玉米大斑病菌LysM家族的鉴定及关键效应因子StLysM1对植物免疫反应的调控作用

Journal of Integrative Agriculture ›› 2025, Vol. 24 ›› Issue (5): 1860-1874.DOI: 10.1016/j.jia.2024.06.006

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玉米大斑病菌LysM家族的鉴定及关键效应因子StLysM1对植物免疫反应的调控作用

收稿日期:2023-10-31 修回日期:2024-06-27 接受日期:2024-04-29 出版日期:2025-05-20 发布日期:2025-04-16

Comprehensive analysis of the LysM protein family and functional characterization of the key LysM effector StLysM1, which modulates plant immunity in Setosphaeria turcica

Xiaodong Gong^{1, 2, 3}, Dan Han^{1, 3}, Lu Zhang^{1, 4}, Guibo Yin^{1, 3}, Junfang Yang^{1, 3}, Hui Jia^{1, 2}, Zhiyan Cao^{1, 4}, Jingao Dong^{1, 4#}, Yuwei Liu^{1, 2, 3#}, Shouqin Gu^{1, 2, 3#}

¹State Key Laboratory of North China Crop Improvement and Regulation, Hebei Agricultural University, Baoding 071000, China
²Hebei Bioinformatic Utilization and Technological Innovation Center for Agricultural Microbes, Hebei Agricultural University, Baoding 071000, China
³College of Life Sciences, Hebei Agricultural University, Baoding 071000, China
⁴College of Plant Protection, Hebei Agricultural University, Baoding 071000, China

Received:2023-10-31 Revised:2024-06-27 Accepted:2024-04-29 Online:2025-05-20 Published:2025-04-16
About author:Xiaodong Gong, Tel: +86-312-7528876, E-mail: gxdjy@126.com; #Correspondence Jingao Dong, Tel: +86-312-7528876, E-mail: dongjingao@126.com; Yuwei Liu, Tel: +86-312-7528876, E-mail: liuyw@hebau.edu.cn; Shouqin Gu, Tel: +86-312-7528876, E-mail: gushouqin@126.com
Supported by:
This work was supported by the State Key Laboratory of North China Crop Improvement and Regulation (NCCIR2023ZZ-17), the Hebei Provincial Central Leading Local Science and Technology Development Fund Project (236Z6508G), the Basic Research Funds for Provincial Universities in Hebei Province (KY2022037, KY2021042) and the Natural Science Foundation of Hebei Province (2023204100, 2021204136).

摘要/Abstract

摘要：

【目的】系统分析玉米大斑病菌LysM家族成员，筛选其中关键效应蛋白，并分析其对植物免疫反应的调控作用，为探究病菌致病性的分子机制奠定基础。【方法】利用生物信息学方法在玉米大斑病菌基因组中鉴定LysM家族成员，对其理化性质、进化关系、结构域及保守位点进行分析；基于转录组数据并结合qRT-PCR技术分析LysM家族基因在病菌侵染玉米过程中的表达模式；通过酵母分泌系统分析信号肽分泌活性；通过本氏烟瞬时表达系统分析关键效应蛋白StLysM1对植物免疫反应的调控作用；利用分子模拟和多糖结合试验检测LysM效应蛋白对几丁质的结合能力。【结果】在玉米大斑病菌基因组中共鉴定得到8个LysM家族成员，分别命名为StLysM1~StLysM8，其中5个成员（StLysM1、StLysM2、StLysM5、StLysM6和StLysM7）为候选效应蛋白，这些效应蛋白均只含有LysM结构域，其他成员则含有额外的保守结构域。系统分析显示，StLysMs可分为真菌/细菌亚类和真菌特异亚类，其中真菌特异亚类的LysM结构域序列包含⁸GDxTC¹²、²⁹WNP³¹基序以及3个高度保守的半胱氨酸残基，而细菌/真菌亚类的LysM结构域序列保守位点较少。StLysM1基因在病菌侵染玉米24 h、72 h过程中持续上调表达，其编码产物为分泌蛋白，且在本氏烟中不能诱导植物的程序性细胞死亡（PCD），但可抑制由BAX/INF1诱导的PCD。StLysM1自身不形成二聚体，但能与几丁质相结合，并抑制由几丁质触发的活性氧爆发及病程相关基因NbPR1和NbPR4的表达，提高了本氏烟对灰葡萄孢的易感性。【结论】在玉米大斑病菌中共鉴定得到8个StLysMs家族成员，其中StLysM1为关键效应因子，能够结合几丁质，进而抑制植物基础免疫并促进病菌侵染。

