The use of individual protective covers (IPCs) to protect newly planted citrus trees from Huanglongbing (HLB) infection is being widely adopted in Florida, an HLB-endemic citrus-producing area. It is known that IPCs positively influence most horticultural traits, increasing tree growth, flush expansion, and leaf size, enabling trees to sustain balanced carbohydrate metabolism by preventing Candidatus Liberibacter asiaticus (CLas) infection, and inducing higher leaf chlorophyll levels. This may result in more productive trees. However, as the tree grows, IPCs eventually are removed, typically between 2 and 3 years after their initial installation. Once IPCs are removed, trees become exposed to the Asian citrus psyllid (ACPs) and ultimately become infected. In this work, we covered Valencia sweet orange trees with IPCs for 30 months, until the trees entered fruit-bearing age. We investigated how the IPC protection of newly planted trees for 30 months influenced the fruit quality and yield of \"Valencia\" trees for three consecutive seasons after IPC removal compared to non-covered trees. The use of IPCs kick-started the newly planted citrus trees, resulting in higher yields and fruits with better internal and external quality. After 30 months of IPC protection, tree canopies were larger and denser, supporting more fruit per tree than non-protected trees for three consecutive seasons, even though by the end of the first season after IPC removal, the trees were HLB-positive. Tree height, scion diameter, canopy volume, and leaf area were significantly improved compared to non-covered trees. Additionally, fruit quality was significantly improved in the three seasons following IPC removal compared to non-covered trees. However, a decline in quality was measurable in fruit from IPC trees after the second harvesting season, with trees affected by HLB. Based on the results from this study, we conclude that the benefits from IPC protection may last for at least three consecutive seasons once trees enter the productive age, despite CLas infection within 12 months after IPC removal.

The Asian citrus psyllid, Diaphorina citri, is the primary vector of the HLB pathogen, Candidatus Liberibacter asiaticus (CLas). The acquisition of CLas shortens the developmental period of nymphs, accelerating the emergence into adulthood and thereby facilitating the spread of CLas. Cuticular proteins (CPs) are involved in insect emergence. In this study, we investigated the molecular mechanisms underlying CLas-promoted emergence in D. citri via CP mediation. Here, a total of 159 CP genes were first identified in the D. citri genome. Chromosomal location analysis revealed an uneven distribution of these CP genes across the 13 D. citri chromosomes. Proteomic analysis identified 54 differentially expressed CPs during D. citri emergence, with 14 CPs exhibiting significant differential expression after CLas acquisition. Five key genes, Dc18aa-1, Dc18aa-2, DcCPR-24, DcCPR-38 and DcCPR-58, were screened from the proteome and CLas acquisition. The silencing of these 5 genes through a modified feeding method significantly reduced the emergence rate and caused various abnormal phenotypes, indicating the crucial role that these genes play in D. citri emergence. This study provides a comprehensive overview of the role of CPs in D. citri and reveals that CLas can influence the emergence process of D. citri by regulating the expression of CPs. These key CPs may serve as potential targets for future research on controlling huanglongbing (HLB) transmission.

São Paulo, Brazil, and Florida, USA, were the two major orange production areas in the world until Huanglongbing (HLB) was discovered in São Paulo in 2004 and Florida in 2005. In the absence of resistant citrus varieties, HLB is the most destructive citrus disease known because of the lack of effective tools to reduce spread of the vector, Diaphorina citri (Asian citrus psyllid), and transmission of the associated pathogen, Candidatus Liberibacter asiaticus. In both countries, a three-pronged management approach was recommended and begun: planting only disease-free nursery trees, effective psyllid control, and removal of all symptomatic trees. In Brazil, these management procedures were continued and improved and resulted in relatively little overall loss of production. In contrast, in Florida the citrus industry has been devastated with annual production reduced by approximately 80%. This review compares and contrasts various cultural and pest management strategies that have been used to reduce infection by the pathogen and increase tolerance of HLB in the main orange-growing regions in the world.

