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

autoimmunity, m6A, poly(A) tail, TIR domain, nanopore .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 23, 2025. ; https://doi.org/10.1101/2025.02.18.638636doi: bioRxiv preprint 2

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 m6A modification is essential for Arabidopsis thaliana viability. However, the molecular basis of the impact of mRNA m6A depletion is poorly understood. Here, we show that disruption of the Arabidopsis mRNA m6A writer complex triggers autoimmunity. Most gene expression changes in m6A writer complex vir-1 mutants grown at 17 °C are explained by defence gene activation and are suppressed at 27 °C, consistent with the established temperature sensitivity of Arabidopsis immunity. Accordingly, w e found enhanced pathogen resistance and increased premature cell death in vir-1 mutants at 17°C but not 2 7°C. Global temperature-sensitive mRNA poly(A) tail length changes accompany these phenotypes. Our results demonstrate that autoimmunity is a major phenotype of mRNA m6A writer complex mutants, which has important implications for interpreting this modification’s role. Furthermore, we open the broader question of whether unmodified RNA triggers immune signalling in plants. .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 23, 2025. ; https://doi.org/10.1101/2025.02.18.638636doi: bioRxiv preprint 3

Distinguishing self from non-self is crucial to ensuring organisms specifically target immune responses against pathogen infection. RNA modifications provide one layer by which this distinction is made in humans (Freund et al, 2019; Karikó et al, 2005). Katalin Karikó, Drew Weissman and colleagues revealed that unmodified RNA stimulates the mammalian innate immunity system by activating the Toll-like receptors (TLRs) TL3, TL7 and TL8 , but incorporating modified nucleosides into RNA ablated this activity (Karikó et al , 2005) . Consistent with this, modified nucleosides have been critical in mRNA therapeutics development, and the first mRNA -based vaccines for COVID -19 were based on 1 -methyl pseudouridine-containing mRNA (Karikó, 2021) . A broader set of factors beyond TLRs function in RNA sensing in humans using RNA structure (including modifications), localisation and availability to distinguish self from non -self (Bartok & Hartmann, 2020). Precision in this process is important because chronic activation of nucleic acid sensing pathways in humans is associated with autoimmune and autoinflammatory conditions (Junt & Barchet, 2015). The most abundant internal modification of mRNA is the methylation of adenosine at the N6 position (m6A) (Murakami & Jaffrey, 2022). Null mutations that eliminate the activity of the corresponding N6 methyladenosine methyltransferase METTL3 are embryonically lethal in mouse, demonstrating that this modification can play essential roles in biology (Geula et al, 2015). A complex of proteins functions with METTL3 to modify mRNA. Orthologs of the human writer complex components, METTL3, METTL14, VIRILIZER, ZC3H13, WTAP and HAKAI, are conserved in Arabidopsis and required for mRNA m6A modification (Růžička et al, 2017; Zhang et al, 2022; Shen et al, 2016). Arabidopsis null mutations in each of these components (except the HAKAI and ZC3H13 orthologs) are not viable, and where they exist, hypomorphic alleles have pleiotropic developmental defects (Růžička et al, 2017; Shen et al, 2016; Wong et al, 2023; Zhang et al, 2022). In humans and Arabidopsis, m6A is predominantly written into the terminal exon of mRNA in a preferred context characterised by the DRm6ACH consensus sequence (Parker et al, 2020; Murakami & Jaffrey, 2022). Reader proteins recognise RNA m 6A modificatio ns and ultimately influence mRNA processing and fate (Zaccara & Jaffrey, 2024, 2020) . The best -characterised m6A reader proteins have a YTH domain, which binds m6A through a cage of aromatic amino acids. Plants and apicomplexans are unique with respect to m6A readers because the conserved CPSF30 component of the cleavage and polyadenylation complex, which binds the AAUAAA poly(A) signal (Chan et al, 2014; Schönemann et al, 2014), has a YTH domain (Stevens et al, 2018). Consistent with this, a major impact of m6A loss on pre-mRNA processing in Arabidopsis writer .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 23, 2025. ; https://doi.org/10.1101/2025.02.18.638636doi: bioRxiv preprint 4 complex mutants is disrupted poly(A) site usage (Parker et al, 2020, 2022; Wong et al, 2023). Not all m6A effects are mediated by YTH reader domains. m6A can affect RNA structure (Roost et al, 2015; Kierzek & Kierzek, 2003) and, through an m6A switch mechanism, influence the association of specific RNA -binding proteins with transcripts in an m 6A-dependent manner (Korn et al, 2021; Wu et al, 2018; Liu et al, 2015). A multilayered innate immunity system mediates defence against pathogens in flowering plants (Locci & Parker, 2024). The first layer consists of t rans-membrane receptor proteins called pattern-recognition receptors (PRRs) that detect pathogens in the external environment and signal an immune response known as pattern-triggered immunity (PTI). The second layer comprises networks of proteins that detect pathogen effectors and their activity inside plant cells and is known as effector-triggered immunity (ETI). ETI is mainly mediated by nucleotide binding/leucine -rich repeat (NLR) receptors. Cross-talk between PTI and ETI potentiates the immune response (Yuan et al, 2021; Ngou et al, 2021; Tian et al, 2021; Pruitt et al, 2021). The diverse immune receptors converge on shared signalling complexes, such as those containing EDS1, to promote an immune response (Locci & Parker, 2024). In cells infected by pathogen s, this can trigger programmed cell death , called the hypersensitive response. Immune responses in neighbouring cells are also activated , but the gene expression pattern differs from those in infected cells (Tang et al, 2023; Jacob et al, 2023). A concentration gradient of the hormone salicylic acid (SA) between the sites of infection and neighbouring cells controls the hypersensitive response and the massive expressi on of defence genes such as PATHOGEN RESPONSIVE 1 (PR1) in surrounding cells and systemic acquired resistance in distal tissues (Betsuyaku et al, 2018; Fu et al, 2012b; Zeier, 2021). PRRs and NLRs are encoded by some of the largest and most rapidly evolving gene families in plants (Barragan & Weigel, 2021). This diversity corresponds to selective pressure not only for pathogen defence but also to dampen immune responses in the absence of infection, reflecting trade -offs between the benefits of disease resistance and the costs of sustained immune responses on development. Like humans, plants can develop autoimmune conditions (Alcázar & Parker, 2011; van Wersch et al, 2016; Freh et al, 2022; Wan et al, 2021). In Arabidopsis, these manifest as compromised development and premature cell death, visible as leaf lesions. Some of the clearest examples of autoimmunity emerge in crosses between different Arabidopsis accessions (Bomblies et al, 2007; Wan et al, 2021). This phenomenon, observed in the first or later generations of plant hybrids, is called hybrid necrosis because of the severe pleiotropic symptoms that compromise development and viability (Bomblies & Weigel, 2007) . A recurring explanation for hybrid nec rosis is simple non -compatible interactions between specific NLRs or other defence genes that activate immune response .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 23, 2025. ; https://doi.org/10.1101/2025.02.18.638636doi: bioRxiv preprint 5 pathways (Wan et al, 2021). Naturally occurring genetic variation (Todesco et al, 2010) or induced mutations (van Wersch et al , 2016; Freh et al , 2022) also trigger Arabidopsis autoimmunity. For example, gain of function mutations that either stabilise the expression or autoactivate NLRs can cause autoimmunity (Zhang et al, 2003; Bonardi et al, 2012), and so can the disruption of signal ling pathways that are either involved in or sensed by defence responses (van Wersch et al, 2016). A characteristic of Arabidopsis autoimmunity is that it can be suppressed by elevated temperature or relatively high humidity , and this property has facilitated the study of autoimmune genotypes (Alcázar & Parker, 2011). Although the Arabidopsis mRNA m6A writer complex is essential for viability, the gene expression changes that explain this are unknown. Here, we asked what groups of genes are affected when the mRNA m6A writer complex is disrupted. We discovered that immune response genes comprise the major class of altered gene expression, but consistent with the temperature-sensitive nature of Arabidopsis immunity, this response was suppressed when plants were grown at elevated temperatures . Furthermore, Arabidopsis mRNA m6A writer complex mutants display temperature-sensitive increased resistance to pathogen infection and increased levels of premature cell death. Therefore, autoimmunity is a major phenotype of Arabidopsis mRNA m6A writer complex mutants. In contrast to cases of hybrid necrosis and some other autoimmune mutants, visible developmental defects of mRNA m6A writer complex mutants were not rescued by growth at elevated temperatures, revealing that the impact of defective mRNA m6A modification on autoimmunity and development is separable. Therefore, as with humans, RNA modifications in Arabidopsis may contribute to distinguishing self from non-self. Our findings suggest that u ncovering how disruption of the mRNA m6A writer complex triggers defence gene expression is fundamental to understanding this RNA modification's role in plant biology. .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 23, 2025. ; https://doi.org/10.1101/2025.02.18.638636doi: bioRxiv preprint 6

Immune gene expression is activated in mRNA m6A writer complex mutants To understand the roles of mRNA m6A in Arabidopsis, we asked what groups of genes were most affected by the loss of function of the m 6A writer complex protein , VIRILIZER. We previously characterised gene expression changes in vir-1 mutants using a combination of Illumina RNA sequencing (RNA-seq) and Oxford Nanopore Technologies direct RNA sequencing (ONT DRS) (Parker et al, 2020). We analysed three genotypes with different VIR activity: a wild-type Col-0 control, a hypomorphic Arabidopsis vir-1 mutant defective in VIR function, and a complementation line expressing VIR fused to Green Fluorescent Protein (GFP) (VIR complemented; VIRc) that partly restores VIR activity in the vir-1 mutant

