Characterization of Cationic Amino Acid Binding Protein from Candidatus Liberibacter Asiaticus and in Silico Study to Identify Potential Inhibitor Molecules.

Cationic amino acid binding protein (CLasArgBP), one of the two amino acid binding receptor in Candidatus Liberibacter asiaticus (CLas), is predominately expressed in citrus psyllids as a part of ATP-binding cassette transport system. The present study describes characterization of CLasArgBP by various biophysical techniques and in silico study, to identify potential inhibitor molecules against CLasArgBP through virtual screening and MD simulations. Further, in planta study was carried out to assess the effect of selected inhibitors on Huanglongbing infected Mosambi plants. The results showed that CLasArgBP exhibits pronounced specificity for arginine, histidine and lysine. Surface plasmon resonance (SPR) study reports highest binding affinity for arginine (Kd, 0.14 µM), compared to histidine and lysine (Kd, 15 µΜ and 26 µΜ, respectively). Likewise, Differential Scanning Calorimetry (DSC) study showed higher stability of CLasArgBP for arginine, compared to histidine and lysine. N(omega)-nitro-L-arginine, Gamma-hydroxy-L-arginine and Gigartinine emerged as lead compounds through in silico study displaying higher binding energy and stability compared to arginine. SPR reports elevated binding affinities for N(omega)-nitro-L-arginine and Gamma-hydroxy-L-arginine (Kd, 0.038 µΜ and 0.061 µΜ, respectively) relative to arginine. DSC studies showed enhanced thermal stability for CLasArgBP in complex with selected inhibitors. Circular dichroism and fluorescence studies showed pronounced conformational changes in CLasArgBP with selected inhibitors than with arginine. In planta study demonstrated a substantial decrease in CLas titer in treated plants as compared to control plants. Overall, the study provides the first comprehensive characterization of cationic amino acid binding protein from CLas, as a potential drug target to manage HLB disease.

CRISPR/Cas12a-Responsive Smart DNA Hydrogel for Sensitive Electrochemiluminescence Detection of the Huanglongbing Outer Membrane Protein Gene.

Citrus Huanglongbing (HLB) is known as the cancer of citrus, where Candidatus Liberibacter asiaticus (CLas) is the most prevalent strain causing HLB. In this study, we report a novel electrochemiluminescence (ECL) biosensor for the highly sensitive detection of the CLas outer membrane protein (Omp) gene by coupling rolling circle amplification (RCA) with a CRISPR/Cas12a-responsive smart DNA hydrogel. In the presence of the target, a large number of amplicons are generated through RCA. The amplicons activate the trans-cleavage activity of CRISPR/Cas12a through hybridizing with crRNA, triggering the response of smart DNA hydrogel to release the encapsulated AuAg nanoclusters (AuAg NCs) on the electrode and therefore leading to a decreased ECL signal. The ECL intensity change (I0 - I) is positively correlated with the concentration of the target in the range 50 fM to 5 nM, with a limit of detection of 40 fM. The performance of the sensor has also been evaluated with 10 samples of live citrus leaves (five HLB negative and five HLB positive), and the result is in excellent agreement with the gold standard qPCR result. The sensing strategy has expanded the ECL versatility for detecting varying levels of dsDNA or ssDNA in plants with high sensitivity.

Comparative Phylogenomic Analysis Reveals Evolutionary Genomic Changes and Novel Toxin Families in Endophytic Liberibacter Pathogens.

Liberibacter pathogens are the causative agents of several severe crop diseases worldwide, including citrus Huanglongbing and potato zebra chip. These bacteria are endophytic and nonculturable, which makes experimental approaches challenging and highlights the need for bioinformatic analysis in advancing our understanding about Liberibacter pathogenesis. Here, we performed an in-depth comparative phylogenomic analysis of the Liberibacter pathogens and their free-living, nonpathogenic, ancestral species, aiming to identify major genomic changes and determinants associated with their evolutionary transitions in living habitats and pathogenicity. Using gene neighborhood analysis and phylogenetic classification, we systematically uncovered, annotated, and classified all prophage loci into four types, including one previously unrecognized group. We showed that these prophages originated through independent gene transfers at different evolutionary stages of Liberibacter and only the SC-type prophage was associated with the emergence of the pathogens. Using ortholog clustering, we vigorously identified two additional sets of genomic genes, which were either lost or gained in the ancestor of the pathogens. Consistent with the habitat change, the lost genes were enriched for biosynthesis of cellular building blocks. Importantly, among the gained genes, we uncovered several previously unrecognized toxins, including new toxins homologous to the EspG/VirA effectors, a YdjM phospholipase toxin, and a secreted endonuclease/exonuclease/phosphatase (EEP) protein. Our results substantially extend the knowledge of the evolutionary events and potential determinants leading to the emergence of endophytic, pathogenic Liberibacter species, which will facilitate the design of functional experiments and the development of new methods for detection and blockage of these pathogens. IMPORTANCELiberibacter pathogens are associated with several severe crop diseases, including citrus Huanglongbing, the most destructive disease to the citrus industry. Currently, no effective cure or treatments are available, and no resistant citrus variety has been found. The fact that these obligate endophytic pathogens are not culturable has made it extremely challenging to experimentally uncover the genes/proteins important to Liberibacter pathogenesis. Further, earlier bioinformatics studies failed to identify key genomic determinants, such as toxins and effector proteins, that underlie the pathogenicity of the bacteria. In this study, an in-depth comparative genomic analysis of Liberibacter pathogens along with their ancestral nonpathogenic species identified the prophage loci and several novel toxins that are evolutionarily associated with the emergence of the pathogens. These results shed new light on the disease mechanism of Liberibacter pathogens and will facilitate the development of new detection and blockage methods targeting the toxins.