5-Azacytidine accelerates mandarin fruit post-ripening and enhances lignin-based pathogen defense through remarkable gene expression activation.

5-Azacytidine (AZ) is a DNA methylation inhibitor that has recently demonstrated potential in regulating fruit quality through exogenous application. In this study, we treated mandarin fruits for 4-day storage. Noteworthy were the induced degreening and the enhanced citrus aroma of fruits under AZ treatment, involving the promotion of chlorophyll degradation, carotenoid biosynthesis, and limonene biosynthesis. Key genes associated with these processes exhibited expression level increases of up to 123.8 times. Additionally, AZ treatment activated defense-related enzymes and altered phenylpropanoid carbon allocation towards lignin biosynthesis instead of flavonoid biosynthesis. The expression levels of lignin biosynthesis-related genes increased by nearly 100 times, leading to fortified lignin that is crucial for citrus defense against Penicillium italicum. Currently, the underlying mechanisms of such intense AZ-induced changes in gene expressions remain unclear and further research could help establish AZ treatment as a viable strategy for citrus preservation.

A Combinatorial Single-Molecule Real-Time and Illumina Sequencing Analysis of Postembryonic Gene Expression in the Asian Citrus Psyllid Diaphorina citri.

Huanglongbing (HLB) is a systemic plant disease caused by 'Candidatus Liberibacter asiaticus (CLas)' and transmitted by Diaphorina citri. D. citri acquires the CLas bacteria in the nymph stage and transmits it in the adult stage, indicating that molting from the nymph to adult stages is crucial for HLB transmission. However, the available D. citri reference genomes are incomplete, and gene function studies have been limited to date. In the current research, PacBio single-molecule real-time (SMRT) and Illumina sequencing were performed to investigate the transcriptome of D. citri nymphs and adults. In total, 10,641 full-length, non-redundant transcripts (FLNRTs), 594 alternative splicing (AS) events, 4522 simple sequence repeats (SSRs), 1086 long-coding RNAs (lncRNAs), 281 transcription factors (TFs), and 4459 APA sites were identified. Furthermore, 3746 differentially expressed genes (DEGs) between nymphs and adults were identified, among which 30 DEGs involved in the Hippo signaling pathway were found. Reverse transcription-quantitative PCR (RT-qPCR) further validated the expression levels of 12 DEGs and showed a positive correlation with transcriptome data. Finally, the spatiotemporal expression pattern of genes involved in the Hippo signaling pathway exhibited high expression in the D. citri testis, ovary, and egg. Silencing of the D. citri transcriptional co-activator (DcYki) gene significantly increased D. citri mortality and decreased the cumulative molting. Our results provide useful information and a reliable data resource for gene function research of D. citri.

C-X-C Motif Chemokine Ligand 1 Promotes Colitis by Modulating the Gut Microbiota.

INTRODUCTION: C-X-C motif chemokine ligand 1 (CXCL1) is a potent neutrophil chemoattractant that plays a pivotal role in recruiting neutrophils during inflammatory conditions. This study explored the role of CXCL1 in modulating the gut microbiota, influencing neutrophil infiltration, and contributing to the development of colitis. METHODS: We employed quantitative PCR to assess CXCL1 expression in colon samples. A mouse model of dextran sulfate sodium (DSS)-induced colitis was utilized to explore the progression of colitis in wild-type (WT) and CXCL1-deficient (CXCL1-/-) mice. RESULTS: Colitis attenuation was evident in CXCL1-/- mice. Significant alterations were observed in the gut microbiome, as revealed by 16S rRNA gene sequencing. Furthermore, CXCL1-/- mice exhibited reduced gut permeability and diminished endotoxin levels in peripheral blood following DSS treatment compared to WT mice. In response to DSS treatment, WT mice showed a clear increase in neutrophil infiltration, while CXCL1-/- mice exhibited lower levels of infiltration. Fecal microbiota transplantation (FMT) using stools from CXCL1-/- mice alleviated DSS-induced colitis. Interestingly, FMT from patients with colitis increased CXCL1 and Ly6G expression in the colons of gut-sterilized mice. Clinical data analysis revealed elevated CXCL1 and CD15 expression in patients with colitis, with a positive correlation between the severity of colitis and the expression of CXCL1 and CD15. CONCLUSION: These findings shed light on the pivotal role of CXCL1 in promoting colitis by modulating the gut microbiota.

