The MYB-like protein MylA contributes to conidiogenesis and conidial germination in Aspergillus nidulans

Myeloblastosis (MYB)-like proteins are a family of highly conserved transcription factors in animals, plants, and fungi and are involved in the regulation of mRNA expression of genes. In this study, we identified and characterized one MYB-like protein in the model organism Aspergillus nidulans. We screened the mRNA levels of genes encoding MYB-like proteins containing two MYB repeats in conidia and found that the mRNA levels of four genes including flbD, cicD, and two uncharacterized genes, were high in conidia. To investigate the roles of two uncharacterized genes, AN4618 and AN10944, deletion mutants for each gene were generated. Our results revealed that AN4618 was required for fungal development. Therefore, we further investigated the role of AN4618, named as mylA, encoding the MYB-like protein containing two MYB repeats. Functional studies revealed that MylA was essential for normal fungal growth and development. Phenotypic and transcriptomic analyses demonstrated that deletion of mylA affected stress tolerance, cell wall integrity, and long-term viability in A. nidulans conidia. In addition, the germination rate of the mylA deletion mutant conidia was decreased compared with that of the wild-type conidia. Overall, this study suggests that MylA is critical for appropriate development, conidial maturation, dormancy, and germination in A. nidulans.


Intro
The intro section L37-72 is too long, please shorten.The section L83-92 is too short.L37 "The myeloblastosis (MYB)-like proteins include the…" change "include" to "contain a highly conserved.." L74 "Lives …even in air" I am not sure it lives suspended in the air, probably simply found in air samples as conidia etc. L75 "higher efficiency in gene manipulation than other Aspergillus species" not sure this is correct nowadays with CRISPR.L80 "possesses ambivalent characteristics and needs to be understood" unclear.What do you mean?L95 "highly expressed in conidia compared with those in hyphae" what do you mean?What are "those" which genes/proteins?Results L104 "using InterPro" it is called interproscan, please correct and provide ref.
L105-add they are shown in Table 1.Fig. 2 please move to the supplementary, it is of secondary importance.L113-126 are confusing and hard to understand.Please rewrite clearly.L122 "the other seven proteins had two MYB repeats having R2 and R3 sequences," I cannot see this in Fig. 2, where is it?L130-There are only seven 2R-mybs.To define their role in conidiogenesis, the best approach is to delete them and look at the phenotype.Trying to understand their involvement via gene expression is a much weaker approach.They may be important for conidiogenesis but unchanged in RNA level etc.This should be noted in the discussion.L137-Please show the verification of the deletion and complementary strains in the supplement.Were several independent isolates verified for the deletion phenotype?Fig 3D-please add the degree of conservation 3E-what are the time-points and growth conditions for V, A, C? please add.L167-According to the methods, conidia were collected after 2 days of growth.Why 2 days, a very short time?Full maturation of conidia takes about 7 days.Fig 5A, please add the number of genes found in each category.L178-This is very general and probably indirect involvement of mylA in these processes.To really find what it does and narrow things down, you need to perform chip-seq analysis to see what genes it binds and possibly controls.Please add in discussion.Fig 5F-you verified only 2 of the 5 genes shown in 5E, what was the verification result for the other 3 genes.Also please add a small map of the trehalose pathway showing the genes involved and the RNAseq changes in their expression in numerical fold change value.Trehalose biosyhtnesis in Aspergilli is regulated by the transcription factors VosA and AtfA.How is their expression affected by mylA deletion?Fig. 6-what about chitin synthesis genes?And mannosylation pathway genes?6C-does your glucan assay measure exposed glucan on the conidial surface?Or do you break up the conidia and separate the cell wall before the assay?Please add.7a-how many total number of genes were found in this cell cycle category?And how many are you showing here from that entire group?Please add.Since mylA is strongly involved in conidiation, how does its deletion affect expression of other conidiation pathway genes (flbB, C, D, BrlA, VosA, AbaA, WetA etc).Where does it function within the known conidiation pathway?Discussion The discussion is too long.Please shorten it to the most important points and try not to repeat the results section.L254 "CicD (transcriptional activator of cichorine)" correct to "CicD (transcriptional activator of cichorine biosynthesis) L257 "the function of MYB-like proteins" correct to "the function of several MYB-like proteins" L259 "we examined the total MYB-like proteins" you only looked at two and focused on one of them.L271 "four conidia-specific" not conidia specific, they are enriched in conidia (vs.hypha) L282-291-Move to results.Provide figure in supplement to show the various binding sites.Add if mylA deletion affects expression of other conidiation pathway genes (flbB, C, D, BrlA, VosA, AbaA, WetA etc).L302-321, add the fold reduction of expression of each of the genes you describe here in your null mutant L325-6 unclear, please rewrite L333-346.Move to results.L345-to prove this you will also need to do overexpression studies for these genes.
