Drosophila suzukii strains
All fly experiments were performed in our well-equipped safety level one (S1) laboratory, which is certified for generating and using genetically modified insects. Wild type D. suzukii from Italy, USA (both kindly provided by Prof. Marc F. Schetelig), and French Alps (Prof. Dr. Nicolas Gompel) as well as the generated transgenic flies were reared on standard Drosophila food and kept at 25 °C throughout this study.
Nucleic acid isolation
Genomic DNA isolation was done from a mix of adult males and females using NucleoSpin® DNA Insect (Macherey-Nagel) according to the manufacturer instructions. Total RNA was isolated from 0 to 24 h embryos enriched for 0–4 h stages using ZR Tissue & Insect RNA MicroPrep (Zymo Research Europe, 79110 Freiburg) according to manufacturer instructions.
All PCR amplifications during the course of this study were performed using Phusion DNA polymerase and Phusion-HF buffer (New England Biolabs GmbH, D-65926 Frankfurt am Main). A list of the used primers is provided in Additional file 3. Plasmid min-preps and PCR products were purified using NucleoSpin® Plasmid and NucleoSpin® Gel and PCR Clean-up kits (Macherey-Nagel GmbH & Co., 52355 Dueren, Germany), respectively. NucleoSpin® Plasmid Transfection-grade (Macherey-Nagel) or QIAGEN Plasmid Plus Midi Kit (QIAGEN GmbH, 40724 Hilden, Germany) were used to prepare plasmids for germline transformation.
Amplification of cDNA ends
To isolate the 5’UTR and the 3’UTR of the early embryonic gene Ds_sryα and the maternal effect gene Ds_nanos, total RNA from 0 to 24 h old (enriched for 0-4 h) D. suzukii embryos was isolated and 1.3 μg were used to generate 5′ RACE-ready cDNA or 3’RACE-ready cDNA using SMARTer™ RACE cDNA amplification kit (Takara Bio Europe SAS, 78100 Saint-Germain-en-Laye, France) according to manufacturer instructions.
The 5’UTR of Ds_sryα and Ds_nanos were recovered by RACE PCR using gene specific primers HM#34 and HM#76, respectively, along with the universal primer (UPM) provided with the kit using Advantage2 DNA polymerase (Takara) with the following program: 94 °C 2 min, (94 °C 30 s, 72 °C 3 min) 5X, (94 °C 30 s, 70 °C 30 s, 72 °C 3 min) 5X, (94 °C 30 s, 68 °C 30 s, 72 °C 3 min) 30X. A single prominent band for each gene was recovered, purified, cloned into pCRII (Thermo Fisher Scientific) to generate pCRII_sryα_5’UTR (HMMA001) and pCRII_nos_5UTR (HMMA012), and sequenced using standard M13 primers.
To recover the 3’UTR of Ds_sryα and Ds_nanos, the gene specific primers HM#42 and HM#77, respectively, along with UPM provided with the kit using Advantage2 DNA polymerase (Takara) were used with the following program: 94 °C 2 min, (94 °C 30 s, 72 °C 3 min) 5X, (94 °C 30 s, 70 °C 30 s, 72 °C 3 min) 5X, (94 °C 30 s, 68 °C 30 s, 72 °C 3 min) 30X. A single prominent band for each gene was recovered, purified, cloned into pCRII (Thermo Fisher Scientific) to generate pCRII_sryα_3UTR (HMMA002) and pCRII_nos_3UTR (HMMA013), and sequenced using standard M13 primer.
Plasmids construction
The plasmid HMMA020 was generated by PCR amplification of the coding sequence of D. suzukii sryα gene plus the 5’UTR using primer pair HM#16/HM#17 and advantage 2 DNA polymerase (Invitrogene) with program 98 °C 3′ followed by [98 °C 30″, 55 °C 30″,72 °C 2′]35X and cloned into the pCRII vector (Invitrogene).
To generate plasmid HMMA021 for in vitro synthesis of RNA probes, the tTA coding sequence was excised from mfs#1215 [10] using EcoRV/BamHI and cloning into pCRII vector digested by the same enzymes.
