To knock down the tomato gene BiP, the gene fragments were amplified from tomato plant DNA using the specific primer pair TRV-Sl-BiP-F/TRV-Sl-BiP-R (Supplementary Table S1 ), respectively. The amplified gene fragment was then cloned into the pDONR221 entry vector (Thermo Fisher Scientific, Waltham, MA, USA) using BP Clonase II enzyme (Thermo Fisher Scientific, Waltham, MA, USA) to create a pDONR221-BiP plasmid clone. Next, the BiP fragment was cleaved from the pDONR221 vector using HpaI and EcoRV restriction enzymes (NEB, Ipswich, MA, USA) and cloned into the vector YL279 using LR Clonase II enzyme (Thermo Fisher Scientific, Waltham, MA, USA). YL279 is a pTRV2 vector that is commonly used for virus-induced gene silencing in plants and was obtained from the Arabidopsis Biological Resource Center (ABRC, Columbus, OH, USA). The resulting plasmid constructs, pTRV2-BiP and pTRV2-GFP (as a control), were transformed into agrobacterium strain C58C1, which was given by Dr. Rose Hammond. Two-week-old tomato plants were then agroinfiltrated with the TRV constructs. Ten days after the TRV inoculation, the tomato plants were infected with PPT phytoplasma via graft inoculation. Newly grown leaves were harvested one month after the PPT phytoplasma infection, and both DNAs and RNAs were extracted for further analysis.
Pdonr221 vector
Manufactured by New England Biolabs
Sourced in United States
The PDONR221 vector is a bacterial expression vector designed for the cloning and expression of recombinant proteins in Escherichia coli. It features a T7 promoter, a lacI gene for inducible expression, and a kanamycin resistance marker for selection.
Automatically generated - may contain errors
2 protocols using pdonr221 vector
1
Knockdown of Tomato Gene BiP
2
Cloning and Expression of GFP-MLKS2 Constructs
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Gene constructs, nomenclature, and sequence information for the clones used in this study are listed in Table S1. GFP-MLKS2 constructs were synthesized (GenScript Biotech Corp.) using the full-length ORF of MLKS2 fused to the C-terminal end of eGFP-FLAG-HA. The synthetic clone (GFP-MLKS2) was obtained in a pUC18 vector, inserted at the BamHI restriction site to produce a plasmid designated pHWBF07. From this vector, the eGFP-FLAG-HA-MLKS2 sequence was PCR amplified with att flanking primers (Table S2) using high fidelity Q5 polymerase (NEB) and cloned into pDONR221 vector by BP cloning (Cat. No. 1235019, Invitrogen) to generate the entry clone designated pHWBF07EC. The eGFP-FLAG-HA-MLKS2 sequence from the entry clone was transferred to vector pH7WG2 [71 ] by Gate LR recombination (Cat. No. 1,235,019, Invitrogen) to obtain the expression vector designated pPK1Fexp. Deletion constructs were generated from the full-length entry clone as a template to PCR-amplify required regions with internal primers containing flanking att sequences and cloned into pDONR211 by Gateway BP cloning method. These deletion constructs were also subcloned into pH7WG2 destination vector by Gateway LR recombination. Full length and domain deletion constructs for ZmSUN2 were generated as described in [35 ].
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