At the cellular level, fungal macroautophagy uses a conserved network of more than 40 ATG-family proteins to sequester cytoplasmic material in a double-membrane phagophore and deliver it to the fungal vacuole for degradation. In yeast and most filamentous fungi, autophagosome assembly is concentrated at a single phagophore assembly site, unlike the multiple ER-associated omegasomes described in mammalian cells. BAR-domain proteins help generate the sharply curved isolation membrane, while branched actin forms a cage around the assembly site that restricts uncontrolled membrane diffusion. After autophagosomes close, molecular motors transport them along microtubules to the vacuole, a particularly demanding task in polarized hyphae where autophagosomes may travel dozens of micrometers.

Membrane lipid signaling and vesicle-trafficking systems add further control over maturation and vacuolar fusion. In M. oryzae, the oxysterol-binding protein-related proteins MoOrp1 and MoOrp2 interact with MoAtg8, localize to the phagophore assembly site and enrich phosphatidylinositol-4-phosphate on autophagosomal membranes. A double gene knockout blocked autophagic flux and was associated with loss of appressorial turgor and host penetration. In Fusarium graminearum, TRAPPIII interacts with the autophagy transmembrane protein FgAtg9 through FgTrs85 and TRAPPC13 while also activating FgRab1 as a guanine nucleotide exchange factor, linking autophagosome biogenesis with ER-to-Golgi and endosome-to-Golgi transport during reproduction and infection.

Trafficking also helps determine which proteases reach the vacuole. The retromer protein MoVps35 in M. oryzae binds the serine protease MoPrb1 on endosomes and directs it into the vacuole, where MoPrb1 assists in localizing MoPep4. Genetic analyses assigned distinct functions to these proteases: MoPrb1 supports degradation of macroautophagy cargo associated with infection structures, and its mutation was linked to failed appressorium development, whereas MoPep4 regulates microautophagy, including peroxisomal degradation, without being required for virulence under the tested conditions. These results indicate that vesicle sorting can differentiate among autophagic pathways.

Selective autophagy remodels organelles and storage reserves in ways that support fungal growth and virulence. Quantitative proteome-turnover analysis in Komagataella phaffii found that changing carbon and nitrogen sources remodeled up to 90% of the pre-existing proteome, with Atg9-dependent bulk autophagy operating alongside cargo-specific pathways. Peroxisomes were cleared through Atg30- and Atg11-dependent pexophagy, while mitochondria underwent slower Atg32-dependent mitophagy. In M. oryzae, the MoDnm1–MoMdv1–MoFis1 complex regulates mitochondrial and peroxisomal fission; disruption impaired mitophagic clearance, conidiation, appressorium-mediated penetration and full virulence.

For lipid droplets in Metarhizium robertsii and Beauveria bassiana, morphological observations indicate partial engulfment by isolation membranes or sequestration through microlipophagy. Deletion of ATG8 homologs was associated with impaired droplet use, conidial germination and host infectivity. Autophagy is also regulated through RNA modifications, transcription and protein modification. In M. oryzae, the m5C methyltransferase Ncl1 modifies specific sites in atg5 and the 3′ untranslated region of atg16, increasing transcript stability and supporting Atg5–Atg16 translation; Pmk1-MAPK phosphorylation and Smt3-dependent SUMOylation also stabilize Ncl1.

The clearest direct link between external stress sensing and autophagy described by the review occurs during appressorium development. The cell-wall-integrity MAP kinase MoMps1 directly phosphorylates the autophagy protease MoAtg4, which processes Atg8 before its lipid modification, and the reported interaction can adjust autophagic flux independently of TOR signaling. The Wee1-family kinase MoSwe1 also coordinates cell-cycle arrest, autophagy and localized cell-wall remodeling during penetration-peg formation. Deletions of ATG genes in Cryptococcus neoformans and Aspergillus fumigatus have also been associated with hypersensitivity to Congo red and echinocandins, supporting a broader association between autophagy and cell-wall stress, although the direct biochemical evidence for MoMps1–MoAtg4 signaling is concentrated in M. oryzae.

At the developmental and host-interaction scale, the review links autophagy to programmed conidial death in M. oryzae. As an appressorium develops, maternal spore cells undergo extensive autophagy, transfer degraded material toward the appressorium and eventually collapse; core ATG mutations can prevent this death and associated transfer. The review also describes lipid peroxidation and ferroportin-1-related iron release as coupled processes, but much of this evidence comes from one fungal model. In C. neoformans, loss of Sgl1 was associated with blocked GFP-Atg8 trafficking, accumulation and extracellular release of ergosteryl-glucoside derivatives, and Mincle-dependent inflammatory responses that cleared the pathogen from lungs in the reported mouse infection model. These experimental results do not establish a clinical intervention.

The review presents Cordyceps militaris as a model for linking host manipulation to multicellular development. During infection, the horizontally transferred trehalase CmTreH1 depletes host trehalose, while the review reports induction of the orexigenic neuropeptide HemaP, hyperphagia and formation of a lipid- and glycogen-rich “fatty pupa.” After host takeover, bulk autophagy, lipophagy and glycophagy are proposed to mobilize nutrients for primordium and fruiting-body growth. Direct functional characterization of core autophagy genes in C. militaris has only begun, so the analogy with the better-characterized FOX-factor program in Sclerotinia sclerotiorum remains a hypothesis. Separately, the review describes Ebselen derivatives reversibly inhibiting the Atg4 catalytic cysteine and reports activity in plant and human fungal models; these findings are preclinical and do not establish a clinical antifungal treatment.

Subject of Research: Review of autophagy in fungal development, pathogenicity and host interactions

Article Title: Autophagy as a multi-scale architect of fungal development and pathogenicity: membrane dynamics, multilayer regulation, and cell wall integrity crosstalk

Article References: Wang, F., Li, J., Chen, H., Zheng, Q., Wei, T., Feng, K., & Chen, B.-X. (2026). Autophagy as a multi-scale architect of fungal development and pathogenicity: membrane dynamics, multilayer regulation, and cell wall integrity crosstalk. IMA Fungus, 17, Article e196033. https://doi.org/10.3897/imafungus.17.196033

Image Credits: AI Generated

DOI: 10.3897/imafungus.17.196033

Keywords: fungal autophagy, membrane remodeling, cell wall integrity, fungal pathogenesis, Magnaporthe oryzae, Cryptococcus neoformans, organelle turnover, antifungal targets, Cordyceps militaris, vesicle trafficking, Autophagy, multi-scale

MoNeo1 belongs to a family of enzymes known as P4-ATPases, or phospholipid flippases. These molecular pumps perform a deceptively simple but profoundly important task: they grab specific phospholipids from the outer, luminal leaflet of a cellular membrane and flip them to the inner, cytosolic leaflet. In doing so, they create what biologists call lipid asymmetry — a deliberate imbalance in the distribution of phospholipids between the two faces of a membrane. That asymmetry is not a cosmetic detail. It changes the physical properties of the membrane, curvature, charge, and fluidity, and it is a prerequisite for the budding of transport vesicles from the trans-Golgi network and endosomal membranes. In budding yeast, the Neo1 protein is essential for life; in humans, mutations in the Neo1 ortholog ATP9A are linked to hypotonia, intellectual disability, and attention deficit hyperactivity disorder. Until now, however, the role of this conserved flippase in plant-pathogenic fungi had not been systematically explored.

The team began by searching the M. oryzae genome for a counterpart of the well-characterized yeast Neo1. A BLAST search against the Kyoto Encyclopedia of Genes and Genomes returned a single strong candidate, the product of gene MGG_04066, which shares 55.28 percent amino acid identity with its yeast ortholog across eighty percent of the protein length. Phylogenetic analysis confirmed that Neo1 is tightly conserved across filamentous fungi, clustering closely with homologs from Botrytis cinerea and Aspergillus oryzae. Structural predictions generated with AlphaFold2, refined against known homologous structures, revealed the canonical P4-ATPase architecture: a transmembrane domain embedded in the lipid bilayer, flanked by the actuator, nucleotide-binding, and phosphorylation domains that together drive the ATP-powered flipping cycle. In short, MoNeo1 looked every bit the genuine flippase, and the researchers were ready to ask what happens when it is removed.

