An integrated proteomic and phosphoproteomic analysis reveals α-crystallin A and vitellogenin A1 as key players involved in CLas infection in Diaphorina citri

An integrated proteomic and phosphoproteomic analysis reveals α-crystallin A and vitellogenin A1 as key players involved in CLas infection in Diaphorina citri | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article An integrated proteomic and phosphoproteomic analysis reveals α-crystallin A and vitellogenin A1 as key players involved in CLas infection in Diaphorina citri Zi-Yi Shangguang, Yi-Hong Zhang, Yun Zhu, Jing-Wen Fan, Jia-Ying Xie, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7919843/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 11 You are reading this latest preprint version Abstract Citrus Huanglongbing (HLB), a severe and destructive plant disease caused by the Gram-negative, phloem-limited bacterium “ Candidatus Liberibacter asiaticus ( C Las)” and transmitted by Diaphorina citri , has been extensively studied. Previous studies have reported that protein post-translational modifications play a crucial role in D. citri response to C Las infection. However, comprehensive phosphoproteomic profiling of D. citri induced by C Las remains underexplored. In this study, a total of 144 differentially expressed proteins (DEPs) and 997 differentially phosphorylated proteins (DPPs) were identified by 4D label-free quantitative proteomics and phosphoproteomics. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that DEPs were mainly associated with molecular binding, structural constituent of cuticle and cytochrome P450, whereas DPPs were predominately involved in acting and calcium binding. A total of thirteen proteins were selected for parallel reaction monitoring (PRM) analysis to validate the reliability of proteomics. Integrated proteomic and phosphoproteomic analyses identified seven co-expressed proteins: vitellogenin-A1 (Vg-A1), alpha-crystallin A chain (αA- crystallin), facilitated trehalose transporter Tret1 (Tret1), LOC103509854, zinc finger protein 319 (ZFP319), LOC113471498 and Protein argonaute-2 (Ago-2). Furthermore, RNA interference (RNAi)-mediated knockdown of vitellogenin-A1 and alpha-crystallin A chain significantly reduced C Las content in D. citri . In conclusion, this study provides the most comprehensive phosphorylation profiles of D. citri in response to C Las infection and identifies two potential targets implicated in C Las infection. Diaphorina citri Proteomics Phosphorproteomics Parallel reaction monitoring CLas pathogen Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Huanglongbing (HLB), a devastating citrus disease, is presumed to be caused by “ Candidatus Liberibacter asiaticus ( C Las)” and transmitted by the Asian citrus psyllid (ACP), Diaphorina citri [ 1 ]. Citrus trees infected with C Las become unproductive and may eventually die over time, leading to substantial economic losses for the citrus industry [ 2 ]. In recent years, peptides selected based on artificial intelligence (AI) has shown considerable potential for the treatment of HLB [ 3 ]. Zhao et al. identified that a 14- amino acid peptide, APP3-14, can target citrus PUB21 (an E3 ubiquitin ligase) to control HLB in greenhouse and field trials [ 4 ]. However, C Las bacteria remains uncultivable in axenic culture, and effective treatments are still lacking. C Las can effectively infect and propagate within D. citri , thus posing significant challenges for the management of HLB [ 5 , 6 ]. The current management of HLB primarily involves the use of chemical insecticides to control its vector, D. citri [ 7 ]. However, the routine application of these chemicals has triggered significant issues-namely, pesticide residues, environmental pollution, and accelerated resistance evolution in pests-creating an urgent demand for innovative approaches to manage phloem-feeding insects. The colonization cycle begins when nymphs of D. citri , acquire C Las bacteria through feeding on infected phloem sap. After ingestion, the bacteria cross the midgut barrier and translocate systemically, ultimately reaching secondary tissues including the hemolymph and salivary glands [ 8 , 9 ]. Fluorescence in situ hybridization (FISH) analysis revealed that C Las was detectable in multiple tissues of D. citri , including the midgut, hemolymph, Malpighian tubules, salivary glands, muscles, and gonads, demonstrating its ability to establish systemic and persistent colonization following infection [ 10 , 11 ]. The acquisition efficiency of C Las was significantly higher in nymphs than in adults. Furthermore, adults following molting were capable of faster transmission [ 12 ]. Therefore, C Las is transmitted in a persistent propagative manner throughout the entire life cycle. Identifying and characterizing the genes or proteins in D. citri that facilitate C Las acquisition and transmission is essential for developing effective HLB management strategies [ 13 ]. Despite evidence that genes including tropomyosin1-X2 , clathrin heavy chain ( Chc ), and adipokinetic hormone ( AKH ) and its receptor ( AKHR ) influence C Las loads in D. citri , [ 14 – 16 ], our understanding of the vector-pathogen interaction remains incomplete when studied solely through this individual gene perspective. Recent advances in omics technologies, coupled with an improved genome annotation for D. citri, have provided comprehensive and accurate gene models, greatly facilitating the exploration of functional genes across diverse biological processes [ 17 ]. High-throughput sequencing serves as a powerful tool for large-scale gene screening, and by enabling comprehensive transcriptomic analysis, it also greatly facilitates the elucidation of interaction mechanisms between C Las and D. citri [ 18 , 19 ]. Our previous transcriptomic analysis of the D. citri midgut identified 778 differentially expressed genes (DEGs), which were functionally annotated to various processes including ubiquitination, immune response, ribosome biogenesis, endocytosis, cytoskeleton organization, and insecticide resistance [ 20 ]. However, the direct functional determinants are the corresponding changes in proteins and their post-translational modifications (e.g., phosphorylation, ubiquitination, and acetylation) [ 5 , 21 , 22 ]. Owing to significant advances in mass spectrometry, proteomics has matured into a discipline-spanning technology. Its capacity for the precise identification of key proteins has proven invaluable for elucidating host-pathogen interactions, including those between insects and pathogens [ 23 – 25 ]. Using iTRAQ-based proteomics, Liu et al. identified 147 DEPs in healthy versus rice stripe virus-infected Laodelphax striatellus , which may contribute to low hatchability and developmental defects through their roles in meiosis and mitosis [ 26 ]. Proteomic profiling of hemolymph from C Las-infected and uninfected D. citri using nano-LC-MS/MS yielded 5531 and 3220 peptides, respectively. Subsequent analysis suggested that a large number of immune defense proteins are absent from hemolymph [ 27 ]. Quantitative isotope-labeled protein interaction reporter (PIR) cross-linkers, coupled with spectral counting-based quantification, was applied to microbe-enriched cellular fractions from both nymph and adult D. citri to characterize in vivo protein interactions [ 28 ]. Among all post-translational modifications of proteins, phosphorylation is the most prevalent and dynamic. It plays a pivotal role in regulating a vast array of biological processes [ 29 ]. Quantitative phosphoproteomic analysis is widely used to comprehensively monitor global phosphorylation modifications of phosphoproteins in signaling pathways and to identify their phosphorylation sites under various conditions [ 30 ]. Meng et al. identified 1144 phosphoproteins from Chilo suppressalis treated with chlorantraniliprole and untreated controls, with the results being validated using parallel reaction monitoring (PRM) [ 31 ]. Although western blot is the gold standard for protein level verification, its application is often hindered by the lack of effective and specific antibodies against target proteins of interest. Currently, PRM has been widely applied in exploratory, large-scale, and precise quantification experiments, demonstrating significant advantages in quantitative analyses [ 32 ]. A comprehensive understanding of the molecular mechanisms of C Las infection in D. citri is currently lacking, especially from an integrated proteomic and phosphoproteomic perspective. This study presents a comprehensive integration of proteomic and phosphoproteomic analyses to elucidate the molecular mechanisms of the D. citri - C Las interaction. We identified and validated DEPs and DPPs from C Las-free and C Las-infected D. citri . Functional enrichment (GO/KEGG) and correlation analyses revealed key pathways and pinpointed seven high-confidence DEPs. Subsequent RNAi silencing of vitellogenin-A1 and alpha-crystallin A chain significantly reduced bacterial titers, validating their functional roles. These findings provide valuable insights into the interaction between C Las bacteria and D. citri . 2. Materials and methods 2.1. D. citri rearing and samples collection D. citri was reared on C Las-free Ponkan mandarin (Citrus reticulata) in insect rearing cages under controlled environmental conditions (27 ± 1℃, 65 ± 5% relative humidity, with a 12 h light: 12 h dark photoperiod), following an established protocol [ 33 ]. To standardize the experimental material, fifth-instar nymphs were transferred onto young shoots of P. mandarin that were either C Las-infected or C Las-free. The infection status of all shoots was verified via polymerase chain reaction (PCR). After five days, heads of adult D. citri were dissected, and genomic DNA was extracted using an Animal Tissue Direct PCR Kit (FOREGENE). PCR amplification targeting a 16S ribosomal DNA fragment with C Las-specific primers OI1 and OI2c (Table S1 ) was performed to confirm C Las infection in D. citri , as previously described [ 5 ]. Subsequently, C Las-free and C Las-infected adult D. citri were collected in RNase-free centrifuge tubes (Fig. 1 ) and stored at -80 ℃. Each group consisted of three biological replicates. 2.2. Total protein extraction and trypsin digestion A total of 1800 C Las-infected D. citri adults were randomly divided into three groups and transferred to sterile centrifuge tubes. An equivalent number of C Las-free D. citri served as the control. Approximately 800 µL of SDT lysis buffer (4% SDS, 100 mM Tris-HCl, 1 mM DTT, pH 7.6) was added to each sample, followed by homogenization on ice using an electric homogenizer. After centrifugation at 12,000 × g for 15 min at 4 ℃, the supernatant containing the protein fraction was collected from each sample, and the centrifugation step was repeated to maximize yield. Protein concentration was determined using a BCA Protein Assay Kit (Bio-Rad, USA). Protein digestion was then carried out with trypsin according to the filter-aided sample preparation (FASP) method described by Wisniewski et al. [ 34 ]. The resulting peptides from each sample were desalted using C18 Cartridges (Empore™ SPE Cartridges C18, standard density, bed I.D. 7 mm, volume 3 mL; Sigma), concentrated by vacuum centrifugation, and reconstituted in 40 µl of 0.1% (v/v) formic acid. 2.3. 