JNK pathway plays a key role in the immune system of the pea aphid and is regulated by microRNA-184

Abstract

Different from holometabolous insects, the hemipteran species such as pea aphid Acyrthosiphon pisum exhibit reduced immune responses with the absence of the genes coding for antimicrobial peptide (AMP), immune deficiency (IMD), peptidoglycan recognition proteins (PGRPs), and other immune-related molecules. Prior studies have proved that phenoloxidase (PO)-mediated melanization, hemocyte-mediated phagocytosis, and reactive oxygen species (ROS) participate in pea aphid defense against bacterial infection. Also, the conserved signaling, Jun N-terminal kinase (JNK) pathway, has been suggested to be involved in pea aphid immune defense. However, the precise role of the JNK signaling, its interplay with other immune responses and its regulation in pea aphid are largely unknown. In this study, using in vitro biochemical assays and in vivo bioassays, we demonstrated that the JNK pathway regulated hemolymph PO activity, hydrogen peroxide concentration and hemocyte phagocytosis in bacteria infected pea aphids, suggesting that the JNK pathway plays a central role in regulating immune responses in pea aphid. We further revealed the JNK pathway is regulated by microRNA-184 in response to bacterial infection. It is possible that in common the JNK pathway plays a key role in immune system of hemipteran insects and microRNA-184 regulates the JNK pathway in animals.

Fig 1. JNK pathway contributes to the pea aphid’s immune defense against bacterial infection.

(A) The JNK signaling cascade of pea aphid based on functional studies from Drosophila melanogaster and Anopheles gambiae. (B) Relative expression levels of JNK, Jun, Fos and Puc in the pea aphids after Gram-positive bacteria M. luteus (M.l) and Gram-negative bacteria P. aeruginosa (P.a) infections with the aphids injected by sterile 0.85% as control groups. The expressions of JNK, Jun, Fos and Puc were normalized with ribosomal protein L7 gene (rpl7) of the pea aphids, and the relative expression of the infection groups were compared to the expression of the control groups at each time point. (C) Efficiency of RNA interference-mediated knockdown of the pea aphid JNK and Puc. The expressions of JNK and Puc were normalized with rpl7 of the pea aphids, and the relative expression of the dsJNK and dsPuc injected groups were compared to the expression of the dsGFP groups at each time point. (D) Effect of JNK and Puc silence on the survival of the pea aphids after M. luteus (M.l) and P. aeruginosa (P.a) infection, n = 20. One representative survival graph from three independent experiments with similar results is shown. The statistical differences between the compared groups were denoted with asterisks. The log-rank (Mantel-Cox) test was used to analyze the pea aphids’ survival curves. *P<0.05; **P<0.01. (E) Effect of JNK and Puc silence on the bacteria loads of the pea aphids after M. luteus (M.l) and P. aeruginosa (P.a) infection, n = 8. Each dot in the graph represents an individual aphid. The horizontal bars indicate mean values and the vertical bars indicate the SEM of the replicates. The statistical differences between the compared groups were denoted with asterisks. P values were determined by Student’s t test. *P<0.05; **P<0.01. For (A) and (B), values shown are the mean (±SEM) of three independent experiments. The statistical differences between the control groups and infection groups were denoted with asterisks (A). The statistical differences between the dsGFP injected groups and dsJNK or dsPuc injected groups were denoted with asterisks (B). P values were determined by Student’s t test. *P<0.05; **P<0.01; ***P<0.001.

Fig 2. JNK pathway regulates ROS metabolism.

