The m6A mark is deposited on RNA by a conserved writer complex. In plants, this complex comprises the methyltransferases MTA and MTB together with FIP37, VIRILIZER-like proteins, and HAKAI, counterparts of the METTL3-METTL14-WTAP machinery familiar from animal cells. The modification preferentially lands on adenosines within the DRACH sequence motif, clustering near stop codons and in 3-prime untranslated regions. Demethylase enzymes known as erasers, including ALKBH9B and ALKBH10B in plants, can strip the mark away, while YTH-domain reader proteins bind methylated transcripts and determine their fate, whether stabilization, degradation, translation, or sequestration. Because the mark is reversible and acts post-transcriptionally, it provides a rapid regulatory layer that operates without altering the underlying genetic sequence. Disrupting core writers such as MTA or FIP37 is lethal to plant embryos, underscoring how deeply this system is woven into normal development, from meristem maintenance to flowering time and cold-stress adaptation.

Whether plant viral RNAs actually carry m6A was unresolved for decades, but since 2017 a series of studies has delivered converging proof. The first biochemical evidence came from Alfalfa mosaic virus, whose RNAs were enriched by immunoprecipitation with anti-m6A antibodies. Subsequent work mapped high-confidence methylation peaks on Wheat yellow mosaic virus, Plum pox virus, Potato virus Y, and Pepino mosaic virus, the latter detected even inside purified viral particles. More recently, antibody-independent nanopore direct RNA sequencing has confirmed m6A on Cucumber mosaic virus genomes, with sites overlapping those found by antibody-based methods, and liquid chromatography coupled to tandem mass spectrometry has verified the modification chemically. In Sugarcane mosaic virus, a potyvirus of maize, researchers pinpointed a single modified adenosine at position 6556, deposited by the maize writer ZmMTA, and showed that a synonymous mutation at that site significantly enhanced infection. Bamboo mosaic virus yielded 122 candidate sites by direct RNA sequencing. Together these techniques leave little doubt that m6A is an authentic feature of diverse plant viral RNAs.

How cytoplasmic plant viruses gain access to a methyltransferase complex that normally resides in the nucleus has become one of the most intriguing mechanistic questions in the field. Unlike nuclear-replicating animal viruses such as influenza or HIV, most plant viruses replicate exclusively in the cytoplasm, seemingly far from the writer machinery. The answer, it turns out, is that viruses actively drag the machinery to themselves. In wheat, the WYMV NIb protein physically interacts with the writer TaMTB and triggers its translocation to cytoplasmic replication sites, where the enzyme methylates viral RNA at a defined position. In Arabidopsis infected with Cucumber mosaic virus, the viral coat protein binds the core writer MTB directly and promotes nuclear-to-cytoplasmic relocation of the entire complex. During Sugarcane mosaic virus infection, ZmMTA co-localizes with viral genomic RNA in cytoplasmic aggregates, though the exact relocation mechanism remains to be worked out. Notably, not every virus succeeds in this recruitment; recent antibody-independent analyses of Chikungunya and Dengue viruses found no detectable m6A, a reminder that epitranscriptomic engagement is selective rather than universal.

Once deposited, the mark can cut in either direction. In several systems m6A behaves as a classic antiviral flag. Hypermethylated Cucumber mosaic virus RNAs are recognized by the reader protein ECT8, which directs them into decay pathways, likely through processing bodies. In Pepino mosaic virus infection of Nicotiana benthamiana, overexpressing the writers MTA or HAKAI boosts viral RNA methylation and restricts infection, while the methylated transcripts are bound by the YTH-domain readers NbECT2A, 2B, and 2C, which recruit nonsense-mediated decay factors such as UPF3 and SMG7 to destroy them. Silencing NbECT2B or the NMD components increases viral RNA stability and susceptibility. In maize, the reader ZmECT23 recognizes methylated Sugarcane mosaic virus RNA and recruits the CCR4-NOT deadenylation complex to destabilize it, and in Nicotiana benthamiana the writer NbMTA targets Potato virus Y genomes for degradation.

