The research, led by Md Tafim Hossain Hrithik and Cecilia Tamborindeguy of the Department of Entomology at Texas A&M University, together with Julien Levy and Taylor Hubbs of the university’s Department of Horticultural Sciences, focuses on the Toll pathway, one of the principal arms of insect innate immunity. Insects lack adaptive immune systems and instead rely on humoral and cellular defenses coordinated by signaling cascades such as Toll, immune deficiency (IMD) and JAK/STAT. In the classic model established in the fruit fly Drosophila melanogaster, the Toll pathway responds to fungi and Gram-positive bacteria, while Gram-negative bacteria activate the IMD pathway. Many hemipteran insects, however, including psyllids and aphids, carry only a partial or entirely absent IMD pathway, which raises intriguing questions about how these insects cope with Gram-negative pathogens such as Lso. Because the potato psyllid encodes the Toll pathway but lacks a complete IMD pathway, understanding Toll signaling becomes central to explaining the insect’s interactions with the bacterium.

As a first step, the team mined the psyllid genome using sequence similarity searches against well-characterized Toll pathway genes from Drosophila melanogaster, the pea aphid Acyrthosiphon pisum and the Asian citrus psyllid Diaphorina citri, applying a stringent E-value cutoff and validating candidates through conserved protein domain analysis with Prosite and InterPro. The search identified six Toll receptor genes, which the researchers named Toll like-1, Toll like-2, Toll like-3, Toll-6, Toll-7 and Tollo, along with ten genes encoding associated pathway components: three Spätzle ligands, the adaptor proteins MyD88, Tube and Pelle, the transcription factor Dorsal, the inhibitor Cactus, and the signaling regulators Traf-6 and Pellino. Phylogenetic analysis using maximum likelihood methods showed that the psyllid Toll proteins cluster most closely with their counterparts in Diaphorina citri, reflecting the evolutionary proximity of these two psyllid species.

The structural analysis of these proteins revealed details with functional implications. Four of the six Toll receptors possess the full canonical architecture, comprising an extracellular leucine-rich repeat domain that mediates ligand recognition and an intracellular Toll/interleukin-1 receptor (TIR) domain that recruits adaptor proteins such as MyD88 to initiate downstream NF-κB-dependent signaling. Toll like-1 and Toll like-3, by contrast, contain only the leucine-rich repeat region and lack the TIR domain, mirroring observations in Diaphorina citri. The authors speculate that these receptors cannot transduce signals independently and may instead serve as ligand-binding components that relay recognition events to other Toll receptors, thereby modulating the strength of the immune response. Tollo, meanwhile, lacks a predicted N-terminal signal peptide, a feature also seen in the citrus psyllid, which may influence where the protein localizes within cells and what it does there.

To determine which of these genes actually participate in immune defense, the researchers turned to a well-established inducer of Toll signaling across insect species: the entomopathogenic fungus Beauveria bassiana, strain Bb-490, a biocontrol agent registered worldwide for insect suppression. In leaf-dipping bioassays, third-instar psyllid nymphs exposed to fungal suspensions of 10^7 conidia per milliliter suffered heavy mortality, with roughly 82 percent dead by the sixth day of the assay, while about 80 percent of control nymphs survived. Quantitative PCR then showed that five of the six Toll receptor genes were significantly up-regulated 48 hours after the fungal challenge, confirming that these receptors are indeed deployed during antifungal defense and providing a functional benchmark for interpreting the responses to bacterial infection.

The heart of the study lies in the response of the Toll genes to the two Lso haplotypes that circulate in the United States, LsoA and LsoB, both of which cause severe disease but differ in their behavior within the insect. The researchers exposed pathogen-free female psyllids to infected tomato plants for acquisition access periods of two, three, five, seven and fourteen days, quantifying bacterial titers by qPCR alongside gene expression. LsoB accumulated more rapidly than LsoA, but the titers of the two haplotypes converged by five days. Strikingly, the gene expression responses were haplotype-specific. During LsoB acquisition, two Toll receptors, Toll like-1 and Toll-7, were up-regulated at the five-day mark, together with the Spätzle-3 ligand. During LsoA acquisition, only Toll-7 responded, and it was the Spätzle gene rather than Spätzle-3 that was induced. The remaining pathway components, including MyD88, Tube, Pelle, Dorsal, Cactus, Traf-6 and Pellino, showed no significant transcriptional change under either condition.