The answer, obtained through targeted gene deletion by homologous recombination, was dramatic. The ΔMoneo1 mutant remained viable — unlike its yeast counterpart — but its growth was visibly stunted across four different culture media compared with the wild-type Guy11 strain and a genetically complemented control. More striking still was the effect on asexual reproduction. Conidia, the spores through which rice blast spreads from plant to plant, were reduced by approximately 87.3 percent in the mutant, and the conidiophores that produce them developed abnormally. Because conidiation is the engine of disease persistence and dissemination in the field, this single defect would be enough to cripple an epidemic. Yet the most consequential phenotype emerged when the researchers turned their attention to infection itself.

M. oryzae initiates infection by germinating a conidium on the leaf surface and building a specialized dome-shaped structure called an appressorium, which generates enormous internal turgor pressure to mechanically breach the rice cuticle. In the ΔMoneo1 mutant, germ tubes elongated abnormally and appressorium formation was delayed, falling by more than fifty percent relative to the wild type at early time points, although the structures eventually formed by twenty-four hours. Formation, however, was not the same as function. Cytorrhysis assays, in which appressoria are challenged with increasing concentrations of glycerol to measure their internal pressure, revealed that mutant appressoria collapsed at significantly higher rates, indicating reduced turgor. Nile red staining showed impaired lipid retention at six and sixteen hours after induction, suggesting a failure in the glycerol generation that fuels the pressure build-up. Consistent with these functional deficits, the mutant produced far fewer invasive hyphae inside rice leaf sheaths, and blast symptoms on both barley leaves and susceptible CO39 rice seedlings were greatly reduced.

The mechanistic thread running through these phenotypes appears to be lipid chemistry. Quantitative lipidomics by high-performance liquid chromatography coupled to mass spectrometry revealed that deletion of MoNEO1 threw the fungus’s phospholipid economy into disarray. Levels of phosphatidylcholine rose by forty-two percent, phosphatidylethanolamine by thirty-eight percent, phosphatidylserine by twenty-one percent, and phosphatidylinositol by nineteen percent, while phosphatidic acid fell by twenty-eight percent and lysophosphatidylethanolamine by seventeen percent. Storage and signaling lipids shifted as well, with triacylglycerol increasing by twenty-three percent and diacylglycerol by fourteen percent. Nearly all of these changes were reversed in the complemented strain, confirming that they were specifically attributable to the loss of MoNeo1. The mutant was also hypersensitive to membrane stress induced by sodium chloride and Calcofluor White, consistent with a compromised membrane barrier. Together, the data paint MoNeo1 as a guardian of phospholipid homeostasis whose absence forces the cell into a compensatory rerouting of lipid metabolism.

Where does MoNeo1 do its work? Live-cell fluorescence microscopy of a MoNeo1-GFP fusion expressed under its native promoter showed the protein as discrete, motile puncta throughout the fungal life cycle. Co-expression with the trans-Golgi network marker MoKex2-mCherry revealed partial co-localization, and labeling of early endosomes with the dye FM4-64 showed transient overlap as well. MoNeo1, in other words, resides at the interface between the trans-Golgi network and the endosomal system — precisely the crossroads through which retrograde cargo traffic must pass. The team then asked how this steady-state distribution is maintained, focusing on the retromer, a conserved pentameric complex that retrieves membrane proteins from endosomes and returns them to the trans-Golgi network, thereby rescuing them from degradation in the vacuole. In yeast, Neo1 is a known retromer cargo, and the researchers suspected the same relationship might hold in M. oryzae.

It did. Co-immunoprecipitation experiments recovered MoNeo1-Flag together with MoVps35-GFP, the cargo-recognition core of the retromer, and live-cell imaging showed the two proteins traveling together on motile puncta through the hyphal cytoplasm. The dependency ran in one direction only: when MoVPS35 was deleted, MoNeo1-GFP vanished from its normal trans-Golgi and endosomal puncta and accumulated instead on the vacuolar membrane, in both vegetative hyphae and conidia. Deleting MoNEO1, by contrast, left the distribution of MoVps35 untouched. The retromer, therefore, sits upstream, continuously recycling MoNeo1 back to its functional address and protecting it from vacuolar degradation. This is a textbook retrograde trafficking relationship, now demonstrated for the first time in a major plant pathogen.

The final piece of the puzzle connects lipid flipping to the secretion of virulence proteins. Affinity purification and mass spectrometry of MoNeo1-Flag complexes identified 521 candidate interacting proteins, among them MoSnc1, an R-SNARE protein known to mediate effector secretion and to travel on the endosome-to-plasma-membrane route. Co-immunoprecipitation confirmed a physical interaction between MoNeo1 and MoSnc1, and the two proteins partially co-localized in living cells. Crucially, in the ΔMoneo1 mutant, the number and size of MoSnc1-positive puncta shrank, and the SNARE was progressively mis-sorted into the vacuole for degradation, while MoNeo1 localization remained normal in a ΔMosnc1 background. The consequence for virulence was direct: the apoplastic effector Bas4 accumulated abnormally in the cytosol and vacuole, and the cytoplasmic effector Pwl2 formed misplaced cytosolic foci instead of being delivered into host cells. The researchers propose that the membrane asymmetry generated by MoNeo1 creates lipid environments — rich in appropriately distributed phosphatidylserine and phosphatidylethanolamine — that are required for the stability, fusion competence, and recycling of MoSnc1-containing transport intermediates. Without that lipid scaffolding, the entire secretion pipeline that arms the fungus for invasion collapses. The study thus reframes MoNeo1 not as a housekeeping enzyme but as a strategic coordinator, integrating retrograde protein sorting with lipid translocation to support appressorium function and host penetration. Because flippases of this family are conserved across fungal pathogens, including the wheat scab fungus Fusarium graminearum and the human pathogen Cryptococcus neoformans, the pathway defined here offers a template for targeted disease control strategies that could protect rice yields without relying on conventional fungicides.

Subject of Research: Role of the P4-ATPase lipid flippase MoNeo1 in vesicle trafficking and pathogenicity of the rice blast fungus Magnaporthe oryzae

Article Title: The lipid flippase MoNeo1 mediates vesicle trafficking and pathogenicity in Magnaporthe oryzae

Article References: Cai, Y., Huang, X., Nie, Y., Luan, Y., Aarti, A., Gong, Q., Sun, P., Abubakar, Y. S., Wang, B., Wang, A., Li, G., Lin, L., & Zheng, W. (2026). The lipid flippase MoNeo1 mediates vesicle trafficking and pathogenicity in Magnaporthe oryzae. Stress Biology, 6(1), Article 28. https://doi.org/10.1007/s44154-026-00305-5

Image Credits: AI Generated

DOI: 10.1007/s44154-026-00305-5

Keywords: Magnaporthe oryzae, rice blast, lipid flippase, P4-ATPase, MoNeo1, vesicle trafficking, retromer, MoVps35, MoSnc1, SNARE, effector secretion, appressorium

Tem1 is a small GTPase, a class of molecular switches that alternate between an active GTP-bound state and an inactive GDP-bound state. In budding yeast, Tem1 sits at the top of the mitotic exit network, or MEN, a signaling cascade that couples the completion of nuclear division to cytokinesis. The Bfa1-Bub2 complex acts as a GTPase-activating protein, or GAP, that hydrolyzes Tem1-bound GTP, keeping the pathway off until the mitotic spindle is correctly positioned. When the spindle pole body, the fungal equivalent of the centrosome, reaches the right location, the polo-like kinase Cdc5 disables the GAP, allowing GTP-loaded Tem1 to trigger a kinase cascade that ultimately releases the phosphatase Cdc14 and drives the cell out of mitosis. Homologs of this pathway exist across fungi, but until now M. oryzae lacked a confirmed Tem1 ortholog, leaving a critical gap in understanding how the pathogen coordinates its unusual cell biology with infection.

