Plant YTHDF proteins are direct effectors of antiviral immunity against an N6 ‐methyladenosine‐containing RNA virus

  1. Mireya Martínez‐Pérez
  2. Frederic Aparicio
  3. Laura Arribas‐Hernández
  4. Mathias Due Tankmar
  5. Sarah Rennie
  6. Sören von Bülow
  7. Kresten Lindorff‐Larsen
  8. Peter Brodersen
  9. Vicente Pallas

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Abstract

In virus–host interactions, nucleic acid‐directed first lines of defense that allow viral clearance without compromising growth are of paramount importance. Plants use the RNA interference pathway as a basal antiviral immune system, but additional RNA‐based mechanisms of defense also exist. The infectivity of a plant positive‐strand RNA virus, alfalfa mosaic virus (AMV), relies on the demethylation of viral RNA by the recruitment of the cellular N6 ‐methyladenosine (m 6 A) demethylase ALKBH9B, but how demethylation of viral RNA promotes AMV infection remains unknown. Here, we show that inactivation of the Arabidopsis cytoplasmic YT521‐B homology domain (YTH)‐containing m 6 A‐binding proteins ECT2, ECT3, and ECT5 is sufficient to restore AMV infectivity in partially resistant alkbh9b mutants. We further show that the antiviral function of ECT2 is distinct from its previously demonstrated function in the promotion of primordial cell proliferation: an ect2 mutant carrying a small deletion in its intrinsically disordered region is partially compromised for antiviral defense but not for developmental functions. These results indicate that the m 6 A‐YTHDF axis constitutes a novel branch of basal antiviral immunity in plants.

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  1. Version published to 10.15252/embj.2022113378
  2. Review Commons

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    Reply to the reviewers

    1. General Statements

            We greatly appreciate the valuable comments from the referees, which have generally been very positive and constructive. The three referees have emphasized the significance of our study that opens a new direction of research regarding the role of RNA modification in viral defense. In addition, the reviewers confirm our view that the audience of our work would be broad.

        The major concerns of the reviewers are limited to four main points:

    1. i) to be clearer in our description on the effect of the m6A-YTHDF axis on the viral infectivity and avoid making assumptions on effects on replication (ref. #1 and #3);

    2. ii) reviewer 1 finds that the title and conclusion of this manuscript defining YTHDF proteins (ECTs) as "direct effectors of antiviral immunity" is misleading. Nonetheless, as detailed below, Reviewer 1 confuses mere knowledge of effects of m6A with those conferred by YTHDF proteins binding to m6A, and indeed overlooks nearly all evidence presented in the paper for how m6A in AMV confers antiviral resistance (i.e. mechanistic insight); iii) the discussion on the relative importance of antiviral RNA silencing and m6A-YTHDF against AMV;

    3. iv) to establish more clearly whether the phase separating capability of IDRs in the reading proteins correlates with the antiviral activity (reviewer 2). We have already completed substantial experimental work to address several of these points. Nonetheless, we find it prudent to ask for an extension of the revision time beyond four weeks to allow for repeats of a few of the infection experiments in question. In the following section, we specify a plan of action for the revisions.

    2. Description of the planned revisions

    • *Regarding the four major concerns raised by the reviewers, we will experimentally address the last two, whereas we think the first two do not need any further experimental work, as explained in section 4. Thus, the working plan for points #3 and #4 will be as follows:

    iii) the discussion on the relative importance of antiviral RNA silencing and m6A-YTHDF against AMV and related viruses

    As we mention in the manuscript (discussion, first chapter), AMV *“is one of only very few studied plant RNA viruses for which no anti-RNAi effector has been identified. In addition, prunus necrotic ringspot virus (PNRSV), a virus genetically and functionally closely related to AMV (Pallas et al, 2013), does not induce easily detectable siRNAs, unlike nearly all other studied plant RNA viruses (Herranz et al, 2015)”. *

    Thus, we do not come up with a strong judgment on whether RNAi is more or less important than m6A-YTHDFs for AMV resistance.