Abstract:

Setosphaeria turcica is limited. In this study, eight StLysM genes are identified and designated as StLysM1 to StLysM8. The analysis of sequence features indicates that five proteins (StLysM1, StLysM2, StLysM5, StLysM6, and StLysM7) are potential effectors. Phylogenetic analysis suggests that the StLysMs are divided into fungal/bacterial and fungus-specific subclasses. Domain architecture analysis reveals that the five StLysM effectors exclusively harbor the LysM domain, whereas the other three StLysM proteins contain additional functional domains. Sequence conservation analysis shows that the fungal-specific LysM domain sequences share the ⁸GDxTC¹² and ²⁹WNP³¹ motifs as well as three highly conserved cysteine residues. Conversely, the LysM domain sequences from the bacterial/fungal branch have few conserved sites. Moreover, expression profiling analysis shows that the StLysM1 gene is significantly upregulated during the infection of maize. Yeast secretion assays and transient expression experiments demonstrate that StLysM1 is a secreted protein that can suppress BAX/INF1-induced programmed cell death in Nicotiana benthamiana. Further functional analysis suggests that StLysM1 cannot interact with itself but it can bind chitin. The transient expression of StLysM1 inhibits the chitin-triggered plant immune response, increasing susceptibility to the phytopathogenic fungus Botrytis cinerea in N. benthamiana. This study reveals that the S. turcica LySM protein family consists of eight members, highlighting the significance of StLysM1 as a vital effector in regulating plant immunity. The results provide insight into StLysMs and establish a foundation for understanding the roles of StLysM proteins in the pathogenic process of S. turcica.

Key words: Setosphaeria turcica , LysM , effector , chitin

Xiaodong Gong, Dan Han, Lu Zhang, Guibo Yin, Junfang Yang, Hui Jia, Zhiyan Cao, Jingao Dong, Yuwei Liu, Shouqin Gu. 玉米大斑病菌LysM家族的鉴定及关键效应因子StLysM1对植物免疫反应的调控作用[J]. Journal of Integrative Agriculture, 2025, 24(5): 1860-1874.

Xiaodong Gong, Dan Han, Lu Zhang, Guibo Yin, Junfang Yang, Hui Jia, Zhiyan Cao, Jingao Dong, Yuwei Liu, Shouqin Gu. Comprehensive analysis of the LysM protein family and functional characterization of the key LysM effector StLysM1, which modulates plant immunity in Setosphaeria turcica[J]. Journal of Integrative Agriculture, 2025, 24(5): 1860-1874.

参考文献

Akcapinar G B, Kappel L, Sezerman O U, Seidl-Seiboth V. 2015. Molecular diversity of LysM carbohydrate-binding motifs in fungi. Current Genetics, 61, 103–113.

Alcântara A, Bosch J, Nazari F, Hoffmann G, Gallei M, Uhse S, Darino MA, Olukayode T, Reumann D, Baggaley L, Djamei A. 2019. Systematic Y2H screening reveals extensive effector-complex formation. Frontiers in Plant Science, 10, 1743.

Bolton M D, van Esse H P, Vossen J H, de Jonge R, Stergiopoulos I, Stulemeijer I J E, van den Berg G C M, Borrás-Hidalgo O, Dekker H L, de Koster C G, de Wit P J, Joosten M H, Thomma BP. 2008. The novel Cladosporium fulvum lysin motif effector Ecp6 is a virulence factor with orthologues in other fungal species. Molecular Microbiology, 69, 119–136.

Buist G, Steen A, Kok J, Kuipers O P. 2008. LysM, a widely distributed protein motif for binding to (peptido) glycans. Molecular Microbiology, 68, 838–847.

