Orchestration of microtubules and the actin cytoskeleton in trichome cell shape determination by a plant-unique kinesin.

Plant materials and growth conditions

A. thaliana ecotype Columbia (Col-0) was used as the genetic background in this study. Among the zwichel ( zwi ) alleles, the zwi-3 allele and zwi-w2 allele were frequently used in previous studies. The zwi-3 allele in Columbia ecotype background, which is expected to produce a truncated ZWI protein (KCBP) at 522 amino acids position lacking the coiled-coil and motor domains, shows the typical, strong zwi chel trichome phenotype ( Oppenheimer et al., 1997 ; Krishnakumar and Oppenheimer, 1999 ). And the zwi-w2 allele in RLD ecotype background shows weaker zwichel trichome phenotype, containing a small portion (about 15.9%) of three-branched trichomes, because sequencing analysis revealed that although a C to T transition results in a stop codon at amino acid position 72, re-initiation of translation likely occurs using the in-frame AUG ∼20 bp downstream from that mutation site as the start codon ( Folkers et al., 2002 ). To find a strong or null KCBP/ZWICHEL allele in Columbia ecotype background, we searched the Salk collection in the Arabidopsis Biological Resource Center, and finally selected the accession of Salk_031704, which contains a T-DNA insertion in the third exon (22 exons in the KCBP/ZWICHEL gene in total) and shows typical, strong zwi trichome phenotype. Coincidently, the Salk_031704 strong allele was used in recent studies and was designated either as kcbp-1 ( Humphrey et al., 2015 ) or as zwiA (accession is N531704), which was ordered from the Nottingham Arabidopsis Stock Centre ( Buschmann et al., 2015 ). The ABD2-GFP marker line is kindly provided by Prof. Shanjin Huang (Tsinghua University). The GFP-TUB6 marker line was described in our recent study ( Liu et al., 2014 ), and the mCherry-TUB6 marker line was generated using the identical method, only replacing the GFP-encoding sequence with the mCherry-encoding sequence. Plasmid construction and generation of GFP-KCBP and rigor-KCBP lines can be found in the following parts, correspondingly. Various cross combinations (the GFP-KCBP line and the mCherry-TUB6 marker line; the kcbp-1 mutant and the GFP-TUB6 marker line; the rigor-KCBP line and the GFP-TUB6 marker line; the kcbp-1 mutant and the ABD2-GFP marker line; the rigor-KCBP line and the ABD2-GFP marker line) were performed to obtain corresponding materials to observe the dynamics of KCBP, MTs, and F-actin, respectively. Plant growth conditions and transformation procedures were as described previously ( Liu et al., 2014 ). The tiny first or second true leaves in the seedlings at 10-day-old stage were dissected and used for live-cell imaging of trichomes at various developmental stages.

