Ecology

Dopamine and its receptor DcDop2 are involved in the mutualistic interaction between ‘Candidatus Liberibacter asiaticus’ and Diaphorina citri

Xiaoge Nian
Jiayun Li
Jilei Huang
Weiwei Yuan
Paul Holford
George Andrew Charles Beattie
Jielan He
Yijing Cen
Yurong He
Songdou Zhang author has email address

Plant Protection Research Institute, Guangdong Academy of Agricultural Sciences, Key Laboratory of Green Prevention and Control on Fruits and Vegetables in South China Ministry of Agriculture and Rural Affairs Guangdong Provincial Key Laboratory of High Technology for Plant Protection, Guangzhou, China
National Key Laboratory of Green Pesticide, Department of Entomology, College of Plant Protection, South China Agricultural University, Guangzhou, China
Instrumental Analysis and Research Center, South China Agricultural University, Guangzhou, China
School of Science, Western Sydney University, Penrith, Australia

https://doi.org/10.7554/eLife.109081.1

Open access
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Figures and data

Effects of CLas on dopamine levels and the expression of DA biosynthesis genes in D. citri.
(A) A volcano plot highlighting differentially-expressed neurotransmitters in CLas+ and CLas- psyllid ovaries, with six being upregulated (red dots) and seven downregulated (green dots). (B) Comparison of dopamine levels in the ovaries of CLas+ and CLas- females 5, 9, 13 days after emergence (DAE). (C) Schematic representation of dopamine biosynthesis and release pathway. (D) A heatmap indicating the temporal expression patterns of six dopamine metabolic pathway genes. Data are presented as means ± SEMs. Significant differences between CLas- and CLas+ psyllids were determined using Student’s t-tests (*p <0.05, **p < 0.01, ***p < 0.001).

Effects of RNAi-mediated silencing of DcHenna1, DcTh, DcDdc, and DcVat1 on energy metabolism and fecundity of CLas+ females
(A) Dopamine titer in the CLas- and CLas+ females post treatment for 48 h with dsDcHenna1, dsDcTh, dsDcDdc, or dsDcVat1. (B-C) TAG and glycogen levels in the fat bodies of CLas- and CLas+ females post treatment for 48 h with dsDcHenna1, dsDcTh, dsDcDdc, or dsDcVat1. (D) Lipid droplets stained with Nile red in fat bodies dissected from CLas+ females treated with dsDcHenna1, dsDcTh, dsDcDdc, or dsDcVat1. Scale bar = 40 μm. (E) Ovary phenotypes in CLas+ females post DcHenna1, DcTh, DcDdc, and DcVat1 treatment. Scale bar = 200 μm. o: ovary, s: spermathecae. (F-H) Comparison of preoviposition period, oviposition period, and fecundity between CLas- and CLas+ females treated with dsDcHenna1, dsDcTh, dsDcDdc, or dsDcVat1. (I) CLas titer in ovaries of CLas+ females treated with dsDcHenna1, dsDcTh, dsDcDdc, or dsDcVat1. (J) Representative confocal images of CLas in reproductive systems of CLas+ females treated with dsDcHenna1, dsDcTh, dsDcDdc, or dsDcVat1. Scale bar = 200 μm. DAPI: cell nuclei were stained with DAPI and visualized in blue. CLas-Cy3: CLas signal visualized in red by staining with Cy3. Merge: merged imaging of co-localization of cell nuclei and CLas. Data shown as means ± SEMs. For A-C, and F-I, significant differences among different treatments are denoted by lowercase letters based on one-way ANOVA followed by the Tukey’s HSD tests at P < 0.05.

Dopamine concentrations mediated by DcDop2 are involved in the mutualistic relationship between CLas and D. citri resulting in increased fecundity.
(A) Temporal expression patterns of DcDop2 between the ovaries of CLas- and CLas+ females. (B) Effects of RNAi on DcDop2 expression in CLas- and CLas+ females treated with dsDcDop2 for 48 h. (C-D) Comparison of TAG and glycogen levels in fat bodies of CLas- and CLas+ females treated with dsDcDop2 for 48 h. (E) Lipid droplets stained with Nile red in fat bodies dissected from CLas+ females treated with dsDcDop2 for 48 h. Scale bar = 40 μm. (F) Ovary phenotypes in dsDcDop2-treated CLas+ females at 48 h. Scale bar = 200 μm. o: ovary, s: spermathecae. (G-I) Comparison of preoviposition period, oviposition period, and fecundity of CLas- and CLas+ adults treated with dsDcDop2 and dopamine rescue. (J) CLas titer in ovaries of CLas+ females treated with dsDcDop2 and DA rescue. (K) Representative confocal images of the reproductive system of CLas+ females treated with dsDcDop2 and dopamine rescue. Scale bar = 200 μm. DAPI: cell nuclei were stained with DAPI and visualized in blue. DcDop2-FITC: DcDop2 signal visualized in green by staining with FITC. CLas-Cy3: CLas signal visualized in red by staining with Cy3. Merge: merged imaging of co-localization of cell nuclei, DcDop2 and CLas. Data presented as means ± SEM. Significant differences between treatments and controls are indicated by asterisks in A-D (Student’ s t-test, *P < 0.05, **P < 0.01, ***P < 0.001). For G-J, significant differences among different treatments are indicated by lowercase letters based on one-way ANOVA followed by Tukey’s HS tests at P < 0.05.

