Fus3, as a Critical Kinase in MAPK Cascade, Regulates Aflatoxin Biosynthesis by Controlling the Substrate Supply in Aspergillus flavus, Rather than the Cluster Genes Modulation

ABSTRACT The Fus3-MAP kinase module is a conserved phosphorylation signal system in eukaryotes that responds to environmental stress and transduction of external signals from the outer membrane to the nucleus. Aspergillus flavus can produce aflatoxins (AF), which seriously threaten human and animal health. In this study, we determined the functions of Fus3, confirmed Ste50-Ste11-Ste7-Fus3 protein interactions and phosphorylation, and explored the possible phosphorylation motifs and potential targets of Fus3. The regulatory mechanism of Fus3 on the biosynthesis of AF was partly revealed in this study. AF production was downregulated in Δfus3, but the transcriptional expression of most AF cluster genes was upregulated. It is notable that the levels of acetyl-CoA and malonyl-CoA, the substrates of AF, were significantly decreased in fus3 defective strains. Genes involved in acetyl-CoA and malonyl-CoA biosynthesis were significantly downregulated at transcriptional or phosphorylation levels. Specifically, AccA might be a direct target of Fus3, which led to acetyl-CoA carboxylase activities were decreased in null-deletion and site mutagenesis strains. The results concluded that Fus3 could regulate the expression of acetyl-CoA and malonyl-CoA biosynthetic genes directly or indirectly, and then affect the AF production that relies on the regulation of AF substrate rather than the modulation of AF cluster genes. IMPORTANCE Aspergillus flavus is an important saprophytic fungus that produces aflatoxins (AF), which threaten food and feed safety. MAP (mitogen-activated protein) kanases are essential for fungal adaptation to diverse environments. Fus3, as the terminal kinase of a MAPK cascade, interacts with other MAPK modules and phosphorylates downstream targets. We provide evidence that Fus3 could affect AF biosynthesis by regulating the production of acetyl-CoA and malonyl-CoA, but this does not depend on the regulation of AF biosynthetic genes. Our results partly reveal the regulatory mechanism of Fus3 on AF biosynthesis and provide a novel AF modulation pattern, which may contribute to the discovery of new strategies in controlling A. flavus and AF contamination.

In this study, the authors determined MAPK pathway genes function, confirmed that Ste50-Ste11-Ste7-Fus3 protein interactions and phosphorylations, explored the possible phosphorylation motifs and potential targets of the terminal kinase Fus3, and illustrated Fus3 responding to diverse environment stresses in Aspergillus. They revealed the mechanism of Fus3 positive regulation on AFs biosynthesis. Δ fus3 mutant showed down-regulation of AFs production, but up-regulation of AFs cluster genes. The substrate of AFs, acetyl-CoA and malonyl-CoA, were significantly decreased in fus3 null-deletion and site-mutagenesis strains, and the genes involved in acetyl-CoA and malonyl-CoA biosynthesis, were significantly down-regulated at transcriptional or phosphorylational levels. This paper is a interesting topic, and the experiments were well designed. It is meaningful for readers in related fields. So my suggestion is acceptable after minor revision. Other comments were followed. 1) All the transcriptome and phosphoproteome data should be submitted to NCBI. 2) In Fig.4, Four mutants (T182A, T182D, T184A, T184D) showed almost the same phenotype, why? Please explain.
3) There are many important MAPK in Aspergillus flavus, why the author chose the Fus3, please explain in Introduction part. 4) Fig.3 showed the transcriptome and phosphoproteome results, but transcriptome only give small partial. Please provide more information on transcriptome. 5) Line 217, "But the expressions of AFs biosynthetic genes were no obvious decrease in fus3 deletion" The author should make clear in which level, transcription or translation? 6) The English writing should be improved by an English native speaker. 7) Fig.8 is not clear, which genes correspond to which phenotype, should be clear and right.