    In any case, although these indirect observations seem to be quite solid, we agree with the reviewer that conclusive evidence to discard RNAi as a defense layer against AMV, at least at the time where ECTs are acting, is lacking. Thus, we plan to evaluate how the absence of the main components of the RNAi machinery affects AMV infection and if this ‘universal’ defense layer interferes/overlaps with the ECTs antiviral defense observed here. Realistically, this will take us 8-10 weeks. The experiments within this topic are based on established and published methods and thus, on solid experience. We do not expect any fallback solution and the results will be conclusive in this sense. We also note that the very time-consuming part of constructing mutants defective in both RNAi and m6A-ECT components (in this case, ect2/ect3/rdr6), as well as a first round of infection assays has already been completed at this point

    iv) To establish more clearly whether the phase separating capability of IDRs in the reading proteins correlates with the antiviral activity (Reviewer 2).

    We agree with Reviewer 2 that this is an interesting and important question. Hence, we have teamed up with the group of Prof. Kresten Lindorff-Larsen, expert in molecular simulations of protein folding and interaction. The Lindorff-Larsen group has recently published a powerful computational approach to simulate phase separation behavior of intrinsically disordered proteins (IDPs) or regions of proteins (IDRs) (Tesei et al., 2021, Accurate model of liquid-liquid phase behavior of intrinsically disordered proteins from optimization of single-chain properties, *PNAS *118, (44) e2111696118). Applying this simulation method to the Arabidopsis ECT proteins establishes two facts that we will incorporate into a revised version:

    • The IDR of ECT2 shows marked phase separation propensity, in agreement with the experimental evidence published in Arribas-Hernández et al., 2018, Plant Cell.
    • The deletion mutant of ECT2 (ΔN5) with defective antiviral activity, yet unaffected ability to accelerate growth of leaf primordia shows markedly reduced phase separation propensity driven, in the main, by the many tyrosine residues in the region deleted in the mutant. These results suggest that phase separation capability indeed correlates with antiviral activity.

    Since not only ECT2, but also ECT3, ECT5 and, to some extent, ECT4, participate in AMV resistance, we plan further simulation work on these proteins during the first two weeks of January 2023 before submission of a revised version of the manuscript.

    3. Description of the revisions that have already been incorporated in the transferred manuscript

      •        All the minor concerns raised by the three reviewers have been addressed and we have incorporated all of their suggestions in this intermediate version.

    4. Description of analyses that authors prefer not to carry out

    • *As previously mentioned, we believe that points 1 and 2 do not require an experimental approach to be addressed for the following reasons:

    i) to be clearer in our description on the effect of the m6A-YTHDF axis on the viral infectivity and avoid making assumptions on effects on replication (ref. #1 and #3)

            We agree with the reviewer that the term 'inhibition of viral replication' was not very appropriate because the idea that was intended to be conveyed was that of viral accumulation.        Hence, we will change this use of language, and we thank the reviewer for pointing out this inaccurate description.

    When it comes to differences between effects on infection in inoculated and non-inoculated leaves, there may be a slight misunderstanding, perhaps because we were not clear enough in our originally submitted version. In reality, there are some differences even in inoculated leaves between wild type and ect mutants, especially in the triple mutant, but the slightly higher accumulation in* ect* mutants is not clearly observed in every experiment and hence, does not always rise to the level of significance. Although it is possible that, at local level, ALKBH9B-mediated m6A would have other ECTs-independent effects, similar to what has been described for some animal viruses (Baquero-Pérez et al., 2021. Viruses), we think that the most likely explanation for this phenomenon is a combination of infection titers and ECT redundancy.