Plasmid construction for genetic complementation experiments

In general, the Phusion DNA polymerase with high-fidelity (New England Biolabs, Beverly, MA, United States) was used to amplify all the required gene products in this study, Fusion PCR was applied to get the GFP-KCBP fusion fragment and various constructs containing individual domain truncations ( Szewczyk et al., 2006 ), and the gateway-based technology was applied to get final expression constructs, please refer to our recent study for detailed description ( Liu et al., 2014 ). To get the GFP-KCBP construct, the genomic fragment of the KCBP gene, including its coding sequence and the 761 bp upstream fragments from the translation initiation codon ATG, was amplified from the genomic DNA with primers of KCBPP-F and GA-KCBP-R. The amplified fragment was cloned into the pENTR/D-TOPO vector by a TOPO-based cloning strategy to get the Entry 1 clone according to manufacturer's instruction (Invitrogen, Carlsbad, CA, United States). Then, a VisGreen version ( Teerawanichpan et al., 2007 ; Liu et al., 2014 ) of GFP tag was added to the NT of KCBP by the following manipulations. The promoter region of the KCBP gene was amplified with primers of KCBPP-F and KCBPP-R; the GFP-encoding sequence was amplified with primers of PGFP-F and GFP-R; the first part of KCBP -coding sequence (1–2050 bp) was amplified with primers of KCBP1X-F and KCBP1X-R. The above-mentioned three PCR fragments were further linked together by Fusion PCR using primers of KCBPP-F and KCBP1X-R. The resulting PCR fragment was subsequently cloned into the pENTR/D-TOPO vector to get the Entry 2 clone. Finally, both the Entry 1 vector and Entry 2 vector were digested by Not I and Xba I, and further ligation reaction was conducted between the gel-purified fragment containing the latter part of KCBP (1951–6023 bp) and the fragment containing the promoter, GFP, and the first part of KCBP genomic sequence, the resulting Entry 3 clone was delivered into pEarleyGate302 by recombination reaction to get the final GFP-KCBP construct. A series of constructs containing various domain truncations were made by the following manipulations. To get the KCBP-∆MyTH4 (KCBP lacking 117–275 amino acids) construct, the fragment containing promoter and 1–435 bp KCBP genomic sequence was amplified with primers of KCBPP-F and KCBP-∆MyTH4-R, and the 992–6023 bp of the KCBP genomic sequence was amplified with primers of KCBP-∆MyTH4-F and GA-KCBP-R, then the two PCR fragments were linked together by Fusion PCR using primers of KCBPP-F and GA-KCBP-R. Finally, the resulting PCR fragment was cloned into pENTR/D-TOPO vector and was then delivered into pEarleyGate302 to get the final KCBP-∆MyTH4 construct. The same strategy was used to generate constructs of KCBP-∆NT (KCBP lacking 2–121 amino acids), KCBP-∆NT-MyTH4 (KCBP lacking 2–275 amino acids), KCBP-∆FERM (KCBP lacking 275–497 amino acids), KCBP-∆MyTH4-FERM (KCBP lacking 116–505 amino acids), KCBP-∆NT-MyTH4-FERM (KCBP lacking 2–505 amino acids), KCBP-∆CC-Motor-CBD (KCBP lacking 532–1266 amino acids), and KCBP-∆CBD (KCBP lacking 1210–1266 amino acids) primers can be found in the Supplementary file 1 . To get the rigor-KCBP construct, we introduced threonine (ACT) 982-to-asparagine (AAC) mutation by the PCR-based mutagenesis. In detail, we designed a pair of overlapping primers (KCBP-T982N-R and KCBP-T982N-F) with the desired nucleotide changes at the target site, and amplified the front half with primers of KCBP2X-F and KCBP-T982N-R, and that latter half with primers of KCBP-T982N-F and GA-KCBP-R, then the two fragments were linked together by Fusion PCR using primers of KCBP2X-F and GA-KCBP-R. The resulting PCR fragment was cloned into the pENTR/D-TOPO vector to get the Entry 4 clone. Finally, both the Entry 1 vector and the Entry 4 vector were digested by Not I and Xba I, and further ligation reaction was conducted between the gel-purified fragment containing the latter part of KCBP containing the threonine 982-to-asparagine mutation and the fragment containing the promoter and the first part of KCBP genomic sequence, the resulting Entry 5 clone was delivered into pEarleyGate302 to get the final rigor-KCBP construct. Genetic complementation and trichome phenotype identification The KCBP (At5G65930) genomic fragment comprising its endogenous promoter and coding region was used for genetic complementation tests. Plasmid construction for the GFP-KCBP construct, the rigor-KCBP construct, and other constructs containing various domain truncations was all based on the above-mentioned KCBP fragment. The constructs were introduced into the kcbp-1 mutant. The T3 homozygous transgenic lines were used to observe trichome phenotype under a fully automated Stereo Microscope (Leica M205 FA) with Leica Application Suite V4.2. The images of trichomes were a maximum z-projection of image series acquired by the Leica LAS Multifocus program.