Reviewer #2 (Remarks to the Author): In this manuscript the authors describe the identification and characterization of MylA, a MYB-like protein which appeared involved in fungal growth, conidiation, stress response and long-term viability of spores in A. nidulans.The study is very interesting and the experimental design is sound.However, I believe the language of the manuscript could use some improvement, in order to better convey the significance of the research.Prior to publication there are several points that should be taken care of: -the abstract and the introduction, more than the rest of the manuscript, would need a thorough English writing screen.(For example, it is unlikely that a protein family IS a transcription factor, as stated in line 24.In line 26 the MYB acronym is explained, but it first appeared in line 24.When talking about one specific protein identified, the correct article is THE and not a , again in line 26.And line 27, "of which" is referring to what? A. nidulans?)After careful reading and re-reading the sense come through, but the lack of correct English makes the text quite difficult to understand and follow.The abstract also does not explain clearly in which order things have been done, I firstly understood that initially the authors identified MylA and by characterizing this protein they connected it to the MYB-like family.
-in the introduction, various references are missing (line 39 and line 40, just to make an example.All the references about first discovery of the MYB domain, and the v-MYB causing leukemia in chickens.Or line 67).
-as non-expert in protein structure, the whole introductory part trying to explain the differences between 1R-2R and 3R MYB actually left me more confused.I acknowledge this might me due to my lack of experience in this particular field, but if the paper should be accessible to a broad spectrum of scientist, I would suggest to simplify a bit this part.
-the paper occasionally suffers from short, repetitive sentences that could be combined for improved readability (especially in the results section).
-for the DEGs analysis, in the first experiment it's specified in the text (line 133) a p-value < 0.5, which seems quite high.Is this a typo, considering than later, in material and methods, the stated pvalue is < 0.05?If not, why this high value?If yes, why write it again in the results if it is generally stated in the material and methods?Or the value stated in the material and methods section is valid only for the analysis mentioned in line 172?-line 200, perhaps is worth mentioning more explicitly in the text that tpsA and ccg9 are threalose biosynthetic genes, otherwise might not be so clear why having those specific two genes there.
-the discussion part is very well written, and consider all aspects reported in the results.To be fair, it bings the argumentation even further, in fact there are parts that are not mentioned at all in the introduction or in the results part (like consideration about abaA deletion strain or LkhA).Maybe, if mentioning these part in the discussion, it would be better to report them as well in the results, and not let them, eventually, only in the supplementary material.
-in the material and methods, the statistical analysis for which particular experiments has been used?For the rest, this part in in general clear and understandable, with the exception of how the mutant strains have been generated.
Overall, this manuscript contains very good data and great science, but the words and style used to deliver the message are not appropriate.The discussion part is well written and thorough, but it seems out of place as the rest of the text is not at the same level.I would suggest to invest a bit more in the introduction and results part, to provide a more uniform style and better convey the nice discoveries achieved in this study.
Reviewer #3 (Remarks to the Author): The paper by Ye-Eun Son et al. identified and characterized a myelobalstosis (MYB)-like protein MylA in the model organism Aspergillus nidulans.Further investigations showed MylA is essential for fungal growth and developtment, and regulating stress tolerance, cell wall integrity and long-term viability.These findings are data-detailed, credible, and innovative.It can be published in after addressing the following concerns.1. Remove Fig. 2 to supplemental files and concise the description for the conserved motifs.2. Line 211-223, fksA was upregulated and β-glucan content higher in ΔmylA strain, indicating MylA inhibits the glucan biosynthesis.Why author thought MylA is required for β-glucan biosynthesis?