Serratia marcescens induces apoptosis in Diaphorina citri gut cells via reactive oxygen species-mediated oxidative stress.

BACKGROUND: Asian citrus psyllid, Diaphorina citri, is a notorious pest in the citrus industry because it transmits Candidatus Liberibacter asiaticus, which causes an uncurable, devastating disease in citrus worldwide. Serratia marcescens is widely distributed in various environments that exhibits toxic effects to many insects. To develop strategies for enhancing the efficiency of pathogen-induced host mortality, a better understanding of the toxicity mechanism of Serratia marcescens on Diaphorina citri is critical. RESULTS: Serratia marcescens KH-001 successfully colonized Diaphorina citri gut by feeding artificial diets, resulting in the damage of cells including nucleus, mitochondria, vesicles, and microvilli. Oral ingestion of Serratia marcescens KH-001 strongly induced apoptosis in gut cells by enhancing levels of Cyt c, p53 and caspase-1 and decreasing levels of inhibitors of apoptosis (IAP) and Bax inhibitor-1 (BI-1). The expression of dual oxidase (Duox) and nitric oxide synthase (Nos) was up-regulated by Serratia marcescens KH-001, which increased hydrogen peroxide (H2 O2 ) levels in the gut. Injection of abdomen of Diaphorina citri with H2 O2 accelerated the death of the adults and induced apoptosis in the gut cells by activating Cyt c, p53 and caspase-1 and suppressing IAP and BI-1. Pretreatment of infected Diaphorina citri with vitamin c (Vc) increased the adult survival and diminished the apoptosis-inducing effect. CONCLUSIONS: The colonization of Serratia marcescens KH-001 in the guts of Diaphorina citri increased H2 O2 accumulation, leading to severe changes and apoptosis in intestinal cells, which enhanced a higher mortality level of D. citr. This study identifies the underlying virulence mechanism of Serratia marcescens KH-001 on Diaphorina citri that contributes to a widespread application in the integrated management of citrus psyllid. © 2023 Society of Chemical Industry.

Erratum: Citrus genomic resources unravel putative genetic determinants of Huanglongbing pathogenicity.

[This corrects the article DOI: 10.1016/j.isci.2023.106024.].

Natural Coumarin Shows Toxicity to Spodoptera litura by Inhibiting Detoxification Enzymes and Glycometabolism.

Coumarin and its derivatives are plant-derived compounds that exhibit potent insecticidal properties. In this study, we found that natural coumarin significantly inhibited the growth and development of Spodoptera litura larvae through toxicological assay. By transcriptomic sequencing, 80 and 45 differentially expressed genes (DEGs) related to detoxification were identified from 0 to 24 h and 24 to 48 h in S. litura after coumarin treatment, respectively. Enzyme activity analysis showed that CYP450 and acetylcholinesterase (AChE) activities significantly decreased at 48 h after coumarin treatment, while glutathione S-transferases (GST) activity increased at 24 h. Silencing of SlCYP324A16 gene by RNA interference significantly increased S. litura larval mortality and decreased individual weight after treatment with coumarin. Additionally, the expression levels of DEGs involved in glycolysis and tricarboxylic acid (TCA) cycle were inhibited at 24 h after coumarin treatment, while their expression levels were upregulated at 48 h. Furthermore, metabonomics analysis identified 391 differential metabolites involved in purine metabolism, amino acid metabolism, and TCA cycle from 0 to 24 h after treated with coumarin and 352 differential metabolites associated with ATP-binding cassette (ABC) transporters and amino acid metabolism. These results provide an in-depth understanding of the toxicological mechanism of coumarin on S. litura.

Alternative splicing of chitin deacetylase 2 regulates chitin and fatty acid metabolism in Asian citrus psyllid, Diaphorina citri.

