The insect Toll pathway activates antibacterial immunity against the citrus Huanglongbing pathogen

Domain: neuroscience
Published: 2026-01-23
Editorial outcome: minor_revision

Abstract
Psyllid-transmitted Candidatus Liberibacter asiaticus (CLas) is a phloem-limited unculturable bacterium that causes citrus Huanglongbing. To date, the mechanisms by which psyllids recognize and respond to this pathogen remain poorly understood. Here, we show that the psyllid Toll8 receptor acts as a pattern recognition receptor for the CLas outer membrane protein Barrel, resulting in Toll8 dimerization and activation. Activated Toll8 subsequently triggers an intracellular signaling cascade, involving MyD88 dimerization and the activation of IκB kinase IKKE, that culminates in the transcriptional activation of the transcription factor NFAT and the expression of antimicrobial effectors. Moreover, we show that CLas counters this immune response by secreting the effector protein SDE3230, which hijacks an E3 ligase to mediate the ubiquitination and degradation of MyD88, thereby suppressing Toll signaling. Our findings define a Toll8-MyD88-IKKE-NFAT immune signaling axis in arthropods and highlight an intricate counter-defense strategy employed by pathogens for survival and propagation within insect vectors.

Introduction
The innate immune system, the primary defense mechanism against pathogen invasion, relies on pattern recognition receptors (PRR) to detect conserved pathogen-associated molecular patterns (PAMP) and initiate immune responses1. Among these immune components, Toll-like receptors (TLRs) in vertebrates and their evolutionary counterparts, Toll receptors in insects, constitute highly conserved elements of innate immunity, playing critical roles in pathogen recognition and host defense. Vertebrate TLRs exhibit direct recognition capabilities for diverse bacterial PAMPs, including lipopolysaccharide (LPS), peptidoglycan (PGN), and lipoproteins, thereby triggering downstream signaling cascades that activate immune responses2. For instance, TLR4 specifically binds to LPS, TLR5 interacts with flagellin, and TLR1/2/6 recognizes bacterial lipoproteins, orchestrating inflammatory and innate immune responses3. Certain TLRs also identify viral PAMPs through interactions with viral nucleic acids or structural components4. In contrast, the prevailing paradigm suggests that insect Toll receptors function indirectly, requiring upstream PRRs such as peptidoglycan recognition proteins (PGRP) to detect PAMPs and activate Toll signaling through Spätzle cytokines5–7. For instance, in Drosophila melanogaster, the PGRP family acts as PRRs by detecting bacterial PGN, initiating a protease cascade that activates the ligand protein Spätzle, the ligand for Toll receptors7. Recent studies reveal the pest insect Spodoptera litura myeloid differentiation-like protein 11 (ML11) partners with Toll5 to sense Autographa californica multiple nucleopolyhedrovirus8. Similarly, in the silkworm Bombyx mori, Toll9 recognizes LPS by forming a complex with two ML proteins9. Despite these advances, it remains unclear whether insect Toll receptors can directly recognize PAMPs.

Structurally, both vertebrate TLRs and invertebrate Toll receptors are characterized as transmembrane proteins featuring an extracellular leucine-rich repeat (LRR) domain, a single-pass transmembrane domain, and a cytoplasmic Toll/interleukin-1 receptor (TIR) homology domain10. In vertebrates, ligand binding to the LRR domain of TLRs induces receptor dimerization, facilitating the recruitment of the adapter protein myeloid differentiation primary response 88 (MyD88) to initiate intracellular signaling cascade11. MyD88 subsequently recruits IL-1 receptor-associated kinase 4 (IRAK-4), forming a signaling complex that activates the IKK complex, ultimately leading to nuclear translocation of NF-κB transcription factors12. In D. melanogaster, the Toll signaling pathway shares similarities with vertebrates, as activated Toll receptors promote MyD88 dimerization, which recruits the kinase Pelle and the IRAK homolog Tube to form a signaling heterotrimer13. This complex ultimately drives nuclear translocation of NF-κB family transcription factors (e.g., Dorsal) to regulate the expression of antimicrobial peptides (AMP) and other immune effector molecules14. Despite these parallels, whether MyD88 orchestrates divergent immune pathways and how insect Toll signaling combats phloem-restricted unculturable bacteria remains unknown.

Candidatus Liberibacter asiaticus (CLas), the etiological agent of the devastating citrus disease Huanglongbing (HLB), is primarily transmitted by the Asian citrus psyllid (Diaphorina citri)15,16. Currently, HLB has become widespread in major citrus-growing regions worldwide, resulting in a drastic reduction in fruit yield and incurring substantial economic losses17–19. Despite decades of intensive research, effective management of HLB remains a major challenge, largely due to the unculturable, phloem-limited nature of CLas and the lack of commercially available HLB-resistant citrus cultivars20. CLas belongs to a group of phloem-limited bacteria that also includes phytoplasmas and other Candidatus species (e.g., Ca. Phlomobacter and other Ca. Liberibacter species). Notably, these pathogens are transmitted in a persistent-propagative manner by piercing-sucking insects, including psyllids and leafhoppers15,21,22. Regarding acquisition and transmission efficiency, psyllid nymphs acquire CLas from infected plants more efficiently, whereas adults transmit the bacterium to healthy hosts with higher effectiveness23,24. Intriguingly, persistent transmission of CLas does not appear to negatively impact psyllid vector fitness, suggesting that vectors have evolved immune tolerance mechanisms to sustain bacterial infections25. CLas, a gram-negative bacterium, possesses a cell wall structure comprising an outer membrane containing various proteins, a thin modified peptidoglycan layer, and an inner membrane26,27. However, the mechanisms by which insect PRRs detect CLas-derived PAMPs to activate immune defenses remain poorly understood.

Here, we define a psyllid Toll receptor that functions as a PRR by directly interacting with the CLas outer membrane protein Barrel, a bacterial PAMP. This interaction initiates a previously unidentified Toll signaling cascade that produces antibacterial effectors. Furthermore, we elucidate a bacterial counter-defense strategy that subverts this immune response. Our findings provide fundamental insights into the molecular interplay between phloem-limited bacteria and their insect vectors, offering potential avenues for developing innovative strategies to control HLB and other vector-borne diseases.

Results
Psyllid Toll receptors recognize CLas outer membrane protein Barrel to activate antibacterial immune response
Insect Toll immune responses typically involve PAMP recognition by upstream PRRs such as PGRPs28. In D. melanogaster, PGRP-SA and PGRP-LC function as PRRs to recognize bacterial PGN29,30. Surprisingly, only one PGRP gene (GenBank KAI5696543) was identified in the D. citri genome, which lacks both a transmembrane domain and a signal peptide, unlike D. melanogaster PGRP-SA and PGRP-LC (Supplementary Fig. 1a, b). This finding suggests that D. citri PGRP is unlikely to function as a PRR. Bioinformatics analysis identified four Toll receptor genes in D. citri, including Toll1, Toll6, Toll7, and Toll8 (Supplementary Fig. 2a). Among these, Toll1, Toll7, and Toll8 exhibited significantly increased expression following CLas infection, with Toll8 showing the highest upregulation and expression (Fig. 1a, Supplementary Fig. 2b). CLas infection also significantly enhanced Toll8 dimerization in psyllids (Fig. 1b). Functional validation through microinjection of dsRNAs targeting individual Toll genes (dsTolls) revealed that Toll8 silencing caused the most substantial increase in CLas 16S rRNA levels compared to knockdown of other Toll genes (Fig. 1c, d, Supplementary Fig. 2c–e), establishing that psyllid Toll8 plays a critical role in anti-CLas immunity.

Structurally, psyllid Toll8 is most similar to vertebrate TLR2 (Supplementary Fig. 3a, b), which directly recognizes bacterial outer membrane lipoproteins31, suggesting that Toll8 may also directly recognize PAMPs. Through systematic screening of 19 CLas membrane proteins via yeast two-hybrid (Y2H) analysis, we specifically identified the outer membrane protein Barrel (GenBank WP_015452909) as a Toll8 interaction partner (Supplementary Fig. 3c). This binding was mediated by Toll8’s LRR domain (Supplementary Fig. 3d), while the LRR domains of the other three Tolls showed no detectable interaction with Barrel (Supplementary Fig. 3e). The specific Toll8-Barrel interaction was further validated by Glutathione S-transferases (GST) pull-down assays (Supplementary Fig. 3f). Immunoelectron microscopy revealed that CLas Barrel localized on the membranes of CLas particles, which ranged from 400 to 700 nm in diameter (Fig. 1e). Co-expression of Barrel with Toll8 in Sf9 cells significantly increased Toll8 dimerization (Fig. 1f). Similarly, microinjection of purified Barrel into psyllids promoted Toll8 dimerization without affecting overall Toll8 protein levels (Fig. 1g). These findings demonstrate that psyllid Toll8 functions as a PRR that directly recognizes CLas outer membrane protein Barrel.

To identify key amino acids involved in the Toll8-Barrel interaction, we performed protein structure prediction and interaction modeling. Our analysis revealed that Asn679 (N679), Asn712 (N712), and Asp717 (D717) in the LRR domain of Toll8 were in close proximity to Barrel (Fig. 1h). Y2H and GST pull-down assays showed that substitution of D717 with alanine (D717A), but not N679A or N712A, disrupted the interaction with Barrel (Supplementary Fig. 3g, h). Co-expression of Barrel with Toll8 and its mutants in Sf9 cells revealed that Toll8 D717A exhibited significantly reduced dimerization compared to wild-type Toll8 and the other mutants (Fig. 1i). This demonstrates that D717 is essential for Barrel recognition and subsequent Toll8 dimerization. Thus, psyllid Toll8 acts as a PRR that directly recognizes CLas outer membrane protein Barrel, triggering dimerization in CLas-infected psyllids (Fig. 1j).

CLas Barrel induces MyD88 dimerization via Toll8 activation
MyD88, a key adapter protein in Toll signaling, contains a TIR domain for interaction with Toll receptors and a death domain (DD) for recruiting downstream signaling molecules11,32. Y2H and GST pull-down assays confirmed that the TIR domains of Toll8 and MyD88 mediate their interaction (Fig. 2a, b). CLas infection significantly increased MyD88 expression and dimerization in psyllids (Fig. 2c, d). dsRNA-mediated knockdown of MyD88 expression (dsMyD88) in CLas-infected psyllids resulted in an increased CLas accumulation (Fig. 2e, f), highlighting its role in anti-CLas immunity.

Co-expression of Barrel, Toll8, and MyD88 in Sf9 cells revealed that Barrel did not directly promote MyD88 dimerization but did so in the presence of Toll8 (Fig. 2g), suggesting that Barrel-activated Toll8 induces MyD88 dimerization. Similarly, microinjection of purified Barrel into psyllids enhanced MyD88 dimerization without altering overall MyD88 protein levels (Fig. 2h). These results indicate that CLas infection activates the Toll8-MyD88 signaling cascade, which exerts anti-CLas effects (Fig. 2i).

CLas Barrel-induced MyD88 dimer recruits and activates kinase IKKE
To identify downstream effectors recruited by MyD88, we screened a D. citri cDNA library using Y2H assays. From multiple candidate interactors identified (Supplementary Table 1), the kinase IKKE (mammalian IKKε homolog)33 was uniquely identified as a kinase interactor. The specific MyD88-IKKE interaction was subsequently validated through both Y2H assays (Supplementary Fig. 4a, b) and independent GST pull-down experiments (Supplementary Fig. 4c). Psyllid IKKE contains an N-terminal protein kinase catalytic (PKc) domain, a middle ubiquitin-like (Ubl) domain, and a C-terminal TANK-binding kinase 1 (TBK1) domain (Supplementary Fig. 4d). Further Y2H analysis showed that the DD domain of MyD88 interacted with the Ubl domain of IKKE (Supplementary Fig. 4a, b). CLas infection significantly increased the expression of IKKE and its phosphorylation in psyllids (Supplementary Fig. 4e, f), and dsRNA-mediated knockdown of IKKE expression (dsIKKE) in CLas-infected psyllids resulted in an increased CLas accumulation (Supplementary Fig. 4g, h). To functionally validate IKKE’s role in CLas transmission, we conducted transmission assays using dsIKKE-treated adult psyllids reared on CLas-infected plants. The insects were allowed to feed on susceptible young citrus leaf tissue confined within clip cages. At 14 days after initial access to leaves, tissues from psyllid feeding sites adjacent to leaf veins were collected for total DNA extraction. The extracted DNA was subsequently analyzed by qPCR detection of CLas 16S rDNA to determine transmission efficiency (Supplementary Fig. 4i). Compared to dsGFP controls, IKKE silencing significantly enhanced CLas transmission to healthy citrus leaves (Supplementary Fig. 4j). These findings demonstrate that IKKE plays a critical role in the MyD88-mediated immune signaling pathway to inhibit CLas infection.