Effect of JNK (A) and Puc (B) silence on the expression of antioxidant genes: OXR1, Cat, GPX and Prx1 of the pea aphids. The expressions of OXR1, Cat, GPX and Prx1 were normalized with rpl7 of the pea aphids. The statistical differences between the dsGFP injected groups and dsJNK or dsPuc injected groups were denoted with asterisks. Effect of JNK (C) and Puc (D) silence on the H₂O₂ concentration in the aphids uninfected and infected by M. luteus (M.l) and P. aeruginosa (P.a). Ten aphids from each group at each time point were used for the measurements of H₂O₂ concentration. The statistical differences between the compared groups were denoted with asterisks. For (A-D), the values shown are the mean (±SEM) of three independent experiments. P values were determined by Student’s t test. *P<0.05; **P<0.01; ***P<0.001.

Fig 3. JNK pathway regulates PPOs expression and PO activity.

Effect of JNK (A) and Puc (B) silence on the expression of PPO1 and PPO2 of pea aphids. The expressions of PPO1and PPO2 were normalized with rpl7 of the pea aphids. The statistical differences between the dsGFP injected groups and dsJNK or dsPuc injected groups were denoted with asterisks. (C) Effect of JNK and Puc silence on the PO activity in the uninfected aphids. (D) Effect of JNK and Puc silence on the PO activity in the aphids infected by M. luteus (M.l) and P. aeruginosa (P.a). For (C) and (D), twenty aphids for the sample per group at each time point were used for measurements of PO activity. The statistical differences between the compared groups were denoted with asterisks. For (A-D), the values shown are the mean (±SEM) of three independent experiments. P-values were determined by Student’s t test. *P<0.05; **P<0.01; ***P<0.001.

Fig 4. JNK pathway mediates hemocytes phagocytosis.

Effect of JNK (A) and Puc (B) silence on the mRNA levels of phagocytosis related genes: TepIII-1, TepIII-2 and YKT6 of the pea aphids. The expressions of TepIII-1, TepIII-2 and YKT6 were normalized with rpl7 of the pea aphids. (C) Ex vivo phagocytosis assay using S. aureus and E. coli AlexaFluor 594 BioParticle (Invitrogen) after knockdown of JNK and Puc. The hemocytes from 20 pea aphids per group were used to perform each experiment. In (A-C), the values shown are the mean (±SEM) of three independent experiments and the statistical differences between the compared groups were denoted with asterisks. P-values were determined by Student’s t test. *P<0.05; **P<0.01; ***P<0.001. (D) The photographs of ex vivo phagocytosis S. aureus and E. coli AlexaFluo 594 BioParticles (Invitrogen) by the hemocytes with the F-actin stained by SF-488 Phalloidin (1/200 diluted, Solarbio) after knockdown of JNK and Puc. The red dots were S. aureus and E. coli, and the green parts were the hemocytes with the F-actin stained. Scale bar: 5 μm.

Fig 5. miR-184a/b target and negatively regulate JNK.

(A) The putative miR-184a and miR-184b target binding site in 3′ UTR of JNK is predicted by using RNAhybrid. The sequences in the lines above were seed region (5′GGACGGA3′) binding sites predicted. (B) Relative expression levels of miR-184a and miR-184b in the pea aphids after M. luteus (M.l) and P. aeruginosa (P.a) infections with the aphids injected by sterile 0.85% as control groups. The expressions of miR-184a and miR-184b were normalized with U6 snRNA of the pea aphids, and the relative expression of the infection groups were compared to the expression of the control groups at each time point. (C) Western blotting of the GFP reporter assays showed that miR-184a and miR-184b directly degrade the 3′UTR of JNK in vitro. The upper arrows point to GFP reporter and the loading control β-actin was pointed by the nether arrows. M: Marker; PAC: pAc-5.1/V5-HisB plasmid was transfected into S2 cells alone, as mock or negative control; GFP: pAc-5.1/V5-HisB-GFP-JNK 3′UTR reporter plasmid was transfected into S2 cells alone, as positive control; mimic: pAc-5.1/V5-HisB-GFP-JNK 3′UTR reporter plasmid and mimic of miR-184a or miR-184b were co-transfected into S2 cells; NC: pAc-5.1/V5-HisB-GFP-JNK 3′UTR reporter plasmid and negative control mimic were co-transfected into S2 cells; mut: mutant mimic of miR-184a or miR-184b (at seed region: 5′-GGACGGA-3′ mutated as 5′-GACAUUC-3′) and pAc-5.1/V5-HisB-GFP-JNK 3′UTR reporter plasmid were co-transfected into S2 cells. (D) Relative expression levels of JNK in pea aphids after injected agomir-184a, agomir-184b and half-dose of agomir-184a plus half-dose of agomir-184b, with the aphids injected with agomir-NC as control group. The expressions of JNK were normalized with rpl7 of the pea aphids, and the relative expression of the agomir-184 groups were compared to the expression of the control groups at each time point. For (B) and (D), the values shown are the mean (±SEM) of three independent experiments and the statistical differences between the compared groups were denoted with asterisks. P values were determined by Student’s t test. *P<0.05; **P<0.01.

