Abstract Fusarium graminearum (F. graminearum) and its derivative mycotoxin deoxynivalenol (DON) are highly concerned with food safety and sustainability worldwide. Although several transcription factors (TFs) had been elucidated, molecular mechanism participates in DON biosynthesis regulation remains largely unrevealed. Here, we first characterized a zinc finger-contained TF in F. graminearum, FgSfp1, which is indispensable for DON production since its depletion resulting in a 95.4% DON yielding reduction. Interestingly, contrast to previous knowledge, all TRI-cluster genes were abnormally upregulated in ΔFgSfp1 while Tri proteins abundance rationally decreased simultaneously. Further evidence show FgSfp1 could coordinate genetic translation pace by manipulating ribosomal biogenesis process. Specifically, FgSfp1-depletion leads to ribosome biogenesis assembly factor (RiBi) expression attenuation along with DON precursor acetyl-CoA synthase reduction since FgSfp1 actively interacts with RNA 2’-O-methylation enzyme FgNop1 revealed by Bi-FC. It subsequently influences mRNA translation pace. In conclusion, we elucidated that the FgSfp1 orchestrates DON biosynthesis via participating RNA posttranscriptional modification for ribosomal RNA maturation, offering insights into the DON biosynthesis regulation. Ultimately, this TF might be a key regulator for DON contamination control in the whole food chain. Subject terms: Fungal biology, Pathogens __________________________________________________________________ Inconsistency between transcriptional and translational in secondary metabolite biosynthesis sheds light on the role of transcription factor FgSfp1 under nutrient-stress condition in filamentous fungi Fusarium graminearum. Introduction A principal concern in the food industry worldwide is the presence of plant pathogens. A prominent example being F. graminearum, that frequently causes devastating Fusarium head blight (FHB) disease in cereal crops, thereby leading to a decrease in crop quality and contamination of food with a variety of mycotoxins^[36]1–[37]3. Mycotoxins, as a group of fungal-derived secondary metabolites, pose significant challenges to food security and safety globally^[38]4. Deoxynivalenol (DON) is a type B trichothecene mycotoxin ubiquitously produced by F. graminearum and other Fusarium genera; it is the most highly abundant mycotoxin detected in the major crops throughout the world and hence causes serious economic problems^[39]5–[40]7. The consumption of DON not only leads to devastating crop mycotoxin contamination, but also has short-term and long-term toxic effects on animals and humans^[41]8,[42]9. Considered the negative impact DON imparts on higher organisms, it is imperative to decipher the enzymatic mechanisms and regulation of the DON biosynthesis pathway. Previous studies have defined a fundamental scheme of this intricate catalyzation process^[43]10,[44]11. Genes that encode DON biosynthetic enzymes form three unique clusters that are distributed on different parts of the F. graminearum genome. The core TRI gene cluster is located at chromosome II, the TRI1 and TRI16 cluster is embedded in chromosome I and the single gene TRI101 locus exists on chromosome IV. The DON biosynthesis process begins with the precursor molecule farnesyl pyrophosphate (FPP), which is derived from acetyl-CoA via the mevalonate pathway^[45]12–[46]14. Through the catalytic processes executed by enzymes encoded by TRI genes, DON is finally generated in the cytosol of F. graminearum^[47]1,[48]10. The DON biosynthesis process is governed by various of environmental factors. Pathogens can adjust their gene expression program in response to adverse living conditions, like improper growth pH, nutrient deficiencies, or competition with other organisms within their ecological niche^[49]15. Transcription factors (TFs) are a group of proteins that can recognize and bind specific DNA sequences to promote the downstream genetic transcription and elongation process and ultimately complete DNA sequence decoding. Previous work has shown that most TFs in F. graminearum contain a DNA binding domain (DBD). Examples include Zn (II)[2]Cys[6], C2H2 zinc finger, GATA, bHLH, and B-ZIP^[50]16,[51]17. These crucial TFs have been deeply investigated, especially for their role in regulating TRI genes. For instance, the C2H2 zinc finger TF Tri6, located inside the TRI-cluster gene region, has been identified as a crucial regulator of TRI-cluster gene expression, binding to their DNA promoters at the TNAGGCCT motif sequence^[52]18. Moreover, recent studies reported pathogens like F. graminearum require finely tuned systems that enable them to sense environmental conditions switch and identify