Role Of Rab3Gene In Cellular Defense Responses Of The Cotton Bollworm,Helicoverpa Armigera

Rab GTPases, a family of the small G protein superfamily, play important roles in several cell processes such as exocytic and endocytic membrane trafficking. Recent studies in Drosophila and shrimp indicated that some Rab proteins are essential for cellular immune responses such as phagocytosis. Recent transcriptome analysis of the cotton boll worm (Helicoverpa armigera) hemocytes identified a Rab GTPase (Ha-Rab3). Rab3is best known for its role in regulating synaptic vesicle trafficking in neurons. Recently, a study in the Chinese mitten crab (Eriocheir sinensis) showed that Rab3may function in innate immunity. In this study, we investigated the role of Ha-Rab3in H. armigera immunity. The results are as follows.1. Identification and sequence analysis of Ha-Rab3.A Rab3cDNA was identified from the transcriptome of H. armigera hemocytes and named Ha-Rab3. This cDNA is678bp long, containing a ORF of657bp. The predicted protein encoded by Ha-Rab3was218aa long and included a Rab domain (20-183aa). Multiple sequence alignment analysis indicated that Ha-Rab3amino acid sequence had significant similarities with the Rab3sequences from other species.2. Tissue distribution of Ha-Rab3mRNAs.Expression of Ha-Rab3mRNAs were examined using RT-PCR in four tissues (epidermis, midguts, fat bodies and hemocytes) of H. armigera larvae. Ha-Rab3was expressed in all tested tissues.3. Expression profiles of Ha-Rab3induced by bacterial or chromatography beads challenge.qRT-PCR was used to examine the expression levels of Ha-Rab3mRNAs in the hemocytes of H. armigera larvae at various time points after the injection of Escherichia coli, Staphylococcus aureus, or chromatography beads. The results showed that both bacterial and bead challenge could up-regulate the expression levels of Ha-Rab3, suggesting that Ha-Rab3gene expression is inducible upon bacterial or beads infection.4. Expression of Ha-Rab3is suppressed by injection of its specific dsRNA.H. armigera larvae were injected with dsRNA (6μg per larva), and the expression levels of Ha-Rab3in larvae hemocytes were analyzed48h post-injection. Ha-Rab3mRNA levels in the hemocytes of dsHa-Rab3-treated larvae were significantly reduced compared to the levels found in control larvae. This result confirmed that Ha-Rab3gene expression levels were repressed by RNAi.5. Knockdown of Ha-Rab3in H. armigera hemocytes significantly impairs the phagocytosis ability of H. armigera hemocytes. In vitro phagocytosis test showed that both phagocytic rate and phagocytic index of H. armigera larval hemocytes against E. coli were impaired when the expression of Ha-Rab3in hemocytes was inhibited by dsHa-Rab3injection. This result indicated that Ha-Rab3may be involved in the phagocytosis response of H. armigera.6. Knockdown of Ha-Rab3in H. armigera hemocytes results in increased bacterial load.Hemolymph bacterial load assay showed that RNAi-treated H. armigera larvae infected with E. coli showed higher bacterial growth in hemolymph compared with infected controls. This result further confirmed that Ha-Rab3plays important roles in defending H. armigera against invading microbe.7. Knockdown of Ha-Rab3in H. armigera hemocytes reduces the spreading ability of hemocytes.Plasmatocytes of dsHa-Rab3-treated larvae were spread in much less frequency than those of control larvae. This result indicated that the spreading ability of plasmatocytes was impaired when the expression of Ha-Rab3was inhibited by dsHa-Rab3.8. Knockdown of Ha-Rab3in H. armigera hemocytes significantly impairs the encapsulation ability of hemocytes.In vivo encapsulation assays showed that the encapsulation ability of H. armigera hemocytes was impaired when the expression of Ha-Rab3was inhibited by dsHa-Rab3. This result indicated that Ha-Rab3may be involved in the encapsulation response of H. armigera.In conclusion, Ha-Rab3gene was identified from H. armigera. RNAi assays showed that Ha-Rab3play important roles in H. armigera cellular immune responses, possibly by mediate plasmatocyte spreading behavior.