Chitin plays an important role in the development and molting of insects. The key genes involved in chitin metabolism were considered promising targets for pest control. In this study, two splice variants of chitin deacetylase 2 (CDA2) from Diaphorina citri were identified, including DcCDA2a and DcCDA2b. Bioinformatics analysis revealed that DcCDA2a and DcCDA2b encoded 550 and 544 amino acid residues with a signal peptide, respectively. Spatio-temporal expression patterns analysis showed that DcCDA2a and DcCDA2b were highly expressed in D. citri wing and nymph stages. Moreover, DcCDA2a and DcCDA2b expression levels were induced by 20-hydroxyecdysone (20E). Silencing DcCDA2a by RNA interference (RNAi) significantly disrupted the D. citri molting and increased D. citri mortality and malformation rate, whereas inhibition of DcCDA2b resulted in a semimolting phenotype. Furthermore, silencing DcCDA2a and DcCDA2b significantly suppressed D. citri chitin and fatty acid metabolism. Our results indicated that DcCDA2 might play crucial roles in regulating D. citri chitin and fatty acid metabolism, and it could be used as a potential target for controlling D. citri.

New Insights into the Plutella xylostella Detoxifying Enzymes: Sequence Evolution, Structural Similarity, Functional Diversity, and Application Prospects of Glucosinolate Sulfatases.

Brassica plants have glucosinolate (GLs)-myrosinase defense mechanisms to deter herbivores. However, Plutella xylostella specifically feeds on Brassica vegetables. The larvae possess three glucosinolate sulfatases (PxGSS1-3) that compete with plant myrosinase for shared GLs substrates and produce nontoxic desulfo-GLs (deGLs). Although PxGSSs are considered potential targets for pest control, the lack of a comprehensive review has hindered the development of PxGSSs-targeted pest control methods. Recent advances in integrative multi-omics analysis, substrate-enzyme kinetics, and molecular biological techniques have elucidated the evolutionary origin and functional diversity of these three PxGSSs. This review summarizes research progress on PxGSSs over the past 20 years, covering sequence properties, evolution, protein modification, enzyme activity, structural variation, substrate specificity, and interaction scenarios based on functional diversity. Finally, we discussed the potential applications of PxGSSs-targeted pest control technologies driven by artificial intelligence, including CRISPR/Cas9-mediated gene drive, transgenic plant-mediated RNAi, small-molecule inhibitors, and peptide inhibitors. These technologies have the potential to overcome current management challenges and promote the development and field application of PxGSSs-targeted pest control.

Sex-specific transcription and DNA methylation landscapes of the Asian citrus psyllid, a vector of huanglongbing pathogens.

The relationship of DNA methylation and sex-biased gene expression is of high interest, it allows research into mechanisms of sexual dimorphism and the development of potential novel strategies for insect pest control. The Asian citrus psyllid, Diaphorina citri Kuwayama, is a major vector for the causative agents of Huanglongbing (HLB), which presents an unparalleled challenge to citrus production worldwide. Here, we identify the X chromosome of D. citri and investigate differences in the transcription and DNA methylation landscapes between adult virgin males and females. We find a large number of male-biased genes on the autosomes and a depletion of such on the X chromosome. We have also characterized the methylome of D. citri, finding low genome-wide levels, which is unusual for an hemipteran species, as well as evidence for both promoter and TE methylation. Overall, DNA methylation profiles are similar between the sexes but with a small number of differentially methylated genes found to be involved in sex differentiation. There also appears to be no direct relationship between differential DNA methylation and differential gene expression. Our findings lay the groundwork for the development of novel epigenetic-based pest control methods, and given the similarity of the D. citri methylome to some other insect species, these methods could be applicable across agricultural insect pests.

Citrus genomic resources unravel putative genetic determinants of Huanglongbing pathogenicity.