Reviewer #4 (Remarks to the Author): Review on 'A MYB-like protein MylA contributes to conidiogenesis and conidial germination in Aspergillus nidulans' by Son YE et al This manuscript focuses on MYB-like proteins of A. nidulans and specifically describes and characterizes the transcription factor MylA and its role in conidia and fungal growth.The manuscript is well organized and pleasant to read.Authors have used several molecular biology and biochemical techniques to identify and characterized the MylA of A. nidulans.They constructed Knock Outs and complementary strains, performed RNA seq, RT-qPCR, conidial trehalose analysis, as well as germination rates, phenotypic comparisons, conidial survival rates, among others.They have also used bioinformatics web servers to compare and describe the MYB-like proteins of A. nidulans.I just have 2 comments that authors might address and others minor suggestions.
Comment 1. Lines 138, 139, 149.In this study, deletion strains (ΔmylA, ΔmylB, ΔmylA/ΔrgsA) and complementary (Cʹ mylA) strains have been generated.A supplementary figure is needed to show how these strains were constructed (schematic overview showing lengths and details of the assembled fragments used to generate the strains) and how they were confirmed (primers or restriction enzymes used for this).
Comment 2. Authors should clarify some details of the RNA seq.
Line 133.Regarding the Fold Change (FC): It is not clear if genes with fold change=2 were considered or not in the RNA seq analysis.It is written |fold change| >2 on line 133 but fold change ≥2 on line 454.
In line 454, authors are talking about DEGs (Differentially Expressed Genes) but is written 'fold change ≥2' which only consider UP-regulated genes.
In line 454 is written 'p value'.Please clarify whether the p-value or the adjusted p-value was used?
Minor suggestions: Lines 333-336.I suggest to add a reference in this sentence (PMID: 15228532).
Line 336.It is important to add in which study the single mutant ΔrgsA was generated to know its parental background.The optimal case for Fig S3 is that the 4 strains compared have the same genetic background.
Line 324.The sentence starting with 'And continuously sequential cell cycle …' needs revision.It is not clear.

Introduction
The intro section L37-72 is too long, please shorten.The section L83-92 is too short.
 Thank you for this comment.Following this comment, we intensively revised the introduction section.
L37 "The myeloblastosis (MYB)-like proteins include the…" change "include" to "contain a highly conserved."  Thank you for this comment.Following this comment, we revised it.
Lines 58-60: The MYB-like proteins contain a highly conserved DNA-binding domain, the MYB domain, which consists of approximately 50 amino acids that are folded into three α-helices.
L74 "Lives …even in air" I am not sure it lives suspended in the air, probably simply found in air samples as conidia etc.
 Thank you for this comment.Following this comment, we revised it.
Lines 77-78: A. nidulans is a saprophytic filamentous fungus that lives ubiquitously in soil, crops and seeds, and even in water 20 .
L75 "higher efficiency in gene manipulation than other Aspergillus species" not sure this is correct nowadays with CRISPR.
 Thank you for this comment.We removed this sentence while editing the introduction section.
L80 "possesses ambivalent characteristics and needs to be understood" unclear.What do you mean?
 Thank you for this comment.We removed this sentence while editing the introduction section.
-Although A. nidulans possesses various characteristics and needs to be understood, the molecular mechanisms underlying fungal physiology remain largely unknown.
L95 "highly expressed in conidia compared with those in hyphae" what do you mean?What are "those" which genes/proteins? Thank you for this comment.We carefully addressed this sentence.
Lines 91-92: characterized one myb gene that is highly expressed in conidia compared to hyphae.

Results
L104 "using InterPro" it is called interproscan, please correct and provide ref.
 Thank you for this comment.Based on this comment, we revised it and added a reference.
Lines 99-100: To identify the MYB-like proteins in A. nidulans, we scanned the MYBlike domain using InterProScan tool 25 .
L105-add they are shown in Table 1.
 We added it.
Fig. 2 please move to the supplementary, it is of secondary importance.
 We appreciate this comment.Based on this comment, we moved Fig. 2 to Supplementary Figure 2.
L113-126 are confusing and hard to understand.Please rewrite clearly.
 We really appreciate this comment.Following this comment, we revised these sentences.
