Disruption of the mRNA m 6 A writer complex triggers autoimmunity in Arabidopsis

  1. Carey L Metheringham
  2. Anjil K Srivastava
  3. Peter Thorpe
  4. Ankita Maji
  5. Matthew T Parker
  6. Geoffrey J Barton
  7. Gordon G Simpson

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Abstract

Distinguishing self from non-self is crucial to direct immune responses against pathogens. Unmodified RNAs stimulate human innate immunity, but RNA modifications suppress this response. mRNA m 6 A modification is essential for Arabidopsis thaliana viability. However, the molecular basis of the impact of mRNA m 6 A depletion is poorly understood. Here, we show that disruption of the Arabidopsis mRNA m 6 A writer complex triggers autoimmunity. Most gene expression changes in m 6 A writer complex vir-1 mutants grown at 17°C are explained by defence gene activation and are suppressed at 27°C, consistent with the frequent temperature sensitivity of Arabidopsis immunity. Accordingly, we found enhanced pathogen resistance and increased premature cell death in vir-1 mutants at 17°C but not 27°C. Global temperature-sensitive mRNA poly(A) tail length changes accompany these phenotypes. Our results demonstrate that autoimmunity is a major phenotype of mRNA m 6 A writer complex mutants, with important implications for interpreting the role of this modification. Furthermore, we open the broader question of whether unmodified RNA triggers immune signalling in plants.

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    Referee #3

    Evidence, reproducibility and clarity

    This study characterizes autoimmunity in mutant lines of Arabidopsis that are lacking components of the m6A methyltransferase complex (MTC). The molecular results and bacterial pathogen resistance of the lines at low temps as compared to high temps support this hypothesis. However, the phenotypic analysis or new complete lack there of (Figure 6), makes the hypothesis and overall story much less convincing. I give some comments for improving the figures below.

    Figure 6 showing the phenotypes in its current set up is very uninformative. More informative pictures and quantitative analyses of specific developmental phenotypes should be added to show the differences between the phenotypes of the mutant and wild-type plants at the two different temperatures. As of now the reader gets a sense of nothing from the figure. Without this Figure demonstrating a major rescue of phenotype at the 27C temperature the reader is not convinced that the autoimmunity is the major cause of the phenotype

    Supplemental Figure 1 is missing from the review file.

    Significance

    It is notable that a couple of recent studies have already shown the increased resistance of MTC component mutants to pathogens (Prall et al. 2024 and Chen et al. 2024), which weakens the impact of the overall findings. In my honest assessment, this study would be well positioned for publication in a mid-tier plant specific journal (e.g. Plant Physiology) based on the currently included results.

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    Referee #2

    Evidence, reproducibility and clarity

    The manuscript by Metheringham et al. reports on interesting new characterizations of phenotypes caused by genetic inactivation of subunits of the methyl transferase complex responsible for N6-adenosine methylation in (pre)-mRNA ("the m6A writer") in the plant Arabidopsis thaliana. The main claim of the paper is that mutants in these subunits exhibit autoimmunity, a claim that is supported by the following lines of evidence:

    • Transcriptome profiling by mRNA-seq shows a gene expression profile with differential expression of many stress- and defense-related genes.
    • The immunity-like gene expression profile is observed under growth at 17{degree sign}C but not at 27{degree sign}C, consistent with the well-known temperature-sensitivity of some (but not all) innate immunity signaling systems in plants.
    • m6A writer mutants show increased resistance to infection by the virulent Pseudomonas syringae DC3000 strain.
    • The primary biochemical defect in m6A writing is not temperature sensitive, excluding the trivial possibility that the mutant alleles chosen for study are simply ts.

    The observations are important and the manuscript is very well written, a pleasure to read: the problem is clearly presented, the experimental results are presented in a clear, logical succession, and the discussion treats important points.

    The study is valuable pending some manuscript revision on the autoimmunity interpretation of the results obtained, and a few suggested edits that can be included if the authors agree that they would improve the paper.

    The finding that an autoimmune-like state is activated in m6A writer mutants is significant because it provides a warning flag on how such mutants should be used for studying the role of m6A in stress response signaling, including reassessment of previously published work. Whether the stress state really is autoimmunity is subject to some debate, particularly because no genetic evidence to support it has been obtained. The results are nonetheless interesting and constitute an important contribution to the community, even if they remain descriptive and with nearly no insight into molecular mechanisms. My suggestions for improvement are summarized below.