4D-Label-free quantitative proteomics analysis 2.3.1. LC-MS/MS analysis LC-MS/MS analysis was conducted using a timsTOF Pro mass spectrometry (Bruker) coupled to a Nanoelute nanoflow UHPLC system (Bruker). Peptides were loaded onto a C18 reversed-phase analytical column (Thermo Scientific) equilibrated with 95% buffer A (0.1% formic acid) and separated with a linear gradient of buffer B (99.9% acetonitrile, 0.1% formic acid) at a flow rate of 300 nL/min. The mass spectrometer was operated in positive ion mode with an electrospray voltage of 1.5 kV. Precursor and fragment ions were analyzed by the TOF analyzer within a mass range of m/z 100–1700. Data were acquired in parallel accumulation-serial fragmentation (PASEF) mode with the following settings: ion mobility coefficient (1/K0) of 0.6–1.6 Vs/cm 2 , and each MS cycle comprising one full MS scan followed by 10 PASEF MS/MS scans. A dynamic exclusion time of 24 s was applied. The mass spectrometry proteomics data have been deposited to the iProX database (accession number: IPX0014247001). 2.3.2. Identification and quantitation of proteins The MS raw data from all samples were collectively processed using MaxQuant software (version 1.6.14) for protein identification. The mass tolerance for precursor ions was set to 20 ppm. Peptide spectrum matches (PSMs) with a confidence level exceeding 99% were considered credible. Only proteins containing at least one unique peptide were retained for subsequent analysis. Both protein and PSM identifications were filtered at a false discovery date (FDR) threshold of 0.01. For protein quantitation, the Mann-Whitney test was applied to compare the C Las-free and C Las-infected groups, with proteins having a P -value 2 or < 0. 5 considered differentially expressed proteins (DEPs). 2.4. 4D-Label-free phosphorproteomic analysis 2.4.1. Phosphopeptides enrichment The labeled samples were reconstituted in an IMAC-Fe column washing solution containing 250 mM acetic acid and 30% acetonitrile. All samples were centrifuged at 12000 × g for 5 min at 4 ℃, and the resulting precipitates were collected. Phosphopeptide enrichment was performed using the High-Select ™ Fe-NTA Phosphopeptide Enrichment Kit (Thermo Scientific) following the manufacturer’s protocol. After lyophilization, the enriched phosphopeptides were redissolved in 20 µL of loading buffer (0.1% formic acid). 2.4.2. LC-MS/MS analysis LC-MS/MS analysis was conducted as outlined in Section 2.3.1, with slight modifications. Briefly, peptides were loaded onto a C18 reversed-phase analytical column (25 cm × 75 µm i.d., 1.9 µm particle size) and equilibrated with buffer A (0.1% formic acid). Separation was achieved using a linear gradient of buffer B (84% acetonitrile, 0.1% formic acid) at a flow rate of 300 nL/min. The mass spectrometer was operated in positive ion mode, acquiring MS spectra across an m/z range of 100–1700 and an ion mobility (1/k0) range of 0.6–1.6. Subsequently, up to 10 PASEF MS/MS scans were triggered with a target intensity of 1.5 k and an intensity threshold of 2500. Dynamic exclusion was activated with a release time of 0.4 min. 2.4.3. Identification and quantitation of phosphorylated proteins Identification and quantitation of phosphorylated proteins was conducted as per Section 2.3.2, with the inclusion of variable modifications for phosphorylation on serine (S), threonine (T), and tyrosine (Y). The criteria for DPP quantitation and identification remained unchanged from the aforementioned section. 2.5. Bioinformatic analysis Hierarchical clustering analysis was performed using Cluster 3.0 ( http://bonsai.hgc.jp/~mdehoon/software/cluster/software.htm ) and results were visualized as a heatmap with Java Treeview software ( http://jtreeview.sourceforge.net ). Motif analysis was carried out with the MEME Suite ( http://meme-suite.org/index.htm ), and protein subcellular localization was predicted using CELLO software ( http://cello.life.nctu.edu.tw/ ). Protein domain signatures were identified by querying the Pfam database via InterProScan. Functional categorization of differentially expressed proteins (DEPs) was assessed through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. Enrichment used a Fisher's exact test with the full set of quantified proteins as the background, and p-values were adjusted for multiple testing using the Benjamini-Hochberg method. 2.6. Validation of DEP by parallel reaction monitoring (PRM) Parallel reaction monitoring (PRM) was employed to validate the DEPs identified by LC-MS/MS. A total of 14 target peptides, along with an isotopically labeled reference peptide (PRTC: ELGQSGVDTYLQTK) spiked into each sample, were analyzed. Analysis consisted of a full-scan event followed by PRM acquisition under the following parameters: resolution, 30,000 (at 200 m/z); automatic gain control (AGC) target, 5e4; maximum injection time, 80 ms; and normalized collision energy, 27%. The signal intensities of the target peptides were quantified and normalized against the internal standard. 2.7. RNA isolation, cDNA synthesis and RT-qPCR analysis Total RNA was extracted from various D. citri samples using TRIzol reagent (Takara, Kusatsu, Japan) following the manufacturer’s instructions. RNA quality was assessed by measuring concentration and purity on a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and evaluating integrity via 1% agarose gel electrophoresis. Only high-quality RNA samples (OD 260/280 : 1.9-2.0; OD 260/230 : ≥1.9) were qualified for cDNA synthesis. cDNA was synthesized from 1 µg of total RNA using a cDNA synthesis kit (YEASEN, Shanghai, China) following the manufacturer's protocol. Briefly, a 15 µL reaction mixture containing 3.0 µL of gDNA digester Mix and RNase-free water was incubated at 42 ℃ for 5 min to remove genomic DNA. Subsequently, 5 µL 4×Hifair®Ⅲ SuperMix plus was added, and the reaction proceeded at 25°C for 5 min and 55°C for 15 min, followed by enzyme inactivation at 85°C for 5 min. The resulting cDNA was stored at − 20°C for subsequent use. qPCR was performed on a Roche LightCycler 96 Automatic Analyzer (Roche Life Science) using the primers listed in Table S1 . Each 20 µL reaction contained 10 µL SYBR Green Mix, 8 µL RNase-free water, 0.5 µL forward primer, 0.5 µL reverse primer, and 1.0 µL cDNA template. The thermal cycling protocol consisted of an initial denaturation at 95 ℃ for 10 min, followed by 40 cycles of 95 ℃ for 10 s, 60 ℃ for 10 s, and 72 ℃ for 15 s. Melting curve analysis was conducted to verify amplification specificity. The relative gene expression levels were calculated using the 2 −ΔΔCt method, with glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) as the reference gene. All samples were amplified in triplicate. 2.8. dsRNA synthesis, microinjection and C Las bacterial acquisition To investigate the roles of vitellogenin-A1 (Vg-A1) and alpha-crystallin A chain (αA-crystallin) in C Las infection, we performed RNAi-mediated silencing of these genes in D. citri . Double-stranded RNA (dsRNA) targeting Vg-A1 (ds Vg-A1 ) and αA-crystallin (ds αA-crystallin ), along with a control ds GFP , were synthesized in vitro using the T7 RioMAXTM Express RNAi System (Promega, Madision, WI, USA) according to the manufacturer’s instructions. Gene-specific primers with T7 promoter sequences are listed in Table S1 . The synthesized dsRNAs were diluted with RNase-free water containing 0.1% red food dye to working concentrations of 500 ng/µL (ds Vg-A1 ) and 1200 ng/µL (ds αA-crystallin ). For microinjection, 5th-instar nymphs were immobilized with a dorsal sticker, and approximately 15 nL of the respective dsRNA solution was delivered into each insect. The injected nymphs were initially maintained on C Las-free Murraya exotica seedlings. After 48 h, silencing efficiency was verified by RT-qPCR, and the nymphs were subsequently transferred to the tender shoots of CLas-infected C. sinensis for an acquisition access period. Surviving adults were collected at 24 h and 48 h post-acquisition and individually stored. The C Las bacterial titer was quantified using a TaqMan-based qPCR assay targeting the CLas 16S rRNA gene, with primers and probe sequences provided in Table S1 . All data were analyzed by one-way ANOVA in SPSS software, with significance levels set at P -values < 0.05 and < 0.01 were considered statistically significant and highly significant, respectively. 3. Results 3.1. Identification and quantification of DEPs and DPPs A 4D-label-free quantitative proteomics analysis was conducted to identify DEPs and DPPs by comparing C Las-free and C Las-infected D. citri . From the 468,270 spectrums acquired, 82213 were successfully matched, resulting in the identification of 20181 peptides (including 18,603 unique peptides) and 3,342 proteins. Subsequently, 3,310 quantifiable proteins were subjected to further differential analysis (Fig. 2 A and Table S2 ). Furthermore, a total of 5,911 phosphopeptides were identified, corresponding to 2,658 phosphoproteins and accounting for 11,521 quantified phosphorylation sites quantified (Fig. 2 B and Table S3 ). Differentially expressed and phosphorylated proteins (DEPs and DPPs) between C Las-free and C Las-infected D. citri were identified using thresholds of a fold change > 2 or < 0.5 and a P -value < 0.05. The results revealed that 63 up- and 81 down-regulated DEPs (Fig. 2 C and Table S4 A), alongside 401 up- and 596 down-regulated DPPs (Fig. 2 D and Table S5 A). Furthermore, 32 DEPs and 52 DPPs were identified exclusively in either C Las-free or C Las infected D. citri from the proteomic and phosphoproteomic databasets, respectively (Table S4 B and Table S5 B). Under a more stringent threshold (fold change > 10 or < 0.1 and P < 0.05), 5 DEPs (up-regulated) and 9 (down-regulated), as well as 21 (up-regulated) and 27 (down-regulated) DPPs, were identified (Fig. 2 C and Fig. 2 D). 3.2. Functional characterization and enrichment of DEPs and DPPs Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and subcellular localization analyses were performed to elucidate the involved biological processes, molecular functions, and signaling pathways. Analysis of subcellular localization revealed that the majority of quantified proteins were distributed in the membrane and nucleus (Fig. 3 A). Gene Ontology (GO) analysis revealed distinct functional pattern for the DEPs. In biological processes (BP), they were primarily enriched in rRNA processing, rRNA metabolic processes and ncRNA processing. For molecular function (MF), the dominant terms were cofactor binding, monooxygenase activity and structural constituent of cuticle. Regarding cellular component (CC), the DEPs were predominantly localized to the nucleolus and nuclear lumen (Fig. 3 B). Domain enrichment analysis revealed distinct profiles for DEPs and DPPs. The most significantly enriched domains were insect cuticle protein and cytochrome P450 for DEPs (Fig. 3 C), and the LIM domain and Immunoglobulin I-set domain for DPPs (Fig. 4 A). Motif analysis of the phosphorylated peptides identified 739 and 439 peptides containing the conserved _S_P_ motifs, respectively (Fig. 4 B). Subcellular localization analysis revealed that the majority of DPPs were distributed in the membrane and nucleus (Fig. 4 C). GO enrichment analysis indicated that these DPPs are primarily associated with functions such as acting binding, calcium ion binding and the actin cytoskeleton (Fig. 4 D). KEGG enrichment analysis further demonstrated that DEPs were predominantly enriched in ABC transporters, whereas DPPs were mainly involved in the calcium signaling pathway (Fig. 5 ). 3.3. Validation of DEPs by PRM To verify the DEPs identified by proteomic analysis, thirteen candidates were selected for parallel reaction (PRM) validation, including LOC103507240, alpha-crystallin A chain (αA-crystallin), cuticle protein 12.5 (CP12.5), phosphoenolpyruvate carboxykinase (PEPCK), clustered mitochondria protein (CMP), E3 ubiquitin ligase PARAQUAT TOLERANCE3 (PQT3), indole-3-acetaldehyde oxidase (IAAld-oxidase), cuticle protein 16.5 (CP16.5), dolichyl-diphosphooligosaccharide-protein glycotransferase (DDOST), mitochondrial protein/calcium exchanger (MCX), vitellogenin-A1 (Vg-A1), ATP-binding cassette sub-family B membrane 3 (ABCB3) and cathepsin L-like (CathL). As shown in Fig. 6 , the expression of LOC103507240, CP12.5, IAAld-oxidase, CP16.5 and CathL was downregulated in D. citri following C Las infection, whereas those of αA-crystallin, CMP, PQT3, MCX, Vg-A1 and ABCB3 was significantly upregulated. The PRM results were highly consistent with the 4D-Label-free proteomics data, confirming the reliability of our proteomics findings. 