Establishment of Neurospora crassa as a model organism for fungal virology

The filamentous fungus Neurospora crassa is used as a model organism for genetics, developmental biology and molecular biology. Remarkably, it is not known to host or to be susceptible to infection with any viruses. Here, we identify diverse RNA viruses in N. crassa and other Neurospora species, and show that N. crassa supports the replication of these viruses as well as some viruses from other fungi. Several encapsidated double-stranded RNA viruses and capsid-less positive-sense single-stranded RNA viruses can be experimentally introduced into N. crassa protoplasts or spheroplasts. This allowed us to examine viral replication and RNAi-mediated antiviral responses in this organism. We show that viral infection upregulates the transcription of RNAi components, and that Dicer proteins (DCL-1, DCL-2) and an Argonaute (QDE-2) participate in suppression of viral replication. Our study thus establishes N. crassa as a model system for the study of host-virus interactions.

1. Since a major question in this study concerns the role of the Neurospora RNAi pathway in the antiviral response, more complete overview of the known Neurospora pathway is warranted in the introduction. In addition, the current study is also highly related to a previous study (Reference 37) which demonstrated the existence of dsRNA-induced transcriptional response, including many RNAi components and putative antiviral genes. This earlier study suggested that this response is a host defense response for viral infection. Moreover, more coverage of the relevant studies in C. parasitica is also needed. 2. Since this study established the system to test the role of potential antiviral genes in Neurospora, the authors probably can check whether some of the putative antiviral genes (Mx, RNA helicase and others) previously identified in reference 37 are involved in antiviral response. It is not necessary to examine many of them, but even if the authors can demonstrate the involvement of just one of the novel antiviral genes will greatly strengthen this study by providing novel mechanistic insights of the antiviral response.. 3. Line 129: Why NiFV1 dsRNA accumulated much less in N. crassa than in N. intermedia? 4. Line 138-140: It will be nice to provide more background on why a helper-5 strain is needed. 5. Line 160 and Figure 2: Why the accumulation of NcPV1 in the RNAi mutants was much lower than those for NcFV1? 6. Figure 3c, more explanation is needed for this result in the text. Did the authors check the induction of other DRAGs previously identified in reference 37? 7. Figure 3e and line 188: It was previously shown that dsRNA expression (37) or DNA damage (which also induced dsRNA from rDNA loci) (Lee et al, 2009) can induced the endogenous QDE-2 levels. It is surprising that the tagged-QDE2 level was not increased and even decreased after viral infection. Can the comparison of NcFV1 and NcPV1 viral sequences reveal anything interesting based on what was previously known viral RNAi suppressors? 8. Figure 4: The dsRNA-induced transcriptional pathway should be incorporated into the figure.
Reviewer #2 (Remarks to the Author): The major claim of the paper is the establishment of a system to study virus-fungus interaction in the model fungus Neurospora crassa, and its use to determine the fungal genes involved in antiviral defense in N. crassa. The authors showed also for the first time the presence of different type viruses in three Neurospora species, and the replication of viruses of Rosellinia necatrix and Chryphonectria parasitica in N. crassa. The authors showed results that will be of interest to others working with mycoviruses and fungus-virus interactions. The results obtained in this work were compared by the authors with previous results obtained in the fungus C. parasitica, that has been extensively used as model to study virus-fungus interactions using mycoviruses of C. parasitica or Editorial Note: This manuscript was transferred from another journal. Mentions of the other journal have been redacted.
of other fungus as Rosellinia necatrix. The level of detail provided in Methods will allow to reproduce some experiments, but for instance for the preparation of protoplasts or for protoplast fusion will be necessary to consult other papers. For a better understanding of the experimental mycoviruses introduction, the authors could draw an explanatory scheme to clarify assays described in materials and methods and results sections. Some specific comments: NcFV1 A type was used for this study, explain the difference with type B and C, and the interest of using A type instead other type. Indicate the natural host of Mycoreovirus 1. In this work it is shown that Neurospora spp. are naturally infected by partitivirus, even if they are infected with Betapartitivirus, the authors should explain why is interesting to use Rosellinia necatrix partitivirus 2 instead another type of virus, to increase the diversity of studied viruses. Some experiments included RnPV2, but not all of them, the inclusion of this mycovirus in all the assays would increase the relevance of the manuscript, since the fungus could be proposed as a general model system for the study of mycovirus-fungus interaction. The authors showed the dsRNA accumulation of RnPV2 and MyRV1, but another method should be used to demonstrate the replication of both viruses in N. crassa and if this replication is stable. According to Extended Data Table 1, Neurospora spp. can also host viruses of the family Togaviridae. Include in the list of viral families in the text.
