Yeast transformation and beta-galactosidase assays were performed following the manufacturer’s instructions (Clontech). Full-length cDNAs for WUS, HAM1, HAM2, HAM3, and HAM4 were cloned into pENTR/D/TOPOor pCR8 (Invitrogen), and then WUS cDNA was Gateway cloned to pDEST32, and HAM1, HAM2, HAM3, and HAM4 cDNAs were Gateway cloned into pDEST22 using standard LR reactions (Invitrogen). All of the deletion derivatives for WUS or HAM1 were generated through overlapping PCR with the primers listed in methods, cloned into pENTR/D-TOPO or pCR8, and cloned into pDEST32 or pDEST22 through LR recombination (Invitrogen). All clones were sequenced to confirm they were in-frame and the corresponding deletions before being transformed into yeast. The bait and prey vectors were transformed into yeast strain MaV203, and three single transformed colonies per genotype were used as triplicate for the LacZ liquid assay in 96 Deep well plates (Thermo) and OD reading was recorded in 96-well plate reader (Tecan). The LacZ activity was calculated as (OD420×1000)/(OD600 × cell volume in μl × assay time in minutes) following the yeast two hybrid handbook (Clontech), including a standard error from three biological replicates.
Pdest22
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PDEST22 is a laboratory instrument designed for precision temperature control. It features a programmable digital interface and supports a range of temperature settings to accommodate various research and testing applications.
Automatically generated - may contain errors
Lab products found in correlation
Pdest32, by Thermo Fisher Scientific (31 mentions)
Proquest two hybrid system, by Thermo Fisher Scientific (4 mentions)
Pdonr221, by Thermo Fisher Scientific (2 mentions)
Pdest22, by Takara Bio (2 mentions)
Yeast protocols handbook, by Takara Bio (2 mentions)
Mav203, by Thermo Fisher Scientific (2 mentions)
Yeast two hybrid handbook, by Takara Bio (2 mentions)
96 deep well plate, by Thermo Fisher Scientific (2 mentions)
Beta galactosidase assays, by Takara Bio (2 mentions)
Pentr d topoor pcr8, by Thermo Fisher Scientific (2 mentions)
Pentr d topo, by Thermo Fisher Scientific (2 mentions)
96 well plate reader, by Tecan (2 mentions)
Ah109, by Takara Bio (2 mentions)
Pdonr222, by Thermo Fisher Scientific (2 mentions)
Pcr8 gw topo, by Thermo Fisher Scientific (2 mentions)
Lr clonase enzyme, by Thermo Fisher Scientific (1 mentions)
Topo pentry, by Thermo Fisher Scientific (1 mentions)
Yeastmaker yeast transformation system 2, by Takara Bio (1 mentions)
Pdest32, by Takara Bio (1 mentions)
Gateway destination vector, by Thermo Fisher Scientific (1 mentions)
45 protocols using pdest22
1
Yeast Transformation and Beta-Galactosidase Assays
2
Genome-wide Yeast Two-Hybrid Assay
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In the Gal4-based Y2H screen, full-length ORFs of S. pneumoniae were shuttled from pENTR221 entry clones (Pathogen Functional Genomics Resource Center [PFGRC]; formerly at the J. Craig Venter Institute [JCVI], Rockville, MD; now maintained by BEI]) into Y2H bait plasmid pDEST22 (Invitrogen, Carlsbad, CA) using Gateway cloning.
Individual bait plasmids were transformed into haploid yeast strain CG-1945 and prey plasmids into Y187 (Clontech, Mountain View, CA) as described previously (39 (link)).
The prey library was created by growing all plasmid strains of the S. pneumoniae entry clone library individually in selective Luria Broth (LB) medium, followed by pooling and plasmid isolation. The resulting entry clone plasmid pool was shuttled into the pGADT7g and pDEST22 prey plasmids by the use of a Gateway LR reaction (Invitrogen). The reaction mixture was then transformed into electrocompetent E. coli DH10B (ElectroMAX; Invitrogen) and grown in selective LB medium, and the plasmids were isolated. Plasmid pools were then transformed into Y187 as described previously (40 (link)) and spread onto 24-by-24-cm dishes containing Synthetic Defined (SD) agar. Finally, all colonies were scratched from the plates, resuspended in 25% glycerol, and stored as 50-μl aliquots at −80°C.
