Mitochondrial protein FgDML1 impacts DON toxin biosynthesis and cyazofamid sensitivity in Fusarium graminearum by affecting mitochondrial homeostasis

Reviewer #2 (Public review):

Summary:

The manuscript entitled "Mitochondrial Protein FgDML1 Regulates DON Toxin Biosynthesis and Cyazofamid Sensitivity in Fusarium graminearum by affecting mitochondrial homeostasis" identified the regulatory effect of FgDML1 in DON toxin biosynthesis and sensitivity of Fusarium graminearum to cyazofamid. The manuscript provides a theoretical framework for understanding the regulatory mechanisms of DON toxin biosynthesis in F. graminearum and identifies potential molecular targets for Fusarium head blight control.

Comments on revised version:

I have no further comments on the revision.

Author response:

The following is the authors’ response to the previous reviews

Public Reviews:

Reviewer #1 (Public review):

Summary:

In their study the authors investigated the F. graminearum homologue of the Drosophila Misato-Like Protein DML1 for a function in secondary metabolism and sensitivity to fungicides.

Strengths:

Generally, the topic of the study is interesting and timely and the manuscript is well written, albeit in some cases details on methods or controls are missing.

Weaknesses:

However, a major problem I see is with the core result of the study, the decrease of the DON content associated with deletion of FgDML1: Although some growth data are shown in figure 6 - indicating a severe growth defect - the DON production presented in figure 3 is not related to biomass. Also, the method and conditions for measuring DON are not described. Consequently, it could well be concluded that the decreased amount of DON detected is simply due to a decreased growth and specific DON production of the mutant remains more or less the same.

To alleviate this concern, it is crucial to show the details on the DON measurement and growth conditions and to relate the biomass formation on the same conditions to the DON amount detected. Only then a conclusion as to an altered production in the mutant strains can be drawn.

We appreciate it very much that you spent much time on my paper and give me good suggestions, we tried our best to revise the manuscript. The point to point responds to the reviewer’s comments are listed as following.

Comments to the revised manuscript:

The authors carefully revised the manuscript and provided explanations for methods in several cases. However, there are still some problems - probably due to misunderstanding - that need revision.

(1) A major problem of the first version of the manuscript was the lack of appropriate description of biomass analysis and the consideration of the respective results for evaluation of production of DON and other metabolites. Although the authors provide some explanation in the response to reviews, I could not find a corresponding explanation or description in the manuscript. It is not sufficient to explain the problem to me, but a detailed explanation and description of the method has to be provided in the manuscript along with the definition of one "unit of mycelium". It is still not entirely clear to me what such a "unit of mycelium" is.

Please clarify this and any other uncertainties that were commented on by me and other reviewers in the manuscript, not only in the response to reviews. Also adjust the reference list accordingly.

Thank you very much for your advice. We appreciate the reviewer’s continued attention to the potential impact of biomass differences on DON production, particularly in light of the reduced growth rate observed in the mutant strain.

We acknowledge that the mutant exhibits slower growth compared to the wild-type strain. However, it is important to emphasize that the reduction in DON levels reported in this study cannot be attributed to decreased fungal biomass. In our experimental design, DON production was normalized to mycelial dry weight, and toxin levels are expressed as μg DON per g dry mycelium. Therefore, differences in total mycelial accumulation among strains were explicitly accounted for and eliminated during data analysis.

By expressing DON production on a per-unit-biomass basis, the measured values reflect the intrinsic DON biosynthetic capacity of the mycelium rather than the overall growth rate or total biomass. Consequently, the observed reduction in DON content in the mutant indicates a genuine impairment in DON biosynthesis per unit of fungal biomass, rather than a secondary effect resulting from reduced mycelial growth.

To avoid ambiguity, we have clarified this point in the revised manuscript by explicitly stating the normalization strategy and the definition of the mycelial unit in the Materials and Methods section, and by emphasizing in the Results/Discussion section that DON levels were compared on a biomass-normalized basis.

We hope that this clarification adequately addresses the reviewer’s concern and clearly distinguishes growth-related effects from alterations in toxin biosynthesis.

