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Screening of Interacting Proteins of MAT in Aspergillus critatus by GST Pull-down |
WU Juan,XU Ning,ZHANG Sheng-hua,ZHANG Xiao-dan,LIU Yuan-yuan,GE Yong-yi**() |
School of Life Sciences, Guizhou University, Guiyang 550025, China |
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Abstract Objective: Aspergillus cristatus is a homothallic fungus, whose sporulation is regulated by osmotic pressure, which is quite different from the light-regulated sporulation mechanism of Aspergillus nidulans. The sexual reproduction of A. cristatus is mainly regulated by MAT1-1-1 and MAT1-2-1, but the regulation mechanism of the MAT gene on the sexual reproduction is still unclear. This study aims to screen the interaction proteins of A. cristatum MAT, and lay the foundation for the further study of the sexual sporulation mechanism of A. cristatum.Methods: This study screened the interaction protein with MAT1-1-1 and MAT1-2-1 by GST pull-down combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). We analyzed the biological information of the interaction protein using ProteinPilot, Gene Ontology and the genome databank of A. cristatus. The study also detected the expression level of SI65_00917 and SI65_03348 in sexual development by RT-qPCR, and used yeast two-hybrid technology to verify their interaction with MAT protein.Results: The GST-MAT1-1-1 and GST-MAT1-2-1 vectors were successfully constructed, and the target bait proteins were induced to express and purify. The bait proteins were used to capture the interacting proteins from the total protein of A. cristatus. The results showed that 56 proteins interacted with MAT1-1-1, and 413 proteins interacted with MAT1-2-1, respectively. GO analysis shows that these interaction proteins are involved in translation regulation, metabolic processes, protein transport, protein binding and other biological processes, and share nucleotide binding activity, catalytic activity, and protein binding activity. The results from RT-qPCR indicated that the interaction proteins SI65_00917 would participate in the sexual development in A.cristatus. Yeast two-hybrid results show that SI65_00917 protein has auto-activation and may be a transcription factor, and SI65_03348 protein interacts with MAT1-1-1 and MAT1-2-1 in yeast.Conclusion: These results indicate that MAT regulates the sexual development of A.cristatus through direct or indirect interaction with the other proteins.
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Received: 30 August 2021
Published: 07 April 2022
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Corresponding Authors:
Yong-yi GE
E-mail: 746560455@qq.com
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[1] |
Ene I V, Bennett R J. The cryptic sexual strategies of human fungal pathogens. Nature Reviews Microbiology, 2014, 12(4):239-251.
doi: 10.1038/nrmicro3236
|
|
|
[2] |
Whittle C A, Nygren K, Johannesson H. Consequences of reproductive mode on genome evolution in fungi. Fungal Genetics and Biology, 2011, 48(7):661-667.
doi: 10.1016/j.fgb.2011.02.005
pmid: 21362492
|
|
|
[3] |
Szewczyk E, Krappmann S. Conserved regulators of mating are essential for Aspergillus fumigatus cleistothecium formation. Eukaryotic Cell, 2010, 9(5):774-783.
doi: 10.1128/EC.00375-09
pmid: 20348388
|
|
|
[4] |
Ni M, Feretzaki M, Sun S, et al. Sex in fungi. Annual Review of Genetics, 2011, 45(1):405-430.
doi: 10.1146/genet.2011.45.issue-1
|
|
|
[5] |
Liu K H, Shen W C. Mating differentiation in Cryptococcus neoformans is negatively regulated by the Crk1 protein kinase. Fungal Genetics and Biology, 2011, 48(3):225-240.
doi: 10.1016/j.fgb.2010.11.005
|
|
|
[6] |
Heitman J, Sun S, James T Y. Evolution of fungal sexual reproduction. Mycologia, 2013, 105(1):1-27.
doi: 10.3852/12-253
pmid: 23099518
|
|
|
[7] |
施笑笑, 王教瑜, 王艳丽, 等. 子囊菌交配型位点与交配型基因研究进展. 微生物学通报, 2020, 47(5):1572-1581.
|
|
|
[7] |
Shi X X, Wang J Y, Wang Y L, et al. Mating type genes in ascomycetes: a review. Microbiology China, 2020, 47(5):1572-1581.
|
|
|
[8] |
Turgeon B G, Yoder O C. Proposed nomenclature for mating type genes of filamentous ascomycetes. Fungal Genetics and Biology, 2000, 31(1):1-5.
pmid: 11118130
|
|
|
[9] |
Ge Y Y, Yu F M, Yang Z J, et al. Genetic basis and function of mating-type genes in Aspergillus cristatus. Mycosphere, 2019, 10(1):622-633.
doi: 10.5943/mycosphere
|
|
|
[10] |
Grognet P, Bidard F, Kuchly C, et al. Maintaining two mating types: structure of the mating type locus and its role in heterokaryosis in Podospora anserina. Genetics, 2014, 197(1):421-432.