Individual bait plasmids were transformed into haploid yeast strain CG-1945 and prey plasmids into Y187 (Clontech, Mountain View, CA) as described previously (39 (link)).
The prey library was created by growing all plasmid strains of the S. pneumoniae entry clone library individually in selective Luria Broth (LB) medium, followed by pooling and plasmid isolation. The resulting entry clone plasmid pool was shuttled into the pGADT7g and pDEST22 prey plasmids by the use of a Gateway LR reaction (Invitrogen). The reaction mixture was then transformed into electrocompetent E. coli DH10B (ElectroMAX; Invitrogen) and grown in selective LB medium, and the plasmids were isolated. Plasmid pools were then transformed into Y187 as described previously (40 (link)) and spread onto 24-by-24-cm dishes containing Synthetic Defined (SD) agar. Finally, all colonies were scratched from the plates, resuspended in 25% glycerol, and stored as 50-μl aliquots at −80°C.
3
Yeast Transformation and Beta-Galactosidase Assays
4
Yeast Two-Hybrid Assay Protocol
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The Y1H reporter strain was obtained as described (Deplancke et al., 2006) .
bZIP17 and bZIP60 full-length and truncated ORFs were cloned into pDEST22 (Invitrogen), to create a fusion with the GAL4 activation domain. TSAR1 and TSAR2 recombined with the destination vector pDEST22 (Invitrogen) had previously been obtained by (Mertens et al., 2016a) . Empty pDEST22 was used as negative control.
The yeast reporter strain was transformed with the preys followed by verification of growth on SD medium lacking His and Trp plates with and without 20 mM 3-amino-1,2,4-triazole after an incubation period of 6 d at 30°C.
bZIP17 and bZIP60 full-length and truncated ORFs were cloned into pDEST22 (Invitrogen), to create a fusion with the GAL4 activation domain. TSAR1 and TSAR2 recombined with the destination vector pDEST22 (Invitrogen) had previously been obtained by (Mertens et al., 2016a) . Empty pDEST22 was used as negative control.
The yeast reporter strain was transformed with the preys followed by verification of growth on SD medium lacking His and Trp plates with and without 20 mM 3-amino-1,2,4-triazole after an incubation period of 6 d at 30°C.
5
Cloning and Subcloning of Pea Symbiotic Genes
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Full-length PsDELLA1, PsKNOX3, and PsBELL1 coding sequences were obtained by amplification of cDNA cv. Finale or cv. Sparkle using specific PCR primer pairs flanking with attB1 and attB2 sequences or CACC in forward primer (Supplementary Table S2 ) for subsequent cloning in pDONR221 or pENTRY-TOPO vectors (Thermo Fisher scientific, United States) according to manufacturer’s protocol.
At the next stage they were finally subcloned into the destination vectors pDEST22 (PREY) or pDEST32 (BAIT) vectors using the LR clonase enzyme (Thermo Fisher scientific). All verified constructs were transferred into MaV203 yeast strain (Thermo Fisher scientific).
Full-length PsNSP2 and PsIPD3/CYCLOPS coding sequences or partial coding sequences upto stop-codon corresponding to those in RisNod14, E69 nsp2 (sym7) or SGEFix--5 [ipd3/cyclops (sym33)] mutants were obtained by amplification of cDNA cv. Finale or Sparkle using specific PCR primer pairs flanking with attB1 and attB2 sequences followed by cloning into pDEST22 (PREY) or pDEST32 (BAIT) vectors.
At the next stage they were finally subcloned into the destination vectors pDEST22 (PREY) or pDEST32 (BAIT) vectors using the LR clonase enzyme (Thermo Fisher scientific). All verified constructs were transferred into MaV203 yeast strain (Thermo Fisher scientific).
Full-length PsNSP2 and PsIPD3/CYCLOPS coding sequences or partial coding sequences upto stop-codon corresponding to those in RisNod14, E69 nsp2 (sym7) or SGEFix--5 [ipd3/cyclops (sym33)] mutants were obtained by amplification of cDNA cv. Finale or Sparkle using specific PCR primer pairs flanking with attB1 and attB2 sequences followed by cloning into pDEST22 (PREY) or pDEST32 (BAIT) vectors.
