATCACGT
qRT-SgbHLH32-R	CACATGCAAATCCACCATCCTC
qRT-SgbHLH33-F	CGTACCCACTACTGCTGCTG
qRT-SgbHLH33-R	TGTCAGGTCCGAAATCGAAGG
qRT-SgbHLH44-F	GTCGAGCAGGGTGAGGAAGTA
qRT-SgbHLH44-R	CATGGCTGCTCCAACATCTTC
UBCE1-F	CAGATCAAGCTGCTGACGAA
UBCE1-R	GAACAAGCGATCATCAGGTTT

基因ID Gene ID	拟南芥编号Arabidopsis number	蛋白质名称Protein name
Unigene15192_SG	AT1G74110.1	细胞色素P450家族蛋白 Cytochrome P450 family protein
CL672.Contig2_SG	AT1G12860.1	bHLH转录因子33 bHLH transcription factor 33
Unigene44350_SG	AT2G42380.2	bZIP转录因子家族蛋白 bZIP transcription factor family protein
CL7251.Contig3_SG	AT5G66540.1	U3小核仁核糖核蛋白 U3 small nucleolar ribonucleoprotein
Unigene29237_SG	AT5G17820.1	过氧化物酶超家族蛋白 Peroxidase superfamily protein
CL1493.Contig6_SG	AT3G25710.1	bHLH转录因子32 bHLH transcription factor 32
Unigene3271_SG	AT3G25230.1	FK506结合蛋白62 FK506 binding protein 62
CL3082.Contig4_SG	AT4G16190.1	半胱氨酸蛋白酶RD19C Cysteine protease RD19C
CL3756.Contig1_SG	AT1G18400.1	bHLH转录因子44 bHLH transcription factor 44

基因ID Gene ID	拟南芥编号Arabidopsis number	蛋白质名称Protein name
Unigene15192_SG	AT1G74110.1	细胞色素P450家族蛋白 Cytochrome P450 family protein
CL672.Contig2_SG	AT1G12860.1	bHLH转录因子33 bHLH transcription factor 33
Unigene44350_SG	AT2G42380.2	bZIP转录因子家族蛋白 bZIP transcription factor family protein
CL7251.Contig3_SG	AT5G66540.1	U3小核仁核糖核蛋白 U3 small nucleolar ribonucleoprotein
Unigene29237_SG	AT5G17820.1	过氧化物酶超家族蛋白 Peroxidase superfamily protein
CL1493.Contig6_SG	AT3G25710.1	bHLH转录因子32 bHLH transcription factor 32
Unigene3271_SG	AT3G25230.1	FK506结合蛋白62 FK506 binding protein 62
CL3082.Contig4_SG	AT4G16190.1	半胱氨酸蛋白酶RD19C Cysteine protease RD19C
CL3756.Contig1_SG	AT1G18400.1	bHLH转录因子44 bHLH transcription factor 44

蛋白 Protein	磷酸化位点 Phosphorylation site	磷酸化肽段 Phosphorylation peptides	可信值Trusted value
SgbHLH32	124 S	LNAIS^* PQHV	0.506
SgbHLH33	393 T	LQPLT^* PTPQ	0.615
SgbHLH44	16 S	QLHNS^* PPPP	0.512
SgbHLH44	47 T	QRQQT^* PNPV	0.533