    1. Although the authors do a lot to support the claim that autoimmunity is an element of m6A writer mutant phenotypes, the study does not include genetic evidence to support this claim. This is important, because if the stress/defense gene activation causes some of the morphological phenotypes of m6A writer mutants, one should be able to suppress such defects by mutation of know immune signaling components such as the appropriate nucleotide-binding leucine-rich repeat proteins, or more generic signaling components such as EDS1, PAD4 and SAG1, common to a subset of such intracellular immune receptors. Resistance to pathogens can be observed in mutants with constitutive stress response signaling, and defense-like gene expression can be induced as a secondary of other primary defects, for instance DNA damage. Similarly, while it is true that some types of immune activation are temperature sensitive, others are not 1, and clearly, elevated temperature changes so much of the physiology of the plant that sensitivity to elevated temperature cannot be used as proof of immune activation. Thus, each of the lines of evidence presented is suggestive, not conclusive. Together, they constitute a good argument, but still not a completely satisfactory proof of the main claim. I do not think that this concern means that a lot of genetic work must be undertaken to make this paper publishable, but I think that the authors should be even more careful about how they interpret their observations. I understand that they favor more or less direct activation of autoimmunity, although even if that were true, it would be unclear what the biochemical triggers of such autoimmunity would be (unmethylated RNA, absence or writer components, excess of free m6A-binding proteins etc). However, given the concerns above, I think the authors should dedicate a small paragraph in the discussion to the possibility that the primary cause of stress/defense-gene expression is unclear and may not result from innate immune surveillance of unmethylated mRNA or components of the m6A pathway as favoured by the authors.
    2. It may be of relevance to search promoters of differentially expressed genes for enrichment of cis-elements. This simple approach identified the W-box in the first papers using transcriptome profiling to characterize the immune state in Arabidopsis 2,3, and could perhaps reveal whether a WRKY-driven transcriptional program drives differential expression or whether several other transcription factor classes may also contribute substantially, as may be expected if a more complex stress-related transcriptional program is activated. I do not think that this is a deal breaker, but some additional useful information from the existing data might be gathered in this way.
    3. Stress response activation has also been clearly described in ect2 ect3 ect4 mutants4 and even if the authors find no evidence for PR1 expression in this mutant, it is still of relevance to include a mention of this result in the discussion, together with the discussion of stress response activation seen in writer mutants in earlier reports 5,6. I would not mind the authors being a bit more explicit about what their results mean for studies that try to conclude on the biological relevance of m6A in different types of stress signaling, using phenotypes writer mutants as their primary line of evidence. But this is of course up to the authors to decide on that.
    4. In the introduction on preferred m6A sequence contexts, please clarify that m6A in plants occurs both DRACH in (G)GAU contexts 7,8.
    5. When mentioning convergence on shared signaling components from immune receptors, please include a tiny bit more information for the reader. For instance, EDS1 is mentioned, but this protein is only required for signaling from (some) TIR-NBS-LRRs, not the class of CC-NBS-LRRs. Indeed, signaling by this latter class may not converge on just one to a few components, as their multimerization appears to form the ion channels required for signaling-inducing ion currents.
    6. Please clarify in the introduction and in later parts that only some forms of autoimmunity can be suppressed by elevated temperature. Sentences like "A hallmark of Arabidopsis autoimmunity is temperature sensitivity..." are a bit misleading. Temperature sensitivity has clearly been used to study some forms of EDS1-dependent immunity, to great effect in the TMV-N interaction for instance, but it is not accurate to call temperature sensitivity a "hallmark of autoimmunity".
    7. In the discussion of possible biochemical triggers of autoimmunity in m6A mutants, please consider the following:

    (A) Mention the possibility that the primary trigger may not be immune receptor-surveillance of some defect induced by lack of m6A in mRNA (as discussed above).

    (B) In connection with the consideration that lack of m6A writer components, not m6A in mRNA, may be a signal, you could include the observation from yeast that Ime4 knockouts have a much stronger phenotype than Ime4 catalytically dead mutants or knockouts of the sole yeast YTH-domain Pho92 9. Indeed, it is a bit of an embarrassment to the plant m6A community that we have not yet examined phenotypes of MTA and MTB catalytically dead mutants, and the present report should further urge the community to finally do this important experiment.

    1. Just a tiny typo on page 15, Pst DC3000, not Pst D3000 (of no relevance to the overall assessment, just a help to eliminate annoying errors before final submission).

    REFERENCES

    1. Demont, H. et al. Downstream signaling induced by several plant Toll/interleukin-1 receptor-containing immune proteins is stable at elevated temperature. Cell Reports 44(2025).
    2. Petersen, M. et al. Arabidopsis MAP kinase 4 negatively regulates systemic acquired resistance. Cell 103, 1111-1120 (2000).
    3. Maleck, K. et al. The transcriptome of Arabidopsis thaliana during systemic acquired resistance. Nature Genetics 26, 403-410 (2000).
    4. Arribas-Hernández, L. et al. The YTHDF proteins ECT2 and ECT3 bind largely overlapping target sets and influence target mRNA abundance, not alternative polyadenylation. eLife 10, e72377 (2021).
    5. Bodi, Z. et al. Adenosine Methylation in Arabidopsis mRNA is Associated with the 3' End and Reduced Levels Cause Developmental Defects. Front Plant Sci 3, 48 (2012).
    6. Prall, W. et al. Pathogen-induced m6A dynamics affect plant immunity. The Plant Cell 35, 4155-4172 (2023).
    7. Arribas-Hernández, L. et al. Principles of mRNA targeting via the Arabidopsis m6A-binding protein ECT2. eLife 10, e72375 (2021).
    8. Wang, G. et al. Quantitative profiling of m6A at single base resolution across the life cycle of rice and Arabidopsis. Nature Communications 15, 4881 (2024).
    9. Ensinck, I. et al. The yeast RNA methylation complex consists of conserved yet reconfigured components with m6A-dependent and independent roles. eLife 12, RP87860 (2023).