To find the missing piece, the team searched the M. oryzae genome using yeast and Fusarium graminearum Tem1 proteins as queries. The search pinpointed MGG_04862, annotated as a septum-promoting GTP-binding protein, which shares 60.21 percent sequence identity with yeast Tem1 and a striking 72.28 percent identity with the Fusarium homolog. Phylogenetic analysis of Tem1 proteins from 24 fungal species placed MoTem1 in a clade with homologs from other devastating plant pathogens, including Botrytis cinerea and Sclerotinia sclerotiorum. The researchers then built a panel of isogenic strains in the Guy11 wild-type background: a knockout lacking the gene entirely, an overexpression strain, and two point mutants locked into opposite states. The Q182L substitution produces a constitutively active GTP-bound protein, while T137N creates a dominant-negative, inactive version. Yeast two-hybrid and pull-down assays confirmed that MoTem1 interacts with the fungal GAP components MoBfa1 and MoBub2 in a state-dependent manner, with the active form recruiting the full complex and the inactive form binding only MoBfa1.

The phenotypes of these mutants revealed that MoTem1’s activity cycling, rather than its mere presence or abundance, is what matters. The constitutively active strain grew about 41.56 percent more slowly than wild type, while the knockout and both locked-state mutants produced dramatically fewer conidia, the three-celled spores that launch infections, with reductions of 35 to 67 percent. Overexpression alone left conidiation untouched, demonstrating that dynamic GDP/GTP cycling is essential for asexual reproduction. Germination assays told a similar story: strains with enhanced or reduced GTPase activity germinated faster than wild type, reaching up to nearly 90 percent germination within two hours, while the constitutively active strain germinated poorly, formed multiple germ tubes, developed thickened cell walls and produced abnormally elongated germ tubes. These defects in pre-infection morphogenesis foreshadowed even more dramatic consequences on the plant itself.

When the researchers spray-inoculated rice seedlings, the virulence patterns were striking. The knockout and constitutively active strains were both attenuated, with the active mutant restricted to small lesion types and never producing the largest class of lesions. The dominant-negative strain, however, was hypervirulent, generating large Type IV lesions that were absent in wild-type infections at the same time point. The authors propose that inactivating MoTem1 promotes invasive hyphal growth, the phase of infection during which the fungus spreads from cell to cell through plasmodesmata, and lesion expansion directly depends on how extensively those hyphae extend. Constitutive activation, by contrast, disrupts the timing of morphogenic programs and likely the deployment of effectors, undermining the finely choreographed sequence of appressorium formation, cuticle penetration and biotrophic establishment that M. oryzae requires.

Stress experiments added another layer of complexity. Under oxidative stress from hydrogen peroxide, the dominant-negative strain was significantly more tolerant than wild type, with a higher half-maximal inhibitory concentration, while the constitutively active strain was hypersensitive. Cell wall stress from Congo Red produced the same split: overexpression conferred tolerance, constitutive activation caused severe sensitivity. Under osmotic stress with sorbitol, the knockout, overexpression and dominant-negative strains all showed enhanced tolerance, but responses to ionic stress with sodium and potassium chloride varied by allele. The picture that emerges is of a cell cycle regulator whose nucleotide-binding state feeds into entirely separate stress signaling pathways, with the GTP-bound form promoting oxidative resilience and the cycling state maintaining cell wall and membrane integrity.

Fluorescence microscopy explained part of the mechanism. EGFP-tagged MoTem1 localized as puncta to spindle pole bodies, confirmed by co-localization with the SPB marker MoAlp6-mCherry. The constitutively active variant accumulated intensely at these structures in both hyphae and conidia, while the inactive variant showed completely diffuse cytoplasmic fluorescence, demonstrating that only the GTP-bound form is recruited to spindle pole bodies. Biochemical assays confirmed that purified MoTem1 has genuine GTP-hydrolyzing activity, roughly four times that of a tag-only control, and that the T137N substitution abolishes it. Critically, both the knockout and the constitutively active strain produced multinucleate hyphal compartments, indicating that nuclear division and septum formation had fallen out of sync. In the knockout, cytokinesis fails outright; in the active mutant, premature MEN activation bypasses the spindle position checkpoint, or SPOC, the surveillance system that normally delays mitotic exit until the spindle is correctly aligned. The dominant-negative strain maintained normal nuclear segregation, which the authors attribute to residual wild-type MoTem1 activity in that background being sufficient for cytokinesis.

Transcriptomic profiling across all five strains revealed the full scope of MoTem1’s influence. Principal component analysis separated the knockout and constitutively active strains sharply from wild type, and differential expression analysis identified 72 genes consistently altered across all mutants, including core cell cycle regulators and stress-responsive transcription factors. Weighted gene co-expression network analysis resolved eight modules, three of which stood out: a brown module correlated with oxidative stress resistance and negatively with growth rate, suggesting a trade-off between stress adaptation and proliferation; a yellow module tied to hyphal growth and enriched for ribosome and glycolysis genes; and a tan module most strongly correlated with pathogenicity, containing 38 virulence-associated genes encoding secreted effectors and cutinases. Among the tan module’s hub genes was MoCHS1, a class V chitin synthase, one of three of M. oryzae’s seven chitin synthases known to be required for pathogenicity.

The link to MoCHS1 proved to be the study’s most consequential finding. Expression of this chitin synthase was downregulated 3.07-fold in the constitutively active strain and upregulated 1.26-fold in the dominant-negative strain, mirroring their virulence phenotypes. Pharmacological validation followed: the chitin synthase inhibitor Polyoxin B suppressed growth of the constitutively active strain by over 40 percent while barely touching wild type, and the dominant-negative strain was strongly resistant. Reactive oxygen species assays on rice leaf disks added a plant immunity dimension. Culture filtrates from the dominant-negative strain elicited the strongest ROS burst in rice tissue, followed by wild type, with the constitutively active strain eliciting the weakest response, and chitinase pretreatment abolished all responses, confirming that chitin oligomers acting as pathogen-associated molecular patterns were responsible. The authors suggest that altered MoCHS1 expression changes how much chitin is exposed or released from the fungal cell wall, modulating the plant’s early immune perception and potentially accelerating the transition to the necrotrophic phase that produces large necrotic lesions.

Taken together, the study repositions MoTem1 from a narrow cell cycle component to a signaling hub with genome-wide reach. Its GTP-dependent recruitment to spindle pole bodies safeguards mitotic fidelity, while its activity states ripple outward through transcriptional networks governing redox balance, cell wall architecture, osmotic adaptation and virulence. The work also highlights evolutionary divergence within the MEN pathway: unlike in Fusarium graminearum, where inactive Tem1 localizes to septa, inactive MoTem1 disperses into the cytoplasm, and M. oryzae’s MoSep1 activates MoMob1 directly, bypassing MoDbf2. For a pathogen responsible for one of agriculture’s most serious diseases, these findings open a conceptually new angle: the machinery that ends mitosis is also a master regulator of how the fungus grows, withstands stress and attacks its host, and perturbing its nucleotide cycle can either cripple the pathogen or, paradoxically, make it more dangerous.

Subject of Research: Role of the mitotic exit GTPase MoTem1 in development, stress adaptation and pathogenicity of the rice blast fungus Magnaporthe oryzae

Article Title: Mutational analysis of the mitotic exit GTPase MoTem1 reveals its role in development, stress adaptation, pathogenicity and global gene regulation in Magnaporthe oryzae

Article References: Pei, M., Xie, X., Cao, Y., Chen, J., Yang, F., Wang, Z., Olsson, S., Lu, G.-D., & Li, Y. (2026). Mutational analysis of the mitotic exit GTPase MoTem1 reveals its role in development, stress adaptation, pathogenicity and global gene regulation in Magnaporthe oryzae. Stress Biology, 6(1), Article 38. https://doi.org/10.1007/s44154-026-00310-8

Image Credits: AI Generated

DOI: 10.1007/s44154-026-00310-8

Keywords: Magnaporthe oryzae, MoTem1, mitotic exit network, GTPase, spindle pole body, SPOC, chitin synthase, MoCHS1, pathogenicity, stress adaptation, transcriptomics, WGCNA

Most efforts to find genes that protect plants from pathogens rely on loss-of-function screens: researchers disable genes one by one and look for plants that become unusually susceptible. But this approach misses a large and valuable class of genes, those whose enhanced activity confers protection. Gain-of-function screens, in which genes are switched on rather than off, are far better at revealing positive regulators of resistance, and they directly nominate candidates that breeders might overexpress to harden crops. In mammalian cells, CRISPR-based activation screens have become routine, but their adoption in plants has lagged, largely because delivering large guide RNA libraries into intact plant tissues is difficult and because screening whole plants at genomic scale is slow and expensive.