(Růžička et al, 2017; Parker et al, 2020). For each genotype grown in sterile conditions at 2 2°C, we sequenced RNA purified from seedlings of at least six biological replicates with Illumina RNA-seq and four with ONT DRS (Supplementary file 1). Using the Illumina RNA-seq data, we identified differentially expressed genes between vir-1 and WT Col-0 by fitting a quasi-likelihood model in edgeR (Chen et al, 2016) (threshold: adj.p 2.0). We found 806 genes significantly upregulated in vir-1 compared to Col-0 and 349 genes significantly downregulated (Supplementary Table 1). We examined GO (gene ontology) term distribution among the differentially expressed genes using gProfiler (Kolberg et al, 2023). The most significantly enriched GO terms were related to response to external stimuli and defence. For example, 92 of the 597 upregulated genes with GO term annotation were annotated with the biological process ‘defense response’ (GO:0006952), a significant enrichment ( adjusted p = 1.32x10 -8) compared to the background of all genes (Figure 1A, Supplementary Table 2). To examine the global expression trends for defence-related genes, in a different way, we identified 1033 genes that included ‘defence’ or ‘defense ’ in their TAIR annotation description (Reiser et al, 2024). Of these genes, 86 were differentially expressed in at least one condition. Plotting the zero-centred fold change of these genes shows the extent of expression recovery in the VIRc complementation line (Figure 1B). For example, expression of the defence marker gene PR1 (AT2G14610) was 311-fold (8.28 log2FC) higher in vir-1 than Col-0 but restored to similar levels as Col -0 in the VIRc complement ation line. The upregulation of PR1 in vir-1 is also detected in the orthogonal ONT DRS data (Figure 1C-E). We next asked if other m6A writer mutants had elevated PR1 expression. We analysed ONT DRS data of fip37-4 mutants that disrupt the Arabidopsis m6A writer complex ortholog of WTAP (Parker et al, 2022). Genes which are significantly upregulated in fip37-4 mutants are .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 23, 2025. ; https://doi.org/10.1101/2025.02.18.638636doi: bioRxiv preprint 7 significantly enriched for GO terms related to defen ce (Supplementary Table 3). Like vir-1 mutants, fip37-4 mutants have elevated PR1 expression (Figure 1F). In contrast, PR1 is not upregulated in fiona1 mutants that dis rupt the Arabidopsis N6 methyladenosine methyltransferase METTL16 ortholog that adds m6A to U6 snRNA (Parker et al, 2022) (Figure 1C). We conclude that a phenotype of Arabidopsis mRNA m6A writer complex mutants is the upregulation of genes involved in defence signalling. Loss of the mRNA m6A writer complex triggers temperature-sensitive autoimmunity The upregulation of defence response genes in vir-1 raised the question of whether autoimmunity might explain the gene expression changes and pleiotropic developmental phenotypes of mRNA m6A writer complex mutants. A hallmark of Arabidopsis autoimmunity is its temperature sensitivity, with autoimmune phenotypes suppressed at elevated ambient temperatures (Alcázar & Parker, 2011) . Therefore, to address whether the gene expression changes we detected in vir-1 mutants reflected an autoimmune response, we compared the gene expression profiles of vir-1 and WT Col-0 seedlings grown in sterile conditions at 17°C and 27 °C. We used a combination of Illumina RNA -seq and ONT DRS to analyse gene expression. We performed four biological replicates with each RNA sequencing technology, genotype and temperature treatment . The resultant sequencing statistics are detailed in Supplementary file 1. We analysed the Illumina RNA-seq data using a quasi-likelihood model (glmQLFit) in edgeR (Chen et al, 2016) and identified genes differentially expressed between vir-1 and WT Col-0 at each temperature. This revealed 1215 genes which were significantly upregulated (adj.p 2.0) in vir-1 at 17°C (Supplementary Table 4). Remarkably, 91% of these genes (1103) were not significantly upregulated in vir-1 at 27°C (Figure 2A), revealing that most gene expression changes in vir-1 are temperature-sensitive. Principal component analysis (PCA) separates the biological replicates by genotype and temperature . The first component, which explains 40% of the variance, captures gene expression changes specific to vir-1 at 17°C. In contrast, vir-1 and Col-0 are indistinguishable at 27°C in this component (Figure 2B). Likewise, correlation matrix analysis reveals that the gene expression features of vir-1 mutants grown at 1 7°C are the most distinct among all the datasets (Figure 2C). We found elevated expression of PR1 in vir-1 grown at 17°C, just as we had previously seen at 22°C, but PR1 expression was at similar levels to WT Col-0 in vir-1 grown at 27 °C (Figure 2D). We also detected this differential PR1 expression pattern with orthogonal ONT DRS data (Supplementary Figure 2A) and RT-qPCR (Supplementary Figure 2B). .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 23, 2025. ; https://doi.org/10.1101/2025.02.18.638636doi: bioRxiv preprint 8 We used a generalised linear model (GLM) to model all conditions simultaneously and identify differential gene expression specific to vir-1 at 17°C. The GLM design contrasts vir-1 at 17°C minus the average of Col-0 at 17°C and 27°C and vir-1 at 27°C. This model identified 931 genes with significantly increased expression (adj.p 2.0) in vir-1 at 17°C compared to the other conditions (Supplementary Figure 2C, Supplementary Table 5). GO- term analysis revealed that t he biological processes most significantly enriched in genes upregulated in vir-1 at 17°C were related to defence responses (Figure 2E, Supplementary Table 6). The defence annotation GO terms were similar in describing the gene expression changes previously detected in vir-1 grown at 2 2°C (Supplementary Figure 2D). As an orthogonal approach, we analysed protein domain enrichment using DAVID (Sherman et al, 2022) (Supplementary Table 6). The most significantly enriched protein domains in genes upregulated in vir-1 at 17 °C were RLP23-like, which is found in receptor-like kinases that function as PRR proteins (15-fold enrichment) and Defensin_plant, a domain found in highly expressed marker proteins of defence ( 10-fold enrichment). Next, we used a different approach to ask how defence gene expression was affected by temperature using the GLM analysis. We plotted the zero-centred log2FC normalised gene expression of 136 genes with defence/defense included in their TAIR annotation in vir-1 at 17°C compared to other conditions. This analysis reveals that the expression level of most genes with a TAIR defence/defense annotation in vir-1 at 17 °C is at WT Col-0 levels when vir-1 mutants are grown at 27°C (Figure 2F). To determine whether the major defence gene expression differences observed in vir- 1 at 17 °C compared to all other conditions might be explained by overlooked pathogen contamination of our experimental material , we examined our RNA -seq data for non - Arabidopsis sequences . We used a ll vir-1 Illumina RNA-sea data to produce a de novo transcriptome assembly and searched the resulting contigs against the GenBank NR (non- redundant) database using BLASTP (Camacho et al, 2009). No significant enrichment of plant pathogen sequences was found in vir-1 17°C samples (Supplementary Table 7). In summary, by exploiting the established temperature sensitivity of Arabidopsis immunity, we discovered that the major annotation terms associated with the upregulated genes in vir-1 mutants at 17°C are related to defence. Therefore, at the gene expression level and strikingly dependent on temperature, we conclude that autoimmunity is a major phenotype of the Arabidopsis mRNA m6A writer complex mutant, vir-1. Genes that function in diverse aspects of immunity are upregulated in vir-1 mutants .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 23, 2025. ; https://doi.org/10.1101/2025.02.18.638636doi: bioRxiv preprint 9 Given the global trend of defence gene activation in vir-1 mutants, we next analysed individual gene expression changes to understand what type of defence gene s were affected. Expression of mRNA encoding the master defence transcription factor SARD1 (Sun et al, 2015; Wang et al, 2011) was upregulated (AT1G73805: log2FC 4.0) in vir-1 at 17°C but not 27°C, consistent with the established temperature sensitivity of SARD1 transcription (Kim et al, 2022) (Figure 3A and Supplementary Figure 3A). The expression of mRNA encoding the FLS2 receptor, which detects the bacterial flagellin flg22 peptide (Gómez-Gómez & Boller, 2000) and is one of the best-characterised Arabidopsis PRR proteins, was upregulated in vir- 1 at 17°C (AT5G46330: log2FC 3.0) but not at 27°C (vir_27 vs Col-0_27: log2FC 1.3) (Figure 3B and Supplementary Figure 3B). We detected the upregulation of 15 annotated NLRs - 13 TIR-NLRs, one C C-NLR (LOV1) and one RPW8-NLR (HR4) at 17 °C but not 27 °C (Supplementary Table 8). For example, the TIR -only TX0 was upregulated (AT1G57630: log2FC 4.1) (Figure 3C and Supplementary Figure 3C ). TX0 can hydrolyse nucleic acids, particularly RNA, and synthesise 2’,3’,-cAMP/cGMP molecules that ultimately signal cell death in the hypersensitive response (Yu et al, 2022). We next asked whether genes previously associated with Arabidopsis autoimmunity were misregulated in vir-1. The TIR-NLR RPS6 (AT5G46470) is recurrently associated with autoimmunity. For example, the extreme phenotypes of Arabidopsis nonsense-mediated RNA decay ( NMD) and mitogen -activated kinase mutants have been attributed to RPS6 (Gloggnitzer et al , 2014; Takagi et al , 2020) , although the mechanisms involved are not understood (Gloggnitzer et al, 2014; Takagi et al, 2020; Parker et al, 2021b). RPS6 expression is not significantly altered in vir-1 RNA-seq data, but ONT DRS analysis indicates that the TIR- only gene located downstream of the RPS6 locus is upregulated (Parker et al , 2021b) (Supplementary Figure 3D). The TIR-NLR SNC1 has been used as a model to understand autoimmune signalling (Zhu et al, 2010); SNC1 was not significantly upregulated in vir-1 at 17°C (log2FC 1.22), but SIDEKICK3, a TIR-NLR required for SNC1-mediated autoimmunity (Dong et al , 2018) , is one of the most upregulated genes (log2FC 8.32) in vir-1 at 17 °C (Supplementary Table 8). Finally, we examined ACD6, which encodes a multipass transmembrane protein with intracellular ankyrin repeats , that mediates a trade -off between growth and defence (Chen et al, 2023). First identified in lab-based mutant screens (Rate et al, 1999; Lu et al, 2003), high-activity ACD6 alleles are frequently found in natural Arabidopsis accessions (Świadek et al, 2017; Zhu et al, 2018; Todesco et al, 2010). ACD6 is upregulated in vir-1 at 17°C (log2FC 6.27), but the expression level is similar to WT Col-0 in vir-1 grown at 27°C (Figure 3D and Supplementary Figure 3E). We asked whether the changes in gene expression between vir-1 and acd6 mutants were related by re-analysing a recently published .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 23, 2025. ; https://doi.org/10.1101/2025.02.18.638636doi: bioRxiv preprint 10 acd6-1 Illumina RNA-seq dataset (Fabian et al, 2023). We found a subset of differentially expressed genes overlap between these two mutants; 205 upregulated genes and 21 down- regulated genes in common (Figure 3E) . However, 931 genes are uniquely upregulated in acd6-1 and 701 genes are uniquely upregulated in vir-1 at these thresholds (adj.p 2.0), indicating that the misregulation of ACD6 alone does not simply explain vir-1 autoimmunity gene expression phenotypes. A group of flowering time genes paralleled the expression of immune response genes. The floral pathway integrator, FT, (Kinoshita & Richter, 2020) is upregulated in vir-1 at 17°C but not 27°C (Supplementary Table 8). In addition, the expression of a group of genes known to function downstream of FT in floral development, including FUL, SOC1, SEP3, SPL4, SPL5, AGL19 and AGL24 phenocopied defence gene expression patterns (Supplementary Table 8, Supplementary Figures 3F-M). In summary, diverse genes attributed to the different ETI and PTI layers of Arabidopsis innate immunity were upregulated in vir-1 mutants at 17°C but not 27°C, together with mRNA encoding the SARD1 master defence transcription factor that controls SA-dependent and SA- independent defence responses. vir-1 mutants exhibit temperature -sensitive pathogen resistance and localised cell death A characteristic of autoimmunity is that plants show enhanced resistance to pathogen s because defence gene expression is already upregulated prior to infection. We, therefore, asked whether the global changes in gene expression detected in vir-1 resulted in a functional impact on pathogen infection. We examined the susceptibility of WT Col -0, vir-1 and fls2 (flagellin sensing 2) mutants to the biotrophic pathogenic bacterium Pseudomonas syringae pv. tomato (Pto) DC3000. The fls2c mutant allele (Zipfel et al, 2004) lacks the receptor for bacterial flagellin and is susceptible to infection, so is a positive control for infection in these experiments. We flooded seedlings grown at 17°C, 21°C and 27°C with P. syringae pv. tomato (Pst) DC3000. We found that fls2c mutants were more susceptible than Col -0 to infection at all temperatures , consistent with previous reports (Zipfel et al , 2004) (Figure 4A, Supplementary Figure 4 , Supplementary Table 9 ). In contrast, vir-1 mutants were more resistant to infection than Col -0 when grown at 17°C and 21°C, but there was no significant difference in infection levels between Col-0 and vir-1 plants grown at 27 °C. Therefore, the temperature-sensitive patterns of defence gene expression detected in vir-1 convert to a corresponding change in immunity. .