Huanglongbing (HLB) is a fatal citrus disease currently threatening all commercially relevant citrus varieties worldwide. In the United States, the putative causative agent, Candidatus Liberibacter asiaticus (CLas), is transmitted by the vector Diaphorina citri, commonly known as the Asian citrus psyllid (ACP). Progress towards understanding CLas and its role in HLB has been hindered by the inability to culture the bacterium and the inability to confidently detect it in diseased trees during the asymptomatic stage. To identify early changes in citrus metabolism in response to inoculation of CLas using its natural psyllid vector, leaves from Madam Vinous sweet orange (Citrus sinensis (L.) Osbeck) trees were exposed to CLas-positive ACP or CLas-negative ACP and longitudinally analyzed using transcriptomics (RNA sequencing), proteomics (liquid chromatography-tandem mass spectrometry), and metabolomics (proton nuclear magnetic resonance). At 4 weeks post-exposure (wpe) to psyllids, the initial HLB plant response was primarily to the ACP and, to a lesser extent, the presence or absence of CLas. Additionally, analysis of 4, 8, 12, and 16 wpe identified 17 genes that were consistently differentially expressed between leaves exposed to CLas-positive ACP versus CLas-negative ACP, one differentially abundant protein, and no differentially accumulated metabolites. The results of this study inform identification of early detection molecular targets and contribute to the broader understanding of vector-transmitted plant pathogen interactions.

Following Citrus sinensis leaf sample collection, the tissue was frozen until ready for NMR preparation.  The frozen ground leaf material was lyophilized, dried leaf material was transferred to 2 mL tubes. One 3.5 mm glass bead was added to each tube before beating and 10 mM phosphate buffer (pH 6.8) pre-heated to 90°C was added to the ground tissue in a 1:20 (w/v) ratio based on each sample's dry weight. Tubes were immediately mixed at 90 °C and then centrifuged at 4°C. Seven hundred fifty microliters of supernatant was collected, transferred to a clean tube, and centrifuged again. Sixty-five microliters of 4.97 mM 4,4-dimethyl-4-silapentane-1-sulfonic acid-d6 (DSS-d6) was added as an internal standard to 585 μL of the resulting supernatant and vortexed. Six hundred microliters of the mixture was transferred to 5 mm NMR tubes and stored at 4°C until NMR data acquisition which took place within 24 hours of NMR sample preparation. ¹H NMR data acquisition was performed using a Bruker Advance 600 MHz NMR spectrometer equipped with a SampleJet using the Bruker “noesypr1d” (RD-90°-t-90°-tm-ACQ) acquisition parameters.

Chenomx NMR suite v8.31 (Chenomx Inc., Edmonton, Alberta, Canada) can be used to open these files.