    Significance

    The finding that an autoimmune-like state is activated in m6A writer mutants is significant because it provides a warning flag on how such mutants should be used for studying the role of m6A in stress response signaling, including reassessment of previously published work. Whether the stress state really is autoimmunity is subject to some debate, particularly because no genetic evidence to support it has been obtained. The results are nonetheless interesting and constitute an important contribution to the community, even if they remain descriptive and with nearly no insight into molecular mechanisms. I wish to congratulate the authors on another valuable contribution to the plant m6A field.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary

    The authors aim to understand the consequences of disrupting N6 methyladenosine, an abundant mRNA modification in plants and other organisms, in Arabidopsis. Genetic ablation of the N6 methyladenosine transferase complex is embryonic lethal in Arabidopsis. Therefore, the authors utilize a hypomorphic allele of VIRILIZER, a component of the complex, to examine gene expression changes and other phenotypes. The authors demonstrate that immune response pathway genes are misregulated in the vir mutant. This transcriptional phenotype is suppressed at higher temperatures, although developmental phenotypes are not. The manuscript provides strong evidence that reduced function of the m6A methyltransferase complex leads to upregulation of immune response genes, although a mechanistic connection between the immune response and m6A in mRNA is not discerned.

    Major comments

    The major claims of the manuscript are that disrupting the m6A writer complex triggers an autoimmune response that is present at 17C and suppressed at 27C (in line with known aspects of Arabidopsis immunity). Consistent with this, they also show that at 17C the vir-1 mutant has more cell death and is more resistant to infection by Pseudomonas syringae. All of these claims are well supported by the data. The authors also claim that polyA tail lengths are different between the two temperatures. They further speculate that mRNAs that lack m6A trigger immune signaling, but this is not directly tested in the study.

    The conclusions about transcriptional activation of the immune response at lower temperatures are sufficiently supported by two types of mRNA sequencing data (direct RNA sequencing and short-read sequences) and appropriate biological replication. The initial profiling was at 22 C, later profiling was at 17C and 27 C. How similar/overlapping were the vir-1 misregulated genes at 17C and 22C? Is the immune response transcriptional signature stronger at 17C than at 22C? The authors sought to determine whether the vir-1 response at 17C was due to pathogen infection of those plants. They used their Illumina RNA-seq data to try and identify pathogen RNAs. They report that there was no significant enrichment of plant pathogen sequences (supplemental table 7). Significant compared to what? Supplemental Table 7 does not indicate that the WT data was assessed and there's no information on significance of enrichment (or nothing obvious, based on column titles). Did the Illumina library prep preparation rely on polyA tails? If so, this is not a sensitive assay to detect bacterial transcripts.

    I found the last section on altered poly(A) tail length and site usage somewhat difficult to follow and the analysis rather cursory. The authors find no difference in polyA site usage in vir-1 at 17C or 27C (although both are different than WT). For Figure 7A, in addition to the histogram of poly A site shift, I would like to see a plot (heatmap?) that compares poly A sites shift for individual mRNAs across samples, instead of only aggregated data. Are there individual mRNAs that differ between 17 and 27C in vir-1?

    A similar comment applies to the data in 7E. Please also compare individual mRNA polyA tail length across samples. What is the significance of the change in polyA tail length? The tails are shorter in vir-1 than Col at 27 C. But vir-1 has a very similar phenotype to WT at 27 C. At 17 C, vir-1 tails are longer than WT. Together, do these two results imply that polyA tail length is unlikely to be related to the observed phenotypes? In other words, if longer tails have no effect, do shorter tails? Is there any relationship between RNAs with altered polyA site usage or tail length and those mRNAs that are misexpressed in the mutant? Are immunity gene mRNAs more likely to be m6A modified than other mRNAs?

    Minor comments

    At times it felt like the authors were stretching to fill seven figure with data. For example, in Figure 1, it was not necessary to show the data on increased PR1 expression in 6 different sub panels (B-F) to convince the reader that PR1 expression was increased. A similar comment applies to Figure 3A-D. In Figure 3 please write the common gene names above the plots.

    In Supplemental Table 3 the Enriched GO Terms tab is blank. Supplementary File 1 appeared to be missing from the submission, so I could not evaluate the sequencing statistics (# of reads per sample, mapping %, etc). Many of the Supplemental Tables would benefit from a readme that describes what analysis was performed and what the different columns mean.

    Significance

    The manuscript provides additional insight on the functional consequences of disrupting adenosine methylation in RNA, identifying features of an autoimmune response. Given the ubiquity of m6A in RNA across eukaryotes, this is a result that will be of interest to basic researchers in the plant RNA modification community and likely those working in other eukaryotes. However, the study is not able to connect the inappropriate expression of immune response genes back to the function m6A in RNA, and the effects might be indirect. Although there is speculation that RNA that lacks m6A might trigger autoimmunity, the presented experiments do not directly test that hypothesis (nor do the authors claim to).

  5. Version published to 10.1101/2025.02.18.638636 on bioRxiv