White Collar 1 Modulates Oxidative Sensitivity and Virulence by Regulating the HOG1 Pathway in Fusarium asiaticum

ABSTRACT Fusarium asiaticum is an epidemiologically important pathogen of cereal crops in east Asia, accounting for both yield losses and mycotoxin contamination problems in food and feed products. FaWC1, a component of the blue-light receptor White Collar complex (WCC), relies on its transcriptional regulatory zinc finger domain rather than the light-oxygen-voltage domain to regulate pathogenicity of F. asiaticum, although the downstream mechanisms remain obscure. In this study, the pathogenicity factors regulated by FaWC1 were analyzed. It was found that loss of FaWC1 resulted in higher sensitivity to reactive oxygen species (ROS) than in the wild type, while exogenous application of the ROS quencher ascorbic acid restored the pathogenicity of the ΔFawc1 strain to the level of the wild type, indicating that the reduced pathogenicity of the ΔFawc1 strain is due to a defect in ROS tolerance. Moreover, the expression levels of the high-osmolarity glycerol (HOG) mitogen-activated protein kinase (MAPK) pathway genes and their downstream genes encoding ROS scavenging enzymes were downregulated in the ΔFawc1 mutant. Upon ROS stimulation, the FaHOG1-green fluorescent protein (GFP)-expressing signal driven by the native promoter was inducible in the wild type but negligible in the ΔFawc1 strain. Overexpressing Fahog1 in the ΔFawc1 strain could recover the ROS tolerance and pathogenicity of the ΔFawc1 mutant, but it remained defective in light responsiveness. In summary, this study dissected the roles of the blue-light receptor component FaWC1 in regulating expression levels of the intracellular HOG-MAPK signaling pathway to affect ROS sensitivity and pathogenicity in F. asiaticum. IMPORTANCE The well-conserved fungal blue-light receptor White Collar complex (WCC) is known to regulate virulence of several pathogenic species for either plant or human hosts, but how WCC determines fungal pathogenicity remains largely unknown. The WCC component FaWC1 in the cereal pathogen Fusarium asiaticum was previously found to be required for full virulence. The present study dissected the roles of FaWC1 in regulating the intracellular HOG MAPK signaling pathway to affect ROS sensitivity and pathogenicity in F. asiaticum. This work thus extends knowledge of the association between fungal light receptors and the intracellular stress signaling pathway to regulate oxidative stress tolerance and pathogenicity in an epidemiologically important fungal pathogen of cereal crops.

would be involved in pathogenicity in different fungi, and here is another example showing that the role of this highly conserved protein is independent of the role in light sensing. Second, there are previous links between light sensing and the HOG pathway (perhaps the best example being in Aspergillus nidulans and red light sensing phytochrome), so this work nicely establishes a link between the blue light pathway and HOG pathway.
Two minor questions for consideration are: Mutation of HOG1 often causes a suite of phenotypes in additional to increased sensitivity to oxidative stress. Are any of these seen in the FaHOG1 mutant, and, to follow on if they are, also in the FaWC1 mutant?
The current model has FaWC1 controlling transcription of FaHOG1; it might be good to look for DNA binding sites in the promoter as another line of evidence for direct regulation.
Minor typographical or editorial considerations.
Ine 44: 'FaWC1 relies on its'. Line 47: 'It's' to 'It was'. Line 50: is due to a defect in ROS'. Lines 61-62: 'but how WCC determines fungal pathogenicity'. Line 67: 'pathogenicity in an important'. Line 93: 'could' to 'can'. Lines 111, 363: 'It's' to 'It is'. Line 134: move 'could' to be 'components could be associated'. Line 141: 'predominant set of pathogens for'. Line 151: 'demonstrated a light-independent role'. Line 183: 'glycol' for 'glycerol'. Line 214: clarify 'The experiment was repeated three times', i.e. does this mean as written done on three independent occasions, or does it refer to biological replicates? Line 215: 'used in the present study'. Line 226: 'cultivar'. Line 227: 'cut with scissors and'. Lines 231-2: grown in a greenhouse'. Line 241: result comes too early, so perhaps 'FaWC1 has a role in oxidative stress tolerance'. Reviewer #2 (Comments for the Author): The white collar complex (WCC) is the main photoreceptor for fungal responses to blue light. WC1 also has light-independent functions. Having shown previously that the virulence and light-sensing functions can be separated, this report brings evidence that WC1 of Fusarium asiaticum (closely related to F. graminearum) promotes virulence through its role in sensing oxidative stress, independently of the flavin-binding LOV domain.