potential threats to adjust their gene expression pattern. For instance, sterol biosynthesis controls TF FgSR^[53]19. The nitrogen resource-sensing TF FgAreA^[54]20 and FgAreB^[55]21, pH-dependent TF FgPacC30^[56]22. Considering the complex composition of TFs and the regulatory complexity of the gene expression pattern under different environmental conditions, it is crucial to understand the mechanisms underlying DON biosynthesis regulation mediated by various TFs. Current studies have successfully established several genetic strategies to confer wheat trichothecenes detoxifying traits by transferring glutathione S-transferase (GST) genes, such as Fhb7 and Fhb1, cloned from Triticeae E genome^[57]23,[58]24. Another perspective in managing DON contamination is uncovering the internal DON biosynthesis regulatory mechanisms to identify potential TFs responsible for DON production. Hopefully, investigation of DON biosynthesis regulatory mechanisms could help in developing feasible solutions to eliminate food threats from mycotoxins produced by pathogens. Although casual association between environmental factors affecting F. graminearum metabolism pattern switch by TFs and mycotoxin DON biosynthesis have been established over the past decade, mechanisms behind this complicated relationship mostly unrevealed. The target-of-rapamycin (TOR) protein is vastly existed and highly conserved in both eukaryotes and prokaryotes. TOR signaling is a nutrient-sensing pathway that regulates cell growth by orchestrating several anabolic processes, namely via ribosome biogenesis, translation initiation^[59]25–[60]27, and nutrient import^[61]28,[62]29, allowing cells adapting to environment alternation. It has been proposed that TOR inhibition in cells could be an effective strategy to extend lifespan when extracellular growth conditions are suboptimal. The regulation of TOR signaling pathway in relation to mycotoxin biosynthesis in F. graminearum is poorly poorly understood. A direct effector of FgTOR (locus tag FGSG_08133) is FgSch9 (locus tag FGSG_00472). Notably, deletion of FgTOR in F. graminearum leads to DON deficiency, however, the mechanism(s) underpinning this phenomenon is unknown^[63]30,[64]31. Therefore, identifying the downstream effectors or transcription factors (TFs) that are governed by FgTOR could provide insight into how DON levels are regulated in F. graminearum. Previous studies have successfully constructed a TF mutant library encompassing 600 putative TFs in F. graminearum that are related to multiple morphological observations including hyphae growth, mycotoxin production, pathogenesis, and stress responses. Interestingly, among the reported TFs, we noticed that a gene (locus tag FGSG_11799) that is predicted to contain two C2H2 zinc finger domains, and its deletion leads to a reduction in DON production. BLASTp analysis revealed that this gene is the counterpart of Sfp1 (Split-finger protein 1) in Saccharomyces cerevisiae, which is considered as a downstream TF of the TOR signaling pathway in response to extracellular stresses^[65]32–[66]34. Previous studies depicted that Sfp1 forms a complex with other TFs, binding to promoter regions of ribosomal protein genes to control ribosome biosynthesis^[67]35,[68]36. However, it is unknown whether there is a link between the regulatory activity of FGSG_11799 and the TRI-cluster genes involved in DON biosynthesis in F. graminearum. Furthermore, the mechanism underpinning the decision-making process fungus undergoes when confronted with adverse living environment, as well as its unique pattern of balancing growth rate and controlling secondary metabolism also remains undefined. Hence in the current study, we investigated the role of the identified FGSG_11799 gene, i) to examine the DON production capability and morphological characteristics of this mutant strain, the phenotypes with the significantly dramatic DON reduction well confirmed. ii) to perform a series of combinatorial approaches, such as complement strain construction, omics-analysis, and interaction network screening. The aim being to identify to uncover the key downstream effectors responsible for DON biosynthesis and elucidate the regulatory mechanisms of upstream signaling pathways related to TRI-cluster genes at both transcriptional and translational levels. iii) Based on the experimental data obtained, establish a hypothetical mechanism illustrating how the FGSG_11799 protein functions as a downstream effector orchestrating DON biosynthesis and anabolic activities in F. graminearum. In total, obtaining a transcription factor would be well helpful for profound understanding of DON biosynthesis in F. graminearum, supporting with the valuable references for DON control