A team led by Jian-Feng Li at Sun Yat-sen University in Guangzhou, working with collaborators at South China Agricultural University, the Chinese University of Hong Kong and the University of Macau, identified two paralogous chitinases in the model plant Arabidopsis thaliana that they named MIC1 and MIC2. Both genes are induced by chitin and by other microbe-associated molecular patterns, including the bacterial flagellin-derived peptide flg22 and the elongation factor Tu-derived peptide elf18. Both proteins are secreted into the apoplast, the fluid-filled space outside the plant cell membrane where fungal contact first occurs, and both possess direct antifungal activity in vitro, breaking down chitin in the fungal cell wall itself. On the surface, the two enzymes look like redundant copies of the same tool.

The resemblance is deceptive. When the researchers generated CRISPR/Cas9 knockout mutants, the two paralogs pulled in opposite directions. Plants lacking MIC1 became markedly more resistant to the grey mould pathogen Botrytis cinerea and to the vascular wilt fungus Verticillium dahliae, while plants lacking MIC2 became more susceptible. The team also showed that the single mutants and the mic1 mic2 double mutant grow normally under standard conditions, which means the antagonistic effects of these enzymes are specific to immune outcomes rather than general developmental defects. The picture that emerged is of two enzymes with the same substrate but opposing roles in tuning the immune response.

The key to this antagonism lies in the products of chitin digestion. Chitin is a polymer of N-acetylglucosamine units, conventionally abbreviated NAG, and the length of the oligomers released by hydrolysis determines their biological meaning. Using high-performance liquid chromatography to track the fate of defined chitin oligomers, the researchers found that MIC2 cleaves long chitin chains into intermediate fragments, principally NAG4 to NAG6, which are precisely the sizes that plant chitin receptors recognize as immunogenic elicitors. These fragments activate mitogen-activated protein kinase signalling, a canonical early readout of chitin-triggered immunity, and pretreatment with NAG4 primed Arabidopsis leaves for enhanced resistance against B. cinerea. MIC2, in other words, is an alarm bell: it chews the fungal wall in a way that generates the molecular signals that rally the plant’s defenses.

MIC1 does the opposite. Rather than stopping at immunogenic intermediates, MIC1 processes chitin all the way down to NAG2 dimers and NAG1 monomers, fragments that are too short to activate receptor signalling. In seedling assays, exogenous chitin normally inhibits root growth, a visible manifestation of constitutive chitin signalling, and MIC1 is the enzyme responsible for preventing this self-inflicted damage. When the researchers tested additional Arabidopsis chitinases from other clades, they found that the MIC1-like degradation pattern, exemplified by the clade IV enzyme chitIV1, is functionally conserved, and that deleting whole clusters of MIC1-like genes reproduced the chitin-hypersensitive growth phenotype. MIC1 and its relatives therefore act as a molecular off-switch, extinguishing the growth-inhibitory signal once the immune alarm has sounded.

What makes one enzyme stop at immunogenic fragments while its near-identical paralog grinds chitin to dust? The answer, uncovered through a combination of mass spectrometry, PNGase digestion assays and site-directed mutagenesis, is N-glycosylation, the attachment of sugar chains to asparagine residues of secreted proteins. Both MIC1 and MIC2 carry N-glycans, and these modifications are not decorative: glycosylation-deficient variants of either protein failed to be secreted efficiently into the apoplast, and a glycosylation-deficient MIC2 could no longer complement the enhanced susceptibility of mic1 mic2 double mutants to B. cinerea. Glycosylation is thus required for these enzymes to reach their site of action at all.

But one glycan does far more than ensure secretion. Mass spectrometry mapped three N-glycosylation sites on MIC1 and two on MIC2, and the decisive difference came down to a single residue: asparagine 137 in MIC1. When the researchers swapped variable segments between the two proteins to create chimeric enzymes, the exchange that transferred the MIC1 region containing Asn137 into MIC2 converted MIC2 into a MIC1-like enzyme, digesting NAG7 into more dimers and monomers and fewer immunogenic pentamers and tetramers. Conversely, the corresponding chimeric version of MIC1 lost its characteristic band shift and behaved more like MIC2. In complementation tests, the chimeric MIC2 carrying the MIC1 glycosylation region partially suppressed chitin-induced growth inhibition, whereas wild-type MIC2 did not, and it slightly increased susceptibility to B. cinerea instead of restoring resistance. Removing the Asn137 glycan from MIC1 converted it into a MIC2-like enzyme, demonstrating that this one sugar attachment dictates the chitinolytic specificity of the entire protein.