Lines 110-117: The sequences of MYB motifs were aligned with those of Arabidopsis thaliana, investigated previously (Supplementary Fig. 1) 16 , and domain structures were predicted using SWISS-MODEL (Supplementary Fig. 2).As shown in Table 1, there were no MYB-like proteins having more than three MYB repeats.A total of 14 proteins had one MYB repeat, where 6 proteins had the R1/R2 motif, and 8 proteins had the R3 motif.In contrast, the other seven proteins had two MYB repeats having R2 and R3 motifs.The repeat size was 50 amino acids on average, and each repeat had three α-helixes.
L122 "the other seven proteins had two MYB repeats having R2 and R3 sequences," I cannot see this in Fig. 2, where is it? Thank you for pointing it out.Based on this comment, we modified this figure .L130-There are only seven 2R-mybs.To define their role in conidiogenesis, the best approach is to delete them and look at the phenotype.Trying to understand their involvement via gene expression is a much weaker approach.They may be important for conidiogenesis but unchanged in RNA level etc.This should be noted in the discussion.
 We really appreciate this valuable comment.Following this comment, we added it in the discussion.We will also further characterize other MYB-like proteins in A. nidulans.
Lines 352-355: Additionally, although we focused on conidia-specific 2R MYB-like TFs, further studies are required to determine the function of other 2R or 1R MYB-like proteins in A. nidulans conidiogenesis.L137-Please show the verification of the deletion and complementary strains in the supplement.Were several independent isolates verified for the deletion phenotype?
 We thank for this critical comment.Following this comment, we added some figures of how mutant strains were generated and confirmed in supplementary Figures 4~6.L167-According to the methods, conidia were collected after 2 days of growth.Why 2 days, a very short time?Full maturation of conidia takes about 7 days.
 Thank you for this valuable comment.We always checked spore viability before we conduct phenotypic and transcriptomic analyses.As shown Figure 4g, the spore viability of 2-days grown ΔmylA conidia was nearly 100%, but the survival rate of 7-or 10-days grown ΔmylA conidia was deceased compared to conidia of WT or complemented strains.The viability of the ΔmylA conidia for 7 days old is only 60%.Therefore, when conducting experiments using this sample, it is difficult to accurately confirm the phenotype and gene expression.Therefore, we used conidia grown for 2 days for the experiments in this manuscript.We hope that this reviewer understands this situation. Thank you for this valuable comment.We also agree with this reviewer's comment on direct roles of MylA in conidia.In current stage, we did not conduct ChIP-seq analysis, so we discussed it in the discussion section.
Lines 349-352: Despite our new findings, to provide novel insights into fungal development and spore properties, we will further investigate the direct regulatory mechanisms of MylA by performing Chromatin-Immuno-Precipitation (ChIP) sequencing and combining RNA-seq and ChIP-seq analyses. We appreciate this valuable comment.Following this comment, we verified mRNA levels of other 3 genes and we added a schematic illustration for trehalose synthetic pathway with RNA seq result.
Trehalose biosynthesis in Aspergilli is regulated by the transcription factors VosA and AtfA.How is their expression affected by mylA deletion?
 Thank you for this valuable comment.We checked expression levels of vosA and atfA in WT and mylA mutant conidia.The log2foldchange of vosA and atfA is about 0.36 and -2.23 in mylA deletion conidia, respectively.From this result, we thought MylA might affect mRNA level of atfA, but not vosA.The detailed mechanism will be further examined in the future. This is a great question.We have already analyzed the expression of chitin synthetic genes but the mRNA levels of most of chitin synthetic genes were not significantly different between WT and ∆mylA conidia (data not shown).For the mannosylation pathway genes, we analyzed the galactomannan biosynthetic pathway genes and presented into Fig.5d.
Lines 220-223: The mRNA expression levels of genes related to galactomannan, hydrophobins, and DHN-melanin biosynthesis were upregulated in mylA deletion conidia (Fig. 5d−5f).These results imply that MylA can be involved in conidial wall composition.
6C-does your glucan assay measure exposed glucan on the conidial surface?Or do you break up the conidia and separate the cell wall before the assay?Please add.
 Thank you for this comment.We did not break up the conidia, so the measured glucan might be exposed glucan on the conidial surface.We briefly mentioned it into the main text.