3.4. Correlation analysis between DEPs and DPPs, and RT-qPCR validation The elucidate the roles of DEPs and DPPs in D. citri following C Las infection, we performed an integrative analysis combining 4D label-free quantitative proteomics and phosphoproteomics data. The results revealed 24 DEPs and 228 DPPs, among which 7 proteins exhibited concurrent alterations at both the expression and phosphorylation levels (Fig. 7 A and 7 B). GO enrichment analysis indicated that in the BP category, DPPs were primarily involved in lipid transport and localization, whereas DEPs were associated with rRNA processing. In the CC category, DPPs were predominantly localized to the actin cytoskeleton and myosin complex, while DEPs were enriched in the nuclear lumen. Regarding MF, DPPs were mainly implicated in calcium ion binding and actin binding, and DEPs were related to monooxygenase activity and structural constituents of the cuticle (Fig. 7 C). KEGG analysis further showed that DPPs were significantly enriched in the calcium signaling pathway, apelin signaling pathway, and cellular senescence, whereas DEPs were primarily involved in ABC transporters (Fig. 7 D). The expression levels of the seven co-expressed proteins identified among the DEPs and DPPs were validated using RT-qPCR. As shown in Fig. 8 , four of them (Vg-A1, αA-crystallin, facilitated trehalose transporter Tret1, and LOC103509854) were significantly upregulated in D. citri following C Las infection, and three proteins (zinc finger protein 319, LOC113471498 and protein argonaute-2) were downregulated, a trend which aligned with the proteomics data. According to the phosphoproteomic dataset, both vitellogenin-A1 and protein argonaute-2 possessed two phosphorylated peptides, and their expression trends were consistent with the proteomic results. In contrast, the remaining five proteins each contained only a single phosphorylated peptide and showed opposing trends to the proteomics data (Table 1 ). Furthermore, we also found that α-crystallin A was expressed only in the C Las-infected groups, while Tret1 was specific to the C Las-free groups (Table 1 ). Table 1 Overlap of co-expressed proteins between quantitative proteomics and phosphoproteomics Protein name Proteomics Phosphoproteomics C Las + / C Las − C Las + / C Las − Vitellogenin-A1 3.7514 DNDYNDDDQKNHQNS(+ 79.97)GSHNNNNHHNSGSNDNK (8.2937) EFFNLATSSQVT(+ 79.97)K (3.6146) α-crystallin A 2.5374 LS(+ 79.97)SDGILSIQAPK (0) Facilitated trehalose transporter Tret1 2.1298 VFTVEEGTVT(+ 79.97)Q (∞) LOC103509854 2.0714 S(+ 79.97)VLLENENVQAISASR (0.1767) Zinc finger protein 319 0.4977 KTTVSTPSTPT(+ 79.97)AASAPVAQPTPPPQQNIVR (4.0917) LOC113471498 0.2281 NEPPEDS(+ 79.97)NDLTNEQR (0.2317) Protein argonaute-2 0.0627 MTIASSSS(+ 79.97)SSSISSAASGAGSK (0.1215) MTIASS(+ 79.97)SSSSSISSAASGAGSK (0.0606) 3.5. Silencing of vitellogenin-A1 and alpha-crystallin A chain effects on C Las content in D. citri Based on the correlation analysis between 4D-label-free quantitative proteomics and phosphoproteomics, two proteins-αA-crystallin and VgA1-were selected for functional validation of their roles in C Las proliferation in D. citri . The results showed that the expression level of Vg-A1 was significantly downregulated at 48 h after ds Vg-A1 injection, while it had no obvious difference at 24 h. In contrast, the relative expression of αA-crystallin was significantly downregulated at both 24 h and 48 h after ds αA-crystallin injection (Fig. 9 A and 9 C). Fifth-instar nymphs treated with ds GFP , ds Vg-A1 , or ds αA-crystallin were subsequently released onto C Las-infected citrus tender tips. The C Las bacterial content was significantly reduced following the silencing of either αA-crystallin or VgA1 (Fig. 9 B and 9 D). These results suggest that both αA-crystallin and Vg-A1 might be involved in C Las infection in D. citri . 4. Discussion To date, the C Las bacteria has not been successfully cultured in vitro , yet it efficiently infects D. citri and proliferates persistently within host cells, facilitating its widespread transmission. This poses a major challenge for the management of HLB. When D. citri feeds on HLB-infected citrus, C Las enters through the stylet's food canal and reaches the gut lumen. D. citri midgut serves as a critical barrier against C Las invasion and proliferation. To investigate the host–pathogen interactions at this gut interface, RNA-seq and proteomic analyses were conducted. The results indicated that C Las infection alters several biological pathways, including the TCA cycle, iron metabolism, insecticide resistance, and the insect immune system [ 35 ]. After crossing the midgut, C Las disseminates via the hemolymph to the salivary glands. The hemolymph also acts as a primary site for initiating cellular and humoral immune responses to cuticular damage and pathogen invasion. Using nano-LC-MS/MS, 5531 and 3220 peptides were identified in the hemolymph of C Las-infected and uninfected D. citri , respectively. Notably, a large number of immune defense proteins were absent from the hemolymph of D. citri [ 36 ]. In our previous research, integrated ubiquitylome and proteome analyses revealed that cytoskeleton-related proteins undergo ubiquitination and play critical roles during C Las infection [ 37 ]. However, phosphorylation-related proteins had not yet been systematically compared between C Las-infected and C Las-free D. citri . During bacterial infection, pathogens often manipulate host protein expression to facilitate their own replication through various strategies, including post-translational modifications (PTMs) such as phosphorylation, ribosylation, and acetylation. These modifications serve as rapid and dynamic regulators of protein function [ 38 ]. Among them, protein phosphorylation is widely recognized as a key mechanism in cellular regulation and signaling [ 39 ]. In this study, we applied 4D-label free quantitative proteomics and phosphoproteomics to investigate the role of phosphorylated proteins in D. citri upon C Las infection. We identified 144 DEPs and 997 DPPs. Domain enrichment analysis revealed that most DEPs were associated with insect cuticle proteins and cytochrome P450 enzymes. Cuticle proteins (CPs) are major structural components of the insect cuticle and are essential for maintaining its integrity and stability [ 40 ]. Here, nine differentially expressed CPs were identified, all of which were significantly downregulated in C Las-infected D. citri . This aligns with previous findings by Yuan et al., who reported that most CP genes were downregulated in C Las-infected D. citri , suggesting that C Las may suppress cuticle formation to facilitate its own proliferation [ 41 ]. Additionally, five P450-related proteins-CYP4C4, CYP4g15-2, CYP6k1-like isoform X1, CYP4g15 and CYP4g15-were significantly upregulated in C Las-infected D. citri . Cytochromes P450 enzymes are critical in the oxidative metabolism of both endogenous and exogenous compounds [ 42 ]. However, conflicting results were reported by Tiwari et al., who observed higher expression of four CYP4 genes ( CYP4DA1 , CYP4C68 , CYP4G70 , and CYP4DB1 ) in uninfected compared to C Las-infected D. citri [ 43 ], indicating a complex role of P450s in D. citri response to C Las infection. Domain analysis further revealed that eight DPPs contained LIM domains, which are known to mediate interactions between the actin cytoskeleton and transcriptional machinery [ 44 ]. Among these, four DPPs (PDZ and LIM domain protein Zasp-like, LIM and actin-binding protein 1, Muscle LIM protein 1, and actin-binding LIM protein 1) were upregulated, while the other four (Four and a half LIM domains protein 2 isoforms X1 and X4, PDZ and LIM domain protein Zasp-like, and LIM domain-containing protein jub-like) were downregulated. Our recent unpublished data suggest that the “four and a half LIM domains protein 2” is involved in C Las proliferation, indicating that LIM domain-containing phosphoproteins may participate in C Las infection. To further elucidate the functional relevance of phosphorylated proteins, we integrated data from 4D-label free quantitative proteomics and phosphoproteomics. Seven overlapping proteins were identified: vitellogenin-A1, alpha-crystallin A chain, facilitated trehalose transporter Tret1, LOC103509854, zinc finger protein 319, LOC113471498, and protein argonaute-2. RT-qPCR validation showed that the Vg-A1 , α-crystallin A , Tret 1 and LOC103509854 were significantly upregulated in C Las-infected D. citri , whereas zinc finger protein 319 ( ZFP319 ), LOC113471498 , and argonaute-2 ( Ago-2 ) were downregulated. Furthermore, silencing of Vg-A1 significantly suppressed C Las proliferation, indicating its involvement in C Las infection. In insects, vitellogenesis are essential for yolk formation and embryonic development [ 45 ]. Multiple studies have reported that C Las infection significantly upregulates egg development-related genes such as vitellogenin 1-like ( Vg-1-like ), vitellogenin A1-like ( Vg-A1-like ) and the vitellogenin receptor ( VgR ) [ 46 ]. Jaiswal et al. also found upregulation of vitellogenin-1 and vitellogenin-2 by 18.4- and 17.0-fold, respectively, in C Las-infected D. citri [ 47 ], underscoring the potential role of Vg-A1 in host response. The α-crystallin, a small heat shock protein with chaperone-like activity, typically consists of αA and αB subunits and helps prevent protein misfolding and aggregation [ 48 ]. We found that inhibition of α-crystallin A significantly reduced C Las titers in D. citri , suggesting its involvement in C Las infection dynamics. 5. Conclusion In conclusion, differentially expressed proteins and differentially phosphorylated proteins were identified by 4D label-free quantitative proteomics and phosphoproteomics. GO and KEGG were performed to reveal the functions of these proteins. Integrated proteomic and phosphoproteomic analyses identified seven co-expressed proteins, and silencing of Vg-A1 and α-crystallin A chain significantly reduced C Las content in D. citri . This study provides initial insights into the role of protein phosphorylation in mediating the interaction between C Las and D. citri . Declarations Competing interests The authors declare no competing interests. Funding This research was funded by the Natural Science Foundation of Jiangxi Province (20224BAB205012), National Natural Science Foundation of China (302260674, 32272549, 32560693) and Gan Poyang Talent Support Program-Training program for academic and technical leaders in major disciplines (20232BCJ23030). Author Contribution Z.L. and H.Y. designed this project; Z.S., Y.Z., and J.X. conducted the experiments; J.F. was involved in the recruitment of participants; Z.S., Y.Z. and H.Y. analyzed the data; Z.L. and H.Y. prepared the initial draft of the manuscript; and all authors have reviewed and approved the manuscript. Data Availability Not applicable. References Li J, He PF, H PB, Li YM, Wu YX, Lu ZJ, et al. Potential of citrus endophyte Bacillus subtilis L1-21 in the control of Candidatus Liberibacter asiaticus in Asian citrus psyllid, Diaphorina citri . Pest Manag Sci. 2022; 78 (12): 5164-5171. Kazemzadeh-Beneh H, Safarnejad MR, Norouzi P, Samsampour D, Alavi SM, Shaterreza P. 