The Chinese team sidestepped this bottleneck by working with rice protoplasts, single plant cells stripped of their cell walls, which can be transfected efficiently and en masse. The researchers built a guide RNA library targeting genes encoding receptor kinases and receptor-like cytoplasmic kinases, a large protein family in rice that spans hundreds of members and includes many known immune regulators. To turn target genes on, they deployed a catalytically dead Cas9 fused to transcriptional activation domains, the dCas9-TV system that Li’s laboratory previously developed for plants. When a guide RNA steers the dCas9-TV activator to a gene’s promoter, that gene’s expression is cranked up without altering the DNA sequence itself.

A clever reporter made the screen readable. The team used the promoter of OsChit6, a chitinase gene that is strongly induced during fungal infection, to drive a reporter construct, so that any guide RNA whose target gene activates this defense program would light up the cell. They adapted the INTACT method, which uses affinity-tagged nuclei, to isolate and enrich protoplasts in which the OsChit6 promoter had been activated. Deep sequencing of guide RNAs recovered from the enriched population then revealed which genes, when overexpressed, switched on the defense marker. The screen converged on a handful of candidates, including several receptor-like cytoplasmic kinases and, most strikingly, OsTV1, a gene with no previously assigned immune function.

Validation followed rapidly through conventional genetics. The researchers generated rice lines in which OsTV1 was overexpressed and independent lines in which it was knocked out using CRISPR/Cas9. The results were unambiguous: overexpression lines activated the OsChit6 defense marker more strongly, while knockout lines failed to mount normal basal and pathogen-induced OsChit6 expression when challenged with Magnaporthe oryzae, the ascomycete fungus responsible for rice blast, the most devastating rice disease worldwide. Protein domain analysis showed that OsTV1 encodes a receptor kinase carrying leucine-rich repeats, a single transmembrane segment and a serine/threonine kinase catalytic domain, an architecture typical of cell-surface signaling receptors. The authors also noted that a segment of the gene was mis-annotated in the standard rice genome annotation database, underscoring how easily such genes can be overlooked.

Confocal microscopy of OsTV1 fused to GFP revealed something unusual about where the protein resides. OsTV1 accumulates specifically at papillae, ring-like cell wall thickenings that epidermal cells build at their periphery as defensive barriers against invading fungi. Papillae are among the earliest and most important physical defenses a plant cell deploys, acting as fortified plugs that block fungal penetration pegs from breaching the cell wall. Remarkably, the papillae-specific localization of OsTV1 held both during normal plant development and following M. oryzae infection, and the fungus did not change the protein’s accumulation or distribution, suggesting OsTV1 functions as a constitutively deployed guardian of these structures rather than a stress-recruited one.

The mechanistic story that emerged centers on silicon, a nutrient that rice is famous for accumulating in prodigious quantities. OsTV1 overexpression increased silicon deposition in leaves and raised leaf rigidity, which the team quantified using nanoindentation assays that measure the mechanical stiffness of leaf tissue. Overexpression lines also showed elevated expression of lignin biosynthesis genes, reinforcing the biochemical armature of the cell wall. Conversely, OsTV1 knockout lines displayed moderately reduced leaf stiffness and weakened lignin gene expression. These findings tie a specific signaling receptor, for the first time, to the physical properties of the leaf that determine how easily a fungal penetration peg can force its way through. Notably, OsTV1 did not alter canonical chitin-triggered immune signaling: MAP kinase activation and reactive oxygen bursts after chitin treatment were unaffected, and co-immunoprecipitation showed no complex formation between OsTV1 and OsCERK1, the central chitin receptor co-receptor. OsTV1 therefore appears to operate on a parallel, physical-defense axis rather than as part of the classical immune signaling machinery.

The agricultural payoff is broad-spectrum. In greenhouse and laboratory assays, OsTV1 overexpression lines resisted M. oryzae more effectively, while knockout lines were markedly more susceptible. The protection extended beyond blast: overexpression lines also tolerated infection by Fusarium fujikuroi, the bakanae pathogen that causes seedling elongation disease, and by Rhizoctonia solani, the sheath blight pathogen that devastates stems and grains under field conditions. Field trials delivered perhaps the most compelling evidence. Under natural disease pressure, OsTV1 overexpression lines restricted neck blast lesions to the mid-panicle region while lesions spread nearly to the panicle base in control plants, and knockout lines suffered severe panicle withering. Against sheath blight, overexpression plants kept green stems and filled their grains normally, whereas knockouts developed extensive stem lesions and shriveled grain. Crucially, all of this protection came at no detectable cost to yield: grain length, grain width and hundred-grain weight were indistinguishable across the genotypes, addressing the long-standing concern that boosting physical defenses, particularly silicon deposition, might stunt growth or reduce harvest.

For molecular breeders, OsTV1 offers an attractive target precisely because it enhances a defense strategy that is intrinsic to rice physiology. Silicon fertilization is already practiced widely in rice agriculture, but its benefits depend on the plant’s capacity to deposit and mobilize the element at the right cellular locations. A receptor kinase that promotes silicon-mediated fortification could be edited, overexpressed or introgressed into elite cultivars to strengthen that capacity from within, potentially reducing fungicide dependence. Just as importantly, the screening platform itself is a significant contribution. Because it operates in protoplasts, it can compress a genome-scale functional search into a rapid cell-based assay, and because it uses dCas9-mediated activation rather than DNA editing, it leaves no permanent scars and can target any promoter sequence. The authors suggest the approach can be adapted to other genes and other cellular processes, from nutrient transport to stress tolerance, and with appropriate reporters and cell types, to other plant species as well. As fungal pathogens continue to evolve resistance to chemical controls and as climate change reshapes disease pressure on staple crops, tools that accelerate the discovery of native resistance mechanisms, and genes like OsTV1 that harness them without penalizing yield, are likely to attract intense attention from both plant scientists and breeders.

Subject of Research: A CRISPR activation screen identifying the papillae-specific receptor kinase OsTV1 as a positive regulator of silicon-mediated, broad-spectrum fungal resistance in rice

Article Title: Cell-based CRISPRa screen identifies a papillae-specific receptor kinase mediating broad-spectrum fungal resistance in rice

Article References: Wang, F.-Z., Bao, Y., Qiu, J., Chen, M.-X., Jiang, H., Li, Z., Xiong, X., Jiang, X., Kou, Y., & Li, J.-F. (2026). Cell-based CRISPRa screen identifies a papillae-specific receptor kinase mediating broad-spectrum fungal resistance in rice. Nature Plants. https://doi.org/10.1038/s41477-026-02385-7

Image Credits: AI Generated

DOI: 10.1038/s41477-026-02385-7

Keywords: rice, CRISPR activation screen, OsTV1, receptor kinase, fungal resistance, rice blast, Magnaporthe oryzae, silicon deposition, papillae, cell wall, lignin, plant immunity

The blast fungus is the most serious disease of cultivated rice, and its reach is expanding. First detected in wheat in Brazil in the 1980s, the pathogen has since spread to parts of Asia and Africa, where it now threatens cereal harvests that millions of people depend upon. Although blast is not currently present in the United Kingdom, researchers caution that a changing climate could eventually allow it to threaten cereal production in northern Europe as well. That combination of global spread and environmental change is precisely why the John Innes Centre team describes their work as a potential intervention against what they call the worst fungal disease of cereals.

To understand why the new findings matter, it helps to look at how the pathogen attacks. Like many plant pathogens, Magnaporthe oryzae inserts small proteins known as effectors into the cells of leaves and stems. Once inside, these effectors manipulate the host’s cellular machinery to suppress defences and promote disease. Plants, in turn, have evolved intracellular immune receptors that can detect these foreign molecules and trigger an immune response. That response typically involves localised cell death around the site of infection, a dramatic but effective sacrifice that cuts off the pathogen’s route to spreading through the rest of the plant.