Lines 216-217: We then evaluated the β-glucan contents in the conidial surface of each strain.7a-how many total number of genes were found in this cell cycle category?And how many are you showing here from that entire group?Please add.
 Thank you for pointing it out.Following this comment, we added it.

Lines 229-233:
To understand the effect of MylA, we investigated the expression levels of 256 genes known to be associated with the mitotic cell cycle.As a result, a total of 95 genes were significantly differently expressed genes between WT and ΔmylA conidia.Among them, about 80 % of them (76 DEGs/95 DEGs) were downregulated in the conidia of the ΔmylA strain (Fig. 6a).
Since mylA is strongly involved in conidiation, how does its deletion affect expression of other conidiation pathway genes (flbB, C, D, BrlA, VosA, AbaA, WetA etc).Where does it function within the known conidiation pathway? This is a great comment.We agreed this valuable comment, so we checked mRNA levels of central development regulators (BrlA, AbaA, and WetA) after asexual induction.We presented this data into Figure 3d.In mylA deletion mutant conidia, we also checked mRNA levels of flbB, flbC, flbD, vosA, brlA, abaA, and wetA.mRNA levels of flbC (Log2FC =2.08), flbD (Log2FC =2.35), and brlA (Log2FC =1.23), were increased, but this is not necessary for in the main manuscript, so we did not mention it.
Lines 147-154: To investigate whether deletion of mylA affects expression of central development regulators, we examined the mRNA levels of brlA, abaA, and wetA after induction of asexual development in the ΔmylA mutant strain.As shown in Fig. 3d, brlA and abaA mRNA levels in ΔmylA strain were significantly increased at 24 h post asexual developmental induction compared to WT and Cʹ mylA strains.And mylA deletion strain showed higher expression levels of wetA than WT and Cʹ mylA strains at 48 h post induction.These results indicated that MylA is needed for proper expression of asexual developmental regulators.

Discussion
The discussion is too long.Please shorten it to the most important points and try not to repeat the results section.
 Thank you for this comment.Based on this comment, we intensively revised and shortened the discussion session.Also, several paragraphs were moved to the results section.
L257 "the function of MYB-like proteins" correct to "the function of several MYB-like proteins"  We revised it.
Lines 266-267: the function of several MYB-like proteins in Aspergillus species remains unknown.
L259 "we examined the total MYB-like proteins" you only looked at two and focused on one of them.
 We revised it.
L271 "four conidia-specific" not conidia specific, they are enriched in conidia (vs.hypha)  We edited it. Thank you for this great comment.Following this comment, we further investigated mRNA levels of the central developmental regulators (BrlA, AbaA, and WetA) in the mylA deletion mutants and described in result session.For the binding sites in the mylA promoter regions, we added it into the supplementary Figure 3.
Lines 147-154 : To investigate whether deletion of mylA affects expression of central development regulators, we examined the mRNA levels of brlA, abaA, and wetA after induction of asexual development in the ΔmylA mutant strain.As shown in Fig. 3d, brlA and abaA mRNA levels in ΔmylA strain were significantly increased at 24 h post asexual developmental induction compared to WT and Cʹ mylA strains.And mylA deletion strain showed higher expression levels of wetA than WT and Cʹ mylA strains at 48 h post induction.These results indicated that MylA is needed for proper expression of asexual developmental regulators.
Lines 279-296 : In this study, we examined the role of MylA in asexual development.
The ΔmylA strain exhibited decreased the amount of conidia and produced abnormal conidiophores (Fig. 3a).In addition, deletion of mylA affects mRNA expression of brlA, abaA, and wetA (Fig. 3d), implying that MylA might affect transcript levels of genes associated with asexual development.To confirm this hypothesis, in-depth mechanistic study will be needed.We found that mRNA level of mylA was specifically high in conidia (Fig. 2e), so we wondered which regulator controls mRNA level of mylA in late phage of development or conidia.To text this, we first investigated the mylA promoter sequence and confirmed that there were one AbaA-response element (5ʹ-CATTCY-3ʹ), one VelB-response element (5ʹ-CCXTGG-3ʹ), and two VosA-response elements (5ʹ-CCXXGG-3ʹ), but not WetA-response element (Supplementary Fig. 3a) 24,39.We next checked mRNA level of mylA in the deletion mutant strains.We checked the mylA transcripts levels with previous RNA-seq data and found no significant difference in levels in the ΔvosA, ΔvelB, and ΔwetA strains 24.We then checked mylA transcript in the ΔabaA strain, which showed increased levels along the asexual development of the ΔabaA strain (Supplementary Fig. 3b).These suggest that AbaA, but not VosA, VelB, and WetA, might control mylA expression during asexual development of A. nidulans.L302-321, add the fold reduction of expression of each of the genes you describe here in your null mutant  Thank you for this comment.Based on this comment, we described the fold change of each gene.