Citrus HLB caused by Candidatus Liberibacter asiaticus is a pathogen-triggered immune disease. Here, we identified putative genetic determinants of HLB pathogenicity by integrating citrus genomic resources to characterize the pan-genome of accessions that differ in their response to HLB. Genome-wide association mapping and analysis of allele-specific expression between susceptible, tolerant, and resistant accessions further refined candidates underlying the response to HLB. We first developed a phased diploid assembly of Citrus sinensis 'Newhall' genome and produced resequencing data for 91 citrus accessions that differ in their response to HLB. These data were combined with previous resequencing data from 356 accessions for genome-wide association mapping of the HLB response. Genes determinants for HLB pathogenicity were associated with host immune response, ROS production, and antioxidants. Overall, this study has provided a significant resource of citrus genomic data and identified candidate genes to be further explored to understand the genetic determinants of HLB pathogenicity.

Quantitative ubiquitylome crosstalk with proteome analysis revealed cytoskeleton proteins influence CLas pathogen infection in Diaphorina citri.

Huanglongbing (HLB), also known as citrus greening disease, is caused by Candidatus Liberbacter asiaticus (CLas) and transmitted by Diaphorina citri. Previous studies reported that CLas infection significantly influences the structure of the D. citri cytoskeleton. However, the mechanisms through which CLas manipulates cytoskeleton-related proteins remain unclear. In this study, we performed quantitative ubiquitylome crosstalk with the proteome to reveal the roles of cytoskeleton-related proteins during the infection of D. citri by CLas. Western blotting revealed a significant difference in ubiquitination levels between the CLas-free and CLas-infected groups. According to ubiquitylome and 4D label-free proteome analysis, 343 quantified lysine ubiquitination (Kub) sites and 666 differentially expressed proteins (DEPs) were identified in CLas-infected groups compared with CLas-free groups. A total of 53 sites in 51 DEPs were upregulated, while 290 sites in 192 DEPs were downregulated. Furthermore, functional enrichment analysis indicated that 18 DEPs and 21 lysine ubiquitinated proteins were associated with the cytoskeleton, showing an obvious interaction. Ubiquitination of D. citri tropomyosin was confirmed by immunoprecipitation, Western blotting, and LC-MS/MS. RNAi-mediated knockdown of tropomyosin significantly increased CLas bacterial content in D. citri. In summary, we provided the most comprehensive lysine ubiquitinome analysis of the D. citri response to CLas infection, thus furthering our understanding of the role of the ubiquitination of cytoskeleton proteins in CLas infection.

Functionally Collaborative Nanostructure for Direct Monitoring of Neurotransmitter Exocytosis in Living Cells.

Neurotransmitter exocytosis of living cells plays a vital role in neuroscience. However, the available amperometric technique with carbon fiber electrodes typically measures exocytotic events from one cell during one procedure, which requires professional operations and takes time to produce statistical results of multiple cells. Here, we develop a functionally collaborative nanostructure to directly measure the neurotransmitter dopamine (DA) exocytosis from living rat pheochromocytoma (PC12) cells. The functionally collaborative nanostructure is constructed of metal-organic framework (MOF)-on-nanowires-on-graphene oxide, which is highly sensitive to DA molecules and enables direct detection of neurotransmitter exocytosis. Using the microsensor, the exocytosis from PC12 cells pretreated with the desired drugs (e.g., anticoronavirus drug, antiflu drug, or anti-inflammatory drug) has been successfully measured. Our achievements demonstrate the feasibility of the functionally collaborative nanostructure in the real-time detection of exocytosis and the potential applicability in the highly efficient assessment of the modulation effects of medications on exocytosis.

Protective effects of safranal on diabetic retinopathy in human microvascular endothelial cells and related pathways analyzed with transcriptome sequencing.