For more than three decades, the dominant characters in this molecular arms race have been a class of receptors called nucleotide-binding and leucine-rich repeat receptors, or NLRs. A considerable body of research has placed NLRs at the forefront of our understanding of how plants mount immune responses to blast and other diseases. But in recent years, scientists studying cereal crops have increasingly turned their attention to a second, newer class of intracellular immune receptors known as tandem kinase proteins, or TKPs. These receptors, first characterised in cereals, appear to operate differently from NLRs, and their full potential has only begun to be explored.

What caught the attention of Professor Mark Banfield, a group leader at the John Innes Centre and an author of the study, was a structural feature that some TKPs share with certain NLRs: an integrated Heavy Metal Associated domain, abbreviated HMA. Previous research had shown that these HMA domains are important for recognising pathogen effectors and for mounting an immune response. If the domain acts as the molecular docking site for effectors, Banfield reasoned, then deliberately altering the amino acids at that binding interface could create novel recognition specificities, effectively reprogramming the receptor to catch effectors it would never notice naturally. That, he suggests, could open a new frontline for plant defence.

To test that idea, the team turned to structural biology. Using biophysical analysis and crystallography, they resolved at high resolution the structural interaction between HMA domains and effectors from the blast pathogen. The structures revealed that HMA domains function as biological baits within TKPs, luring pathogen effectors into a binding interaction. When the effector takes the bait, the receptor is activated and the plant’s immune response follows. Seeing the interface in atomic detail gave the researchers exactly the map they needed: they could see which residues on the HMA domain contact the effector and therefore which positions to target when engineering new binding properties.

The engineering itself delivered the study’s most striking result. Guided by the structural knowledge, the researchers successfully modified TKP immune receptors so that they acquired dual specificity, binding to effectors associated with infection of both wheat and barley. Such broad recognition rarely occurs in nature, where receptors tend to be tuned to particular pathogen molecules. Demonstrating that a cereal immune receptor can be redesigned to respond to multiple effectors simultaneously is a powerful indication that TKPs are amenable to bioengineering, and it sets the stage for work on the many TKPs that have recently been discovered in diverse cereal crops. In principle, the approach could allow scientists to custom-engineer disease resistance receptors that respond to several effectors at once, making it much harder for the pathogen to escape detection.

It is important to note that these proof of principle experiments were carried out in protoplasts, individual wheat cells used as surrogates for whole plant tissue. The next stage for the group is to translate the findings from these surrogate assays into glasshouse plants, where the engineered receptors must function within intact immune systems and provide genuine protection against infection. Dr Daniel Yu, the first author of the study, said the work shows that this new class of resistance proteins is amenable to engineering purposes, and that tools previously used to engineer NLR receptors may also work with this new class. That continuity with established methods could accelerate progress considerably, since the plant science community already has decades of experience in manipulating NLRs.

The structural understanding of effector-receptor binding may also be amplified by other emerging technologies. Precision breeding and artificial intelligence approaches could help researchers design novel specificities into HMA domains more rapidly and accurately than trial-and-error screening would allow. Machine learning tools trained on protein structures are increasingly capable of predicting how changes at a binding interface will alter molecular recognition, and combining such predictions with the crystallographic data from this study could compress the design cycle for new disease resistance genes from years to months.

Perhaps the most strategically significant aspect of the work lies in the relationship between the two receptor classes. Professor Banfield noted that NLRs and TKPs are probably activated in different ways, which opens the possibility of more robust immunity in the field because breeders would not be putting all their eggs in one basket. Further down the road, he suggested, techniques may allow genes encoding TKPs and NLRs to be stacked together against the same disease, producing what he described as a super-resistant crop. Because the two receptor classes would trigger immunity through different mechanisms, it is likely to be substantially more difficult for the pathogen to evolve around the combined defence. For a fungus that already claims enough rice each year to feed 60 million people and is steadily extending its range into wheat and barley, that kind of layered, engineered resistance may be exactly the intervention global food security requires.

Subject of Research: Bioengineering of tandem kinase plant immune receptors to broaden recognition of cereal blast fungus effectors

Article Title: Can this biotech innovation halt the destructive march of a cereal killer?

Article References: Can this biotech innovation halt the destructive march of a cereal killer?. (n.d.). Original publication

Image Credits: AI Generated

DOI: Not provided

Keywords: blast fungus, Magnaporthe oryzae, tandem kinase proteins, NLR receptors, HMA domain, plant immunity, wheat, rice, barley, effector recognition, bioengineering, food security

The study, published in the journal Stress Biology, focuses on a long-standing puzzle in plant immunity: how the actin cytoskeleton, a dynamic meshwork of protein filaments just beneath the cell membrane, communicates with the signaling pathways that mobilize defense. Scientists have known for years that when a plant detects pathogen-associated molecular patterns, or PAMPs, through cell-surface receptors, the actin network rapidly reorganizes. Filament density, orientation and bundling all shift within minutes. Disrupting this network, whether chemically or genetically, typically leaves plants more vulnerable to infection. What remained unclear was which molecular players orchestrate the remodeling and how it connects to downstream immune outputs such as reactive oxygen species production.

To answer these questions, the team turned to the rice-Magnaporthe oryzae pathosystem, a classic model in which fungal spores land on the leaf surface, germinate, and build dome-shaped infection structures called appressoria. These structures generate enormous turgor pressure through glycerol accumulation, allowing the fungus to mechanically puncture the plant cuticle. The host cytoskeleton and cell wall at the infection site are critical barriers to this penetration, making them natural focal points for studying basal immunity.

The researchers first confirmed the importance of actin polymerization using latrunculin B, a macrolide compound derived from marine sponges that binds monomeric actin and prevents filament elongation. When seedlings of the japonica cultivars TP309 and Nipponbare were pretreated with the drug before spray inoculation with a virulent M. oryzae isolate, they became markedly more susceptible to blast, with higher concentrations producing stronger effects. Quantitative PCR revealed greater fungal growth in treated plants. The drug also broadly suppressed defense hormone pathways: genes in the salicylic acid pathway, including OsPAL1, OsICS1 and PR4, and genes in the jasmonic acid and ethylene pathways, including OsAOS2 and OsERF1, were all downregulated. In other words, a structurally intact actin network is not merely a passive barrier but an active prerequisite for full immune signaling.

That result pointed the investigators toward BUI1, also known as RMD or OsFH5, a class II formin protein previously famous for its role in rice development. Loss of BUI1 function causes bent uppermost internodes, dwarfism, wavy panicles and abnormal seeds, all traced to impaired cell elongation and disrupted cytoskeletal arrays. The protein carries three conserved domains: a PTEN domain that targets it to the chloroplast surface, a proline-rich FH1 domain that interacts with profilin to promote actin polymerization, and an FH2 domain that binds the growing ends of actin filaments. Notably, the team found that latrunculin B treatment downregulated BUI1 expression, hinting that the gene sits within the very regulatory circuit the drug perturbs.

Testing the bui1 mutant, derived from the moderately resistant cultivar Zhejing 22, confirmed the suspicion. After punch inoculation with M. oryzae, the mutant developed significantly larger lesions than wild type, and field trials in a blast nursery showed dramatically reduced resistance under natural infection pressure. Using a GFP-tagged fungal strain, the researchers tracked infection in real time and found that appressorium formation and maturation were both more efficient on bui1 leaf sheath cells, suggesting that BUI1-mediated actin organization actively impedes this critical early step of fungal invasion. BUI1 expression itself was upregulated upon fungal inoculation, consistent with a positive role in defense.

The most striking finding came when the team visualized actin filaments directly. Using fluorescent phalloidin staining and confocal microscopy, they showed that treatment with the PAMPs chitin or flg22 triggered a rapid, transient increase in actin filament bundling in wild-type leaf sheath cells within five minutes. In the bui1 mutant, this response was severely attenuated, and the baseline cytoskeleton appeared disorganized. During actual fungal infection, wild-type cells accumulated dense actin bundles around infection sites by twelve hours post-inoculation, while the mutant failed to mount this defense-associated remodeling. BUI1, the data showed, is essential for PAMP-triggered actin reorganization, placing a formin protein squarely within the earliest tier of the plant immune response.

