Journal: Viruses
Article Title: Evidence of Adaptive Evolution in Wolbachia -Regulated Gene DNMT2 and Its Role in the Dipteran Immune Response and Pathogen Blocking
doi: 10.3390/v13081464
Figure Lengend Snippet: IPOD regulates DNMT2 expression in Drosophila. ( A ) maximum-likelihood tree of the interaction partner of DNMT2 ( IPOD ) gene present in multiple Drosophila species was constructed using RAxML using a multiple sequence alignment of IPOD nucleotide sequences. The sequence of the IPOD orthologs from Lucilia cuprina , Musca domestica , and Sarcophaga bullata were used as outgroups. Scale bars represent branch lengths. ( B ) Inter-protein co-evolutionary analyses of DNMT2 and IPOD orthologs were performed using the TreeCmp software packages. Red dashed lines connect the same Drosophila taxon. ( C ) Shannon conservation plot representing the degree of conservation ( Y -axis) of IPOD orthologs present at every amino acid position ( X -axis) across Drosophilids . The horizontal dashed line indicates the mean conservation score (0.46) across all amino acid positions. Colored boxes represent three InterPro domains identified across all IPOD orthologs in Drosophilids , including the N-terminal signal peptide (depicted in orange), followed by a C-terminal non-cytoplasmic domain (depicted in white) consisting of a conserved domain of unknown function (DUF4766, depicted in yellow) and a glycine-rich disordered region (depicted in green) present at the C-terminal end. ( D , E ) IPOD is an upstream regulator of Mt2 expression in Drosophila melanogaster . ( D ) IPOD expression was knocked down in Wolbachia w Mel-colonized Drosophila melanogaster (TRiP line# 60092) by driving expression of a targeting short-hairpin RNA (shRNA) against the target IPOD mRNA. Relative expression of the target IPOD mRNA and Mt2 mRNA was assessed via quantitative RT-PCR using total RNA derived from age-matched females. Siblings lacking the shRNA were used as the negative control. Two-tailed t -tests on log-transformed values; IPOD : p < 0.05, t = 3.678, df = 8.00, Mt2 : p < 0.05, t = 2.454, df = 8.00. Error bars represent the standard error of the mean (SEM) of experimental replicates ( n = 5). ( E ) Mt2 expression was knocked down in Wolbachia w Mel-colonized Drosophila melanogaster by driving expression of a targeting short-hairpin RNA (shRNA) against the target mRNA. Relative expression of the target Mt2 mRNA and IPOD mRNA was assessed via quantitative RT-PCR using total RNA derived from age-matched females. Siblings lacking the shRNA were used as the negative control. Two-tailed t -tests on log-transformed values; Mt2 : p < 0.01, t = 2.576, df = 12.00, IPOD : p = 0.717969, t = 0.3686, df = 14.00. Error bars represent the standard error of the mean (SEM) of experimental replicates ( n = 6–8). ( F ) Effect of IPOD knockdown on Wolbachia- mediated virus inhibition. Age-matched Wolbachia- colonized female flies, either wild type or expressing IPOD -targeting shRNA, were intrathoracically injected with the SINV-nLuc virus. At indicated times post-infection ( X -axis), flies were harvested and snap-frozen before homogenization. Homogenized lysates were used to measure luciferase expression (RLU, Y -axis), which was subsequently used as a proxy to quantify virus replication. Two-way ANOVA of multivariate comparisons with Sidak’s post hoc test; IPOD knockdown: p < 0.01, Time: p < 0.01. Error bars represent the standard error of the mean (SEM) of experimental replicates ( n = 3/time point). * p < 0.05, ** p < 0.01, ns = not-significant.
Article Snippet: For IPOD, domains were defined based on Pfam and InterPro domain prediction results obtained using Drosophila melanogaster IPOD as an input query [ , ].
Techniques: Expressing, Construct, Sequencing, Software, shRNA, Quantitative RT-PCR, Derivative Assay, Negative Control, Two Tailed Test, Transformation Assay, Knockdown, Virus, Inhibition, Injection, Infection, Homogenization, Luciferase