Cen K, Li B, Lu Y, Zhang S, Wang C. 2017. Divergent LysM effectors contribute to the virulence of Beauveria bassiana by evasion of insect immune defenses. PLOS Pathogens, 13, e1006604.

Chen C, Chen Y, Jian H, Yang D, Dai Y, Pan L, Shi F, Yang S, Liu Q. 2018. Large-scale identification and characterization of Heterodera avenae putative effectors suppressing or inducing cell death in Nicotiana benthamiana. Frontiers in Plant Science, 15, 2062.

Cheng Y, Wu K, Yao J, Li S, Wang X, Huang L, Kang Z. 2017. PSTha5a23, a candidate effector from the obligate biotrophic pathogen Puccinia striiformis f. sp. tritici, is involved in plant defense suppression and rust pathogenicity. Environmental Microbiology, 19, 1717–1729.

Crooks G E, Hon G, Chandonia J M, Brenner S E. 2004. WebLogo: A sequence logo generator. Genome Research, 14, 1188–1190.

Cui L, Zhao L, Wang B, Han Z, Hu Y. 2022. Genetic diversity, population structure and mating type distribution of Setosphaeria turcica on corn in midwestern China. Journal of Fungi, 8, 1165.

De J R, Thomma B P. 2009. Fungal LysM effectors: extinguishers of host immunity? Trends Microbiol, 17, 151–157.

Dölfors F, Holmquist L, Dixelius C, Tzelepis G. 2019. A LysM effector protein from the basidiomycete Rhizoctonia solani contributes to virulence through suppression of chitin-triggered immunity. Molecular Genetics and Genomics, 294, 1211–1218.

Dubey M, Vélëz H, Broberg M, Jensen D F, Karlsson M. 2020. LysM proteins regulate fungal development and contribute to hyphal protection and biocontrol traits in Clonostachys rosea. Frontiers in Microbiology, 11, 679.

Edgar R C. 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research, 32, 1792–1797.

Gruber S, Vaaje-Kolstad G, Matarese F, López-Mondéjar R, Kubicek C P, Seidl-Seiboth V. 2011. Analysis of subgroup C of fungal chitinases containing chitin-binding and LysM modules in the mycoparasite Trichoderma atroviride. Glycobiology, 21, 122–133.

Gu B, Kale S D, Wang Q, Wang D, Pan Q, Cao H, Meng Y, Kang Z, Tyler B M, Shan W. 2011. Rust secreted protein Ps87 is conserved in diverse fungal pathogens and contains a RXLR-like motif sufficient for translocation into plant cells. PLoS ONE, 6, e27217.

Harishchandra D L, Zhang W, Li X, Chethana K W T, Hyde K D, Brooks S, Yan J, Peng J. 2020. A LysM domain-containing protein LtLysM1 is important for vegetative growth and pathogenesis in woody plant pathogen Lasiodiplodia theobromae. The Plant Pathology Journal, 36, 323–334.

Hu S P, Li J J, Dhar N, Li J P, Chen J Y, Jian W, Dai X F, Yang X Y. 2021. Lysin motif (LysM) proteins: interlinking manipulation of plant immunity and fungi. International Journal of Molecular Sciences, 22, 3114.

Jones J D G, Dangl J L. 2006. The plant immune system. Nature, 444, 323–329.

De Jonge R, Thomma B P H J. 2009. Fungal LysM effectors: extinguishers of host immunity? Trends in Microbiology, 17, 151–157.

De Jonge R, Peter van Esse H, Kombrink A, Shinya T, Desaki Y, Bours R, van der Krol S, Shibuya N, Joosten M H A J, Thomma B P H J. 2010. Conserved fungal LysM effector Ecp6 prevents chitin-triggered immunity in plants. Science, 329, 953–955.

Kim S, Chen J, Cheng T, Gindulyte A, He J, He S, Li Q, Shoemaker B A, Thiessen P A, Yu B, Zaslavsky L, Zhang J, Bolton E E. 2021. PubChem in 2021: new data content and improved web interfaces. Nucleic Acids Research, 49, D1388–D1395.