Broad-spectrum resistance is the holy grail of crop protection, and BUI1 delivered on that front too. The mutant proved more susceptible not only to blast but also to bacterial blight, developing longer lesions after inoculation with the Xoo strain PXO99A, and to sheath blight when tested in CRISPR-generated knockout lines in the Nipponbare background. Conversely, plants overexpressing BUI1 under its native promoter showed enhanced bacterial blight resistance proportional to expression level. Time-course transcriptome analysis after Xoo infection revealed extensive transcriptional reprogramming in wild-type plants that was largely absent in the mutant, with key defense pathways, including phenylpropanoid biosynthesis, MAPK signaling and plant-pathogen interaction networks, failing to activate without BUI1.

Perhaps most intriguingly, BUI1 turned out to act downstream of ROD1, a calcium sensor previously shown to suppress rice immunity by activating catalase-mediated ROS scavenging, whose loss confers broad-spectrum resistance. When the researchers knocked out BUI1 in the rod1 background, the strong resistance of rod1 plants was significantly compromised against blast, bacterial blight and sheath blight alike, though the double mutants remained less susceptible than fully wild-type plants. Yeast two-hybrid assays detected no direct physical interaction between BUI1 and ROD1 or the ROD1-interacting catalase OsCatB, indicating the relationship is genetic rather than structural. DAB staining and hydrogen peroxide measurements showed that the double mutants accumulated lower ROS levels than rod1 alone, pointing to ROS homeostasis as the mechanistic bridge.

RNA sequencing after M. oryzae infection explained why. In the bui1 knockout background, genes encoding peroxidases and metallothioneins, including OsPOD, POXA, OsCatB, OsMT1d and OsMT1f, were strongly upregulated, while ROS-producing genes such as RbohA and RbohB were unchanged. The mutant exhibited higher peroxidase activity and correspondingly lower hydrogen peroxide accumulation. BUI1, it appears, maintains immune competence primarily by restraining ROS-scavenging pathways, allowing the oxidative burst that hallmark of pattern-triggered immunity to reach effective levels. The protein also contains intrinsically disordered regions predicted to mediate liquid-liquid phase separation, and truncated variants lacking these regions failed to undergo phase separation in vitro, suggesting that BUI1 may form biomolecular condensates that serve as organizational hubs integrating cytoskeletal dynamics with defense signaling. For breeders, the message is clear: a single formin gene links the cell’s structural skeleton, its oxidative chemistry and its hormone signaling into one coordinated defense program, and tuning that gene could yield rice lines that shrug off multiple diseases without yield penalties.

Subject of Research: The role of the rice formin gene BUI1 in coordinating actin cytoskeleton remodeling and reactive oxygen species homeostasis during broad-spectrum plant disease resistance.

Article Title: BUI1 coordinates actin cytoskeleton remodeling and ROS homeostasis to confer broad-spectrum disease resistance in rice

Article References: Lin, H., Cui, K., Wang, J., Jin, Y., Zhang, X., Lu, Y., Wu, X., Zhang, L., Liu, S., Liu, J., Li, Q., Deng, Y., Yang, W., He, Z., & Gao, M. (2026). BUI1 coordinates actin cytoskeleton remodeling and ROS homeostasis to confer broad-spectrum disease resistance in rice. Stress Biology, 6(1), Article 48. https://doi.org/10.1007/s44154-026-00321-5

Image Credits: AI Generated

DOI: 10.1007/s44154-026-00321-5

Keywords: BUI1, rice immunity, actin cytoskeleton, ROS homeostasis, Magnaporthe oryzae, pattern-triggered immunity, formin, ROD1, broad-spectrum resistance, Xanthomonas oryzae, peroxidase, plant pathology

Rice blast, caused by the fungus Magnaporthe oryzae, is widely regarded as the most devastating disease of rice worldwide. The pathogen attacks leaves, stems, and critically the grain-bearing panicles, and under favorable conditions it can wipe out a substantial share of a harvest. Farmers have long relied on fungicides and resistant cultivars, but the fungus is notorious for its ability to mutate and overcome single-gene defenses. That makes the search for broad-spectrum and stacked resistance genes a central priority for rice breeders everywhere, and it is precisely what makes the new findings so significant.

The research team, led by Thokchom Nepolian Singh and colleagues at the College of Agriculture, Central Agricultural University, Imphal, set out to determine which of the well-characterized blast resistance genes are already present in the region’s germplasm. Fifty-eight genotypes collected from across Northeastern India were evaluated, alongside a susceptible control genotype known as HR-12 and a resistant check variety called Tetep, a famous donor of blast resistance originally from Vietnam. The team grew all entries in a uniform blast nursery, an experimental setting designed to apply intense, natural disease pressure so that the true resistance reactions of each genotype could be observed and scored.

In parallel with the field evaluation, the researchers employed fourteen gene-specific molecular markers to detect the presence of known resistance genes in each genotype’s DNA. This marker-assisted screening approach exploits the fact that most major resistance genes can be tracked through tightly linked DNA sequences, allowing breeders to identify valuable genes without waiting years for field validation. The molecular analysis revealed a strikingly high frequency of resistance genes across the collection, confirming that Northeastern Indian rice landraces represent a largely untapped reservoir of blast resistance.

Among the genes surveyed, Pik-p emerged as the most widely distributed across the screened genotypes. It was followed closely by Piz, detected in 96.66 percent of the material, Pi40(t) at 93.33 percent, and Pi-ta at 90.00 percent. Several other important genes also appeared at notable frequencies, including Piz-t at 86.66 percent, Pi40 at 83.33 percent, and Pik at 73.33 percent. These genes are not interchangeable; each encodes a different intracellular immune receptor of the nucleotide-binding, leucine-rich repeat class that recognizes specific secreted proteins from the blast fungus, triggering a defensive hypersensitive response that halts pathogen spread.

The standout discoveries, however, were two genotypes named Itanagar and Tomila, which carried eleven different blast resistance genes each. That level of natural gene stacking is rare and makes these landraces exceptionally valuable as donor parents for breeding programs aiming to engineer durable resistance. Gene stacking is widely considered the most reliable strategy against blast because the fungus would need to simultaneously overcome multiple immune recognition systems to successfully infect the plant, a far more difficult evolutionary feat than defeating a single gene.

Close behind the top performers, seven additional genotypes each harbored ten resistance genes: Chakhao Poreiton, Chakhao Chandel-2, Liangneng Chakhao, Barbite rice, Hungyo, Chamiyak, and Lamyanba. The presence of multiple resistance genes in these traditional aromatic and local varieties is particularly encouraging because several of them, such as the Chakhao cultivars prized in Manipur for their distinctive aroma and cultural value, are already of economic and gastronomic importance. Improving their agronomic performance while retaining their intrinsic qualities becomes far easier when they already carry elite disease-resistance genetics.

The technical workflow behind these findings reflects standard best practice in modern plant pathology and genetics. High-molecular-weight genomic DNA was isolated from young leaf tissue, and polymerase chain reactions were run using primers specific to each resistance gene locus. Amplified fragments were then separated on agarose gels, and the presence or absence of the expected band sizes served as evidence for each gene. Combining this molecular passport with disease scores from the uniform blast nursery allowed the team to link genetic potential with actual field performance, a critical validation step that pure molecular screens sometimes lack.

The broader context of the work is equally important. Rice is the staple crop for billions of people, and Northeastern India is recognized as one of the centers of rice genetic diversity, home to landraces adapted to steep terrains, variable rainfall, and diverse pest pressures. Much of this diversity remains genetically uncharacterized, and as modern high-yielding varieties displace traditional cultivars, unique alleles risk being lost before their value is known. Studies like this one serve as both a rescue mission and a roadmap, documenting which resistance genes exist where, before that information and the germplasm itself disappear.