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Supplementary Files TableS1.docx TableS2.xlsx TableS3.xlsx TableS4.xlsx TableS5.xlsx UncroppedGel.tif Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 23 Apr, 2026 Reviews received at journal 22 Apr, 2026 Reviews received at journal 12 Apr, 2026 Reviewers agreed at journal 23 Mar, 2026 Reviewers agreed at journal 23 Mar, 2026 Reviews received at journal 14 Feb, 2026 Reviewers agreed at journal 29 Jan, 2026 Reviewers invited by journal 27 Jan, 2026 Editor assigned by journal 23 Dec, 2025 Submission checks completed at journal 22 Dec, 2025 First submitted to journal 22 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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The distinction between \u003cem\u003eC\u003c/em\u003eLas-free and \u003cem\u003eC\u003c/em\u003eLas-infected \u003cem\u003eD. citri\u003c/em\u003e groups was verified by PCR detection.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7919843/v1/22c10cb7280f0f0275548b52.png"},{"id":101507746,"identity":"ec1410b7-97ed-476b-99a2-1a58c9849442","added_by":"auto","created_at":"2026-01-30 14:42:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":265082,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of the proteomic and phosphoproteomic datasets. (A) Histogram of identification and quantification of proteins; (B) Histogram of identification and quantification of phosphoproteins; (C) Statistics of differentially expressed proteins (DEPs); (D) Statistics of differentially phosphoproteins proteins (DPPs).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7919843/v1/cc6a4684693cfcfe32387db7.png"},{"id":101507713,"identity":"dcb3981b-f391-40db-8d84-eafd815abb8a","added_by":"auto","created_at":"2026-01-30 14:42:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":823203,"visible":true,"origin":"","legend":"\u003cp\u003eSubcellular localization and enrichment analyses of differentially expressed proteins (DEPs) between \u003cem\u003eC\u003c/em\u003eLas-free and \u003cem\u003eC\u003c/em\u003eLas-infected groups. (A) Subcellular localization of DEPs; (B) GO enrichment analysis of DEPs; (C) Domain enrichment analysis of DEPs.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7919843/v1/9ef9ccd03003ce9cde83c033.png"},{"id":101507882,"identity":"0422e7b4-b947-42f3-a0f9-90d07b11a604","added_by":"auto","created_at":"2026-01-30 14:43:08","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":703151,"visible":true,"origin":"","legend":"\u003cp\u003eDomain enrichment, motif numbers, subcellular localization and GO enrichment analysis of differentially phosphorylated proteins (DPPs) between \u003cem\u003eC\u003c/em\u003eLas-free and \u003cem\u003eC\u003c/em\u003eLas-infected groups. (A) Domain enrichment analysis of DPPs; (B) Motif numbers analysis of DPPs; (C) Pie chart of subcellular localization of DPPs; (D) GO enrichment analysis of DPPs.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7919843/v1/5749df34678af6c3d25b4db3.png"},{"id":101507797,"identity":"818f0f96-d235-4f92-8a94-b303ea54427b","added_by":"auto","created_at":"2026-01-30 14:42:55","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":526169,"visible":true,"origin":"","legend":"\u003cp\u003eKEGG enrichment analysis of differentially expressed proteins (DEPs) and differentially phosphorylated proteins (DPPs) between \u003cem\u003eC\u003c/em\u003eLas-free and \u003cem\u003eC\u003c/em\u003eLas-infected groups. (A) KEGG enrichment analysis of DPPs; (B) KEGG enrichment analysis of DPPs.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7919843/v1/fef6274486e80f95dd4a6726.png"},{"id":101507767,"identity":"dce7fbfc-8669-4b66-b56f-fcccd33e6034","added_by":"auto","created_at":"2026-01-30 14:42:48","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":551863,"visible":true,"origin":"","legend":"\u003cp\u003ePRM validation of thirteen differentially expressed proteins (DEPs) in \u003cem\u003eC\u003c/em\u003eLas-free and \u003cem\u003eC\u003c/em\u003eLas-infected groups, including LOC103507240, α-crystallin A chain, cuticle protein 12.5, phosphoenolpyruvate carboxykinase, clustered mitochondria protein, E3 ubiquitin ligase PARAQUAT TOLERANCE 3, Indole-3-acetaldehyde oxidase, cuticle protein 16.5, dolichyl-diphosphooligosaccharide-protein glycotransferase, mitochondrial protein/calcium exchanger, vitellogenin-A1, ATP-binding cassette sub-family B member 3 and cathepsin L-like.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7919843/v1/14f0920fb9fbb02785a637c4.png"},{"id":101507783,"identity":"aa90a007-fe23-460b-a4b2-89aba7817d95","added_by":"auto","created_at":"2026-01-30 14:42:52","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1645773,"visible":true,"origin":"","legend":"\u003cp\u003eIntegrated analysis of co-expressed proteins from proteomic and phosphoproteomic data. (A) Correlation analysis between differentially expressed proteins (DEPs) and differentially phosphorylated proteins (DPPs); (B) Venn diagram showing the overlap of proteins between datasets; (C) GO enrichment analysis of co-expressed proteins; (D) KEGG enrichment analysis of co-expressed proteins.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-7919843/v1/690917e8c5ef5fddf30c3d43.png"},{"id":101507743,"identity":"dd2588f2-782d-4961-9570-fb9581b80bd4","added_by":"auto","created_at":"2026-01-30 14:42:47","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":521549,"visible":true,"origin":"","legend":"\u003cp\u003eExpression level analysis of seven co-expressed proteins in \u003cem\u003eC\u003c/em\u003eLas-free and \u003cem\u003eC\u003c/em\u003eLas-infected \u003cem\u003eD. citri\u003c/em\u003e by RT-qPCR. Relative expression levels were calculated using the 2\u003csup\u003e-∆∆Ct\u003c/sup\u003e method. Statistical analysis was conducted using SPSS software. Significant differences are indicated by ** (\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-7919843/v1/81261479787f4246b7a015ef.png"},{"id":101507788,"identity":"8e84f86a-d77e-4fa9-a655-5469c68c42d2","added_by":"auto","created_at":"2026-01-30 14:42:52","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":715067,"visible":true,"origin":"","legend":"\u003cp\u003eDetection of \u003cem\u003eC\u003c/em\u003eLas content in \u003cem\u003eD. citri\u003c/em\u003eafter silencing \u003cem\u003eVg-A1\u003c/em\u003e and \u003cem\u003eαA-crystallin\u003c/em\u003eby RNA interference (RNAi). (A) and (C): Expression level analysis of \u003cem\u003eVg-A1\u003c/em\u003eand \u003cem\u003eαA-crystallin\u003c/em\u003e at 24 h and 48 h after injection of ds\u003cem\u003eVg-A1\u003c/em\u003e and ds\u003cem\u003eαA-crystallin\u003c/em\u003e, respectively; (B) and (D): Detection of CLas content in \u003cem\u003eD. citri\u003c/em\u003e after silencing \u003cem\u003eVg-A1\u003c/em\u003e and \u003cem\u003eαA-crystallin\u003c/em\u003e by RNAi. The control groups were treated with ds\u003cem\u003eGFP\u003c/em\u003e. Statistical analysis was conducted using SPSS software. Significant differences are indicated by ** (\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"Figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-7919843/v1/945382ac69fa97eb37caf8e9.png"},{"id":101756051,"identity":"fcefb1e0-09b7-416b-b552-4102df96d5f1","added_by":"auto","created_at":"2026-02-03 10:56:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9950437,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7919843/v1/e95004dd-87da-4ed8-9046-a91b99cd46f4.pdf"},{"id":101507717,"identity":"e02e773a-0abd-4fc2-abe2-feab2d25b6ca","added_by":"auto","created_at":"2026-01-30 14:42:42","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":17737,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7919843/v1/db0703a690d8831d05cdb328.docx"},{"id":101752325,"identity":"d83cb144-4de7-4e5e-a6dc-a2bb80f979a2","added_by":"auto","created_at":"2026-02-03 10:26:47","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":3553841,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7919843/v1/f7d59a4a1d43da942ca35f04.xlsx"},{"id":101507749,"identity":"56572947-be57-407c-85fb-bab9531eb018","added_by":"auto","created_at":"2026-01-30 14:42:48","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":2912322,"visible":true,"origin":"","legend":"","description":"","filename":"TableS3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7919843/v1/d63899d2b879f0f89373e522.xlsx"},{"id":101507863,"identity":"99462c47-f52f-498c-a0a5-7e7fbc23a073","added_by":"auto","created_at":"2026-01-30 14:43:04","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":728751,"visible":true,"origin":"","legend":"","description":"","filename":"TableS4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7919843/v1/b742c0c02ac26b6c112b39cb.xlsx"},{"id":101507747,"identity":"8f9e7983-b0b6-4a65-a45b-206192bdb2fd","added_by":"auto","created_at":"2026-01-30 14:42:48","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":3095776,"visible":true,"origin":"","legend":"","description":"","filename":"TableS5.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7919843/v1/dc02a783aae1ec4b83fc02da.xlsx"},{"id":101507715,"identity":"bc471f6f-c4b7-48aa-b815-f02f916b2e1e","added_by":"auto","created_at":"2026-01-30 14:42:41","extension":"tif","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":613794,"visible":true,"origin":"","legend":"","description":"","filename":"UncroppedGel.tif","url":"https://assets-eu.researchsquare.com/files/rs-7919843/v1/1c3234bfe9515c361e53bc13.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"An integrated proteomic and phosphoproteomic analysis reveals α-crystallin A and vitellogenin A1 as key players involved in CLas infection in Diaphorina citri","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eHuanglongbing (HLB), a devastating citrus disease, is presumed to be caused by \u0026ldquo;\u003cem\u003eCandidatus\u003c/em\u003e Liberibacter asiaticus (\u003cem\u003eC\u003c/em\u003eLas)\u0026rdquo; and transmitted by the Asian citrus psyllid (ACP), \u003cem\u003eDiaphorina citri\u003c/em\u003e [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Citrus trees infected with \u003cem\u003eC\u003c/em\u003eLas become unproductive and may eventually die over time, leading to substantial economic losses for the citrus industry [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In recent years, peptides selected based on artificial intelligence (AI) has shown considerable potential for the treatment of HLB [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Zhao et al. identified that a 14- amino acid peptide, APP3-14, can target citrus PUB21 (an E3 ubiquitin ligase) to control HLB in greenhouse and field trials [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. However, \u003cem\u003eC\u003c/em\u003eLas bacteria remains uncultivable in axenic culture, and effective treatments are still lacking. \u003cem\u003eC\u003c/em\u003eLas can effectively infect and propagate within \u003cem\u003eD. citri\u003c/em\u003e, thus posing significant challenges for the management of HLB [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The current management of HLB primarily involves the use of chemical insecticides to control its vector, \u003cem\u003eD. citri\u003c/em\u003e [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. However, the routine application of these chemicals has triggered significant issues-namely, pesticide residues, environmental pollution, and accelerated resistance evolution in pests-creating an urgent demand for innovative approaches to manage phloem-feeding insects.