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. 2024 Feb 27:14:1360680.
doi: 10.3389/fcimb.2024.1360680. eCollection 2024.

Odorant binding protein 18 increases the pathogen resistance of the imported willow leaf beetle, Plagiodera versicolora

Haoling Rong  1 , Xin He  1 , Yipeng Liu  1 , Mei Liu  1 , Xiaolong Liu  1 , Min Lu  1
Affiliations

Odorant binding protein 18 increases the pathogen resistance of the imported willow leaf beetle, Plagiodera versicolora

Haoling Rong et al. Front Cell Infect Microbiol. .

Abstract

Background: Insect odorant-binding proteins (OBPs) are a class of small molecular weight soluble proteins. In the past few years, OBPs had been found to work as carriers of ligands and play a crucial role in olfaction and various other physiological processes, like immunity. A subset of insect OBPs had been found to be expressed differently and play a function in immunity of fungal infection. However, there are few studies on the role of OBPs in immunity of bacterial infection.

Methods: To identify the immune-related OBPs of Plagiodera versicolora after infected by Pseudomonas aeruginosa, we determined the mortality of P. versicolora to P. aeruginosa and selected the time point of 50% mortality of larvae to collect samples for RNA-seq. RNAi technology was used to investigate the function of immune-related OBPs after P. aeruginosa infection.

Results: RNA-seq data shows that PverOBP18 gene significantly up-regulated by 1.8-fold and further RT-qPCR affirmed its expression. Developmental expression profile showed that the expression of PverOBP18 was highest in the pupae, followed by the female adults, and lower in the 1st-3rd larvae and male adults with lowest in eggs. Tissue expression profiling showed that PverOBP18 was dominantly expressed in the epidermis. RNAi knockdown of PverOBP18 significantly reduced the expression of bacterial recognition receptor gene PGRP and antibacterial peptide gene Attacin and reduced the resistance of P. versicolora to P. aeruginosa infection.

Conclusion: Our results indicated that PverOBP18 gene increased the pathogen resistance of P. versicolora by cooperating with the immune genes and provided valuable insights into using OBPs as targets to design novel strategies for management of P. versicolora.

Keywords: Plagiodera versicolora; Pseudomonas aeruginosa; insect immunity; odorant binding protein; pathogen infection; pathogen resistance.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
The survival curves and transcriptomic changes of P. versicolora larvae infected with P. aeruginosa (A) Kaplan–Meier survival curves of second-instar P. versicolora larvae treated with P. aeruginosa. The log-rank test was used to assess the significance of differences between two survival curves. *** P < 0.001. (B) Distribution of unigene size in the P. versicolora transcriptome assembly (C) KEGG classification of differentially expressed unigenes between control (PACK) group and P. aeruginosa infected (PA) group.
Figure 2
Figure 2
Differential expression analysis between control (PACK) group and P. aeruginosa infected (PA) group and the expression profiles of up-regulated gene PverOBP18. (A) Volcano plot. The log2 FC indicates the mean expression level for each gene. Each dot represents one gene. After P. aeruginosa infection, gray dots represent no significant unigenes between PACK and PA, the blue dots represent down-regulated genes and red dots represent up-regulated genes. (B) Expression levels of PverOBP18 gene at three time points, day 1 (1D), day 2 (2D) and day 5 (5D) assessed by RT-qPCR and normalized to the reference gene RPS18 expression level. Data are means ± SD (n = 3). The letters above the bar indicate the significance of differences as determined by t-test. ns, not significant; * P < 0.05.
Figure 3
Figure 3
Temporal and spatial expression profiles of PverOBP18. (A) Expression levels of PverOBP18 gene at different development stages. L1 - L3, 1st instar larvae – 3rd instar larvae; MA, male adults; FA, female adults. (B) Expression levels of PverOBP18 gene at different tissues of 3rd instar larvae. EP, epidermis; FG, foregut; MG, midgut; HG, hindgut; FB, fat body; MT, Malpighian tubule; Relative expression levels were analyzed using RT-qPCR and normalized to the reference gene RPS18 expression level. Data are means ± SD (n = 3). The letters above the bar indicate the significance of differences as determined by one-way ANOVA (LSD, P < 0.05).
Figure 4
Figure 4
The synergistic effect of dsRNA and the entomopathogenic P. aeruginosa on P. versicolora larvae. (A) Silencing efficiency of PverOBP18 in P. versicolora treated with dsPverOBP18. RPS18 was used as a reference gene to normalize the relative expression of the indicated genes. Data are means ± SD (n = 3). The letters above the bar indicate the significance of differences as determined by t-test. ** P < 0.01. (B) Comparison of mortality of P. aeruginosa infected P. versicolora in response to the administration of dsPverOBP18. Kaplan–Meier survival curves of second-instar P. versicolora larvae. The log-rank test was used to assess the significance of differences between two survival curves. ns, not significant; *** P < 0.001; ** P < 0.01; * P < 0.05.
Figure 5
Figure 5
Immunity-related genes analysis of PverOBP18-silenced and H2O control P. versicolora larvae to P. aeruginosa infection by RT-qPCR. PGRP, peptidoglycan-recognition protein. RPS18 was used as a reference gene to normalize the relative expression of the indicated genes. Data are means ± SD (n = 3). The letters above the bar indicate the significance of differences as determined by one-way ANOVA (LSD, P < 0.05).
Figure 6
Figure 6
A simple diagram illustrating the mechanism and outcome of how the studied OBP increased the resistance of P. versicolora against P. aeruginosa..
See this image and copyright information in PMC

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