Aim: To determine the effect of safranal on diabetic retinopathy in vitro and its possible mechanisms. Methods: We used human retinal microvascular endothelial cells (HRMECs) to test the influence of safranal in vitro. High glucose damage was established and an safranal was tested at various concentrations for its potential to reduce cell viability using the MTT assay. We also employed apoptosis detection, cell cycle detection, a transwell test, and a tube formation assay to look into safranal's inhibitory effects on high glucose damage at various doses. Furthermore, mRNA transcriptome sequencing was performed. mRNA expression levels in a high glucose damage model, a high glucose damage model treated with safranal, and a blank control were compared to find the possible signaling pathway. Western blotting was used to confirm the expressions of several molecules and the levels of phosphorylation in each for the newly discovered pathway. Results: Cell proliferation was inhibited under a high glucose condition but could be protected by safranal at different concentrations (P<0.001). Flow cytometry results suggested safranal also protected cells from apoptosis (P=0.006). A transwell test demonstrated reduced invasiveness of safranal-treated cells in a high glucose condition (P<0.001). In a tube formation investigation, there were noticeably more new branches in the high gloucose group compared to a high glucose treated with safranal group (P<0.001). In mRNA expression patterns on transcriptome sequencing, the MAPK signaling pathway showed an expression ratio. With western blotting, the phosphorylation level of p38-AKT was elevated under a high glucose condition but could be inhibited by safranal. The expression of molecules associated with cell adhesion, including E-cadherin, N-cadherin, Snail, Twist, and fibronectin also changed significantly after safranal treatment under a high glucose condition. Conclusion: Safranal can protect diabetic retinopathy in vitro, and the p38-AKT signaling pathway was found to be involved in the pathogenesis of diabetic retinopathy and could be inhibited by safranal. This pathway may play a role by influencing cell migration and adhesion.

Analysis of Effect of Compound Salt Stress on Seed Germination and Salt Tolerance Analysis of Pepper (Capsicum annuum L.).

To determine the salt tolerance and physiological mechanism of pepper (Capsicum annuum L.) at the germination stage, the Hongtianhu 101 and Xinxiang 8 varieties, which have large differences in salt tolerance, are employed as the study materials. Six mixed salt concentrations of 0, 3, 5, 10, 15, and 20 g/L derived using equal molar ratios of Na2CO3, NaHCO3, NaCl, CaCl2, MgCl2, MgSO4, and Na2SO4 are used. To determine their effects, the related indexes of seed germination, seedling growth, and physiology are measured, and salt tolerance is comprehensively evaluated using membership function analysis. The results show that as the mixed salt concentration increases, the germination potential, germination index, germination rate, seed germination vigor index, root length, and root fresh weight of the two cultivars significantly decrease, whereas the relative salt rate gradually increases. The hypocotyl length and fresh weight aboveground increase first and then decrease, while the malondialdehyde (MDA), proline (Pro) content, catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) activity decrease and then increase. The germination potential, germination index, germination rate, seed germination vigor index, root length, root fresh weight, MDA and Pro content, and CAT activity of the Hongtianhu 101 seeds are higher than those of Xinxiang 8 for all salt concentrations employed here. However, hypocotyl length, fresh weight aboveground, and relative salt rate are lower in Hongtianhu 101 than in Xinxiang 8. The comprehensive evaluation of salt tolerance reveals that the total weighted values of the two membership function indexes increase first and then decrease as the mixed salt concentration increases. Compared with 5 g/L, which has the highest membership function value, the index under salt concentrations of 3 g/L, 10 g/L, and 15 g/L decreases by 4.7%-11.1%, 25.3%-28.3%, and 41.4%-45.1%, respectively. This study provides theoretical guidance for the breeding of salt-tolerant varieties of pepper and an analysis of the physiological mechanisms involved in salt tolerance and salt-tolerant cultivation.

Antibody-Based Methods Reveal the Protein Expression Properties of Glucosinolate Sulfatase 1 and 2 in Plutella xylostella.