Two main issues would need to be addressed, both related to the WC1-Hog1 genetic interaction. First, rescue of wc1 phenotypes by Hog1 overexpression supports the hypothesis that the Hog1 MAPK may be acting downstream of WC1, or co-regulating some of the same genes. The critical factor though for MAP kinases is their phosphorylation level, not necessarily their expression level. Second, although much evidence is gained from a line overexpressing Hog1, no data are provided to conclude that Hog1 is overexpressed. These concerns could be addressed by immmunoblot assays; for overexpression, strong support could be obtained, at least, by qPCR showing increased expression levels of Hog1 in the overexpression line. The qPCR assays are already in place ( Figure 4) but it seems the OE line was not yet tested.
Other comments: major: in Figures 2 and 5, only single spore images are shown. More images, or quantitation would be needed to strengthen the conclusions that GFP nuclear retention is changed by the treatments. line 246-248 Growth rate inhibition is stronger in the wc1 mutant. This implies that WCC is not the photoreceptor for this particular light effect, an interesting finding but not discussed either here or in the Discussion.
line 255 -sensitivity to ROS or ROS production? Figure 2A looks like ROS production.
line 277 typo into to line 238 -all these four -the pattern differs between the different genes, please give a little more detail line 293 (and in another instance as well) "dwarf expression" better "decreased expression" line 344-5 can replace "Meanwhile" with "Futhermore" and also rewrite for example as follows -..strains caused levels of stem rot similar to or higher than the WT strain ( Figure 8). line 638 (references) delete "dagger" Staff Comments:

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Dear Editors and Reviewers,
Thank you very much for your message dated on 4 th Feb 2023 with very constructive comments and suggestions concerning our manuscript entitled "White Collar 1 modulates oxidative sensitivity and virulence via regulating the HOG1 pathway in Fusarium asiaticum" (Spectrum05206-22). Those comments are helpful for revising and improving our paper, and the kind suggestions are also of crucial significance to our future research.
We have carefully studied all the comments, carried out additional experiments as suggested, and made revisions in the updated manuscript, which we hope to satisfactorily meet with approval. All specific revisions have been marked in red font in the revised manuscript, and the detailed explanations are addressed in the following

Point-by-Point Responses to Reviewers' Comments -Reviewer #1:
The research defines the basis by which a blue light sensor impacts the pathogenicity of the plant pathogen Fusarium asiaticum, a close relative of F. graminearum, and a major problem in east Asia. Some of the authors had previously established the key part of FaWC1 for pathogenicity is its DNA binding zinc finger domain. Here they discover the downstream components involved, tied to a decrease in oxidative stress tolerance as mediated via the HOG signaling pathway. The findings should be of wide interest to those exploring signal transduction pathways in fungi. First, for many years it was puzzling why a light receptor would be involved in pathogenicity in different fungi, and here is another example showing that the role of this highly conserved protein is independent of the role in light sensing. Second, there are previous links between light sensing and the HOG pathway (perhaps the best example being in Aspergillus nidulans and red light sensing phytochrome), so this work nicely establishes a link between the blue light pathway and HOG pathway.
Response: Thanks for the reviewer's general comments on this manuscript. Two minor questions for consideration are: Mutation of HOG1 often causes a suite of phenotypes in additional to increased sensitivity to oxidative stress. Are any of these seen in the FaHOG1 mutant, and, to follow on if they are, also in the FaWC1 mutant?
Response: Thanks very much for this comment. The Fahog1 knock-out mutant indeed shows a suit of defects, including but not limited to hyphal growth, resistance to oxidative agent, osmotic stress tolerance, sexual and asexual development, carotenoid synthesis and pathogenicity. Some of the ΔFahog1 defects, including resistance to oxidative agent, carotenoid synthesis, sexual development, and pathogenicity, were also seen in the ΔFawc1 mutant. Overexpressing Fahog1 in the ΔFawc1 mutant could only restore the defects in resistance to oxidative agent and pathogenicity, but not the defects in sexual development and carotenoid synthesis. These findings may suggest that the transcript abundancy of Fahog1 pathway is regulated by FaWC1 in response to oxidative stimulus and host infection, although the FaHOG1 and FaWC1 can still have their own independent roles in regulating stress responses and metabolisms.