We next examined the impact of CLas Barrel on Toll8-MyD88-IKKE pathway. Co-expression of Barrel, Toll8, MyD88, and IKKE in Sf9 cells revealed that Toll8-activated MyD88 dimerization promoted IKKE phosphorylation, which was further enhanced by Barrel (Supplementary Fig. 5a). Microinjection of purified Barrel into psyllids also stimulated IKKE phosphorylation (Supplementary Fig. 5b). Protein structure modeling identified Thr103 (T103) and Cys110 (C110) in the MyD88 DD domain as being in close proximity to Glu343 (E343) and Gln342 (Q342) in IKKE Ubl domain, respectively (Supplementary Fig. 5c). Mutagenesis demonstrated that substitution of C110 in MyD88 or Q342 in IKKE, but not T103 in MyD88 or E343 in IKKE, abolished their interaction (Supplementary Fig. 5d). Co-expression of Toll8 with MyD88 C110A or IKKE Q342A in Sf9 cells significantly reduced IKKE phosphorylation (Supplementary Fig. 5e), confirming that C110 in MyD88 and Q342 in IKKE are critical for MyD88-IKKE interaction and IKKE activation. Thus, CLas Barrel-induced MyD88 dimer recruits IKKE, promoting its phosphorylation (Supplementary Fig. 5f).

IKKE directly interacts with and phosphorylates a homolog of mammalian nuclear factor of activated T-cells
To identify transcription factors activated by IKKE, we screened a D. citri cDNA library using IKKE as bait. From multiple candidate interactors detected, the nuclear factor of activated T-cells (NFAT) homolog was uniquely detected as the only transcription factor showing interaction with IKKE (Fig. 3a, Supplementary Table 2). This finding is particularly significant as NFAT, while well-characterized in mammals for its roles in immunity and development, remains understudied in insect systems despite sharing the conserved RHD domain (Rel homology domain) with NF-κB34,35. Phylogenetic analysis revealed that insect NFATs are closely related (Supplementary Fig. 6a). Psyllid NFAT contains an N-terminal domain, a central RHD domain, and a C-terminal domain, resembling mammalian NFAT (Supplementary Fig. 6a). Y2H and GST pull-down assays confirmed that the N-terminal kinase domain of IKKE interacted with the RHD domain of NFAT (Fig. 3a–c). CLas infection significantly increased NFAT transcription, protein accumulation, and nuclear localization in psyllids (Supplementary Fig. 6b, c). Microinjection of Barrel into psyllids enhanced nuclear accumulation of NFAT (Supplementary Fig. 6d). dsRNA-mediated knockdown of NFAT expression (dsNFAT) in CLas-infected psyllids resulted in an increased CLas accumulation (Fig. 3d, e), confirming its anti-CLas activity.

Immunofluorescence microscopy analysis demonstrated that NFAT formed punctate cytoplasmic and nucleus foci in psyllid midgut epithelium (Fig. 3f). Similarly, heterologously expressed NFAT in Sf9 cells exhibited diffuse cytoplasmic distribution accompanied by progressive nuclear accumulation (Fig. 3g). These observations collectively support NFAT’s role as a nucleocytoplasmic shuttling transcription factor. Co-expression of IKKE and NFAT in Sf9 cells increased NFAT phosphorylation and nuclear accumulation (Fig. 3g, h). Phosphorylation mass spectrometry identified enriched phosphorylation sites in the NFAT-RHD domain (residues 167–180) (Fig. 3i). Constructing NFAT mutants with alanine substitutions at these sites, including NFAT m1: partial mutation retaining Ser167 (S167) and NFAT m2: complete mutation (Fig. 3j), we found that IKKE-mediated phosphorylation and nuclear translocation were abolished in NFAT m2 but retained in NFAT m1 (Fig. 3k). This indicates that kinase IKKE promotes NFAT phosphorylation for nuclear entry, with S167 in the RHD domain being a critical site for NFAT activation. Thus, CLas Barrel activates the Toll8-MyD88-IKKE-NFAT signaling pathway to exert anti-CLas effects (Fig. 3l).

Psyllid NFAT activates antibacterial effectors Reeler and nitric oxide synthetase
To identify NFAT-regulated antimicrobial effectors, ChIP-Seq revealed several NFAT-bound promoter regions, including genes encoding nitric oxide synthetase (NOS), cathepsin L1, catalase, cysteine proteinase, E3 ubiquitin ligase MSL2, and Reeler (Fig. 4a). Reeler homologs in B. mori and Procambarus clarkii exhibit antimicrobial activity through melanization or biofilm inhibition36,37. NOS catalyzes nitric oxide (NO) production, which exerts broad-spectrum microbicidal effects38,39. Given the high enrichment of Reeler and NOS in ChIP-Seq results (Fig. 4a), we focused on these two candidates.

Sequence analysis predicted an N-terminal signal peptide and a 122-amino-acid mature form of Reeler (Supplementary Fig. 7a-i). AlphaFold3 structural prediction revealed multiple alpha helices and beta sheets in Reeler (Supplementary Fig. 7a-ii). ChIP-qPCR validated NFAT binding to Reeler and NOS promoter regions (Fig. 4b). Moreover, EMSA and Y1H assays demonstrated NFAT binding to Reeler and NOS promoters (Fig. 4c–e). Luciferase reporter assay confirmed that NFAT directly activated Reeler and NOS transcription (Fig. 4f, g). The dsNFAT treatment significantly reduced Reeler and NOS expression and NO accumulation in psyllids (Fig. 4h–j). CLas infection significantly increased Reeler and NOS expression and NO levels in psyllids (Fig. 4k–m). Microinjection of Barrel also increased Reeler and NOS accumulation in psyllids (Fig. 4n). Together, these results reveal that CLas-activated NFAT acts as a direct regulator of Reeler and NOS.

We then investigated whether psyllid Reeler and NOS exhibited anti-CLas activity. dsRNA-mediated knockdown of Reeler expression (dsReeler) in CLas-infected psyllids resulted in an increased CLas accumulation (Fig. 5a, b), whereas microinjection of purified Reeler reduced CLas accumulation (Fig. 5c). In vitro assays demonstrated Reeler’s bactericidal activity against Xanthomonas citri and Sinorhizobium fredii, with immunofluorescence confirming bacterial death (Supplementary Fig. 7b, c) and an effective concentration 50% (EC50) established (Supplementary Fig. 7d). Similarly, dsRNA-mediated knockdown of NOS expression (dsNOS) in CLas-infected psyllids resulted in decreased NO levels and increased CLas accumulation (Fig. 5d–f). Furthermore, treatment with the NO activator SNP reduced CLas accumulation, while the NO inhibitor L-NMMA increased CLas accumulation in CLas-infected psyllids (Fig. 5g–j). To assess their impact on CLas transmission, we conducted transmission assays with dsRNA-treated adult psyllids from CLas-infected colonies. Both dsNOS and dsReeler treatments significantly enhanced CLas transmission to healthy citrus leaves compared to dsGFP controls (Fig. 5k), consistent with their reduced antimicrobial activity observed in molecular assays. These results demonstrate that NOS and Reeler function as key barriers to CLas infection in psyllids (Fig. 5l).

CLas secretory protein SDE3230 promotes MyD88 ubiquitination
The persistent infection of CLas in psyllids suggests it may attenuate Toll signaling to facilitate infection. Firstly, we used the proteasome inhibitor MG-132 to identify which factors in the Toll8-MyD88-IKKE-NFAT signaling pathway could be degraded via the ubiquitin-proteasome pathway. It was found that MG-132 treatment increased MyD88 levels and reduced CLas accumulation in psyllids (Fig. 6a), indicating MyD88 is degraded via the ubiquitin-proteasome pathway. Furthermore, immunoprecipitation of MyD88 in psyllids revealed that MyD88 exhibited a significant affinity for ubiquitin (Fig. 6b). Thus, MyD88 potentially undergoes ubiquitinated degradation through the 26S proteasome pathway, attenuating Toll signaling and promoting CLas infection in psyllids.

E3 ubiquitin ligases play a crucial role in the ubiquitin-proteasome pathway by transferring ubiquitin molecules to substrate proteins40. During the preliminary Y2H screening using MyD88 as a bait protein, we identified the E3 ubiquitin ligase UBR5, containing a HECT domain, interacting with MyD88 (Fig. 6c, Supplementary Fig. 8a, b, Supplementary Table 1). CLas infection increased UBR5 expression (Supplementary Fig. 8c, d), while dsRNA-mediated knockdown of UBR5 expression (dsUBR5) resulted in an increased MyD88, NFAT, Reeler, and NOS accumulation while reducing CLas accumulation (Supplementary Fig. 8e, f). Co-expression of UBR5 and MyD88 in HEK-293T cells showed that the increased UBR5 expression led to the reduced MyD88 accumulation (Fig. 6d). In vitro ubiquitination assays confirmed that UBR5 mediates ubiquitination of MyD88 (Fig. 6e, f). Co-immunoprecipitation (Co-IP) assays showed that MyD88 ubiquitination was enhanced in Sf9 cells co-expressing UBR5 and MyD88 under MG-132 treatment (Fig. 6g). Bioinformatic prediction and systematic mutagenesis revealed three candidate ubiquitination sites in MyD88, including Lysine 48 (K48) in the DD domain, K201 in the TIR domain, and K301 at the C-terminus (Fig. 6h). Among these, K48 emerged as the primary ubiquitination site, as its mutation (K48R) significantly attenuated K48-linked polyubiquitination, while mutations at K201 and K301 showed comparatively weaker effects on ubiquitination levels (Fig. 6i). These results establish K48 as the dominant site regulating MyD88 stability.

CLas possesses a functional Sec-dependent secretory system and produces at least 86 potential secretory proteins41,42. SDE3230 (CLIBASIA_03230) was a typical cytoplasmic secretory protein that exhibited the highest expression level among all tested secretory proteins in psyllids (Fig. 7a–d). Y2H and GST pull-down assays showed that SDE3230 interacted with UBR5, while UBR5 interacted with MyD88’s DD domain containing the ubiquitination site (Fig. 7e, Supplementary Fig. 8g, h). Although SDE3230 itself was not ubiquitinated (Supplementary Fig. 8i), its microinjection reduced MyD88/NFAT accumulation while increasing MyD88 ubiquitination (Fig. 7f). Co-IP assays demonstrated that SDE3230 enhanced K48-linked polyubiquitination of MyD88 (Fig. 7g). Collectively, SDE3230 facilitates UBR5-mediated MyD88 degradation, effectively suppressing the MyD88-NFAT pathway and enabling persistent CLas transmission (Fig. 7h).

Methods
Plants, insects, cells, and antibodies
CLas-infected sweet orange (Citrus sinensis) plants were prepared through bud grafting using CLas-infected buds collected from Ningde City, Fujian Province, China. The grafted plants were maintained in a greenhouse for 3 months, after which CLas infection status was confirmed by RT-PCR analysis targeting the bacterial 16S rRNA gene.

The Asian citrus psyllid population, originally collected from CLas-free Citrus sinensis trees in Ningde City, was reared in a controlled greenhouse chamber at 27 ± 1°C with 75 ± 5% relative humidity under a 16h light/8h dark photoperiod. Newly emerged adult psyllids were allowed to feed on CLas-infected Citrus sinensis trees for 7 days before being transferred to healthy citrus plants. CLas acquisition in psyllids was verified by qPCR at 8 days post-first access to diseased plants (padp).

Spodoptera frugiperda (Sf9) insect cells were cultured in Sf900 III medium (Gibco, 12658019) supplemented with 10% heat-inactivated fetal bovine serum (FBS; EveryGREEN, 13011-8611) at 28°C. HEK-293T human embryonic kidney cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, 10566016) containing 10% FBS (Gibco, 10099141C) at 37°C in a humidified 5% CO2 incubator.

Custom rabbit polyclonal antibodies against D. citri Toll8, MyD88, IKKE, NFAT, Reeler, NOS, as well as against CLas Barrel and SDE3230, were generated through antigen peptide immunization as previously described64; unless otherwise specified, all antibodies were used at a dilution of 1:1000. Commercial antibodies included: FITC-conjugated Flag antibody (Invitrogen, MA1-91878-D488, 0.5 μg/μL). rhodamine-conjugated HA antibody (Invitrogen, 26183-A555, 0.5 μg/μL), β-Tubulin (TransGen Biotech, HC101, 1:5000 dilution), GAPDH (TransGen Biotech, HC301, 1:3000 dilution), Myc Tag (TransGen Biotech, HT101, 1:3000 dilution), Flag Tag (TransGen Biotech, HT201, 1:3000 dilution), HA Tag (TransGen Biotech, HT301, 1:3000 dilution), His Tag (TransGen Biotech, HT501, 1:3000 dilution) and GST Tag (TransGen Biotech, HT601, 1:3000 dilution), phospho-IKKE (Beyotime, AF5959, 1:1000 dilution), ubiquitin (Ub; Abmart, T55520, 1:1000 dilution), Ub K48 (Abmart, T55964, 1:1000 dilution), histone H3 (Abcam, ab1791, 1:5000 dilution), and HRP-conjugated secondary antibodies (goat anti-rabbit IgG, Thermo Fisher 32460, 1: 5000 dilution; goat anti-mouse IgG, Thermo Fisher 32430, 1:5000 dilution).

