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Journal of Integrative Agriculture
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TbNACα negatively regulates Trichoderma breve T069 synthesis of ethyl caffeate and enhances antagonism of Sclerotium rolfsii
Zhen Liu1, 2*, Ning Xu1*, Jumei Hou1, 2, Tong Liu1, 2#

1 Key Laboratory of Green Prevention and Control of Tropical Diseases and Pests, Hainan University, Haikou 570228, China

2 School of Breeding and Multiplication (Sanya Institute of Breeding and Multiplication), Hainan University, Sanya 572025, China

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摘要  木霉(Trichoderma spp.一种广泛用于防治各种植物病害的有益微生物。白绢病由齐整小核菌(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NACNascent 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TbNACαT. 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。TbNACαT. 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EC50107.15 μg·mL-1qPCR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) is involved in various biological functions in eukaryotes which have been extensively studied in animals and plants, but its role in the biocontrol action of microorganisms needs to be better understood. In this study, we investigated the function of TbNACα, one of the subunits of NAC, in the biocontrol activity of Trichoderma breve T069 against Sclerotium rolfsii. The TbNACα gene was deleted from T. breve T069, and the ΔTbNACα mutant showed significantly reduced mycelial growth, spore production, and spore germination. Additionally, volatile substances from ΔTbNACα had no significant effect on S. rolfsii, while non-volatile substances significantly inhibited the growth of S. rolfsii. Transcriptome sequencing results showed that compared to wild-type T069, the ΔTbNACα mutant had 3,398 differentially expressed genes, mainly regulating the expression of genes related to 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. Moreover, the metabolic substances of ethyl caffeate had the strongest activity against S. rolfsii, with an EC50 of 107.15 μg·mL-1. Quantitative Real-time PCR (qPCR) analysis revealed that genes involved in the ethyl caffeate synthesis pathway were significantly upregulated in ΔTbNACα strains. This study provides a basis for the negative regulation of ethyl caffeate synthesis and elucidates the antagonistic inhibition mechanism of TbNACα in T. breve T069.
Keywords:  Trichoderma breve              Sclerotium rolfsii              NACα              antagonism              metabolism              ethyl caffeate  
Online: 05 March 2024  
About author:  Zhen Liu, E-mail: liuzhenhenan@163.com; Ning Xu, E-mail: ningxu1998@163.com; #Correspondence Liu Tong, E-mail: liutongamy@sina.com *Both authors contributed equally to this paper.

Cite this article: 

Zhen Liu, Ning Xu, Jumei Hou, Tong Liu. 2024. TbNACα negatively regulates Trichoderma breve T069 synthesis of ethyl caffeate and enhances antagonism of Sclerotium rolfsii. Journal of Integrative Agriculture, Doi:10.1016/j.jia.2024.01.030

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