Xenorhabdus hominickii, an entomopathogenic bacterium, inhibits eicosanoid biosynthesis of target insects to suppress their immune responses by inhibiting phospholipase A2 (PLA2) through binding to a damage-associated molecular pattern (DAMP) molecule called dorsal switch protein 1 (DSP1) from Spodoptera exigua, a lepidopteran insect. However, the signalling pathway between DSP1 and PLA2 remains unknown. The objective of this study was to determine whether DSP1 could activate Toll immune signalling pathway to activate PLA2 activation and whether X. hominickii metabolites could inhibit DSP1 to shutdown eicosanoid biosynthesis. Toll-Spätzle (Spz) signalling pathway includes two Spz (SeSpz1 and SeSpz2) and 10 Toll receptors (SeToll1-10) in S. exigua. Loss-of-function approach using RNA interference showed that SeSpz1 and SeToll9 played crucial roles in connecting DSP1 mediation to activate PLA2. Furthermore, a deletion mutant against SeToll9 using CRISPR/Cas9 abolished DSP1 mediation and induced significant immunosuppression. Organic extracts of X. hominickii culture broth could bind to DSP1 at a low micromolar range. Subsequent sequential fractionations along with binding assays led to the identification of seven potent compounds including 3-ethoxy-4-methoxyphenol (EMP). EMP could bind to DSP1 and prevent its translocation to plasma in response to bacterial challenge and suppress the up-regulation of PLA2 activity. These results suggest that X. hominickii inhibits DSP1 and prevents its DAMP role in activating Toll immune signalling pathway including PLA2 activation, leading to significant immunosuppression of target insects.

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Salicylic acid is a plant hormone that can mediate various plant physiological processes. Salicylic acid can bind to human high mobility group box 1 (HMGB1) and interrupt its role in mediating immune responses. Dorsal switch protein 1 (DSP1) is an insect homolog of HMGB1. In this study, a DSP1 (Se-DSP1) encoded in Spodoptera exigua, a phytophagous insect, was characterized, and its potential role in immune response was explored. Upon bacterial challenge, Se-DSP1 was localized in the nucleus and released into the hemolymph. The released Se-DSP1 could mediate both cellular and humoral immune responses by activating eicosanoid biosynthesis. Salicylic acid could bind to Se-DSP1 with a high affinity. The immune responses of S. exigua were significantly interrupted by SA feeding. Larvae reared on tomatoes with high endogenous SA levels became more susceptible to entomopathogens. Taken together, these results suggest a tritrophic defensive role of plant SA against phytophagous insects.

High‐mobility group box 1 (HMGB1) is a nuclear protein highly conserved in eukaryotes and ubiquitously expressed to regulate transcription and chromatin remodeling. Dorsal switch protein 1 (DSP1) is its insect homolog. A lepidopteran DSP1 acts as a damage‐associated molecular pattern (DAMP) in response to immune challenge. The objective of this study was to determine the role of DAMP in the mealworm beetle, Tenebrio molitor, a coleopteran insect. DSP1 of T. molitor (Tm‐DSP1) encodes 536 amino acids and shares sequence similarities with Homo sapiens HMGB1 (56.3%) and Spodoptera exigua DSP1 (59.2%). An antisera raised against S. exigua DSP1 was cross‐reactive to Tm‐DSP1. Like other insect DSPs, Tm‐DSP1 has a relatively long N‐terminal extension in addition to two conserved HMG box domains. It was expressed in all developmental stages of T. molitor and different larval tissues. Upon immune challenge, its expression level was upregulated. Its RNA interference (RNAi) treatment resulted in a significant reduction in immune responses measured by hemocyte nodule formation against bacterial infection. In addition, the induction of some antimicrobial peptide genes to the immune challenge was suppressed by its RNAi treatment. Interestingly, phospholipase A2 associated with eicosanoid biosynthesis was significantly suppressed in its catalytic activity by the RNAi treatment specific to Tm‐DSP1 expression. Without any pathogen infection, injection of a lepidopteran DSP1 induced both cellular and humoral immune responses. These results suggest that Tm‐DSP1 in T. molitor can act as a DAMP molecule and mediate immune responses upon immune challenge.

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