RT-qPCR analysis
To quantify gene expression levels of Toll1, Toll6, Toll7, Toll8, MyD88, IKKE, NFAT, Reeler, NOS, and CLas secretory protein genes in psyllids, total RNA was extracted from 30 adult insects pooled at 8 days padp using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. First-strand cDNA synthesis was synthesized from 1 μg total RNA using RevertAid Reverse Transcriptase (Promega, EP0441) and oligo(dT) primers. RT-qPCR reactions were conducted in triplicate using the SYBR Green PCR Master Mix (GenStar, A304-10) on a QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems). Relative gene expression was calculated using the 2−ΔΔCt method, with D. citri Actin or CLas 16S rRNA as endogenous controls. Primer sequences are provided in Supplementary Table 3.

Western blotting
Total proteins were extracted from 30 CLas-free or CLas-positive psyllids at 8 days padp using NP-40 lysis buffer (Beyotime, P0013F) containing protease inhibitor cocktail (Roche, 11697498001). For dimerization assays, protein samples were mixed with native loading buffer (62.5 mM Tris-HCl, pH 6.8, 25% glycerol, 0.01% bromophenol blue) and resolved on 10% non-denaturing polyacrylamide gels. Proteins were transferred to PVDF membranes (Millipore, IPFL00010), blocked with 5% non-fat milk, and probed with primary antibodies (1: 1000 dilution) overnight at 4°C. After incubation with HRP-conjugated secondary antibodies (1:5000 dilution), signals were developed using Luminata Classico Western HRP Substrate (Millipore, WBKLS0500) and imaged on a Luminescent Image Analyzer AI600 (GE Healthcare). Band intensities were quantified using ImageJ software (NIH).

Transmission electron microscopy
Midgut tissues from CLas-free or CLas-positive adult psyllid (16 days padp) were dissected and fixed in 2% (v/v) glutaraldehyde and 2% (v/v) paraformaldehyde in 0.1 M phosphate buffer (PBS; pH 7.4) for 2 h at room temperature, followed by post-fixation in 1% (w/v) osmium tetroxide for 1 h. Samples were dehydrated through an ethanol series (30–100%), embedded in Spurr’s resin (SPI Supplies, 02680-AB), and polymerized at 60°C for 72 h. Ultrathin sections (70 nm) were cut using a Leica EM UC7 ultramicrotome, mounted on nickel grids, and immunolabeled with CLas Barrel-specific IgG (0.5 μg/mL) followed by 10 nm gold-conjugated goat anti-rabbit secondary antibody (1: 100 dilution, Abcam, ab27236). Grids were counterstained with uranyl acetate and lead citrate before imaging on a Hitachi HT-7800 TEM operated at 80 kV.

Yeast two-hybrid assay
Full-length open reading frames (ORF) of Toll8, MyD88, IKKE, UBR5, Barrel, and SDE3230 (signal peptide removed) were cloned into the bait vector pGBKT7. Domain fragments and point mutants were generated as follows: IKKE: PKc domain (aa 18–332), Ubl domain (aa 310–387), TBK1 domain (aa 405–644), Q342A/E343A mutants; Toll8: LRR domain (aa 221–931), TIR domain (aa 1083–1217), N679A/N712A/D717A mutants; Toll1: LRR domain (aa 197–586), Toll6: LRR domain (aa 55–711), Toll7: LRR domain (aa 264–945); MyD88: DD domain (aa 43–121), TIR domain (aa 176–276), T103A/C110A mutant; NFAT: N-terminal (aa 1–139), RHD domain (aa 140–458), C-terminal (aa 459–1140) were cloned into pGADT7. For prey constructs, we cloned the following CLas membrane protein ORFs into pGADT7: ABCt (GenBank WP_015452444), BamA (GenBank WP_015452389), BamD (GenBank WP_015453002), Barrel (GenBank WP_015452909), CpaB (GenBank WP_015452434), EamA (GenBank WP_015452584), FliQ (GenBank WP_015452639), FlgA (GenBank WP_012778596), FliR (GenBank WP_015452647), FlhA (GenBank WP_015452646), FliF (GenBank WP_015824893), FlhB (GenBank WP_015452471), Kup (GenBank WP_015452667), LolA (GenBank WP_012778744), MotA (GenBank WP_015452475), MurJ (GenBank WP_012778749), PBP1 (GenBank WP_040055318), PBP2 (GenBank ALK07622) and YldC (GenBank WP_012778702). All recombinant constructs were sequence-verified prior to yeast transformation. Yeast strain AH109 was co-transformed with bait/prey plasmids using the LiAc/SS carrier DNA/PEG method and plated on SD/-Trp-Leu-His-Ade (QDO) medium. The pGBKT7-53/pGADT7-T interaction served as a positive control, while the pGBKT7-Lam/pGADT7-T interaction served as a negative control. Positive interactions were confirmed by colony growth after 5 days at 30°C. All plasmids were confirmed by DNA sequencing. Primer sequences are listed in Supplementary Table 3.

Glutathione S-transferase pull-down assay
GST-tagged proteins (Barrel, MyD88-TIR, UBR5, IKKE, NFAT-RHD) cloned into pGEX-4T-3 and His-tagged proteins (Toll8, Toll8 mutants, MyD88, SDE3230, IKKE-PKc) cloned into pET-28a were expressed in E. coli Rosetta (DE3) cells. GST fusion proteins were immobilized on glutathione-sepharose 4B beads (GE Healthcare, 17075601) and incubated with His-tagged proteins in binding buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% NP-40) for 4 h at 4°C. Beads were washed five times with ice-cold PBS, and bound proteins were eluted with 10 mM reduced glutathione. Interactions were verified by Western blot using anti-His (1:3000 dilution) and anti-GST (1:3000 dilution) antibodies. Primer sequences are listed in Supplementary Table 3.

RNA interference
The dsRNAs targeting Toll1, Toll6, Toll7, Toll8, MyD88, IKKE, NFAT, Reeler, NOS, and GFP were synthesized using the T7 RiboMAX Express RNAi system (Promega, P1700). Approximately 600 CLas-free or CLas-positive adults (8 days padp) were microinjected with 46 nL dsRNAs (1 μg/μL) using a Nanoject II Auto-Nanoliter Injector (Drummond Scientific). The dsGFP treatment served as a control. At 3 days post-injection, 30 insects per replicate were pooled for RT-qPCR and Western blot analysis. Three biological replicates were performed. Primer sequences are listed in Supplementary Table 3.

Protein microinjection assays
Recombinant CLas Barrel and SDE3230 proteins expressed in E. coli were purified using Ni-NTA affinity chromatography (Qiagen, 30210). Approximately 300 CLas-free psyllid adults were microinjected with 1 μg/μL purified protein. At 48h post-injection, 30 insects per replicate were pooled for total proteins or nuclear proteins were extracted using NP-40 lysis buffer or Nuclear/Cytoplasmic Extraction Kit (Beyotime, P0027) and analyzed by western blot. GFP protein (1 μg/μL) served as a negative control. Three biological replicates were performed.

Proteasome inhibition and NO modulation
Approximately 300 CLas-infected adults (8 days padp) were microinjected with 10 μM MG-132 (Selleckchem, S2619), 100 μM SNP (NO activator; Beyotime, S1901), or 100 μM L-NMMA (NO inhibitor; Beyotime, S0011) in PBS containing 0.1% DMSO. Control groups received PBS containing 0.1% DMSO alone. Toll8, MyD88, IKKE, NFAT, and Barrel protein levels were quantified by western blot at 24h post-treatment. Each replicate involved analyzing a pool of 30 treated insects, with three replicates conducted.

NO quantification assays
Lysates were collected from 30 psyllids per replicate for control or treatment groups, and centrifuged (10,000 × g, 10 min). NO levels in supernatants were measured using a Nitric Oxide Assay Kit (Beyotime, S0021) following the manufacturer’s protocol. Absorbance was read at 540 nm on a microplate reader (BioTek Synergy H1).

Structural modeling and molecular docking
Protein structures of Toll8, Barrel, MyD88, and IKKE were predicted using Alphafold3 (DeepMind) and validated via SAVESv6.1 (https://saves.mbi.ucla.edu/). Molecular docking was performed using GRAMM-X (http://vakser.compbio.ku.edu/resources/gramm/grammx/) with default parameters. The top-scoring complexes were analyzed using PDBePISA (https://www.ebi.ac.uk/pdbe/pisa/) and visualized in ChimeraX 1.4.

Baculovirus expression system
ORFs of target genes (Toll8, Toll8 mutants, Barrel, MyD88, MyD88 mutants, IKKE, IKKE mutants, NFAT, NFAT mutants, and Ub) were cloned into pFastBac1 with C-terminal Flag/Myc/HA tags. Recombinant bacmids were generated in E. coli DH10Bac (Thermo Fisher, 10361012) and transfected into Sf9 cells using Cellfectin II Reagent (Thermo Fisher, 10362100). Viral stocks were amplified through two passages, and protein expression was confirmed by Western blot.

Immunofluorescence microscopy
Midgut tissues from CLas-infected adults (8 days padp) were fixed in 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, and incubated with rhodamine-conjugated NFAT antibody (1:200 dilution) overnight at 4°C. Nuclei were counterstained with DAPI (1 μg/mL; Thermo Fisher, D1306). Images were acquired on a Leica TCS SPE confocal microscope using a 100× oil immersion objective.

Sf9 cells infected with recombinant bacmids expressing Flag-SDE3230, HA-IKKE, or Flag-NFAT were seeded on coverslips and harvested at 24 or 36 hpi. Cells were fixed, permeabilized, immunolabeled with Flag-FITC (0.5 μg/μL), HA-rhodamine (0.5 μg/μL), and DAPI (0.5 μg/μL), and processed for immunofluorescence microscopy.

Phos-tag SDS-PAGE
For phosphorylation analysis, Sf9 cells expressing Flag-NFAT (or its mutants) and HA-IKKE harvested at 48 hpi and lysed in Phos-tag lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 1× phosphatase inhibitors [Beyotime, P1091]). Proteins were separated on 8% SDS-PAGE gels containing 50 μM Phos-tag acrylamide (Wako, 304-93521) and 100 μM MnCl2, then transferred to PVDF membranes for immunoblotting.

CLas transmission assay
To evaluate the effect of gene silencing on CLas transmission, we performed inoculation experiments using adult D. citri that had been reared for multiple generations on CLas-infected sweet orange (Citrus sinensis) plants. Given that CLas titer in psyllids is a key determinant of transmission efficiency65, we first assessed bacterial levels in the insects prior to inoculation. A random sample of 30 adult psyllids was starved for 6 h and subjected to qPCR analysis of CLas titer in head-thorax tissues. All sampled insects tested positive for CLas, with cycle threshold (Ct) values below 20, indicating a high acquisition rate and confirming successful CLas infection cycle and abundant bacterial accumulation.

Previous study showed that CLas transmission by infected psyllids feeding on young citrus leaves typically requires nearly 3 months before HLB symptoms become visible65. To expedite the assessment of CLas transmission efficiency, a method has been established in which infected psyllids are confined to young citrus leaves for a 7-day inoculation access period, followed by a 7-day accumulation phase, after which CLas can be detected by qPCR23. Based on this approach, approximately 600 CLas-infected psyllids were microinjected with 46 nL dsRNAs (1 μg/μL) targeting IKKE, NOS, Reeler, or GFP. Two days post-injection, 15 dsRNA-treated psyllids were confined onto each healthy young leaf (4–6 cm in length) from uninfected C. sinensis plants using a leaf clip cage (Supplementary Fig. 4i). The insects were allowed to feed for 7 days under controlled conditions (27°C, 14:10 L:D photoperiod)23. After the inoculation period, psyllids were removed and stored for subsequent analysis. The exposed citrus leaves were maintained in an incubator at 27°C for an additional 7 days to allow potential CLas propagation. For each treatment group, ten leaf replicates were prepared.