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 23, 2025. ; https://doi.org/10.1101/2025.02.18.638636doi: bioRxiv preprint 11 Localised premature cell death is a phenotype of plant immune responses to infection, and leaf lesions are a feature of some autoimmune genotypes (van Wersch et al, 2016; Freh et al , 2022) . To investigate whether vir-1 mutants exhibited elevated levels of cell death compared to WT Col-0 in the absence of pathogen infection , we stained seedlings grown in sterile conditions with the vitality marker trypan blue (TB). We recorded microscopy images and quantified the levels of detectable TB staining for 10 individual leaves of each genotype grown in each condition using ImageJ (Schneider et al, 2012). We detected the highest levels of cell death in vir-1 grown at 17°C (Figure 4B and 4C, Supplementary Table 10). However, at 27°C, cell death patterns in vir-1 were at negligible levels, comparable to those detected in WT Col-0 at 17°C and 27°C. We, therefore, conclude that vir-1 mutants show elevated levels of premature cell death at 17°C when immune response pathways are autoactivated. Overall, vir-1 mutants’ temperature-sensitive response to two orthogonal analyses of autoimmunity - enhanced pathogen resistance and increased premature cell death - is consistent with the global patterns of gene expression changes we detect at the RNA level. These findings suggest that the mRNA m6A writer complex is required to dampen defence pathway signalling to prevent autoimmunity in the absence of pathogen s but not for the defence responses that suppress P. syringae pv. tomato ( Pto) DC3000 infection. We, therefore, conclude that vir-1 mutants have a temperature-sensitive autoimmune phenotype. mRNA m6A levels in vir-1 mutants are not temperature-sensitive An ethyl methanesulfonate-induced 5’ splice site mutation in intron 5 (G+1 to A) causes the vir-1 allele, resulting in cryptic splicing events within exon 5 that disrupt the VIR open reading frame (Růžička et al, 2017). Since mutations that disrupt splic e sites can be temperature - sensitive (Sablowski & Meyerowitz, 1998) , we asked if the expression of VIR mRNA was restored at 27°C. However, we found no evidence from our RNA-seq data to support this idea (Supplementary 5A and 5B). We next asked whether mRNA m6A levels in vir-1 mutants were restored to wild -type levels at 27°C. We first used liquid chromatography -tandem mass spectrometry (LC-MS/MS) to analyse the m 6A/A (adenosine) ratio in poly(A)+ RNA purified from Col-0 and vir-1 grown at 17°C and 27°C. The level of poly(A)+ RNA m6A modification in the hypomorphic vir-1 allele was reduced to approximately 10% of that detected in Col-0 at both 17°C and 27°C (Figure 5A, Supplementary Table 11), consistent with previous reports of reduced m6A levels in vir-1 mutants (Růžička et al, 2017; Parker et al, 2020). In addition to LC-MS/MS, we used the ONT DRS data to examine mRNA m6A levels. We have previously mapped m6A in ONT DRS data using the Differr and Yanocomp programs, which depend upon comparing WT and mutant genotypes (Parker et al , 2020, 2021a) . We supplemented .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 23, 2025. ; https://doi.org/10.1101/2025.02.18.638636doi: bioRxiv preprint 12 Yanocomp analysis with m6Anet, a neural-network-based method that can call read-level m6A stoichiometry without genotype comparison (Hendra et al, 2022). We found no restoration of m6A levels identified by m6Anet (Figure 5B) or Yanocomp analysis (Supplementary Figure 5C) in vir-1 at 27°C compared to 17 °C. We conclude that the suppression of immune gene expression detected in vir-1 at 27°C is not explained by a n accompanying change in mRNA m6A modification. The visible developmental defects of vir-1 mutants are separable from autoimmune gene expression Arabidopsis autoimmune mutants characteristically show developmental defects that can be rescued by growth at elevated temperatures (Alcázar & Parker, 2011). We asked whether the impact of autoimmunity might explain the developmental phenotypes of vir-1. We examined the development of Col-0 and vir-1 mutant plants from germination to flowering and seed-set at 17 °C and 27 °C. We included acd6-1 as a positive control for an autoimmune mutant compromised in development at 17 °C, which appears more like WT Col-0 when grown at 27°C. We found that the short stature and lack of apical dominance characterising the visible developmental phenotypes of vir-1 mutants were not rescued by growth at 27°C (Figure 6A). However, we could replicate the previously reported developmental rescue of acd6-1 mutants at 27 °C compared to 17 °C (Supplementary Figure 6A). We, therefore, conclude that the impact that loss of the mRNA m 6A writer complex has on development and defence gene expression programmes is separable. Next, we asked if we could detect gene expression changes that might underpin developmental change in vir-1 mutants. We identified significant gene expression differences consistently affected by the vir-1 mutation across all our datasets, irrespective of temperature (adj.p 2.0) (Supplementary Table 12). The flowering time control gene, FLC, was the only gene significantly downregulated across all our vir-1 experimental conditions. Only 58 genes were consistently upregulated. However, the most enriched GO term biological processes for these genes were “defence response ”, “systemic acquired resistance”, and “defence response to other organism” (Supplementary Figure 6B, Supplementary Table 13), indicating that defence gene upregulation remains an important vir-1 gene expression phenotype. For example, RNA encoding the flavin-dependent monooxygenase, FMO1, was upregulated in vir-1 relative to WT Col-0 in all tested conditions (Supplementary Figure 6C). FMO1 is a critical regulator of systemic acquired resistance to pathogen infection (Hartmann et al, 2018). We also ident ified consistent upregulation of the AtNUDT24 nudix hydrolase. AtNUDT24 is uncharacterised, but other members of this protein family function to modify .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 23, 2025. ; https://doi.org/10.1101/2025.02.18.638636doi: bioRxiv preprint 13 signalling nucleotides in plant defence (Yu et al , 2022; Bartsch et al , 2006) or in RNA decapping and hydrolysis, among other roles (Laudenbach et al, 2021; Carreras-Puigvert et al, 2017). Overall, we conclude that although defence gene activation in vir-1 mutants is suppressed at 27°C, visible developmental defects are not, and autoimmune gene expression programmes remain a component of the vir-1 mutant phenotype. Altered poly(A) tail length distributions are a temperature-sensitive phenotype of vir-1 mutants To understand how the loss of mRNA m 6A might trigger an autoimmune response , we next asked if RNA processing was affected in vir-1 mutants grown at different temperatures . Consistent with our previous reports (Parker et al , 2020, 2022) , we found that genetic disruption of the mRNA m6A writer complex resulted in a global shift to proximal poly(A) site usage in diverse mRNAs (Figure 7A). The scale of this effect was similar in vir-1 mutants grown at 17°C and 27°C. Altered 3’ end formation may trigger autoimmunity, bu t the subsequent signalling might be suppressed at 27°C, so we addressed this question differently. If alternative poly(A) site usage triggered the autoimmune response, it might be mediated by the CPSF30-YTH m6A reader. We examined gene expression changes in cpsf30-yth mutants using ONT DRS . The cpsf30-yth mutants express a truncated protein comprising the N - terminal Zinc-Finger domains that bind the AAUAAA poly(A) signal, but lack the C-terminal YTH domain. We found PR1 is not upregulated in cpsf30-yth, although it is upregulated in fip37-4 mutants analysed alongside here as a positive control (Figure 7B). Therefore, this combination of data does not provide evidence that altered poly(A) site usage triggers the autoimmunity phenotypes of Arabidopsis mRNA m6A writer complex mutants. The cytoplasmic YTH reader domain proteins likely mediate specific impacts of m6A on RNA fate (Brodersen & Arribas-Hernández, 2024). The most abundant of these are ECT2, 3 and 4 (Arribas-Hernández et al, 2018). We analysed a triple mutant in each gene, te234 (Flores-Téllez et al, 2023) using ONT DRS. We found no evidence that PR1 was upregulated in te234. In contrast, PR1 upregulation was again detected in fip37-4 mutants included here as a positive control (Figure 7C). Finally, we asked if poly(A) tail length was altered in vir-1 mutants. We have previously reported a change in poly(A) tail length profiles at specific genes in vir-1 (Parker et al, 2020), and we asked if this was a more widespread phenotype. We used the ONT software Dorado to estimate transcript poly(A) tail length in our ONT DRS data. This analysis reveals a characteristic periodicity in estimated lengths of read poly(A) tails that likely reflects the .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 23, 2025. ; https://doi.org/10.1101/2025.02.18.638636doi: bioRxiv preprint 14 footprint of binding of multiple poly(A) binding proteins (PABPs) (Baer & Kornberg, 1983; Passmore & Coller, 2022). We found that the distribution of estimated poly(A) tail lengths was markedly different in vir-1 compared to WT Col -0 - at 17°C; relatively fewer transcripts with short poly(A) tails and more with longer poly(A) tails are detected in vir-1 compared to WT- Col-0 (Figure 7D). At 27°C, vir-1 mutant mRNA poly(A) tail length profiles are different again, with a distribution more enriched in short poly(A) tails compared to WT Col -0 (Figure 7D). In contrast, the 30 nt poly(A) tail of Saccharomyces cerevisiae ENOLASE II RNA, used here as a spike-in calibration standard during ONT DRS library preparation , is consistent across all samples, with an estimated median tail length of 33 nt (Supplementary Figure 7A). Changes in the global distributions of poly(A) tail length may be partly due to differences in the sets of genes expressed at detectable levels in the different conditions. To examine the differences in tail length per gene and exclude genes that were only expressed in single conditions, we used the Wasserstein distance metric to quantify shifts in mean per gene tail length distributions between conditions (Parker et al, 2021b). This analysis identified a shift towards longer mean poly(A) tails in vir-1 at 17°C compared to Col-0 and a shift towards shorter mean poly(A) tails in vir-1 at 27 °C, consistent with the different distributions of estimated poly(A) tails lengths (Figure 7E, Supplementary Table 14). Temperature-sensitive changes in mean poly(A) tail length in Col -0 were modest by comparison (Figure 7F). We asked if the shift in mean poly(A) tail length was directly associated with the loss of m 6A modification. We found that while genes with m6Anet-predicted m6A sites had shorter poly(A) tails, the temperature -dependent differences in poly(A) tail length distribution were seen in genes predicted to be either m6A-modified or non-modified (Supplementary Figure 7B). In summary, our findings reveal global changes in poly(A) tail length distributions as the primary temperature-sensitive mRNA processing phenotype of vir-1 mutants. This global phenotype is not restricted to genes that function in defence responses or to those transcripts predicted to have lost m6A modification. .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 23, 2025. ; https://doi.org/10.1101/2025.02.18.638636doi: bioRxiv preprint 15