In the third section of results, the conclusion is that the two Dicers DCL-1/2 and one Argonaut QDE-2 play a major role in anti-viral RNA silencing, but according with the results this is true for the fusarivirus. For the partitivirus, even when the differences are no significant between wt and mutants, DCL-1/2 has no role in anti-viral silencing, only in (Dqde-2/Dsms-2) and (Dqde-1/DSad-1/Drrp-3) there is an increase of NcPV1 accumulation. The partitivirus is a dsRNA virus, and the fusarivirus is a ssRNA (+) virus, and dsRNA accumulation has been used to determine the effect of the mutation of genes involved in silencing pathways, the results are clear, but maybe a northern blot would be a complementary analysis for this purpose. The Northern blot in Figure 3a has a strong background and it is difficult to see the bands, maybe the authors should substitute the photo in the figure. Figure 3c shows different gene expression, but this analysis was performed using "12 possible virus-infected strains and 12 highly likely virus-free strains" (pag. 17 in material and methods), if the authors can no assure that the fungal strains are o no infected with mycoviruses, this analysis should not be considered in the result section.
In the discussion section the authors summarized the results and only discussed with other studies developed with in C. parasitica, but another studies have been published about the roles of argonautes and dicers, for instance in Sclerotinia sclerotiorum, that maybe could be mentioned.
Reviewer #3 (Remarks to the Author): The study shows that several double stranded RNA viruses and newly identified ss(+)RA viruses can be transfected to Neurospora crassa. The authors also show that RNAi genes, DCL-1/2 and QDE-2, were induced upon virus infection. One argonaute mutant, Hqde-2, had an increase in accumulation of NcFV1. Probed by NcFV1, they also showed that the two dicer genes work redundantly in antiviral defense, and that the other RNAi genes are not playing redundant roles in antiviral RNAi. The RNA-Seq data showed differential expression of dcl-2, qde-2, and rrp-3 genes in the virus infected wild-type strain, as well as the corresponding changes in protein accumulation in a virus-specific manner. The transfections were achieved by multiple methods, indicating that N. crassa will be a very good model system to study virus-host interactions and co-infections of dsand ss(+) viral genome types. Additionally, the manuscript presented the evidence to support the model of antiviral RNA silencing pathway. The manuscript contains extensive data/analysis and provides novel insights into virus-host interaction in a model system, which sets the foundation for a lot of in depth investigation on a genetic system with ease, such as potential RNA silencing suppressor targeting argonaute to induce autophagy, as eluded by the authors. The system can also be applicable to understanding other agricultural important fungal pathogens as hosts of viruses, especially for fungi belonging to Ascomycetes. I think the authors can elaborate more on the use of N. crassa as a model system and how specific pathogen control or antiviral strategies can be devised. Overall, the last paragraph of discussion can be an opportunity to emphasize the importance of such a system and should be strengthened. Suggestions on minor edits: L30: should be "one gene-one enzyme" L32: probably "because of unconfirmed virus infection despite numerous …." would sound better L47~48: two "generally" in one sentence: maybe change to "commonly" for one of them L50: delete "relatively" to make it sound better L53: add hyphen to "fusion-based" L107: belong into should be "belong to"

REVI EWER COM M ENTS
Reviewer #1 (Remar ks to the Author ): Neurospora crassa has been an important model organism for RNAi research but the antiviral function of the RNAi pathway was not previously demonstrated. In this study, the authors demonstrated that Neurospora is a natural viral host by discovering multiple dsRNA viruses in different Neurospora isolates. In addition, the authors developed the method to demonstrate that N. crassa can be used as an experimental system to examine viral replication and antiviral response. The results showed that the RNAi components Dicer and QDE-2 suppress viral replication in Neurospora. Furthermore, viral infection induces RNAi components transcriptionally. Together, these results represents an important advance in fungal antiviral research by laying the foundation in a model organism. Although the study was well carried out, there are a few issues the author should address before publication. Response: Thank you for the reviewer' s overall positive comments on our work. We must confess that our descriptions of most subdivision are limited due largely to length limitations set by [redacted] which we originally submitted our paper to. We have added explanations to some methods and expand ed the Results and Discussion sections.
1. Since a major question in this study concerns the role of the Neurospora RNAi pathway in the antiviral response, more complete overview of the known Neurospora pathway is warranted in the introduction. In addition, the current study is also highly related to a previous study (Reference 37) which demonstrated the existence of dsRNA-induced transcriptional response, including many RNAi components and putative antiviral genes. This earlier study suggested that this response is a host defense response for viral infection. Moreover, more coverage of the relevant studies in C. parasitica is also needed. Response: The Introduction and associated sections have greatly been expanded to introduce the N. crassa RNA silencing pathways and the C. parasitica antiviral RNA silencing (page 3, 2 nd paragraph).