doi: 10.1534/genetics.113.159988
pmid: 24558260
|
|
|
[11] |
Kanamori M, Kato H, Yasuda N, et al. Novel mating type-dependent transcripts at the mating type locus in Magnaporthe oryzae. Gene, 2007, 403(1-2):6-17.
pmid: 17881155
|
|
|
[12] |
Yong M L, Yu J J, Pan X Y, et al. Two mating-type genes MAT1-1-1 and MAT1-1-2 with significant functions in conidiation, stress response, sexual development, and pathogenicity of rice false smut fungus Villosiclava virens. Current Genetics, 2020, 66(5):989-1002.
doi: 10.1007/s00294-020-01085-9
|
|
|
[13] |
Becker K, Beer C, Freitag M, et al. Genome-wide identification of target genes of a mating-type α-domain transcription factor reveals functions beyond sexual development. Molecular Microbiology, 2015, 96(5):1002-1022.
doi: 10.1111/mmi.2015.96.issue-5
|
|
|
[14] |
Dyer P S, Paoletti M. Reproduction in Aspergillus fumigatus: sexuality in a supposedly asexual species. Medical Mycology, 2005, 43(S1):S7-S14.
|
|
|
[15] |
Böhm J, Hoff B, O’Gorman C M, et al. Sexual reproduction and mating-type-mediated strain development in the penicillin-producing fungus Penicillium chrysogenum. PNAS, 2013, 110(4):1476-1481.
doi: 10.1073/pnas.1217943110
|
|
|
[16] |
Arnaise S, Debuchy R, Picard M. What is a bona fide mating-type gene? Internuclear complementation of mat mutants in Podospora anserina. Molecular and General Genetics: MGG, 1997, 256(2):169-178.
|
|
|
[17] |
Kim H, Wright S J, Park G, et al. Roles for receptors, pheromones, G proteins, and mating type genes during sexual reproduction in Neurospora crassa. Genetics, 2012, 190(4):1389-1404.
doi: 10.1534/genetics.111.136358
|
|
|
[18] |
Whiteway M S, Wu C, Leeuw T, et al. Association of the yeast pheromone response G protein beta gamma subunits with the MAP kinase scaffold Ste5p. Science, 1995, 269(5230):1572-1575.
pmid: 7667635
|
|
|
[19] |
Chol K Y, Satterberg B, Lyons D M, et al. Ste5 tethers multiple protein kinases in the MAP kinase cascade required for mating in S. cerevisiae. Cell, 1994, 78(3):499-512.
doi: 10.1016/0092-8674(94)90427-8
|
|
|
[20] |
任春光, 谭玉梅, 任秀秀, 等. 冠突曲霉veA基因缺失型与野生型的差异代谢物研究. 菌物学报, 2018, 37(2):193-204.
|
|
|
[20] |
Ren C G, Tan Y M, Ren X X, et al. Differential metabolite analysis of the veA gene deletion and wild type strains of Aspergillus cristatus. Mycosystema, 2018, 37(2):193-204.
|
|
|
[21] |
余春芳, 熊庆, 李文仿, 等. 冠突散囊菌nsdD基因超表达菌株的构建及表型分析. 基因组学与应用生物学, 2017, 36(3):900-905.
|
|
|
[21] |
Yu C F, Xiong Q, Li W F, et al. Construction and phenotypic analysis of over-expression strain of nsdD gene in Eurotium cristatum. Genomics and Applied Biology, 2017, 36(3):900-905.
|
|
|
[22] |
Lehti-Shiu M D, Panchy N, Wang P P, et al. Diversity, expansion, and evolutionary novelty of plant DNA-binding transcription factor families. Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, 2017, 1860(1):3-20.
doi: 10.1016/j.bbagrm.2016.08.005
|
|
|
[23] |
Nolting N, Pöggeler S. A MADS box protein interacts with a mating-type protein and is required for fruiting body development in the homothallic ascomycete Sordaria macrospora. Eukaryotic Cell, 2006, 5(7):1043-1056.
pmid: 16835449
|
|
|
[24] |
Jacobsen S, Wittig M, Pöggeler S. Interaction between mating-type proteins from the homothallic fungus Sordaria macrospora. Current Genetics, 2002, 41(3):150-158.
pmid: 12111096
|
|
|
[25] |
郑欣欣. 茯砖茶中“金花”菌产孢机制及其功能性研究. 西安: 陕西科技大学, 2015.
|
|
|
[25] |
Zheng X X. Study on sporulation mechanism and function of ‘Jinhua’ fungi in fuzhuan brick tea. Xi’an: Shanxi University of Science & Technology, 2015.
|
|
|
[26] |
Rao X Y, Huang X L, Zhou Z C, et al. An improvement of the 2-ΔΔCt method for quantitative real-time polymerase chain reaction data analysis . Biostatistics,Bioinformatics and Biomathematics, 2013, 3(3):71-85.
|
|
|
[27] |
Wissmueller S, Font J, Liew C W, et al. Protein-protein interactions: analysis of a false positive GST pulldown result. Proteins, 2011, 79(8):2365-2371.
doi: 10.1002/prot.23068
|
|
|
|
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