We have discovered that disruption of the mRNA m 6A modification complex triggers autoimmunity in Arabidopsis. Using a combination of Illumina RNA -seq and ONT DRS, we reveal, in unprecedented detail, the temperature -sensitive changes in RNA expression, modification and processing caused by mRNA m 6A writer complex depletion. At 17°C, most gene expression class changes in vir-1 mRNA m6A writer complex mutants are explained by defence gene activation. By exploiting the established temperature sensitivity of Arabidopsis immunity, we found that the vast majority of these gene expression changes did not occur when we grew the vir-1 mRNA m6A writer mutant at 27°C. We used orthogonal experimental approaches to examine the biological impact of this defence gene activation. We found temperature-sensitive enhanced levels of premature cell death and resistance to P. syringae Pst D3000 pathogen infection in vir-1 mutants consistent with the idea that these gene expression changes convert into functional consequences for immunity. Not all gene expression changes , or all developmental phenotypes of vir-1 mutants, were rescued by elevated temperature, demonstrating that the impacts of m 6A-dependent changes on gene expression are separable. Together, these different lines of evidence all point to the disruption of the mRNA m6A writer complex triggering autoimmunity in Arabidopsis. The autoimmune phenotypes of Arabidopsis m 6A writer complex mutants have not previously been described. However, while analysing the impact of mRNA m6A on plant pathogen infection, it was recently reported that Arabidopsis mutants defective in METTL3, VIR and WTAP orthologs, and a line ectopically overexpressing the mRNA m6A demethylase ALKBH10B, were all more resistant to infection by P. syringae DC3000, P. syringae pv maculicola and a fungal pathogen Botrytis cinerea (Prall et al, 2023). Consistent with this, genes commonly upregulated in METTL3 ortholog mutant and ectopic ALKBH10B expression lines are enriched in GO term annotations for d efence signalling (Prall et al , 2023) . Furthermore, early microarray analysis of METTL3 ortholog mutants reported a general enrichment among the overexpressed genes for GO terms related to stress responses (Bodi et al, 2012). These independent findings are consistent with our analysis of fip37-4 and vir-1 and generalise the idea that autoimmunity is a major Arabidopsis mRNA m6A writer complex mutant phenotype. These discoveries raise the key question of how disruption of mRNA m 6A writer complexes triggers autoimmunity. Our analysis does not explain how defence pathways are autoactivated in Arabidopsis mRNA m6A writer complex mutants. Remarkably, given its importance, the mechanism by which modified RNAs ablate TLR signalling in humans is also .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 23, 2025. ; https://doi.org/10.1101/2025.02.18.638636doi: bioRxiv preprint 16 unknown. We first considered whether m 6A readers might mediate the autoactivation of defence gene expression. However, we found no evidence that either cpsf30-yth mutants or te234 mutants phenocopied the defence gene expression of vir-1 mutants. There are 13 m6A- reading YTH domain-containing protein s encoded in the Arabidopsis genome. Therefore, approaches that resolve redundancy in their function (Flores-Téllez et al, 2023) will be required to test an association with autoimmunity further . The major temperature -sensitive RNA phenotype we detected in vir-1 mutants was a global change in mRNA poly(A) tail length: we found relatively fewer RNAs with short poly(A) tails (100nt) compared to WT Col-0 at 17°C but this phenotype was reversed at 27°C. This finding could indicate that the trimming of longer poly(A) tails - an essential feature of newly transcribed mRNAs (Passmore & Coller, 2022) - is defective and/or mRNAs with short poly(A) tails are more susceptible to decay in vir-1 mutants at 17°C. Poly(A) tails can reduce innate immun e responses of human cells to RNA (Koski et al , 2004) . Furthermore, autoimmunity and pleiotropic developmental defects are phenotypes of Arabidopsis mutants defective in the nuclear poly(A) polymerase, PAP1, that catalyses mRNA poly(A) tail formation (Vi et al, 2013). However, the inter-relatedness of the phenotypes we detect here is not yet clear. Poly(A) tail length changes were not restricted to transcripts with predicted m 6A sites, indicating this change is not a direct result of m6A loss. Nor were poly(A) tail length changes restricted to transcripts involved in defence functions. Increased poly(A) tail length is a stress response phenotype of different species (Tudek et al , 2021; Yamagishi et al , 2024) and promotes stress granule formation in humans (Yamagishi et al , 2024; Tsai et al , 2025) . Therefore, the temperature -sensitive poly(A) tail length changes we detect here may be a previously unappreciated autoimmunity phenotype. An aspect of RNA biology we have not explored is whether changes in condensate association caused by loss of mRNA m6A might trigger autoimmunity. mRNA m6A modifications can influence separation into biomolecular condensates such as human stress granules (Ries et al, 2023). Significantly, the buffering of self RNA by condensates regulates human innate immune responses (Maharana et al, 2022). Analysing other proteins more closely connected to Arabidopsis poly(A) tail processing and RNA fate (Sasse et al , 2024) could help unravel connections between RNA modification, poly(A) tail length, altered RNA homeostasis and the causes or consequences of autoimmunity. Different receptors detect RNA as a molecular signature of pathogen infection in humans, and RNA's availability, localisation, and structure (including sequence and modification) are essential criteria for distinguishing self and non -self (Schlee & Hartmann, 2016; Bartok & Hartmann, 2020) . For example, uridine mononucleotides and di or .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 23, 2025. ; https://doi.org/10.1101/2025.02.18.638636doi: bioRxiv preprint 17 trinucleotides are bound on different sites of TLR8 in monocyte endosomes in a manner that depends on upstream RNAse 2 and T2 processing of pathogen RNA (Ostendorf et al, 2020). RNAs purified from P. syringae DC3000 bacteria or transcribed in vitro and infiltrated into Arabidopsis leaves activate innate immunity, demonstrating that non -self RNAs can trigger immune responses in Arabidopsis (Lee et al, 2016). Aside from the RNAi machinery, which plays crucial roles in viral defence in plants (Lopez-Gomollon & Baulcombe, 2022), receptors that recognise pathogen RNAs are poorly characterised in plants. However, plant TIR domains have recently been found to hydrolyse RNA (Yu et al, 2022). Compared to DNA, RNA is the preferred substrate for TIR domains to synthesise 2’,3’ ,-cAMP/cGMP molecules that signal cell death in the hypersensitive response (Yu et al, 2022). Crucially, a mutation that disrupts this synthetase activity is sufficient to block cell death signalling (Yu et al, 2022). TIR domains are frequently found in Arabidopsis NLR proteins and can also be expressed as TIR-only proteins (Essuman et al, 2022) encoded by TIR -only genes or the widespread prematurely terminated transcripts of NLR genes (Parker et al, 2021b). We found that mRNAs encoding TIR domain proteins, including TX0, for which RNA hydrolysing activity has been demonstrated (Yu et al, 2022), were significantly upregulated in vir-1 mutants. The natural RNA substrates of Arabidopsis TIR domains are unknown. An important question is whether TIR domains sense non -self RNAs or perturbed RNA homeostasis that indicates pathogen activity. The nudix hydrolase family member NUDT7 acts as a phosphodiesterase to modify 2’3’-cAMP/cGMP and thus modulates signalling through EDS1 (Yu et al, 2022). Notably, one of the most consistently upregulated genes in vir-1 is the uncharacterised nudix hydrolase, AtNUDT24. It may not be the loss of mRNA m6A itself that triggers autoimmunity. ETI functions to detect pathogen activity that disrupts host cell proteins or processes and activates an immune response. In this way, NLRs act as guards, with the effector-targeted host cell proteins or processes being guardees (Dangl & Jones, 2001; Van der Biezen & Jones, 1998; Remick et al, 2023). It is possible that the mRNA m6A writer complex is a guardee and that disruption of the writer complex, rather than the absence of mRNA m 6A, is detected and triggers autoimmune signalling. Therefore, defence signalling pathways in vir-1 mutants may directly detect non-modified RNA, a disrupted mRNA m6A writer complex, poly(A) tail perturbation, or changed RNA homeostasis resulting from decay or condensate association . However, the connection between disrupted m6A writer complex and autoimmunity may be even more indirect. Loss of m 6A is associated with diverse changes in gene expression and pleiotropic developmental changes. Therefore, if the changes in gene expression, RNA processing, or development found in mRNA m6A writer complex mutants phenocopy features of pathogen .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 23, 2025. ; https://doi.org/10.1101/2025.02.18.638636doi: bioRxiv preprint 18 infection, they may indirectly trigger immune pathway activation. Consequently, understanding how immune gene expression is activated is crucial to understanding the directness by which mRNA m6A impacts these diverse RNA and developmental phenotypes. Our findings, therefore, have practical implications for studying the impact of mRNA m6A on plant biology. Since most gene expression changes in mRNA m6A writer complex mutants at lower ambient temperatures are caused by autoactivation of defence gene expression, the interpretation of the effects of mRNA m6A will be complicated by indirect changes that vary according to environmental conditions (such as temperature), which may differ between studies. Mutating defence signalling hubs in mRNA m6A writer complex mutant backgrounds might suppress autoimmunity but not necessarily comprehensively block autoimmune signalling. Our global transcriptome analysis only provides snapshots of gene expression changes in Arabidopsis seedlings . However, u nderstanding how mRNA m 6A directly influences mRNA processing and fate and, hence, development and autoimmunity will require alternative experimental approaches . Determining immediate gene expression changes following mRNA m6A writer complex shutdown using, for example, proteolysis targeting chimaeras (PROTACS) (Békés et al, 2022) in defined cell types and developmental contexts may help us understand the direct roles of mRNA m6A. In conclusion, our study establishes a new conceptual framework for analysing the impact of mRNA m6A on plant biology. The molecular basis of the events that trigger mRNA m6A writer complex-dependent autoimmunity is unknown, but uncovering this should lead to fundamental insights into the role of mRNA m6A in plant biology and how defence gene signalling occurs. .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 23, 2025. ; https://doi.org/10.1101/2025.02.18.638636doi: bioRxiv preprint 19

Plant Material Wild-type Arabidopsis thaliana accession Col-0 and te234 (Arribas-Hernández et al, 2020) were obtained from the Nottingham Arabidopsis Stock Centre. The vir-1 and VIR - complemented (VIR::GFP -VIR) lines (Růžička et al , 2017) were from K. R ůžička, Brno , Czechia; acd6-1 was from J. Greenberg, Chicago, USA; fip37-4 was from R. Fray, Nottingham, UK; fls2c (SAIL_691C4) (Zipfel et al, 2004) was from P. Hemsley, Dundee, UK; cpsf30-yth (GK477H04) was from Ł. Szewc, Poznań, Poland. Plant Growth Conditions Seeds were sown on MS10 medium plates, stratified at 4°C for 2 days, germinated in a controlled environment at 22°C under 16 hr light/8 hr dark conditions and harvested for RNA purification 14 days after transfer to 22°C. For temperature assays, plant growth chambers were set to either 17°C or 27 °C, with all other conditions the same as above. Seedlings were harvested 14 days after germination during the first two hours of the light period following an 8-hour dark phase. Four-week-old plants were used for phenotyping the adult plants at 17°C or 27 °C. Trypan Blue Staining Trypan blue staining was performed on leaves from 4-week-old plants of WT Col-0 and vir-1 grown at 17°C and 27°C. Leaves were stained in a solution of Tris-EDTA equilibrated phenol (pH 8) (25%), glycerol (25% v/v), lactic acid (25% v/v) with trypan blue ( 10mg/ml). Leaves were treated with staining solution for 10 minutes at 95 °C then incubated overnight at room temperature. Leaves were destained in chloral hydrate solution twice, for 4 h and overnight. Stained leaves were imaged under a Zeiss histology microscope at 10x magnification. Images were imported into ImageJ (Schneider et al, 2012), and the total stained area was measured in pixels, with the stained area expressed as a percentage of the total leaf area. Data collected from 10 leaves per condition was plotted, and a two-way ANOVA test with post hoc Turkey’s HSD tests was used to assess the effects of genotype and temperature and their interaction on the percentage of leaf stained. Genotyping of Arabidopsis Individual mutants and progeny from crosses were genotyped by PCR analysis of purified DNA using a combination of derived cleaved amplified polymorphic sequence (dCAPS) .