Toll‐9 mediates <i>Drosophila melanogaster</i> melanization in a PPO3‐dependent manner

Dear Editor, The innate immune system of insects relies on two major branches: the humoral responses, which involve antimicrobial peptides (AMPs) regulated by the nucleic factor kappa B (NF-κB) signaling pathways, and the cellular responses, which include hemocyte-mediated phagocytosis, encapsulation, and melanization (Lavine & Strand, 2002; Lemaitre & Hoffmann, 2007). Among these, melanization is a fast-acting defense mechanism that results in the deposition of melanin at sites of pathogen invasion or wounding, promoting pathogen killing and wound healing through reactive intermediates (Hillyer, 2016). In Drosophila melanogaster (fruit fly), the melanization cascade is driven by the enzymatic activity of phenoloxidases (POs), which are activated from prophenoloxidases (PPOs). Three PPOs, including PPO1, PPO2, and PPO3, have been identified in Drosophila. PPO1 and PPO2 are mainly expressed in plasmatocytes and lamellocytes, while PPO3 is specifically expressed in crystal cells (Yang et al., 2021). The Drosophila genome encodes nine Toll receptor family members, among which Toll-1 (also known as Toll), first characterized for its developmental roles (Anderson et al., 1985), is the canonical receptor that activates NF-κB-dependent AMP production upon recognition of some types of Gram-positive bacteria or fungi (Lemaitre et al., 1996). The other eight types of Toll receptors (from Toll-2 to Toll-9) have been demonstrated to execute potential roles in immunity, but some are still unclear or debated. Instead, these eight Toll receptors are thought to play more critical roles in development than immunity. Recently, we and colleagues identified Toll-9 as an essential regulator in the fly antitumor immunity (Ji & Hoffmann, 2024). In addition, Toll-9 has also been demonstrated to mediate cell competition, neurodegeneration, apoptosis, and antiviral immunity in Drosophila (Meyer et al., 2014; Shields et al., 2022; Sakakibara et al., 2023; Chauhan et al., 2025). However, the potential role of Toll-9 in the host melanization reaction has not been previously explored in depth. We examined the impact of Toll-9 on Drosophila melanization at multiple developmental stages. It was previously demonstrated that loss of Toll-9 does not affect the melanization reaction in adult flies (Narbonne-Reveau et al., 2011). In addition, the Toll-9 loss-of-function mutants survived normally without apparent melanization reactions at both the larval and pupal stages (Fig. S1A, B). We next used the act-Gal4 driver and carried out genetic crossings to generate Toll-9 overexpression (OE) flies (referred to as act>Toll-9 OE). Interestingly, Toll-9 OE resulted in striking melanotic masses, which was evident from the larval to pupal stages (Fig. 1A, B). We did not observe significant melanization reactions in the controls (act>LacZ OE) (Fig. 1A, B). Of note, these melanized Toll-9 OE pupae displayed apparent developmental defects (thin and short, Fig. 1A) and failed to undergo eclosion. This lethality phenotype was consistent with a previous study in which they used the da-Gal4 to drive Toll-9 OE in Drosophila (Yagi et al., 2010). Toll receptors are type I integral membrane glycoproteins, consisting of the N-terminal leucine-rich repeat (LRR) domain, the transmembrane (TM) domain, and the C-terminal intracellular Toll/interleukin-1 receptor/resistance protein (TIR) domain (Gay & Gangloff, 2007). To determine the structural requirement for Toll-9's regulatory role in Drosophila melanization, we subsequently generated three types of transgenic flies overexpressing the Toll-9 extracellular LRR domain (referred to as act>Toll-9LRR OE), the TM domain (referred to as act>Toll-9TM OE), or the intracellular TIR domain (referred to as act>Toll-9TIR OE), respectively (Fig. 1C). Strikingly, only the intracellular TIR domain was sufficient to induce robust melanization, while the LRR and TM domains alone had no observable effects (Fig. 1D, E). The above findings suggest that Toll-9 mediates Drosophila melanization through intracellular signaling mechanisms involving the TIR domain. To elucidate the molecular mechanism underlying Toll-9-induced melanization in Drosophila, we performed liquid chromatography plus tandem mass spectrometry (LC-MS/MS)-based quantitative proteomics on Toll-9 OE flies (Fig. 2A). We detected 3236 proteins/peptides, among which 647 were upregulated (more than 2-fold) and 679 were downregulated (less than 0.5-fold) by Toll-9 OE, respectively (Fig. 2B). Gene ontology (GO) enrichment analyses revealed that these upregulated proteins were associated with protein maturation, response to fungus, and amine metabolic process (Fig. 2C). In contrast, most of the downregulated candidates were enriched in functions related to molting, reproductive process, and membrane organization (Fig. 2D). Among the differentially regulated proteins, some melanization-related genes were significantly upregulated (Fig. 2E and Table S1) by Toll-9 OE. For instance, PPO3 and PPO2 were increased by more than 100-fold and 2-fold, respectively (Fig. 2E). No significant changes were observed for PPO1 (Fig. 2E). Additional upregulated proteins included serine proteases and immune modulators such as Spätzle-processing enzyme (SPE), Grass, and Persephone (Psh) (Fig. 2E), suggesting a broad activation of the melanization-related pathways. In Drosophila, the melanization cascade is modulated by POs, which are activated from PPOs, including PPO1, PPO2, and PPO3. Therefore, we performed reverse transcription plus polymerase chain reaction (RT-qPCR) experiments to monitor the transcript levels of all PPO genes in Toll-9 OE larvae. The PPO3 mRNA level was increased by more than 70-fold relative to that in the controls (P < 0.001) (Fig. 3A), consistent with the observed protein upregulation (Fig. 3B). However, the expression of PPO1 and PPO2 remained unchanged in both Toll-9 OE and control animals (Fig. 3A, B). These data suggest that Toll-9 may regulate PPO3 expression at the transcriptional or post-transcriptional level. To test whether PPO3 is functionally required for the Toll-9-induced melanization phenotype, we knocked down PPO3 in the genetic background of Toll-9 OE. Notably, PPO3 knockdown nearly fully suppressed the melanotic phenotype at both the larval and pupal stages (Fig. 3C, D). We also observed in Toll-9 OE larvae an increased PO activity, which was reversed by PPO3 silencing (Fig. 3E). Of note, silencing of PPO1 or PPO2 hardly affected the enhanced melanization phenotype by Toll-9 OE (Fig. 3C, D). These results demonstrate that PPO3, but neither PPO1 nor PPO2, is essential for the melanization response downstream of Toll-9 (Fig. 3F). Our study identifies Toll-9 receptor protein as a previously unrecognized regulator of melanization in Drosophila, operating through a PPO3-dependent mechanism. While the canonical Toll-1 receptor is well known for mediating NF-κB-dependent AMP expression, Toll-9 appears to control a distinct arm of immunity via the melanization cascade. The fact that Toll-9 overexpression, but not loss-of-function, induces melanotic masses suggests that Toll-9 activity is tightly regulated under physiological conditions and may be context-dependent. Importantly, we demonstrate that the intracellular TIR domain of Toll-9 is necessary for the melanization phenotype, indicating that downstream signaling components (potentially involving Myd88 or other TIR-adapter proteins) are recruited in this process. Notably, when we overexpressed Toll-9 by using the hml-Gal4, we detected melanization reactions (Fig. S2A), which were much weaker than what we observed in act>Toll-9 OE animals. We reason that the hml-Gal4 may drive relatively weaker expression compared to the act-Gal4 or the potential role of Toll-9 in lamellocytes would be more responsible for the melanization phenotype than that in hemocytes. Our proteomic profiling reveals a clear upregulation of PPO3 and related proteolytic factors involved in melanization reaction, highlighting Toll-9's role in orchestrating a pro-melanization transcriptional program. Further analyses did not indicate a significant change in lamellocyte numbers upon Toll-9 overexpression (Fig. S2B), suggesting that the effect is not due to expansion of the lamellocyte population but rather to transcriptional activation of PPO3. The strong correlation between Toll-9-induced PPO3 expression and melanization, and the complete suppression of the phenotype upon PPO3 knockdown, collectively argue that PPO3 is the key effector in this pathway. The implications of this finding extend to broader questions about how Toll receptors diversify their immune functions. Toll-9 may act in a non-redundant, damage- or context-specific manner, potentially integrating environmental cues such as tissue injury or cellular stress. Interestingly, prior studies have shown that Toll-9 is upregulated in neurodegenerative models and tumor suppression contexts, both of which are associated with chronic inflammation or cellular dysregulation (Meyer et al., 2014; Shields et al., 2022; Sakakibara et al., 2023; Chauhan et al., 2025). Melanization is not only a defense mechanism but also participates in wound repair and encapsulation, suggesting that Toll-9 might contribute to tissue integrity beyond pathogen clearance. It remains to be determined whether Toll-9 requires extracellular ligands such as Spätzle (Spz) for activation or whether it can signal constitutively or via endogenous damage-associated ligands. Future studies should explore the upstream activators and downstream effectors of Toll-9 signaling in both immune and non-immune contexts. Taken together, our findings introduce a new role for Toll-9 in the Drosophila innate immune reaction and establish a direct mechanistic link between Toll receptor signaling and the melanization cascade, thereby adding to our understanding of the functional repertoire of Toll and/or Toll-like receptors in other insect species. This study was supported by the National Natural Science Foundation of China (32100702). All authors have seen and agreed with the contents of the manuscript. There is no conflict of interests, including specific financial interests and affiliations relevant to the subject of the manuscript. Fig. S1 Loss of Toll-9 does not affect the melanization reaction in Drosophila. Fig. S2 Overexpression of Toll-9 in hemocytes causes melanization in Drosophila. Table S1 Increased candidates in the proteomic analysis. Table S2 Oligos used for RT-qPCR experiments. 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