Yet some viruses have turned the same mark to their advantage. When the WYMV NIb protein lures TaMTB to replication complexes, the resulting site-specific methylation stabilizes the viral RNA and prevents its degradation, and mutating the methylation site reduces viral stability and pathogenicity. A parallel strategy appears in the plant rhabdovirus Barley yellow striate mosaic virus, whose P6 messenger RNA is hypermethylated; loss of the mark reduces P6 stability and infectivity. Intriguingly, barley fights back with its own eraser, HvALKBH1B, which binds P6 mRNA into cytoplasmic condensates through liquid-liquid phase separation, a process requiring the protein’s intrinsically disordered region, thereby stripping the viral advantage. The lesson is that m6A is not inherently antiviral or proviral; it is a regulatory currency that whichever side controls it can spend.

The mark also shapes viral systemic movement, not merely local accumulation. Alfalfa mosaic virus depends on the host demethylase ALKBH9B removing m6A from its RNAs for efficient long-distance spread, an interaction mediated by the viral coat protein. Excessive methylation reduces phloem transport and restricts movement through the plant, so a balanced level of the mark is essential for the virus to travel. Consistent with this, infection by Plum pox virus and Potato virus Y lowers global m6A levels in Nicotiana benthamiana, and knocking down ALKBH9 homologs diminishes accumulation of both viruses. On the defensive side, the YTHDF-family readers ECT2, ECT3, and ECT5 bind Alfalfa mosaic virus RNAs in an m6A-dependent manner and act as restriction factors suppressing viral accumulation. Strikingly, this regulation is specific to ALKBH9B, since disrupting the related homologs ALKBH9A or ALKBH9C leaves infection untouched, revealing a fine-grained specificity in how plants deploy their epitranscriptomic arsenal.

Viruses, unsurprisingly, have evolved countermeasures. The 2b protein of Cucumber mosaic virus, already infamous as a suppressor of RNA silencing, competitively binds the writer components HAKAI and MTB, impairing the integrity of the writer complex in both nucleus and cytoplasm. This suppresses methylation not only on viral RNAs but across the host transcriptome; plants lacking m6A are more susceptible to a 2b-deficient mutant but not to wild-type virus, and simply expressing 2b in transgenic plants lowers global m6A levels. Pepino mosaic virus takes a different route: its RNA-dependent RNA polymerase interacts with the tomato writer SlHAKAI and the autophagy protein SlBeclin1, forming cytoplasmic granules that route SlHAKAI to autophagic destruction, thereby stripping methylation from viral RNA. In maize, the Sugarcane mosaic virus protease NIa-Pro hijacks the initiation factor ZmeIF4A3 into viral replication complexes, sterically blocking ZmMTA-mediated methylation and sparing viral RNA from ZmECT23-directed decay. Each case illustrates a mutually antagonistic arms race fought at the level of RNA chemistry.

Viral infection also rewrites the host’s own methylation landscape in ways that reshape immunity. Tobacco mosaic virus lowers m6A levels in tobacco, coinciding with upregulation of the eraser NbALKBH5, while Rice stripe virus and Rice black-streaked dwarf virus raise methylation levels in rice, enriching marks on transcripts involved in RNA silencing and hormone defense, including the antiviral genes OsAGO18 and OsSLRL1. In watermelon facing Cucumber green mottle mosaic virus, susceptible plants show early hypermethylation of stress-related transcripts, whereas resistant cultivars exhibit later hypomethylation that preserves defense gene expression. In Arabidopsis, CMV-induced hypomethylation upregulates the salicylic acid regulators NPR3 and CBP60a, and Potato virus Y triggers a transient rise in methylation at five to ten days post-inoculation that falls by day fourteen, accompanied by activation of a transcription factor that boosts NbMTA expression and accelerates viral RNA degradation. These virus-specific, time-dependent shifts integrate m6A into the broader networks of hormone signaling and stress adaptation.

The review’s authors argue that these findings position m6A as a promising lever for crop protection, whether through overexpressing writer components, modulating reader activity, or breeding viral methylation sites out of susceptible hosts. Significant questions remain, however. Recent work warns that false-positive signals can arise in both antibody-based and some antibody-independent mapping methods, making rigorous negative controls essential, and emerging single-molecule technologies are expected to sharpen the resolution of modification detection. Researchers still do not fully understand what sequence or structural features direct methylation to particular viral transcripts, how the temporal choreography of methylation unfolds during infection, or how reliably these mechanisms can be engineered into durable field resistance. What is already clear is that the epitranscriptome has moved from the margins of plant virology to its center, and that the contest over a single methyl group on adenosine may help decide the outcome of infections that devastate staple crops worldwide.

Subject of Research: The role of m6A RNA modification in plant-virus interactions

Article Title: The expanding role of m6A RNA modification in plant-virus dynamics: friend, foe, or both?