\u003c/p\u003e \u003cp\u003eThe colonization cycle begins when nymphs of \u003cem\u003eD. citri\u003c/em\u003e, acquire \u003cem\u003eC\u003c/em\u003eLas bacteria through feeding on infected phloem sap. After ingestion, the bacteria cross the midgut barrier and translocate systemically, ultimately reaching secondary tissues including the hemolymph and salivary glands [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Fluorescence \u003cem\u003ein situ\u003c/em\u003e hybridization (FISH) analysis revealed that \u003cem\u003eC\u003c/em\u003eLas was detectable in multiple tissues of \u003cem\u003eD. citri\u003c/em\u003e, including the midgut, hemolymph, Malpighian tubules, salivary glands, muscles, and gonads, demonstrating its ability to establish systemic and persistent colonization following infection [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The acquisition efficiency of \u003cem\u003eC\u003c/em\u003eLas was significantly higher in nymphs than in adults. Furthermore, adults following molting were capable of faster transmission [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Therefore, \u003cem\u003eC\u003c/em\u003eLas is transmitted in a persistent propagative manner throughout the entire life cycle. Identifying and characterizing the genes or proteins in \u003cem\u003eD. citri\u003c/em\u003e that facilitate \u003cem\u003eC\u003c/em\u003eLas acquisition and transmission is essential for developing effective HLB management strategies [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Despite evidence that genes including \u003cem\u003etropomyosin1-X2\u003c/em\u003e, \u003cem\u003eclathrin heavy chain\u003c/em\u003e (\u003cem\u003eChc\u003c/em\u003e), and \u003cem\u003eadipokinetic hormone\u003c/em\u003e (\u003cem\u003eAKH\u003c/em\u003e) and its receptor (\u003cem\u003eAKHR\u003c/em\u003e) influence \u003cem\u003eC\u003c/em\u003eLas loads in \u003cem\u003eD. citri\u003c/em\u003e, [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], our understanding of the vector-pathogen interaction remains incomplete when studied solely through this individual gene perspective. Recent advances in omics technologies, coupled with an improved genome annotation for D. citri, have provided comprehensive and accurate gene models, greatly facilitating the exploration of functional genes across diverse biological processes [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. High-throughput sequencing serves as a powerful tool for large-scale gene screening, and by enabling comprehensive transcriptomic analysis, it also greatly facilitates the elucidation of interaction mechanisms between \u003cem\u003eC\u003c/em\u003eLas and \u003cem\u003eD. citri\u003c/em\u003e [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Our previous transcriptomic analysis of the \u003cem\u003eD. citri\u003c/em\u003e midgut identified 778 differentially expressed genes (DEGs), which were functionally annotated to various processes including ubiquitination, immune response, ribosome biogenesis, endocytosis, cytoskeleton organization, and insecticide resistance [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. However, the direct functional determinants are the corresponding changes in proteins and their post-translational modifications (e.g., phosphorylation, ubiquitination, and acetylation) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOwing to significant advances in mass spectrometry, proteomics has matured into a discipline-spanning technology. Its capacity for the precise identification of key proteins has proven invaluable for elucidating host-pathogen interactions, including those between insects and pathogens [\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Using iTRAQ-based proteomics, Liu et al. identified 147 DEPs in healthy versus rice stripe virus-infected \u003cem\u003eLaodelphax striatellus\u003c/em\u003e, which may contribute to low hatchability and developmental defects through their roles in meiosis and mitosis [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Proteomic profiling of hemolymph from \u003cem\u003eC\u003c/em\u003eLas-infected and uninfected \u003cem\u003eD. citri\u003c/em\u003e using nano-LC-MS/MS yielded 5531 and 3220 peptides, respectively. Subsequent analysis suggested that a large number of immune defense proteins are absent from hemolymph [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Quantitative isotope-labeled protein interaction reporter (PIR) cross-linkers, coupled with spectral counting-based quantification, was applied to microbe-enriched cellular fractions from both nymph and adult \u003cem\u003eD. citri\u003c/em\u003e to characterize in vivo protein interactions [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Among all post-translational modifications of proteins, phosphorylation is the most prevalent and dynamic. It plays a pivotal role in regulating a vast array of biological processes [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Quantitative phosphoproteomic analysis is widely used to comprehensively monitor global phosphorylation modifications of phosphoproteins in signaling pathways and to identify their phosphorylation sites under various conditions [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Meng et al. identified 1144 phosphoproteins from \u003cem\u003eChilo suppressalis\u003c/em\u003e treated with chlorantraniliprole and untreated controls, with the results being validated using parallel reaction monitoring (PRM) [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Although western blot is the gold standard for protein level verification, its application is often hindered by the lack of effective and specific antibodies against target proteins of interest. Currently, PRM has been widely applied in exploratory, large-scale, and precise quantification experiments, demonstrating significant advantages in quantitative analyses [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. A comprehensive understanding of the molecular mechanisms of \u003cem\u003eC\u003c/em\u003eLas infection in \u003cem\u003eD. citri\u003c/em\u003e is currently lacking, especially from an integrated proteomic and phosphoproteomic perspective.\u003c/p\u003e \u003cp\u003eThis study presents a comprehensive integration of proteomic and phosphoproteomic analyses to elucidate the molecular mechanisms of the \u003cem\u003eD. citri\u003c/em\u003e-\u003cem\u003eC\u003c/em\u003eLas interaction. We identified and validated DEPs and DPPs from \u003cem\u003eC\u003c/em\u003eLas-free and \u003cem\u003eC\u003c/em\u003eLas-infected \u003cem\u003eD. citri\u003c/em\u003e. Functional enrichment (GO/KEGG) and correlation analyses revealed key pathways and pinpointed seven high-confidence DEPs. Subsequent RNAi silencing of \u003cem\u003evitellogenin-A1\u003c/em\u003e and \u003cem\u003ealpha-crystallin A chain\u003c/em\u003e significantly reduced bacterial titers, validating their functional roles. These findings provide valuable insights into the interaction between \u003cem\u003eC\u003c/em\u003eLas bacteria and \u003cem\u003eD. citri\u003c/em\u003e.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\"\u003e\n \u003ch2\u003e2.1. \u003cem\u003eD. citri\u003c/em\u003e rearing and samples collection\u003c/h2\u003e\n \u003cp\u003e\u003cem\u003eD. citri\u003c/em\u003e was reared on \u003cem\u003eC\u003c/em\u003eLas-free \u003cem\u003ePonkan mandarin\u003c/em\u003e (Citrus reticulata) in insect rearing cages under controlled environmental conditions (27\u0026thinsp;\u0026plusmn;\u0026thinsp;1℃, 65\u0026thinsp;\u0026plusmn;\u0026thinsp;5% relative humidity, with a 12 h light: 12 h dark photoperiod), following an established protocol [\u003cspan\u003e33\u003c/span\u003e]. To standardize the experimental material, fifth-instar nymphs were transferred onto young shoots of \u003cem\u003eP. mandarin\u003c/em\u003e that were either \u003cem\u003eC\u003c/em\u003eLas-infected or \u003cem\u003eC\u003c/em\u003eLas-free. The infection status of all shoots was verified via polymerase chain reaction (PCR). After five days, heads of adult \u003cem\u003eD. citri\u003c/em\u003e were dissected, and genomic DNA was extracted using an Animal Tissue Direct PCR Kit (FOREGENE). PCR amplification targeting a 16S ribosomal DNA fragment with \u003cem\u003eC\u003c/em\u003eLas-specific primers OI1 and OI2c (Table \u003cspan\u003eS1\u003c/span\u003e) was performed to confirm \u003cem\u003eC\u003c/em\u003eLas infection in \u003cem\u003eD. citri\u003c/em\u003e, as previously described [\u003cspan\u003e5\u003c/span\u003e]. Subsequently, \u003cem\u003eC\u003c/em\u003eLas-free and \u003cem\u003eC\u003c/em\u003eLas-infected adult \u003cem\u003eD. citri\u003c/em\u003e were collected in RNase-free centrifuge tubes (Fig. \u003cspan\u003e1\u003c/span\u003e) and stored at -80 ℃. Each group consisted of three biological replicates.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\"\u003e\n \u003ch2\u003e2.2. Total protein extraction and trypsin digestion\u003c/h2\u003e\n \u003cp\u003eA total of 1800 \u003cem\u003eC\u003c/em\u003eLas-infected \u003cem\u003eD. citri\u003c/em\u003e adults were randomly divided into three groups and transferred to sterile centrifuge tubes. An equivalent number of \u003cem\u003eC\u003c/em\u003eLas-free \u003cem\u003eD. citri\u003c/em\u003e served as the control. Approximately 800 \u0026micro;L of SDT lysis buffer (4% SDS, 100 mM Tris-HCl, 1 mM DTT, pH 7.6) was added to each sample, followed by homogenization on ice using an electric homogenizer. After centrifugation at 12,000 \u0026times; g for 15 min at 4 ℃, the supernatant containing the protein fraction was collected from each sample, and the centrifugation step was repeated to maximize yield. Protein concentration was determined using a BCA Protein Assay Kit (Bio-Rad, USA). Protein digestion was then carried out with trypsin according to the filter-aided sample preparation (FASP) method described by Wisniewski et al. [\u003cspan\u003e34\u003c/span\u003e]. The resulting peptides from each sample were desalted using C18 Cartridges (Empore\u0026trade; SPE Cartridges C18, standard density, bed I.D. 7 mm, volume 3 mL; Sigma), concentrated by vacuum centrifugation, and reconstituted in 40 \u0026micro;l of 0.1% (v/v) formic acid.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\"\u003e\n \u003ch2\u003e2.3. 4D-Label-free quantitative proteomics analysis\u003c/h2\u003e\n \u003cdiv id=\"Sec6\"\u003e\n \u003ch2\u003e2.3.1. LC-MS/MS analysis\u003c/h2\u003e\n \u003cp\u003eLC-MS/MS analysis was conducted using a timsTOF Pro mass spectrometry (Bruker) coupled to a Nanoelute nanoflow UHPLC system (Bruker). Peptides were loaded onto a C18 reversed-phase analytical column (Thermo Scientific) equilibrated with 95% buffer A (0.1% formic acid) and separated with a linear gradient of buffer B (99.9% acetonitrile, 0.1% formic acid) at a flow rate of 300 nL/min. The mass spectrometer was operated in positive ion mode with an electrospray voltage of 1.5 kV. Precursor and fragment ions were analyzed by the TOF analyzer within a mass range of m/z 100\u0026ndash;1700. Data were acquired in parallel accumulation-serial fragmentation (PASEF) mode with the following settings: ion mobility coefficient (1/K0) of 0.6\u0026ndash;1.6 Vs/cm\u003csup\u003e2\u003c/sup\u003e, and each MS cycle comprising one full MS scan followed by 10 PASEF MS/MS scans. A dynamic exclusion time of 24 s was applied. The mass spectrometry proteomics data have been deposited to the iProX database (accession number: IPX0014247001).\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec7\"\u003e\n \u003ch2\u003e2.3.2. Identification and quantitation of proteins\u003c/h2\u003e\n \u003cp\u003eThe MS raw data from all samples were collectively processed using MaxQuant software (version 1.6.14) for protein identification. The mass tolerance for precursor ions was set to 20 ppm. Peptide spectrum matches (PSMs) with a confidence level exceeding 99% were considered credible. Only proteins containing at least one unique peptide were retained for subsequent analysis. Both protein and PSM identifications were filtered at a false discovery date (FDR) threshold of 0.01.\u003c/p\u003e\n \u003cp\u003eFor protein quantitation, the Mann-Whitney test was applied to compare the \u003cem\u003eC\u003c/em\u003eLas-free and \u003cem\u003eC\u003c/em\u003eLas-infected groups, with proteins having a \u003cem\u003eP\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and fold change (FC)\u0026thinsp;\u0026gt;\u0026thinsp;2 or \u0026lt;\u0026thinsp;0. 