Spinning-disc confocal microscopy and motor motility analysis

Live-cell imaging was carried out under a spinning disk confocal microscope (UltraView VoX, Perkin Elmer, Beaconsfield, Buckinghamshire, UK) equipped with the Yokogawa Nipkow CSU-X1 spinning disk scanner, Hamamatsu EMCCD 9100-13, Nikon TiE inverted microscope with the Perfect Focus System as described previously ( Liu et al., 2014 ). Acquired images were processed and analyzed using Volocity (Perkin Elmer), Image J ( http://rsbweb.nih.gov/ij ), MetaMorph (Molecular Devices, Sunnyvale, CA, United States). The run-length distribution and the velocity distribution of GFP-KCBP were calculated in Origin software (OriginLab) by frequency counts. The mean values and 95% confidence interval were calculated in SAS (SAS Software), as described previously ( Kong et al., 2015 ).

Purification of GFP-tagged motor-less KCBP with various domain truncations

The full-length cDNA fragments of KCBP were amplified from the Arabidopsis cDNA with primers of KCBP1X-F and GA-KCBP-R, and the GFP-coding sequence amplified with primers of GFP-F and GFP-R, then the two PCR fragments were linked together by Fusion PCR using primers of GFP-F and GA-KCBP-R. The resulting PCR fragment was cloned into the pENTR/D-TOPO vector to get the Entry 6 clone. The cDNA fragments encoding polypeptide of GFP-NT-MyTH4-FERM-CC (1–749 amino acids), GFP-NT-MyTH4-FERM (1–614 amino acids), GFP-NT-MyTH4 (1–275 amino acids), GFP-MyTH4 (116–275 amino acids), GFP-FERM (276–503 amino acids), GFP-NT(1–115 amino acids), and GFP were generated based on the Entry 6 clone. The truncated fragments were digested with Sal I and Not I and were reconstructed into the pET-28a vector to get final expression constructs. Primers can be found in the Supplementary file 1 . Finally, the expression constructs were transformed into Escherichia coli strain Transetta (DE3, TransGen Biotech, Beijing, China) to induce expression. The recombinant proteins were purified using nickel-nitrilotriacetic acid (Ni-NTA) resin following procedures described by the manufacturer (Qiagen, Hilden, Germany). Fractions containing the protein were collected, combined, and dialyzed overnight against PEM buffer (80 mM PIPES, 1 mM EGTA (ethylene glycol tetraacetic acid), and 1 mM MgSO 4 , pH 6.9). Protein concentration was determined by a Bio-Rad protein assay kit. Protein samples of 3 μg were analyzed by SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electropheresis). Single-molecule (particle) imaging assay The purified porcine brain tubulin labeled with NHS-rhodamine was kindly provided from Prof. Tonglin Mao (China Agricultural University). Taxol-stabilized NHS-rhodamine MTs were incubated with 1 μM GFP-NT, 1 μM GFP-MyTH4, 1 μM GFP-FERM, 1 μM GFP-NT-MyTH4, 1 μM GFP-NT-MyTH4-FERM, 1 μM GFP-NT-MyTH4-FERM-CC, and 1 μM control GFP, respectively, in PEM buffer at equal molar ratios for 30 min at room temperature, modified from a previous study ( Liu et al., 2013 ).

Fluorescent images of MTs and various

GFP-KCBP truncated proteins were visualized using a Zeiss inverted fluorescence microscope (Axio Observer Z1) with a Zeiss Plan-Apochromat 100× oil immersion objective (NA = 1.4). The rabbit skeletal muscle actins were provided by Prof. Shanjin Huang (Tsinghua University). F-actin (3 µM) was incubated with 1 μM GFP-NT, 1 μM GFP-MyTH4, 1 μM GFP-FERM, 1 μM GFP-NT-MyTH4, 1 μM GFP-NT-MyTH4-FERM, 1 μM GFP-NT-MyTH4-FERM-CC, and 1 μM control GFP at the indicated concentrations at room temperature for 30 min and then labeled with 3 µM Alexa561-phalloidin (Invitrogen). Actin filaments were subsequently diluted to a final concentration of 10 nM in fluorescence buffer (10 mM imidazole, pH 7.0, 50 mM KCl, 2 mM MgCl 2 , 1 mM EGTA, 100 mM DTT (Dithiothreitol), 100 µg/ml Glucoxidase, 15 mg/ml Glucose, 20 µg/ml catalase, and 0.5% methylcellulose), modified from a previous study ( Wu et al., 2010 ). The diluted samples were visualized using a Zeiss inverted fluorescence microscope (Axio Observer Z1) with a Zeiss Plan-Apochromat 100× oil immersion objective (NA = 1.4).