Reviewer #1:
The mycology and transcriptome and phosphoproteome methodological were well carried out.
In this study, the authors determined MAPK pathway gene function, confirmed that Ste50-Ste11-Ste7-Fus3 protein interactions and phosphorylations, explored the possible phosphorylation motifs and potential targets of the terminal kinase Fus3, and illustrated Fus3 responding to diverse environment stresses in Aspergillus. They revealed the mechanism of Fus3 positive regulation on AFs biosynthesis. Δfus3 mutant showed down-regulation of AFs production, but up-regulation of AFs cluster genes. The substrate of AFs, acetyl-CoA and malonyl-CoA were significantly decreased in fus3 null-deletion and site-mutagenesis strains, and the genes involved in acetyl-CoA and malonyl-CoA biosynthesis, were significantly down-regulated at transcriptional or phosphorylation levels. This paper is an interesting topic, and the experiments were well designed. It is meaningful for readers in related fields. So, my suggestion is acceptable after minor revision.

Response:
Thank you for the kind summary and review on our manuscript. And in this revised edition, the context was optimized, more information, including Fus3-TAP and ACCase analyses, were added, the language was revised by the native speaker, and some minor mistakes were corrected. The revised manuscript was more logical, and more information was supplied about the regulation of Fus3 on AFs biosynthesis. Thanks again for your serious and careful review on our manuscript. It is truly helpful to improve our manuscript.
Other comments were followed. 1) All the transcriptome and phosphoproteome data should be submitted to NCBI.

Response:
Thanks for your comment. The transcriptome data was submitted to NCBI and the transcriptome data ID is PRJNA777400 (Line 531). In the newest edition, the phosphoroteome data was submitted to iProX database, and the ID is IPXO003678000 with the share link (https://www.iprox.cn/page/SSV024.html;url=1638843223734VUiQ, Psssword: qZIU) (Line

Response:
Thanks for your comment. In this study, the two phosphorylation sites, T182 and Y184, were identified by the phosphoproteome, which is accordant with Yang et al. reported. By generating the site mutagenesis strains, we noticed that the phenotypes of fus3 T182A and fus3 Y184A strains were impaired in conidia production, sclerotia formation and AFs biosynthesis (Fig. 6). Because T182 and Y184 are the critical phosphorylation sites of Fus3, fus3 T182A and fus3 Y184A abolished the phosphorylation function of Fus3, and their phenotypes are similar with the fus3 null-deleted strain. But unexpectedly, fus3 T182D and fus3 Y184D did not recover the WT phenotypes, but still consisted with the Δfus3 phenotypes (Fig. 6).
A lot of phosphorylation studies found that the replacements of Thr/Tyr residues with Asp are not always perfectly mimic the constitutively phosphorylation phenotypes. The possible reason is that the variation of amino acid leads to the changes in protein high-level structure and proteins interaction, which results in the defective protein functions and the mutant phenotypes. In our research, we also noticed that the interactions of Fus3 T182A , Fus3 T182D , Fus3 Y184A and Fus3 Y184D with MAPK proteins. As shown, Fus3 T182D could not interact with the other MAPK modules, suggested that the replacement of threonine with aspartic acid obviously affected the interactions and the phosphorylation signal transduction, which abolished the normally function of Fus3.
3) There are many important MAPK in Aspergillus flavus, why the author chose the Fus3, please explain in Introduction part.
Fus3, as the terminals kinase of MAPK pathway, could directly affect the expressions and the functions of several regulators, for example VeA, SteA (Bayram et al., 2012) (Line 90-91). More important, AFs, the most critical secondary metabolism of A. flavus, are positively regulated by Fus3 (Frawley et al., 2019;Yang et al., 2020) (Line 104-106). And our previous research also found the inhibition of eugenol on AFs is closely relevant with Fus3 (Lv et al., 2018). But the specific regulatory mechanism of Fus3 on AFs biosynthesis is still unclear. Therefore, we focused on the function and regulation of Fus3 in this study.  Fig.3 showed the transcriptome and phosphoproteome results, but transcriptome only give small partial. Please provide more information on transcriptome.

