Qiaprep miniprep

DNA amplification was obtained by PCR with either KAPA HiFi HotStart ReadyMix (Roche KK2601) or Q5 High-Fidelity 2X Master Mix (New England Biosystems #M0492) following manufacturer protocol unless otherwise noted. PCR products were digested with 1 μL DpnI (NEB #R0176L) for 1 hour and purified by agarose gel extraction (MinElute Gel Extraction Kit, Qiagen #28606). Plasmid constructs were created by Gibson cloning with NEBuilder HiFi DNA Assembly Master Mix (NEB #E2621L) or Gibson Assembly Master Mix (NEB E2611L). Assembled plasmids were transformed into Mach1 (Invitrogen #C862003) or STBL3 (from Q3 Macrolab at University of California, Berkeley) competent cells for amplification and were purified with QIAprep miniprep (Qiagen #27104).

All restriction digest reactions were performed with enzymes provided by NEB according to the user's manual. T4 DNA Ligase (NEB) was used for ligation reactions. Chemically competent DH5α E. coli (Life Technologies) was used for transformation following the manufacturer’s instructions. QIAprep Miniprep or Midiprep Kits (Qiagen) were used for plasmid purification. Cloning was verified by PCR followed by gel electrophoresis and sequencing.
To make the rescuing mir-1 transgene, primer pair ‘celmir1fwd2/rvs2’ was used to insert the mir-1 coding region into vector L3781 downstream of gfp. The mir-1 promoter was then cloned 5' to gfp using the primer pair ‘m1p fwd/rvs’. To make muscle-expressed vha-13 constructs, the vha-13 cDNA was amplified with Kpn1 overhangs using primers vha-13 fwd/rvs and cloned into vector pDEST R4-R3 to give myo-3p::flag::HA::mCherry::vha-13cDNA::unc-54 3'UTR. The unc-54 3'UTR was excised with Not1/BglI and replaced with the vha-13 3'UTR using primers vha-13U fwd/rvs to generate myo-3p::flag::HA::mCherry::vha-13cDNA::vha-13 3'UTR. Primers and plasmids are listed in the Key resources table and Supplementary file 1h.

Candida antarctica ATCC 28323 was grown in YPD medium with 1% (w/v) peptone, 2% (w/v) yeast extract and 2% (w/v) glucose, pH 4.5. Submerged cultivation was carried out at 30 °C for 72 h, at 300 rpm in a 300 ml Erlenmeyer flask. Genomic DNA (gDNA) was isolated using QIAamp DNA Mini Kit (Qiagen). Primers for the lipase A gene PCR (Supplementary Table 1) were designed as recommended by pETBlue-2 (Novagen). PCR products were separated by electrophoresis on 1% (w/v) agarose gel. Cloning was performed using Perfectly Blunt Cloning Kits (Novagen). Plasmid DNA was isolated from the NovaBlue Singles Competent Cells using QIAprep Miniprep (Qiagen), and a clone expressing recombinant protein was selected from transformed E. coli Tuner (DE3)pLacI competent cells. Plasmid DNA was isolated and the lipase A gene was sequenced using a Sanger DNA sequencing method. This plasmid DNA was used as a template in the epPCR reactions.

Specific primers for MiETR1 were designed (Genbank ID: AF227742.1; Table 1). Conserved regions of ERS-like sequences from woody plants and Arabidopsis were identified by alignments to design degenerate primers. Nested PCRs were performed to verify sequence specificity before cloning. The PCR products were then ligated into the pGEM-T vector (Promega, VIC, Australia) following the manufacturer's recommendations. After blue-white selection, a colony PCR with gene specific primers (Table 1) was performed to verify positive clones for subsequent plasmid extraction (QIAPrep Miniprep, Qiagen, Germany) and sequencing (GATC, Germany). Using degenerate primers to identify the homolog to the Arabidopsis AtERS1, three different versions of mango ERS1 were detected and confirmed by sequencing: a version with the full length sequence (MiERS1) that is comparable to the AtERS1, a medium sized MiERS1m with a length of 1203 nucleotides, and a short MiERS1s with a length of 561 nucleotides. The sequences were confirmed to be MiERS1-like by BLAST search using the NCBI online tool (http://blast.ncbi.nlm.nih.gov) and following the recommendations of Samach (2012 (link)).

To collect biomass for metagenomic sequencing, we removed larger particles and eukaryotes such as Dunaliella salina, by pre-filtering samples through 3-μm filters (cellulose-nitrate, Millipore). For environmental DNA extraction, we mixed the water samples (120 L) and used 40 L for biomass collection using two strategies. Ten liters of the sample were centrifuged at 4500 rpm (3260×g) for 60 min, and retentate was used for DNA extraction using QIAprep® Miniprep (Qiagen). The second strategy was biomass collection on polycarbonate membrane filters with 0.22-μm pore size (Isopore Membrane Filter, Isopore™ Millipore), and finally DNA extraction from filters using Qiagen DNeasy (Qiagen). The quantity and quality of the extracted DNA were analyzed by a NanoDrop™ One C Microvolume UV–Vis Spectrophotometer and agarose gel electrophoresis.