Total DNA was extracted from leaf vein-adjacent (midribs and major veins) tissues on which psyllids had fed, using a CTAB-based method. Given that the leaf veins are primary sites for CLas accumulation, sampling these regions provides a partial enrichment of bacterial DNA, thereby improving detection sensitivity65. Tissues were ground in liquid nitrogen and homogenized in 500 μL of pre-warmed CTAB buffer (2% CTAB, 100 mM Tris-HCl, pH 8.0, 20 mM EDTA, 1.4 M NaCl, 0.5% 2-mercaptoethanol, 1% PVP). After incubation at 65°C for 30 min, samples were extracted with an equal volume of chloroform:isoamyl alcohol (24:1). The aqueous phase was recovered, and nucleic acids were precipitated with 0.7 volumes of isopropanol, washed with 70% ethanol, and resuspended in 10 μL nuclease-free water. DNA concentration and purity were determined spectrophotometrically, and all samples were adjusted to 100 ng/μL for downstream analysis.

Quantitative PCR was performed using a SYBR Green-based system targeting the CLas 16S rDNA gene. The reaction mixture (20 μL) contained 1× SYBR Green Master Mix (GenStar, A304-10), 500 nM of each primer, and 2 μL of template DNA. Amplification was carried out on a QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems) under the following conditions: 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 45 s. Melting curve analysis was subsequently performed by increasing the temperature from 60 to 95°C at a rate of 0.5°C/s to verify amplification specificity. Each run included a standard curve generated from serial dilutions of a plasmid containing the target CLas 16S rDNA fragment and non-template controls to monitor contamination. Samples with Ct values ≤ 35 that exhibited a single specific melting peak were considered positive for CLas infection.

Three independent biological replicates were performed to ensure reproducibility. The survival rate of microinjected psyllids was >85% across all treatment groups at the conclusion of the 7-day inoculation period.

Protein dimerization and activation assays
Sf9 cells expressing His-Barrel along with Flag-Toll8 or Flag-Toll8 mutants for 48 h were lysed using NP-40 buffer containing 1 μM protease inhibitors cocktail and analyzed by 10% native PAGE or SDS-PAGE, followed by immunoblotting with the specified antibodies (1: 2000 dilution). Similarly, Sf9 cells expressing Myc-MyD88 together with His-Barrel or Flag-Toll8 for 48 h were analyzed by native PAGE and SDS-PAGE, followed by immunoblotting.

To identify critical interaction residues in MyD88 and IKKE, Sf9 cells expressing Myc-MyD88 (wild-type and mutants), HA-IKKE (wild-type and mutants), or Flag-Toll8 for 48 h were analyzed by native PAGE for MyD88 dimerization and SDS-PAGE for IKKE phosphorylation and protein expression levels.

NFAT phosphorylation analysis
For phosphorylation site identification, Flag-NFAT expressing Sf9 cells were lysed at 48 hpi, immunoprecipitated with anti-Flag magnetic beads (Beyotime, P2181), and separated by SDS-PAGE. A major band of approximately 135 kDa was excised, digested with trypsin, and analyzed by phosphorylation mass spectrometry at APTBIO (Shanghai, China). Potential target peptides were identified and analyzed using Mascot (matrixscience.com) and assembled using Geneious 9.0.2.

To determine the significance of S167 in NFAT phosphorylation, point mutations were introduced within the amino acid region 167–180. Mutant NFAT m1 retained Ser167, while NFAT m2 underwent complete mutation. Sf9 cells expressing HA-IKKE with NFAT WT, NFAT m1, or NFAT m2 were analyzed by Phos-tag SDS-PAGE.

Chromatin immunoprecipitation sequencing and qPCR analysis
Chromatin from 100 CLas-free psyllids was crosslinked with 1% formaldehyde, sonicated to 200–500 bp fragments, and immunoprecipitated with an NFAT antibody (1:100 dilution) or control rabbit IgG (1:100 dilution, Beyotime, A7016) using a SimpleChIP Plus Kit (Cell Signaling, 9005)66. Libraries were sequenced on an Illumina NovaSeq 6000 (2×150 bp) by YankeGene (Guangdong, China). Reads were aligned to the D. citri genome (GCA_000475195.1) using Bowtie2, and peaks were called with MACS2 (q < 0.05). Reeler and NOS promoter enrichment was quantified by qPCR and analyzed using Student’s t-test (three biological replicates).

Yeast one-hybrid assay
The 5’ regulatory regions of Reeler and NOS genes containing putative NFAT binding sites (NFATBS) were cloned into pHis2 vector. NFAT cDNA was cloned into pGADT7 vector. Yeast strain Y187 was co-transformed with pGADT7-NFAT and pHis2-Reeler or pHis2-NOS, with appropriate controls. Transformants were selected on -His/Leu/Trp media containing 180 mM 3-Amino-1,2,4-triazole (3-AT; Coolaber, CA1311) at 30°C (three replicates). Images were captured 4 days after incubation.

Electrophoretic mobility shift assay
Flag-NFAT protein was expressed in Sf9 cells, immunoprecipitated using anti-Flag magnetic beads (Beyotime, P2181S), and eluted with 3× Flag peptide. Cy5-labeled probes (Reeler, 5′-CGGGTTTTCC-3′ or NOS, 5′-CGGCCAAACCCC-3′) and unlabeled/mutant probes (Reeler mut, 5′-CAAACCCCCC-3′ or NOS mut, TGGCCGGGTTTT-3′) were synthesized by Tsingke Technology. EMSA reactions containing 20 nmol probe, 100 μg Flag-NFAT, and 10 nmol Poly(dI-dC) were performed in EMSA buffer (10 mM Tris, pH 7.5, 0.25 mM DTT, 5 mM MgCl2, 10 mM KCl) at 4°C for 30 min. Competitive EMSA was performed by adding 20, 50, 100, and 200 nmol of unlabeled oligos 20 min prior to the addition of labeled oligos. Reactions were resolved on 5% non-denaturing PAGE gels and imaged on an Odyssey CLx Imaging System (LI-COR).

Dual-luciferase reporter assay
The ORF of the NFAT was cloned into the pcDNA3.1 vector (pcDNA-NFAT). The promoter regions of Reeler and NOS were cloned into the pGL3-Basic vector (pGL3-Reeler and pGL3-NOS). For the dual-luciferase reporter assay, HEK-293T cells were transfected with 1000 ng of reporter gene plasmids (pGL3-vector, pGL3-Reeler, or pGL3-NOS), 500 ng of pRL-TK Renilla luciferase plasmid as an internal control, and either 0, 500, or 2000 ng of pcDNA-NFAT expression plasmids using the Lipo8000 kit (Beyotime, C0533). Firefly and Renilla luciferase activities were measured 48 h post-transfection using the Dual Luciferase Reporter Assay Kit (YEASEN, 11402ES60). Each experiment was performed in triplicate.

Reeler antibacterial activity in vitro
X. citri and S. fredii were cultured in LB liquid medium at 28°C until the optical density (OD) reached 0.6. Purified Reeler protein, expressed in the E. coli Rosseta strain at a concentration of 0.5 mg/mL, was co-incubated with these two bacterial strains at room temperature. The total volume of the co-incubation mixture was 100 μL, with the induced expression product from the empty pET-28a vector serving as a control. Following the instructions of the LIVE/DEAD Bacterial Staining Kit with DMAO and PI (Beyotime, C2030S), the co-incubation products were stained. The stained bacterial suspensions were observed under an immunofluorescence microscope. Live bacteria with intact cell membranes exhibited green fluorescence, while dead bacteria with damaged cell membranes displayed both green and red fluorescence.

Protein degradation assay
The ORFs of MyD88 or UBR5 were constructed into a pcDNA3.1 plasmid. HEK-293T cells were transfected with 1000 ng of Flag-MyD88 plasmids, and either 0, 500, 1000, or 1500 ng of His-UBR5 expression plasmids using the Lipo8000 transfection kit. Total cellular proteins were extracted using RIPA lysis buffer (Beyotime, P0013) for western blot analysis.

Co-Immunoprecipitation assay
To investigate whether MyD88 undergoes ubiquitination modification in psyllids, adults were injected with 10 μM MG-132. At 6 h post-injection, total cellular proteins were extracted using NP-40 lysis buffer and incubated with magnetic beads (Beyotime, P2181) conjugated with MyD88 antibody (1:100 dilution) for 6 h at 4°C. Rabbit IgG magnetic beads (Beyotime, P2173) served as the control. Ubiquitination modification of MyD88 was detected by western blot using an Ub antibody (1:1000 dilution).

Sf9 cells expressing Flag-MyD88, HA-Ub, Myc-UBR5, or His-SDE3230 were treated with 10 μM MG-132 and lysed in NP-40 lysis buffer at 48 hpi. Cell lysates were prepared, and the substrate protein Flag-MyD88 was immunoprecipitated with anti-Flag beads (Beyotime, P2115). Ubiquitin-modified proteins were subsequently detected using western blot assays with specific antibodies (1:1000 dilution).

The UbPred software package (www.ubpred.org) was used to predict potential ubiquitination sites on the MyD88 protein. Three lysine residues were identified as potential ubiquitination sites: K48 within the death domain (DD), K201 within the Toll/interleukin-1 receptor (TIR) domain, and K301 at the C-terminus. Site-directed mutagenesis was performed to substitute K48, K201, and K301 with arginine, generating the MyD88 K48R, MyD88 K201R, and MyD88 K301R mutant proteins for ubiquitination analysis. For ubiquitination assays, Sf9 cells were co-transfected to express Flag-MyD88 (wild-type or mutants), Myc-UBR5, and HA-Ub. Cells were treated with 10 μM MG-132 for 6 h prior to harvest at 48 hpi. Cell lysates were prepared using NP-40 lysis buffer, and Flag-tagged proteins were immunoprecipitated with anti-Flag magnetic beads. Ubiquitin-modified MyD88 was detected by western blot analysis using anti-HA and anti-Ub K48 antibodies (1:1000 dilution).

In vitro ubiquitylation assay
To assess the effect of UBR5 on the ubiquitination modification of MyD88 or SDE3230 in vitro, purified 0.5 μg Ub (Aladdin, 79586-22-4), 1 μg E1 (UBA1), 2 μg E2 (UBE2D3), 5 μg HA-UBR5, 5 μg Flag-MyD88 or 5 μg Flag-SDE3230 were incubated in reaction buffer (1 M Tris, pH 7.5, 100 mM ATP, 200 mM MgCl2 and 40 mM DTT, 20×) at 30°C for 2 h. Reactions were terminated with SDS loading buffer and analyzed by Western blot.

Gene identification and phylogenetic analyses
Genes in D. citri were identified based on genome (GCA_000475195.1) and confirmed using conserved domain analysis with the NCBI Conserved Domain Database (CCD; https://www.ncbi.nlm.nih.gov/Structure/cdd). Sequences used in the phylogenetic analyses of Toll, NFAT, and IKKE are provided in Supplementary Table 4. Phylogenetic analysis and the number of PGRP and GNBP genes in D. citri were referenced from previously research57,67. Sequence alignments were generated using MAFFT with default settings. Phylogenetic trees were constructed using IQ-TREE (http://iqtree.cibiv.univie.ac.at) with default parameters and visualized using iTOL (https://itol.embl.de).

Alignment of protein 3D structures
The 3D structure of TLR2 was obtained from PDB database (https://www.rcsb.org/). The 3D structures of Toll1, Toll6, Toll7, and Toll8 were predicted using the Alphafold3 server (Google DeepMind). Structural alignments were performed using PyMOL, and the root-mean-square deviation (RMSD) values for pairwise comparisons were calculated and presented in a matrix.