Response:
Thanks for your comment. Firstly, Fus3 as a phosphokinase, would directly regulate the phosphorylation levels of the downstream genes. The variations in transcriptional level in Δfus3 should be resulted from the indirect regulation of Fus3. In order to find the potential target of Fus3, we paid more attention in phosphoproteome analysis. But we still discovered a lot of useful information from the transcriptome data. The transcriptional level changes of AFs related genes and developmental genes in Δfus3 partly illustrated the function and regulation of Fus3. And the transcriptome data supported our conclusion (Fig. 5).
So, as your suggestion, the transcriptome results were added in the revised manuscript as the supplementary materials (Table S1). 5) Line 217, "But the expressions of AFs biosynthetic genes were no obvious decrease in fus3 deletion" The author should make clear in which level, transcription or translation?

Response:
Sorry for the writing mistake. The sentence was corrected as "It is paradoxical that AFs cluster genes were up-regulated at transcriptional level, but AFs production was decreased in Δfus3" in Line 256.
6) The English writing should be improved by an English native speaker.

Response:
Thank you for your suggestion. We have carefully checked the context and improved our language by the native speaker. Fig.8 is not clear, which genes correspond to which phenotype, should be clear and right

Response:
Thanks for your comment. In the revised manuscript, the figure was divided into two parts, the regulation of Fus3 on fungal development and the regulation on AFs biosynthesis. And the directions of arrows are more explicit and certain, which is more easily to read and understand.

Reviewer #2:
In this manuscript, the authors examined the roles of Fus3 in Aspergillus flavus. Fus3 is a key component in MAPK pathway and play a various role in fungal development and mycotoxin productions. To examine the role of Fus3, the authors carried out transcriptomic and phosphoproteomic analyses. Although the authors present lots of results, these results are difficult to support their conclusions. If the authors analyze these results carefully, it is thought that meaningful conclusions will be presented.

Response:
Thank you for your kind summary and review on our manuscript. In the revised manuscript, we changed some imprecise statements and conclusions, paid more attention to the regulation of Fus3 on AFs biosynthesis, and added more experiments to support our conclusions. The mainly conclusion in the revised manuscript is "Fus3 could regulate carbon metabolism genes' expressions at transcriptional and phosphorylation level, especially AccA, then control the levels of AFs biosynthetic precursors, acetyl-CoA and malonyl-CoA, and subsequently affect the AFs production" (Line 423-426). And the consecution and logical relationship of the context were adjusted. The revised manuscript is more credible and logical than before.
To be specific, we constructed the SBP::Fus3::GFP strain, and performed the Fus3-TAP (tandem affinity purification) analysis, which supplied the reliable evidences of Fus3 interactions.
Fus3, Ste7, Ste11, Ste50 and AccA were identified by Fus3-TAP analysis (Table S3). And the analyses of ACCase quantity and ACCase indicated that the defective Fus3 significantly inhibit the ACCase activity, which confirmed the regulations of Fus3 on AccA, malonyl-CoA and AFs production.
Thanks again for your serious and careful review on our manuscript. Your suggestions are truly helpful to improve our manuscript.
1. The results of protein interacting experiments do not fully support the results presented by the authors. In vivo protein interacting experiment such as IP assay should be needed.

Response:
Thanks for your suggestion. To better investigating the Fus3 interactions, we constructed the SBP::Fus3::GFP strain, with SBP and GFP tags for twice protein purification. The Fus3-TAP results were shown in Table S3. Total of 862 proteins were found by LC-MS/MS. Ste50, Ste7 and Ste11, recognized in the list, which confirmed the direct interactions between Fus3 and the other MAPK modules (Line 139-140). AccA was also identified as the potential target of Fus3, which supported that Fus3 could regulate AFs biosynthesis depending on the modulation of AFs substrate supplement (Line 291-293). The TAP analysis is helpful to support our conclusion.
2. It's not clear why the authors combine the results of phosphoproteomic and transcriptomic analyses. They selected 66 proteins which decreased phosphorylation and mRNA expression, but its' not reasonable.

