短梗木霉TbNACα负调控咖啡酸乙酯合成增强拮抗白绢病菌的研究

doi:10.1016/j.jia.2024.01.030

摘要/Abstract

摘要：

木霉（Trichoderma spp.）是一种广泛用于防治各种植物病害的有益微生物。白绢病是由齐整小核菌（Sclerotium rolfsii Sacc.）引起的植物病害，能够造成严重的经济损失。新生多肽相关复合物NAC（Nascent polypeptide-associated complex）对于维持蛋白质内稳态起关键作用，然而在生防微生物中的功能尚不明确。因此，本研究从Trichoderma breve T069拮抗白绢病菌出发，探究TbNACα在T. breve T069拮抗白绢病菌过程中的功能，分析TbNACα参与调控木霉次生代谢产物的作用机制。通过生物信息学对TbNACα进行保守结构域分析发现蛋白N端（49-115）含有NAC保守结构域，C端（178-214）含有UBA结构域，属于典型的NAC蛋白结构域。TbNACα在T. breve T069拮抗白绢病菌过程中显著下调表达，暗示TbNACα在木霉拮抗白绢病菌过程中可能起负调控作用。进一步对TbNACα功能分析发现ΔTbNACα突变体的菌丝生长速度受到抑制、产孢量减少、孢子萌发时间延长。值得注意的是，ΔTbNACα的非挥发性物质能显著抑制白绢病菌的生长，而回补突变体能够恢复上述功能，表明TbNACα可能参与调控非挥发性拮抗活性物质的合成。通过比较转录组分析筛选到3,398个差异表达基因，经过功能富集分析之后发现，TbNACα基因敲除后显著影响了短梗木霉的代谢过程，主要调控次生代谢物生物合成酶、水解酶和膜转运蛋白相关基因的表达。另外，利用UHPLC-OE-MS技术对ΔTbNACα关键代谢物质鉴定，发现ΔTbNACα正离子模式（POS）共有27个上调代谢物；负离子模式（NEG）共有23个上调代谢物，对其中6个显著差异代谢物质进行拮抗活性验证，发现咖啡酸乙酯对白绢病菌活性最强，其EC₅₀为107.15 μg·mL^-1。qPCR分析发现ΔTbNACα突变体中咖啡酸乙酯合成通路关键基因关显著上调表达。综上所述，TbNACα基因的缺失提高T. breve T069咖啡酸乙酯合成通路基因上调表达，促进咖啡酸乙酯的积累，增强ΔTbNACα突变体拮抗白绢病菌的能力。本研究明确TbNACα基因负调控咖啡酸乙酯合成，揭示了木霉拮抗白绢病菌的新机制。

Abstract:

The nascent polypeptide-associated complex (NAC) plays crucial roles in various biological functions in eukaryotes and has been extensively studied in animals and plants; however, its role in the biocontrol mechanisms of microorganisms requires further investigation. This study examined the function of TbNACα, a NAC subunit, in the biocontrol activity of Trichoderma breve T069 against Sclerotium rolfsii. Following deletion of the TbNACα gene from T. breve T069, the ΔTbNACα mutant exhibited significantly reduced mycelial growth, spore production, and spore germination. While volatile substances from ΔTbNACα showed no significant effect on S. rolfsii, non-volatile substances demonstrated significant inhibition of S. rolfsii growth. Transcriptome sequencing analysis revealed 3,398 differentially expressed genes in the ΔTbNACα mutant compared to wild-type T069, primarily regulating genes associated with secondary metabolite biosynthetic enzymes, hydrolases, and membrane transport proteins. Untargeted metabolomics identified 50 upregulated metabolites (27 in positive ion mode and 23 in negative ion mode) in crude extracts from ΔTbNACα mutant metabolite broth. Among these metabolic substances, ethyl caffeate demonstrated the strongest activity against S. rolfsii, with an EC₅₀ of 107.15 μg mL^–1. Quantitative real-time PCR (qPCR) analysis indicated significant upregulation of genes involved in the ethyl caffeate synthesis pathway in ΔTbNACα strains. This research establishes the negative regulation of ethyl caffeate synthesis and elucidates the antagonistic inhibition mechanism of TbNACα in T. breve T069.

Key words: Trichoderma breve , Sclerotium rolfsii , NACα , antagonism , metabolism , ethyl caffeate

Zhen Liu, Ning Xu, Jumei Hou, Tong Liu. 短梗木霉TbNACα负调控咖啡酸乙酯合成增强拮抗白绢病菌的研究[J]. Journal of Integrative Agriculture, 2025, 24(11): 4324-4341.

