Generation and Characterization of Myosin XI-K Marker Lines

A genomic copy of the myosin XI-K gene (AT5G20490) tagged by insertion of the YFP ORF (XI-K:YFP) was generated as described for the FLAG-tagged XI-K (Peremyslov et al., 2010 (link)). The YFP ORF was PCR amplified and inserted in a pMDC32 binary vector carrying a genomic copy of the XI-K using overlapping PCR. The resulting plasmid was mobilized into Agrobacterium tumefaciens GV3101 and used to transform the 3KO xi-k xi-1 xi-2 plants (Peremyslov et al., 2010 (link)) by floral dipping. The identifiers for the T-DNA insertion lines used to generate 3KO plants were Salk_067972, Salk_019031, and Salk_055785, respectively. Transgenic plants designated 3KOR were selected using Hygromycin-containing medium and YFP imaging. Immunoblotting using a rabbit polyclonal XI-K antibody, rabbit polyclonal antibody to ER binding protein (BiP; a gift from Dr. J. Denecke), or mouse monoclonal GFP antibody (Roche) was done as described (Peremyslov et al., 2008 (link)). All four originally selected, independent lines of transformed plants showed very similar levels of XI-K:YFP expression and virtually indistinguishable phenotypes. One of these lines was selected for all following analyses. To compare the expression levels of myosin XI-K in Columbia-0 to that of XI-K:YFP in 3KOR line plants, the immunoblots were quantified by measuring mean band intensity normalized to that of a loading control (BiP; n = 4 for each variant). The phenotypes of plant lines presented in Figures 1D–F were characterized as described (Peremyslov et al., 2010 (link)); statistical analyses of the data, including standard deviations shown as error bars and t-tests, were done using Microsoft Excel package. The p values, corresponding to pairwise comparisons of data sets, are presented in the text under Results.
To generate transgenic lines expressing compartment-specific markers, a binary vector pCB301 was modified to accommodate an UBQ10 promoter (Geldner et al., 2009 (link)) and a polyadenylation signal, and used to generate N-terminal fusions of the mTurquoise fluorescent protein (Goedhart et al., 2010 (link)) with the actin-binding domain LifeAct (Goedhart et al., 2010 (link)), the transmembrane domain of N-acetylglucosaminyl transferase I (NAG; Grebe et al., 2003 (link)), or SCAMP2 (Toyooka et al., 2009 (link)). An ER-targeted CFP was described before (Peremyslov et al., 2010 (link)). The marker-expressing 3KOR plants were selected using glufosinate. The transgenic plant lines generated in Columbia or 3KOR genetic background had normal developmental phenotypes under optimal growth conditions. This was also the case for the LifeAct-mTurquoise plants in accord with recent independent work (van der Honing et al., 2011 (link)).