Multisite gateway three fragment vector construction kit

A middle entry vector carrying dendra2-rab3 was generated by fusing the rab3 open reading frame of pBHUAS-Rab3-YFP (kindly provided by Michael Nonet) [45 (link)], and the dendra2 sequence from pDendra2-N1 (kindly provided by Jean-René Huynh, Institut Curie, Paris) via PCR amplification. The middle entry vector was combined with a standard p5’UAS vector (Tol2kit) and a standard p3’SV40pA using the MultiSite Gateway Three-Fragment Vector Construction Kit (ThermoFisher Scientific) to obtain pUAS-dendra2-rab3-pApUAS-dendra2-rab3-pA.

The viral sensor construct and the gfp::sta-1 construct were generated by Gateway cloning using the MultiSite Gateway three-fragment vector construction kit (Life Technologies). Gateway entry clones containing each of the following were generated by standard techniques: sur-5 promoter, sdz-6 promoter, sta-1 coding sequence, eGFP(F64L/S65T) (eGFP stands for enhanced green fluorescent protein), and tbb-2 3′ untranslated region (3′UTR). The single-copy transgene was generated by transposase-mediated integration (MosSCI), as described previously (42 (link), 43 (link)), at insertion site ttTi5605 on chromosome II. Injection mixes contained 20 ng/µl of vector, 20 ng/µl of Mos1 transposase (only for MosSCI), and 5 ng/μl of a pharynx marker. Integration of the extrachromosomal array was performed by ethyl methanesulfonate (EMS) treatment (50 mM EMS for 4 h).

The set-2 transcriptional reporter includes a ∼400 bp region located at the 5′ end of the set-2 gene amplified using the following primers: 5′-ccgatgcacagtagaaatctg-3′ and 5′-gcaaacttcatatccagaccata-3′. The PCR product was cloned into pD95.75mCherry. Tissue-specific rescue constructs were made using a MultiSite Gateway Three-Fragment Vector Construction Kit (Life Technologies) as described previously (Mariani et al., 2016 (link)). The set-2 cDNA was a kind gift from Francesca Palladino (École normale supérieure de Lyon, France).

Molecular biology was performed according to standard protocols. The Multisite Gateway Three-Fragment Vector Construction Kit (Life Technologies, Grand Island, NY) was used to construct donor plasmids. Fragments were then recombined into the MosSCI destination vectors pCFJ150 or pCFJ212 (Frøkjær-Jensen et al., 2008 (link), 2012 (link)). For constructs in which two fragments were ligated by PCR, fusion PCR was performed as in Hobert (2002) (link). Primers used for generating constructs are listed in Table 3, and plasmid construction strategies are summarized in Table 4.

The psur-5::GFP::OrsayRNA2::tbb-2 and psur-5::GFP::OrsayRNA1::tbb-2 constructs were generated by using the MultiSite Gateway Three-Fragment Vector Construction kit (Life Technologies). psur-5 in the first position was a gift from the Ahringer laboratory, and green fluorescent protein (GFP; cloned from pPD95.75) was placed in the second position. PCR fusion was used to generate the OrsayRNA::tbb-2 fragments that were placed in the third position. All three fragments were combined in an LR reaction into the pCFJ150 vector. The sequences of the primers used to amplify viral segments from cDNA are available on request.

Deletion of the hcpA gene was carried out in P. chrysogenum strain DS54555, which lacks penicillin cluster genes and the ku70 gene [18] (link). This strain was kindly provided by the DSM Biotechnology Center (Delft, Netherlands). A deletion plasmid was constructed by amplifying the flanking regions of the targeted gene with the Multisite Gateway^®Three-Fragment Vector Construction Kit according to the procedure described by Invitrogen using pDEST R₄-R₃p as template. Primers used for the construction of the deletion plasmid pDEST R₄-R₃p PcHcpA (Figure S1) are listed in Table S1. Escherichia coli DH5α (F– Φ80lacZΔM15 Δ(lacZYA-argF) U169 recA1 endA1 hsdR17 (rK–, mK+) phoA supE44 λ– thi-1 gyrA96 relA1) was used as host strain for high frequency transformation and plasmid DNA amplification [21] . All the strains were grown on yeast nitrogen base-glucose-yeast extract (YGG)-medium for protoplasts formation and transformation [22] (link). Both mutant and host strains of P. chrysogenum were grown on secondary metabolite production medium as described previously [18] (link).