Lines 299-317: When we further analyzed the mitotic cell cycle-related genes, we found most genes to be downregulated in the conidia of the ΔmylA strain (Fig. 7a), among which LAMMER kinase A (LkhA; Log2FC = -1.03)affects the emergence of germ tube and hyphal polarity by modulating the transcript levels of nimx cdc2 , which controls the S phase and G2/M DNA damage checkpoint 43 .NimO (Never in mitosis; Log2FC = -1.57) is required to check a cell cycle linking S and M phases and is essential for proper DNA synthesis after progression of the S phase.nimO mutants exhibited much more delayed germination rate compared to that of WT strain 44.The putative NimO-binding partner Cdc7 (Log2FC = -1.05)also affects proper DNA replication 45 .As an anaphase-promoting complex/cyclosome (APC/C), BimA (Blocked-in-mitosis; Log2FC = -1.81) is essential for spindle poles to promote the onset of anaphase among mitosis.Loss-of-function mutations in bimA resulted in growth-arrested cells with condensed chromatin and a short mitotic spindle 46 .BimC, a motor protein (kinesins; Log2FC = -3.10),plays a role in spindle pole body separation and mitotic spindle formation 47. SepA (Log2FC =2.07) and SepH (Log2FC = -1.13)enable the formation of cortical actin rings at the surrounding septation and around the tips.In addition, PabA (Log2FC = -1.29) is required for proper septation during septation and cytokinesis in A. nidulans 48 .Altogether, MylA is required for activating almost all stages of mitosis, including the interphase and mitosis.L325-6 unclear, please rewrite  We revised this paragraph.
Lines 319-325: Dormant conidia form germ tubes and synthesize septum after the first nuclear division.With our results, we hypothesize that MylA causes normal spore germination in A. nidulans.In fact, the absence of mylA led to a much less rate of conidial germination compared to that in the WT and Cʹ mylA strains (Fig. 7c).These findings indicate that MylA plays a crucial role in conidial germination by regulating the cell cycle.Nonetheless, further studies are required to investigate the detailed molecular mechanisms of MylA in cell cycle.

L333-346. Move to results.
 We really appreciate this valuable comment.Following this comment, we moved these sentences and revised the figure legend.

Relationship between MylA and RgsA
It has been demonstrated that RgsA, a regulator of G-protein signaling, downregulates spore germination and vegetative growth by turning off the GanB-mediated signaling, which activates vegetative growth through the cAMP/PKA signaling pathway.We focused on the fact that the ΔrgsA mutant exhibited increased germination rates, unlike the ΔmylA mutant.To reveal the potential relationship between RgsA and MylA, we generated double deletion mutants of mylA and rgsA (Fig. 7a).As shown in Fig. 7b, the conidia of ΔmylA or ΔmylAΔrgsA strains appeared to exhibit poor spore germination ability.A quantitative analysis showed that the conidia of ΔmylA or ΔmylAΔrgsA strains exhibited considerably reduced germination rates, whereas the conidia of the ΔrgsA strain germinated rather quickly compared with the conidia of the WT strain (Fig. 7c).Remarkably, the germination rate of the double deletion mutant was similar to that of the ΔmylA mutant.These results suggested that MylA regulates conidial germination unaffected by deletion of rgsA, and the relationship between MylA and GanB-mediated signaling should be confirmed through additional studies.
L345-to prove this you will also need to do overexpression studies for these genes.
 Thank you for this comment.We agreed this comment and revised this sentence.