6
Yeast Two-Hybrid Screening for Autophagy Protein Interactions
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The entry clone containing FYVE3 cDNA was recombined with the destination vector pDEST22 (Thermo Fisher Scientific) via the LR Clonase II reaction. The resulting pDEST22-FYVE3 and pDEST32 expression vectors for bait proteins fused to SAR1B, SAR1C, ATG8A, ATG8E, ATG8F, ATG8I, ATG18A and FYVE2 (Kim et al., 2022 (link)) were used for transformation of the AH109 strain and Y187 strain through the Yeastmaker Yeast Transformation System 2 (Clontech, Mountain View, CA, USA). To determine protein interaction, yeast colonies isolated from Trp- and Leu- deficient medium were transferred to Trp-, Leu-, and His- deficient medium containing 0 and 1 mM 3-amino-1,2,4-triazole.
7
Yeast Two-Hybrid Protein Interaction Analysis
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8
Yeast Two-Hybrid Analysis of GhMYB4 and GhMYB66
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To characterize the interaction between GhMYB4 and GhMYB66 protein, a bait vector carrying the full-length GhMYB4 ORF fused to the GAL4 DNA-binding domain in a pDEST32 (Invitrogen) vector and a prey vector carrying full-length GhMYB66 ORF fused to the GAL4 DNA activation domain in a pDEST22 (Invitrogen) vector were generated according to the manufacturer’s instructions (ProQuest Two-Hybrid System; Invitrogen). pDEST32 and pDEST22 were introduced into AH109 and Y189 strains, respectively. The primers used in the study are listed in Supplemental Table S5 .
9
Lettuce RNA Extraction and cDNA Library
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Lactuca sativa cv. Olof seedlings were sprayed with water, 0.1 mg ml−1 benzothiadiazole (BTH) solution, B. lactucae race Bl:24 (compatible interaction) or isolate F703 (incompatible interaction) spore suspension. RNA was extracted using phenol/chloroform extraction at 24 h after BTH treatment and 3 days after infection; 2 mg of RNA (0.5 mg per treatments) was used to construct a three‐frame uncut cDNA library in prey vector pDEST22 by Invitrogen Custom Services (Invitrogen, Carlsbad, CA, USA).
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Lactuca sativa cv. Olof seedlings were sprayed with water, 0.1 mg ml−1 benzothiadiazole (BTH) solution, B. lactucae race Bl:24 (compatible interaction) or isolate F703 (incompatible interaction) spore suspension. RNA was extracted using phenol/chloroform extraction at 24 h after BTH treatment and 3 days after infection; 2 mg of RNA (0.5 mg per treatments) was used to construct a three‐frame uncut cDNA library in prey vector pDEST22 by Invitrogen Custom Services (Invitrogen, Carlsbad, CA, USA).
10
Yeast Two-Hybrid Screening for DY1 Interactors
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The yeast two-hybrid screening was performed largely according to the MatchmakerTM GAL4 Two-Hybrid System 3 & Libraries User Manual (TaKaRa). Full length ORF of DY1 was cloned into pDEST32 (Invitrogen, Carlsbad, CA, United States) and then transformed into the yeast (Saccharomyces cerevisiae) strain AH109. A. thaliana cDNA library constructed in pDEST22 (Invitrogen) was maintained in the yeast strain Y187. After mating, diploid yeast cells were screened on SD/-Leu/-Trp/-His triple dropout medium. From positive colonies, cDNA insertions encoding the prey proteins were amplified by PCR and sequenced. Candidate genes were further tested for pair-wise interaction using a pGAD-T7/pGBK-T7 system (TaKaRa). Briefly, the coding sequences of DY1 and candidate genes were cloned into pGAD-T7 and pGBK-T7, respectively. Plasmids were co-transformed into yeast strain AH109 and selected on SD/-Leu/-Trp double drop out plates. Colonies were serial diluted and further tested on SD/-Leu/-Trp/-His/-Ade quadruple dropout plates for detecting protein–protein interactions.
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