Response:
Fus3, as a phosphokinase, could positively regulated the phosphorylation level of downstream targets. The transcriptional variations must be result from the indirect regulations of Fus3. So, we focused on the down-regulated phosphorylation level genes in fus3 deletion strain.
Phosphoproteome analysis were performed using the method of Pierce TiO 2 Phosphopeptide Enrichment. The genes' expression must disturb the result of phosphopeptide enrichment. So, to reduce this disturbance, we searched the Fus3 potential targets from the proteins, which the phosphorylation levels were decreased, but the transcriptional levels were not significantly decreased in the mean while. The similar analysis method was also used in the previous study (Mattos et al., 2020).
And in the revised manuscript, the phrase was changed as "To reduce the interferences from the decrease in mRNA level, total of 1033 down-regulated phosphorylation proteins in Δfus3 were regarded as the potential Fus3 targets, excluding 66 proteins with significant down-regulation at 3. The authors generated various phospho-mutant strains. However, it is difficult to make conclusions based on the results obtained from this experiment.

Response:
In this study, we generated the phosphor-site mutagenesis, and sever experiments were performed to investigate the variation of Fus3. We believe that these investigations about the site mutagenesis strains supported our conclusion.
Firstly, the phenotypes of the phosphate-site strains were similar with ∆fus3, demonstrating that these phosphate-site mutagenesis directly abolish the normal function of Fus3. The variations of the critical phosphate-site directly disturb the phosphate signaling transmission.
Secondly, the ACCase activity of the phosphate-site mutagenesis strains were significantly decreased, which indicated the phosphorylation function of Fus3 is critical for ACCase activity.
The previous study reported the phosphorylation level of AccA affects its activity (Choi et al., 2014;Jia et al., 2017). So, the phosphate-site mutagenesis strains confirmed the regulation of Fus3 on AccA at phosphorylation level. Furtherly, combined the phosphoproteome, TAP, and the phosphorylation motif analyses, we speculated that AccA might be a direct target of Fus3.
Taken, the investigations of phosphate-site mutagenesis strains were useful to support our conclusion.  Importantly, we also identified that AccA, not as a TF, might be potential target of Fus3. The phosphorylation level of AccA was down-regulated in Δfus3, the direct interaction of AccA and Fus3 were noticed by Fus3-TAP analysis, and the phosphorylation motif [RxxSP] was also identified in AccA. All information suggested that AccA could be a direct target of Fus3. Furtherly, the phosphorylation level of AccA affects ACCase activity (Jia et al., 2017). In our research, the ACCase activities in Fus3 defective strains were significantly decreased, while the ACCase activity of fus3 T182D was partly recovered (Fig. 6G). So, in our research, Fus3 could directly regulate the phosphorylation of AccA, and then affect the ACCase activity and malonyl-CoA level, subsequently positively modulate the AFs production. This is the mainly conclusion in our manuscript.
In addition, we also noticed the down-regulated phosphorylation level of other TFs, such as AtfA, AtfB, AP-1. Previous reports showed that phosphorylation levels are critical for their transcriptional activated functions (Choi, 2014;Sanchez-Mir et al., 2020). But in our research, Fus3 regulated AFs independent on AFs cluster genes and these regulators. So, we did not perform the further investigations, and we moved these TFs information to the supplementary materials in the revised manuscript (Table S2). We might discover more Fus3 targets and study more Fus3 regulations in future research.  Thank you for submitting your revision and addressing the reviewer comments. While we are willing to consider a revised version of this paper at Spectrum, our editorial assessment is that the writing still needs substantial improvement. As I am certain you can appreciate, this is also in your best interest so that issues with incorrect wording do not discourage others from reading your work. One option is that you could ask a colleague who is a native English speaker to provide you comprehensive feedback on the writing. We would alternatively suggest that you are also welcome to use one of the services recommended by ASM: https://journals.asm.org/content/language-editing-services As these revisions are quite minor, I expect that you should be able to turn in the revised paper in less than 30 days, if not sooner.
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Reviewer #1:
The mycology and transcriptome and phosphoproteome methodological were well carried out.
In this study, the authors determined MAPK pathway gene function, confirmed that Ste50-Ste11-Ste7-Fus3 protein interactions and phosphorylations, explored the possible phosphorylation motifs and potential targets of the terminal kinase Fus3, and illustrated Fus3 responding to diverse environment stresses in Aspergillus. They revealed the mechanism of Fus3 positive regulation on AFs biosynthesis. Δfus3 mutant showed down-regulation of AFs production, but up-regulation of AFs cluster genes. The substrate of AFs, acetyl-CoA and malonyl-CoA were significantly decreased in fus3 null-deletion and site-mutagenesis strains, and the genes involved in acetyl-CoA and malonyl-CoA biosynthesis, were significantly down-regulated at transcriptional or phosphorylation levels. This paper is an interesting topic, and the experiments were well designed. It is meaningful for readers in related fields. So, my suggestion is acceptable after minor revision.