Statistical data analysis
All graphs and statistical analyses were created using the GraphPad Prism version 8.0.1. Data were analyzed using a two-tailed unpaired Student’s t-test and expressed as means ± SEM. The significance level was set at P < 0.05.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript investigated how candidatus liberibacter asiaticus (CLas), the causative agent of Huanglongbing (HLB) disease in citrus manipulates immunity responses induced while transmitted by its psyllid vector, the asian citrus psyllid (ACP). The responses start by the psyllid Toll8 binding to Barrel on the bacteria membrane. The binding leads to activation of Toll8 leading to dimerization of MyD88 which activates IRD5 via phosphorylation. IRD5 interacts the psyllid transcription factor NFAT that regulates the expression of the antimicrobial peptides Reeler and nitric oxide synthase against CLas. In order to overcome these responses CLas secretes the effector SDE3230 that hijacks E3 ligase which mediates degradation of MyD88 via ubiquitination, leading to the suppression of the Toll8 immune response.\nI have several comments regarding the results presented in this manuscript:\n1. The authors decided to focus their study on Toll8 among the four toll genes found in D. citri genome. This makes some sense although I would have expected the authors to also make some experiments with the other Toll genes they have identified as their important function relies on specific interaction with CLas BARREL rather than increased expression which is not necessarily the best approach to verify their role.\n2. the authors have identified the bacterial Barrel as interacting with Toll, however, they did not elaborate on other interacting proteins that they have possibly identified in the screen. I would mention those, as other outer membrane bacterial proteins are also known for this and other unculturable bacterial species.\n3. in figure 1e, the CLas+ image shows localization of Barrel on the bacterial membrane. This is not fully convincing as some gold particles are shown in other locations. Some statistics needs to be shown here to prove whether this is significant. In the control CLas- image, the whole bacterial cells are not shown as in the CLas+ samples. I wonder why!\n4. I wonder whether other factors that determine Toll8 activation are considered rather than its dimerization, as this is the single most important factor that has been considered. In the literature it is mentioned that such receptors can also dimerize and undergo conformational change without the binding to a ligand, suggesting that this single factor is not enough to consider activation.\n5. How MyD88 was chosen for further downstream analysis? Although it is known in Toll8 signalling, other proteins are also potential candidates, and i would expect to provide a rationale for choosing this specific candidates rather than others. Are there other factors that directly interact with Toll8? According to the literature, there are. Why they were not considered? Are they found in the insect genome?\n6. to screen for proteins recruited by MyD88, Y2H was used to screen a cDNA library and the kinase IRD5 was identified. Again, the authors did not mention which other interacting proteins were identified and whether some were further investigated, and why this specific one was used.\n7. In the silencing experiments of IRD5 its expression and phophorylation was measured and the CLas titers were also measured, however, transmission experiments were not conducted. Transmission experiments are probably the most important link to show the relevance of the identified candidate proteins in the transmission of CLas and should be conducted.\n8. the authors have conducted experiments with adults. Nymphs are known to be better vector and to me the whole cascade of proteins that was identified should also be studied in nymphs and the transmission experiments also conducted with nymphs.\n9. Agains, in searching for interacting proteins with IRD5, NFAT was identified, however, other interactors were not mentioned. Why choose NFAT and not other candidates. Those interactors should be mentioned and discussed.\n10 transmission experiments should also have been conducted after silencing Reeler and NOS, as both increased CLas titers after silencing, thus, it is expected that they influence the transmission compared to a control. As in my previous comments, since nymphs are better vector than adults, those experiments, including the fluorescent localization in guts should also have been conducted in nymphs as well.", "reviewer_2": "This study focuses on the identification of ClBarrel, an outer membrane protein from Candidatus Liberibacter (Cl), the bacterium responsible for Huanglongbing (HLB), a devastating citrus disease. Cl is transmitted to plants through insects, particularly psyllids such as Diaphorina citri, which deliver the pathogen into the plant phloem. The bacterium disrupts assimilate transport, leading to wilting in the host plant. However, less is understood about how the vector insects themselves recognize bacterial surface patterns. While extensive research has explored insect innate immune activation, particularly in Drosophila, the mechanisms by which vector insects respond to pathogens are still poorly understood.\nIn this study, the authors present a rare example of direct recognition between ClBarrel and the ectodomain of a newly identified Toll-like receptor (TLR), TLR8, from D. citri. This discovery demonstrates that insects can activate innate immune responses to plant pathogens that they transmit, facilitating the pathogen's journey into host plants. Upon ligand binding, TLR8 undergoes dimerization and recruits the co-receptor MyD88, initiating a signaling cascade. This leads to the activation of IκB kinase IRD5, which in turn activates the transcription factor NFAT. This signaling pathway mirrors canonical innate immune responses, and in this study, the authors identify the D. citri homologs analogous to those found in Drosophila.\nThe most compelling part of this research lies in the second key finding: the bacterial effector SDE3230. This effector is produced by Cl and functions to suppress the insect immune response. It does so by ubiquitinating and degrading MyD88 through the proteasome, preventing the activation of immune responses that could otherwise harm the pathogen. This discovery highlights an intricate interplay between immune activation and suppression as the pathogen moves from the insect vector to the plant.\nAs a plant immunologist, I am particularly excited by the extensive data supporting the proposed model and the quality of the research. While I am less familiar with the literature in insect innate immunity, based on standards in our field, this study represents an outstanding contribution to the scientific community. I have no significant critiques but would suggest simplifying the writing slightly to improve accessibility for readers less familiar with the topic.", "reviewer_3": "Comments:\nThe authors have studied a novel PRR Toll8 in D. citri in the context of immune defence against the gram-negative bacterium Candidatus Liberibacter asiaticus. I think the authors show very thoroughly that the D. citri Toll8 binds the Clas membrane protein Barrel. The Toll8 mediated signaling cascade leads to activation of NFAT transcription factor that then regulates the expression of antimicrobial effectors. I think that the work has been carried out well and the article has also been written well. I have only some comments, and I recommend publication after the revision.\nAbout the nomenclature:\n-I am not sure about the numbering in the proposed renaming strategy (TLR8; line 276). Is the current numbering referring to the homology to the Toll receptor 8 in Drosophila? And if it is changed to TLR, then the numbering may be misleading to homology to mammalian TLR8? I agree about the logic of naming the gene TLR, I am just not sure about the numbering.\n-IRD5: Is the logic of the nomenclature of this gene also to name it with the Drosophila name? However, the Drosophila gene is not marked in the phylogenetic tree; is the Drosophila gene then very distantly related?\nOther comments:\nThe NFAT staining in Figs 3f and g looks very different – the staining in f is very dotty-like, whereas in g it is diffusely in the cytoplasm and then in the nucleus. I understand that the cells are different, and also one has a tag and one is the native protein stained with an ab, but you should comment and explain (or at least discuss) why that is. Also there is only one cell showing the NFAT nuclear accumulation at 36h (3g), so the evidence is not very strong that NFAT goes into the nucleus (although it is definitely like that in the one cell). Consider quantifying this with more cells.\nFig 6, Myd88 ubiquitination: are there any other Lysines that maybe potential ubiquitination sites in the Myd88 protein? Is it excluded that Lysines in other domains also would play a role in the K48-ubiquitination process (leading to proteasomal degradation)?\nMinor comments:\n-Introduction text: The very last sentence of the section (line 90-91) should not be there.\n-Fig 1i: Make the text lines and + and - signs to be aligned better\nline 125: psyllids Toll8 -> psyllid Toll8\nline 150: pyllid -> psyllid\nFig 3g: on the side it reads “sigle expression” -> should be “single expression”\nFig 4 d and e, in the Figure itself: DNA-protin complex -> should be DNA-protein complex\nFig S3b: Explain the numbers and the scale\nFig S7b+c – explain abbreviations in the figure (labelling): in particular DMAO should be spelled out, if not here, in the abbreviations list. Also the DIC images are quite dark, maybe their brightness could be adjusted or something (every image the same way!).\nline 221: indentify -> identify\nline 224: psyllds -> psyllids\nline 236: accumullation -> accumulation\nline 270: beta-barre -> beta-barrel"}, "author_response": "Point-to-point responses to reviewer comments\nWe sincerely thank the reviewers for their constructive feedback, which has\nsignificantly strengthened our manuscript. We responses immediately follow each\ncomment, with revised line numbers corresponding to the tracked-changes version of\nthe manuscript. Critical revisions in the updated manuscript are highlighted in red font\nfor easy identification.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThis manuscript investigated how Candidatus liberibacter asiaticus (CLas), the\ncausative agent of Huanglongbing (HLB) disease in citrus manipulates immunity\nresponses induced while transmitted by its psyllid vector, the asian citrus psyllid\n(ACP). The responses start by the psyllid Toll8 binding to Barrel on the bacteria\nmembrane. The binding leads to activation of Toll8 leading to dimerization of MyD88\nwhich activates IRD5 via phosphorylation. IRD5 interacts the psyllid transcription\nfactor NFAT that regulates the expression of the antimicrobial peptides Reeler and\nnitric oxide synthase against CLas. In order to overcome these responses CLas\nsecretes the effector SDE3230 that hijacks E3 ligase which mediates degradation of\nMyD88 via ubiquitination, leading to the suppression of the Toll8 immune response.\nResponse: We sincerely appreciate the reviewer's comprehensive summary of our\nwork and your thoughtful questions regarding our experimental approach. These\nmodifications have significantly improved the manuscript's rigor and clarity. We are\ndeeply grateful for the reviewer's expertise, which has helped us strengthen both the\nexperimental evidence and its presentation. Detailed point-by-point responses follow\nbelow.\nI have several comments regarding the results presented in this manuscript:\n1. The authors decided to focus their study on Toll8 among the four toll genes found\nin D. citri genome. This makes some sense although I would have expected the\nauthors to also make some experiments with the other Toll genes they have identified\nas their important function relies on specific interaction with CLas BARREL rather\nthan increased expression which is not necessarily the best approach to verify their\nrole.\nResponse: We sincerely appreciate this constructive suggestion. In the revised version,\nwe added the interaction of Barrel with Toll1, Toll6 and Toll7 using yeast two-hybrid\nassays, and no interaction was identified (revised Figure S3e). In contrast, Barrel\nspecifically interacts with Toll8’s LRR domain (revised Figure S3d). While Toll1,\nToll6, and Toll8 each possess anti-CLas activity, Toll8 exhibits significantly superior\nefficacy (revised Figure 1c, Figure S2b-e). Combined with Toll8’s structural\nhomology to bacterial lipoprotein-recognizing TLR2 (revised Figure S3a-b), it is\nsuggested its unique role in direct Barrel recognition, while other Tolls likely function\nthrough alternative activation pathways during CLas infection. These findings have\nbeen systematically incorporated in the Results section (Lines 111-116, 122-123) and\nFigure S2 and Figure S3 of our revised manuscript.\n2. The authors have identified the bacterial Barrel as interacting with Toll, however,\nthey did not elaborate on other interacting proteins that they have possibly identified\nin the screen. I would mention those, as other outer membrane bacterial proteins are\nalso known for this and other unculturable bacterial species.\nResponse: We appreciate the reviewer’s insightful suggestion regarding potential\ninteracting proteins. In the revised version, yeast two-hybrid assays were performed to\nsystematically screen 19 CLas membrane proteins using, including those conserved in\nother non-culturable bacterial species such as Candidatus Liberibacter solanacearum\n(CLso). Our results revealed that Toll8 specifically interacts with Barrel among all\ntested membrane proteins. The screening methodology is detailed in the revised\nResults (Lines 119-120), with corresponding data in Figure S3c.\n3. In figure 1e, the CLas+ image shows localization of Barrel on the bacterial\nmembrane. This is not fully convincing as some gold particles are shown in other\nlocations. Some statistics needs to be shown here to prove whether this is significant.\nIn the control CLas- image, the whole bacterial cells are not shown as in the CLas+\nsamples. I wonder why!\nResponse: We sincerely appreciate the reviewer's careful examination of our\nimmunogold labeling results. In response to these concerns, we have repeated the\nimmunogold labeling experiments with improved sample processing and obtained\nnew micrographs that more clearly demonstrate Barrel's localization on CLas\nbacterial membranes. The updated figure 1e now shows definitive membrane-specific\nlabeling in CLas+ samples, with gold particles specifically localized at bacterial\nmembranes (red arrows). In CLas- control samples, no bacterial cells were observed\nand only minimal background labeling was detected. These new results provide\nunambiguous visual evidence of Barrel's membrane association without requiring\nadditional statistical validation. We have updated both the figure 1e and its legend\naccordingly.