Lines 256-258: These results suggested that MylA regulates conidial germination unaffected by deletion of rgsA, and the relationship between MylA and GanB-mediated signaling should be confirmed through additional studies.

Reviewer #2 (Remarks to the Author):
In this manuscript the authors describe the identification and characterization of MylA, a MYB-like protein which appeared involved in fungal growth, conidiation, stress response and long-term viability of spores in A. nidulans.The study is very interesting, and the experimental design is sound.However, I believe the language of the manuscript could use some improvement, in order to better convey the significance of the research.
Prior to publication there are several points that should be taken care of:  We really appreciate this reviewer's valuable comments for our manuscript.We carefully checked reviewer's comments and revised the manuscript following comments.We hope that we have improved the manuscript to a level of reviewer's satisfaction.
-the abstract and the introduction, more than the rest of the manuscript, would need a thorough English writing screen.(For example, it is unlikely that a protein family IS a transcription factor, as stated in line 24.In line 26 the MYB acronym is explained, but it first appeared in line 24.When talking about one specific protein identified, the correct article is THE and not a , again in line 26.And line 27, "of which" is referring to what? A. nidulans?)After careful reading and re-reading the sense come through, but the lack of correct English makes the text quite difficult to understand and follow.The abstract also does not explain clearly in which order things have been done, I firstly understood that initially the authors identified MylA and by characterizing this protein they connected it to the MYB-like family.
 We really appreciate to comment.Following this comment, we carefully revised the abstract and the introduction sections.In addition, a professional editor carefully checked and revised the entire manuscript.
Lines 24-37: Myeloblastosis (MYB)-like proteins are a family of highly conserved transcription factors in animals, plants, and fungi, regulating the mRNA expression of genes.In this study, we identified and characterized one of the MYB-like proteins in the model organism Aspergillus nidulans.We screened mRNA levels of genes encoding the MYB-like proteins containing two MYB repeats in conidia, and found that mRNA levels of four genes including flbD, cicD, and two uncharacterized genes were high in conidia.To investigate the roles of two uncharacterized genes, AN4618 and AN10944, the deletion mutants for each gene were generated and found that AN4618 was required for fungal development.Therefore, we further investigated the roles of AN4618, named as mylA, encoding the MYB-like protein containing two MYB repeats.Functional studies showed that MylA is essential for normal fungal growth and development.Phenotypic and transcriptomic analyses demonstrated that deletion of mylA affected stress tolerance, cell wall integrity, and long-term viability in A. nidulans conidia.
-in the introduction, various references are missing (line 39 and line 40, just to make an example.All the references about first discovery of the MYB domain, and the v-MYB causing leukemia in chickens.Or line 67).
 We thank for this valuable comment.Following this, we added appropriate references in the introduction.
-as non-expert in protein structure, the whole introductory part trying to explain the differences between 1R-2R and 3R MYB actually left me more confused.I acknowledge this might me due to my lack of experience in this particular field, but if the paper should be accessible to a broad spectrum of scientist, I would suggest to simplify a bit this part.
 We agreed this valuable comment, so we revised this part for the general reader.
Lines 58-69: The MYB-like proteins contain a highly conserved DNA-binding domain, the MYB domain, which consists of approximately 50 amino acids that are folded into three α-helices.Based on the number of imperfect repeats, MYB-like proteins are subdivided into four groups; 1R-MYB (one repeat), 2R-MYB (two repeats), 3R-MYB (three repeats), and 4R-MYB (four repeats).The 1R-MYB proteins are composed of a partial or single MYB repeat (R1/R2 or R3) and contribute to morphogenesis and development in plants 13,14 .The 2R-MYB group is composed of two MYB motifs (R2 and R3 repeats), and most of these proteins are related to the determination of cell fate and regulation of cellular development, stress response, and primary and secondary metabolism in plants 15 .The 3R-MYB proteins have three MYB motifs, called R1, R2, and R3 repeats; this group regulates the cell cycle in humans, animals, and plants [16][17][18] .
-the paper occasionally suffers from short, repetitive sentences that could be combined for improved readability (especially in the results section).
 We appreciate this comment.Following this comment, we revised the entire manuscript.
