Screening of Proteins Interacting with CsSSK1 in Colletotrichum siamense by Yeast Two-hybrid Technique

doi:10.3969/j.issn.1000-2561.2021.01.027

Abstract

Abstract:

Colletotrichum siamense is the main pathogen of anthracnose of rubber tree or other tropical crops. A two component system is involved in the adaptation of fungi to external environmental changes, drug resistance and virulence in pathogenic fungi. In pathogenic fungi, it includes receptors and SSK1 (SLF-interacting SKP1-like1) response regulatory protein components. To obtain proteins interacting with SSK1 in C. siamense, a homologue gene CsSSK1, 2750 bp long with cDNA 2190 bp, was cloned. It contained a cheY homologs receiving domains and 10 complex structures. Then the pGBKT7-CsSSK1 vector was constructed. By an yeast two hybrid system, ten proteins, including transketolase, β-lactamase, alcohol dehydrogenase, ubiquitin, tubulin and 5 unknown or hypothetical proteins, maight interacting with CsSSK1, were identified after sequencing and bioinformatics analysis. This result would provide a basis for further study of the functions, interaction proteins and regulatory mechanism of CsSSK1.

Key words: Colletotrichum siamense, two component system, response regulatory protein SSK1, interaction protein

CLC Number:

S763.7

FANG Siqi, LI Zedong, WANG Jiyuan, HE Qiguang, LI Xiao, LIU Wenbo, ZHANG Yu, LIN Chunhua, MIAO Weiguo. Screening of Proteins Interacting with CsSSK1 in Colletotrichum siamense by Yeast Two-hybrid Technique[J]. Chinese Journal of Tropical Crops, 2021, 42(1): 198-204.

Figures/Tables 7

Fig. 1 Agarose gel electrophoresis of PCR products of CsSSK1 Marker: DL5000 DNA Marker; 1: The amplified product of the CsSSK1 gene using DNA as the template; 2: The amplified product of the CsSSK1 gene using the cDNA as the template.

Fig. 2 SMART analysis of CsSSK1 protein structure

Fig. 3 Enzyme digestion verification diagram of pGBKT7-SSK1 The restriction enzymes are EcoRⅠ and BamHⅠ.

Fig. 4 Growth of yeast clone with pGBKT7-CsSSK1 and pGADT7 on different media A: SD / -Trp-Leu medium; B: SD / -Trp-Leu-His-Ade medium.

Fig. 5 PCR verification of positive clones obtained from yeast two-hybrid using CsSSK1 as bait protein M: The DL2000 DNA Marker; 1-24: PCR results for the positive clones using primers pGBKT7-F/R.

Tab. 1 Information on putative CsSSK1-interacting proteins identified by the Y2HGlod system

编号 Number	片段大小 Fragment length/bp	同源蛋白和物种 Homologous protein and origin	E值 E-Value	同源性 Homology	分值 Score	功能注释 Function annotation
SSK1-23	441	ELA24698.1 Colletotrichum fructicola Nara gc5	2E-48	0.9639	168	金属内酰胺酶
SSK1-20	725	ELA29973.1 Colletotrichum fructicola Nara gc5	3E-88	0.9718	285	转酮醇酶
SSK1-18	828	ADD85280.1 Calonectria reteaudii	3E-09	0.5556	62	微管蛋白
SSK1-12	770	ELA24752.1 Colletotrichum fructicola Nara gc5	2E-70	0.9083	229	锌乙醇脱氢酶
SSK1-14	678	XP_018514083.1 Brassica rapa	0.072	0.2941	41.6	泛素亚型X1
SSK1-7	459	ELA25416.1 Colletotrichum fructicola Nara gc5	2E-72	0.9262	225	bys1 domain- containing protein
SSK1-15	351	EQB56384.1 Colletotrichum gloeosporioides Cg-14	3E-37	0.8608	138	假定蛋白
SSK1-1	598	EXF74198.1 Colletotrichum fioriniae PJ7	5E-07	0.9615	54.7	假定蛋白
SSK1-8	560	XP_022478823.1 Colletotrichum orchidophilum	3E-05	0.3303	53.5	假定蛋白
SSK1-13	629	XP_023628327.1 Ramularia collocygni	2.7	0.4167	40.8	未知

Fig. 6 Confirmation of interaction between 10 positive prey plasmids and pGBKT7-CsSSK1 by Y2HGlod. pGBKT7: P53+pGADT7:Rec-T is the positive control; pGBKT7+pGADT7 is the negative control; Yeast-1-Yeast-23 are yeast cells who were co-tranformed with pGBKT7-CsSSK1 and each of the 10 putatively positive prey plasmids (Tab. 1) and plated on SD/-Trp-Leu-His-Ade plates.