Identification and validation of the targeting of DcDop2 by miR-31a.
(A) Prediction of potential miRNA binding sites in the 3’-UTR of DcDop2 using miRanda and RNAhybrid. (B) Assessment of miRNA-mediated regulation through dual-luciferase reporter assays in HEK293T cells co-transfected with miRNA agomir and recombinant pmirGLO vectors containing binding sites for miR-275, novel-miR-15 and miR-31a in the 3’ UTR of DcDop2. (C) Dual-luciferase reporter assays in HEK293T cells co-transfected with miR-31a agomir and recombinant pmirGLO vectors containing will-type or mutated DcDop2-3’UTR. (D) Tissue-specific expression of miR-31a in CLas+ female adults 7 DAE in the head, ovary, fat body, and midgut. (E) Comparison of temporal expression patterns of miR-31a in the ovaries of CLas- and CLas+ females. (F) Impact of miR-31a agomir and antagomir treatments on DcDop2 mRNA level in the ovaries of CLas- and CLas+ psyllids after 48 h. (G) In vivo assessment of miR-31a targeting DcDop2 through RNA immunoprecipitation assay. Data are presented as means ± SEM. For figures C, D, and F, significant differences among the different treatments are indicated by lowercase letters based on one-way ANOVA followed by Tukey’s Honest Significant Difference tests at P < 0.05. Significant differences between treatments and controls are denoted by asterisks in B and E-G (Student’s t-test, **p < 0.01, ***p < 0.001).

Involvement of miR-31a in the mutualistic interactions between D. citri and CLas.
(A-B) Comparative analysis of TAG and glycogen levels in the fat bodies of CLas- and CLas+ females following agomir-31a treatment for 48 h. (C) Visualization of lipid droplets stained with Nile red in fat bodies extracted from CLas+ females treated with agomir-31a for 48 h. Scale bar = 40 μm. (D) Ovarian phenotypes of CLas+ female treated with agomir-31a for 48 h. Scale bar = 200 μm. o: ovary, s: spermathecae. (E-G) Comparison of the preoviposition period, oviposition period, and fecundity between CLas- and CLas+ adult females treated with agomir-31a. (H) Quantification of CLas titer in the ovaries of CLas+ females treated with agomir-31a for 48 h. (I) Representative confocal images showing DcDop2 and CLas in the reproductive system of CLas+ females treated with agomir-31a for 48 h. Scale bar = 200 μm. The signals of DAPI, DcDop2-FITC, and CLas-Cy3 are consistent with described in Figure 2. Data are shown as means ± SEM. Significant differences between treatments and controls indicated by asterisks (Student’s t-test, ***p < 0.001).

The modulation of the AKH and JH signaling pathway by the DA pathway enhances fecundity in CLas+ D. citri.
(A) JH titer in the abdomen of CLas+ females treated with dsDcDop2 for 48 h. (B) Influence of dsDcDop2 treatment on mRNA levels of key genes in the AKH and JH signaling pathway in fat bodies of CLas+ females. (C) Impacts of dsDcDop2 treatment on mRNA levels of key genes in the AKH and JH signaling pathway in the ovaries of CLas+ females. (D-E) Rescue of the JH analogue and AKH on the fecundity and CLas titer phenotypes caused by the DcDop2 knockdown. (F) JH titers in the abdomens of Clas+ females after agomir-31a treatment for 48 h. (G) Effects of agomir-31a treatment on mRNA level of key genes in the AKH and JH signaling pathway in fat bodies of CLas+ females. (H) Effects of agomir-31a treatment on mRNA levels of key genes in the AKH and JH signaling pathway in the ovaries of CLas+ females. (I-J) Rescue of the JH analogue and AKH on the fecundity and CLas titer phenotypes caused by the agomir-31a treatments. Data are shown as means ± SEM. For figures D, E, I, and J, significant differences among the different treatments are indicated by lowercase letters based on one-way ANOVA followed by Tukey’s Honest Significant Difference tests at P < 0.05. Significant differences between treatments and controls are denoted by asterisks (Student’s t-test, ***p < 0.001).

Diagram illustrating the involvement of DA and its receptor, DcDop2, in the mutualistic interaction between CLas and D. citri.
CLas infection initially triggers the upregulation of DA biosynthesis genes, leading to increased dopamine levels. Subsequently, DA activates its receptor DcDop2, acting as an upstream regulator of AKH/JH signaling, governing energy metabolism and reproductive development. In order to maintain the appropriate level of DcDop2 expression, miR-31a exerts negative regulation by targeting the 3’-UTR. Ultimately, CLas infection enhances the host’s energy metabolism, thereby boosting the host’s reproductive capacity and promoting its own proliferation and dissemination, achieving a win-win situation for both.

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