PvC3H72 (Phytozome accession no.: Pavir.J07041.1) was previously identified as a CCCH-type Znf family gene and classified in the clade-XIV as a stress-responsive gene [18 (link)]. The full length gene was amplified from the gDNA of a selected line ‘HR8’ from switchgrass lowland ecotype ‘Alamo’ [47 (link)], and its encoded amino acid sequence is shown in Additional file 3 : Figure S2. The gene was cloned into the Gateway entry vector pENTR/D (Invtirogen, Carlsbad, CA). The gene was subcloned into p2GWF7 [48 (link)], pGBKT7 (Invitrogen) and pVT1629 [47 (link)] through LR reaction (Invitrogen).
Pgbkt7
Manufactured by Thermo Fisher Scientific
Sourced in Australia, United States
The PGBKT7 is a laboratory equipment manufactured by Thermo Fisher Scientific. It is designed for performing various experimental procedures in a controlled environment. The core function of this product is to provide a secure and reliable platform for conducting scientific investigations. However, a detailed description of its specific features and intended use cannot be provided while maintaining an unbiased and factual approach.
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Lab products found in correlation
Lr reaction, by Thermo Fisher Scientific (2 mentions)
Pentr d, by Thermo Fisher Scientific (2 mentions)
Pgadt7, by Thermo Fisher Scientific (2 mentions)
P2gwf7, by Thermo Fisher Scientific (1 mentions)
Pgex4t3, by Thermo Fisher Scientific (1 mentions)
Pcrtm2.1 topo, by Thermo Fisher Scientific (1 mentions)
Yeast protocols handbook, by Takara Bio (1 mentions)
Y2h gold yeast cells, by Takara Bio (1 mentions)
Gateway system, by Thermo Fisher Scientific (1 mentions)
Pgadt7, by Takara Bio (1 mentions)
Pgbkt7, by Takara Bio (1 mentions)
8 protocols using pgbkt7
1
CCCH-type Znf gene cloning and expression
2
Cloning and Characterization of PvC3H69 Gene
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The full-length gene of PvC3H69 (Phytozome accession no.: Pavir.J04795.1), with 394 amino acids without intron was amplified from the gDNA of a lowland ecotype ‘Alamo’ switchgrass37 (link). The gene was firstly cloned into the Gateway entry vector pENTR/D (Invitrogen). It was subcloned into p2GWF7.038 (link), pGBKT7 (Invitrogen), and pVT162937 (link) through LR reaction (Invitrogen). The primers used for PvC3H69 cloning are listed in Table S1 .
3
Yeast two-hybrid assay for Rad1 mutants
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Yeast two hybrid was performed in order to assess the ability of rad1-E349K and rad1-E706K to interact with Rad10 and Slx4. RAD10 and SLX4 were cloned into pGADT7 and RAD1 was cloned into in the pGBKT7 (Invitrogen). The rad1-E349K and rad1-E706K single point mutations were introduced in the pGBKT7-RAD1 by site-directed mutagenesis. Plasmids were transformed into opposite mating types (a or α) and mated to create diploid strains harboring both plasmids. Interactions were assayed according to manufacturer’s protocol.
4
Yeast Two-Hybrid Screening of Maize and Wheat NF-YB Factors
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The CDS of ZmNF-YB2a (accession no. NP_001105435) was cloned into the vector pGBKT7 (Invitrogen, Victoria, Australia) for Y2H screening. ZmNF-YB2a was used because maize ZmNF-YB2 cDNA could not be isolated based on the sequence submitted to public databases by Nelson et al. (2007) . The only protein sequence difference between the two is an insert of seven amino acid residues (GKTIPAN) in the HFD of ZmNF-YB2, which is absent in ZmNF-YB2a and similar NF-YB subunits from wheat and other grasses. ZmNF-YB2a was used as bait to screen WGL and WENDL (Lopato et al., 2006 (link)), WHSL (Eini et al., 2013 (link)), WGD (developing grain at 0–6 days after pollination collected from the wheat cv. RAC875 subjected to drought at flowering), and WRDL (roots of wheat cv. RAC875 seedlings grown in soil and subjected to drought) cDNA libraries as previously described (Eini et al., 2013 (link)). A large number of independent clones containing full-length or partial coding regions of the TaNF-YC15 cDNA were isolated. Because of self-activating properties of the full-length TaNF-YC15, a truncated version of the protein beginning at residue Ala97 was used to re-screen the same cDNA libraries. Clones containing inserts encoding two different full-length NF-YB TFs, designated as TaNF-YB2 (18 independent clones) and TaNF-YB4 (22 independent clones), were isolated.