“DON toxin was measured using a Wise Science ELISA-based kit (Wise Science, Jiangsu, China) (Li et al., 2019; Zheng et al., 2018). Under toxin-producing conditions (28 °C, 145 rpm), fungal strains were cultured in TBI medium for 7 days. Cultures were initiated using freshly grown mycelia. After incubation, mycelia and culture filtrates were separated by filtration. The culture filtrates were collected for DON determination, while the mycelia were harvested for biomass analysis. The collected mycelia were washed with sterile distilled water and dried at 60 °C to constant weight. The dry weight of mycelia was recorded and used for normalization of DON production. One mycelial unit was defined as 1 g of dry mycelial biomass. DON concentration in the culture filtrates was quantified using an enzyme-linked immunosorbent assay (ELISA). Briefly, 50 μL of culture filtrate or DON standard solution was added to wells of a 96-well microplate pre-coated with DON antigen, followed by the addition of enzyme conjugate and antibody working solution according to the manufacturer’s instructions. After incubation and washing, color development was achieved using substrate solution and terminated by stop solution. Absorbance was measured at 450 nm using a microplate reader. A standard curve was generated using log₁₀-transformed DON concentrations of the standards and the corresponding percentage absorbance values. DON concentrations in the samples were calculated based on the standard curve. Total DON production was calculated according to the culture volume (30 mL) and subsequently normalized to mycelial dry weight. DON production was expressed as μg DON per g dry mycelium. Each treatment group contains three biological replicates and three technical replicates.”

(2) Another problem was, that the authors considered FgDML1 a regulator of DON production. As mentioned by me and reviewer 3, FgDML1 is crucial to numerous functions in F. graminearum and its lack causes a plethora of problems for fungal physiology. Hence, although it is clear that the lack of FgDML1 causes alterations in DON production, it is not appropriate to designate this factor as a "regulator".

It seems to me that the authors are afraid that if FgDML1 would not be a "regulator" that this would decrease the value of their study, which is not the case. This is a matter of correct wording. Therefore, please revise the wording accordingly, starting with the title:

...FgDML1 impacts DON toxin biosynthesis...

Moreover, for sure the manuscript might benefit from more detailed description of the whole cascade leading from FgDML1 to DON biosynthesis and production of the other metabolites that change upon deletion. Such explanation can help the reader grasp the relevance of FgDML for regulatory processes as well as on more general versus specific effects.

Thank you very much for your advice. We fully agree that, given the pleiotropic functions of FgDML1 in F. graminearum and the broad physiological defects caused by its deletion, it is not appropriate to designate FgDML1 as a direct or specific “regulator” of DON biosynthesis.

We acknowledge that the use of the term “regulator” in the previous version was imprecise. Following the reviewer’s suggestion, we have revised the wording throughout the manuscript to more accurately reflect the role of FgDML1. Specifically, we now describe FgDML1 as a factor that impacts or affects DON toxin biosynthesis rather than directly regulating it. The title has been revised accordingly to read:

“Mitochondrial protein FgDML1 impacts DON toxin biosynthesis and cyazofamid sensitivity in F. graminearum by affecting mitochondrial homeostasis”

Importantly, we would like to emphasize that our intention was not to overstate the specificity of FgDML1 in DON regulation, but rather to highlight its influence on secondary metabolism in the context of its broader biological functions. To address this more clearly, we have expanded the Discussion section to provide a more detailed and cautious interpretation of the potential cascade linking FgDML1 deletion to altered DON biosynthesis and changes in other metabolites.

'Secondary metabolite biosynthesis is generally regarded as an energy-intensive process that is tightly coupled to cellular energy metabolism. ATP serves as the primary energy currency supporting enzymatic reactions, macromolecule synthesis, and subcellular organization required for secondary metabolism. Disruption of ATP generation has been shown to directly impair toxin biosynthesis: for example, silencing of ATP synthase subunit α (AtpA) significantly reduces ATP synthesis and inhibits the production of the TcdA and TcdB toxins(Marreddy et al., 2024). Similarly, in plants, ATP depletion leads to a metabolic shift in which growth and basic physiological processes are prioritized at the expense of energetically costly secondary metabolites, including toxins(Xiao et al., 2024). Together, these findings highlight ATP availability as a key determinant of secondary metabolite production across biological systems.

In filamentous fungi, mitochondria play a central role in sustaining cellular ATP levels through oxidative phosphorylation and are therefore critical for biosynthetic and stress-adaptive processes. In F. graminearum, mutants defective in mitochondrial components, such as the voltage-dependent anion channel (mitochondrial porin), exhibit aberrant mitochondrial morphology, reduced ATP production, and markedly decreased DON accumulation and virulence (Han et al., 2022). These observations establish a direct link between mitochondrial energy metabolism and secondary metabolite output, supporting the notion that intact mitochondrial function and adequate ATP supply are prerequisites for robust DON production.