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 23, 2025. ; https://doi.org/10.1101/2025.02.18.638636doi: bioRxiv preprint 20 markers (Michaels & Amasino, 1998), T-DNA insertion markers and sequencing. Genotyping primer pairs used in the study are listed in Supplementary Table 15. Pathogenesis Assays Arabidopsis Col-0, vir-1 and fls2c seedlings were treated with Pseudomonas syringae pv tomato (Pto) DC3000 using flood inoculation. Bacteria were cultured on MG agar supplemented with rifampicin at 28 °C for 24-48 h. Four-week-old seedlings flood-inoculated with a bacterial suspension of P. syringae DC3000 (5x106 Colony-Forming Units (CFU)/ml) containing 0.025% Silwet L-77. Sterilised seedlings were grown on half-strength MS agar for 2-3 weeks at 17°C, 21°C and 27°C before inoculation. The bacterial suspension was applied to the Arabidopsis seedlings, and plates were incubated at room temperature for 2-3 minutes. Excess liquid was drained, and seedlings were maintained at growing temperatures for three days. Bacterial growth was calculated using serial dilution of material from three seedlings per plate and recorded as CFU/mg. The significance of changes in bacterial growth in the differing conditions was tested using ANOVA. The experiment was repeated to confirm results. RNA Isolation Total RNA was isolated using the RNeasy Plant Mini kit (Qiagen) and treated with RNAse-free DNase (Promega-M6101). RNA concentration and integrity were measured using a NanoDrop one spectrophotometer and Agilent 4150 Tapestation. Gene expression analysis by RT-qPCR Total RNA was extracted from 14-day-old seedlings. Total RNAs were treated with RNAse - free DNase (Promega-M6101). First-strand cDNAs were synthesised using SuperScript™ III Reverse Transcriptase (Thermo Fisher Scientific -12574026). qPCR was carried out on a LightCycler® 96 Instrument using Brilliant III Ultra -Fast SYBR Green qRT -PCR Master Mix (Agilent-600886). Three biological replicates (independently harvested samples) with three technical replicates for each were analysed. Relative expression levels were determined using the 2 −ΔΔCT method. Arabidopsis UBQ10 (AT4G05320) was used as internal control. qPCR primer pairs are listed in Supplementary Table 15. Preparation of libraries for Illumina RNA-sequencing Illumina RNA-seq libraries were prepared by Genewiz (Azenta LifeScience) using NEB Next Ultra Directional Library Prep Kit according to the manufacturer’s instructions. Paired -end sequencing with a read length of 150bp was carried out on the Illumina NovaSeq X following .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 23, 2025. ; https://doi.org/10.1101/2025.02.18.638636doi: bioRxiv preprint 21 the manufacturer’s instructions. Raw sequence data was converted to fastq and de - multiplexed using Illumina bcl2fastq version 2.20. Processing of Illumina RNA-seq data and differential gene expression analysis Quality assessment of RNA-seq reads was performed using Fast QC (Andrews, 2017). For digital expression, the Salmon index was built using Arabidopsis Araport11 transcript annotations (Cheng et al , 2017) . Transcript and gene -level counts were estimated using Salmon (with gtf option and Araport 11 annotation) (version 1.9.0) (Krishnakumar et al, 2015). Differential expression analysis was performed in edgeR (version 4.2.2) using a quasi - likelihood generalised linear model (glmQLFit). Annotation of genes of interest, categorising them as defence, flowering or other and returning GO annotation with further annotation was performed using custom scripts: https://github.com/bartongroup/PT_Arabidopsis_names_to_annot. To visualise dimensional reduction in the context of RNA -seq quality control , PCA plots were created using ggplot2 (version 3.5.1). Correlation and heatmap plots were generated with the ptr script from the Trinity RNAseq package (version 2.15.2) (Grabherr et al, 2011). GO enrichment heatmap were made using msbio (metascape)(v3.5.20240901) (Zhou et al, 2019). Functional enrichment analysis was performed using a combination of Goseq (version 1.42.0) (Young et al, 2010) and g:Profiler (version e111_eg58_p18_f463989d) with the g:SCS multiple testing correction method and a significance threshold < 0.05 (Kolberg et al, 2023). Domain enrichment analysis was performed in DAVID (Sherman et al, 2022) using a FDR significance threshold of < 0.05. To determine whether pathogen contamination was present in the vir-1 RNA-seq samples, reads were mapped to the TAIR10 genome, and from the resulting bam file, unmapped reads were returned using STAR (version 2.7.11b) (Dobin et al, 2013). BBnorm (October 19, 2017) was then used to normalise the “unmapped” reads with the following setting “ target=75 min=3 ” (Bushnell, 2022) . The normalised reads were assembled using Trinity (Grabherr et al , 2011) (version 2.15.2) with –trimmomatic –no_normalise. The transcriptome assembly was processed using cd-hit-est (version 4.8.1) (-c 0.90 -n 8 -T 24 -M 0) (Fu et al, 2012a) to reduce redundancy at 90%. The resulting final transcriptome assembly was then searched against Genbank NR with Diamond-BLASTP using Diamond (version v2.0.5.143 ) (Buchfink et al , 2021) . The diamond BLASTP output was post -taxonomically annotated using https://github.com/peterthorpe5/public_scripts/tree/master/Diamond_BLAST_add_taxonomic _info. The final taxonomically assigned BLAST output was then interrogated for the presence .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 23, 2025. ; https://doi.org/10.1101/2025.02.18.638636doi: bioRxiv preprint 22 of plant pathogens, as defined here ( https://phytopathdb.org/pathogens_eg/). Digital expression per condition to this assembl y was estimated using Salmon (Patro et al, 2017), and differential expression analysis was performed as described above. ONT DRS Library Preparation Total RNA was isolated , as detailed above. Poly(A)+ RNA was purified from approximately 100µg of total RNA using the Dynabeads mRNA purification kit (Thermo Fisher Scientific) following the manufacturer’s instructions. The quality and quantity of mRNA were assessed using the Nanodrop one spectrophotometer (Thermo Fisher Scientific) and Tape station 4150 (Agilent Technologies). ONT DRS libraries were prepared from 100ng poly(A)+ RNA for the WT Col-0 - vir-1 comparison at 17°C and 27°C. All other ONT DRS libraries were prepared from 500ng poly(A)+ RNA. Libraries were made using the Direct RNA sequencing kit (SQK - RNA002; Oxford Nanopore Technologies) according to the manufacturer’s instructions. The poly(T) adapter was ligated to the mRNA using T4 DNA ligase (New England Biolabs) in the Quick Ligase reaction buffer (New Eng land Biolabs) for 15 min at room temperature. First - strand cDNA was synthesised by SuperScript III Reverse Transcriptase (Thermo Fisher Scientific) using the oligo(dT) adapter. The RNA –cDNA hybrid was then purified using Agencourt RNAClean XP magnetic beads (Beckman Coulter). The sequencing adapter was ligated to the mRNA using T4 DNA ligase (New England Biolabs) in the Quick Ligase reaction buffer (New England Biolabs) for 15 min at room temperature followed by a second purification step using Agencourt beads (as described above). Libraries were loaded onto R9 version SpotON Flow Cells (Oxford Nanopore Technologies) and sequenced using a GridION device at the Tayside Centre for Genomic Analysis, School of Medicine, University of Dundee, for a 48-hour runtime. Four biological replicates were performed for each genotype. ONT DRS mapping Reads were basecalled using Dorado version 0.5.3 (Oxford Nanopore Technologies) using the rna002_70bps_hac@v3 high accuracy model. Reads were aligned to the Araport11 transcriptome (Cheng et al, 2017) and the TAIR10 Arabidopsis genome (Lamesch et al, 2012) using minimap2 version 2.17 (Li, 2018) configured for spliced alignment . The following parameters were used for both alignments: --end-seed-pen=15 for end seed penalties, -A1, - B1, -O2,32, -E1,0 and -C9 to tune alignment scoring. For genomic alignment, splice junction information was incorporated using the --junc-bed parameter, which referenced the annotated introns BED file. A junction bonus of 10 (--junc-bonus=10) was applied to prioritise alignments that utili sed known splice junctions, increasing alignment accuracy for spliced reads . The .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 23, 2025. ; https://doi.org/10.1101/2025.02.18.638636doi: bioRxiv preprint 23 spliced alignment was optimi sed with parameters -k14, -uf, -w5, --splice, and -g2000, along with a maximum intron size of 200,000 ( -G200000), --splice-flank=yes for spliced alignment flanking detection, and -z200 for seeding thresholds. Alignments were converted to BAM files and indexed using samtools version 1.18. Prediction of m6A in ONT DRS data using m6Anet Event information was extracted from raw signal data and transcriptome alignments using the f5c implementation of eventalign with event scaling (Gamaarachchi et al, 2020). Aligned event files were processed using m6anet (Hendra et al, 2022). Data preparation and inference was performed using the pretrained Arabidopsis model with the default read probability threshold of 0.9 (Hendra et al , 2022). The distribution of predicted modification ratios for all sites passing this threshold was plotted for each condition. Analysis of poly(A) site usage in ONT DRS data Differential 3’ analysis was performed on bam files using the d3pendr tool as described previously (Parker et al, 2021b). The statistical significance of the 3’ shift was assessed by permuting read alignments between the control and treatment distributions to determine the maximum distance achieved by random sampling. Estimation of poly(A) tail length in ONT DRS data The length of poly(A) tails per read was estimated using “--no-trim --estimate-poly-a” with the following model: rna002_70bps_hac@v3 in Dorado (version 0.5.3). Reads were mapped to the Araport 11 transcriptome using minimap2 (see above), and the resulting bam file was used to generate a read-to transcript table for further interrogation. Differences in mean poly(A) tail length per gene between conditions were calculated as previously described (Parker et al, 2020). In brief , poly(A) tail lengths were aggregated by gene ID , and where genes were present with at least ten reads in both conditions, the distributions of poly(A) tail lengths were compared using the Wasserstein distance. Significance was assessed using a permutation test with 999 bootstraps. Genes were classed as m6A-modified if they had a t least one site above the probability-modified threshold of >0.9 in at least one Col-0 sample. Gene tracks Gene track figures were generated using Matplotlib (versio n 3.9.2) from normalised bigwig files of Illumina RNA-Seq coverage and pooled bam files of reads per condition. For tracks .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 23, 2025. ; https://doi.org/10.1101/2025.02.18.638636doi: bioRxiv preprint 24 with >100 ONT DRS reads per condition, a random subsample of 100 reads per track w as plotted. m6A liquid chromatography with tandem mass spectrometry m6A analysis using tandem liquid chromatography-mass spectroscopy (LC /MS-MS) was performed as previously described (Parker et al, 2020, 2021b). LC/MS-MS was carried out by the FingerPrints Proteomics facility at the University of Dundee. A two-way ANOVA test was used to assess the effects of genotype and temperature and their interaction on the ratio of m6A to A. Code and data availability Illumina FASTQ and ONT FAST5 files for the 17ºC and 27ºC dataset are deposited in the ENA under accession code PRJEB85795. ONT FAST5 for the te234 and cpsf30-yth datasets are deposited in the ENA under accession codes PRJEB85859 and PRJEB85860 respectively. Source data for LC-MS, flood inoculation and image staining are available in supporting files. Source code, R notebooks and Snakemake (Mölder et al, 2021) pipelines are available at github.com/bartongroup/m6a_arabidopsis_autoimmunity.