    The suggestion to use protoplasts is very accurate, but it would not resolve any doubt in this scenario, because ECTs are mainly expressed in mitotically active cells (Arribas-Hernández et al, 2020, 2018) and, since mature tissues make up the better part of the leaves used to isolate protoplasts, only few of the isolated cells would be useful. In addition, we previously showed that AMV accumulation is reduced in alkbh9b protoplasts compared to WT (Martínez-Pérez et al., 2021. Front. Microbiol.), which suggests that m6A levels of vRNAs are critical for the first stages of the infection, but in that case no problems with the expression pattern of the demethylase were expected.

    ii) The title and conclusion of this manuscript defined YTHDF proteins (ECTs) as "direct effectors of antiviral immunity", which is misleading. Effector molecules of an antiviral immunity cannot be identified when the effector mechanism is unknown;

    In this regard, we have a very different vision from the one the reviewer proposes. We believe that it is not correct to say that the effector molecules of an antiviral immunity cannot be identified until its mechanism is demonstrated. In fact, RNA silencing effectors were discovered long before their mechanism was elucidated in detail. One molecular interpretation of the Flor’s seminal gene-for-gene model, in terms of receptor/effector recognition, is that specific interaction between the receptor and its recognized (cognate) effector protein triggers resistance.

    Furthermore, we strongly believe that we provide enough arguments to propose a model, although, as we comment in the end of the discussion, “we view this model as a conceptual framework of value in the design of future experiments to test its validity”. The reasoning that we show here is the following:

    1. The m6A binding proteins are necessary for the antiviral response.
    2. At least ECT2 recognizes AMV RNAs in vivo and that its m6A-binding capacity is necessary to play a role in AMV infection.
    3. Simply losing methylase activity – with the same developmental defects as ect2/3/ – does not lead to the same degree of loss of resistance, and you can affect AMV resistance without affecting developmental functions of ECT2. Altogether, these observations justify the proposal that m6A exerts antiviral effects by acting as binding sites of ECT proteins in viral RNA, which we consider a clear mechanistic advance.

    Bearing in mind that m6A-modified vRNAs might concentrate in replication complexes and that MeRIP-seq methodology to map m6A revealed site multiplicity in the genome of some RNA viruses (Gokhale et al., 2016. Cell Host&Microb; Martínez-Pérez et al., 2017; Lichinchi et al., 2016. Nat Microbiol; Lichinchi et al., 2016. Cell Host&Microb; Marquez-Molins et al, 2022), our results recalled the previously proposed model in which m6A sites multiplicity causes the phase separation of these RNAs through the interaction of the IDRs of the YTH proteins (Ries et al, 2019; Fu & Zhuang, 2020; Gao et al, 2019). Now, with the new simulations of phase separation behavior, although still a model that requires further experimental tests, we have better evidence to support the model that it is related to LLPS of ECT-bound viral RNA. Therefore, we firmly believe that our title conceptually reflects the basic concepts of resistance induction in virus-plant interactions.

  3. Review Commons

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    Referee #3

    Evidence, reproducibility and clarity

    Previously, the authors showed evidence that m6A modifications of AMV RNAs erased by the host ALKBH9b enhances AMV spread in Arabidopsis. In this paper, the authors show by transcriptome analysis and RT-qPCR that the accumulation of m6A "reader" proteins, ECT2, 3 and 5 are increased during AMV infection in Arabidopsis. Combined mutations of ect2,3 and 5 led to increased AMV accumulation, suggesting that ECT2,3,5 are critical in inhibition of AMV accumulation in systemic leaves of Arabidopsis. Mutagenesis of ECT2 putative m6A-binding pocket did not restore AMV resistance in double-mutant de23 plant, arguing that m6A reader function of this protein is needed to provide resistance against AMV. Then, proximity-labeling was used to show that ECT2 binds to AMV RNA2 and likely RNA1 in planta. Finally, debilitating both the m6A eraser (ALKBH9b) and the reader (ect2,3,5) restored susceptibility to AMV infection in Arabidopsis, thus providing evidence that the m6A reader proteins are critical for resistance against AMV and that AMV exploits ALKBH9b to fight against ECT2,3,5 in plants. Altogether, these are novel and important findings. The paper is well-written.