Fig 6. miR-184a/b regulate ROS metabolism, PO activity and hemocytes phagocytosis.

Effect of injection of agomir-184a, agomir-184b and half-dose agomir-184a plus half-dose agomir-184b on H₂O₂ concentration (A) and the PO activity (B) in the aphids uninfected and infected by M. luteus (M.l) and P. aeruginosa (P.a) and the hemocytes phagocytosis (C-D). Ten aphids for the sample per group at each time point were used for the measurements of H₂O₂ concentration. Twenty aphids for the sample per group at each time point were used for measurements of PO activity. The hemocytes from 20 pea aphids per group were used to perform each experiment. For (A-C), the values shown are the mean (±SEM) of three independent experiments and the statistical differences between the compared groups were denoted with asterisks. P-values were determined by Student’s t test. *P<0.05; **P<0.01; ***P<0.001. (D) The photographs of ex vivo phagocytosis S. aureus and E. coli AlexaFluo 594 BioParticle (Invitrogen) by the hemocytes with the F-actin stained by SF-488 Phalloidin (1/200 diluted, Solarbio) after injection of agomir-184a, agomir-184b and half-dose agomir-184a plus half-dose agomir-184b. The red dots were S. aureus and E. coli, and the green parts were the hemocytes with the F-actin stained. Scale bar: 5 μm.

Fig 7. miRNA-184a/b affected pea aphid survival and bacteria multiplication after infection.

(A) Effect of injection of agomir-184a, agomir-184b and half-dose agomir-184a plus half-dose agomir-184b on the survival of pea aphids after M. luteus (M.l) and P. aeruginosa (P.a) infection with the aphids injected by sterile 0.85% as control groups, n = 20. Survival graphs show one representative experiment out of three independent experiments with similar results. The statistical differences between the compared groups were denoted with asterisks. The log-rank (Mantel-Cox) test was used to analyze pea aphids’ survival curves. *P<0.05. (B) Effect of injection of agomir-184a, agomir-184b and half-dose agomir-184a plus half-dose agomir-184b on the bacterial load of pea aphids after M. luteus (M.l) and P. aeruginosa (P.a) infection, n = 8. Each dot in the graph represents an individual aphid. The horizontal bars indicate mean values and the vertical bars indicate the SEM of the replicates. The statistical differences between the compared groups were denoted with asterisks. P values were determined by Student’s t test. *P<0.05.

Fig 8. A schematic summary of the role of JNK pathway that modulates pea aphid immune signaling and is negatively regulated by miR-184.

The transcription factor AP-1 is activated through Jun and Fos that are phosphorylated by JNK. AP-1 upregulates PO activity, increases hemocytes phagocytosis, and upregulates genes expression for ROS detoxification. JNK expression is negatively regulated by miRNA-184a/b. Bacterial infection downregulates synthesis of miRNA-184, leading to deliverance of JNK pathway and activation of immune responses consequently, to protect pea aphid from infection and oxidative stress.