In fusion snap assembly master mix

The wild-type (WT) human NPR2 cDNA was obtained from NCBI. To confirm the possible consequences of the splice site variant, an in vitro analysis was performed using a minigene splicing assay based on the pSPL3 exon trapping vector (Invitrogen) [17 ]. The minigene included the 169-bp exon 15, the 147-bp exon 16, the 124-bp exon 17, the 69-bp exon 18, and the introns of 15, 16, 17 and 18. The minigene sequence was then inserted into the multiple cloning site of the pSPL3 vector (restriction sites: EcoRI and BamHI). The pSPL3-WT plasmid contained the gene of interest. Subsequently, the c.2643G > A variant was introduced into the sequence using PCR with mutagenic primers (5’-CCCATGCAAGTGAGAGCCAT-3’), and the PCR products were ligated using the In-Fusion® Snap Assembly Master Mix (TaKara, Beijing, China).

Plant material accession A056 was utilized for total RNA extraction and reverse transcription polymerase chain reaction to generate cDNA sequences (an identical method to the described qPCR procedure). Primers were designed based on the CDS of StNHX1, StNHX2, StNHX3, StNHX5, StNHX6, StNHX7, and StCHX19 from potato DM genomes. Detailed primer sequences can be found in Supplementary Table S2. In order to clone the coding region sequences of the StNHX1, StNHX2, StNHX3, StNHX5, StNHX6, StNHX7, and StCHX19 genes, the cDNA of the A056 material was employed as a template for PCR amplification using the Supplementary Table S2 primers. The sequences of the StNHX1, StNHX2, StNHX3, StNHX5, StNHX6, StNHX7, and StCHX19 genes were amplified and inserted into the P416 vector using the seamless cloning method (In-Fusion® Snap Assembly Master Mix) (TaKaRa). Finally, vector construction was verified by sequencing. The constructed vectors included P416-StNHX1, P416-StNHX2, P416-StNHX3, P416-StNHX5, P416-StNHX6, P416-StNHX7, and P416-StCHX19.

The plasmids used in this study are listed in Table S3. Plasmids were constructed using a conventional cloning method with restriction endonucleases and T4 DNA ligase, the gap repair cloning method (51 (link), 52 (link)), In-Fusion cloning using an In-Fusion Snap Assembly Master Mix (Takara Bio Inc), or a seamless cloning method described by Liu et al. (53 (link)). Oligonucleotide primers used for plasmid constructions were listed in Table S4. DNA fragments used for plasmid construction via gap repair cloning and seamless cloning were listed in Tables S5 and S6, respectively. The detailed methods for the preparation of individual plasmids are described in the Supporting Information.

The fabp10-GRAB_ATP and fabp10-GRAB_Ado plasmids were constructed using PCR with KOD plus Neo (Toyobo) and In-Fusion Snap Assembly Master Mix (Takara) to produce vectors with Tol2 transposon sites. For fabp10-GRAB_ATP/-GRAB_Ado, the promoter sequence for hepatocytes was amplified using PCR and used to replace elavl3-GRAB_ATP/-GRAB_Ado. Multisite Gateway cloning was performed with the destination vector pDestTol2, the 5′-entry vector containing the fabp10 promoter, the middle entry vector containing pME-mCherry, and the 3′-entry vector containing p3E-polyA. Fabp10:mCherry was used to create fabp10 promoter elements. The zebrafish strains used were as follows: wild-type (AB strain; ZFIN, Eugen, OR, USA), fabp10-GRAB_ATP, and fabp10-GRAB_Ado. The plasmid was injected along with the transposon into the one-cell-stage embryo of wild-type zebrafish, as described previously^{49 (link)}. The plasmid insertion was confirmed by observing heart mCherry in 2 dpf larvae. Injected embryos were raised, and adult zebrafish 2 mpf were identified by amplifying the EGFP gene using PCR with the primers listed in Table 1. They were then outcrossed with wild-type zebrafish to obtain the next generation. Further, to confirm GFP expression in GRAB zebrafish, GRAB_ATP and GRAB_Ado larvae were embedded in E3 medium with ATP (5 mM) or Ado (6 mM) solutions. Live images were captured as described below.

PsAF5, PsPHB2, PsATG8, PsMC, PsCytc, PsAIF B, PsRab5, PsRab7, and SKL genes were amplified from P. sojae cDNA and ligated into pYF3 (G418) or pYF3OX (oxathiapiprolin) using In-Fusion Snap Assembly Master Mix (Takara) to express them as Flag, Myc, GFP, BFP, His, HA or mCherry fusion proteins. The amplified primer information is provided in Supplementary Table 10, and the tag sequences are provided in Supplementary Table 11. Mitochondrial targeting signal (MTS) sequences were amplified from P. infestans β-ATPase and ligated to pYF3 through enzymatic digestion to generate pYF3-MTS-mCherry, pYF3-MTS-GFP, and pYF3-MTS-mCherry-GFP constructs^{43 (link)}. The Flag-PsAF5^ΔANK and Flag-PsAF5^ΔFYVE constructs were expressed in pYF3 and verified through PCR and western blot. Supplementary Table 12 lists all primers used for PsAF5 mutant vector construction.