Structural modelling with AlphaFold showed that MIC1 and MIC2 are highly similar in sequence and fold, with their catalytic glutamates aligned and their glycosylation sites positioned on surface loops, including the Loop III region implicated in substrate handling in GH19 family chitinases. The Asn137 glycan apparently reshapes the local architecture of the catalytic cleft in a way that changes how many NAG units the enzyme removes per cleavage event. This is a remarkable example of post-translational modification acting as a functional diversification code: two genes born of duplication, nearly interchangeable in sequence, are sorted into opposing immune roles by a single covalently attached oligosaccharide. The authors describe this as an N-glycosylation code governing the functional specialization of paralogous chitinases.

The implications extend well beyond Arabidopsis. A bioinformatic survey revealed that MIC1-like chitinases are widespread across the plant kingdom, and the Asn137-equivalent residue is highly conserved among MIC1 orthologs from different Arabidopsis ecotypes. The team turned to rice, the world’s most important staple crop, and generated CRISPR knockout mutants of the rice ortholog OsMIC1. The osmic1 mutants showed enhanced resistance to rice blast and to bacterial blight, the latter an intriguing bonus suggesting the mechanism is not strictly antifungal, while their grain length, grain width and thousand-grain weight remained indistinguishable from wild-type plants. Like their Arabidopsis counterparts, however, osmic1 seedlings did exhibit chitin-induced root growth inhibition, confirming that the dampening function of OsMIC1 is conserved.

For breeders, this is the rare kind of result that points directly at a molecular target. Fungal pathogens destroy a substantial fraction of global crop yields each year, and conventional resistance breeding often trades yield for defense. The MIC1/MIC2 system suggests a third path: tune the enzymatic processing of a universal pathogen-derived signal so that immunity is amplified without the growth penalty that constitutive immune signalling imposes. Editing or selecting variants of MIC1-like chitinases, or manipulating the glycosylation machinery that controls them, could in principle produce crops that detect and respond to fungal attack more vigorously while growing as if no pathogen were present. A single sugar on a single asparagine, it turns out, may hold the balance between feast and famine in the plant kingdom.

Subject of Research: N-glycosylation-dependent functional divergence of paralogous plant chitinases balancing immunity and growth

Article Title: N-glycosylation code diversifies paralogous chitinases to balance plant immunity and growth

Article References: Li, C., Lin, X.-X., Tian, L., Sun, W., Liu, J., Liu, Q., Du, Y., Jiang, X., Liang, Z., Deng, Y. Z., Au, S. W. N., Wan, J.-B., & Li, J.-F. (2026). N-glycosylation code diversifies paralogous chitinases to balance plant immunity and growth. Nature Plants. https://doi.org/10.1038/s41477-026-02420-7

Image Credits: AI Generated

DOI: 10.1038/s41477-026-02420-7

Keywords: chitinases, N-glycosylation, plant immunity, chitin signalling, Arabidopsis thaliana, MIC1, MIC2, growth-defense trade-off, fungal resistance, rice breeding, apoplast, CRISPR

The review focuses on green synthesis, a method that uses plant extracts, bacteria, yeast and fungi to manufacture nanoparticles rather than relying on the toxic chemicals and energy-intensive processes of conventional production. Materials with diameters between 1 and 100 nanometres behave fundamentally differently from their bulk counterparts because of their enormous surface-to-volume ratio, and the researchers argue that plants are the most practical biological factories for making them. Plant-derived synthesis is more economical than chemical routes, reduces pollution, and enhances environmental and human health safety. Crucially, the surface chemistry of each nanoparticle depends on the plant extract used, meaning that the choice of leaf, rhizome or peel extract can tune the final material’s properties. The bottom-up approach, in which nanoparticles are assembled atom by atom from precursor solutions, is generally preferred because it produces more uniform products with fewer defects and easier control over fabrication parameters.