This asymmetry carries considerable biological weight. Because the differential expression peaked at five days, a point at which LsoA and LsoB titers in the insects were equivalent, the authors argue that the altered gene expression reflects the timing and nature of infection rather than the bacterial load. The distinct Spätzle response patterns further suggest that individual Spätzle ligands may play specialized roles in activating Toll signaling against different Lso haplotypes, echoing findings in Drosophila where single Toll receptors can bind multiple Spätzle proteins. The data also align with earlier transcriptomic work showing that LsoB triggers more extensive gene expression changes in the psyllid than LsoA does, indicating that the two bacterial haplotypes interact differently with the vector’s physiology despite their close relationship.

Perhaps the most consequential finding concerns persistence. The team compared Toll gene expression among fifth-instar nymphs, adult males and adult females from colonies permanently infected with LsoA or LsoB and found that none of the six Toll receptors was regulated during persistent infection, regardless of developmental stage. A two-way analysis of variance confirmed significant effects of developmental stage on the expression of Toll like-1, Toll like-2, Toll-6 and Toll-7, but neither infection status nor the interaction between stage and infection had a statistically significant effect. The researchers interpret this silence as evidence that Lso may actively manipulate or suppress the psyllid’s Toll pathway once infection is established, and they emphasize that immune activation is best captured during the early acquisition phase rather than in persistently infected insects.

An important caveat frames the study’s conclusions: even when Toll genes were induced during early acquisition, the response failed to stop the pathogen. Both LsoA and LsoB are successfully transmitted by potato psyllids in a circulative and propagative manner, meaning the bacterium multiplies within the insect as it moves through the body. The authors suggest that the Toll-mediated defense, while real, is either insufficient in magnitude or too restricted in scope to block Lso, much as the robust Toll activation triggered by Beauveria bassiana could not prevent the fungal infection from killing the nymphs. The psyllid’s missing IMD pathway may compound this vulnerability, potentially explaining its permissiveness to this Gram-negative bacterium, although endosymbiont-mediated defenses or cellular immune mechanisms may also contribute, as has been proposed for Diaphorina citri.

The practical implications reach toward disease management. With no economically viable genetic resistance to Lso in affected crops, current control depends heavily on insecticide applications to suppress psyllid populations, a strategy with well-known limitations. By defining the Toll pathway components of Bactericera cockerelli and demonstrating their differential engagement with LsoA and LsoB, the study identifies molecular targets that could be exploited to disrupt transmission, whether through breeding, RNA interference or microbiome-based approaches. The finding that Beauveria bassiana potently activates the psyllid’s antifungal Toll genes while killing nymphs also reinforces interest in fungal biocontrol as a component of integrated strategies. As the authors note, this work marks the beginning of understanding the molecular mechanisms of Toll signaling in the potato psyllid, and that understanding could ultimately help protect potato and tomato fields from one of their most destructive disease complexes.

Subject of Research: Toll pathway gene expression in the potato psyllid during infection with 'Candidatus Liberibacter solanacearum'

Article Title: Identification and gene expression profiling of toll genes in the potato psyllid (Bactericera cockerelli) in response to ‘Candidatus liberibacter solanacearum’

Article References: Identification and gene expression profiling of toll genes in the potato psyllid (Bactericera cockerelli) in response to ‘Candidatus liberibacter solanacearum’. (n.d.). https://doi.org/10.1007/s11033-026-12777-9

Image Credits: AI Generated

DOI: 10.1007/s11033-026-12777-9

Keywords: potato psyllid, Bactericera cockerelli, Candidatus Liberibacter solanacearum, Toll pathway, zebra chip disease, innate immunity, gene expression, Beauveria bassiana, Spätzle, insect vector, plant pathogen, RT-qPCR