The current model has FaWC1 controlling transcription of FaHOG1; it might be good to look for DNA binding sites in the promoter as another line of evidence for direct regulation.
Response: Thanks for the constructive comment. We fully agree that it is worth to explore whether direct regulation exist between FaWC1 and the Hog1 pathway genes.
Actually, we did try ChIP assay with the ΔFawc1::FaWC1-GFP strain, however, we didn't succeed in obtaining the immunoprecipitated DNA library using the GFP antibody, probably because the absolute expression abundancy of the FaWC1-GFP protein is low in the fungus. Alternatively, we carried out the in vitro strategy, Electrophoretic mobility shift assay (EMSA). There are two predicted motifs, TCTTCCTCCTC and CCATCTAT, in the Fahog1 promoter region that can be bound by FaWC1. Subsequently, EMSA assay confirmed that FaWC1 could indeed bind to the promoter regions of Fahog1 (Figure 7 in the revised manuscript). Although, in vivo evidence for direct regulation of FaWC1 on transcription of Fahog1 is still lacking, the genetic and in vitro biochemical data obtained in this study could support the assumption that appropriate expression patterns of Fahog1 in response to stresses is regulated by FaWC1.
Minor typographical or editorial considerations.
The white collar complex (WCC) is the main photoreceptor for fungal responses to blue light. WC1 also has light-independent functions. Having shown previously that the virulence and light-sensing functions can be separated, this report brings evidence that WC1 of Fusarium asiaticum (closely related to F. graminearum) promotes virulence through its role in sensing oxidative stress, independently of the flavin-binding LOV domain.
Two main issues would need to be addressed. First, rescue of wc1 phenotypes by Hog1 overexpression supports the hypothesis that the Hog1 MAPK may be acting downstream of WC1, or co-regulating some of the same genes. The critical factor though for MAP kinases is their phosphorylation level, not necessarily their expression level.
Response: Thank you very much for this comment. We totally agree that a critical factor for MAP kinases is their phosphorylation level. Additional Western Blot assay was thus carried out, using the anti-Hog1p and anti-phospho-p38 antibodies. The result showed that loss of FaWC1 could also alter the phosphorylation level of FaHOG1 in response to stresses ( Figure 8 in the revised manuscript).
Second, although much evidence is gained from a line overexpressing Hog1, no data are provided to conclude that Hog1 is overexpressed. These concerns could be addressed by immmunoblot assays; for overexpression, strong support could be obtained, at least, by qPCR showing increased expression levels of Hog1 in the overexpression line. The qPCR assays are already in place (Figure 4) but it seems the OE line was not yet tested.
Response: Thanks for the suggestion. We have detected Fahog1 expression levels in the ∆Fawc1::Fahog1 OE by qPCR assays, and added the data in the revised manuscript as a supplementary Figure S1.
In Figures 2 and 5, only single spore images are shown. More images, or quantitation would be needed to strengthen the conclusions that GFP nuclear retention is changed by the treatments.
Response: Thank you very much for this important comment. We have added less magnified figures showing more spores in each view as supplementary Figure S2 to indicate the overall GFP nuclear retention status of each sample. 4. Line 199 and elsewhere: "testify" better "tested" Response: It has been revised as suggested.
5. Line 184 -50 mg/ml -probably micrograms/ml? Response: Thanks for this comment. Actually, 50 mg/ml is the concentration of the geneticin mother solution, the working concentration in selection medium is 50 μg/ml.
We have revised it accordingly.
Lines 197-198 there seems to be a typo in the wavelengths, DAPI is detected by UVA excitation and blue fluorescence emission Response: Thank you very much for your comment. I am sorry that it is a typo in the wavelengths. The Zeiss microscope with the filter set for excitation of DAPI ranges from 358-360 nm, and emission spectrum is 460-461 nm. Accordingly, the wavelengths for detecting DAPI have been revised in the text.
Line 200 -better: photorepair of UV damage Response: Thanks for the helpful comment. We have revised it as suggested.
Line 247 -FaWC2 should be FaWC1, if I understood correctly Response: Thank you very much for the comment. We have corrected it.
Line 246-248 Growth rate inhibition is stronger in the wc1 mutant. This implies that WCC is not the photoreceptor for this particular light effect, an interesting finding but not discussed either here or in the Discussion.
Response: Thank you very much for this comment. The related discussion has thus been revised accordingly in the re-submitted manuscript (Line 405-409).
Line 255 -sensitivity to ROS or ROS production? Figure 2A looks like ROS production.