The Black cells phenotype is caused by a point mutation in the Drosophila pro-phenoloxidase 1 gene that triggers melanization and hematopoietic defects

Melanization contributes to arthropod-specific innate immunity through deposition of melanin at wound sites or around parasites, with concomitant release of microbicidal reactive oxygen species. Melanization requires sequential activation of proteolytic enzymes in the hemolymph, including the final enzyme pro-phenoloxidase. Black cells (BC) is a mutation causing spontaneous melanization of Drosophila crystal cells, a hemocyte cell type producing phenoloxidases. BC individuals exhibit circulating black spots but fail to melanize upon injury. Although BC is widely used as a loss-of-function mutant of phenoloxidases, the mutation causing BC remained unknown. Here, we identified a single point mutation in the prophenoloxidase I (PPO1) gene of Bc flies causing an Alanine to Valine change in the C-terminal domain of PPO1, predicted to affect the conformation of the N-terminal pro-domain cleavage site at a distance and causing uncontrolled catalytic activity. Genomic insertion of a PPO1(A480V)transgene phenocopies Black cells, proving that A480V is indeed the causal mutation of the historical Bc phenotype. (C) 2014 Elsevier Ltd. All rights reserved.

The melanization reaction in Drosophila: More than black or white

Jan Paul Dudzic

The melanization reaction is a rapid and important immune mechanism in arthropods. It results in the production of melanin at the site of injury and around invading microbes. The enzymes responsible for melanogenesis are phenoloxidases (PO), which catalyze the oxidation of phenols to quinones, which then polymerize into melanin. A by-product of melanogenesis are cytotoxic compounds that can pose a threat to the host organism itself due to their unspecific effects. Melanogenesis is therefore tightly regulated: POs are produced in an inactive form as prophenoloxidases (PPOs), which get activated by the sequential cleavage of an extracellular serine protease (SP) cascade. The aims of this PhD thesis were to better understand the melanization reaction in Drosophila melanogaster, at both the effector and the regulation levels. D. melanogaster has three PPO genes. We generated for the first time a mutant for the third PPO gene, PPO3, and analyzed its function. We demonstrated that PPO3 has an important role in the melanotic encapsulation reaction, a defense mechanism against parasitization. Additionally, we extended our knowledge about the other PPOs, PPO1 and PPO2. We confirmed their role in the defense against septic infections and ascribed a new role for PPO2 in the melanotic encapsulation. The use of single or combined mutations allowed us to show that each PPO mutant has a specific phenotype, and that knocking out two of three genes is required to abolish a particular function completely. Thus, Drosophila PPOs have partially overlapping functions to optimize melanization. Finally, we demonstrated that PPO3 is the result of a gene duplication of PPO2, restricted to a subgroup of Drosophila, and likely evolved as an additional defense mechanism in the cellular encapsulation process, probably due to the evolutionary pressure from parasitoid wasps. In the second part of this thesis, we re-addressed the roles and functions of three SPs involved in the melanization process. We developed a novel screening method for defects in melanization by infecting flies with a low-dose of S. aureus. We found that only one of the three SPs, Sp7, is involved in survival upon septic infections. Additionally, we demonstrated that the melanization reaction resulting in the clearance of systemic infections is regulated by extracellular components of the Toll pathway. While a connection between the SPs regulating the melanization and the Toll pathway was found in other insects, our study provides the first demonstration in D. melanogaster. We also present evidence of a disconnect between the melanin production at wound sites and the melanization reaction resulting in the clearance of infections, indicating a role of cytotoxic compounds in the killing of microbes. Finally, we ascribed a new role to Hayan, another SP implicated in melanization. Hayan is dispensable for the clearance of septic infections, but is important for wound melanization. We also demonstrated that Hayan plays an important role in the activation of the Toll pathway. Hayan, together with the SP Persephone, is necessary to activate Toll after infection. We propose that both Hayan and Persephone are the result of a recent gene duplication event. This can explain why they still have overlapping functions, but these genes also show signs of early sub-functionalization. Collectively, our work provides important insights on both melanization and the activation of the Toll pathway.

EPFL2018

Genomic Variation and Its Impact on Gene Expression in Drosophila melanogaster

Monica Albarca Aguilera, Bart Deplancke, Jeffrey David Jensen, Andreas Massouras, Sebastian Martin Waszak

Understanding the relationship between genetic and phenotypic variation is one of the great outstanding challenges in biology. To meet this challenge, comprehensive genomic variation maps of human as well as of model organism populations are required. Here, we present a nucleotide resolution catalog of single-nucleotide, multi-nucleotide, and structural variants in 39 Drosophila melanogaster Genetic Reference Panel inbred lines. Using an integrative, local assembly-based approach for variant discovery, we identify more than 3.6 million distinct variants, among which were more than 800,000 unique insertions, deletions (indels), and complex variants (1 to 6,000 bp). While the SNP density is higher near other variants, we find that variants themselves are not mutagenic, nor are regions with high variant density particularly mutation-prone. Rather, our data suggest that the elevated SNP density around variants is mainly due to population-level processes. We also provide insights into the regulatory architecture of gene expression variation in adult flies by mapping cis-expression quantitative trait loci (cis-eQTLs) for more than 2,000 genes. Indels comprise around 10% of all cis-eQTLs and show larger effects than SNP cis-eQTLs. In addition, we identified two-fold more gene associations in males as compared to females and found that most cis-eQTLs are sex-specific, revealing a partial decoupling of the genomic architecture between the sexes as well as the importance of genetic factors in mediating sex-biased gene expression. Finally, we performed RNA-seq-based allelic expression imbalance analyses in the offspring of crosses between sequenced lines, which revealed that the majority of strong cis-eQTLs can be validated in heterozygous individuals.

Public Library of Science2012

The melanization reaction in Drosophila: More than black or white

Jan Paul Dudzic

EPFL2018