Copper nanoparticles receive extensive attention, partly because copper is far cheaper than silver or gold, which matters enormously for agricultural deployment. Copper has been used against fungal disease since 1761, when seed grains soaked in mild copper sulfate solution were found to resist seed-borne fungi. The review documents striking results: a colloidal suspension containing just 7 parts per million of copper nanoparticles showed strong activity against Corticium salmonicolor, the agent of pink disease in rubber trees, when sprayed on infected plantations. In tea plantations, copper nanoparticles biosynthesised by the bacterium Streptomyces griseus achieved a 52.7 percent disease reduction at a dose of 2.5 parts per million, outperforming bulk copper, which managed only 45.3 percent. Nanoparticles made from Celastrus paniculatus leaf extract inhibited the mycelial growth of the wilt pathogen Fusarium oxysporum by up to 76.29 percent, while copper nanoparticles tested at 450 parts per million suppressed fungal growth by 93.98 percent over nine days of incubation.

Zinc oxide nanoparticles emerge as another standout, particularly attractive because zinc is generally safe for humans and causes little to no allergic response. The review reports that zinc oxide nanoparticles inhibited the growth of Aspergillus flavus by 37.81 percent at 500 parts per million and by 63.57 percent at 1000 parts per million, and that they strongly suppressed Rhizoctonia solani and Fusarium oxysporum, achieving 63.4 and 88.9 percent antifungal activity respectively at a concentration of 100 milligrams per millilitre. Against the post-harvest pathogens Botrytis cinerea and Penicillium expansum, doses above 3 millimoles per litre significantly inhibited growth. The proposed mechanisms are technically rich: zinc oxide nanoparticles generate reactive oxygen species on the fungal cell wall, potentially damaging DNA and denaturing proteins, while their ionic interaction with the wall leads to structural destruction. They may also disrupt membrane permeability, blocking the incorporation of lipids and proteins into the cell membrane, and possibly interfere with mitotic spindle division by targeting microtubules and inhibiting DNA transcription.

Silver nanoparticles, the most widely cited nanomaterial in the antimicrobial literature, occupy a special place in the review. Silver is toxic to fungi at very low concentrations, as little as 0.2 parts per million, while human health is largely unaffected at low doses, which explains its use in medical device coatings, orthopaedic and dental graft materials, wound dressings, water sanitisation and even textiles. The mechanistic detail is particularly compelling. Quantitative analysis of antifungal effects against Ustilaginoidea virens, the rice false smut fungus, showed that 2-nanometre silver particles were markedly more potent than 15-nanometre particles, with inhibition increasing dose-dependently between 0.5 and 10 micrograms per millilitre. Treated fungal cells showed altered ratios of phosphatidylcholine to phosphatidylethanolamine, a shift that destroys membrane integrity and function, along with a depletion of C18:2 phospholipids suggesting impaired desaturation. In Candida albicans, silver nanoparticles arrested the cell cycle at the G2/M phase, triggering reactive oxygen species production and depleting metal-based antioxidant enzymes.

The review also surveys less familiar candidates. Gold nanoparticles, valued for their chemical stability, oxidation resistance and biocompatibility, can be fabricated as nanorods, nanostars, nanobelts, nanospheres, nanocages and nanoprisms. Gold nanoparticles synthesised from Annona muricata showed zones of inhibition ranging from 30 to 66 percent against test fungi, with Penicillium camemberti most susceptible at 4 milligrams per litre, while gold particles made from Pongamia pinnata achieved 80 percent inhibition of a plant pathogenic fungus at just 0.8 milligrams per millilitre. Iron oxide nanoparticles, existing as magnetite, maghemite and hematite phases, showed activity against Candida albicans with a zone of inhibition of 53.67 millimetres when synthesised by the fungus Aspergillus terreus, and chitosan-coated iron oxide particles inhibited Fusarium solani, Candida albicans and Aspergillus niger. Magnesium oxide nanoparticles carry a Generally Recognised as Safe designation from the US Food and Drug Administration, making them especially promising, and particles derived from Citrus aurantium peel extract inhibited Candida albicans with a 26-millimetre zone of inhibition.