Zhen Liu, Ning Xu, Jumei Hou, Tong Liu. TbNACα negatively regulates Trichoderma breve T069 synthesis of ethyl caffeate and enhances antagonism of Sclerotium rolfsii[J]. Journal of Integrative Agriculture, 2025, 24(11): 4324-4341.

参考文献

Bae Y, Knudsen G R. 2000. Cotransformation of Trichoderma harzianum with β-glucuronidase and green fluorescent protein genes provides a useful tool for monitoring fungal growth and activity in natural soils. Applied and Environmental Microbiology, 66, 810–815.

Bansal R, Mukherjee P K. 2016. Identification of novel gene clusters for secondary metabolism in Trichoderma genomes. Microbiology, 85, 185–190.

Bello F, Montironi I D, Medina M B, Munitz M S, Ferreira F V, Williman C, Vázquez D, Cariddi L N, Musumeci M A. 2022. Mycofumigation of postharvest blueberries with volatile compounds from Trichoderma atroviride IC-11 is a promising tool to control rots caused by Botrytis cinerea. Food Microbiology, 106, 104040.

Boregowda N, Jogigowda S C, Bhavya G, Sunilkumar C R, Geetha N, Udikeri S S, Chowdappa S, Govarthanan M, Jogaiah S. 2022. Recent advances in nanoremediation: Carving sustainable solution to clean-up polluted agriculture soils. Environmental Pollution, 297, 118728.

Coppola M, Diretto G, Digilio M C, Woo S L, Giuliano G, Molisso D, Pennacchio F, Lorito M, Rao R. 2019. Transcriptome and metabolome reprogramming in tomato plants by Trichoderma harzianum strain T22 primes and enhances defense responses against aphids. Frontiers in Physiology, 10, 745.

Dahal D, Pich A, Braun H P, Wydra K. 2010. Analysis of cell wall proteins regulated in stem of susceptible and resistant tomato species after inoculation with Ralstonia solanacearum: A proteomic approach. Plant Molecular Biology, 73, 643–658.

Ding J, Mei J, Huang P, Tian Y, Liang Y, Jiang X, Li M. 2020. Gα3 subunit Thga3 positively regulates conidiation, mycoparasitism, chitinase activity, and hydrophobicity of Trichoderma harzianum. AMB Express, 10, 1–9.

Ding Y, Jia Y, Shi Y, Zhang X, Song C, Gong Z, Yang S. 2018. OST1‐mediated BTF3L phosphorylation positively regulates CBFs during plant cold responses. The EMBO Journal, 37, e98228.

Dwivedi S K, Prasad G. 2016. Integrated management of Sclerotium rolfsii: An overview. European Journal of Biomedical and Pharmaceutical Sciences, 3, 137–146.

George R, Walsh P, Beddoe T, Lithgow T. 2002. The nascent polypeptide-associated complex (NAC) promotes interaction of ribosomes with the mitochondrial surface in vivo. FEBS Letters, 516, 213–216.

Hussien R A, Gnedy M M, Sayed A A, Bondok A, Alkhalifah D H M, Elkelish A, Tawfik M M. 2022. Evaluation of the fungicidal effect of some commercial disinfectant and sterilizer agents formulated as soluble liquid against Sclerotium rolfsii infected tomato plant. Plants, 11, 3542.

Jacob S, Sajjalaguddam R R, Sudini H K. 2018. Streptomyces sp. RP1A-12 mediated control of peanut stem rot caused by Sclerotium rolfsii. Journal of Integrative Agriculture, 17, 892–900.

Jomaa A, Gamerdinger M, Hsieh H, Wallisch A, Chandrasekaran V, Ulusoy Z, Scaiola A, Hegde R S, Shan S, Ban N. 2022. Mechanism of signal sequence handover from NAC to SRP on ribosomes during ER-protein targeting. Science, 375, 839–844.

Kaido M, Inoue Y, Takeda Y, Sugiyama K, Takeda A, Mori M, Tamai A, Meshi T, Okuno T, Mise K. 2007. Downregulation of the NbNACa1 gene encoding a movement-protein-interacting protein reduces cell-to-cell movement of Brome mosaic virus in Nicotiana benthamiana. Molecular Plant–Microbe Interactions, 20, 671–681.

Kator L, Hosea Z Y, Oche O D. 2015. Sclerotium rolfsii: Causative organism of southern blight, stem rot, white mold and sclerotia rot disease. Annals of Biological Research, 6, 78–89.

Keller N P. 2019. Fungal secondary metabolism: Regulation, function and drug discovery. Nature Reviews Microbiology, 17, 167–180.