2.
Since this study established the system to test the role of potential antiviral genes in Neurospora, the authors probably can check whether some of the putative antiviral genes (Mx, RNA helicase and others) previously identified in reference 37 are involved in antiviral response. It is not necessary to examine many of them, but even if the authors can demonstrate the involvement of just one of the nove antiviral genes will greatly strengthen this study by providing novel mechanistic insights of the antiviral response. Response: Thank you for this suggestion. We have added data on Hex1 (page 9, Supplementary Fig. 6) whose orthologue in Fusarium graminearum was previously identified as a host factor for the replication and symptom induction of a fusarivirus of (Son et al., JVI, 2013). [redacted].
3. Line 129: Why NiFV1 dsRNA accumulated much less in N. crassa than in N. intermedia? Response: We assume that the less NiFV1 dsRNA accumulation in N. crassa may be explained by the greater adaptability of NiFV1 to N. intermedia. As mentioned on page 6, similar phenomena were previously observed after virus introduction into new experimental hosts.
4. Line 138-140: It will be nice to provide more background on why a helper-5 strain is needed.
Response: More explanation has been added in the main text and legend of a supplementary figure (Fig. 4) illustrating an experimental procedure for horizontal virus transfer using the helper-5 strain. Figure 2: Why the accumulation of NcPV1 in the RNAi mutants was much lower than those for NcFV1? Response: Please note that these viruses belong to totally different virus families: Partitiviridae and proposed Fusariviridae. Different members even within the same Parttiviridae show different susceptibility to antiviral RNA silencing (Chiba et al., JVI, 2013;Virus Res, 2016;Aulia et al., Curr Res Virol Sci, 2020). Some viruses accumulate much more in RNA silencing-deficient mutants than in wild-type strains, whereas other do not. The current virology cannot explain this phenomenon well.

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6. Figure 3c, more explanation is needed for this result in the text. Did the authors check the induction of other DRAGs previously identified in reference 37? Response: Other dsRNA-induced genes such as NCU04490 (6-16 family), NCU07036 (3' -5' exonuclease), NCU04472 (RNA helicase), NCU09495 (set-6), and NCU00947 (unknown function) have also been confirmed to be induced. This has briefly been touched in the text on page 8. We would rather not include these data in the current manuscript, but report them together with deep virological data in the near future.
7. Figure  Reviewer #2 (Remar ks to the Author ): The major claim of the paper is the establishment of a system to study virus-fungus interaction in the model fungus Neurospora crassa, and its use to determine the fungal genes involved in antiviral defense in N. crassa. The authors showed also for the first time the presence of different type viruses in three Neurospora species, and the replication of viruses of Rosellinia necatrix and Chryphonectria parasitica in N. crassa. The authors showed results that will be of interest to others working with mycoviruses and fungus-virus interactions. The results obtained in this work were compared by the authors with previous results obtained in the fungus C. parasitica, that has been extensively used as model to study virus-fungus interactions using mycoviruses of C. parasitica or of other fungus as Rosellinia necatrix.
The level of detail provided in Methods will allow to reproduce some experiments, but for instance for the preparation of protoplasts or for protoplast fusion will be necessary to consult other papers. For a better understanding of the experimental mycoviruses introduction, the authors could draw an explanatory scheme to clarify assays described in materials and methods and results sections. Response: The reviewer' s positive evaluation of our study is appreciated. The detailed procedures have been described for protoplast fusion (page 12) and illustrated for helper-5 mediated horizontal virus transfer (Supplementary Fig. 4).
Some specific comments: NcFV1 A type was used for this study, explain the difference with type B and C, and the interest of using A type instead other type. Response: NcFV1 subgroups A to C can be differentiated by their phylogenetic relationships and sequence identity. This has been added on page 3.
Indicate the natural host of Mycoreovirus 1. Response: Original host of MyRV1 has been described on page 5.
In this work it is shown that Neurospora spp. are naturally infected by partitivirus, even if they are infected with Betapartitivirus, the authors should explain why is interesting to use Rosellinia necatrix partitivirus 2 instead another type of virus, to increase the diversity of studied viruses. Some experiments included RnPV2, but not all of them, the inclusion of this mycovirus in all the assays would increase the relevance of the manuscript, since the fungus could be proposed as a general model system for the study of mycovirus-fungus interaction.