    I do not have main concerns.

    Minor points:

    • Abstract: "AMV replication" should be replaced by "AMV infection" or "AMV spread", because the locally infected leaves show similar AMV replication/accumulation in de23 and wt Arabidopsis.

    • Alkbh9b mutation causes inhibition of local and systemic movement of AMV, whereas de23 mutant increases AMV accumulation only in systemic leaves (Fig. 1). What is the explanation that de23 does not affect local movement of AMV?

    • The last chapter (p19-20) is too speculative and too long, it does not make the paper more interesting: I recommend shortening it and to minimize speculation.

    Significance

    This paper shows evidence for a new antiviral strategy present in plants. Overall, this is a significant new finding.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary

    In this manuscript, the authors asked the important question of how RNA modification is associated with viral defense in plants. Based on the previous findings that the infectivity of AMV relies on demethylation of viral RNA by recruitment of the cellular m6A demethylase ALKBH9B, in this study, they showed that inactivation of the m6A reader proteins, ECT2, ECT3, and ECT5, is sufficient to restore AMV infectivity in partially resistant alkbh9b mutants. Considering the potential roles of m6A modification in viral defense but the limited knowledge on this topic, the current study opens a new direction of research regarding the role of RNA modification in viral defense.

    Major comments

    • It is interesting to see that the IDR of ECT2 harbors two separable activities employed to achieve different goals: one that stimulates cellular proliferation by binding to endogenous m6A-containing mRNA, and one that effects basal antiviral resistance when ECT2 binds to hypermethylated viral RNA. Considering that the IDR of a protein contributes the chaperone activity of the protein and then can increase the binding capacity of the protein to different substrates, it would be more informative if the authors discuss whether the RNA chaperone activity of IDR of ECT2 is possibly involved in the different processes.

    • It is interesting to propose a working model that m6A site multiplicity in AMV RNA may be a key factor distinguishing it from endogenous mRNA. In that sense, it would be clearer if the authors describe how many m6A sites are present in AMV RNA. Are these m6A sites clustered in certain regions of the viral RNA important for viral replication?

    • It is not clear what the correlation between the phase separation capability of ECT proteins and viral infection is.

    Minor comments

    • In page 6, what is the alfalfa mosaic virus (AMV) RNA 3? Are there AMV RNA 1 and 2? What are their differences? Is the m6A-YTH module specific to AMV RNA 3?

    • Name of the mutants; it would be better if same name was used for the mutant throughout the manuscript and in figures; for instance, ect2-1/ect5-2 and de25, as well as ect2-1/ect3-1/ect5-4 and te235 were used, which is not easy to follow.

    • Figure 4G legend is missing.

    Significance

    Considering the potential roles of m6A modification in viral defense but the limited knowledge on this topic, the current study opens a new direction of research regarding the role of RNA modification in viral defense.

    The audience will be broad, including any persons who are working on epitranscriptomics, plant sciences, viral infection, and clinical application. I am working on epitranscriptomic RNA modification in plant development and abiotic stress responses.

  5. Review Commons

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    Referee #1

    Evidence, reproducibility and clarity

    The authors have shown previously that local and systemic infection of alfalfa mosaic virus (AMV) is inhibited and the relative abundance of m6A in the viral RNAs is increased in mutant Arabidopsis plants defective in the m6A demethylase gene ALKBH9B. Here the authors show that genetic inactivation of 2 or 3 m6A-binding proteins (ECTs or m6A readers) enhances the systemic, but not the local, infection of AMV. Notably, the systemic virus resistance of the demethylase mutant plants is largely eliminated by inactivating the same set of ECTs. Moreover, the authors detected in vivo association of ECT2 with AMV RNAs and identified a N-terminal motif of ECT2 that is necessary for AMV resistance, but dispensable for its role in endogenous developmental functions. These findings together provide further evidence to support their earlier conclusion for an antiviral role of m6A methylation of viral RNAs. Unfortunately, several key questions remain unknown and should be addressed to justify publication in EMBO J.