To generate plasmid HMMA339 for in vitro synthesis of RNA probe against φC31 integrase mRNA, 800 bp of the coding sequence was digested out from plasmid HMMA98 using SmaI/NotI and cloned into pCRII plasmid digested by EcoRV/NotI.
The plasmid FCMH01 was generated by PCR amplification of 800 bp of Cas9 coding sequence using primers pair HM#560/HM#561 with program 98 °C 3′ followed by [98 °C 30″, 64 °C 30″, 72 °C 30″] 5X [98 °C 30″, 72 °C 1′] 35X, digested by and cloned into XhoI/BamHI sites of pCRII vector.
To generate piggyBac transformation vector HMMA185 and HMMA186, first plasmid HMMA006 [52] was digested by AscI to remove sryα-tTA, and the backbone was ligated to give rise to HMMA007. attP220 was PCR amplified from HMMA007 using primer pair HM#368/HM369 and program 98 °C 3’followed by [98 °C 30″, 58 °C 30″, 72 °C 20″] 5X [98 °C 30″,72 °C 1′] 35X and cloned into EcoRV cut site of HMMA007 to give rise to HMMA185. To generate HMMA186 the EcoRI/HpaI fragment PUb::nlsEGFP from mfs#1213 [51] was cloned into the EcoRI/HpaI sites of HMMA185.
For the generation of piggyBac transformation vectors HMMA330 and HMMA331, first Gibson assembly was performed to clone EGFPSV40 and the 3XP3 promoter into the piggyBac backbone of HMMA007 digested by EcoRI to give rise to HMMA227, in which the EGFP gene was then replaced by DsRed.T3 from HMMA007 by AgeI/NotI to give rise to HMMA228. Then the attP220 was PCR amplified from HMMA007 using primer pair HM#131/HM#117 with PCR program 98 °C 3’followed by [98 °C 30″, 60 °C 30″, 72 °C 20″] 35X and cloned into EcoRI site of HMMA227 and HMMA228 giving rise to HMMA304 and HMMA305, respectively. Finally, the AscI/AgeI fragments from mfs#1213 and mfs#1214 [51] containing the PUb promoter were cloned into AscI/AgeI sites of HMMA304 and HMMA305 to give rise to HMMA330 and HMMA331, respectively.
To generate the spermatogenesis specific driver construct HMMA389, 1 kb upstream region of D. suzukii Ds-β2t gene including the 5’UTR was PCR amplified from genomic DNA of the wild type Italian strain using primer pair HM#35/HM#36 with program 98 °C 3′ [98 °C 30″, 61 °C 30″, 72 °C 30″] 5X [98 °C 30″,67 °C 30″72 °C 30″] 35X and cloned in NcoI/XbaI sites of mfs#1215 [10] giving rise to HMMA015. The Dm-β2t 3UTR was then PCR amplified from gDNA of wild type D. melanogaster strain OreR using primer pair HM#706/HM#707 with program 98 °C 3′ [98 °C 30″, 63 °C 30″, 72 °C 20″] 5X [98 °C 30″,70 °C 30″72 °C 20″] 35X and cloned into HMMA015 to give rise to HMMA253. Finally, the AscI fragment from HMMA253 was cloned into the AscI site of the transformation vector HMMA331.
To generate attB integration vector HMMA182 which can be used to integrate a plasmid into single attP site, the 5-piggyBac region was PCR amplified from plasmid HMMA006 using primer pair T7/mfs#370, with program 98 °C 3′ [98 °C 30″, 51 °C 30″, 72 °C 20″] 40X digested by EcoRV and cloned into the blunted BamHI site of HMMA172, giving rise to HMMA181. Then the EcoRI/ApaI fragment containing the PUb::nlsEGFP was excised mfs#1213 [51] and cloned into EcoRI/ApaI of HMMA181.