For breeders, the practical implications are immediate. The multi-gene genotypes identified here can be crossed with elite varieties and their offspring tracked using the same gene-specific markers, enabling precise introgression of resistance cassettes without dragging along undesirable traits. Gene pyramiding through marker-assisted backcrossing has already proven effective in improving Basmati and japonica rice cultivars elsewhere, and the Northeastern Indian donors identified in this study offer locally adapted, genetically diverse material for the same strategy. As blast continues to threaten rice production in a changing climate, the humble landraces of the region’s terraced hills may prove to be among the most important allies in keeping one of the world’s most vital food crops safe.

Subject of Research: Molecular screening of blast resistance genes in Northeastern Indian rice germplasm (Oryza sativa L.)

Article Title: Molecular Screening for Identification of Blast Resistance Genes in Northeastern Indian Rice Germplasm (Oryza sativa L.)

Article References: Singh, T. N., Saharia, D. D., Biswas, D., Devi, O. P., Pyngrope, A. H., Singh, N. B., Kh., P., Manjunath, P., Devi, T. R., Phurailatpam, S., Ngangkham, U., Devi, E. L., & Chongtham, S. K. (2026). Molecular Screening for Identification of Blast Resistance Genes in Northeastern Indian Rice Germplasm (Oryza sativa L.). Indian Journal of Genetics and Plant Breeding, 86(3), 292-302. https://doi.org/10.1007/s44489-026-00032-1

Image Credits: AI Generated

DOI: 10.1007/s44489-026-00032-1

Keywords: rice blast, Magnaporthe oryzae, blast resistance genes, Northeastern India, rice germplasm, marker-assisted selection, gene pyramiding, Oryza sativa, Pik-p, Pi40(t), landraces, plant breeding

Rice blast has long been regarded as the most important fungal disease of rice, a staple crop that feeds roughly half of the global population. The pathogen, Magnaporthe oryzae, infects leaves, stems, and panicles, producing the characteristic diamond-shaped lesions that can decimate yields under favorable humid conditions. Conventional control relies heavily on fungicide applications and the deployment of resistance genes in cultivars, but the fungus is notorious for its adaptive capacity, repeatedly overcoming single resistance genes in the field. This has driven scientists to look beyond the plant’s own genome for partners in defense, and the rhizosphere microbiome has emerged as a compelling frontier.

The rhizosphere—the narrow zone of soil influenced by root exudates—harbors some of the densest and most metabolically active microbial communities on Earth. Plant roots actively recruit and nourish specific microbes through the release of sugars, organic acids, and signaling compounds, and in return, certain bacteria can suppress pathogens, modulate plant hormones, or prime immune responses. Whether the rhizosphere communities of disease-resistant rice cultivars are fundamentally different from those of susceptible ones, and how those communities respond when the blast pathogen attacks, had remained incompletely resolved. The new greenhouse study set out to answer precisely those questions.

The research team, led by Tingting Yang and Di Han of the College of Plant Protection at Shenyang Agricultural University, together with colleagues at Liaoning Academy of Agricultural Sciences, grew blast-resistant and blast-susceptible rice cultivars under controlled greenhouse conditions and compared their rhizobacterial communities both before and after challenge with M. oryzae. Using 16S rDNA amplicon sequencing—a technique that catalogs bacterial taxa by amplifying a conserved genetic marker—they profiled the diversity and composition of the root-associated bacteria across the experimental treatments.

The sequencing results revealed statistically significant differences in rhizobacterial diversity and community composition between resistant and susceptible cultivars, confirming that the plant genotype leaves a measurable imprint on which bacteria congregate around its roots. More striking was what happened after pathogen infection: both cultivar types underwent distinct community shifts, but the direction and magnitude of those shifts differed between resistant and susceptible plants. The researchers detected differential microbial enrichment patterns across cultivars and disease states, indicating that the rhizosphere is not a passive bystander in the rice–blast interaction but a dynamic environment that responds to both plant genotype and pathogen pressure.

Among the taxa whose abundance changed, one genus stood out. Bacillus species were consistently enriched in the rhizospheres of healthy plants of resistant cultivars and, tellingly, in infected plants of susceptible cultivars. This dual pattern suggests that Bacillus populations may be associated with disease status in both contexts—either helping to maintain health in resistant plants or responding to infection in susceptible ones. Bacillus species are well known in agricultural microbiology for their ability to produce antimicrobial compounds, form protective biofilms on roots, and induce systemic resistance in host plants, which makes their enrichment patterns particularly noteworthy for biocontrol-oriented research.

To probe the structure of these microbial communities more deeply, the team applied network analysis, a computational approach that maps co-occurrence and potential ecological interactions among taxa. The analysis showed that cultivar resistance was associated with measurable differences in the architecture of the rhizosphere microbial community. In practical terms, resistant and susceptible rice varieties appear to host rhizobacterial networks with different organization, hinting that community structure—not merely the presence or absence of particular species—may contribute to the disease-suppressive potential of the root environment.

The study then moved from community profiling to the isolation and characterization of individual candidates. From the rhizosphere of healthy rice plants, the researchers recovered a bacterial strain designated Bacillus velezensis H2. The strain was characterized through a combination of morphological and biochemical tests, 16S rRNA gene sequencing, and single-nucleotide polymorphism (SNP) profiling to confirm its taxonomic identity. B. velezensis is a species that has attracted considerable attention in biocontrol research because many of its strains carry extensive biosynthetic machinery for antimicrobial secondary metabolites.

Whole-genome analysis using the antiSMASH platform—an algorithmic pipeline that scans bacterial genomes for biosynthetic gene clusters—identified multiple clusters in the H2 genome involved in secondary metabolite production. Among these were genes encoding nonribosomal peptide synthetases, the giant multifunctional enzymes responsible for assembling many of the lipopeptide antibiotics that Bacillus species are famous for, such as members of the surfactin, iturin, and fengycin families. The presence of these clusters provides a genomic rationale for the strain’s observed biological activity and suggests that H2 is genetically equipped to interfere with fungal pathogens in the root zone.

Laboratory assays confirmed that promise in practice. In vitro tests demonstrated that B. velezensis H2 exhibits strong antifungal activity against Magnaporthe oryzae, directly inhibiting the growth of the blast pathogen. While in vitro inhibition does not guarantee field-level disease suppression—greenhouse and field validation remain essential next steps—the combination of rhizosphere origin, rich biosynthetic potential, and demonstrable antagonism makes H2 a compelling candidate for development as a biocontrol agent. If it can colonize rice roots effectively and express its antifungal arsenal under realistic soil conditions, it could contribute to reducing the chemical fungicide burden in rice production systems.

The broader significance of the study lies in its integrated perspective. Rather than treating plant immunity and the microbiome as separate domains, the work links cultivar resistance, rhizobacterial community structure, pathogen-induced shifts, and a concrete antagonistic isolate into a single narrative. It reinforces a growing consensus in plant pathology: breeding for resistance and engineering beneficial microbiomes may be complementary strategies. Rice blast remains a moving target, but evidence that resistant cultivars cultivate distinct and potentially protective bacterial communities—and that strains like B. velezensis H2 can directly oppose the pathogen—offers a scientifically grounded path toward more sustainable, microbiome-aware crop protection.

Subject of Research: Rhizosphere bacterial community shifts in rice blast-resistant cultivars and the antagonistic activity of Bacillus velezensis against the rice blast pathogen

Article Title: Rhizosphere Bacterial Community Shifts in Rice Blast–Resistant Cultivars and the Antagonistic Activity of Bacillus velezensis

Article References: Yang, T., Han, D., Ding, A., Du, S., Wang, W., Huang, Y., & Ahsan, T. (2026). Rhizosphere Bacterial Community Shifts in Rice Blast–Resistant Cultivars and the Antagonistic Activity of Bacillus velezensis. Microbial Ecology. https://doi.org/10.1007/s00248-026-02858-4

Image Credits: AI Generated

DOI: 10.1007/s00248-026-02858-4

Keywords: rhizosphere microbiome, rice blast disease, Bacillus velezensis, Magnaporthe oryzae, biocontrol, 16S rDNA sequencing, secondary metabolites, nonribosomal peptide synthetases, antiSMASH, disease resistance, Rhizosphere, Bacterial

Epigenetic control in plants operates through three principal channels: DNA methylation, histone modification, and non-coding RNAs. DNA methylation in rice occurs in three sequence contexts, CG, CHG and CHH, and is maintained by distinct enzymes. Chromomethylase3 handles CHG methylation, DNA Methyltransferase 1 manages CG methylation, and Domains Rearranged Methyltransferases catalyze CHH methylation. When a pathogen attacks, these methylation patterns shift, silencing genes that would otherwise help the invader and activating defense programs that would otherwise stay dormant. The review emphasizes that this is not a passive process; it is a dynamic, energy-conscious strategy that lets the plant balance the metabolic cost of immunity against the demands of growth.