5
Cloning and Transformation of Switchgrass NAP Genes
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The CDS of PvSSG PvNAP1 and PvNAP2 was amplified from the cDNA of a selected line "HR8" of the tetraploid switchgrass (P. virgatum L.) ecotype "Alamo" (Xu et al., 2011a) , cloned into the Gateway entry vector pENTR/D (Invitrogen Life Technologies, Carlsbad, CA, USA), and then subcloned into destination vectors, such as p2GWF7.0 for subcellular localization assay (Karimi et al., 2002) , pGBKT7 and pGADT7 (Invitrogen) for Y2H and transactivity assays, pVT1629 (Xu et al., 2011b) , for genetic transformation of switchgrass, and pEarlygate103 and pTA7001 for the transformation of Arabidopsis (Arabidopsis thaliana). A PvSSG-unique fragment was selected for RNAi (Supplemental Table S2) by generating a hairpin structure in the RNAi Entry vector, pGM-kannibal, and then subcloned the hairpin structure into the destination vector, pVT1629. The primers used for cloning and vector construction are shown in Supplemental Table S3.
6
Reverse Genetic Studies in Plasmodium Species
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Plasmids pCRTM2.1-TOPO®, pQE30, pGEX4T3, pGADT7, and pGBKT7 were purchased from Invitrogen, Qiagen, Life Sciences and Clontech, respectively. Plasmids used in reverse genetic studies in P. falciparum, pCAM-BSD and pCAM-BSD-HA, were kind gifts from Prof. C. Doerig (Monash University, Melbourne, VIC, Australia). Plasmids used in P. berghei reverse genetic studies, p-TRAD4Ty-TetO7-HA-hDHFR and pBS-DHFR, were given by Prof. D. Soldati-Favre (University of Geneva, Switzerland) and Prof. R. Tewari (University of Nottingham, United Kingdom), respectively.
All primers used in this study are indicated in Supplementary Table1 .
All primers used in this study are indicated in Supplementary Table
7
Yeast-based Transactivation Assay for IbBBX29
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The transactivation assay was carried out according to the Yeast Protocols Handbook (Clontech). The full-length IbBBX29 coding sequence and fragments corresponding to amino acids 1-50 and 51-190 were individually cloned into the NdeI and EcoRI sites of pGBKT7 (Invitrogen). The empty pGBKT7 vector was used as a negative control, while pGBKT7-53 was used as a positive control. The constructs were transformed into the Y2H Gold yeast strain by the PEG/lithium acetate method. The transformed yeast colonies were screened on SD medium lacking tryptophan (-Trp) and transferred to SD medium lacking tryptophan, histidine, and adenine (-Trp -His -Ade).
8
Yeast Two-Hybrid Analysis of IbNAC3 Interactions
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The full-length coding sequence of IbNAC3 was inserted into the prey vector pGADT7 (Clontech) and the fulllength coding sequence of ANAC011, ANAC072, ANAC083, ANAC100, and NAP was separately cloned into the bait vector pGBKT7 (Clontech) by Gateway system (Invitrogen). Then the negative control, positive control, and indicated co-transformed recombinant constructs were introduced into the Y2HGold yeast cells according to the manufacturer's instructions (Clontech). A series of dilutions were transferred onto DDO (SD/-Trp-Leu), QDO (SD/-Trp-Leu-His-Ade), QDO/Aba (QDO with 200 ng/mL Aba), QDO/3-AT (QDO with 2 mM 3-AT), and QDO/Aba/ABA (QDO/Aba with 20 μM ABA) medium and were incubated at 30°C for 3-5 days to detect their survival.
For additional Y2H assays to map the protein domain of IbNAC3 that are required for each specific interaction with ANAC011, ANAC072, ANAC083, ANAC100, and NAP, all the truncated IbNAC3 sequences were amplified by PCR by the indicated primers and inserted into the pGBKT7 vector by Gateway system (Invitrogen). Different combinations of the recombinant pGADT7 and pGBKT7 plasmids were co-transformed into the yeast strain Y2HGold and the interactions were detected as above. Each interaction had at least three biological replicates with similar results, related primers were listed in Supplemental Table S6.
For additional Y2H assays to map the protein domain of IbNAC3 that are required for each specific interaction with ANAC011, ANAC072, ANAC083, ANAC100, and NAP, all the truncated IbNAC3 sequences were amplified by PCR by the indicated primers and inserted into the pGBKT7 vector by Gateway system (Invitrogen). Different combinations of the recombinant pGADT7 and pGBKT7 plasmids were co-transformed into the yeast strain Y2HGold and the interactions were detected as above. Each interaction had at least three biological replicates with similar results, related primers were listed in Supplemental Table S6.
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