We thank Prof. Steven Spoel (University of Edinburgh) for Pseudomonas syringae pv. Tomato (Pto) DC3000. We thank Dr. Martin Balcerowicz (University of Dundee) for providing access to temperature-controlled environment cabinets and Dr. Rachel Taylor (University of Leeds) for temperature-controlled plant growth. We are grateful to Katie Dempsey, whose experiments led us to investigate the temperature sensitivity of vir-1 mutants. We thank Dr. Martin Balcerowicz, Prof. Brendan Davies and Dr. Davide Bulgarelli for helpful comments on the manuscript. We thank the University of Dundee HPC and Research Computing at the James Hutton Institute for providing computational resources and technical support through the BBSRC-funded “UK’s Crop Diversity Bioinformatics HPC” (BB/S019669/1 and BB/X019683/1). This work was supported by awards from the BBSRC (BB/V010662/1 and BB/M010060/1 to GGS and GJB; BB/W007673/1 to GGS ). The FingerPrints Proteomics facility at the University of Dundee is supported by a Wellcome Trust Technology Platform Award (097945/B/11/Z). Competing Interests The authors declare no competing interests. .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 23, 2025. ; https://doi.org/10.1101/2025.02.18.638636doi: bioRxiv preprint 25 FIGURES Figure 1: Immune gene expression is activated in m6A writer complex mutants. A) Top 10 gene ontology terms enriched in the set of 597 genes significantly upregulated (FDR 2.0) in vir-1 mutants compared to Col-0 WT at 20°C. B) Heatmap showing the TMM-FPKM normalised log2 centred fold expression between vir-1, Col-0 and VIRc for all genes with TAIR annotations including the term ‘defense/defence’. PR1 (AT2G14610) is highlighted with an arrow. C) Normalised log2 counts per million of PR1 (AT2G14610) in Col- .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 23, 2025. ; https://doi.org/10.1101/2025.02.18.638636doi: bioRxiv preprint 26 0 (n = 7), vir-1 (n = 6) and VIRc (n = 6) in Illumina RNA-seq. Boxes represent the interquartile range of the logged values. D) Normalised log2 counts per million of PR1 (AT2G14610) in Col-0, Col-0, vir-1 and VIRc in ONT DRS reads (n= 4 samples per genotype). E) Upregulation of PR1 (AT2G14610) in vir-1 at 20°C, shown by a gene track of Illumina RNA -seq and downsampled ONT DRS reads. C) Normalised log2 counts per million of PR1 (AT2G14610) in Col-0, fip37-4 and fio-1 at 20°C in ONT DRS, showing the upregulation of PR1 in fip37-4 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 23, 2025. ; https://doi.org/10.1101/2025.02.18.638636doi: bioRxiv preprint 27 Figure 2: Loss of m6A triggers a temperature-sensitive autoimmune response. A) Upset plot showing the overlap in significantly upregulated and downregulated genes (log2FC +/- 2.0 FDR < 0.001) in vir-1 at 17°C compared to Col-0 at 17°C and vir-1 at 27ºC compared to Col-0 at 27ºC. B) Principal component analysis showing the clustering of samples by experimental condition (including genotype) and temperature . C) Correlation matrix and hierarchical clustering of expression profiles for each condition. The clustering shows that gene expression patterns are distinct for all conditions. In addition, biological replicates within conditions cluster together. However, the gene expression patterns detected in vir-1 separate .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 23, 2025. ; https://doi.org/10.1101/2025.02.18.638636doi: bioRxiv preprint 28 as the most different of all possible comparisons . D) Significant upregulation of PR1 (AT2G14610) in vir-1 at 17°C, shown by a boxplot of normalised expression (log 2 counts per million) in Illumina RNA-seq data (n= 4 samples per genotype). E) Most enriched GO terms among genes significantly upregulated (FDR < 0.001, log2FC<2.0) in vir-1 at 17°C compared to the average of: Col-0 17°C, 27°C and vir-1 at 27°C. Source data available in Supplementary Table 6. F) Heatmap showing the TMM -FPMK normalised log2 centred fold change for all differentially regulated genes in vir-1 at 17 °C (log2FC +/ - 2.0 FDR < 0.001) with TAIR annotations including ‘defense/defence’ for conditions vir-1 at 17°C, 27°C, Col-0 at 17°C and 27°C. PR1 (AT2G14610) is highlighted with an arrow. .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 23, 2025. ; https://doi.org/10.1101/2025.02.18.638636doi: bioRxiv preprint 29 Figure 3: Defence genes that function in diverse aspects of immunity are upregulated in vir-1 mutants A) Significant upregulation of SARD1 (AT1G73805) in vir-1 at 17°C, shown by a boxplot of normalised expression (log2 counts per million) in Illumina RNA-seq data (n = 4 samples per condition). B) Significant upregulation of FLS2 (AT5G46330) in vir-1 at 17°C, shown by a boxplot of normalised expression (log 2 counts per million) in Illumina RNA -seq data (n = 4 samples per condition). C) Significant u pregulation of TX0 (AT1G57630) in vir-1 at 17°C, shown by a boxplot of normalised expression (log 2 counts per million) in Illumina RNA -seq data (n = 4 samples per condition). D) Significant upregulation of ACD6 (AT4G14400) in vir-1 at 17°C, shown by a boxplot of normalised expression (log 2 counts per million) in Illumina RNA-seq data (n = 4 samples per condition). E) Upset plot showing modest overlap in differentially upregulated genes between vir-1 at 17°C and previously published acd6-1 RNA- seq data (Fabian et al, 2023). .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 23, 2025. ; https://doi.org/10.1101/2025.02.18.638636doi: bioRxiv preprint 30 Figure 4: vir-1 mutants exhibit temperature-sensitive pathogen resistance and localised cell death. A) Four-week-old Col-0 WT , vir-1 and fls2c seedlings flood-inoculated with a bacterial suspension of Pst DC3000 (5x106 CFU/ml) and 0.025% v/v Silwet L-77. Bacterial populations were quantified at 3 days post inoculation (dpi) (n = 3 per condition). One way ANOVA tests on each genotype revealed a significant effect of temperature in the vir-1 genotype (F = 37.23, p = 0.0 358) which was not present in Col-0 WT. Source data available in Supplementary Table 9. This experimental analysis was replicated independently in Supplementary Figure 4. B) Trypan blue staining of Col-0 WT and vir-1 mutant leaves imaged with a Zeiss histology microscope at 10x magnification. C) Estimation of trypan blue staining patterns using ImageJ (n = 10 per condition). Two-way ANOVA revealed significant effects of temperature (F = 22.34, p = 3.45 × 10⁻⁵), genotype (F = 22.18, p = 3.63 × 10⁻⁵), and their interaction (F = 22.16, p = 3.66 × 10 ⁻⁵). Post hoc comparisons using Tukey’s HSD test indicated that vir-1 at 17°C significantly differed from all other conditions ( p < 0.0001) . Source data available in Supplementary Table 10. .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 23, 2025. ; https://doi.org/10.1101/2025.02.18.638636doi: bioRxiv preprint 31 Figure 5: m6A levels in vir-1 mutants are not temperature-sensitive. A) LC-MS/MS analysis showing the significant effect of genotype on the m6A/A ratio and the lack of significant interaction between genotype and temperature on m 6A levels (two-way ANOVA; p 0.9 predicted by m6Anet. Individual replicates are plotted as solid lines, with the combined density of a condition (genotype and temperature) plotted as a dashed line. 3545 sites were predicted to have an m6A modified site in at least one Col-0 sample, compared to only 327 in vir-1. .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 23, 2025. ; https://doi.org/10.1101/2025.02.18.638636doi: bioRxiv preprint 32 Figure 6: The primary visible developmental defects of vir-1 mutants are not rescued by growth at 27°C A) Developmental phenotype of Arabidopsis WT Col-0 and mutant vir-1 grown at 17°C and 27°C. Plants are 28 days old and were grown at the indicated temperatures throughout their development following a 2-day stratification treatment. .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 23, 2025. ; https://doi.org/10.1101/2025.02.18.638636doi: bioRxiv preprint 33 Figure 7: Poly(A) tail length distributions of vir-1 are disrupted in a temperature sensitive manner A) Shifts towards upstream (promoter -proximal) poly(A) site usage are detected in vir-1 mutants at 17°C and 27°C compared to Col -0 WT. Source data available in Supplementary Table 14. B) Normalised expression (log 2 counts per million) of PR1 (AT2G14610) in ONT DRS analysis of cpsf30-yth mutants at 20°C (n = 4 per condition). C) Normalised expression (log 2 counts per million) of PR1 (AT2G14610) in ONT DRS analysis of te234 triple mutants at 20°C ( n = 4 per condition). D) Density distribution of poly(A) tail lengths in Col-0 and vir-1 at 17°C and 27°C. The distribution of each replicate is plotted individually. E) Histograms depicting the distribution of Wasserstein distance metric for significant changes in mean poly(A) tail length per gene between Col-0 and vir-1 at 17°C and 27°C, and between Col-0 at 27°C and 17°C. At 17°C, 13 genes have significantly shorter poly(A) tails in vir-1, while 7,894 genes have significantly longer tails. At 27°C, 6,669 genes displayed significantly shorter poly(A) tails in vir-1, whereas 7 genes had significantly longer tails. In Col-0 there are 1,425 genes with significantly shorter mean poly(A) tails at 17ºC compared to 27ºC. These findings are derived from data pooled across all replicates. .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 23, 2025. ; https://doi.org/10.1101/2025.02.18.638636doi: bioRxiv preprint 34 Supplementary Figure 2 – Linked to Figure 2 A) Normalised logged counts per million of PR1 (AT2G14610) in Col -0 and vir-1 17°C and 27ºC (n = 3-4 per condition). B) RT-qPCR showing the upregulation of PR1 in vir-1 at 17°C (n = 3 per condition). C) Volcano plot showing the log2 fold change and adjusted p -value of differential gene expression in vir-1 at 17°C contrasted to the average expression in vir-1 at 27°C and Col-0 at 17°C and 27°C. Genes with log2FC > 2 and p < 0.001 are coloured in red, genes which only pass the p-value threshold are coloured in black, and genes which only pass the log2FC threshold are coloured in blue. Non-significant changes (NS) are coloured in grey. Source data available in Supplementary Table 5. D) Overlap in enriched GO terms between genes upregulated at 17°C contrasted to the average expression in vir-1 at 27°C and Col-0 at 17°C and 27°C, and genes which were significantly upregulated in vir-1 at 22ºC contrasted to Col-0 at 22ºC. .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 23, 2025. ; https://doi.org/10.1101/2025.02.18.638636doi: bioRxiv preprint 35 Supplementary Figure 3: Linked to Figure 3 A) Upregulation of SARD1 (AT1G73805) in vir-1 at 17°C, shown by a boxplot of normalised expression (log 2 counts per million) in ONT DRS data (n = 3 -4 samples per condition). B) Upregulation of FLS2 (AT5G46330) in vir-1 at 17°C, shown by a boxplot of normalised expression (log 2 counts per million) in ONT DRS data (n = 3 -4 samples per condition). C) Upregulation of TX10 (AT1G57630) in vir-1 at 17°C, shown by a boxplot of normalised .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 23, 2025. ; https://doi.org/10.1101/2025.02.18.638636doi: bioRxiv preprint 36 expression (log 2 counts per million) in ONT DRS data (n = 3 -4 samples per condition). D) Gene track of ONT DRS data showing the upregulation of a novel TIR domain-containing gene (annotated as Novel gene) downstream of RPS6 (AT5G46470) in vir-1 at 17 °C (n = 3 -4 samples per condition). E) Upregulation of ACD6 (AT4G14400) in vir-1 at 17°C, shown by a boxplot of normalised expression (log 2 counts per million) in ONT DRS data (n = 4 samples per condition). F-M) Boxplots showing the normalised log 2 counts per million (as produced by edgeR) for the flowering genes; FT (AT1G65480), FUL (AT5G60910), SOC1 (AT2G45660), SEP3 (AT1G24260), SPL4 (AT1G53160), SPL5 (AT3G15270), AGL19 (AT4G22950) and AGL24 (AT4G24540), in Illumina RNA-seq of vir-1 and Col-0 at 17ºC and 27ºC (n = 4 samples per condition). .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 23, 2025. ; https://doi.org/10.1101/2025.02.18.638636doi: bioRxiv preprint 37 Supplementary Figure 4: Linked to Figure 4 Four-week-old Col-0 WT , vir-1 and fls2c seedlings flood-inoculated with a bacterial suspension of Pst DC3000 (5x106 CFU/ml) and 0.025% v/v Silwet L-77. Bacterial populations were quantified at 3 days post inoculation (dpi) (n = 3 per condition). One way ANOVA tests on each genotype revealed a significant effect of temperature in the vir-1 genotype (F = 23.02, p = 0.00197) which was not present in Col -0 WT or fls2. Source data available in Supplementary Table 9. This experimental analysis represents an independent replication of the experiment presented in Figure 4A. 17°C 20°C 27°C Col−0 fls2c vir−1 Col−0 fls2c vir−1 Col−0 fls2c vir−1 1e+06 1e+08 1e+10 Genotype CFU/mg genotype Col−0 fls2c vir−1 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 23, 2025. ; https://doi.org/10.1101/2025.02.18.638636doi: bioRxiv preprint 38 Supplementary Figure 5: Linked to Figure 5 A) Gene track showing the expression of VIRILIZER in Illumina RNA-seq VIR expression in vir-1 mutants at 17°C and 27°C. B) Magnified portion of the VIRILIZER gene track showing the combined coverage and alignment of Illumina RNA-seq around the EMS point mutation in vir-1. The vir-1 mutation affects the 5’ splice site of intron 5 (G+1 to A), which leads to the activation of cryptic 5’ splice sites upstream in exon 5 detected only in vir-1 (denoted by an arrow). No suppression of this cryptic splicing is found at 27 °C. Aligned reads were subsampled to 200 reads per condition. C) Density distribution of the ratio of modification predicted by Yanocomp, for modifications with an FDR < 0.05. Predicted modification ratios for vir-1 and Col-0 at 17°C were obtained by comparisons of vir-1 at 17°C and Col-0 at 17°C. Predicted modification ratios for vir-1 and Col-0 at 27°C were obtained by comparing vir-1 at 27°C and Col-0 at 27°C. .