UNASSIGNED: Huanglongbing (HLB), a disease that\'s ubiquitous worldwide, wreaks havoc on the citrus industry. The primary culprit of HLB is the gram-negative bacterium Candidatus Liberibacter asiaticus (CLas) that infects the phloem, but its damaging mechanism is yet to be fully understood.
UNASSIGNED: In this study, a multitude of tools including weighted correlation network analysis (WGCNA), protein-protein interaction (PPI) network analysis and gene expression profiling are employed to unravel the intricacies of its pathogenesis. The investigation pinpoints various central genes, such as the ethylene-responsive transcription factor 9 (ERF9) and thioredoxin reductase 1 (TrxR1), that are associated with CLas invasion and resultant disturbances in numerous biological operations. Additionally, the study uncovers a range of responses through the detection of differential expressed genes (DEGs) across different experiments. The discovery of core DEGs leads to the identification of pivotal genes such as the sieve element occlusion (SEO) and the wall-associated receptor kinase-like 15 (WAKL15). PPI network analysis highlights potential vital proteins, while GO and KEGG pathway enrichment analysis illustrate a significant impact on multiple defensive and metabolic pathways. Gene set enrichment analysis (GSEA) indicates significant alterations in biological processes such as leaf senescence and response to biotic stimuli.
UNASSIGNED: This all-encompassing approach extends valuable understanding into the pathogenesis of CLas, potentially aiding future research and therapeutic strategies for HLB.

The bifunctional enzyme, 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase/inosine monophosphate (IMP) cyclohydrolase (ATIC) is involved in catalyzing penultimate and final steps of purine de novo biosynthetic pathway crucial for the survival of organisms. The present study reports the characterization of ATIC from Candidatus Liberibacer asiaticus (CLasATIC) along with the identification of potential inhibitor molecules and evaluation of cell proliferative activity. CLasATIC showed both the AICAR Transformylase (AICAR TFase) activity for substrates, 10-f-THF (Km, 146.6 μM and Vmax, 0.95 μmol/min/mg) and AICAR (Km, 34.81 μM and Vmax, 0.56 μmol/min/mg) and IMP cyclohydrolase (IMPCHase) activitiy (Km, 1.81 μM and Vmax, 2.87 μmol/min/mg). The optimum pH and temperature were also identified for the enzyme activity. In-silico study has been conducted to identify potential inhibitor molecules through virtual screening and MD simulations. Out of many compounds, HNBSA, diosbulbin A and lepidine D emerged as lead compounds, exhibiting higher binding energy and stability for CLasATIC than AICAR. ITC study reports higher binding affinities for HNBSA and diosbulbin A (Kd, 12.3 μM and 34.2 μM, respectively) compared to AICAR (Kd, 83.4 μM). Likewise, DSC studies showed enhanced thermal stability for CLasATIC in the presence of inhibitors. CD and Fluorescence studies revealed significant conformational changes in CLasATIC upon binding of the inhibitors. CLasATIC demonstrated potent cell proliferative, wound healing and ROS scavenging properties evaluated by cell-based bioassays using CHO cells. This study highlights CLasATIC as a promising drug target with potential inhibitors for managing CLas and its unique cell protective, wound-healing properties for future biotechnological applications.

The bacterium responsible for a disease that infects citrus plants across Asia facilitates its own proliferation by increasing the fecundity of its host insect.

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is the key vector insect transmitting the Candidatus Liberibacter asiaticus (CLas) bacterium that causes the devastating citrus greening disease (Huanglongbing, HLB) worldwide. The D. citri salivary glands (SG) exhibit an important barrier against the transmission of HLB pathogen. However, knowledge on the molecular mechanism of SG defence against CLas infection is still limited. In the present study, we compared the SG transcriptomic response of CLas-free and CLas-infected D. citri using an illumine paired-end RNA sequencing. In total of 861 differentially expressed genes (DEGs) in the SG upon CLas infection, including 202 upregulated DEGs and 659 downregulated DEGs were identified. Functional annotation analysis showed that most of the DEGs were associated with cellular processes, metabolic processes, and the immune response. Gene ontology and Kyoto Encyclopaedia of Genes and Genomes enrichment analyses revealed that these DEGs were enriched in pathways involving carbohydrate metabolism, amino acid metabolism, the immune system, the digestive system, the lysosome, and endocytosis. A total of 16 DEGs were randomly selected to further validate the accuracy of RNA-Seq dataset by reverse-transcription quantitative polymerase chain reaction. This study provides substantial transcriptomic information regarding the SG of D. citri in response to CLas infection, which may shed light on the molecular interaction between D. citri and CLas, and provides new ideas for the prevention and control of citrus psyllid.