Article References: Liu, J.-H., Yu, H., & Duan, C.-G. (2026). The expanding role of m6A RNA modification in plant-virus dynamics: friend, foe, or both?. Advanced Biotechnology, 4(1), Article 7. https://doi.org/10.1007/s44307-026-00100-3

Image Credits: AI Generated

DOI: 10.1007/s44307-026-00100-3

Keywords: m6A, RNA modification, epitranscriptome, plant viruses, plant immunity, methyltransferase, YTH-domain proteins, RNA decay, viral countermeasures, crop resistance, Cucumber mosaic virus, RNA methylation

A new study published in MicrobiologyOpen has now combined two cutting-edge technologies, CRISPR interference and nanopore native RNA sequencing, to systematically probe the epitranscriptome of Escherichia coli. The research, led by Miranda E. Pitt of the University of Technology Sydney and Lachlan J. M. Coin of the University of Melbourne, together with colleagues, demonstrates a workflow in which individual RNA modification enzymes are switched off in living bacteria, and the resulting loss of chemical marks is read out directly at the single-molecule level. The approach not only confirmed several well-known modification sites on ribosomal RNA but also revealed candidate modification sites scattered across bacterial messenger RNA, a frontier that has remained largely unmapped.

The technical challenge the team faced is formidable. Traditional methods for detecting RNA modifications, such as immunoprecipitation or chemical treatments like bisulfite conversion, are laborious, require highly purified and concentrated RNA, and generally capture only one modification type at a time. Bacterial RNA adds further complications: rigorous lysis procedures and complex cell membranes accelerate RNA degradation, and bacterial transcripts have notoriously short half-lives, in many cases under one minute. Enzymes called RNases can rapidly destroy extracted RNA unless inhibited immediately. Direct RNA sequencing, released by Oxford Nanopore Technologies in 2017, offers a faster alternative because it reads native RNA molecules as they pass through protein nanopores, preserving the chemical modifications that conventional methods destroy when RNA is converted to complementary DNA. However, the chemistry was originally incompatible with bacterial transcripts, which lack the polyadenylated tails found in eukaryotic messenger RNA.

The researchers overcame this barrier by artificially adding poly(A) tails to bacterial transcripts before sequencing, a technique they had refined in previous work. To create the perturbations needed to detect modifications, they turned to CRISPR interference, or CRISPRi. Unlike standard CRISPR gene editing, which cuts DNA, CRISPRi uses a catalytically inactive form of the Cas9 protein, dCas9, that binds to a target gene guided by a matching guide RNA and simply blocks its transcription. This knocks down, rather than knocks out, gene expression, avoiding the toxicity and off-target DNA disruption associated with genome cutting while allowing inducible, tunable silencing. The team used a plasmid system optimized for E. coli, in which dCas9 expression is triggered by the small molecule anhydrotetracycline.

Five genes encoding known ribosomal RNA modification enzymes were targeted: rlmF and rlmJ, both responsible for N6-methyladenosine marks on 23S ribosomal RNA; rsmF, which deposits 5-methylcytosine on 16S ribosomal RNA; rsmG, which installs 7-methylguanosine on 16S ribosomal RNA; and rluD, a pseudouridine synthase that modifies three positions in a functionally critical stem-loop of the 23S subunit. A sixth gene, add, encoding adenosine deaminase, was included to explore whether it might carry previously unrecognized RNA editing activity. The knockdown system was tested across multiple E. coli reference strains, including the type strain ATCC 11775, ATCC 8739, ATCC 25922, and a multidrug-resistant isolate, ATCC BAA-2452. Quantitative reverse transcription PCR confirmed that most target genes were repressed by more than 80 percent, although rsmG proved harder to silence, reaching only about 54 percent reduction, and the resistant isolate could not be transformed with the plasmid without a severe fitness cost.

With knockdown strains in hand, the researchers sequenced both total RNA and ribosomal RNA-depleted, messenger RNA-enriched samples on nanopore flow cells, using computational tools to compare ionic current signals between knockdown and control conditions. The software nanocompore, which does not require prior training and is agnostic to modification type, compared each perturbed sample against two controls: the unmodified original isolate and a plasmid control expressing dCas9 without a guide RNA. For rluD, all three known pseudouridine sites in the 23S ribosomal RNA were detected with strong statistical significance, and the sequence motifs surrounding those sites matched published reports. The rsmG knockdown likewise produced a clear signal at the known 7-methylguanosine position in 16S ribosomal RNA. In contrast, the sites modified by rlmF, rlmJ, and rsmF yielded weak signals that were difficult to distinguish from noise, echoing the findings of earlier benchmarking studies showing that certain modification types, particularly 5-methylcytosine and some N6-methyladenosine sites, are inherently harder to detect with current nanopore basecalling and current-difference tools.