Response:
Thank you for the kind summary and review on our manuscript. And in this revised edition, the context was optimized, more information, including Fus3-TAP and ACCase analyses, were added, the language was revised by the native speaker, and some minor mistakes were corrected. The revised manuscript was more logical, and more information was supplied about the regulation of Fus3 on AFs biosynthesis. Thanks again for your serious and careful review on our manuscript. It is truly helpful to improve our manuscript.
Other comments were followed.
1) All the transcriptome and phosphoproteome data should be submitted to NCBI.

Response:
Thanks for your comment. The transcriptome data was submitted to NCBI and the transcriptome data ID is PRJNA777400 (Line 523). In the newest edition, the phosphoroteome data was submitted to iProX database, and the ID is IPXO003678000 with the share link (https://www.iprox.cn/page/SSV024.html;url=1638843223734VUiQ, Psssword: qZIU) (Line

Response:
Thanks for your comment. In this study, the two phosphorylation sites, T182 and Y184, were identified by the phosphoproteome, which is accordant with Yang et al. reported. By generating the site mutagenesis strains, we noticed that the phenotypes of fus3 T182A and fus3 Y184A strains were impaired in conidia production, sclerotia formation and AFs biosynthesis (Fig. 6). Because T182 and Y184 are the critical phosphorylation sites of Fus3, fus3 T182A and fus3 Y184A abolished the phosphorylation function of Fus3, and their phenotypes are similar with the fus3 null-deleted strain. But unexpectedly, fus3 T182D and fus3 Y184D did not recover the WT phenotypes, but still consisted with the Δfus3 phenotypes (Fig. 6).
A lot of phosphorylation studies found that the replacements of Thr/Tyr residues with Asp are not always perfectly mimic the constitutively phosphorylation phenotypes. The possible reason is that the variation of amino acid leads to the changes in protein high-level structure and proteins interaction, which results in the defective protein functions and the mutant phenotypes. In our research, we also noticed that the interactions of Fus3 T182A , Fus3 T182D , Fus3 Y184A and Fus3 Y184D with MAPK proteins. As shown, Fus3 T182D could not interact with the other MAPK modules, suggested that the replacement of threonine with aspartic acid obviously affected the interactions and the phosphorylation signal transduction, which abolished the normally function of Fus3.  Fig.3 showed the transcriptome and phosphoproteome results, but transcriptome only give small partial. Please provide more information on transcriptome.