5 considered differentially expressed proteins (DEPs).\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\"\u003e\n \u003ch2\u003e2.4. 4D-Label-free phosphorproteomic analysis\u003c/h2\u003e\n \u003cdiv id=\"Sec9\"\u003e\n \u003ch2\u003e2.4.1. Phosphopeptides enrichment\u003c/h2\u003e\n \u003cp\u003eThe labeled samples were reconstituted in an IMAC-Fe column washing solution containing 250 mM acetic acid and 30% acetonitrile. All samples were centrifuged at 12000 \u0026times; g for 5 min at 4 ℃, and the resulting precipitates were collected. Phosphopeptide enrichment was performed using the High-Select\u003csup\u003e\u0026trade;\u003c/sup\u003e Fe-NTA Phosphopeptide Enrichment Kit (Thermo Scientific) following the manufacturer\u0026rsquo;s protocol. After lyophilization, the enriched phosphopeptides were redissolved in 20 \u0026micro;L of loading buffer (0.1% formic acid).\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec10\"\u003e\n \u003ch2\u003e2.4.2. LC-MS/MS analysis\u003c/h2\u003e\n \u003cp\u003eLC-MS/MS analysis was conducted as outlined in Section 2.3.1, with slight modifications. Briefly, peptides were loaded onto a C18 reversed-phase analytical column (25 cm \u0026times; 75 \u0026micro;m i.d., 1.9 \u0026micro;m particle size) and equilibrated with buffer A (0.1% formic acid). Separation was achieved using a linear gradient of buffer B (84% acetonitrile, 0.1% formic acid) at a flow rate of 300 nL/min. The mass spectrometer was operated in positive ion mode, acquiring MS spectra across an m/z range of 100\u0026ndash;1700 and an ion mobility (1/k0) range of 0.6\u0026ndash;1.6. Subsequently, up to 10 PASEF MS/MS scans were triggered with a target intensity of 1.5 k and an intensity threshold of 2500. Dynamic exclusion was activated with a release time of 0.4 min.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec11\"\u003e\n \u003ch2\u003e2.4.3. Identification and quantitation of phosphorylated proteins\u003c/h2\u003e\n \u003cp\u003eIdentification and quantitation of phosphorylated proteins was conducted as per Section 2.3.2, with the inclusion of variable modifications for phosphorylation on serine (S), threonine (T), and tyrosine (Y). The criteria for DPP quantitation and identification remained unchanged from the aforementioned section.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\"\u003e\n \u003ch2\u003e2.5. Bioinformatic analysis\u003c/h2\u003e\n \u003cp\u003eHierarchical clustering analysis was performed using Cluster 3.0 (\u003cspan\u003e\u003cspan\u003ehttp://bonsai.hgc.jp/~mdehoon/software/cluster/software.htm\u003c/span\u003e\u003c/span\u003e) and results were visualized as a heatmap with Java Treeview software (\u003cspan\u003e\u003cspan\u003ehttp://jtreeview.sourceforge.net\u003c/span\u003e\u003c/span\u003e). Motif analysis was carried out with the MEME Suite (\u003cspan\u003e\u003cspan\u003ehttp://meme-suite.org/index.htm\u003c/span\u003e\u003c/span\u003e), and protein subcellular localization was predicted using CELLO software (\u003cspan\u003e\u003cspan\u003ehttp://cello.life.nctu.edu.tw/\u003c/span\u003e\u003c/span\u003e). Protein domain signatures were identified by querying the Pfam database via InterProScan. Functional categorization of differentially expressed proteins (DEPs) was assessed through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. Enrichment used a Fisher\u0026apos;s exact test with the full set of quantified proteins as the background, and p-values were adjusted for multiple testing using the Benjamini-Hochberg method.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\"\u003e\n \u003ch2\u003e2.6. Validation of DEP by parallel reaction monitoring (PRM)\u003c/h2\u003e\n \u003cp\u003eParallel reaction monitoring (PRM) was employed to validate the DEPs identified by LC-MS/MS. A total of 14 target peptides, along with an isotopically labeled reference peptide (PRTC: ELGQSGVDTYLQTK) spiked into each sample, were analyzed. Analysis consisted of a full-scan event followed by PRM acquisition under the following parameters: resolution, 30,000 (at 200 m/z); automatic gain control (AGC) target, 5e4; maximum injection time, 80 ms; and normalized collision energy, 27%. The signal intensities of the target peptides were quantified and normalized against the internal standard.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\"\u003e\n \u003ch2\u003e2.7. RNA isolation, cDNA synthesis and RT-qPCR analysis\u003c/h2\u003e\n \u003cp\u003eTotal RNA was extracted from various \u003cem\u003eD. citri\u003c/em\u003e samples using TRIzol reagent (Takara, Kusatsu, Japan) following the manufacturer\u0026rsquo;s instructions. RNA quality was assessed by measuring concentration and purity on a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and evaluating integrity via 1% agarose gel electrophoresis. Only high-quality RNA samples (OD\u003csub\u003e260/280\u003c/sub\u003e: 1.9-2.0; OD\u003csub\u003e260/230\u003c/sub\u003e: \u0026ge;1.9) were qualified for cDNA synthesis.\u003c/p\u003e\n \u003cp\u003ecDNA was synthesized from 1 \u0026micro;g of total RNA using a cDNA synthesis kit (YEASEN, Shanghai, China) following the manufacturer\u0026apos;s protocol. Briefly, a 15 \u0026micro;L reaction mixture containing 3.0 \u0026micro;L of gDNA digester Mix and RNase-free water was incubated at 42 ℃ for 5 min to remove genomic DNA. Subsequently, 5 \u0026micro;L 4\u0026times;Hifair\u0026reg;Ⅲ SuperMix plus was added, and the reaction proceeded at 25\u0026deg;C for 5 min and 55\u0026deg;C for 15 min, followed by enzyme inactivation at 85\u0026deg;C for 5 min. The resulting cDNA was stored at \u0026minus;\u0026thinsp;20\u0026deg;C for subsequent use.\u003c/p\u003e\n \u003cp\u003eqPCR was performed on a Roche LightCycler 96 Automatic Analyzer (Roche Life Science) using the primers listed in Table \u003cspan\u003eS1\u003c/span\u003e. Each 20 \u0026micro;L reaction contained 10 \u0026micro;L SYBR Green Mix, 8 \u0026micro;L RNase-free water, 0.5 \u0026micro;L forward primer, 0.5 \u0026micro;L reverse primer, and 1.0 \u0026micro;L cDNA template. The thermal cycling protocol consisted of an initial denaturation at 95 ℃ for 10 min, followed by 40 cycles of 95 ℃ for 10 s, 60 ℃ for 10 s, and 72 ℃ for 15 s. Melting curve analysis was conducted to verify amplification specificity. The relative gene expression levels were calculated using the 2\u003csup\u003e\u0026minus;\u0026Delta;\u0026Delta;Ct\u003c/sup\u003e method, with \u003cem\u003eglyceraldehyde-3-phosphate dehydrogenase\u003c/em\u003e (\u003cem\u003eGAPDH\u003c/em\u003e) as the reference gene. All samples were amplified in triplicate.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\"\u003e\n \u003ch2\u003e2.8. dsRNA synthesis, microinjection and \u003cem\u003eC\u003c/em\u003eLas bacterial acquisition\u003c/h2\u003e\n \u003cp\u003eTo investigate the roles of vitellogenin-A1 (Vg-A1) and alpha-crystallin A chain (\u0026alpha;A-crystallin) in \u003cem\u003eC\u003c/em\u003eLas infection, we performed RNAi-mediated silencing of these genes in \u003cem\u003eD. citri\u003c/em\u003e. Double-stranded RNA (dsRNA) targeting \u003cem\u003eVg-A1\u003c/em\u003e (ds\u003cem\u003eVg-A1\u003c/em\u003e) and \u003cem\u003e\u0026alpha;A-crystallin\u003c/em\u003e (ds\u003cem\u003e\u0026alpha;A-crystallin\u003c/em\u003e), along with a control ds\u003cem\u003eGFP\u003c/em\u003e, were synthesized in vitro using the T7 RioMAXTM Express RNAi System (Promega, Madision, WI, USA) according to the manufacturer\u0026rsquo;s instructions. Gene-specific primers with T7 promoter sequences are listed in Table \u003cspan\u003eS1\u003c/span\u003e. The synthesized dsRNAs were diluted with RNase-free water containing 0.1% red food dye to working concentrations of 500 ng/\u0026micro;L (ds\u003cem\u003eVg-A1\u003c/em\u003e) and 1200 ng/\u0026micro;L (ds\u003cem\u003e\u0026alpha;A-crystallin\u003c/em\u003e).\u003c/p\u003e\n \u003cp\u003eFor microinjection, 5th-instar nymphs were immobilized with a dorsal sticker, and approximately 15 nL of the respective dsRNA solution was delivered into each insect. The injected nymphs were initially maintained on \u003cem\u003eC\u003c/em\u003eLas-free \u003cem\u003eMurraya exotica\u003c/em\u003e seedlings. After 48 h, silencing efficiency was verified by RT-qPCR, and the nymphs were subsequently transferred to the tender shoots of CLas-infected \u003cem\u003eC. sinensis\u003c/em\u003e for an acquisition access period. Surviving adults were collected at 24 h and 48 h post-acquisition and individually stored. The \u003cem\u003eC\u003c/em\u003eLas bacterial titer was quantified using a TaqMan-based qPCR assay targeting the CLas 16S rRNA gene, with primers and probe sequences provided in Table \u003cspan\u003eS1\u003c/span\u003e. All data were analyzed by one-way ANOVA in SPSS software, with significance levels set at \u003cem\u003eP\u003c/em\u003e-values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and \u0026lt;\u0026thinsp;0.01 were considered statistically significant and highly significant, respectively.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Identification and quantification of DEPs and DPPs\u003c/h2\u003e \u003cp\u003eA 4D-label-free quantitative proteomics analysis was conducted to identify DEPs and DPPs by comparing \u003cem\u003eC\u003c/em\u003eLas-free and \u003cem\u003eC\u003c/em\u003eLas-infected \u003cem\u003eD. citri\u003c/em\u003e. From the 468,270 spectrums acquired, 82213 were successfully matched, resulting in the identification of 20181 peptides (including 18,603 unique peptides) and 3,342 proteins. Subsequently, 3,310 quantifiable proteins were subjected to further differential analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Furthermore, a total of 5,911 phosphopeptides were identified, corresponding to 2,658 phosphoproteins and accounting for 11,521 quantified phosphorylation sites quantified (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). Differentially expressed and phosphorylated proteins (DEPs and DPPs) between \u003cem\u003eC\u003c/em\u003eLas-free and \u003cem\u003eC\u003c/em\u003eLas-infected \u003cem\u003eD. citri\u003c/em\u003e were identified using thresholds of a fold change\u0026thinsp;\u0026gt;\u0026thinsp;2 or \u0026lt;\u0026thinsp;0.5 and a \u003cem\u003eP\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The results revealed that 63 up- and 81 down-regulated DEPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eA), alongside 401 up- and 596 down-regulated DPPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD and Table \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003eA). Furthermore, 32 DEPs and 52 DPPs were identified exclusively in either \u003cem\u003eC\u003c/em\u003eLas-free or \u003cem\u003eC\u003c/em\u003eLas infected \u003cem\u003eD. citri\u003c/em\u003e from the proteomic and phosphoproteomic databasets, respectively (Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eB and Table \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003eB). Under a more stringent threshold (fold change\u0026thinsp;\u0026gt;\u0026thinsp;10 or \u0026lt;\u0026thinsp;0.1 and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), 5 DEPs (up-regulated) and 9 (down-regulated), as well as 21 (up-regulated) and 27 (down-regulated) DPPs, were identified (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Functional characterization and enrichment of DEPs and DPPs\u003c/h2\u003e \u003cp\u003eGene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and subcellular localization analyses were performed to elucidate the involved biological processes, molecular functions, and signaling pathways. Analysis of subcellular localization revealed that the majority of quantified proteins were