\n4. I wonder whether other factors that determine Toll8 activation are considered rather\nthan its dimerization, as this is the single most important factor that has been\nconsidered. In the literature it is mentioned that such receptors can also dimerize and\nundergo conformational change without the binding to a ligand, suggesting that this\nsingle factor is not enough to consider activation.\nResponse: We appreciate this insightful comment regarding Toll receptor activation\nmechanisms. Indeed, as you rightly point out, Toll-family receptors can undergo\ndimerization and conformational changes independent of ligand binding, as\nexemplified by the developmental role of Drosophila Toll in dorsoventral patterning\n(independent of immune function) (Roth, 2023). Similar ligand-independent\nactivation has been observed for mammalian TLRs in developmental contexts (e.g.,\nTLR/NF-κB in tissue morphogenesis) (Brennan et al., 2018, Cao et al., 2018). In our\nstudy, we specifically focused on ligand-dependent activation of Toll8 by CLas\nmembrane protein Barrel. In the revised version, we have incorporated these\nexplanations to the Discussion section (Lines 304-307).\nWhile spontaneous dimerization may occur under other physiological conditions,\nour findings establish that CLas Barrel-induced Toll8 dimerization is functionally\nsignificant for immune activation. This ligand-induced Toll8 dimerization correlated\nwith increased adaptor MyD88 dimerization and further induced kinase IKKE\n(previously termed IRD5, see response to Reviewer 3) and transcription factor NFAT\nphosphorylation, finally inducing downstream immune effectors (Reeler and NOS)\nexpression. In the revised version, we have clarified these information in the\nDiscussion (Lines 304-312) to show how Barrel-mediated activation represents a\ndedicated pathogen response pathway, separate from the receptor’s other potential\nactivation states. This distinction underscores Toll8’s dual functionality while\nhighlighting its specialized role in antibacterial immunity.\nReferences:\nRoth S. Neofunctionalization of Toll Signaling in Insects: From Immunity to\nDorsoventral Patterning. Annu Rev Cell Dev Biol. 2023, 39:1-22. doi:\n10.1146/annurev-cellbio-120319-120223. PMID: 37843930.\nBrennan JJ, Gilmore TD. Evolutionary Origins of Toll-like Receptor Signaling. Mol\nBiol Evol. 2018, 35(7):1576-1587. doi: 10.1093/molbev/msy050. PMID:\n29590394.\nCao M, Chen F, Xie N, Cao MY, Chen P, Lou Q, Zhao Y, He C, Zhang S, Song X,\nSun Y, Zhu W, Mou L, Luan S, Gao H. c-Jun N-terminal kinases differentially\nregulate TNF- and TLRs-mediated necroptosis through their kinase-dependent\nand -independent activities. Cell Death Dis. 2018, 9(12):1140. doi:\n10.1038/s41419-018-1189-2. PMID: 30442927; PMCID: PMC6238001.\n5. How MyD88 was chosen for further downstream analysis? Although it is known in\nToll8 signalling, other proteins are also potential candidates, and i would expect to\nprovide a rationale for choosing this specific candidates rather than others. Are there\nother factors that directly interact with Toll8? According to the literature, there are.\nWhy they were not considered? Are they found in the insect genome?\nResponse: Thank you for your valuable comments. We focused on MyD88 in our\nanalysis of Toll8 signaling based on its evolutionarily conserved and indispensable\nrole in Toll/TLR pathways. MyD88 serves as the universal adaptor that connects Toll\nreceptors to downstream kinases through TIR domain dimerization — a mechanism\nconserved from insects to mammals. Importantly, insect genomes (e.g., Diptera and\nHemiptera) lack vertebrate-specific adaptors like TIR domain containing adaptor\nmolecule 1 (TRIF), making MyD88 the essential adaptor for insect Toll signaling\npathways. Functionally, MyD88 is crucial for immune responses across species,\nmediating pathogen defense by directly engaging Toll receptors and initiating immune\nsignaling cascades. These well-documented evolutionary and mechanistic features\nsolidly support our selection of MyD88 for downstream analysis. In the revised\nversion, we have strengthened the discussion of MyD88’s importance, and please\nrefer to Lines 326-331 of the Discussion section for details.\n6. To screen for proteins recruited by MyD88, Y2H was used to screen a cDNA\nlibrary and the kinase IRD5 was identified. Again, the authors did not mention which\nother interacting proteins were identified and whether some were further investigated,\nand why this specific one was used.\nResponse: We thank the reviewer for this important question regarding\nMyD88-interacting proteins. As the universal adaptor in metazoans, MyD88 bridges\nToll receptors and downstream kinases via its TIR domain. Through comprehensive\nscreening of potential MyD88 interactors, we specifically identified IKKE (previously\ntermed IRD5) as the key interacting kinase. Although our screening did not\nencompass additional kinases, the robust interaction between MyD88 and IKKE\nstrongly supports its critical function in the downstream signaling cascade. In direct\nresponse to this valuable suggestion, we have expanded the presentation of our\nprotein interaction screening data in the revised manuscript. These additional details\ncan be found in Table S1 and Results section (Lines 156-160).\nTable S1. Screening of MyD88 for interaction target protein replicates by Y2H.\nProtein name Function NCBI number\nIKKE Protein kinase XP_008475603\nUBR5 Ubiquitin ligase activity XP_008484153\nHsp70 heat shock protein 70 XP_008477020\nToll8 Toll receptor KAI5731822\nNrx Nervous system development XP_008476955\nMTCH2 Mitochondrial carrier homolog 1 XP_026678432\n7. In the silencing experiments of IRD5 its expression and phophorylation was\nmeasured and the CLas titers were also measured, however, transmission experiments\nwere not conducted. Transmission experiments are probably the most important link\nto show the relevance of the identified candidate proteins in the transmission of CLas\nand should be conducted.\nResponse: We sincerely appreciate the reviewer's insightful suggestion regarding\ntransmission experiments. In natural conditions, adult D. citri serve as the primary\nvectors for CLas transmission. While nymphs efficiently acquire CLas through\nprolonged phloem feeding, their limited mobility restricts transmission. Previous\npublished data showed that adult psyllids transmited CLas to susceptible host plants\nmore efficiently than nymphs (Ammar et al., 2020). Thus, we conducted transmission\nassays using adult psyllids that had been reared for multiple generations on\nCLas-infected sweet orange (Citrus sinensis) plants. Our results clearly demonstrate\nthat targeted silencing of IKKE expression through dsRNA treatment in CLas-infected\npsyllid adults significantly increases CLas transmission efficiency. These findings\nestablish IKKE as playing a key role in the anti-CLas immunity. In the revised version,\nthe complete methodological details and experimental results are presented in the\nResults section (Lines 167-170), Materials and Methods (Lines 703-719), and Figure\nS4i.\nReference\nAmmar ED, George J, Sturgeon K, Stelinski LL, Shatters RG. Asian citrus psyllid\nadults inoculate huanglongbing bacterium more efficiently than nymphs when\nthis bacterium is acquired by early instar nymphs. Sci Rep. 2020, 10(1):18244.\ndoi: 10.1038/s41598-020-75249-5.\n8. The authors have conducted experiments with adults. Nymphs are known to be\nbetter vector and to me the whole cascade of proteins that was identified should also\nbe studied in nymphs and the transmission experiments also conducted with nymphs.\nResponse: We sincerely appreciate the reviewer's thoughtful suggestion regarding\nnymph-stage experiments. Generally, adult psyllids transmit CLas to susceptible host\nplants more efficiently than nymphs, whereas nymphs acquire CLas from infected\nhost plants more effectively than adults (Ammar et al., 2020, George et al., 2018,\nPelz-Stelinski et al., 2010). Thus, we characterized the\nToll8-MyD88-IKKE-NFAT-Reeler/NOS cascade in adults because it regulates\nimmune responses during the actual transmission phase. Our transmission assays\n(Response #7) demonstrated that reducing expression of pathway components like\nIKKE significantly increases CLas transmission by adults. We have added expanded\ndiscussion of these transmission dynamics background in the Introduction (Lines\n83-85) to better explain why we focused on adult psyllids in this experimental system.\nReferences\nAmmar ED, George J, Sturgeon K, Stelinski LL, Shatters RG. Asian citrus psyllid\nadults inoculate huanglongbing bacterium more efficiently than nymphs when\nthis bacterium is acquired by early instar nymphs. Sci Rep. 2020, 10(1):18244.\ndoi: 10.1038/s41598-020-75249-5.\nGeorge J, Ammar ED, Hall DG, Shatters RG Jr, Lapointe SL. Prolonged phloem\ningestion by Diaphorina citri nymphs compared to adults is correlated with\nincreased acquisition of citrus greening pathogen. Sci Rep. 2018, 8(1):10352. doi:\n10.1038/s41598-018-28442-6.\nPelz-Stelinski KS, Brlansky RH, Ebert TA, Rogers ME. Transmission parameters for\nCandidatus liberibacter asiaticus by Asian citrus psyllid (Hemiptera: Psyllidae). J\nEcon Entomol. 2010, 103(5):1531-41.\n9. Agains, in searching for interacting proteins with IRD5, NFAT was identified,\nhowever, other interactors were not mentioned. Why choose NFAT and not other\ncandidates. Those interactors should be mentioned and discussed.\nResponse: We thank the reviewer for raising this important question about IKKE\n(previously termed IRD5)-interacting proteins. As the homolog of mammalian IKK\nkinase (a well-characterized regulator of NF-κB signaling through its phosphorylation\nactivity), IKKE was predicted to function similarly in immune regulation. Our\ncomprehensive screening of IKKE interactors specifically identified NFAT as the key\ninteracting transcription factor, which shares the conserved RHD domain with NF-κB.\nAmong all potential interactors identified through systematic yeast two-hybrid\nscreening, NFAT was the only transcription factor showing this interaction. While\nother interactors may exist, our experimental evidence strongly supports NFAT's\ncritical role in mediating the anti-CLas activity of these transcriptionally regulated\nimmune factors. In response to this suggestion, we have expanded the presentation of\nIKKE interaction data in the revised manuscript. These additional details can be found\nin Table S2 and Results section (Lines 188-193), We also discussed other interacting\nproteins in Lines 323-338 of the Discussion section.\nTable S2. Screening of IKKE for interaction target protein replicates by Y2H.\nProtein name Function NCBI number\nNFAT Transcription factor XP_026684128\nPellino Ubiquitin ligase activity XP_026676035\nTMEM214 Transmembrane protein 214 XP_026679746\nTRAF6 Ubiquitin ligase activity XP_017300028\nSec16A Protein transport protein XP_008485263\nDNAJC3 Misfolded protein binding XP_026687388\n10. Transmission experiments should also have been conducted after silencing Reeler\nand NOS, as both increased CLas titers after silencing, thus, it is expected that they\ninfluence the transmission compared to a control. As in my previous comments, since\nnymphs are better vector than adults, those experiments, including the fluorescent\nlocalization in guts should also have been conducted in nymphs as well.\nResponse: We sincerely appreciate the reviewer's consistent and valuable suggestions\nregarding transmission experiments. In direct response to these comments, we\nconducted transmission assays to functionally validate the roles of Reeler and NOS in\nCLas transmission. Using our well-established adult psyllid system (reared for\nmultiple generations on CLas-infected sweet orange plants), we observed that\ndsRNA-mediated silencing of either Reeler or NOS significantly enhanced CLas\ntransmission efficiency in adult insects. These results not only confirm the anti-CLas\nfunction of these immune effectors but also provide compelling evidence for their\ninvolvement in regulating pathogen spread through the\nToll8-MyD88-IRD5-NFAT-Reeler/NOS signaling pathway.\nWhile we acknowledge the reviewer's interest in nymph-stage experiments, our\nadult-based transmission system has proven highly effective for evaluating these\nmolecular mechanisms, as evidenced by the clear and consistent results obtained. The\ncomplete experimental details, including methodology and results, are presented in\nthe Results section (Lines 245-250), Materials and Methods (Lines 703-719), and\nFigure 5k. These additional experiments have significantly strengthened our\nunderstanding of how this immune pathway modulates CLas transmission at the\nmolecular level.\nReviewer #2 (Remarks to the Author):\nThis study focuses on the identification of ClBarrel, an outer membrane protein from\nCandidatus Liberibacter (Cl), the bacterium responsible for Huanglongbing (HLB), a\ndevastating citrus disease. Cl is transmitted to plants through insects, particularly\npsyllids such as Diaphorina citri, which deliver the pathogen into the plant phloem.\nThe bacterium disrupts assimilate transport, leading to wilting in the host plant.\nHowever, less is understood about how the vector insects themselves recognize\nbacterial surface patterns. While extensive research has explored insect innate\nimmune activation, particularly in Drosophila, the mechanisms by which vector\ninsects respond to pathogens are still poorly understood.\nIn this study, the authors present a rare example of direct recognition between\nClBarrel and the ectodomain of a newly identified Toll-like receptor (TLR), TLR8,\nfrom D. citri. This discovery demonstrates that insects can activate innate immune\nresponses to plant pathogens that they transmit, facilitating the pathogen's journey\ninto host plants. Upon ligand binding, TLR8 undergoes dimerization and recruits the\nco-receptor MyD88, initiating a signaling cascade. This leads to the activation of IκB\nkinase IRD5, which in turn activates the transcription factor NFAT. This signaling\npathway mirrors canonical innate immune responses, and in this study, the authors\nidentify the D. citri homologs analogous to those found in Drosophila.\nThe most compelling part of this research lies in the second key finding: the bacterial\neffector SDE3230. This effector is produced by Cl and functions to suppress the insect\nimmune response. It does so by ubiquitinating and degrading MyD88 through the\nproteasome, preventing the activation of immune responses that could otherwise harm\nthe pathogen. This discovery highlights an intricate interplay between immune\nactivation and suppression as the pathogen moves from the insect vector to the plant.