Khan R A A, Najeeb S, Hussain S, Xie B, Li Y. 2020. Bioactive secondary metabolites from Trichoderma spp. against phytopathogenic fungi. Microorganisms, 8, 817.

Kirstein Miles J, Scior A, Deuerling E, Morimoto R I. 2013. The nascent polypeptide‐associated complex is a key regulator of proteostasis. The EMBO Journal, 32, 1451–1468.

Kogan G L, Gvozdev V A. 2014. Multifunctional nascent polypeptide-associated complex (NAC). Molecular Biology, 48, 189–196.

Kong W, Ni H, Wang W, Wu X. 2022. Antifungal effects of volatile organic compounds produced by Trichoderma koningiopsis T2 against Verticillium dahliae. Frontiers in Microbiology, 13, 1013468.

Kubicek C P, Herrera-Estrella A, Seidl-Seiboth V, Martinez D A, Druzhinina I S, Thon M, Zeilinger S, Casas-Flores S, Horwitz B A, Mukherjee P K. 2011. Comparative genome sequence analysis underscores mycoparasitism as the ancestral life style of Trichoderma. Genome Biology, 12, R40.

Kubicek C P, Steindorff A S, Chenthamara K, Manganiello G, Henrissat B, Zhang J, Cai F, Kopchinskiy A G, Kubicek E M, Kuo A. 2019. Evolution and comparative genomics of the most common Trichoderma species. BMC Genomics, 20, 485.

Lee S, Kim H, Ahn J. 2021. Biosynthesis of ethyl caffeate via caffeoyl-CoA acyltransferase expression in Escherichia coli. Applied Biological Chemistry, 64, 1–6.

Li N, Alfiky A, Wang W, Islam M, Nourollahi K, Liu X, Kang S. 2018. Volatile compound-mediated recognition and inhibition between Trichoderma biocontrol agents and Fusarium oxysporum. Frontiers in Microbiology, 9, 2614.

Li X, Guo M, Xu D, Chen F, Zhang H, Pan Y, Li M, Gao Z. 2015. The nascent-polypeptide-associated complex alpha subunit regulates the polygalacturonases expression negatively and influences the pathogenicity of Sclerotinia sclerotiorum. Mycologia, 107, 1130–1137.

Liu Z, Xu N, Pang Q, Khan R A A, Xu Q, Wu C, Liu T. 2023. A salt-tolerant strain of Trichoderma longibrachiatum HL167 is effective in alleviating salt stress, promoting plant growth, and managing fusarium wilt disease in cowpea. Journal of Fungi, 9, 304.

Malmierca M G, Mccormick S P, Cardoza R E, Alexander N J, Monte E, Gutiérrez S. 2015. Production of trichodiene by Trichoderma harzianum alters the perception of this biocontrol strain by plants and antagonized fungi. Environmental Microbiology, 17, 2628–2646.

Moya P, Girotti J R, Toledo A V, Sisterna M N. 2018. Antifungal activity of Trichoderma VOCs against Pyrenophora teres, the causal agent of barley net blotch. Journal of Plant Protection Research, 58, 45–53.

Mukherjee P K, Buensanteai N, Moran-Diez M E, Druzhinina I S, Kenerley C M. 2012. Functional analysis of non-ribosomal peptide synthetases (NRPSs) in Trichoderma virens reveals a polyketide synthase (PKS)/NRPS hybrid enzyme involved in the induced systemic resistance response in maize. Microbiology, 158, 155–165.

Mukhopadhyay R, Kumar D. 2020. Trichoderma: A beneficial antifungal agent and insights into its mechanism of biocontrol potential. Egyptian Journal of Biological Pest Control, 30, 133.

Ott A, Locher L, Koch M, Deuerling E. 2015. Functional dissection of the nascent polypeptide-associated complex in Saccharomyces cerevisiae. PLoS ONE, 10, e143457.

Rajani P, Aiswarya H, Vasanthakumari M M, Jain S K, Bharate S B, Rajasekaran C, Ravikanth G, Uma Shaanker R. 2019. Inhibition of the collar rot fungus, Sclerotium rolfsii Sacc. by an endophytic fungus Alternaria sp.: Implications for biocontrol. Plant Physiology Reports, 24, 521–532.

Rodrigues A O, May De Mio L L, Soccol C R. 2023. Trichoderma as a powerful fungal disease control agent for a more sustainable and healthy agriculture: Recent studies and molecular insights. Planta, 257, 31.

Ruangwong O, Wonglom P, Suwannarach N, Kumla J, Thaochan N, Chomnunti P, Pitija K, Sunpapao A. 2021. Volatile organic compound from Trichoderma asperelloides TSU1: Impact on plant pathogenic fungi. Journal of Fungi, 7, 187.

