Pdonr222

The specific primers SaHsfA4a-F/R and SaHsfA4c-F/R (Supplementary Table S2) were used to amplify the open reading frames in S. alfredii. The purified PCR products were then cloned into the Gateway entry vector pDONR222 (Invitrogen, Carlsbad, USA), and recombined into pYES-DEST52 to generate pYES-DEST52-SaHsfA4a and pYES-DEST52-SaHsfA4c, respectively. The ∆ycf1 strain was transformed with each construct, using the lithium acetate method [33 (link),52 (link)]. The empty pYES2.0 vector was used as control. To perform the Cd tolerance test, the transgenic yeast lines were grown up to OD₆₀₀ = 1, and then serially diluted (OD₆₀₀ = 10⁰, 10⁻¹, 10⁻², 10⁻³, 10⁻⁴, and 10⁻⁵), spotted on SG-U agar plates supplemented with 0, 15, and 30 µM CdCl₂, and incubated at 28 °C for 3 days [33 (link),50 (link)]. Additionally, the relative growth of transformants was determined by measuring OD₆₀₀ at 12 h intervals. For the Cd-uptake assay, yeast cells that were transformed with the empty, pYES-DEST52-SaHsfA4a or pYES-DEST52-SaHsfA4c vector were grown at 28 °C on SG-U, supplemented with 15 µM CdCl₂ for 96 h, and finally, the Cd accumulation was measured [50 (link)].

The primers used for the following plasmid constructs are shown in Supporting Information Additional file 1: Table S1. The various mutants of PvRxLR16 for PVX assay were amplified using combinations of primers documented in Additional file 1: Table S1. The PCR products were cut with Xma I and Sal I restriction enzymes and ligated into the PVX::flag vector. For GFP fusion constructs, PvRxLR16, PvRxLR16:NES and PvRxLR16:nes were amplified with gene-specific primers modified to contain the Gateway (Invitrogen) attB recombinantion sites. The PCR amplicons were cloned into entry vector pDONR222 (Invitrogen) via BP reactions and subsequently recombined into the binary vector pH7FWG2, 0 using Gateway LR recombination. To make the constructs for virus induced gene silencing (VIGS) in N. benthamiana, partial sequences of purpose genes were amplified from cDNA of N. benthamiana and subsequently ligated into pTRV2 vector using the Xma I and Kpn I restriction sites. All the generated plasmids were validated by sequencing by Majorbio, Inc. (Shanghai, China). The schematic diagrams of constructs used in this study were shown in Additional file 2: Figure S1.

The specific primers SaGLIP8-F/R were used to amplify the open reading frame of SaGLIP8 (Table S1). The purified PCR product was first inserted into the entry vector pDONR222 (Invitrogen, Carlsbad, CA, USA), and then yeast expression vector pYES-DEST52-SaGLIP8 were constructed by gateway LR reaction. The empty vector pYES2.0 was used as a control. Two vectors, expression vector pYES-DEST52-SaGLIP8 as well as empty vector pYES2.0, were transformed into the Cd sensitive mutant strain Saccharomyces cerevisiae (Δycf1) using the lithium acetate method (Liu et al., 2016 ). Positive colony selection was performed in the solid medium with 50 µg ml⁻¹ ampicillin and PCR reaction. The selected positive clones in the yeast liquid were cultured to an OD600 value of 0.8–1.0, and then spotted on SG-U (synthetic galactose-uracil) solid medium containing concentrations of 0, 15 and 30 µM CdCl₂. The strains in SG-U liquid medium were diluted (OD₆₀₀ = 100, 1/10, 1/100, 1/1000, 1/10000 and 1/100000), then incubated in a 28 °C incubator for 3 d (Chen et al., 2017 (link); Liu et al., 2016 ). In addition, two transformed yeast cell strains were cultured on liquid SG-U medium containing 30 µM CdCl₂ for 96 h at 28 °C to determine the Cd accumulation levels by the Inductively Coupled Plasma Mass Spectrometry (ICP-MS, NexIon 300D, Perkin Elmer, Shelton, CT, USA).

