Study Of The Role And Regulation Of JNK Pathway In The Immune System Of Pea Aphid,Acyrthosiphon Pisum

The aphids are seriously damaged agricultural pests.Using natural enemies of the aphids such as parasitic wasps and microbial pathogens to control aphids plays an important role in IPM（Integrated Pest Management）strategies.The immune defense system of the aphids will be activated,if they are infected by microbial pathogens or parasitized by wasps.The activated defense system induces a series of immune responses to deal with infection and parasitism.A deep insight into the aphids’immune defense mechanism is better for biological control of aphids.Pea aphid,Acyrthosiphon pisum,is an important model to study aphids and hemiptera insects.However,to date,little is known about the mechanism of pea aphid immune system against microbial pathogens infection.The conserved Jun N-terminal kinase（JNK）signaling pathway is activated by adverse environmental stresses and microbial pathogens infection to protect organisms from damages.The JNK pathway of insects plays important roles in wound healing,defensing against adverse environmental stresses and regulation of immune responses to infection.Genomic analysis suggests that the genes coding antimicrobial peptides（AMPs）,peptidoglycan recognition proteins（PGRPs）,immune deficiency（IMD）and other immune related genes are missing in the pea aphid.However,previous studies suggested that the JNK pathway might be involved in the pea aphid defense against bacterial infection.Studies also clearly showed that the phenoloxidase（PO）-mediated melanization,the production reactive oxygen species（ROS）,and the hemocytes-mediated phagocytosis participate in the defense of pea aphid.Although pea aphid has an instact miRNA synthesis pathway and 163 miRNAs have been identified,specific functions of these miRNAs are largely unclear.In this study,we revealed the mechanism of JNK pathway regulating pea aphid immune responses against bacterial infection and the function of the miRNAs targeting JNK.The main results are as follows:（1）Bacterial infection activates the JNK pathway and the JNK pathway plays an important role in protection of pea aphid against infection.The key genes in JNK pathway including JNK,Jun,Fos and Puc were all significantly up-regulated after infection by bacteria.The JNK and Puc were silenced by using RNA interference technique and the JNK silencing decreased Puc m RNA level.These results suggest that JNK pathway is responsive to microbial challenge and regulates espression of downstream genes in pea aphid.Compared to the control groups,knockdown of JNK resulted in higher mortalities of the aphids after bacterial infection and higher loads of bacteria inside the aphids;in contrast,knockdown of Puc resulted in lower mortalities and lower bacterial loads inside the aphids.This indicates that the JNK pathway plays an important role in the defense against bacterial infection in pea aphid.（2）Pea aphid JNK pathway regulates ROS metabolism,PO activity of hemolymph and hemocytes phagocytosis.Knockdown of JNK decreased OXR1（Oxidation resistance 1）,Cat（Catalase）,GPX（Glutathione peroxidase）,and Prx1（Peroxiredoxin 1）m RNA levels significantly,whereas silencing of Puc significantly increased the expression of these genes significantly.H₂O₂ levels were higher in JNK knockdown aphids;in contrast,H₂O₂ levels were much lower in Puc knockdown aphids.These results indicate that the JNK pathway mediates ROS metabolism in pea aphid.Knockdown of JNK significantly decreased PPO1 and PPO2 m RNA levels,whereas knockdown of Puc showed the opposite effects.Knockdown of JNK decreased PO activity,whereas knockdown of Puc increased PO activity.These results suggest that the pea aphid JNK pathway regulates the expression of PPOs and PO activity of activity.Knockdown of JNK significantly decreased Tep III-1,Tep III-2,and YKT6 m RNA levels;in contrast,knockdown of Puc significantly increased the m RNA levels of these genes.Knockdown of JNK decreased phagocytosis,whereas knockdown of Puc showed the opposite effects.These results clearly demonstrate that the JNK pathway mediates hemocytes phagocytosis in pea aphid.（3）JNK is negatively regulated by miRNA-184a/b in pea aphid;miRNA-184a/b regulates ROS metabolism,PO activity,and hemocyte phagocytosis through negatively regulating the expression of JNK,affecting the defense against bacterial infection.JNK m RNA is likely targeted by miR-184a/b,predicted in silico.In vitro,reporter gene assay confirmed that JNK m RNA is targeted and negatively regulated by miRNA-184a/b.In vivo,the JNK m RNA level significantly decreased in the miRNA-184a/b mimics injected pea aphids.H₂O₂ levels were higher and PO activity was lower in agomir-184a/b injected aphids than in aphids from the control groups.In the aphids injected with agomir-184a/b,hemocytes exhibited reduced phagocytosis.In bacterial infected conditions,agomir-184a/b injected aphids showed higher mortality and higher loads of infected bacteria.In addition,sequence alignment showed that miRNA-184is highly conserved among animal species.In silico prediction showed that JNK is likeky regulated by miRNA-184 in other insect species,zebrafish,frog,mouse,and human.Therefore,the regulation of the JNK pathway by miRNA-184 is likely a universal mechanism in animal.In conclusion,in this study,we demonstrated that bacterial infection activates the JNK pathway of pea aphid and the activated JNK pathway regulates ROS metabolism,PO activity of hemolymph and hemocytes phagocytosis to defend against bacterial infection.Mi RNA-184a/b regulates ROS metabolism,PO activity,and hemocyte phagocytosis through regulating JNK pathway negatively,affecting the defense against bacterial infection.We clearly revealed the role of JNK pathway in pea aphid immune defense system and regulation of of the JNK pathway by miRNA.This study provides ingignts into our understanding of immune defense mechanism of hemipteran insects,and establishes foundation for effective controlling of aphids using pathogenic microorganisms.