Selenium nanoparticles add a further dimension, since selenium is an essential trace element central to antioxidant defence in animal cells. Selenium nanoparticles fabricated with Bacillus subtilis showed stronger activity against Aspergillus species than against Candida, while particles synthesised by the fungus Trichoderma atroviride inhibited the mycelial growth of Colletotrichum capsici and Alternaria solani at 50 and 100 parts per million. Applied to tomato leaf blight caused by Alternaria alternata, selenium nanoparticles achieved an inhibition rate of 89.6 percent at 100 micrograms per millilitre, and at 80 parts per million they completely suppressed early blight disease in potatoes. Notably, the surface coating matters: selenium nanoparticles stabilised with poly-L-lysine showed the strongest antifungal action against the crop pathogen Sclerotinia sclerotiorum compared with polyacrylic acid or polyvinylpyrrolidone coatings. Copper oxide nanoparticles, meanwhile, achieved inhibition rates above 95 percent at low concentrations against Fusarium solani, Neofusicoccum species and Fusarium oxysporum.

Doping strategies push performance further. When zinc oxide is doped with metals such as iron, copper, cobalt or magnesium, or with non-metals and rare-earth elements, its electronic and optical properties change, often boosting reactive oxygen species generation. Iron-doped zinc oxide nanoparticles made with Hibiscus rosa-sinensis leaf extract inhibited Candida albicans with a 16.5-millimetre zone of inhibition, compared with 12.4 millimetres for undoped particles. In comparative studies, iron-doped zinc oxide at 40 microlitres matched the standard antifungal mycostatin against Aspergillus niger, magnesium-doped zinc oxide performed comparably to clotrimazole against Candida albicans, and biogenic silver-doped zinc oxide nanoparticles approached the activity of amphotericin B against Aspergillus flavus. Copper incorporation into zinc oxide structures was shown to enhance reactive oxygen species production and thereby increase antifungal efficacy against agricultural pathogens.

Underpinning all of these results is a coherent mechanistic framework. The review identifies three principal routes of fungal killing. The first is physical disruption: nanoparticles bind to the fungal cell wall and penetrate the membrane, causing structural damage and leakage of intracellular contents. Scanning electron microscopy comparisons against Candida glabrata revealed that silver nanoparticles created pores and distorted membranes, whereas gold nanoparticles induced no notable morphological changes, and gold particles have additionally been shown to cause nuclear condensation and DNA fragmentation in Candida albicans. The second route is oxidative stress: reactive oxygen species generated on particle surfaces, including hydroxyl radicals and hydrogen peroxide, damage DNA, proteins and lipids, with silver ions also complexing with thiols to prevent fungal detoxification. The third route is interference with cell signalling pathways, exemplified by chitosan nanoparticles, which interact with fungal cell walls and modulate pathways such as Toll-like receptor activation and the PI3K/AKT/mTOR cascade. In practice, these mechanisms often operate in combination, as with titanium dioxide nanoparticles, which both disrupt membranes and generate reactive oxygen species.

The authors are candid about the limitations. Almost all evidence to date comes from in vitro experiments, and in vivo studies are urgently needed to understand how these materials behave against fungal pathogens in living organisms. Safety evaluation must take centre stage, since metal and metal oxide nanoparticles can be toxic at high concentrations, as silver’s effects on freshwater and marine organisms demonstrate. The review calls for the synthesis and assessment of bimetallic and trimetallic nanoparticles, which may possess properties distinct from monometallic ones, along with standardised testing protocols to allow meaningful comparison across studies. Still, the overall message is one of genuine promise: nanoparticles exploit multiple simultaneous mechanisms of action, which lowers the risk of resistance development, and their small size, high surface area and tunable surface chemistry allow them to overcome the twin limitations of conventional antifungals, namely drug resistance and toxicity. If the safety and in vivo questions can be answered, plant-powered nanomaterials may soon protect both the world’s crops and its patients.

Subject of Research: Green-synthesized metal and metal oxide nanoparticles as antifungal agents

Article Title: Green-synthesized metal and metal oxide nanoparticles as emerging antifungal agents: current advances, mechanisms, and future perspectives

Article References: Vijayalakshmi, K., Swaramanjari, T., Shanmugavel, M., & Gnanamani, A. (2025). Green-synthesized metal and metal oxide nanoparticles as emerging antifungal agents: current advances, mechanisms, and future perspectives. Discover Biotechnology, 2(1), Article 18. https://doi.org/10.1007/s44340-025-00024-z

Image Credits: AI Generated

DOI: 10.1007/s44340-025-00024-z

Keywords: nanoparticles, green synthesis, antifungal agents, metal oxides, silver nanoparticles, zinc oxide, copper nanoparticles, reactive oxygen species, fungal resistance, plant pathogens, Candida albicans, nanotechnology

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