Human clones in Gateway donor vectors (pENTR 223.1 or pDONR 222, Invitrogen) were transferred into the vector pGW-HA.attB (same as fly cDNAs noted above) using Gateway LR clonase enzyme (11791019, Invitrogen). For clones not supplied in a Gateway-compatible system, the cDNA was PCR amplified from the parent plasmid using primers that hybridized to either end of the cDNA sequence with a Gateway sequence added to their 5′ ends. The amplified fragment was then cloned into pENTR 223.1 using Gateway BP clonase enzyme (11789020, Invitrogen). When a cDNA did not contain a stop codon at its 3′ end and one was biologically necessary, then an in-frame stop was engineered into the PCR 3′ reverse primer. After the clonase reaction, the mix was electroporated into DH5α Escherichia coli and plated with an antibiotic for the selection of transformants. The 5′ and 3′ ends of each human cDNA cloned into pGW-HA.attB were sequenced. Confirmed pGW-HA.attB cDNA clones were injected at BestGene. Each cDNA was inserted into a landing site on chromosome 2 (BL9752 or BL9723) and chromosome 3 (BL9750). For each cDNA at each landing site, a homozygous line was produced. One of the two stocks was deposited at Bloomington, and both were deposited in Kyoto. Note that when no tag is present, then the genotype suffix is N instead of HA. A complete list of UAS.human cDNA stocks is in Supplementary Table 2b.

RNAs from female (MSM−) and male (MSM+) gonads at 15, 18, 22, 26, 30, 35, 40, 47, 58, 70, 110, 200, and 360 dph were pooled and reverse-transcribed. The purified double strand cDNAs were cloned into pDONR222 (Invitrogen) by BP Clonase II enzyme mix (Invitrogen) and the library titre was 1.12 × 10⁷ cfu (colony-forming units) (S13A, S13B and S13E Fig). Then, these cDNAs were transferred from plasmid pDONR222 to pPR3-N-DEST (Dualsystems Biotech) by BP Clonase II enzyme mix (Invitrogen) and the library titre was 3.40 × 10⁷ cfu (colony-forming units) (S13A, S13C and S13F Fig). The mature peptide sequence of gsdf-A (from 95 to 186 amino acids) was cloned into pBT3-SUC vector (Dualsystems Biotech) as bait (pBT3-SUC -Gsdf-A) and yeast library screening was performed according to protocol of the DUAL membrane starter kits User Manual (Dualsystems Biotech).

The expression constructs for LANA (pcDNA3.1/LANA) and KSHV cluster miRNAs (pcDNA3.1/cluster) were described in previous reports [24 ,33 (link)]. For construction of vFLIP and vCyclin expression vectors, Gateway Cloning method was used. After ORFs of vFLIP and vCyclin were amplified by PCR and cloned into entry vector (pDONR222, Invitrogen) using BP recombination, ORFs were cloned into pLenti6/V5-DEST (Invitrogen) using LR recombination following the manufacturer’s procedure to create pLenti6/vFLIP and pLenti6/vCyclin. Reporter vector containing the promoter of miR-17-92 cluster upstream of luciferase vector was kindly provided from Dr. De Guire [19 (link)]. pGL3-SBE4 (Promega) contains four SMAD binding elements upstream of luciferase gene, activated by TGF-β ligand. pCMV-Renilla (Promega), expressing renilla luciferase, was used for normalization of firefly luciferase activity.

The full-length cDNA of PcNRAMP1 was amplified and ligated into Gateway entry vector pDONR222 (Invitrogen™, Shanghai, China). After sequencing, the cDNA was directionally cloned into pK7WGF to generate PcNRAMP1-pK7WGF, in which PcNRAMP1 was fused to the C-terminal of EGFP under the control of the CaMV35S promoter (GFP-PcNRAMP1) as described elsewhere [5 (link)]. The primer sequences used were listed in Table S1. Then, GFP-PcNRAMP1 was co-transformed with pMDC32-1A CAN2b-mCherry (a plasma membrane marker) into Nicotiana benthamiana leaf epidermal cells by agroinfiltration [5 (link)]. After infiltration for 48 h, the subcellular localization of PcNRAMP1 was detected by a confocal laser scanning microscope (LSM880, Carl Zeiss, Jena, Germany). The excitation/emission wavelengths were 488 nm/510 to 530 nm for green fluorescence, and 587 nm/610 nm for red fluorescence.