-for the DEGs analysis, in the first experiment it's specified in the text (line 133) a pvalue < 0.5, which seems quite high.Is this a typo, considering than later, in material and methods, the stated p-value is < 0.05?If not, why this high value?If yes, why write it again in the results if it is generally stated in the material and methods?Or the value stated in the material and methods section is valid only for the analysis mentioned in line 172? We are so sorry for the confusion.We revised method for RNA-seq analysis.
Lines 448-450: DEGs were defined based on absolute value (log2(fold change) ≥ 1 and p-value <0.05, and GO analyses were performed using the R package and FungiDB.
-line 200, perhaps is worth mentioning more explicitly in the text that tpsA and ccg9 are trehalose biosynthetic genes, otherwise might not be so clear why having those specific two genes there.
 Thank you for this valuable comment.We further checked mRNA levels of other genes involved in trehalose biosynthesis and added the results into the Figure 5f.
-the discussion part is very well written, and consider all aspects reported in the results.
To be fair, it brings the argumentation even further, in fact there are parts that are not mentioned at all in the introduction or in the results part (like consideration about abaA deletion strain or LkhA).Maybe, if mentioning these part in the discussion, it would be better to report them as well in the results, and not let them, eventually, only in the supplementary material.
 Thank you for this valuable comment.Following this comment, we moved some sentences to the results section.We also revised the discussion section to mention the relationship between mylA and other genes.
Lines 246-258: Relationship between MylA and RgsA/GanB signaling pathway in spore germination It has been demonstrated that RgsA, a regulator of G-protein signaling, downregulates spore germination and vegetative growth by turning off the GanB-mediated signaling, which activates vegetative growth through the cAMP/PKA signaling pathway 37,38 .We focused on the fact that the ΔrgsA mutant exhibited increased germination rates, unlike the ΔmylA mutant.To reveal the potential relationship between GanB/RgsA signaling and MylA, we generated double deletion mutants of mylA and rgsA (Fig. 7a).As shown in Fig. 7b, the conidia of ΔmylA or ΔmylAΔrgsA strains appeared to exhibit poor spore germination ability.A quantitative analysis showed that the conidia of ΔmylA or ΔmylAΔrgsA strains exhibited considerably reduced germination rates, whereas the conidia of the ΔrgsA strain germinated rather quickly compared with the conidia of the WT strain (Fig. 7c).Remarkably, the germination rate of the double deletion mutant was similar to that of the ΔmylA mutant.Collectively, we suggest that MylA acts downstream of GanB-mediated signaling for regulating conidial germination (Fig. 7d).
-in the material and methods, the statistical analysis for which particular experiments has been used?For the rest, this part in in general clear and understandable, with the exception of how the mutant strains have been generated.
 Thank you for this valuable comment.Following this, we added the statistical analysis for the experiments in the methods section.
Lines 502-505: All data except mutant construction were reported as mean ± standard deviation, and Student's unpaired t-test was used to evaluate statistical differences between the WT and ΔmylA strains.p < 0.05 was considered significant.All statistical analyses were conducted using the GraphPad Prism software (version 5).. Overall, this manuscript contains very good data and great science, but the words and style used to deliver the message are not appropriate.The discussion part is well written and thorough, but it seems out of place as the rest of the text is not at the same level.I would suggest to invest a bit more in the introduction and results part, to provide a more uniform style and better convey the nice discoveries achieved in this study.
 We truly appreciate this reviewer's positive and encouraging comments.We thoroughly revised the entire manuscript to understand the general readers.We hope that the revised version is suitable for publication in Communication Biology.

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Fig 3D-please add the degree of conservation

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Fig 5A, please add the number of genes found in each category.

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Fig 5F-you verified only 2 of the 5 genes shown in 5E, what was the verification result for the other 3 genes.Also please add a small map of the trehalose pathway showing the genes involved and the RNA seq changes in their expression in numerical fold change value.

Lines 272- 274 :
In the present study, we selected two uncharacterized MYB-like proteins, MylA and MylB, which contain two MYB motifs (R2 and R3 repeats) based on RNA-seq data (Fig 2a).L282-291-Move to results.Provide figure in supplement to show the various binding sites.Add if mylA deletion affects expression of other conidiation pathway genes (flbB, C, D, BrlA, VosA, AbaA, WetA etc).