Extending the analysis to messenger RNA, the team identified significant modification-site changes on several bacterial transcripts shared across strains. Genes harboring candidate modification sites included ompC, which encodes an outer membrane porin; lpp1; salPB; cspC; dbhA and dbhB; and secY, which encodes a core component of the protein secretion machinery. For the rluD and rsmG knockdowns, which had produced the strongest ribosomal RNA signals, the researchers searched messenger RNA for sequence motifs matching those seen at the known ribosomal sites. Strikingly, a significant change was found on the well-characterized pseudouridine motif in the gene encoding elongation factor Tu, the workhorse protein of translation, as well as on transcripts of lpp1, gapA, and fusA. For rsmG, significant sites matching the known 7-methylguanosine motif appeared in hupA, lpp1, rplT, rpsA, and ompC. Because these messenger RNA sites were detected in more than one strain background, they represent plausible, though not yet chemically verified, targets of bacterial modification enzymes acting beyond the ribosome.

The study also examined the downstream consequences of losing these modifications, measuring bacterial growth and profiling the proteome by quantitative mass spectrometry. Growth effects varied by strain: activation of dCas9 itself imposed a measurable burden in ATCC 11775, which carries an extra plasmid, while ATCC 8739 tolerated the system well. Statistically significant growth delays were observed for knockdowns of rluD and rsmF in one strain and rlmF, rluD, and rsmF in another, consistent with prior reports that the phenotypic impact of ribosomal RNA modification loss depends heavily on genetic background. Proteomic analysis detected roughly 1,800 to 2,400 proteins per strain and revealed modest but reproducible changes, including alterations in citrate lyase, the trehalose-specific phosphotransferase component TreB, the xylose-binding protein XylF, the sodium/proline symporter PutP, formate hydrogenlyase subunit HycB, and the oligopeptide transporter OppC. The authors note these changes require further validation and may only become pronounced under stress conditions such as antibiotic exposure or temperature shifts, which previous work has shown can reshape the bacterial epitranscriptome.

The broader significance of the work lies in its implications for antimicrobial resistance and bacterial virulence. RNA modifications are already known to help pathogens evade entire antibiotic classes, including aminoglycosides, chloramphenicol, and macrolides, and to contribute to antibiotic tolerance in organisms such as Vibrio cholerae. Loss of tRNA modification enzymes can impair a bacterium’s ability to infect and cause disease in a host. A reliable, rapid, genome-wide method for mapping modification dynamics in bacteria could therefore illuminate how pathogens respond to clinical interventions at the level of their RNA. The authors caution that fine-tuning is still needed, particularly for messenger RNA modification detection across diverse bacterial species and under varying growth conditions, and newer nanopore chemistry promises higher accuracy and greater yield. Yet as a proof of principle, pairing CRISPR interference with native RNA sequencing offers a scalable blueprint for interrogating the bacterial epitranscriptome, one silenced enzyme and one molecule of RNA at a time.

Subject of Research: Mapping RNA modifications in the Escherichia coli epitranscriptome using CRISPR interference gene knockdown and nanopore direct RNA sequencing

Article Title: Interrogating the Escherichia coli Epitranscriptome Via CRISPR Interference and Nanopore Native RNA Sequencing

Article References: Pitt, M. E., Zhang, J., Nguyen, A. N. T., Hall, M. B., Jebeli, L., Featherstone, L. A., Myers, G. S. A., Scott, N. E., & Coin, L. J. M. (2026). Interrogating the Escherichia coli Epitranscriptome Via CRISPR Interference and Nanopore Native RNA Sequencing. MicrobiologyOpen, 15(5), Article e70413. https://doi.org/10.1002/mbo3.70413

Image Credits: AI Generated

DOI: 10.1002/mbo3.70413

Keywords: epitranscriptome, E. coli, CRISPR interference, nanopore sequencing, direct RNA sequencing, RNA modifications, ribosomal RNA, messenger RNA, pseudouridine, m6A, antimicrobial resistance, MicrobiologyOpen