Sardi J D C O, Gullo F P, Freires I A, de Souza Pitangui N, Segalla M P, Fusco-Almeida A M, Rosalen P L, Regasini L O, Mendes-Giannini M J S. 2016. Synthesis, antifungal activity of caffeic acid derivative esters, and their synergism with fluconazole and nystatin against Candida spp. Diagnostic Microbiology and Infectious Disease, 86, 387–391.

Shenouda M L, Ambilika M, Cox R J. 2021. Trichoderma reesei contains a biosynthetic gene cluster that encodes the antifungal agent ilicicolin H. Journal of Fungi, 7, 1034.

Silva-Campos M, Callahan D L, Cahill D M. 2022. Metabolites derived from fungi and bacteria suppress in vitro growth of Gnomoniopsis smithogilvyi, a major threat to the global chestnut industry. Metabolomics, 18, 74.

Sridharan A P, Thankappan S, Karthikeyan G, Uthandi S. 2020. Comprehensive profiling of the VOCs of Trichoderma longibrachiatum EF5 while interacting with Sclerotium rolfsii and Macrophomina phaseolina. Microbiological Research, 236, 126436.

Stracquadanio C, Quiles J M, Meca G, Cacciola S O. 2020. Antifungal activity of bioactive metabolites produced by Trichoderma asperellum and Trichoderma atroviride in liquid medium. Journal of Fungi, 6, 263.

Tamizi A, Mat-Amin N, Weaver J A, Olumakaiye R T, Akbar M A, Jin S, Bunawan H, Alberti F. 2022. Genome sequencing and analysis of Trichoderma (Hypocreaceae) isolates exhibiting antagonistic activity against the papaya dieback pathogen. Erwinia mallotivora. Journal of Fungi, 8, 246.

Tarekegn G, Sakhuja P K, Swart W J, Tamado T. 2007. Integrated management of groundnut root rot using seed quality and fungicide seed treatment. International Journal of Pest Management, 53, 53–57.

Vicente I, Baroncelli R, Hermosa R, Monte E, Vannacci G, Sarrocco S. 2022. Role and genetic basis of specialised secondary metabolites in Trichoderma ecophysiology. Fungal Biology Reviews, 39, 83–99.

Wang P, Wu P, Huang R, Chung K. 2020. The role of a nascent polypeptide-associated complex subunit alpha in siderophore biosynthesis, oxidative stress response, and virulence in Alternaria alternata. Molecular Plant–Microbe Interactions, 33, 668–679.

Wang T, Pan M, Xiao N, Wu J, Wang Q, Cheng T, Yan G, Wu D, Li N, Shao J. 2021. In vitro and in vivo analysis of monotherapy and dual therapy with ethyl caffeate and fluconazole on virulence factors of Candida albicans and systemic candidiasis. Journal of Global Antimicrobial Resistance, 27, 253–266.

Wang X, Xie X, Liu J, Wang G, Qiu D. 2020. Nascent polypeptide-associated complex involved in the development and pathogenesis of Fusarium graminearum on Wheat. Engineering, 6, 546–552.

Yan L, Wang Z, Song W, Fan P, Kang Y, Lei Y, Wan L, Huai D, Chen Y, Wang X. 2021. Genome sequencing and comparative genomic analysis of highly and weakly aggressive strains of Sclerotium rolfsii, the causal agent of peanut stem rot. BMC Genomics, 22, 1–15.

Yan S, Tang Z, Su W, Sun W. 2005. Proteomic analysis of salt stress‐responsive proteins in rice root. Proteomics, 5, 235–244.

Yang K, Kim H, Jin U, Lee S S, Park J, Lim Y P, Pai H. 2007. Silencing of NbBTF3 results in developmental defects and disturbed gene expression in chloroplasts and mitochondria of higher plants. Planta, 225, 1459–1469.

Zeilinger S, Gruber S, Bansal R, Mukherjee P K. 2016. Secondary metabolism in Trichoderma - Chemistry meets genomics. Fungal Biology Reviews, 30, 74–90.

Zhang M, Davie J. 2015. Deregulation of NAC complex inhibits muscle differentiation and blocks apoptosis in rhabdomyosarcoma cells. Cancer Research, 75, 28.

Zhang Y, Zhuang W. 2022. MAPK Cascades mediating biocontrol activity of Trichoderma brevicrassum strain TC967. Journal of Agricultural and Food Chemistry, 70, 2762–2775.

Zin N A, Badaluddin N A. 2020. Biological functions of Trichoderma spp. for agriculture applications. Annals of Agricultural Sciences, 65, 168–178.