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  • William Reese posted an update 2 weeks ago

    Ative humidity. Sinorhizobium meliloti 2011 harboring a hemA:lacZ construct and strain 1021 harboring the mCherry-expressing construct have been applied for inoculation. Agrobacterium rhizogenes MSU440 was used for hairy root transformation, and Agrobacterium tumefaciens EHA105 was used for transient expression in Nicotiana benthamiana. DNA Manipulation and Plasmid Construction Point mutations of ROP10 were introduced into plasmids pMD18-T (Takara) by PCR amplification employing primers containing the indicated mutations plus the Takara MutanBEST Kit (Takara). To make expression constructs for the analysis of M. truncatula root hairs, pENTR ROP10, ROP10CA, ROP10DN, and ROP10CA C197+203S were used for LR recombination reactions with pUB-GW-GFP (Maekawa et al., 2008), producing pUbi:ROP10, pUbi:ROP10CA, pUbi:ROP10DN, and pUbi:ROP10CAC197+203S plasmids, respectively. To create ROP10:GFP, ROP10CA:GFP, ROP10DN:GFP, ROP10C203S: GFP, Ebselen Formula ROP10C197S:GFP, and ROP10C197+203S:GFP constructs, the region containing the CaMV 35S promoter and GFP was released from plasmid pA7-GFP (Voelker et al., 2006) by HindIII and EcoRI and inserted into pCAMBIA 2300S to acquire the control vector 2300S-GFP. Then, ROP10 and derivatives have been amplified by PCR and inserted into the 2300S-GFP vector working with SalI and SpeI. To make expression constructs for BiFC analysis, ROP10, ROP10CA, and ROP10DN as well as the kinase domains of NFP (NFP PK; amino acids 312 to 582) and LYK3 (LYK3 PK; amino acids 323 to 620) had been amplified by PCR, inserted into pENTR through TOPO reaction (Invitrogen), after which utilized for the LR recombination reaction (Invitrogen) with either pCCFP-X or pNYFP-Y (Bai et al., 2007) to get CFPC:ROP10, CFPC:ROP10CA, CFP C :ROP10DN, YFP N :NFP PK, and YFP N :LYK3 PK constructs, respectively. To make expression constructs for colocalization in planta, NFP and LYK3 had been fused for the mCherry gene (Clontech; pmCherry Vector) by overlap extension PCR and then inserted into pA7-GFP using XhoI and SmaI. Then, the HindIII and EcoRI fragment was inserted into pCAMBIA 2300S to acquire the NFP:mCherry and LYK3:mCherry constructs, respectively. To create BD and AD constructs for GAL4-based Y2H assays, the sequences of ROP10, ROP10CA, and ROP10DN had been amplified by PCR and cloned with the aid of EcoRI and XhoI in to the pGBKT7 DNA BD vector (Clontech). NFP full length (NFP FL), NFP kinase domain (NFP PK), LYK3 complete length (LYK3 FL), and LYK3 kinase domain (LYK3 PK) were amplified by PCR and cloned with EcoRI and XhoI into the pGADT7 AD vector (Clontech). To create AD and BD constructs for LexA-based Y2H assays, the sequences of ROP10, ROP10CA, and ROP10DN had been amplified by PCR and cloned with all the help of EcoRI and XhoI into the pB42 AD vector (Clontech). NFP complete length (NFP FL), NFP kinase domain (NFP PK), and LYK3 kinase domain (LYK3 PK) had been amplified by PCR and cloned with EcoRI and XhoI into the pLexA DNA BD vector (Clontech). To create expression constructs for pull-down assays, the sequences of ROP10, ROP10CA, and ROP10DN have been amplified by PCR and cloned with EcoRI and XhoI in to the pGEX-6P-1 vector (GE Healthcare), which was then applied for GST-tagged protein expression in Escherichia coli. Similarly, the NFP kinase domain (NFP PK) and LYK3 kinase domain (LYK3 PK) were amplified by PCR from pENTR/SD/D-NFP PK and pENTR/SD/D-LYK3 PK and cloned with EcoRI and XhoI into pET-28a(+) (Novagen) for His-tagged protein expression.

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