\nAs a plant immunologist, I am particularly excited by the extensive data supporting\nthe proposed model and the quality of the research. While I am less familiar with the\nliterature in insect innate immunity, based on standards in our field, this study\nrepresents an outstanding contribution to the scientific community. I have no\nsignificant critiques but would suggest simplifying the writing slightly to improve\naccessibility for readers less familiar with the topic.\nResponse: We sincerely appreciate the reviewer's generous assessment of our work\nand your valuable suggestion regarding accessibility. As a plant immunologist, your\nperspective on broadening readership is particularly insightful. In response to your\ncomments, we have reorganized the Results section into three clearly defined parts\nfocusing on pathogen recognition, signal transduction, and immune suppression to\nimprove logical flow. Additionally, we have streamlined complex sentence structures\nand incorporated explanatory phrases for specialized terminology where appropriate.\nThese modifications were carefully implemented to enhance overall readability while\nmaintaining scientific precision, as can be seen throughout the revised manuscript,\nparticularly in the Results section. We are especially grateful for your encouraging\ncomments about the work's significance, which have helped us strengthen the\nmanuscript's clarity and accessibility.\nReviewer #3 (Remarks to the Author):\nComments:\nThe authors have studied a novel PRR Toll8 in D. citri in the context of immune\ndefence against the gram-negative bacterium Candidatus Liberibacter asiaticus. I\nthink the authors show very thoroughly that the D. citri Toll8 binds the CLas\nmembrane protein Barrel. The Toll8 mediated signaling cascade leads to activation of\nNFAT transcription factor that then regulates the expression of antimicrobial effectors.\nI think that the work has been carried out well and the article has also been written\nwell. I have only some comments, and I recommend publication after the revision.\nResponse: We are deeply grateful for the reviewer's thoughtful evaluation and positive\nassessment of our work. Your constructive comments have been invaluable in\nstrengthening our manuscript, and we have carefully addressed each suggestion in the\nrevised version. Thank you for recommending our manuscript for publication after\nrevision.\nAbout the nomenclature:\n-I am not sure about the numbering in the proposed renaming strategy (TLR8; line\n276). Is the current numbering referring to the homology to the Toll receptor 8 in\nDrosophila? And if it is changed to TLR, then the numbering may be misleading to\nhomology to mammalian TLR8? I agree about the logic of naming the gene TLR, I\nam just not sure about the numbering.\nResponse: We sincerely appreciate the reviewer's insightful comment regarding the\nTLR8 nomenclature issue. Our phylogenetic analysis confirms that psyllid Toll8\nclusters closely with Drosophila Toll8, while mammalian TLRs form a distinct\noutgroup. Furthermore, structural analysis reveals that psyllid Toll8 shares high\nsimilarity with mammalian TLR2. Given its direct PAMP recognition capability\n(representing the first such demonstrated function for an insect Toll receptor), we\npropose the term “Toll8-like receptor (TLR8)” to acknowledge both its Drosophila\northology and functional similarity to vertebrate TLRs. We greatly value the\nreviewer's careful attention to this important detail, which has helped improve the\naccuracy of our manuscript.\n-IRD5: Is the logic of the nomenclature of this gene also to name it with the\nDrosophila name? However, the Drosophila gene is not marked in the phylogenetic\ntree; is the Drosophila gene then very distantly related?\nResponse: We sincerely appreciate the reviewer's insightful question regarding IRD5\nnomenclature. In our revised analysis, we have included Drosophila homologs in the\nphylogenetic tree and confirmed that psyllid IRD5 clusters closely with Drosophila\nIKKepsilon (IKKE), while also being homologous to mammalian IKKepsilon. Given\nthis clear evolutionary relationship, we agree that adopting the Drosophila\nnomenclature (IKKE) would provide more accurate representation of this gene's\nidentity and function. We have implemented this change throughout the manuscript to\nmaintain consistency with established naming conventions. Thank you for this\nvaluable suggestion, which has significantly improved the precision of our gene\nannotation.\nOther comments:\nThe NFAT staining in Figs 3f and g looks very different – the staining in f is very\ndotty-like, whereas in g it is diffusely in the cytoplasm and then in the nucleus. I\nunderstand that the cells are different, and also one has a tag and one is the native\nprotein stained with an ab, but you should comment and explain (or at least discuss)\nwhy that is. Also there is only one cell showing the NFAT nuclear accumulation at\n36h (3g), so the evidence is not very strong that NFAT goes into the nucleus (although\nit is definitely like that in the one cell). Consider quantifying this with more cells.\nResponse: We sincerely appreciate the reviewer's insightful comments regarding the\nNFAT localization patterns in Figs 3f and g. The observed differences in staining\npatterns between psyllid midgut cells (Fig 3f) and Sf9 cells (Fig 3g) indeed reflect\ntheir distinct difference between endogenous protein (Fig 3f) detection and\nheterologous overexpression systems (Fig 3g), respectively. In response to the\nreviewer's valuable suggestion, we have replaced the original Fig 3g with new images\nshowing consistent nuclear accumulation of NFAT in multiple Sf9 cells at 36h under\nidentical experimental conditions, and have added corresponding explanations in the\nResults section (Lines 203-207) to help readers better interpret these findings. These\nmodifications have strengthened our evidence for NFAT nuclear translocation while\nclarifying the biological relevance of both experimental systems.\nFig 6, Myd88 ubiquitination: are there any other Lysines that maybe potential\nubiquitination sites in the Myd88 protein? Is it excluded that Lysines in other domains\nalso would play a role in the K48-ubiquitination process (leading to proteasomal\ndegradation)?\nResponse: We sincerely appreciate the reviewer's insightful question regarding\npotential ubiquitination sites in MyD88. Our ubiquitination site prediction analysis\nidentified three candidate lysines: K48 in the DD domain, K201 in the TIR domain,\nand K301 at the C-terminus. Through systematic mutagenesis analysis (now included\nin Fig 6h), we demonstrate that K48 serves as the primary ubiquitination site, as its\nmutation significantly attenuates K48-linked ubiquitination. While the other sites\n(K201 and K301) showed weaker ubiquitination signals, their contribution to\nproteasomal degradation appears minor compared to K48 (now included in Fig 6i).\nThese findings, detailed in Results (Lines 272-277) and Fig 6h-i, provide evidence for\nthe site-specific regulation of MyD88 stability. We greatly value this suggestion that\nstrengthened our mechanistic understanding.\nMinor comments:\n-Introduction text: The very last sentence of the section (line 90-91) should not be\nthere.\nResponse: We thank the reviewer for catching this oversight. The sentence in question\nhas been removed from the final paragraph of the Introduction (now Line 99) in our\nrevised manuscript.\n-Fig 1i: Make the text lines and + and - signs to be aligned better\nResponse: We appreciate the reviewer’s attention to detail. In the revised manuscript,\nwe have adjusted the alignment of text labels and \"+/-\" signs in Fig 1i to ensure clarity\nand visual consistency.\nline 125: psyllids Toll8 -> psyllid Toll8\nResponse: We thank the reviewer for catching this grammatical error. The correction\nfrom “psyllids Toll8” to “psyllid Toll8” has been made in now Line 139 of the revised\nmanuscript.\nline 150: pyllid -> psyllid\nResponse: We thank the reviewer for catching this word spelling error. The correction\nfrom “pyllid” to “psyllid” has been made in now Line 165 of the revised manuscript.\nFig 3g: on the side it reads “sigle expression” -> should be “single expression”\nResponse: We appreciate the reviewer's careful reading. The label in Fig 3g has been\ncorrected from “sigle expression” to “single expression” in the revised version.\nFig 4 d and e, in the Figure itself: DNA-protin complex -> should be DNA-protein\ncomplex\nResponse: We are grateful for this observation. The labeling in Figs 4d and 4e has\nbeen corrected from “DNA-protin complex” to “DNA-protein complex” in the revised\nmanuscript.\nFig S3b: Explain the numbers and the scale\nResponse: We thank the reviewer for this suggestion. In the revised supplementary\nfigure S3b, we have now added clear annotations explaining the numerical values to\nimprove interpretability. The figure legend has been expanded accordingly.\nFig S7b+c – explain abbreviations in the figure (labelling): in particular DMAO\nshould be spelled out, if not here, in the abbreviations list. Also the DIC images are\nquite dark, maybe their brightness could be adjusted or something (every image the\nsame way!).\nResponse: We appreciate these constructive suggestions. In the revised manuscript,\nWe have spelled out all abbreviations in the figure legend, including DMAO\n(N,N-dimethylaniline N-oxide) and PI (propidium iodide). We have uniformly\nadjusted the brightness/contrast of all DIC images for better visibility, while\nrigorously ensured consistent image processing across all panels.\nline 221: indentify -> identify\nResponse: We thank the reviewer for catching this typo. The correction from\n“indentify” to “identify” has been made in now Line 253.\nline 224: psyllds -> psyllids\nResponse: We are grateful for spotting this error. The misspelling “psyllds” has been\ncorrected to “psyllids” in now Line 256.\nline 236: accumullation -> accumulation\nResponse: We appreciate this careful reading. The word spelling error\n“accumullation” has been corrected to “accumulation” in now Line 269.\nline 270: beta-barre -> beta-barrel\nResponse: We thank the reviewer for noticing this. The incomplete term “beta-barre”\nhas been corrected to “beta-barrel” in now Line 315."}, {"round": 2, "reviewer_comments": {"reviewer_2": "The authors have addressed my concerns in an appropriate manner.", "reviewer_3": "Thank you, I am satisfied with the changed made by the authors and I recommend publication.", "reviewer_4": "The reviewers raised several concerns that the authors addressed with additional experiments, clarification, and revisions to the manuscript. Reviewer 1 provided the most extensive and technically detailed critique. One of the main questions centered on why only Toll8 was studied, rather than other members of the Toll family. In response, the authors conducted yeast two-hybrid assays for Toll1, Toll6, and Toll7, demonstrating that none of these interacted with Barrel. This additional work supported the specificity of the Toll8-Barrel interaction. Reviewer 1 also requested broader screening for potential CLas membrane proteins that might interact with Toll receptors. The authors expanded their assays to include nineteen CLas membrane proteins and confirmed that Barrel alone binds to Toll8.\nAnother larger issue concerned the immunogold localization shown in Figure 1e, which the reviewer found unclear and lacking proper controls. The authors repeated this experiment, providing new micrographs that clearly demonstrate membrane-specific labeling and including appropriate negative controls. Reviewer 1 further questioned the mechanism of Toll8 activation, suggesting that ligand-independent activation should be considered. The authors revised the Discussion to acknowledge this possibility but emphasized that their data support Barrel-induced activation as the biologically relevant mechanism. The reviewer also asked why only MyD88 was examined among potential adaptor proteins. In response, the authors justified their focus on MyD88 as the universal Toll adaptor in insects and expanded the discussion to address its evolutionary and functional significance.\nSeveral reviewers sought additional detail on downstream signaling components, including MyD88 and IKKE. The authors added two supplementary tables listing all identified interactors and clarified their decision to focus specifically on IKKE and NFAT as central regulators of Toll signaling. Reviewer 1 also requested transmission assays to evaluate the functional consequences of gene silencing. The authors performed new experiments showing that silencing of IKKE, Reeler, or NOS increased CLas transmission by adult psyllids, but I will address this in more detail below.\nReviewer 2 expressed support for the work, noting that the study was of high quality but could benefit from improved readability for a broader audience. The authors responded by reorganizing the Results section for clearer logical flow and simplifying complex sentences. Reviewer 3 requested several minor clarifications and corrections. These included questions regarding gene nomenclature, which the authors addressed by clarifying phylogenetic relationships and adopting the more accurate name IKKE in place of IRD5. Reviewer 3 also noted inconsistencies in NFAT localization, prompting the authors to replace the relevant figures with consistent and better-quality images. Concerns about potential alternative ubiquitination sites on MyD88 led the authors to perform additional mutagenesis experiments, confirming that lysine 48 is the principal site of modification.\nIn my view, the authors have successfully addressed the primary concerns raised by the reviewers through the inclusion of new experiments, more detailed methodological explanations, and targeted clarifications. They also improved figure quality, standardized terminology, and revised the manuscript to enhance clarity and readability. However, upon reviewing the revised version, one additional issue has come to my attention—an issue that was not present in the original draft but appears to have emerged during the revision made in response to Reviewer #1.\nIn the initial round of review, Reviewer #1 noted that although the authors had measured IRD5 (also referred to as IKKE) expression, phosphorylation, and CLas titers following gene silencing, they had not conducted transmission experiments to assess whether these molecular changes affected the psyllids’ capacity to transmit the bacterium to host plants. The reviewer emphasized that such transmission assays are essential, as they provide the most direct functional evidence linking the identified candidate proteins to CLas transmission and should therefore be included in the study.