Kombrink A, Thomma B P H J. 2013. LysM effectors: secreted proteins supporting fungal life. PLoS Pathogens, 9, e1003769.

Kombrink A, Rovenich H, Shi-Kunne X, Rojas-Padilla E, van den Berg G C M, Domazakis E, de Jonge R, Valkenburg D, Sánchez-Vallet A, Seidl M F, Thomma B P. 2017. Verticillium dahliae LysM effectors differentially contribute to virulence on plant hosts. Molecular Plant Pathology, 18, 596–608.

Lacomme C, Santa Cruz S. 1999. Bax-induced cell death in tobacco is similar to the hypersensitive response. Proceedings of the National Academy of Sciences, 96, 7956–7961.

Lee W S, Rudd J J, Hammond-Kosack K E, Kanyuka K. 2014. Mycosphaerella graminicola LysM effector-mediated stealth pathogenesis subverts recognition through both CERK1 and CEBiP homologues in wheat. Molecular Plant-Microbe Interactions®, 27, 236–243.

Liu Y, Yang X, Gan J, Chen S, Xiao Z X, Cao Y. 2022. CB-Dock2: improved protein–ligand blind docking by integrating cavity detection, docking and homologous template fitting. Nucleic Acids Research, 50, W159–W164.

Marshall R, Kombrink A, Motteram J, Loza-Reyes E, Lucas J, Hammond-Kosack K E, Thomma B P H J, Rudd J J. 2011. Analysis of two in planta expressed LysM effector homologs from the fungus Mycosphaerella graminicola reveals novel functional properties and varying contributions to virulence on wheat. Plant Physiology, 156, 756–769.

Martinez D A, Oliver B G, Gräser Y, Goldberg J M, Li W, Martinez-Rossi N M, Monod M, Shelest E, Barton R C, Birch E, Brakhage A A, Chen Z, Gurr S J, Heiman D, Heitman J, Kosti I, Rossi A, Saif S, Samalova M, Saunders C W, et al 2012. Comparative genome analysis of Trichophyton rubrum and related dermatophytes reveals candidate genes involved in infection. mBio, 3, e00259-12.

Mentlak T A, Kombrink A, Shinya T, Ryder L S, Otomo I, Saitoh H, Terauchi R, Nishizawa Y, Shibuya N, Thomma B P, Talbot N J. 2012. Effector-mediated suppression of chitin-triggered immunity by Magnaporthe oryzae is necessary for rice blast disease. Plant Cell, 24, 322–335.

Mistry J, Finn R D, Eddy S R, Bateman A, Punta M. 2013. Challenges in homology search: HMMER3 and convergent evolution of coiled-coil regions. Nucleic Acids Research, 41, e121–e121.

Mueller D S, Wise K A, Sisson A J, Allen T W, Bergstrom G C, Bosley D B, Bradley C A, Broders K D, Byamukama E, Chilvers M I, Collins A, Faske T R, Friskop A J, Heiniger R W, Hollier C A, Hooker D C, Isakeit T, Jackson-Ziems T A, Jardine D J, Kelly H M, et al. 2016. Corn yield loss estimates due to diseases in the United States and Ontario, Canada from 2012 to 2015. Plant Health Progress, 17, 211–222.

Oguiza J A. 2022. LysM proteins in mammalian fungal pathogens. Fungal Biology Reviews, 40, 114–122.

Price M N, Dehal P S, Arkin A P. FastTree 2--approximately maximum-likelihood trees for large alignments. PloS ONE 5, e9490.

Rossi R L D, Reis E M. 2014. Semi-selective culture medium for Exserohilum turcicum isolation from corn seeds. Summa Phytopathologica, 40, 163–167.

Sánchez-Vallet A, Mesters J R, Thomma B P. 2015. The battle for chitin recognition in plant-microbe interactions. FEMS Microbiology Reviews, 39, 171–183.

Sánchez-Vallet A, Saleem-Batcha R, Kombrink A, Hansen G, Valkenburg D J, Thomma B P H J, Mesters J R. 2013. Fungal effector Ecp6 outcompetes host immune receptor for chitin binding through intrachain LysM dimerization. eLife, 2, e00790.