    Major comments

    1. The authors reported in 2017 that both the local and systemic infection of AMV is inhibited in the m6A demethylase mutant plants (PNAS 114:10755-60). In this work, they show that inactivation of ECTs enhances only the systemic AMV infection, but has no effect on the local infection (Fig. 2). Studies presented in neither Fig. 5 nor Fig. 6 examined possible effects on the local virus infection. Moreover, line 1 of page 13 mentioned a model that ECT binding "causes inhibition of viral replication". To resolve these contradictory descriptions on the step of the virus infection cycle targeted by m6A RNA methylation, it is essential to perform protoplast replication assays to determine whether the mutation of either the demethylase gene or ECTs affects viral RNA replication at single-cell level and to examine local infection for the studies presented in Figs 5 & 6.

    2. The title and conclusion of this manuscript defined YTHDF proteins (ECTs) as "direct effectors of antiviral immunity", which is misleading. It remains completely unknown why m6A methylation of viral RNAs is inhibitory to virus infection. The available data suggest that it may act by blocking virus replication and/or movement or by enhancing any of the several known antiviral responses. Effector molecules of an antiviral immunity cannot be identified when the effector mechanism is unknown.

    3. At the end of page 13 and elsewhere in this manuscript, the authors conclude that "the m6A-ECT axis constitutes a first, basal layer of antiviral defense", a conclusion that is not supported by the evidence presented. This conclusion will be incorrect if m6A methylation of viral RNAs does not inhibit virus accumulation levels in the protoplast virus replication assays as requested above.

    4. In the first paragraph of page 17 and elsewhere in this manuscript, the authors question the relative importance of antiviral RNA silencing against AMV and related viruses as compared to m6A RNA methylation. It is important to determine if AMV becomes more virulent in RNAi-defective mutant plants such as dcl2/4 double mutant and if m6A RNA methylation also confers AMV resistance in RNAi-defective mutant plants.

    Minor comments:

    1. State the time post-inoculation when the samples were taken for the RNA-seq analysis in Fig.1.

    2. State whether all of the northern blotting experiments used RNA extracted from single plant or pooled plants and had been repeated.

    3. Verify that the statistical analysis method used in virus titer quantitative analysis is student t-test or one-way ANOVA.

    4. The legend to Fig. 4F should be Fig. 4F and 4G.

    5. Explain what ∆2 means in Fig. 6B.

    6. If both the left and right bars correspond to 1 cm, 9-DAG-old plants would be much bigger than 16-DAG-old plants, which cannot be true.

    Significance

    Advance:

    The authors have shown previously that local and systemic infection of alfalfa mosaic virus (AMV) is inhibited and the relative abundance of m6A in the viral RNAs is increased in mutant Arabidopsis plants defective in the m6A demethylase gene ALKBH9B. Here the authors show that genetic inactivation of 2 or 3 m6A-binding proteins (ECTs or m6A readers) enhances the systemic, but not the local, infection of AMV. Notably, the systemic virus resistance of the demethylase mutant plants is largely eliminated by inactivating the same set of ECTs. Moreover, the authors detected in vivo association of ECT2 with AMV RNAs and identified a N-terminal motif of ECT2 that is necessary for AMV resistance, but dispensable for its role in endogenous developmental functions. These findings together provide further evidence to support their earlier conclusion for an antiviral role of m6A methylation of viral RNAs.

    Audience:

    Broad, including those interested in RNA modifications, antiviral immunity, and plant biology.

    Your expertise:

    Antiviral immunity, plant biology

  6. Version published to 10.1101/2022.10.19.512835v1 on bioRxiv