Response:
Thanks for your comment. Firstly, Fus3 as a phosphokinase, would directly regulate the phosphorylation levels of the downstream genes. The variations in transcriptional level in Δfus3 should be resulted from the indirect regulation of Fus3. In order to find the potential target of Fus3, we paid more attention in phosphoproteome analysis. But we still discovered a lot of useful information from the transcriptome data. The transcriptional level changes of AFs related genes and developmental genes in Δfus3 partly illustrated the function and regulation of Fus3. And the transcriptome data supported our conclusion (Fig. 5).
So, as your suggestion, the transcriptome results were added in the revised manuscript as the supplementary materials (Table S1). 5) Line 217, "But the expressions of AFs biosynthetic genes were no obvious decrease in fus3 deletion" The author should make clear in which level, transcription or translation?

Response:
Sorry for the writing mistake. The sentence was corrected as "It is paradoxical that AFs cluster genes were up-regulated at transcriptional level, but AFs production was decreased in Δfus3" in Line 251.
6) The English writing should be improved by an English native speaker.

Response:
Thank you for your suggestion. We have carefully checked the context and improved our language by the native speaker. Fig.8 is not clear, which genes correspond to which phenotype, should be clear and right

Response:
Thanks for your comment. In the revised manuscript, the figure was divided into two parts, the regulation of Fus3 on fungal development and the regulation on AFs biosynthesis. And the directions of arrows are more explicit and certain, which is more easily to read and understand.

Reviewer #2:
In this manuscript, the authors examined the roles of Fus3 in Aspergillus flavus. Fus3 is a key component in MAPK pathway and play a various role in fungal development and mycotoxin productions. To examine the role of Fus3, the authors carried out transcriptomic and phosphoproteomic analyses. Although the authors present lots of results, these results are difficult to support their conclusions. If the authors analyze these results carefully, it is thought that meaningful conclusions will be presented.

Response:
Thank you for your kind summary and review on our manuscript. In the revised manuscript, we changed some imprecise statements and conclusions, paid more attention to the regulation of Fus3 on AFs biosynthesis, and added more experiments to support our conclusions. The mainly conclusion in the revised manuscript is "Fus3 could regulate the expression of carbon metabolism genes at the transcriptional and phosphorylation level, especially AccA, then control the levels of AF biosynthetic precursors, acetyl-CoA and malonyl-CoA, and subsequently affect AF production" (Line 418-421). And the consecution and logical relationship of the context were adjusted. The revised manuscript is more credible and logical than before.
To be specific, we constructed the SBP::Fus3::GFP strain, and performed the Fus3-TAP (tandem affinity purification) analysis, which supplied the reliable evidences of Fus3 interactions.
Fus3, Ste7, Ste11, Ste50 and AccA were identified by Fus3-TAP analysis (Table S3). And the analyses of ACCase quantity and ACCase indicated that the defective Fus3 significantly inhibit the ACCase activity, which confirmed the regulations of Fus3 on AccA, malonyl-CoA and AFs production.
Thanks again for your serious and careful review on our manuscript. Your suggestions are truly helpful to improve our manuscript.
1. The results of protein interacting experiments do not fully support the results presented by the authors. In vivo protein interacting experiment such as IP assay should be needed.

Response:
Thanks for your suggestion. To better investigating the Fus3 interactions, we constructed the SBP::Fus3::GFP strain, with SBP and GFP tags for twice protein purification. The Fus3-TAP results were shown in Table S3. A total of 862 proteins were found by LC-MS/MS. Ste50, Ste7 and Ste11, recognized in the list, which confirmed the direct interactions between Fus3 and the other MAPK modules (Line 133-135). AccA was also identified as the potential target of Fus3, which supported that Fus3 could regulate AFs biosynthesis depending on the modulation of AFs substrate supplement (Line 287-288). The TAP analysis is helpful to support our conclusion.
2. It's not clear why the authors combine the results of phosphoproteomic and transcriptomic analyses. They selected 66 proteins which decreased phosphorylation and mRNA expression, but its' not reasonable.