Sánchez-Vallet A, Tian H, Rodriguez-Moreno L, Valkenburg D J, Saleem-Batcha R, Wawra S, Kombrink A, Verhage L, de Jonge R, van Esse H P, Zuccaro A, Croll D, Mesters J R, Thomma B P H J. 2020. A secreted LysM effector protects fungal hyphae through chitin-dependent homodimer polymerization. PLoS Pathogens, 16, e1008652.

Savary S, Willocquet L, Pethybridge S J, Esker P, McRoberts N, Nelson A. 2019. The global burden of pathogens and pests on major food crops. Nature Ecology & Evolution, 3 (3): 430–439.

Seidl-Seiboth V, Zach S, Frischmann A, Spadiut O, Dietzsch C, Herwig C, Ruth C, Rodler A, Jungbauer A, Kubicek C P. 2013. Spore germination of Trichoderma atroviride is inhibited by its LysM protein TAL6. The FEBS journal, 280, 1226–36.

Suarez-Fernandez M, Aragon-Perez A, Lopez-Llorca L V, Lopez-Moya F. 2021. Putative LysM effectors contribute to fungal lifestyle. International Journal of Molecular Sciences, 22, 3147.

Takahara H, Hacquard S, Kombrink A, Hughes H B, Halder V, Robin G P, Hiruma K, Neumann U, Shinya T, Kombrink E, Shibuya N, Thomma B P, O'Connell R J. 2016. Colletotrichum higginsianum extracellular LysM proteins play dual roles in appressorial function and suppression of chitin-triggered plant immunity. New Phytologist, 211, 1323–1337.

Tian H, Fiorin G L, Kombrink A, Mesters J R, Thomma B P H J. 2022. Fungal dual-domain LysM effectors undergo chitin-induced intermolecular, and not intramolecular, dimerization. Plant Physiology, 190, 2033–2044.

Tian H, MacKenzie C I, Rodriguez-Moreno L, van den Berg G C M, Chen H, Rudd J J, Mesters J R, Thomma B P H J. 2021. Three LysM effectors of Zymoseptoria tritici collectively disarm chitin-triggered plant immunity. Molecular Plant Pathology, 22, 683–693.

Xu D, Zhang Y. 2011. Improving the physical realism and structural accuracy of protein models by a two-step atomic-level energy minimization. Biophysical Journal, 101, 2525–2534.

Xu Q, Wang J, Zhao J, Xu J, Sun S, Zhang H, Wu J J, Tang C, Kang Z, Wang X. 2020. A polysaccharide deacetylase from Puccinia striiformis f. sp. tritici is an important pathogenicity gene that suppresses plant immunity. Plant Biotechnology Journal, 18, 1830-1842.

Yin W, Wang Y, Chen T, Lin Y, Luo C. 2018. Functional evaluation of the signal peptides of secreted proteins. Bio Protocol, 8, e2839.

Zhang H, Kim M S, Huang J, Yan H, Yang T, Song L, Yu W, Shim W B. 2022. Transcriptome analysis of maize pathogen Fusarium verticillioides revealed FvLcp1, a secreted protein with type-D fungal LysM and chitin-binding domains, that plays important roles in pathogenesis and mycotoxin production. Microbiological Research, 265, 127195.

Zhang H, Wen S H, Li P H, Lu L Y, Yang X, Zhang C J, Guo L Y, Wang D, Zhu X Q. 2024. LysM protein BdLM1 of Botryosphaeria dothidea plays an important role in full virulence and inhibits plant immunity by binding chitin and protecting hyphae from hydrolysis. Frontiers in Plant Science, 8, 1–14. 1320980.

Zhang X C, Cannon S B, Stacey G. 2009. Evolutionary genomics of LysM genes in land plants. BMC Evolutionary Biology, 9, 183.

Zhou X, Zheng W, Li Y, Pearce R, Zhang C, Bell E W, Zhang G, Zhang Y. 2022. I-TASSER-MTD: a deep-learning-based platform for multi-domain protein structure and function prediction. Nature Protocols, 17, 2326–2353.

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