References 23

[1]	刘凯悦. 双组分系统基因MoSLN1和MoSKN7在稻瘟病菌生长发育与致病过程中的功能研究[D]. 南京: 南京农业大学, 2011.
[2]	Bahn Y S. Master and commander in fungal pathogens: The two-component system and the HOG signaling pathway[J]. Eukaryotic Celly, 2008,7(12):2017-2036.
[3]	吴雪昌, 胡森杰, 酵母HOG-MAPK途径[J]. 细胞生物学杂志, 2005,27(3):247-252.
[4]	Oide S, Liu J, Yun S-H, et al. Histidine kinase two-component response regulator proteins regulate reproductive development, virulence, and stress responses of the fungal cereal pathogens Cochliobolus heterostrophus and Gibberella zeae[J]. Eukaryotic Cell, 2010,9(12):1867-1880. DOI URL PMID
[5]	Shor E, Chauhan N, Goldman W E A. Case for two-component signaling systems as antifungal drug targets[J]. PLoS Pathogens, 2015,11(2):e1004632. DOI URL PMID
[6]	Posas F, Saito H. Activation of the yeast SSK2 MAP kinase kinase kinase by the SSK1 two-component response regulator[J]. The EMBO Journal, 1998,17(5):1385-1394. DOI URL PMID
[7]	Porter S W, West A H. A common docking site for response regulators on the yeast phosphorelay protein YPD1[J]. BBA - Proteins and Proteomics, 2005,1748(2):138-145. DOI URL PMID
[8]	Rispail N, Soanes D M, Ant C, et al. Comparative genomics of MAP kinase and calcium-calcineurin signalling components in plant and human pathogenic fungi[J]. Fungal Genetics and Biology, 2009,46(4):287-298. DOI URL PMID
[9]	Baltanás R, Bush A, Couto A, et al. Pheromone-induced morphogenesis improves osmoadaptation capacity by activating the HOG MAPK pathway[J]. Science Signaling, 2013,6(272):26.
[10]	Yan L, Yang Q, Jiang J, et al. Involvement of a putative response regulator Brrg-1 in the regulation of sporulation, sensitivity to fungicides, and osmotic stress in Botrytis cinereal[J]. Applied Microbiology and Biotechnology, 2011,90(1):215-226. DOI URL PMID
[11]	Horie T, Tatebayashi K, Yamada R, et al. Phosphorylated SSK1 prevents unphosphorylated SSK1 from activating the SSk2 mitogen-activated protein kinase kinase kinase in the yeast high-osmolarity glycerol osmoregulatory pathway[J]. Molecular and Cellular Biology, 2008,28(17):5172-5183. DOI URL PMID
[12]	Maeda T, Takekawa M, Saito H. Activation of yeast PBS2 MAPKK by MAPKKKs or by binding of an SH3-containing osmosensor[J]. Science, 1995,269(5223):554-558. DOI URL PMID
[13]	Zheng J Y, Tang C, Deng C L, et al. Involvement of a response regulator VdSSK1 in stress response, melanin biosynjournal and full virulence in Verticillium dahliae[J]. Frontiers in Microbiology, 2019,10:606. DOI URL PMID
[14]	林春花, 杨欢, 赵晓宇, 等. 海南橡胶树炭疽菌Colletotrichum siamense和C. fructicola的鉴定及系统发育分析[J]. 热带作物学报, 2018,39(1):129-136.
[15]	Liu X B, Li B X, Cai J M, et al. Colletotrichum species causing anthracnose of rubber trees in China[J]. Scientific Reports, 2018,8(1):10435. DOI URL PMID
[16]	Sharma G, Pinnaka A K, Shenoy B D. Resolving the Colletotrichum siamense species complex using ApMat marker[J]. Fungal Diversity, 2015,71(1):247-264.
[17]	Lin C H, Huang G X, Zheng F C, et al. Functional characterization of CgPBS2, a MAP kinase kinase in Colletotrichum gloeosporioides, using osmotic stress sensitivity as a selection marker[J]. Eruopean Journal of Plant Pathology, 2018,152(3):801-813.
[18]	Letunic I, Bork P. 20 years of the SMART protein domain annotation resource[J]. Nucleic Acids Research, 2018,46(D1):493-496.
[19]	Chauhan N, Inglis D, Roman E, et al. Candida albicans response regulator gene SSK1 regulates a subset of genes whose functions are associated with cell wall biosynjournal and adaptation to oxidative stress[J]. Eukaryot Cell, 2003,2(5):1018-1024. DOI URL PMID
[20]	Branscum K M, Menon S K, Foster C A, et al. Insights revealed by the co-crystal structure of the Saccharomyces cerevisiae histidine phosphotransfer protein Ypd1 and the receiver domain of its downstream response regulator Ssk1[J]. Protein Science, 2019,28(12):2099-2111. DOI URL PMID
[21]	Fernandez J, Marroquin-Guzman M, Wilson R A. Evidence for a transketolase-mediated metabolic checkpoint governing biotrophic growth in rice cells by the blast fungus Magnaporthe oryzae[J]. PLoS Pathogens, 2014,10(9):1004354.
[22]	赵斌, 霍静倩, 张哲, 等. 以植物转酮醇酶为靶标的除草活性物质筛选及其生物活性测定[C]//病虫害绿色防控与农产品质量安全——中国植物保护学会2015年学术年会论文集, 北京: 中国农业科学技术出版社, 2015: 672.
[23]	鞠林成, 史亚兴, 苗东旭, 等. 金属β内酰胺酶及其抑制剂的研究进展[J]. 医学综述, 2019,25(6):1222-1227.