To generate the helper plasmid HMMA098, the coding sequence of φC31 was PCR amplified from plasmid mfs#1289 [39] using primers pair MK153/HM#123 with program 98 °C 3′ [98 °C 30″, 72 °C 1′ 20″] 35X. The reverse primer introduces the SV40 nuclear localization sequence at the C-terminus, which can improve the efficiency of φC31 integrase [64]. A second round of PCR using primer pair MK153/HM#203 was used to amplify φC31nls using 1 μl of the first PCR reaction as a template with program 98 °C 3′ [98 °C 30″, 67 °C 30″, 72 °C 1′] 5X [98 °C 30″,72 °C 1′ 20″] 35X and clone into HMMA051 NcoI/NotI replacing the piggyBac transposase coding sequence and giving rise to HMMA098. The piggyBac helper HMMA051 was generated by cloning the SV40 3’UTR digested from CH#705 by HindIII/NotI into HMMA050 HindIII/NotI. sites. The latter was made by PCR amplification of Ds-hsp70 promoter [52] from gDNA using primer pair HM73/HM#74 and program 98 °C 3’followed by [98 °C 30″, 58 °C 30″, 72 °C 30″] 5X [98 °C 30″, 66 °C 30″, 72 °C 30′] 35X and cloning into EcoRI site of HMMA049,which was generated by cloning the piggyBac transposase coding sequence excised from MK004 [23] by EcoRI/NotI into the shuttle vector pSLaf1180af [65].
To generate φC31 integrase based RMCE donor plasmids, HMMA253 and HMMA254, the annealed oligos HM#101/HM#337 generating the bacterial attachment site attB were cloned into SpeI site of pCRII vector (Invitrogene) giving rise to HMMA172. The gypsy insulators were digested out using SpeI/EcoRI from a fragment amplified from mfs#1213 [51] using primer pair HM#469/HM#470 with program 98 °C 3’followed by [98 °C 30″, 70 °C 30″, 72 °C 2′] 35X and cloned into the cut site of HMMA172 to give rise to HMMA189. The EcoRI/NotI fragments PUb::nlsEGFP and PUb::DsRed.T3 were excised from HMMA186 and HMMA185, respectively, and cloned into HMMA189 to give rise to HMMA190 and HMMA191, respectively. Finally, SV40 was PCR amplified from HMMA007 using primer pair HM#179/HM#124 and program 98 °C 3’followed by [98 °C 30″, 62 °C 30″, 72 °C 20″] 5X [98 °C 30″, 68 °C 30″, 72 °C 20″] 35X and cloned along with annealed oligos HM#101/HM#108 into HMMA190 and HMMA191 NotI/XbaI-blunted.
To generate HMMA336, for φC31-RMCE, the tetracycline responsive element TRE along with the P-element basal promoter was PCR amplified from CH 727 [9] using primers pair HM#584/ CH6R [9] with PCR program 98 °C 3’followed by [98 °C 30″, 69 °C 30″, 72 °C 30″] 35X and cloned into EcoRI/ClaI sites of HMMA56 [52] replacing the hsp70 promoter giving rise to HMMA317 then the AscI fragment containing Cas9 fused to the TREp and the SV40 3’UTR was clone into AscI site of HMMA253.
To generate self-docking transformation plasmid HMMA223 the AscI fragment containing nosE/P-φC31-nos was excised from the shuttle vector HMMA221 and cloned into AscI site of HMMA185. HMMA221 was generated by replacement of Cas9 coding sequence in plasmid HMMA167 by φC31 integrase CDS. To make HMMA167, first the 3UTR of Ds-nanos was PCR amplified from HMMA013 using primer pair HM#94/HM95 with program 98 °C 3’followed by [98 °C 30″, 66 °C 30″, 72 °C 30] 5X [98 °C 30″, 72 °C 1′] 35X and cloned into the shuttle vector pSLaf1180af [65] XbaI/AflII sites giving rise to HMMA062. Then Cas9 CDS was excised from HMMA056 [52] and cloned into ClaI/XbaI sites of HMMA062 giving rise to HMMA165. Then the palindromic (self-complementary) oligo HM#102 was annealed to itself to introduce the 2X BbsI recognition site and cloned into the ClaI site of HMMA165 to give rise to HMMA166. Finally, a 2 Kb upstream regulatory region of Ds-nanos gene including the 5’UTR was PCR amplified from gDNA using primer pair HM#345/HM#113 and program 98 °C 3’followed by [98 °C 30″, 72 °C 1′ 30″] 35X and cloned into HMMA166 BbsI site by golden gate resulting in HMMA167.