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 23, 2025. ; https://doi.org/10.1101/2025.02.18.638636doi: bioRxiv preprint 39 Supplementary Figure 6: Linked to Figure 6 A) Phenotype of Col-0, vir-1, fip37-4, cpsf30-yth, te234 and acd6-1 grown at 17ºC and 27ºC. B) Gene ontology biological process terms enriched in the 58 genes consistently significantly upregulated (log2FC +/- 2.0 FDR < 0.001) in vir-1 across 17°C, 20°C and 27°C compared to Col-0 and VIRc. Source data available in Supplementary Table 13. C) Upregulation of FMO1 (AT1G19250) in vir-1 at both 17°C and 27°C in Illumina RNA-seq and ONT DRS data, shown by gene tracks and boxplots of normalised expression (log 2 counts per million). .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 23, 2025. ; https://doi.org/10.1101/2025.02.18.638636doi: bioRxiv preprint 40 Supplementary Figure 7: Linked to Figure 7 A) Density distribution of poly(A) tail lengths of Saccharomyces cerevisiae ENOLASE II spike-in sequences in Col-0 and vir-1 at 17°C and 27°C. ENOLASE II transcripts with a poly(A) tail length of 30 nt are included as the RNA calibration standard during ONT DRS library preparation. B) Density distribution of poly(A) tails in Col-0 and vir-1 at 17°C and 27°C, divided into those belonging to genes with a predicted m6A modification in Col-0 and those with no predicted modification. The distribution of poly(A) tails is plotted individually for each replicate. .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 23, 2025. ; https://doi.org/10.1101/2025.02.18.638636doi: bioRxiv preprint 41

Alcázar R & Parker JE (2011) The impact of temperature on balancing immune responsiveness and growth in Arabidopsis. Trends Plant Sci 16: 666–675 Andrews S (20 17). FastQC: A quality control tool for high throughput sequence data. v3. BabrahamBioinformatics. https://www.bioinformatics.babraham.ac.uk/projects/fastqc/. Arribas-Hernández L, Bressendorff S, Hansen MH, Poulsen C, Erdmann S & Brodersen P (2018) An m6A -YTH module controls developmental timing and morphogenesis in Arabidopsis. Plant Cell 30: 952–967 Arribas-Hernández L, Simonini S, Hansen MH, Paredes EB, Bressendorff S, Dong Y, Østergaard L & Brodersen P (2020) Recurrent requirement for the m6A - ECT2/ECT3/ECT4 axis in the control of cell proliferation during plant organogenesis. Development 147: dev189134 Baer BW & Kornberg RD (1983) The protein responsible for the repeating structure of cytoplasmic poly(A)-ribonucleoprotein. J Cell Biol 96: 717–721 Barragan AC & Weigel D (2021) Plant NLR diversity: the known unknowns of pan-NLRomes. Plant Cell 33: 814–831 Bartok E & Hartmann G (2020) Immune sensing mechanisms that discriminate self from altered self and foreign nucleic acids. Immunity 53: 54–77 Bartsch M, Gobbato E, Bednarek P, Debey S, Schultze JL, Bautor J & Parker JE (2006) Salicylic acid -independent ENHANCED DISEASE SUSCEPTIBILITY1 signaling in Arabidopsis immunity and cell death is regulated by the monooxygenase FMO1 and the Nudix hydrolase NUDT7. Plant Cell 18: 1038–1051 Békés M, Langley DR & Crews CM (2022) PROTAC targeted protein degraders: the past is prologue. Nat Rev Drug Discov 21: 181–200 Betsuyaku S, Katou S, Takebayashi Y, Sakakibara H, Nomura N & Fukuda H (2018) Salicylic acid and jasmonic acid pathways are activated in spatially different domains around the infection site during effector-triggered immunity in Arabidopsis thaliana. Plant Cell Physiol 59: 8–16 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 23, 2025. ; https://doi.org/10.1101/2025.02.18.638636doi: bioRxiv preprint 42 Bodi Z, Zhong S, Mehra S, Song J, Graham N, Li H, May S & Fray RG (2012) Adenosine methylation in Arabidopsis mRNA is associated with the 3’ end and reduced levels cause developmental defects. Front Plant Sci 3: 48 Bomblies K, Lempe J, Epple P, Warthmann N, Lanz C, Dangl JL & Weigel D (2007) Autoimmune response as a mechanism for a Dobzhansky -Muller-type incompatibility syndrome in plants. PLoS Biol 5: e236 Bomblies K & Weigel D (2007) Hybrid necrosis: autoimmunity as a potential gene-flow barrier in plant species. Nat Rev Genet 8: 382–393 Bonardi V, Cherkis K, Nishimura MT & Dangl JL (2012) A new eye on NLR proteins: focused on clarity or diffused by complexity? Curr Opin Immunol 24: 41–50 Brodersen P & Arribas -Hernández L (2024) The m6A -YTH regulatory system in plants: A status. Curr Opin Plant Biol 82: 102650 Buchfink B, Reuter K & Drost H -G (2021) Sensitive protein alignments at tree -of-life scale using DIAMOND. Nat Methods 18: 366–368 Bushnell B (2022) BBMap. Retrieved from https://sourceforge.net/projects/bbmap Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K & Madden TL (2009) BLAST+: architecture and applications. BMC Bioinformatics 10: 421 Carreras-Puigvert J, Zitnik M, Jemth A -S, Carter M, Unterlass JE, Hallström B, Loseva O, Karem Z, Calderón-Montaño JM, Lindskog C, et al (2017) A comprehensive structural, biochemical and biological profiling of the human NUDIX hydrolase family. Nat Commun 8: 1541 Chan SL, Huppertz I, Yao C, Weng L, Moresco JJ, Yates JR 3rd, Ule J, Manley JL & Shi Y (2014) CPSF30 and Wdr33 directly bind to AAUAAA in mammalian mRNA 3’ processing. Genes Dev 28: 2370–2380 Chen J, Li L, Kim JH, Neuhäuser B, Wang M, Thelen M, Hilleary R, Chi Y, Wei L, Venkataramani K, et al (2023) Small proteins modulate ion -channel-like ACD6 to regulate immunity in Arabidopsis thaliana. Mol Cell 83: 4386-4397.e9 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 23, 2025. ; https://doi.org/10.1101/2025.02.18.638636doi: bioRxiv preprint 43 Chen Y, Lun ATL & Smyth GK (2016) From reads to genes to pathways: differential expression analysis of RNA -Seq experiments using Rsubread and the edgeR quasi -likelihood pipeline. F1000Res 5: 1438 Cheng C-Y, Krishnakumar V, Chan AP, Thibaud -Nissen F, Schobel S & Town CD (2017) Araport11: a complete reannotation of the Arabidopsis thaliana reference genome. Plant J 89: 789–804 Dangl JL & Jones JD (2001) Plant pathogens and integrated defence responses to infection. Nature 411: 826–833 Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, Batut P, Chaisson M & Gingeras TR (2013) STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29: 15– 21 Dong OX, Ao K, Xu F, Johnson KCM, Wu Y, Li L, Xia S, Liu Y, Huang Y, Rodriguez E, et al (2018) Individual components of paired typical NLR immune receptors are regulated by distinct E3 ligases. Nat Plants 4: 699–710 Essuman K, Milbrandt J, Dangl JL & Nishimura MT (2022) Shared TIR enzymatic functions regulate cell death and immunity across the tree of life. Science 377: eabo0001 Fabian M, Gao M, Zhang X-N, Shi J, Vrydagh L, Kim S-H, Patel P, Hu AR & Lu H (2023) The flowering time regulator FLK controls pathogen defense in Arabidopsis thaliana. Plant Physiol 191: 2461–2474 Flores-Téllez D, Tankmar MD, von Bülow S, Chen J, Lindorff -Larsen K, Brodersen P & Arribas-Hernández L (2023) Insights into the conservation and diversification of the molecular functions of YTHDF proteins. PLoS Genet 19: e1010980 Freh M, Gao J, Petersen M & Panstruga R (2022) Plant autoimmunity -fresh insights into an old phenomenon. Plant Physiol 188: 1419–1434 Freund I, Eigenbrod T, Helm M & Dalpke AH (2019) RNA modifications modulate activation of innate Toll-like receptors. Genes 10: 92 Fu L, Niu B, Zhu Z, Wu S & Li W (2012a) CD -HIT: accelerated for clustering the next - generation sequencing data. Bioinformatics 28: 3150–3152 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 23, 2025. ; https://doi.org/10.1101/2025.02.18.638636doi: bioRxiv preprint 44 Fu ZQ, Yan S, Saleh A, Wang W, Ruble J, Oka N, Mohan R, Spoel SH, Tada Y, Zheng N, et al (2012b) NPR3 and NPR4 are receptors for the immune signal salicylic acid in plants. Nature 486: 228–232 Gamaarachchi H, Lam CW, Jayatilaka G, Samarakoon H, Simpson JT, Smith MA & Parameswaran S (2020) GPU accelerated adaptive banded event alignment for rapid comparative nanopore signal analysis. BMC Bioinformatics 21: 343 Geula S, Moshitch -Moshkovitz S, Dominissini D, Mansour AA, Kol N, Salmon -Divon M, Hershkovitz V, Peer E, Mor N, Manor YS, et al (2015) m 6 A mRNA methylation facilitates resolution of naïve pluripotency toward differentiation. Science 347: 1002– 1006 Gloggnitzer J, Akimcheva S, Srinivasan A, Kusenda B, Riehs N, Stampfl H, Bautor J, Dekrout B, Jonak C, Jiménez -Gómez JM, et al (2014) Nonsense -mediated mRNA decay modulates immune receptor levels to regulate plant antibacterial defense. Cell Host Microbe 16: 376–390 Gómez-Gómez L & Boller T (2000) FLS2: an LRR receptor -like kinase involved in the perception of the bacterial elicitor flagellin in Arabidopsis. Mol Cell 5: 1003–1011 Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, Adiconis X, Fan L, Raychowdhury R, Zeng Q, et al (2011) Full-length transcriptome assembly from RNA- Seq data without a reference genome. Nat Biotechnol 29: 644–652 Hartmann M, Zeier T, Bernsdorff F, Reichel -Deland V, Kim D, Hohmann M, Scholten N, Schuck S, Bräutigam A, Hölzel T, et al (2018) Flavin monooxygenase-generated N- hydroxypipecolic acid is a critical element of plant systemic immunity. Cell 173: 456- 469.e16 Hendra C, Pratanwanich PN, Wan YK, Goh WSS, Thiery A & Göke J (2022) Detection of m6A from direct RNA sequencing using a multiple instance learning framework. Nat

19: 1590–1598 Jacob P, Hige J, Song L, Bayless A, Russ D, Bonardi V, El Kasmi F, Wünsch L, Yang Y, Fitzpatrick CR, et al (2023) Broader functions of TIR domains in Arabidopsis immunity. Proc Natl Acad Sci U S A 120: e2220921120 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 23, 2025. ; https://doi.org/10.1101/2025.02.18.638636doi: bioRxiv preprint 45 Junt T & Barchet W (2015) Translating nucleic acid-sensing pathways into therapies. Nat Rev Immunol 15: 529–544 Karikó K (2021) Modified uridines are the key to a successful message. Nat Rev Immunol 21: 619 Karikó K, Buckstein M, Ni H & Weissman D (2005) Suppression of RNA recognition by Toll - like receptors: the impact of nucleoside modification and the evolutionary origin of RNA. Immunity 23: 165–175 Kierzek E & Kierzek R (2003) The thermodynamic stability of RNA duplexes and hairpins containing N6 -alkyladenosines and 2 -methylthio-N6-alkyladenosines. Nucleic Acids Res 31: 4472–4480 Kim JH, Castroverde CDM, Huang S, Li C, Hilleary R, Seroka A, Sohrabi R, Medina -Yerena D, Huot B, Wang J, et al (2022) Increasing the resilience of plant immunity to a warming climate. Nature 607: 339–344 Kinoshita A & Richter R (2020) Genetic and molecular basis of floral induction in Arabidopsis thaliana. J Exp Bot 71: 2490–2504 Kolberg L, Raudvere U, Kuzmin I, Adler P, Vilo J & Peterson H (2023) g:Profiler-interoperable web service for functional enrichment analysis and gene identifier mapping (2023 update). Nucleic Acids Res 51: W207–W212 Korn SM, Ulshöfer CJ, Schneider T & Schlundt A (2021) Structures and target RNA preferences of the RNA-binding protein family of IGF2BPs: An overview. Structure 29: 787–803 Koski GK, Karikó K, Xu S, Weissman D, Cohen PA & Czerniecki BJ (2004) Cutting edge: innate immune system discriminates between RNA containing bacterial versus eukaryotic structural features that prime for high-level IL-12 secretion by dendritic cells. J Immunol 172: 3989–3993 Krishnakumar V, Hanlon MR, Contrino S, Ferlanti ES, Karamycheva S, Kim M, Rosen BD, Cheng C-Y, Moreira W, Mock SA, et al (2015) Araport: the Arabidopsis information portal. Nucleic Acids Res 43: D1003-9 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 23, 2025. ; https://doi.org/10.1101/2025.02.18.638636doi: bioRxiv preprint 46 Lamesch P, Berardini TZ, Li D, Swarbreck D, Wilks C, Sasidharan R, Muller R, Dreher K, Alexander DL, Garcia -Hernandez M, et al (2012) The Arabidopsis Information Resource (TAIR): improved gene annotation and new tools. Nucleic Acids Res 40: D1202-10 Laudenbach BT, Krey K, Emslander Q, Andersen LL, Reim A, Scaturro P, Mundigl S, Dächert C, Manske