Citric acid impairs type B trichothecene biosynthesis of Fusarium graminearum but enhances its growth and pigment biosynthesis: transcriptomic and proteomic analyses

Abstract

Fusarium graminearum is a pathogenic fungus that causes devastating plant diseases such as Fusarium head blight, leading to significant food waste and posing a threat to human health due to the accumulation of mycotoxins. As a soil-borne fungus, F. graminearum interacts closely with plants and soil, completing its life cycle in an intricate environment. In this investigation, citric acid (CA), a plant root exudate and heavy metal chelator, was investigated for its effect on F. graminearum at concentrations of 2.5, 5, 10, and 20 mM. The fungus treated with this CA gradient exhibited different growth conditions and mycelial colors. Integrative analysis of transcriptomics and proteomics found that the growth of the fungus was accelerated by the upregulation of carbon source metabolism-related enzymes such as phosphoenolpyruvate carboxykinase (PEPCK), Glpk, and LAI12, and the mycelial pigments were altered by polyketide biosynthetic enzymes like AurF, AurJ, and AurT, which were responsible for the production of aurofusarin and rubrofusarin. Interestingly, many Tri genes and their corresponding proteins along with type B trichothecene biosynthesis were significantly downregulated, though the fungal growth was promoted. In this study, the CK, 2.5, and 5 mM groups had similar expression patterns of RNA and protein, while the 10 and 20 mM groups were alike. With the validation of CK, 5, and 10 mM groups, the discovery of CA as a type B trichothecene biosynthesis inhibitor and growth promoter was expected to facilitate its reasonable application and contribute to further research for the prevention of F. graminearum.

Importance: Fusarium graminearum is a challenging phytopathogen that causes plant disasters worldwide, such as Fusarium head blight and ear rot in small grain crops like wheat and maize. Besides, the invasion of the fungus on plants is often accompanied by the production of health-threatening mycotoxins-trichothecenes. Therefore, the control of Fusarium graminearum has always been a hot area of research. However, currently, the prevalence of these plant diseases around the world and the mycotoxin accumulation beyond safety limits indicate the necessity for more related research. In the last decades, researchers have sought to identify the key substances associated with the growth, propagation, and mycotoxin production of the fungus, such as carbon and nitrogen sources. Organic acids have always been considered antifungal agents due to their abundant H+ content. However, their comprehensive impact on fungi has been rarely investigated. This research focused on the effect of citric acid, a type of crop exudate and a common heavy-metal chelator, on the metabolism of Fusarium graminearum. The result was expected to provide a theoretical basis for further studies on plants-soil-fungi interactions and the reasonable utilization of citric acid in agriculture.

Keywords: F. graminearum; citric acid; proteomic; transcriptomic; trichothecene.

Fig 1

Morphology of F. graminearum grown on TBI solid medium. V, vertical view; L, lateral view.

Fig 2

(A) Venn plots of mutual and different genes among the five groups. (B) Venn plots of mutual and different proteins among the five groups. (C) Principal component analysis of the transcriptomes among the five groups. (D) Principal component analysis of the proteomes among the five groups.

Fig 3

KEGG enrichment analyses of differentially expressed genes between (A) 2.5 mM and CK, (B) 5 mM and CK, (C) 10 mM and CK, and (D) 20 mM and CK.

Fig 4

KEGG enrichment analyses of differentially expressed proteins between (A) 2.5 mM and CK, (B) 5 mM and CK, (C) 10 mM and CK, and (D) 20 mM and CK.

Fig 5

Association analysis of transcriptome and proteome. (A) Venn plot of genes and proteins detected. (B) Cluster analysis among five groups. (C) Correlation analysis between proteins and genes. (D) Differentially expressed genes and proteins. NDEPs/NDEGs: not differentially expressed genes or proteins.

Fig 6

Schematic diagram of type B trichothecene biosynthesis, along with the RNA-seq and RT-qPCR results of several genes required for important steps.

Fig 7

RT-qPCR validation. (A) RNA-seq and RT-qPCR results for Tri6, Tri10, and Tri12 of the 5 and 10 mM groups. (B) Fit curve of RNA-seq and RT-qPCR for 11 Tri genes of the 5 and 10 mM groups.

Fig 8

PRM validation of selected proteins related to growth, pigment biosynthesis, and mycotoxin biosynthesis. *P < 0.05.