UNASSIGNED: Asian citrus psyllid (ACP, Diaphorina citri) is an important transmission vector of \"Candidatus Liberibacter asiaticus\" (CLas), the causal agent of Huanglongbing (HLB), the most destructive citrus disease in the world. As there are currently no HLB-resistant rootstocks or varieties, the control of ACP is an important way to prevent HLB. Some viruses of insect vectors can be used as genetically engineered materials to control insect vectors.
UNASSIGNED: To gain knowledge on viruses in ACP in China, the prevalence of five RNA and DNA viruses was successfully determined by optimizing reverse transcription polymerase chain reaction (RT-PCR) in individual adult ACPs. The five ACP-associated viruses were identified as follows: diaphorina citri bunyavirus 2, which was newly identified by high-throughput sequencing in our lab, diaphorina citri reovirus (DcRV), diaphorina citri picorna-like virus (DcPLV), diaphorina citri bunyavirus (DcBV), and diaphorina citri densovirus-like virus (DcDV).
UNASSIGNED: DcPLV was the most prevalent and widespread ACP-associated virus, followed by DcBV, and it was detected in more than 50% of all samples tested. DcPLV was also demonstrated to propagate vertically and found more in salivary glands among different tissues. Approximately 60% of all adult insect samples were co-infected with more than one insect pathogen, including the five ACP-associated viruses and CLas.
UNASSIGNED: This is the first time these viruses, including the newly identified ACP-associated virus, have been detected in individual adult ACPs from natural populations in China\'s five major citrus-producing provinces. These results provide valuable information about the prevalence of ACP-associated viruses in China, some of which have the potential to be used as biocontrol agents. In addition, analysis of the change in prevalence of pathogens in a single insect vector is the basis for understanding the interactions between CLas, ACP, and insect viruses.

Huanglongbing (HLB) is one of the most devastating diseases of citrus worldwide. The phloem-restricted bacterium Candidatus Liberibacter asiaticus (CLas) is considered to be the main pathogen responsible for HLB. There is currently no effective practical strategy for the control of HLB. Our understanding of how pathogens cause HLB is limited because CLas has not been artificially cultured. In this study, 15 potential virulence factors were predicted from the proteome of CLas through DeepVF and PHI-base searches. One among them, FlgI, was found to inhibit yeast growth when expressed in Saccharomyces cerevisiae. The expression of the signal peptide of FlgI fused with PhoA in Escherichia coli resulted in the discovery that FlgI was a novel Sec-dependent secretory protein. We further found that the carboxyl-terminal HA-tagged FlgI was secreted via outer membrane vesicles in Sinorhizobium meliloti. Fluoresence localization of transient expression FlgI-GFP in Nicotiana benthamiana revealed that FlgI is mainly localized in the cytoplasm, cell periphery, and nuclear periphery of tobacco cells. In addition, our experimental results suggest that FlgI has a strong ability to induce callose deposition and cell necrosis in N. benthamiana. Finally, by screening a large library of compounds in a high-throughput format, we found that cyclosporin A restored the growth of FlgI-expressing yeast. These results confirm that FlgI is a novel Sec-dependent effector, enriching our understanding of CLas pathogenicity and helping to develop new and more effective strategies to manage HLB.

Ribonuclease HI (RNase HI) is well conserved across prokaryotes and eukaryotes, and has long been known to localize in the nucleic acid-containing cellular compartments for acting as an R-loop eraser but has never been determined to be a secreted protein. \"Candidatus Liberibacter asiaticus\" (CLas) is a fastidious α-proteobacterium that causes Huanglongbing (HLB), a devastating citrus disease often associated with flowering out of season. In this study, using the SecretomeP program coupled with an Escherichia coli-based alkaline phosphatase assay, we demonstrated that the CLas RNase HI (LasRNHⅠ) was a non-classically secreted protein. Further experiments identified that LasRNHⅠ could interact with a citrus B-box zinc finger protein CsBBX28 in the plant nucleolus. The in vitro assays indicated that CsBBX28 dramatically enhanced the R-loop-degrading activity of LasRNHⅠ. Remarkably, co-expression of CsBBX28 and LasRNHⅠ in Arabidopsis thaliana led to a much later flowering time than that of wild-type Arabidopsis, as well as that of the transgenic A. thaliana expressing only CsBBX28 or LasRNHⅠ, and lastingly and significantly repressed transcription of FLOWERING LOCUS T (FT), a floral pathway integrator. Similarly, ectopic expression of LasRNHⅠ in citrus greatly reduced the transcription level of FT. The data together disclosed the extracellular secretion of LasRNHⅠ, and that LasRNHⅠ physically interacted with CsBBX28 and served as a flowering repressor through suppressing the FT expression, suggesting a novel role of RNase HI in the bacteria interacting with the host plants.