The study, published in PLOS Genetics by Christopher J. Johnson, Joshua Kavaler, Timethea Slade, Kayla Wilson, Christina D. Cota, and Alberto Stolfi, focuses on the tunicate Ciona, one of the most tractable experimental models for studying chordate development. Tunicates occupy a pivotal position in the tree of life: they are the sister group to vertebrates within the chordate phylum, meaning they share a more recent common ancestor with us than any other invertebrate lineage does. Yet unlike vertebrates, tunicates evolved a biphasic lifecycle, alternating between a motile larval stage and a sedentary adult stage. The papillae are the key organ governing the transition between these two phases, making them a fascinating case study in how evolution repurposes embryonic machinery for new anatomical innovations.

The papillae of most tunicate larvae are arranged as a group of three morphologically identical organs. During embryogenesis, they arise from the anterior neural plate border, a region of the developing embryo nestled between the ventral epidermis and the more dorsal and posterior progenitors of the neural tube. This embryonic address matters. Based on its location and its molecular signature, the anterior neural plate border in tunicates has been evolutionarily linked to the vertebrate placodes, the thickened patches of embryonic ectoderm that give rise to sensory structures such as the inner ear, the nose, and the lens of the eye. Understanding how the papillae are patterned therefore offers a window into how sensory organs were built in the common ancestor of all chordates.

Previous work had established that a signaling pathway called BMP, short for bone morphogenetic protein, is essential for the specification, patterning, and morphogenesis of the papilla region in the Ciona embryo. BMP signaling is one of the great workhorses of developmental biology, used across the animal kingdom to draw boundaries between tissues and to instruct cells about their fates. But knowing that BMP is required is only the beginning of the story. Signaling pathways are blunt instruments; they must be interpreted by transcription factors, the proteins that switch specific genes on or off in specific cells. The downstream mechanisms that translate BMP activity into the actual anatomy of the papillae remained poorly understood, and that is the gap the new study set out to close.

The researchers identified a transcription factor called NK4, the Ciona ortholog of the vertebrate NKX2-3, NKX2-5, and NKX2-6 genes, as a central player in this process. NK4 belongs to the NKX2 family of homeobox transcription factors, a group of DNA-binding proteins with deep evolutionary roots and well-documented roles in organ development across the animal kingdom. In vertebrates, the closest relatives of NK4 are famous for their roles in building the heart and the thyroid, among other structures. The new work shows that in the tunicate embryo, this ancient gene family has been recruited to a very different task: carving a single field of cells into three discrete sensory organs.

The evidence places NK4 squarely downstream of BMP signaling. The team found that NK4 is activated by BMP in the embryonic region that will give rise to the papillae, meaning that BMP does not act alone but instead works through this transcription factor to execute its patterning program. This kind of pathway-to-transcription-factor relay is a recurring theme in development, allowing a broadly active signal to be converted into precise, cell-type-specific gene expression. In this case, the relay allows a diffuse gradient of BMP activity to be transformed into the sharply defined anatomy of three separate papillae.

What does NK4 actually do once it is switched on? The study provides a compelling answer: it acts as a transcriptional repressor, and its principal target appears to be a regulatory gene called Foxg. In normal embryos, Foxg is expressed in exactly three cell clusters, and those clusters are the ones that give rise to the three papillae. NK4’s job is to restrict Foxg expression to those three spots. The logic is elegant: rather than activating the papilla program directly in three places, the embryo uses NK4 to repress the program everywhere except where other influences allow it to persist. Repression, in developmental biology, is often as creative as activation, and this system is a textbook example.

The functional tests drive the point home with striking clarity. When the researchers disrupted NK4 function, the boundary control was lost and the embryo formed a single large papilla instead of three separate ones. The papilla program, freed from repression, spread across the entire region and merged into one organ. The opposite experiment produced the mirror-image result: when NK4 was overexpressed, its repressive power overwhelmed the system, Foxg was shut down everywhere, and the papillae were eliminated entirely. Together, these gain-of-function and loss-of-function experiments show that the level of NK4 activity is precisely calibrated, and that the tripartite anatomy of the organ depends on keeping that calibration within narrow limits.

How does the embryo achieve that calibration? The study points to two opposing influences that fine-tune NK4’s reach. On one side, the expression of NK4 is restricted dorsally by Chordin, a well-known antagonist of BMP signaling. Chordin binds BMP molecules and prevents them from activating their receptors, so in regions where Chordin is present, the BMP-to-NK4 relay is interrupted. On the other side, the researchers found that Msx, a transcription factor expressed on the ventral side of the embryo, can alleviate the repressive effect of NK4. In other words, where Msx is present, NK4’s grip on Foxg is loosened, allowing the papilla program to proceed. The interplay between Chordin’s dorsal restriction and Msx’s ventral counteraction potentially explains the characteristic tripartite patterning of the papillae: three narrow zones where the balance of these forces tips in favor of Foxg expression, surrounded by territory where NK4 keeps the program silenced.

Beyond its immediate findings, the study raises broader questions about the evolution of sensory organs in chordates. The anterior neural plate border that produces the papillae is thought to correspond to the territory that, in vertebrates, generates placodes and the neural crest, two innovations that underpin the elaborate sensory and cranial structures of animals with backbones. If a BMP-dependent, NKX2-family-mediated repressive circuit helps carve sensory territories out of this border region in tunicates, similar logic may have operated in the last common ancestor of tunicates and vertebrates, or may have been independently redeployed in each lineage from an ancient toolkit. Either way, the work demonstrates that the difference between one organ and three can hinge on a single repressor and its antagonists, a reminder that the grand architecture of animal bodies is built from molecular switches of exquisite sensitivity. For a larva whose survival depends on landing in the right place at the right time, that precision is everything.

Subject of Research: BMP-dependent NKX2-family transcriptional regulation patterning the tripartite sensory-adhesive papillae of tunicate larvae

Article Title: An NKX2-3/5/6 homolog is required downstream of BMP signaling to pattern the sensory-adhesive organ of a tunicate larva

Article References: Johnson, C. J., Kavaler, J., Slade, T., Wilson, K., Cota, C. D., & Stolfi, A. (2026). An NKX2-3/5/6 homolog is required downstream of BMP signaling to pattern the sensory-adhesive organ of a tunicate larva. PLOS Genetics, 22(10), e1012334. https://doi.org/10.1371/journal.pgen.1012334

Image Credits: AI Generated

DOI: 10.1371/journal.pgen.1012334

Keywords: tunicates, Ciona, BMP signaling, NKX2 transcription factors, NK4, Foxg, Chordin, Msx, papillae, neural plate border, embryonic patterning, chordate evolution

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