Response:
Fus3, as a phosphokinase, could positively regulated the phosphorylation level of downstream targets. The transcriptional variations must be result from the indirect regulations of Fus3. So, we focused on the down-regulated phosphorylation level genes in fus3 deletion strain.
Phosphoproteome analysis were performed using the method of Pierce TiO 2 Phosphopeptide Enrichment. The genes' expression must disturb the result of phosphopeptide enrichment. So, to reduce this disturbance, we searched the Fus3 potential targets from the proteins, which the phosphorylation levels were decreased, but the transcriptional levels were not significantly decreased in the mean while. The similar analysis method was also used in the previous study (Mattos et al., 2020).
And in the revised manuscript, the phrase was changed as "To reduce the interferences from the decrease in mRNA level, total of 1033 down-regulated phosphorylation proteins in Δfus3 were regarded as the potential Fus3 targets, excluding 66 proteins with significant down-regulation at transcriptional level" in Line 163-166. 3. The authors generated various phospho-mutant strains. However, it is difficult to make conclusions based on the results obtained from this experiment.

Response:
In this study, we generated the phosphor-site mutagenesis, and sever experiments were performed to investigate the variation of Fus3. We believe that these investigations about the site mutagenesis strains supported our conclusion.
Firstly, the phenotypes of the phosphate-site strains were similar with ∆fus3, demonstrating that these phosphate-site mutagenesis directly abolish the normal function of Fus3. The variations of the critical phosphate-site directly disturb the phosphate signaling transmission.
Secondly, the ACCase activity of the phosphate-site mutagenesis strains were significantly decreased, which indicated the phosphorylation function of Fus3 is critical for ACCase activity.
The previous study reported the phosphorylation level of AccA affects its activity (Choi et al., 2014;Jia et al., 2017). So, the phosphate-site mutagenesis strains confirmed the regulation of Fus3 on AccA at phosphorylation level. Furtherly, combined the phosphoproteome, TAP, and the phosphorylation motif analyses, we speculated that AccA might be a direct target of Fus3.
Taken, the investigations of phosphate-site mutagenesis strains were useful to support our conclusion.

Response:
Thanks for your comment. We found that defect of fus3 obviously leaded to the impairment of fungal development, such as conidia production and sclerotia formation. And based on the transcriptome data, we notice a lot of developmental genes, such as con6, con10, rodA, brlA and nsdD, showed significantly decrease in Δfus3. We believe that these reductions are due to the phosphorylation level decrease of the upstream regulators, such as VeA, SteA, Con7, NsdD. The phosphorylation level of these regulator could affect their transcriptional regulating activity (Odenbach et al., 2007). Therefore, the transcriptional expressions of critical genes related development are accordant with the phenotype variations. In this study, we mainly focused on the regulation of Fus3 on AFs regulation. So, our research is lack of more solid information about the regulation of Fus3 on fungal. We only described a possible regulatory pathway of Fus3 on fungal development. And in the revised manuscript, we deleted the overstate expressions and the uncertain conclusions. The revised manuscript is more rigorous than before. Thanks for your suggestions again.

Response:
Thank you for your comments. Based on our phosphoproteome data, we noticed that several TFs showed the down-regulated phosphorylation levels in Δfus3, indicating these TFs might be directly or indirectly regulated by Fus3 at phosphorylation levels. At first, we summarized the down-regulated phosphorylation TFs to investigate the Fus3 regulations and functions.
Several TFs have been regarded as the direct target of Fus3, such as VeA, Con7, and SteA (Odenbach et al., 2007;Bayram et al., 2012;Katayama T et al., 2021). Deletion of fus3 leads to the down-regulated phosphorylation level, and furtherly results in the decreased transcription of downstream genes and the defect development phenotype. And according to the potential targets, we predicted that the phosphorylation motif of Fus3 might be the [RxxSP] in A. flavus.
Importantly, we also identified that AccA, not as a TF, might be potential target of Fus3. The phosphorylation level of AccA was down-regulated in Δfus3, the direct interaction of AccA and