The glucosinolates (GLs) and myrosinase defensive systems in cruciferous plants were circumvented by Plutella xylostella using glucosinolate sulfatases (PxGSSs) during pest-plant interaction. Despite identifying three duplicated GSS-encoding genes in P. xylostella, limited information regarding their spatiotemporal and induced expression is available. Here, we investigated the tissue- and stage-specific expression and induction in response to GLs of PxGSS1 and PxGSS2 (PxGSS1/2) at the protein level, which shares a high degree of similarity in protein sequences. Western blotting (WB) analysis showed that PxGSS1/2 exhibited a higher protein level in mature larvae, their guts, and gut content. A significantly high protein and transcript levels of PxGSS1/2 were also detected in the salivary glands using WB and qRT-PCR. The immunofluorescence (IF) and immunohistochemistry (IHC) results confirmed that PxGSS1/2 is widely expressed in the larval body. The IHC was more appropriate than IF when autofluorescence interference was present in collected samples. Furthermore, the content of PxGSS1/2 did not change significantly under treatments of GL mixture from Arabidopsis thaliana ecotype Col-0, or commercial ally (sinigrin), 4-(methylsulfinyl)butyl, 3-(methylsulfinyl)propyl, and indol-3-ylmethyl GLs indicating that the major GLs from leaves of A. thaliana Col-0 failed to induce the expression of proteins for both PxGSS1 and PxGSS2. Our study systemically characterized the expression properties of PxGSS1/2 at the protein level, which improves our understanding of PxGSS1/2-center adaptation in P. xylostella during long-term insect-plant interaction.

Preferable timing of intraductal ultrasonography during endoscopic retrograde cholangiopancreatography lithotomy: A prospective cohort study.

Aim: Intraductal ultrasonography (IDUS) is a highly sensitive and non-invasive detective method that can be used to detect complete calculus clearance during endoscopic retrograde cholangiopancreatography (ERCP). In this study, we examined the preferable timing of IDUS during ERCP lithotomy. Methods: From 2017 to 2020, patients with choledocholithiasis were randomized into IDUS-BL (IDUS performed before lithotomy) group, IDUS-ALC (cholangiography and IDUS performed after lithotomy) group, and IDUS-AL group (IDUS performed after lithotomy) group. The influence of IDUS on the accuracy of prejudgment, the incidence of residual stones, the need for repeated lithotomy (RL), and fluoroscopy time were analyzed. Results: A total of 184 patients were enrolled. No residual stones were found during follow-up in any of the three groups. There was no difference in prejudgment accuracy rate on size and number of stones between different groups (all P > 0.05). RL were performed in 5, 9, and 9 cases of IDUS-BL, IDUS-ALC, and IDUS-AL group, respectively (P > 0.05). IDUS-AL group had a shorter fluoroscopy time than the other two groups (1.5 ± 0.6 vs. 2.8 ± 1.2, 2.5 ± 1.0 min, P < 0.05). Incidence of RL was related to the location of calculus [middle or lower part of common bile duct (CBD)], lithotripsy, dilated CBD (2.12 ± 0.46 vs. 1.78 ± 0.40 cm, P < 0.01), and inaccuracy prejudgment. Conclusion: IDUS performed after lithotomy is preferable for shorten fluoroscopy time during ERCP. IDUS is a reliable solution for the stone omission, which may be more valuable for patients with high-risk factors of RL.

MCPIP-1-Mediated Immunosuppression of Neutrophils Exacerbates Acute Bacterial Peritonitis and Liver Injury.

Monocyte chemotactic protein-1-induced protein-1 (MCPIP-1) is highly expressed in activated immune cells and negatively regulates immune responses, while the mechanisms underlying the immunoregulation of neutrophils in acute bacterial infection and liver injury remain elusive. Here, we examined the role of MCPIP-1 in regulating neutrophil functions during acute bacterial peritonitis and liver injury. Mice with myeloid cell-specific overexpression (McpipMye-tg) or knockout (McpipΔMye) of MCPIP-1 were generated. We found that reactive oxygen species and myeloperoxidase production, formation of neutrophil extracellular traps, and migratory capacity were deficient in McpipMye-tg neutrophils but enhanced in McpipΔMye neutrophils. The recruitment of neutrophils and pathogen clearance were markedly suppressed in McpipMye-tg mice following intraperitoneal infection with Salmonella typhimurium while intensified in McpipΔMye mice. Severe acute S. typhimurium-infected peritonitis and liver injury occurred in McpipMye-tg mice but were alleviated in McpipΔMye mice. RNA sequencing, RNA-binding protein immunoprecipitation and qPCR analysis revealed that MCPIP-1 downregulated the protective functions of neutrophils via degrading the mRNA of cold inducible RNA-binding protein. Consistently, MCPIP-1 was highly expressed in neutrophils of patients with acute infectious diseases, especially in those with liver injury. Collectively, we uncover that MCPIP-1 negatively regulates the antibacterial capacities of neutrophils, leading to exacerbating severe acute bacterial peritonitis and liver injury. It may serve as a candidate target for maintaining neutrophil homeostasis to control acute infectious diseases.

