Identification and characterization of four Drosophila suzukii cellularization genes and their promoters

Abstract

Background The spotted-wing Drosophila (Drosophila suzukii) is a widespread invasive pest that causes severe economic damage to fruit crops. The early development of D. suzukii is similar to that of other Drosophilids, but the roles of individual genes must be confirmed experimentally. Cellularization genes coordinate the onset of cell division as soon as the invagination of membranes starts around the nuclei in the syncytial blastoderm. The promoters of these genes have been used in genetic pest-control systems to express transgenes that confer embryonic lethality. Such systems could be helpful in sterile insect technique applications to ensure that sterility (bi-sex embryonic lethality) or sexing (female-specific embryonic lethality) can be achieved during mass rearing. The activity of cellularization gene promoters during embryogenesis controls the timing and dose of the lethal gene product. Results Here, we report the isolation of the D. suzukii cellularization genes nullo, serendipity-α, bottleneck and slow-as-molasses from a laboratory strain. Conserved motifs were identified by comparing the encoded proteins with orthologs from other Drosophilids. Expression profiling confirmed that all four are zygotic genes that are strongly expressed at the early blastoderm stage. The 5′ flanking regions from these cellularization genes were isolated, incorporated into piggyBac vectors and compared in vitro for the promoter activities. The Dsnullo promoter showed the highest activity in the cell culture assays using D. melanogaster S2 cells. Conclusions The similarities in the gene coding and 5′ flanking sequence as well as in the expression pattern of the four cellularization genes between D. melanogaster and D. suzukii, suggest that conserved functions may be involved in both species. The high expression level at the early blastoderm stage of the four cellularization genes were confirmed, thus their promoters can be considered in embryonic lethality systems. While the Dsnullo promoter could be a suitable candidate, all reported promoters here are subject to further in vivo analyses before constructing potential pest control systems.

A transgenic female killing system for the genetic control of Drosophila suzukii

“…The development of effective TESS constructs for D. suzukii benefits from the inclusion of homologous regulatory elements with predictable activity. Accordingly, we previously tested the promoters from four D. suzukii cellularization genes, revealing that the Dsnullo promoter achieved the strongest reporter gene expression in D. melanogaster S2 cells 28 . The sry-α promoter is also widely used for TESS constructs in fruit fly species 18 – 20 .…”

“…Each reaction was heated to 98 °C for 3 min followed by 40 cycles of 98 °C for 10 s and 60 °C for 30 s. The primers are listed in Table S2 . Three biological and three technical replicates were carried out for each reaction, and results were normalized to the TATA binding protein ( TBP ) reference gene using the 2 -∆∆Ct method as previously described 28 , 29 .…”

Effects of antibiotics on the in vitro expression of tetracycline-off constructs and the performance of Drosophila suzukii female-killing strains

“…We previously characterized promoters from four Drosophila suzukii cellularization genes, including nullo , serendipity-α ( sry-α ), bottleneck ( bnk ), and slow-as-molasses ( slam ). The Dsnullo promoter regulated the strongest DsRed-NLS gene expression in Drosophila melanogaster S2 cells ( Yan et al, 2020a ). We also previously generated piggyBac vectors containing a bicistronic gene cassette in which EGFP-NLS (or DsRed-NLS) and DsRed-CAAX (or EGFP-CAAX) were co-expressed by a 2A peptide (DrosCV-2A or TaV-2A) ( Schwirz et al, 2020 ).…”

“…Bicistronic gene cassettes containing EGFP-NLS (or DsRed-NLS) and DsRed-CAAX (or EGFP-CAAX), which were separated by a 2A peptide (DrosCV-2A or TaV-2A), were excised from V220 _pBXLII_attP_PUb-AmCyan_PUb-DsRed-NLS-DrosCV-2A-EGFP-CAAX-SV40 ,V221 _pBXLII_attP_PUb-AmCyan_PUb-DsRed-NLS-TaV-2A-EGFP- CAAX-SV40 ,V222 _pBXLII_attP_PUb-AmCyan_PUb-EGFP-NLS-DrosCV-2A-DsRed-CAAX-SV40 , or V223 _pBXLII_attP_PUb-AmCyan_PUb-EGFP-NLS-TaV-2A-DsRed-CAAX-SV40 ( Schwirz et al, 2020 ) and used to replace the DsRed-NLS in the vector V206 _pXLBacII-attP-PUb-AmCyan_Dsnullo_DsRed-NLS-SV40 ( Yan et al, 2020a ) at the Bsu36I and MluI restriction sites, to generate V355 _pBXLII_attP_PUb-AmCyan_Dsnullo-DsRed-NLS-DrosCV-2A-EGFP-CAAX-SV40 ,V356 _pBXLII_attP_PUb-AmCyan_Dsnullo-DsRed-NLS- TaV-2A-EGFP-CAAX-SV40 , V357 _pBXLII_attP_PUb-AmCyan_Dsnullo-EGFP-NLS-DrosCV-2A-DsRed-CAAX-SV40 , and V358 _pBXLII_attP_PUb-AmCyan_Dsnullo-EGFP-NLS-TaV-2A-DsRed-CAAX-SV40 , respectively. Similarly, the excised bicistronic gene cassette from V220, V221, V222, or V223 was used to replace the effector gene cassette Dshid Ala4 -CctraF in the vector V229 _pBXLII_attP_PUbAmCyan_Dsnullo-tTA-SV40_TREhs43-Dshid Ala4 -CctraF-SV40 ( Schetelig et al, 2021 ) at the Bsu36I and MluI restriction sites to generate V359 _pBXLII_attP_PUbAmCyan_Dsnullo-tTA-SV40_TREhs43-DsRed-NLS-DrosCV-2A-EGFP-CAAX-SV40 , V360 _pBXLII_attP_PUbAmCyan_Dsnullo-tTA-SV40_TREhs43-DsRed-NLS-TaV-2A-EGFP-CAAX-SV40 , V361 _pBXLII_attP_PUbAmCyan_Dsnullo-tTA-SV40_TREhs43-EGFP-NLS-DrosCV-2A-DsRed-CAAX-SV40 , and V362 _pBXLII_attP_PUbAmCyan_Dsnullo-tTA-SV40_TREhs43-EGFP-NLS-TaV-2A-DsRed-CAAX-SV40 , respectively.…”

“…The cell transfection and counting were carried out as previously described ( Yan et al, 2020a ; Schetelig et al, 2021 ). Briefly, Drosophila Schneider 2 (S2) cells were grown on Schneider’s medium containing 10% heat-inactivated fetal bovine serum (Hi-FBS) and 1% penicillin/streptomycin in closed-capped flasks without CO 2 at 25°C.…”