distributed in the membrane and nucleus (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Gene Ontology (GO) analysis revealed distinct functional pattern for the DEPs. In biological processes (BP), they were primarily enriched in rRNA processing, rRNA metabolic processes and ncRNA processing. For molecular function (MF), the dominant terms were cofactor binding, monooxygenase activity and structural constituent of cuticle. Regarding cellular component (CC), the DEPs were predominantly localized to the nucleolus and nuclear lumen (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Domain enrichment analysis revealed distinct profiles for DEPs and DPPs. The most significantly enriched domains were insect cuticle protein and cytochrome P450 for DEPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), and the LIM domain and Immunoglobulin I-set domain for DPPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Motif analysis of the phosphorylated peptides identified 739 and 439 peptides containing the conserved _S_P_ motifs, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Subcellular localization analysis revealed that the majority of DPPs were distributed in the membrane and nucleus (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). GO enrichment analysis indicated that these DPPs are primarily associated with functions such as acting binding, calcium ion binding and the actin cytoskeleton (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). KEGG enrichment analysis further demonstrated that DEPs were predominantly enriched in ABC transporters, whereas DPPs were mainly involved in the calcium signaling pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Validation of DEPs by PRM\u003c/h2\u003e \u003cp\u003eTo verify the DEPs identified by proteomic analysis, thirteen candidates were selected for parallel reaction (PRM) validation, including LOC103507240, alpha-crystallin A chain (αA-crystallin), cuticle protein 12.5 (CP12.5), phosphoenolpyruvate carboxykinase (PEPCK), clustered mitochondria protein (CMP), E3 ubiquitin ligase PARAQUAT TOLERANCE3 (PQT3), indole-3-acetaldehyde oxidase (IAAld-oxidase), cuticle protein 16.5 (CP16.5), dolichyl-diphosphooligosaccharide-protein glycotransferase (DDOST), mitochondrial protein/calcium exchanger (MCX), vitellogenin-A1 (Vg-A1), ATP-binding cassette sub-family B membrane 3 (ABCB3) and cathepsin L-like (CathL). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, the expression of LOC103507240, CP12.5, IAAld-oxidase, CP16.5 and CathL was downregulated in \u003cem\u003eD. citri\u003c/em\u003e following \u003cem\u003eC\u003c/em\u003eLas infection, whereas those of αA-crystallin, CMP, PQT3, MCX, Vg-A1 and ABCB3 was significantly upregulated. The PRM results were highly consistent with the 4D-Label-free proteomics data, confirming the reliability of our proteomics findings.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Correlation analysis between DEPs and DPPs, and RT-qPCR validation\u003c/h2\u003e \u003cp\u003eThe elucidate the roles of DEPs and DPPs in \u003cem\u003eD. citri\u003c/em\u003e following \u003cem\u003eC\u003c/em\u003eLas infection, we performed an integrative analysis combining 4D label-free quantitative proteomics and phosphoproteomics data. The results revealed 24 DEPs and 228 DPPs, among which 7 proteins exhibited concurrent alterations at both the expression and phosphorylation levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). GO enrichment analysis indicated that in the BP category, DPPs were primarily involved in lipid transport and localization, whereas DEPs were associated with rRNA processing. In the CC category, DPPs were predominantly localized to the actin cytoskeleton and myosin complex, while DEPs were enriched in the nuclear lumen. Regarding MF, DPPs were mainly implicated in calcium ion binding and actin binding, and DEPs were related to monooxygenase activity and structural constituents of the cuticle (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). KEGG analysis further showed that DPPs were significantly enriched in the calcium signaling pathway, apelin signaling pathway, and cellular senescence, whereas DEPs were primarily involved in ABC transporters (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe expression levels of the seven co-expressed proteins identified among the DEPs and DPPs were validated using RT-qPCR. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e, four of them (Vg-A1, αA-crystallin, facilitated trehalose transporter Tret1, and LOC103509854) were significantly upregulated in \u003cem\u003eD. citri\u003c/em\u003e following \u003cem\u003eC\u003c/em\u003eLas infection, and three proteins (zinc finger protein 319, LOC113471498 and protein argonaute-2) were downregulated, a trend which aligned with the proteomics data. According to the phosphoproteomic dataset, both vitellogenin-A1 and protein argonaute-2 possessed two phosphorylated peptides, and their expression trends were consistent with the proteomic results. In contrast, the remaining five proteins each contained only a single phosphorylated peptide and showed opposing trends to the proteomics data (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Furthermore, we also found that α-crystallin A was expressed only in the \u003cem\u003eC\u003c/em\u003eLas-infected groups, while Tret1 was specific to the \u003cem\u003eC\u003c/em\u003eLas-free groups (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eOverlap of co-expressed proteins between quantitative proteomics and phosphoproteomics\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eProtein name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProteomics\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePhosphoproteomics\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eC\u003c/em\u003eLas\u003csup\u003e+\u003c/sup\u003e/\u003cem\u003eC\u003c/em\u003eLas\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eC\u003c/em\u003eLas\u003csup\u003e+\u003c/sup\u003e/\u003cem\u003eC\u003c/em\u003eLas\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eVitellogenin-A1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e3.7514\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDNDYNDDDQKNHQNS(+\u0026thinsp;79.97)GSHNNNNHHNSGSNDNK (8.2937)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEFFNLATSSQVT(+\u0026thinsp;79.97)K (3.6146)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eα-crystallin A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.5374\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLS(+\u0026thinsp;79.97)SDGILSIQAPK (0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFacilitated trehalose transporter Tret1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.1298\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVFTVEEGTVT(+\u0026thinsp;79.97)Q (\u0026infin;)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLOC103509854\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.0714\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eS(+\u0026thinsp;79.97)VLLENENVQAISASR (0.1767)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZinc finger protein 319\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.4977\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eKTTVSTPSTPT(+\u0026thinsp;79.97)AASAPVAQPTPPPQQNIVR (4.0917)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLOC113471498\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.2281\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNEPPEDS(+\u0026thinsp;79.97)NDLTNEQR (0.2317)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eProtein argonaute-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.0627\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMTIASSSS(+\u0026thinsp;79.97)SSSISSAASGAGSK (0.1215)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMTIASS(+\u0026thinsp;79.97)SSSSSISSAASGAGSK (0.0606)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Silencing of \u003cem\u003evitellogenin-A1\u003c/em\u003e and \u003cem\u003ealpha-crystallin A chain\u003c/em\u003e effects on \u003cem\u003eC\u003c/em\u003eLas content in \u003cem\u003eD. citri\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eBased on the correlation analysis between 4D-label-free quantitative proteomics and phosphoproteomics, two proteins-αA-crystallin and VgA1-were selected for functional validation of their roles in \u003cem\u003eC\u003c/em\u003eLas proliferation in \u003cem\u003eD. citri\u003c/em\u003e. The results showed that the expression level of \u003cem\u003eVg-A1\u003c/em\u003e was significantly downregulated at 48 h after ds\u003cem\u003eVg-A1\u003c/em\u003e injection, while it had no obvious difference at 24 h. In contrast, the relative expression of \u003cem\u003eαA-crystallin\u003c/em\u003e was significantly downregulated at both 24 h and 48 h after ds\u003cem\u003eαA-crystallin\u003c/em\u003e injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA and \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eC). Fifth-instar nymphs treated with ds\u003cem\u003eGFP\u003c/em\u003e, ds\u003cem\u003eVg-A1\u003c/em\u003e, or ds\u003cem\u003eαA-crystallin\u003c/em\u003e were subsequently released onto \u003cem\u003eC\u003c/em\u003eLas-infected citrus tender tips. The \u003cem\u003eC\u003c/em\u003eLas bacterial content was significantly reduced following the silencing of either \u003cem\u003eαA-crystallin\u003c/em\u003e or \u003cem\u003eVgA1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB and \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eD). These results suggest that both αA-crystallin and Vg-A1 might be involved in \u003cem\u003eC\u003c/em\u003eLas infection in \u003cem\u003eD. citri\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eTo date, the \u003cem\u003eC\u003c/em\u003eLas bacteria has not been successfully cultured \u003cem\u003ein vitro\u003c/em\u003e, yet it efficiently infects \u003cem\u003eD. citri\u003c/em\u003e and proliferates persistently within host cells, facilitating its widespread transmission. This poses a major challenge for the management of HLB. When \u003cem\u003eD. citri\u003c/em\u003e feeds on HLB-infected citrus, \u003cem\u003eC\u003c/em\u003eLas enters through the stylet's food canal and reaches the gut lumen. \u003cem\u003eD. citri\u003c/em\u003e midgut serves as a critical barrier against \u003cem\u003eC\u003c/em\u003eLas invasion and proliferation. To investigate the host\u0026ndash;pathogen interactions at this gut interface, RNA-seq and proteomic analyses were conducted. The results indicated that \u003cem\u003eC\u003c/em\u003eLas infection alters several biological pathways, including the TCA cycle, iron metabolism, insecticide resistance, and the insect immune system [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. After crossing the midgut, \u003cem\u003eC\u003c/em\u003eLas disseminates via the hemolymph to the salivary glands. The hemolymph also acts as a primary site for initiating cellular and humoral immune responses to cuticular damage and pathogen invasion. Using nano-LC-MS/MS, 5531 and 3220 peptides were identified in the hemolymph of \u003cem\u003eC\u003c/em\u003eLas-infected and uninfected \u003cem\u003eD. citri\u003c/em\u003e, respectively. Notably, a large number of immune defense proteins were absent from the hemolymph of \u003cem\u003eD. citri\u003c/em\u003e [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In our previous research, integrated ubiquitylome and proteome analyses revealed that cytoskeleton-related proteins undergo ubiquitination and play critical roles during \u003cem\u003eC\u003c/em\u003eLas infection [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. However, phosphorylation-related proteins had not yet been systematically compared between \u003cem\u003eC\u003c/em\u003eLas-infected and \u003cem\u003eC\u003c/em\u003eLas-free \u003cem\u003eD. citri\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eDuring bacterial infection, pathogens often manipulate host protein expression to facilitate their own replication through various strategies, including post-translational modifications (PTMs) such as phosphorylation, ribosylation, and acetylation. These modifications serve as rapid and dynamic regulators of protein function [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Among them, protein phosphorylation is widely recognized as a key mechanism in cellular regulation and signaling [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In this study, we applied 4D-label free quantitative proteomics and phosphoproteomics to investigate the role of phosphorylated proteins in \u003cem\u003eD. citri\u003c/em\u003e upon \u003cem\u003eC\u003c/em\u003eLas infection. We identified 144 DEPs and 997 DPPs. Domain enrichment analysis revealed that most DEPs were associated with insect cuticle proteins and cytochrome P450 enzymes. Cuticle proteins (CPs) are major structural components of the insect cuticle and are essential for maintaining its integrity and stability [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Here, nine differentially expressed CPs were identified, all of which were significantly downregulated in \u003cem\u003eC\u003c/em\u003eLas-infected \u003cem\u003eD. citri\u003c/em\u003e. This aligns with previous findings by Yuan et al., who reported that most CP genes were downregulated in \u003cem\u003eC\u003c/em\u003eLas-infected \u003cem\u003eD. citri\u003c/em\u003e, suggesting that \u003cem\u003eC\u003c/em\u003eLas may suppress cuticle formation to facilitate its own proliferation [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Additionally, five P450-related proteins-CYP4C4, CYP4g15-2, CYP6k1-like isoform X1, CYP4g15 and CYP4g15-were significantly upregulated in \u003cem\u003eC\u003c/em\u003eLas-infected \u003cem\u003eD. citri\u003c/em\u003e. Cytochromes P450 enzymes are critical in the oxidative metabolism of both endogenous and exogenous compounds [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. However, conflicting results were reported by Tiwari et al., who observed higher expression of four CYP4 genes (\u003cem\u003eCYP4DA1\u003c/em\u003e, \u003cem\u003eCYP4C68\u003c/em\u003e, \u003cem\u003eCYP4G70\u003c/em\u003e, and \u003cem\u003eCYP4DB1\u003c/em\u003e) in uninfected compared to \u003cem\u003eC\u003c/em\u003eLas-infected \u003cem\u003eD. citri\u003c/em\u003e [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], indicating a complex role of P450s in \u003cem\u003eD. citri\u003c/em\u003e response to \u003cem\u003eC\u003c/em\u003eLas infection. Domain analysis further revealed that eight DPPs contained LIM domains, which are known to mediate interactions between the actin cytoskeleton and transcriptional machinery [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Among these, four DPPs (PDZ and LIM domain protein Zasp-like, LIM and actin-binding protein 1, Muscle LIM protein 1, and actin-binding LIM protein 1) were upregulated, while the other four (Four and a half LIM domains protein 2 isoforms X1 and X4, PDZ and LIM domain protein Zasp-like, and LIM domain-containing protein jub-like) were downregulated. Our recent unpublished data suggest that the \u0026ldquo;four and a half LIM domains protein 2\u0026rdquo; is involved in \u003cem\u003eC\u003c/em\u003eLas proliferation, indicating that LIM domain-containing phosphoproteins may participate in \u003cem\u003eC\u003c/em\u003eLas infection.\u003c/p\u003e \u003cp\u003eTo further elucidate the functional relevance of phosphorylated proteins, we integrated data from 4D-label free quantitative proteomics and phosphoproteomics. Seven overlapping proteins were identified: vitellogenin-A1, alpha-crystallin A chain, facilitated trehalose transporter Tret1, LOC103509854, zinc finger protein 319, LOC113471498, and protein argonaute-2. RT-qPCR validation showed that the \u003cem\u003eVg-A1\u003c/em\u003e, \u003cem\u003eα-crystallin A\u003c/em\u003e, \u003cem\u003eTret\u003c/em\u003e1 and \u003cem\u003eLOC103509854\u003c/em\u003e were significantly upregulated in \u003cem\u003eC\u003c/em\u003eLas-infected \u003cem\u003eD. citri\u003c/em\u003e, whereas \u003cem\u003ezinc finger protein 319\u003c/em\u003e (\u003cem\u003eZFP319\u003c/em\u003e), \u003cem\u003eLOC113471498\u003c/em\u003e, and \u003cem\u003eargonaute-2\u003c/em\u003e (\u003cem\u003eAgo-2\u003c/em\u003e) were downregulated. Furthermore, silencing of \u003cem\u003eVg-A1\u003c/em\u003e significantly suppressed \u003cem\u003eC\u003c/em\u003eLas proliferation, indicating its involvement in \u003cem\u003eC\u003c/em\u003eLas infection. In insects, vitellogenesis are essential for yolk formation and embryonic development [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Multiple studies have reported that \u003cem\u003eC\u003c/em\u003eLas infection significantly upregulates egg development-related genes such as \u003cem\u003evitellogenin 1-like\u003c/em\u003e (\u003cem\u003eVg-1-like\u003c/em\u003e), \u003cem\u003evitellogenin A1-like\u003c/em\u003e (\u003cem\u003eVg-A1-like\u003c/em\u003e) and the \u003cem\u003evitellogenin receptor\u003c/em\u003e (\u003cem\u003eVgR\u003c/em\u003e) [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Jaiswal et al. also found upregulation of vitellogenin-1 and vitellogenin-2 by 18.4- and 17.0-fold, respectively, in \u003cem\u003eC\u003c/em\u003eLas-infected \u003cem\u003eD. citri\u003c/em\u003e [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], underscoring the potential role of Vg-A1 in host response. The α-crystallin, a small heat shock protein with chaperone-like activity, typically consists of αA and αB subunits and helps prevent protein misfolding and aggregation [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. We found that inhibition of \u003cem\u003eα-crystallin A\u003c/em\u003e significantly reduced \u003cem\u003eC\u003c/em\u003eLas titers in \u003cem\u003eD. citri\u003c/em\u003e, suggesting its involvement in \u003cem\u003eC\u003c/em\u003eLas infection dynamics.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn conclusion, differentially expressed proteins and differentially phosphorylated proteins were identified by 4D label-free quantitative proteomics and phosphoproteomics. GO and KEGG were performed to reveal the functions of these proteins. Integrated proteomic and phosphoproteomic analyses identified seven co-expressed proteins, and silencing of \u003cem\u003eVg-A1\u003c/em\u003e and \u003cem\u003eα-crystallin A\u003c/em\u003e chain significantly reduced \u003cem\u003eC\u003c/em\u003eLas content in \u003cem\u003eD. citri\u003c/em\u003e. This study provides initial insights into the role of protein phosphorylation in mediating the interaction between \u003cem\u003eC\u003c/em\u003eLas and \u003cem\u003eD. citri\u003c/em\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research was funded by the Natural Science Foundation of Jiangxi Province (20224BAB205012), National Natural Science Foundation of China (302260674, 32272549, 32560693) and Gan Poyang Talent Support Program-Training program for academic and technical leaders in major disciplines (20232BCJ23030).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eZ.L. and H.Y. designed this project; Z.S., Y.Z., and J.X. conducted the experiments; J.F. was involved in the recruitment of participants; Z.S., Y.Z. and H.Y. analyzed the data; Z.L. and H.Y. prepared the initial draft of the manuscript; and all authors have reviewed and approved the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLi J, He PF, H PB, Li YM, Wu YX, Lu ZJ, et al. 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Int J Mol Sci. 2022; 23(16):9347.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Diaphorina citri, Proteomics, Phosphorproteomics, Parallel reaction monitoring, CLas pathogen","lastPublishedDoi":"10.21203/rs.3.rs-7919843/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7919843/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCitrus Huanglongbing (HLB), a severe and destructive plant disease caused by the Gram-negative, phloem-limited bacterium \u0026ldquo;\u003cem\u003eCandidatus\u003c/em\u003e Liberibacter asiaticus (\u003cem\u003eC\u003c/em\u003eLas)\u0026rdquo; and transmitted by \u003cem\u003eDiaphorina citri\u003c/em\u003e, has been extensively studied. Previous studies have reported that protein post-translational modifications play a crucial role in \u003cem\u003eD. citri\u003c/em\u003e response to \u003cem\u003eC\u003c/em\u003eLas infection. However, comprehensive phosphoproteomic profiling of \u003cem\u003eD. citri\u003c/em\u003e induced by \u003cem\u003eC\u003c/em\u003eLas remains underexplored. In this study, a total of 144 differentially expressed proteins (DEPs) and 997 differentially phosphorylated proteins (DPPs) were identified by 4D label-free quantitative proteomics and phosphoproteomics. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that DEPs were mainly associated with molecular binding, structural constituent of cuticle and cytochrome P450, whereas DPPs were predominately involved in acting and calcium binding. A total of thirteen proteins were selected for parallel reaction monitoring (PRM) analysis to validate the reliability of proteomics. Integrated proteomic and phosphoproteomic analyses identified seven co-expressed proteins: vitellogenin-A1 (Vg-A1), alpha-crystallin A chain (αA- crystallin), facilitated trehalose transporter Tret1 (Tret1), LOC103509854, zinc finger protein 319 (ZFP319), LOC113471498 and Protein argonaute-2 (Ago-2). Furthermore, RNA interference (RNAi)-mediated knockdown of \u003cem\u003evitellogenin-A1\u003c/em\u003e and \u003cem\u003ealpha-crystallin A chain\u003c/em\u003e significantly reduced \u003cem\u003eC\u003c/em\u003eLas content in \u003cem\u003eD. citri\u003c/em\u003e. In conclusion, this study provides the most comprehensive phosphorylation profiles of \u003cem\u003eD. citri\u003c/em\u003e in response to \u003cem\u003eC\u003c/em\u003eLas infection and identifies two potential targets implicated in \u003cem\u003eC\u003c/em\u003eLas infection.\u003c/p\u003e","manuscriptTitle":"An integrated proteomic and phosphoproteomic analysis reveals α-crystallin A and vitellogenin A1 as key players involved in CLas infection in Diaphorina citri","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-30 14:41:18","doi":"10.21203/rs.3.rs-7919843/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-23T10:02:30+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-22T18:04:06+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-12T15:05:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"39205250336790492828775724754883316117","date":"2026-03-23T18:40:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"246247536952600951643447734228657331447","date":"2026-03-23T06:23:01+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-14T14:07:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"81475063241669474707221493654737770214","date":"2026-01-30T02:40:20+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-27T18:58:44+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-23T19:57:33+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-22T09:05:03+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Genomics","date":"2025-12-22T08:53:26+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9bb7d17f-c9e2-4045-b372-d8e71e7e7c88","owner":[],"postedDate":"January 30th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-04-23T10:09:58+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-30 14:41:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7919843","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7919843","identity":"rs-7919843","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}