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