At the heart of this regulatory architecture sits the RNA-directed DNA methylation pathway, or RdDM. In this pathway, the plant-specific RNA polymerases Pol IV and Pol V generate transcripts that RNA-DEPENDENT RNA POLYMERASE 2 converts into double-stranded RNA, which DICER-LIKE 3 then processes into 24-nucleotide small interfering RNAs. ARGONAUTE 4 binds these siRNAs and uses Pol V transcripts as scaffolds to recruit DRM2, the enzyme that lays down new cytosine methylation marks. Pathogen infection triggers the production of small RNAs that silence transposable elements, genomic parasites whose activation is linked to instability and disease susceptibility. Silencing these elements enhances resistance, and remarkably, the trait can be inherited by the next generation. In rice, the largest subunit of Pol IV is encoded by OsNRPD1a and OsNRPD1b; knocking these genes down produces dwarfed plants with increased tillering, reduced panicle length and, through a still-unknown pathway, enhanced disease resistance.

The review highlights WRKY45 as a striking example of multilayered epigenetic control. This transcription factor, a hub of quantitative resistance to bacterial blight, is regulated simultaneously by DNA methylation, histone modifications and small RNA pathways. A transposon-derived small RNA called TE-siR815 modulates the WRKY45 locus, and demethylation at that locus alters expression and, with it, susceptibility to Xoo. The authors argue that WRKY45-1 and WRKY45-2 function as molecular bridges connecting epigenetic control to the wholesale transcriptional reprogramming that defines the immune response against both bacterial and fungal attackers.

Concrete examples of methylation-driven resistance abound in the literature the review surveys. The famous resistance gene Xa21, which encodes a receptor kinase conferring defense against diverse Xoo strains, has a relative called Xa21G that was isolated through methylation-sensitive amplification polymorphism screening in a mutant line generated with the DNA methylation inhibitor 5-aza-2′-deoxycytidine. In wild-type plants the Xa21G promoter is methylated and its transcripts are undetectable, but in the demethylated mutant the gene is expressed. Similarly, the blast resistance gene Pib, which encodes a nucleotide-binding leucine-rich repeat protein, carries CG methylation at two promoter sites, and the PigmS gene is governed by demethylation of two tandem miniature transposons, MIT1 and MITE2, that balance tissue-specific transcription against yield. The de novo methyltransferase gene OsDRM2 regulates defense against M. oryzae, while OsRos1a, a repressor of silencing, is vital for resistance to bacterial leaf streak caused by Xanthomonas oryzae pv. oryzicola.

Histone chemistry adds a second, equally intricate layer. Activating marks such as trimethylation of histone H3 lysine 4 open chromatin and permit transcription, whereas repressive marks such as H3K27me3 seal genes shut. Rice possesses twenty Jumonji C domain histone demethylases, and several are now firmly implicated in immunity. JMJ704 promotes resistance to bacterial blight by removing H3K4me2/3 marks from negative regulators of defense, including OsWRKY62 and the susceptibility gene OsSWEET14. JMJ705, induced by stress and infection, strips H3K27me3 from defense and jasmonic acid-responsive genes; its overexpression produces a lesion-mimic phenotype and heightened Xoo resistance, while its loss diminishes resistance. On the acetylation side, the histone acetyltransferase OsHAC704 activates defense genes, whereas the deacetylases OsHDAC1 and OsHDT701 act as brakes. Knocking down OsHDAC1 enhances broad-spectrum blast resistance without stunting the plant, and the transcription factor OsGRAS30 boosts resistance by inhibiting OsHDAC1 and elevating H3K27ac levels. OsHDT701, working with the RNase P subunit Rpp30, deacetylates histone H4 at pattern-recognition and defense genes, dampening responses to both M. oryzae and Xoo. The sirtuin-like deacetylase OsSRT1 suppresses jasmonic acid biosynthesis genes, and its downregulation triggers lesion formation and hypersensitive-response gene expression, tying histone modification directly to hormone-mediated defense signaling.

Pathogens, for their part, have learned to weaponize the host’s epigenetic machinery. The secreted effector Uv1809 from the false smut fungus Ustilaginoidea virens enhances OsSRT2-mediated histone deacetylation, lowering H4K5ac and H4K8ac levels and crippling the transcriptional activation of defense genes. Host-induced silencing of Uv1809 restores resistance, demonstrating that fungal effectors actively suppress immunity by hijacking chromatin regulators. Other fungal and bacterial effectors induce histone hypoacetylation more broadly, underscoring an escalating arms race fought at the level of chromatin.

Non-coding RNAs form the third pillar. The microRNA miR7695 targets OsNRAMP6, and its overexpression increases blast resistance, while miR160a targets the auxin signaling component ARF16 and, when overexpressed, limits fungal growth and boosts hydrogen peroxide accumulation and defense gene expression. Conversely, M. oryzae induces miR319, which suppresses OsTCP21 and compromises jasmonic acid synthesis, a clear example of a pathogen exploiting a host microRNA. In the antiviral arena, ARGONAUTE 18 sequesters miR168, freeing AGO1 for antiviral RNA interference, and AGO1a overexpression confers resistance to Xoo. Viral infection suppresses miR528, unleashing its target L-ascorbate oxidase to generate reactive oxygen species for defense. Beyond microRNAs, RNA modifications are emerging as a frontier: the m6A methyltransferase MTA1 is essential for virulence in the blast fungus, and in rice the ac4C RNA modification, written by OsNAT10, undergoes infection-triggered changes that reprogram translation and activate jasmonic acid biosynthesis, a mechanism the review flags as a key discovery in plant immunity.

Perhaps the most consequential theme is epigenetic memory. DNA methylation patterns associated with resistance can persist for generations without the original pathogen stimulus, and rice plants exposed to M. oryzae show heritable methylation and histone changes that activate defense genes in their progeny. Methyl jasmonate-induced priming elevates H3K4 and H3K9 acetylation at defense gene promoters and creates chromatin-based memory of wounding. The review argues that this molecular memory, combined with emerging tools such as CRISPR-based epigenome editing, epi-breeding with natural epialleles, and high-throughput biomarker screening using bisulfite sequencing and ChIP-seq, could deliver broad-spectrum, durable resistance without the yield penalties that plague conventional approaches. Challenges remain, including the complexity of interacting epigenetic layers, potential off-target effects of editing systems, and the difficulty of validating biomarkers in a dynamic epigenome. But the authors are clear about the destination: identifying resistance-associated epigenetic markers and integrating them with traditional breeding and biotechnology offers a sustainable path to protecting rice, and global food security, against pathogens that will not stop evolving.

Subject of Research: Epigenetic regulation of rice immunity against bacterial and fungal pathogens

Article Title: Regulatory role of epigenetics in rice immunity against bacterial and fungal pathogens

Article References: Shah, G., Ullah, H., Chen, L., Shah, A. Z., Shaheen, R., Liu, J., & Kong, W. (2026). Regulatory role of epigenetics in rice immunity against bacterial and fungal pathogens. Crop Health, 4(1), Article 18. https://doi.org/10.1007/s44297-026-00080-9

Image Credits: AI Generated

DOI: 10.1007/s44297-026-00080-9

Keywords: rice immunity, epigenetics, DNA methylation, histone modification, non-coding RNAs, RdDM, Magnaporthe oryzae, Xanthomonas oryzae, disease resistance, epigenetic breeding, CRISPR epigenome editing, food security