Potential of citrus endophyte Bacillus subtilis L1-21 in the control of Candidatus Liberibacter asiaticus in Asian citrus psyllid, Diaphorina citri.

BACKGROUND: Asian citrus psyllid (ACP), also known as Diaphorina citri, is the natural vector of Candidatus Liberibacter asiaticus (CLas), which is responsible for Huanglongbing (HLB), a devastating citrus disease. Previously, the pathogen was successfully excluded from diseased citrus plants by using the indigenous endophyte Bacillus subtilis L1-21. However, the pathogen elimination and colonization potential of B. subtilis L1-21 in the carrier vector ACP, as well as the recruitment of native microbial communities of psyllid in the presence of endophytes, are still unknown. RESULTS: Initially, we suggested that endophyte L1-21 reduced the CLas copies in ACP from 6.58 × 106 to 5.04 × 104 per insect after 48 h, however, the pathogen copies remained stable in the negative control. The endophyte was stable for 48 h after application. Among the bacterial genera those highlighted in ACP were Candidatus Liberibacter, Pseudomonas, Candidatus Profftella, Methylobacterium-Methylorubrum, Pantoea, Curtobacterium, Wolbachia, Actinomycetospora, and Bacillus. Interestingly, B. subtilis L1-21 easily colonizes the midgut of ACP but cannot be detected in eggs. When ACP with endophyte L1-21 was allowed to feed on new citrus leaves, the highest colonization was observed. We also found that psyllids carrying endophyte L1-21 after feeding on citrus leaves reduced the CLas copies in leaves on the 0, 3rd and 5th day from 8.18 × 10,4 2.6 × 10,3 and 0 pathogen copies/g fresh midvein, respectively. CONCLUSIONS: We propose that B. subtilis L1-21 is a native endophyte in citrus and psyllid, which efficiently reduces the CLas pathogen in both citrus and psyllids, provides a more protective effect by increasing the number of cultivable endophytes, and successfully colonizes the midgut of ACP. © 2022 Society of Chemical Industry.

Silencing of Glycogen Synthase Kinase 3 Significantly Inhibits Chitin and Fatty Acid Metabolism in Asian Citrus Psyllid, Diaphorina citri.

Glycogen is a predominant carbohydrate reserve in various organisms, which provides energy for different life activities. Glycogen synthase kinase 3 (GSK3) is a central player that catalyzes glucose and converts it into glycogen. In this study, a GSK3 gene was identified from the D. citri genome database and named DcGSK3. A reverse transcription quantitative PCR (RT-qPCR) analysis showed that DcGSK3 was expressed at a high level in the head and egg. The silencing of DcGSK3 by RNA interference (RNAi) led to a loss-of-function phenotype. In addition, DcGSK3 knockdown decreased trehalase activity, glycogen, trehalose, glucose and free fatty acid content. Moreover, the expression levels of the genes associated with chitin and fatty acid synthesis were significantly downregulated after the silencing of DcGSK3. According to a comparative transcriptomics analysis, 991 differentially expressed genes (DEGs) were identified in dsDcGSK3 groups compared with dsGFP groups. A KEGG enrichment analysis suggested that these DEGs were primarily involved in carbon and fatty acid metabolism. The clustering analysis of DEGs further confirmed that chitin and fatty acid metabolism-related DEGs were upregulated at 24 h and were downregulated at 48 h. Our results suggest that DcGSK3 plays an important role in regulating the chitin and fatty acid metabolism of D. citri.