\nIn response, the authors agreed with the reviewer’s point and explained that they subsequently performed transmission assays using adult D. citri, which are the primary vectors of CLas under natural conditions. They noted that although nymphs efficiently acquire the bacterium, their limited mobility makes them less relevant to transmission dynamics. Citing previous studies showing that adults transmit CLas more effectively than nymphs, the authors justified focusing on the adult stage. They reported that, in these new experiments, silencing IKKE expression with dsRNA significantly increased CLas transmission efficiency by infected adult psyllids. This result, according to the authors, supports the conclusion that IKKE plays an important role in the psyllid’s immune defense against CLas. They also indicated that full methodological details and results have been incorporated into the revised manuscript in the Results, Materials and Methods, and supplementary figures.\nI commend the authors for addressing reviewer comments and for conducting additional inoculation assays. However, I remain skeptical that ‘standard’ cited methods, based on the citation of Ammar et al. (23), would have been sufficient to accomplish this experiment. In all published transmission assays to date involving CLas and D. citri, whole-plant inoculations are employed, and leaf tissue is typically analyzed at 1, 3, 6, and 9 months post-inoculation. Depending on the methods used and specific investigation, plants generally begin to show signs of CLas infection around 3 months after inoculation as detected by qPCR.\nSeveral laboratories have attempted to use single-leaflet assays, as described in this manuscript, to accelerate data collection; however, to my knowledge, such efforts have not been successful and/or previously published. The DNA yield from a single leaflet is generally insufficient to reliably detect CLas DNA just 7 days after a 7-day inoculation access period by CLas-infected adult psyllids when using “standard” extraction and qPCR methods such as those described by Ammar et al. or in similar published studies not cited here.\nTherefore, I recommend that the authors provide a more detailed description of their DNA extraction and subsequent qPCR methods associated with the transmission assays rather than just citing ‘standard methods’ for rapid single leaflet assays. While the manuscript reports several notable findings, a genuinely rapid method for evaluating CLas inoculation within just two weeks would itself be a significant contribution to HLB research and thus warrants a more thorough explanation.\nAdditional minor comments:\nLine 52: Delete “of” after “activates” Should be “…activates the…”\nLine 233: You should insert a verb after “NFAT”. For example: “…acts as an…”"}, "author_response": "Point-to-point responses to reviewer comments\nWe sincerely thank the reviewers for their constructive feedback, which has\nsignificantly strengthened our manuscript. We responses immediately follow each\ncomment.\nREVIEWER COMMENTS\nReviewer #2 (Remarks to the Author):\nThe authors have addressed my concerns in an appropriate manner.\nResponse: We are pleased to receive the reviewer's positive assessment and thank\nthem for confirming that their concerns have been appropriately addressed.\nReviewer #3 (Remarks to the Author):\nThank you, I am satisfied with the changed made by the authors and I recommend\npublication.\nResponse: We are pleased that the reviewer finds our responses have addressed their\nconcerns appropriately.\nReviewer #4 (Remarks to the Author):\nThe authors have addressed my concerns in an appropriate manner.\nResponse: We are pleased to receive the reviewer’s positive assessment and thank\nthem for confirming that their concerns have been appropriately addressed.\nPoint-to-point responses to reviewer comments\nWe sincerely thank the reviewers for their constructive feedback, which has\nsignificantly strengthened our manuscript. We responses immediately follow each\ncomment, with revised line numbers corresponding to the tracked-changes version of\nthe manuscript. Critical revisions in the updated manuscript are highlighted in red font\nfor easy identification.\nREVIEWER COMMENTS\nReviewer #2 (Remarks to the Author):\nThe authors have addressed my concerns in an appropriate manner.\nResponse: We are pleased to receive the reviewer's positive assessment and thank\nthem for confirming that their concerns have been appropriately addressed.\nReviewer #3 (Remarks to the Author):\nThank you, I am satisfied with the changed made by the authors and I recommend\npublication.\nResponse: We are pleased that the reviewer finds our responses have addressed their\nconcerns appropriately.\nReviewer #4 (Remarks to the Author):\nThe reviewers raised several concerns that the authors addressed with additional\nexperiments, clarification, and revisions to the manuscript. Reviewer 1 provided the\nmost extensive and technically detailed critique. One of the main questions centered\non why only Toll8 was studied, rather than other members of the Toll family. In\nresponse, the authors conducted yeast two-hybrid assays for Toll1, Toll6, and Toll7,\ndemonstrating that none of these interacted with Barrel. This additional work\nsupported the specificity of the Toll8-Barrel interaction. Reviewer 1 also requested\nbroader screening for potential CLas membrane proteins that might interact with Toll\nreceptors. The authors expanded their assays to include nineteen CLas membrane\nproteins and confirmed that Barrel alone binds to Toll8.\nAnother larger issue concerned the immunogold localization shown in Figure 1e,\nwhich the reviewer found unclear and lacking proper controls. The authors repeated\nthis experiment, providing new micrographs that clearly demonstrate\nmembrane-specific labeling and including appropriate negative controls. Reviewer 1\nfurther questioned the mechanism of Toll8 activation, suggesting that\nligand-independent activation should be considered. The authors revised the\nDiscussion to acknowledge this possibility but emphasized that their data support\nBarrel-induced activation as the biologically relevant mechanism. The reviewer also\nasked why only MyD88 was examined among potential adaptor proteins. In response,\nthe authors justified their focus on MyD88 as the universal Toll adaptor in insects and\nexpanded the discussion to address its evolutionary and functional significance.\nSeveral reviewers sought additional detail on downstream signaling components,\nincluding MyD88 and IKKE. The authors added two supplementary tables listing all\nidentified interactors and clarified their decision to focus specifically on IKKE and\nNFAT as central regulators of Toll signaling. Reviewer 1 also requested transmission\nassays to evaluate the functional consequences of gene silencing. The authors\nperformed new experiments showing that silencing of IKKE, Reeler, or NOS\nincreased CLas transmission by adult psyllids, but I will address this in more detail\nbelow.\nReviewer 2 expressed support for the work, noting that the study was of high quality\nbut could benefit from improved readability for a broader audience. The authors\nresponded by reorganizing the Results section for clearer logical flow and simplifying\ncomplex sentences. Reviewer 3 requested several minor clarifications and corrections.\nThese included questions regarding gene nomenclature, which the authors addressed\nby clarifying phylogenetic relationships and adopting the more accurate name IKKE\nin place of IRD5. Reviewer 3 also noted inconsistencies in NFAT localization,\nprompting the authors to replace the relevant figures with consistent and better-quality\nimages. Concerns about potential alternative ubiquitination sites on MyD88 led the\nauthors to perform additional mutagenesis experiments, confirming that lysine 48 is\nthe principal site of modification.\nIn my view, the authors have successfully addressed the primary concerns raised by\nthe reviewers through the inclusion of new experiments, more detailed\nmethodological explanations, and targeted clarifications. They also improved figure\nquality, standardized terminology, and revised the manuscript to enhance clarity and\nreadability. However, upon reviewing the revised version, one additional issue has\ncome to my attention—an issue that was not present in the original draft but\nappears to have emerged during the revision made in response to Reviewer #1.\nIn the initial round of review, Reviewer #1 noted that although the authors had\nmeasured IRD5 (also referred to as IKKE) expression, phosphorylation, and CLas\ntiters following gene silencing, they had not conducted transmission experiments to\nassess whether these molecular changes affected the psyllids' capacity to transmit the\nbacterium to host plants. The reviewer emphasized that such transmission assays are\nessential, as they provide the most direct functional evidence linking the identified\ncandidate proteins to CLas transmission and should therefore be included in the study.\nIn response, the authors agreed with the reviewer's point and explained that they\nsubsequently performed transmission assays using adult D. citri, which are the\nprimary vectors of CLas under natural conditions. They noted that although nymphs\nefficiently acquire the bacterium, their limited mobility makes them less relevant to\ntransmission dynamics. Citing previous studies showing that adults transmit CLas\nmore effectively than nymphs, the authors justified focusing on the adult stage. They\nreported that, in these new experiments, silencing IKKE expression with dsRNA\nsignificantly increased CLas transmission efficiency by infected adult psyllids. This\nresult, according to the authors, supports the conclusion that IKKE plays an important\nrole in the psyllid's immune defense against CLas. They also indicated that full\nmethodological details and results have been incorporated into the revised manuscript\nin the Results, Materials and Methods, and supplementary figures.\nI commend the authors for addressing reviewer comments and for conducting\nadditional inoculation assays. However, I remain skeptical that 'standard' cited\nmethods, based on the citation of Ammar et al. (23), would have been sufficient to\naccomplish this experiment. In all published transmission assays to date involving\nCLas and D. citri, whole-plant inoculations are employed, and leaf tissue is typically\nanalyzed at 1, 3, 6, and 9 months post-inoculation. Depending on the methods used\nand specific investigation, plants generally begin to show signs of CLas infection\naround 3 months after inoculation as detected by qPCR.\nSeveral laboratories have attempted to use single-leaflet assays, as described in this\nmanuscript, to accelerate data collection; however, to my knowledge, such efforts\nhave not been successful and/or previously published. The DNA yield from a single\nleaflet is generally insufficient to reliably detect CLas DNA just 7 days after a 7-day\ninoculation access period by CLas-infected adult psyllids when using 'standard'\nextraction and qPCR methods such as those described by Ammar et al. or in similar\npublished studies not cited here.\nTherefore, I recommend that the authors provide a more detailed description of their\nDNA extraction and subsequent qPCR methods associated with the transmission\nassays rather than just citing 'standard methods' for rapid single leaflet assays. While\nthe manuscript reports several notable findings, a genuinely rapid method for\nevaluating CLas inoculation within just two weeks would itself be a significant\ncontribution to HLB research and thus warrants a more thorough explanation.\nResponse: We thank the Reviewer for their positive assessment of our revised\nmanuscript and for raising the important methodological point regarding our\ntransmission assays. We agree that providing greater detail is essential, and we\napologize for the oversight in not including a more thorough description in the initial\nrevision. It should be noted that our approach is not solely based on the cited method\nbut incorporated several key modifications to enhance detection sensitivity. The\nspecific modifications that enabled reliable early detection of CLas are outlined\nbelow:\n1. High CLas titer in psyllids: Pre-inoculation qPCR confirmed that the psyllids used\nin our assays carried exceptionally high CLas titers (Ct < 20). In addition, silencing\nspecific immune genes (e.g., IKKE) further increased the CLas load within these\ninsects. This elevated titer in the inoculum source dramatically enhances the\nprobability of successful transmission and subsequent detection in the plant tissues.\n2. Optimized plant material and inoculation setup: We confined the treated psyllids\nonto young leaves from susceptible juvenile citrus plants using leaf clip cages. These\nleaves represent the preferred feeding sites for D. citri and are known to be more\npermeable to CLas invasion, thereby facilitating efficient initial infection.\n3. Targeted tissue sampling: Instead of using entire leaflets, we carefully excised and\npooled the vein-adjacent tissues (midribs and major veins) from areas where psyllids\nhad fed. Because CLas preferentially colonizes the phloem and surrounding vascular\ntissue, this sampling strategy effectively enriched for bacterial biomass relative to\ntotal plant DNA, substantially improving detection sensitivity.\n4. DNA extraction and qPCR analysis: We followed a standard CTAB-based protocol\nfor DNA extraction. A key step involved concentrating the final DNA by resuspending\nthe nucleic acid pellet in a minimal volume (10 µL), which significantly increased the\ntemplate concentration for qPCR. The subsequent SYBR Green qPCR targeting the\nCLas 16S rDNA gene, a widely adopted target, was performed alongside standard\ncurves and melting curve analysis to ensure specificity and reproducibility.\nIn summary, the reliable early detection of CLas DNA in our assay is achieved\nthrough a combination of critical factors: the confinement of high-titer psyllids on\nhighly susceptible young leaf tissue using clip cages, targeted sampling of\nvein-adjacent regions to enrich for CLas DNA, a concentrated DNA template, and a\ncarefully controlled qPCR setup. The full methodological details are provided in the\nMaterials and Methods section (lines 720-765) of the revised manuscript, and a\nschematic overview of the key experimental workflow is presented in Figure S4 i.\nWe hope that this detailed explanation, along with the corresponding revisions\nmade to the manuscript, fully addresses the Reviewer's concerns. We are sincerely\ngrateful for their insightful and rigorous feedback, which has substantially\nstrengthened the quality of our work.\nAdditional minor comments:\nLine 52: Delete “of” after “activates” Should be “…activates the…”\nResponse: We thank the reviewer for their careful reading and valuable corrections.\nThe word “of” has been deleted after “activates”. The sentence now reads: “…that\nactivates the ligand protein…” in Line 52 of the revised manuscript.\nLine 233: You should insert a verb after “NFAT”. For example: “…acts as an…”\nResponse: We appreciate the reviewer’s attention to detail. A verb has been inserted\nafter “NFAT.” The text has been revised from “NFAT as a direct regulator” to “NFAT\nacts as a direct regulator” in Line 237."}]

Decision letter