K, Moser M, et al (2021) NUDT2 initiates viral RNA degradation by removal of 5’-phosphates. Nat Commun 12: 6918 Lee B, Park Y-S, Lee S, Song GC & Ryu C-M (2016) Bacterial RNAs activate innate immunity in Arabidopsis. New Phytol 209: 785–797 Li H (2018) Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics 34: 3094– 3100 Liu N, Dai Q, Zheng G, He C, Parisien M & Pan T (2015) N(6) -methyladenosine-dependent RNA structural switches regulate RNA-protein interactions. Nature 518: 560–564 Locci F & Parker JE (2024) Plant NLR immunity activation and execution: a biochemical perspective. Open Biol 14: 230387 Lopez-Gomollon S & Baulcombe DC (2022) Roles of RNA silencing in viral and non-viral plant immunity and in the crosstalk between disease resistance systems. Nat Rev Mol Cell Biol 23: 645–662 Lu H, Rate DN, Song JT & Greenberg JT (2003) ACD6, a novel ankyrin protein, is a regulator and an effector of salicylic acid signaling in the Arabidopsis defense response. Plant Cell 15: 2408–2420 Maharana S, Kretschmer S, Hunger S, Yan X, Kuster D, Traikov S, Zillinger T, Gentzel M, Elangovan S, Dasgupta P, et al (2022) SAMHD1 controls innate immunity by regulating condensation of immunogenic self RNA. Mol Cell 82: 3712-3728.e10 Michaels SD & Amasino RM (1998) A robust method for detecting single-nucleotide changes as polymorphic markers by PCR. Plant J 14: 381–385 Mölder F, Jablonski KP, Letcher B, Hall MB, Tomkins -Tinch CH, Sochat V, Forster J, Lee S, Twardziok SO, Kanitz A, et al (2021) Sustainable data analysis with Snakemake. F1000Res 10: 33 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 23, 2025. ; https://doi.org/10.1101/2025.02.18.638636doi: bioRxiv preprint 47 Murakami S & Jaffrey SR (2022) Hidden codes in mRNA: Control of gene expression by m6A. Mol Cell 82: 2236–2251 Ngou BPM, Ahn H -K, Ding P & Jones JDG (2021) Mutual potentiation of plant immunity by cell-surface and intracellular receptors. Nature 592: 110–115 Ostendorf T, Zillinger T, Andryka K, Schlee -Guimaraes TM, Schmitz S, Marx S, Bayrak K, Linke R, Salgert S, Wegner J, et al (2020) Immune sensing of synthetic, bacterial, and protozoan RNA by toll -like receptor 8 requires coordinated processing by RNase T2 and RNase 2. Immunity 52: 591-605.e6 Parker MT, Barton GJ & Simpson GG (2021a) Yanocomp: robust prediction of m6A modifications in individual nanopore direct RNA reads. bioRxiv: 2021.06.15.448494 Parker MT, Knop K, Sherwood AV, Schurch NJ, Mackinnon K, Gould PD, Hall AJ, Barton GJ & Simpson GG (2020) Nanopore direct RNA sequencing maps the complexity of Arabidopsis mRNA processing and m6A modification. Elife 9: e49658 Parker MT, Knop K, Zacharaki V, Sherwood AV, Tomé D, Yu X, Martin PG, Beynon J, Michaels SD, Barton GJ, et al (2021b) Widespread premature transcription termination of Arabidopsis thaliana NLR genes by the spen protein FPA. Elife 10: e65537 Parker MT, Soanes BK, Kusakina J, Larrieu A, Knop K, Joy N, Breidenbach F, Sherwood AV, Barton GJ, Fica SM, et al (2022) m6A modification of U6 snRNA modulates usage of two major classes of pre-mRNA 5’ splice site. Elife 11: e78808 Passmore LA & Coller J (2022) Roles of mRNA poly(A) tails in regulation of eukaryotic gene expression. Nat Rev Mol Cell Biol 23: 93–106 Patro R, Duggal G, Love MI, Irizarry RA & Kingsford C (2017) Salmon provides fast and bias- aware quantification of transcript expression. Nat Methods 14: 417–419 Prall W, Sheikh AH, Bazin J, Bigeard J, Almeida -Trapp M, Crespi M, Hirt H & Gregory BD (2023) Pathogen-induced m6A dynamics affect plant immunity. Plant Cell 35: 4155– 4172 Pruitt RN, Locci F, Wanke F, Zhang L, Saile SC, Joe A, Karelina D, Hua C, Fröhlich K, Wan W-L, et al (2021) The EDS1 –PAD4–ADR1 node mediates Arabidopsis pattern - triggered immunity. Nature 598: 495–499 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 23, 2025. ; https://doi.org/10.1101/2025.02.18.638636doi: bioRxiv preprint 48 Rate DN, Cuenca JV, Bowman GR, Guttman DS & Greenberg JT (1999) The gain-of-function Arabidopsis acd6 mutant reveals novel regulation and function of the salicylic acid signaling pathway in controlling cell death, defenses, and cell growth. Plant Cell 11: 1695–1708 Reiser L, Bakker E, Subramaniam S, Chen X, Sawant S, Khosa K, Prithvi T & Berardini TZ (2024) The Arabidopsis Information Resource in 2024. Genetics 227: 1–11 Remick BC, Gaidt MM & Vance RE (2023) Effector -triggered immunity. Annu Rev Immunol 41: 453–481 Ries RJ, Pickering BF, Poh HX, Namkoong S & Jaffrey SR (2023) m6A governs length - dependent enrichment of mRNAs in stress granules. Nat Struct Mol Biol 30: 1525– 1535 Roost C, Lynch SR, Batista PJ, Qu K, Chang HY & Kool ET (2015) Structure and thermodynamics of N6-methyladenosine in RNA: a spring-loaded base modification. J Am Chem Soc 137: 2107–2115 Růžička K, Zhang M, Campilho A, Bodi Z, Kashif M, Saleh M, Eeckhout D, El-Showk S, Li H, Zhong S, et al (2017) Identification of factors required for m6 A mRNA methylation in Arabidopsis reveals a role for the conserved E3 ubiquitin ligase HAKAI. New Phytol 215: 157–172 Sablowski RW & Meyerowitz EM (1998) Temperature-sensitive splicing in the floral homeotic mutant apetala3-1. Plant Cell 10: 1453–1463 Sasse A, Ray D, Laverty KU, Tam CL, Albu M, Zheng H, Lyudovyk O, Dalal T, Nie K, Magis C, et al (2024) Reconstructing the sequence specificities of RNA -binding proteins across eukaryotes. bioRxiv 2024.10.15.618476 Schlee M & Hartmann G (2016) Discriminating self from non-self in nucleic acid sensing. Nat Rev Immunol 16: 566–580 Schneider CA, Rasband WS & Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9: 671–675 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 23, 2025. ; https://doi.org/10.1101/2025.02.18.638636doi: bioRxiv preprint 49 Schönemann L, Kühn U, Martin G, Schäfer P, Gruber AR, Keller W, Zavolan M & Wahle E (2014) Reconstitution of CPSF active in polyadenylation: recognition of the polyadenylation signal by WDR33. Genes Dev 28: 2381–2393 Shen L, Liang Z, Gu X, Chen Y, Teo ZWN, Hou X, Cai WM, Dedon PC, Liu L & Yu H (2016) N(6)-Methyladenosine RNA modification regulates shoot stem cell fate in Arabidopsis. Dev Cell 38: 186–200 Sherman BT, Hao M, Qiu J, Jiao X, Baseler MW, Lane HC, Imamichi T & Chang W (2022) DAVID: a web server for functional enrichment analysis and functional annotation of gene lists (2021 update). Nucleic Acids Res 50: W216–W221 Stevens AT, Howe DK & Hunt AG (2018) Characterization of mRNA polyadenylation in the apicomplexa. PLoS One 13: e0203317 Sun T, Zhang Y, Li Y, Zhang Q, Ding Y & Zhang Y (2015) ChIP -seq reveals broad roles of SARD1 and CBP60g in regulating plant immunity. Nat Commun 6: 10159 Świadek M, Proost S, Sieh D, Yu J, Todesco M, Jorzig C, Rodriguez Cubillos AE, Plötner B, Nikoloski Z, Chae E, et al (2017) Novel allelic variants in ACD6 cause hybrid necrosis in local collection of Arabidopsis thaliana. New Phytol 213: 900–915 Takagi M, Iwamoto N, Kubo Y, Morimoto T, Takagi H, Takahashi F, Nishiuchi T, Tanaka K, Taji T, Kaminaka H, et al (2020) Arabidopsis SMN2/HEN2, encoding DEAD-box RNA helicase, governs proper expression of the resistance gene SMN1/RPS6 and is involved in dwarf, autoimmune phenotypes of mekk1 and mpk4 mutants. Plant Cell Physiol 61: 1507–1516 Tang B, Feng L, Hulin MT, Ding P & Ma W (2023) Cell -type-specific responses to fungal infection in plants revealed by single-cell transcriptomics. Cell Host Microbe 31: 1732- 1747.e5 Tian H, Wu Z, Chen S, Ao K, Huang W, Yaghmaiean H, Sun T, Xu F, Zhang Y, Wang S, et al (2021) Activation of TIR signalling boosts pattern-triggered immunity. Nature 598: 500– 503 Todesco M, Balasubramanian S, Hu TT, Traw MB, Horton M, Epple P, Kuhns C, Sureshkumar S, Schwartz C, Lanz C, et al (2010) Natural allelic variation underlying a major fitness trade-off in Arabidopsis thaliana. Nature 465: 632–636 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 23, 2025. ; https://doi.org/10.1101/2025.02.18.638636doi: bioRxiv preprint 50 Tsai Y -C, Uechi H, Millar SR, Kadijk E, Schreiber KJ, Minkovich A, Huang JQ, Zhang Z, Barsacchi R, Möbius C, et al (2025) Dynamic proximal interactomics and chemical genetic screening reveal CCR4-NOT sequestration in stress granules as a mechanism for transcript stabilization. bioRxiv 2025.02.04.636058 Tudek A, Krawczyk PS, Mroczek S, Tomecki R, Turtola M, Matylla -Kulińska K, Jensen TH & Dziembowski A (2021) Global view on the metabolism of RNA poly(A) tails in yeast Saccharomyces cerevisiae. Nat Commun 12: 4951 Van der Biezen EA & Jones JD (1998) Plant disease -resistance proteins and the gene -for- gene concept. Trends Biochem Sci 23: 454–456 Vi SL, Trost G, Lange P, Czesnick H, Rao N, Lieber D, Laux T, Gray WM, Manley JL, Groth D, et al (2013) Target specificity among canonical nuclear poly(A) polymerases in plants modulates organ growth and pathogen response. Proc Natl Acad Sci U S A 110: 13994–13999 Wan W-L, Kim S -T, Castel B, Charoennit N & Chae E (2021) Genetics of autoimmunity in plants: an evolutionary genetics perspective. New Phytol 229: 1215–1233 Wang L, Tsuda K, Truman W, Sato M, Nguyen LV, Katagiri F & Glazebrook J (2011) CBP60g and SARD1 play partially redundant critical roles in salicylic acid signaling: Role of CBP60 proteins in salicylic acid signaling. Plant J 67: 1029–1041 van Wersch R, Li X & Zhang Y (2016) Mighty dwarfs: Arabidopsis autoimmune mutants and their usages in genetic dissection of plant immunity. Front Plant Sci 7: 1717 Wong CE, Zhang S, Xu T, Zhang Y, Teo ZWN, Yan A, Shen L & Yu H (2023) Shaping the landscape of N6-methyladenosine RNA methylation in Arabidopsis. Plant Physiol 191: 2045–2063 Wu B, Su S, Patil DP, Liu H, Gan J, Jaffrey SR & Ma J (2018) Molecular basis for the specific and multivariant recognitions of RNA substrates by human hnRNP A2/B1. Nat Commun 9: 420 Yamagishi R, Inagaki H, Suzuki J, Hosoda N, Sugiyama H, Tomita K, Hotta T & Hoshino S -I (2024) Concerted action of ataxin -2 and PABPC1 -bound mRNA poly(A) tail in the formation of stress granules. Nucleic Acids Res 52: 9193–9209 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 23, 2025. ; https://doi.org/10.1101/2025.02.18.638636doi: bioRxiv preprint 51 Young MD, Wakefield MJ, Smyth GK & Oshlack A (2010) Gene ontology analysis for RNA - seq: accounting for selection bias. Genome Biol 11: R14 Yu D, Song W, Tan EYJ, Liu L, Cao Y, Jirschitzka J, Li E, Logemann E, Xu C, Huang S, et al (2022) TIR domains of plant immune receptors are 2’,3’ -cAMP/cGMP synthetases mediating cell death. Cell 185: 2370-2386.e18 Yuan M, Jiang Z, Bi G, Nomura K, Liu M, Wang Y, Cai B, Zhou J-M, He SY & Xin X-F (2021) Pattern-recognition receptors are required for NLR -mediated plant immunity. Nature 592: 105–109 Zaccara S & Jaffrey S (2024) Understanding the redundant functions of the m6A -binding YTHDF proteins. RNA 30: 468–481 Zaccara S & Jaffrey SR (2020) A unified model for the function of YTHDF proteins in regulating m6A-modified mRNA. Cell 181: 1582-1595.e18 Zeier J (2021) Metabolic regulation of systemic acquired resistance. Curr Opin Plant Biol 62: 102050 Zhang M, Bodi Z, Mackinnon K, Zhong S, Archer N, Mongan N, Simpson G & Fray R (2022) Two zinc finger proteins with functions in m6A writing interact with HAKAI. Nat Commun 13: 1127 Zhang Y, Goritschnig S, Dong X & Li X (2003) A gain -of-function mutation in a plant disease resistance gene leads to constitutive activation of downstream signal transduction pathways in suppressor of npr1-1, constitutive 1. Plant Cell 15: 2636–2646 Zhou Y, Zhou B, Pache L, Chang M, Khodabakhshi AH, Tanaseichuk O, Benner C & Chanda SK (2019) Metascape provides a biologist -oriented resource for the analysis of systems-level datasets. Nat Commun 10: 1523 Zhu W, Zaidem M, Van de Weyer A-L, Gutaker RM, Chae E, Kim S-T, Bemm F, Li L, Todesco M, Schwab R, et al (2018) Modulation of ACD6 dependent hyperimmunity by natural alleles of an Arabidopsis thaliana NLR resistance gene. PLoS Genet 14: e1007628 Zhu Y, Qian W & Hua J (2010) Temperature modulates plant defense responses through NB- LRR proteins. PLoS Pathog 6: e1000844 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 23, 2025. ; https://doi.org/10.1101/2025.02.18.638636doi: bioRxiv preprint 52 Zipfel C, Robatzek S, Navarro L, Oakeley EJ, Jones JDG, Felix G & Boller T (2004) Bacterial disease resistance in Arabidopsis through flagellin perception. Nature 428: 764–767 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 23, 2025. ; https://doi.org/10.1101/2025.02.18.638636doi: bioRxiv preprint