No Evidence of Apoptotic Response of the Potato Psyllid Bactericera cockerelli to “ Candidatus Liberibacter solanacearum” at the Gut Interface

Abstract

“Candidatus Liberibacter solanacearum” is a pathogen transmitted by the potato psyllid Bactericera cockerelli (Šulc) (Hemiptera: Triozidae) in a persistent manner. In this study, we investigated the molecular interaction between “Ca. Liberibacter solanacearum” and the potato psyllid at the gut interface. Specifically, we focused on the apoptotic response of potato psyllids to the infection by two “Ca. Liberibacter solanacearum” haplotypes, LsoA and LsoB. To this end, we first quantified and localized “Ca. Liberibacter solanacearum” in the gut of adult psyllids. We then evaluated the existence of an apoptotic response in the insect gut using microscopy analyses to visualize the nuclei and the actin cytoskeleton of the gut cells and DNA fragmentation analyses by agarose gel electrophoresis. We also performed annexin V cell death assays to detect apoptosis. Finally, we annotated apoptosis-related genes from the potato psyllid transcriptome and evaluated their expression in response to “Ca. Liberibacter solanacearum” infection. The results showed no cellular markers of apoptosis despite the large amount of “Ca. Liberibacter solanacearum” present in the psyllid gut. In addition, only three genes potentially involved in apoptosis were regulated in the psyllid gut in response to “Ca. Liberibacter solanacearum”: the apoptosis-inducing factor AIF3 was downregulated in LsoA-infected psyllids, while the inhibitor of apoptosis IAPP5 was downregulated and IAP6 was upregulated in LsoB-infected psyllids. Overall, no evidence of apoptosis was observed in the gut of potato psyllid adults in response to either “Ca. Liberibacter solanacearum” haplotype. This study represents a first step toward understanding the interactions between “Ca. Liberibacter solanacearum” and the potato psyllid, which is crucial to developing approaches to disrupt their transmission.

Acquisition and transmission of two ‘Candidatus Liberibacter solanacearum’ haplotypes by the tomato psyllid Bactericera cockerelli

“…These immune responses could affect the replication rates of Lso in the tomato psyllid gut. If indeed, different responses are elicited by each haplotype, it is possible that the response only affects Lso accumulation upon infection or in adult psyllids, because, adults that acquired Lso as nymphs have similar LsoA or LsoB titers in their guts 23 .…”

“…Next, the guts were permeabilized by adding 0.1% Triton X-100 (Calbiochem/EMD Chemicals, Gibbstown, NJ) for 30 min at room temperature, and washed three times with PBS containing 0.05% Tween 20 (PBST) prior to a 1-h blocking incubation at room temperature with blocking buffer (PBST with 1% [w/v] bovine serum albumin). Lso immunolocalization was performed using a rabbit-derived polyclonal antibody (GenScript Corp, Piscataway, NJ) directed against Lso OMP-B (Ab-OMP-B) which detects both Lso haplotypes 23 . The guts were incubated with the antibody (diluted 1:500) overnight at 4 °C.…”

Concanavalin A Toxicity Towards Potato Psyllid and Apoptosis Induction in Midgut Cells

“…L. asiaticus' (CLas) determined that the presence of CLas-induced apoptosis in adult psyllid midgut cells [44]. However, this phenomenon does not appear to occur in the potato psyllid in response to Lso [32]. One hypothesis to explain the absence of detectable apoptosis in the gut of Lso-infected adults is that the Lso-induced intracellular immune response did not reach or exceed the threshold to trigger an intracellular apoptotic immune reaction [45].…”

“…The caspase genes involved in apoptosis pathways were identified by searching the psyllid transcriptome datasets [31] and verified by sequencing [32]. The primers for sequence validation can be found in Table S1.…”

“…In total, three caspase genes (caspase 1-3) were identified in the psyllid transcriptome dataset [31]. The domains of caspases were identified in our previous study [32]. Caspase 1 is a putative effector caspase with a short pro-domain, while caspase 2 is a putative initiator caspase with a caspase recruitment domain (CARD).…”