Germline transformation
All piggyBac germline transformation experiments were performed using transformation vector and helper plasmid MK006 [23] at a final concentration of 500 ng/μL and 200 ng/μL respectively. For φC31-mediated site-specific transformation and φC31-mediated RMCE, the donor vectors were injected along with the helper plasmid HMMA098 at a concentration of 500 ng/μL and 300 ng/μL, respectively. The materials and the procedure of germline transformation were as described previously [23, 52]. Emerged G0 flies were crossed individually to three wild type flies of the opposite sex.
Generation of RNA probes
To generate DIG-labelled antisense RNA probes for in situ hybridization against Ds_sryα, Ds_nanos, tTA, Cas9, or φC31 integrase, DNA templates for in vitro transcription were prepared by restriction enzyme linearization of pCRII vectors containing either the whole gene pCRII_Ds-sryα (HMMA020), the 3’RACE fragment pCRII_Ds-nos_3UTR (HMMA013), the coding sequence pCRII_tTA (HMMA021), or 800 bp of the coding sequence of in case of pCRII_Cas9 (FCMH01) and pCRII_φC31 (HMMA399) using XhoI, BamHI, NotI, NotI, or EcoRI, respectively. The antisense RNA labelling reaction was done using the DIG-labelling kit (Thermo Fisher Scientific) according to manufacturer instructions using 1 μg of DNA as template in a total reaction mix of 20 μL. The reaction was allowed to proceed for 3 h at 37 °C followed by Turbo DNaseI treatment (Thermo Fisher Scientific) for 30 min to remove template DNA. Two microliter of 0.2 M EDTA were used to inactivate the reaction. The probes were then ethanol precipitated and resuspended in 100 μL RNA resuspension buffer (5:3:2 H2O: 20X SSC: formaldehyde) and stored at − 80 °C.
Testes, ovary, and embryo whole mount in situ hybridization
Testes from 3 to 5 days old males from wild type D. suzukii, spermatogenesis specific driver line 389_M25M1, or progeny of the cross of the driver 389_M25M1 to the responder line 366_F3F1 were dissected in ice cold 1X phosphate buffered saline (PBS). Fixation and in situ hybridization were performed according to protocol by Lecuyer [66]. Anti-sense DIG labelled RNA probe against tTA was used to detect the expression driven by the Ds-β2t E/P. The Cas9 anti-sense RNA probe was used to detect the expression of Cas9 in the progenies arising from the cross testing the tet-off system. Anti-sense and sense probes previously described [52] were used as control.
To confirm the expression of the isolated Ds-nanos gene and the φC31 integrase driven by the regulatory regions of Ds-nanos in the ovaries of D. suzukii wild type flies and the transgenic self-docking line 223_F7M1, respectively, we collected 3–5 days old female flies and dissected the ovaries in ice-cold 1X PBS. The fixation and the in situ hybridization were performed as described [66].
To confirm the endogenous cellularization-specific expression of Ds_sryα in wild type embryos. and whether the 349 bp of its upstream regulatory region including the 5’UTR are enough to drive expression of tTA in the transgenic driver line 06_F5M2 in a similar pattern, we performed embryo whole mount in situ hybridization using respective anti-sense DIG-labelled RNA probes in 0–24 h old embryos. Fixation and in situ hybridization were performed according to Lecuyer [66].
Microscopy
To observe and image testes, ovaries, and embryos, Zeiss Imager.Z2 equipped with two cameras, Axiocam 506 mono and Axiocam 305 colour (Zeiss, 73447 Oberkochen, Germany) was used. Images were taken using Axiocam 305 with bright field or DIC settings.
Screening for transgenic flies and fluorescence imaging were performed using Leica M205 FA fluorescence stereomicroscope equipped with camera Q imaging Micropublisher 5.0 RTV (Leica Mikrosysteme Vertrieb Gmb, Wetzlar, 35578 Germany). Transgenic flies were screened using filter sets RFP (excitation: ET546/10, emission: ET605/70) or GFP-LP (excitation: ET480/40, emission: ET510 LP), respectively, and imaged using cold light (Fig. 3A′,B′), filter sets RFP (Fig. 3A″,B″), or EYFP (excitation: ET500/20, emission: ET535/30; Fig. 3A‴,B‴).