Consistent with this energy-dependent framework, biosynthesis of the mycotoxin DON in F. graminearum requires substantial ATP input. In the present study, ATP content in the ΔFgDML1 mutant was significantly lower than in the wild-type PH-1 and the complemented strain ΔFgDML1-C, and DON production was concomitantly reduced (Fig. 4A). Importantly, DON levels were normalized to mycelial dry weight, indicating that the observed reduction reflects a decreased biosynthetic capacity per unit biomass rather than a secondary consequence of reduced fungal growth. This distinction demonstrates that impaired DON production in the ΔFgDML1 mutant arises primarily from metabolic limitations.

At the cellular level, ATP depletion compromises multiple energy-dependent steps required for DON biosynthesis. The formation of toxisomes, which are specialized subcellular structures responsible for the spatial organization of DON biosynthetic enzymes, is essential for efficient mycotoxin production and is an ATP-dependent process. Reduced ATP levels disrupt toxisome assembly, and accordingly, the ΔFgDML1 mutant was unable to form functional toxisomes (Fig. 4C). In parallel, western blot analysis revealed a marked reduction in the abundance of the DON biosynthetic enzyme FgTri1 (Fig. 4D). In addition, ATP-dependent processes are directly involved in the biogenesis of the DON biosynthetic machinery: the ATPase activity of myosin I (FgMyo1) is required for efficient translation of key DON biosynthetic enzymes, and disruption of its ATPase function results in reduced DON production(Tang et al., 2018). These findings further underscore the dependence of DON biosynthesis on cellular energy status.

DON production is also regulated at the transcriptional level by the TRI gene cluster, with Tri5 and Tri6 serving as core components of the biosynthetic pathway. Tri5 encodes trichodiene synthase, which catalyzes the first committed step of DON biosynthesis. In the ΔFgDML1 mutant, expression levels of FgTri5 and FgTri6 were significantly downregulated (Fig. 4B), suggesting that impaired energy metabolism indirectly affects transcription of DON biosynthetic genes. Although no direct regulatory role of DML family proteins in gene expression has been reported in Saccharomyces cerevisiae or Drosophila melanogaster, their established functions in cell division and microtubule organization raise the possibility that FgDML1 indirectly influences gene expression through effects on chromatin organization or cell-cycle progression(Schulze and Wallrath, 2007).

In addition to reduced ATP levels, deletion of FgDML1 resulted in a significant decrease in acetyl-CoA content (Fig. 5C), a key precursor for trichothecene biosynthesis. Acetyl-CoA links central carbon metabolism with secondary metabolite production, and its depletion further constrains DON biosynthesis by limiting substrate availability. Broader metabolomic studies support this relationship, showing that perturbations in TCA cycle intermediates and central carbon metabolism are closely associated with altered DON production, reinforcing a mechanistic linkage between energy generation and toxin biosynthesis(Atanasova-Penichon et al., 2018).

“Taken together, these results support a model in which FgDML1 influences DON production indirectly by maintaining mitochondrial energy metabolism. Reduced ATP availability in the ΔFgDML1 mutant restricts energy-dependent biosynthetic processes, disrupts toxisome formation, diminishes DON biosynthetic enzyme abundance and gene expression, and limits precursor supply, ultimately leading to a substantial reduction in DON biosynthesis that is independent of fungal biomass effects.” (in L284-350). In this revised discussion, we explicitly distinguish between general physiological effects caused by the loss of FgDML1 and more specific consequences on secondary metabolic pathways.

We believe that this revised wording and the expanded mechanistic discussion more accurately reflect the biological role of FgDML1 and improve the conceptual clarity of the manuscript, without overstating its function as a dedicated regulator of DON production.

Reviewer #2 (Public review):

Summary:

The manuscript entitled "Mitochondrial Protein FgDML1 Regulates DON Toxin Biosynthesis and Cyazofamid Sensitivity in Fusarium graminearum by affecting mitochondrial homeostasis" identified the regulatory effect of FgDML1 in DON toxin biosynthesis and sensitivity of Fusarium graminearum to cyazofamid. The manuscript provides a theoretical framework for understanding the regulatory mechanisms of DON toxin biosynthesis in F. graminearum and identifies potential molecular targets for Fusarium head blight control. The paper in innovative, but there are issues in the writing that need to be added and corrected.

Comments on revisions:

The author has addressed my questions.

We appreciate it very much that you spent much time on my paper and give me good suggestions.