Response: An explanation has been added as to why RnPV2 was used. Response: Thank you for this suggestion. We feel that it is too early to conclude based solely on the partial genomic sequence that the detected toga-like virus is a member of the family Togaviridae. The partially sequenced toga-like mycoviruses has much shorter genomic elements of <4.0 kb compared to a typical togavirus with a genome of ~10 kb. It is unknown whether the toga-like mycovirus is multi-segmented, or whether it forms spherical particles like animal togaviruses. Therefore, it is safe to wait until the full-genome sequence of the toga-like virus from N. crassa strain D23 is determined. Taking these into account, " alpha-like supergroup," broadly including toga-like, virga-like, and other related viruses, has been added in the text (page 6, Supplementary Table 1).
In the third section of results, the conclusion is that the two Dicers DCL-1/2 and one Argonaut QDE-2 play a major role in anti-viral RNA silencing, but according with the results this is true for the fusarivirus. For the partitivirus, even when the differences are no significant between wt and mutants, DCL-1/2 has no role in anti-viral silencing, only in (Dqde-2/Dsms-2) and (Dqde-1/DSad-1/Drrp-3) there is an increase of NcPV1 accumulation. The partitivirus is a dsRNA virus, and the fusarivirus is a ssRNA (+) virus, and dsRNA accumulation has been used to determine the effect of the mutation of genes involved in silencing pathways, the results are clear, but maybe a northern blot would be a complementary analysis for this purpose. Response: RT-qPCR has been performed on (+)ssRNA of all the three viruses, NcFV1, NcPV1, and RnPV2. The results have been shown in Supplementary Fig. 5 which has a trend similar to their dsRNA accumulation. Whereas over 25-fold elevation of NcFV1 (+)ssRNA was observed in Δdcl-1/2, Δqde-2 and Δqde-2/Δsms-2, no over 4-fold augmentation of the partitivirus (+)ssRNA was observed in any mutant. These observations have been described and interpreted on pages 6 and 11. Figure 3c shows different gene expression, but this analysis was performed using " 12 possible virus-infected strains and 12 highly likely virus-free strains" (pag. 17 in material and methods), if the authors can no assure that the fungal strains are o no infected with mycoviruses, this analysis should not be considered in the result section. Response: We have deleted two N. crassa strains that were not confirmed to be dsRNA positive by our analyses. Thus, Fig. 3c now has included the data of 10 fungal strains and shown similar induction patters to the previous data.
In the discussion section the authors summarized the results and only discussed with other studies developed with in C. parasitica, but another studies have been published about the roles of argonautes and dicers, for instance in Sclerotinia sclerotiorum, that maybe could be mentioned. Response: The discussion section has been expanded to include discussion on the RNA silencing pathways in other filamentous fungi such as S. sclerotiorum and F. graminearum (page 10).
Reviewer #3 (Remar ks to the Author ): The study shows that several double stranded RNA viruses and newly identified ss(+)RA viruses can be transfected to Neurospora crassa. The authors also show that RNAi genes, DCL-1/2 and QDE-2, were induced upon virus infection. One argonaute mutant, Δqde-2, had an increase in accumulation of NcFV1. Probed by NcFV1, they also showed that the two dicer genes work redundantly in antiviral defense, and that the other RNAi genes are not playing redundant roles in antiviral RNAi. The RNA-Seq data showed differential expression of dcl-2, qde-2, and rrp-3 genes in the virus infected wild-type strain, as well as the corresponding changes in protein accumulation in a virus-specific manner. The transfections were achieved by multiple methods, indicating that N. crassa will be a very good model system to study virus-host interactions and co-infections of ds-and ss(+) viral genome types. Additionally, the manuscript presented the evidence to support the model of antiviral RNA silencing pathway.
The manuscript contains extensive data/analysis and provides novel insights into virus-host interaction in a model system, which sets the foundation for a lot of in depth investigation on a genetic system with ease, such as potential RNA silencing suppressor targeting argonaute to induce autophagy, as eluded by the authors. The system can also be applicable to understanding other agricultural important fungal pathogens as hosts of viruses, especially for fungi belonging to Ascomycetes. I think the authors can elaborate more on the use of N. crassa as a model system and how specific pathogen control or antiviral strategies can be devised. Overall, the last paragraph of discussion can be an opportunity to emphasize the importance of such a system and should be strengthened.
Suggestions on minor edits: L30: should be " one gene-one enzyme" L32: probably " because of unconfirmed virus infection despite numerous …." would sound better L47~48: two " generally" in one sentence: maybe change to " commonly" for one of them L50: delete " relatively" to make it sound better L53: add hyphen to " fusion-based" L107: belong into should be " belong to" Response: All of the suggestions have been incorporated. The Discussion has been expanded to discuss the use of N. crassa, as suggested page 11.