Identification of T-DNA insertion in kcbp-1 mutant

A standard PCR-based method was used to identify the T-DNA insertion in kcbp-1 ( Humphrey et al., 2015 ), as described by Kong et al. (2015) . Gene-specific primers (031704-LP and 031704-RP) were used to amplify an approximate 1000 bp DNA fragment in KCBP . The T-DNA insertion was detected using 031704-RP and the left-border primer (LBb1.3). For RT-PCR analysis of transcription level of KCBP in the kcbp-1 mutant, total RNA was extracted using Trizol reagent Invitrogen and was used for first strand cDNA synthesis by the SuperScript III First-Strand Synthesis System (Life Technologies, Carlsbad, CA, United States) with oligo (dT)18 primers. Then, the cDNA was used as a template for PCR reactions using primers shown in the primer list. UBQ5 was used as control.

Plant materials and growth conditions

A. thaliana ecotype Columbia (Col-0) was used as the genetic background in this study. Among the zwichel ( zwi ) alleles, the zwi-3 allele and zwi-w2 allele were frequently used in previous studies. The zwi-3 allele in Columbia ecotype background, which is expected to produce a truncated ZWI protein (KCBP) at 522 amino acids position lacking the coiled-coil and motor domains, shows the typical, strong zwi chel trichome phenotype ( Oppenheimer et al., 1997 ; Krishnakumar and Oppenheimer, 1999 ). And the zwi-w2 allele in RLD ecotype background shows weaker zwichel trichome phenotype, containing a small portion (about 15.9%) of three-branched trichomes, because sequencing analysis revealed that although a C to T transition results in a stop codon at amino acid position 72, re-initiation of translation likely occurs using the in-frame AUG ∼20 bp downstream from that mutation site as the start codon ( Folkers et al., 2002 ). To find a strong or null KCBP/ZWICHEL allele in Columbia ecotype background, we searched the Salk collection in the Arabidopsis Biological Resource Center, and finally selected the accession of Salk_031704, which contains a T-DNA insertion in the third exon (22 exons in the KCBP/ZWICHEL gene in total) and shows typical, strong zwi trichome phenotype. Coincidently, the Salk_031704 strong allele was used in recent studies and was designated either as kcbp-1 ( Humphrey et al., 2015 ) or as zwiA (accession is N531704), which was ordered from the Nottingham Arabidopsis Stock Centre ( Buschmann et al., 2015 ). The ABD2-GFP marker line is kindly provided by Prof. Shanjin Huang (Tsinghua University). The GFP-TUB6 marker line was described in our recent study ( Liu et al., 2014 ), and the mCherry-TUB6 marker line was generated using the identical method, only replacing the GFP-encoding sequence with the mCherry-encoding sequence. Plasmid construction and generation of GFP-KCBP and rigor-KCBP lines can be found in the following parts, correspondingly. Various cross combinations (the GFP-KCBP line and the mCherry-TUB6 marker line; the kcbp-1 mutant and the GFP-TUB6 marker line; the rigor-KCBP line and the GFP-TUB6 marker line; the kcbp-1 mutant and the ABD2-GFP marker line; the rigor-KCBP line and the ABD2-GFP marker line) were performed to obtain corresponding materials to observe the dynamics of KCBP, MTs, and F-actin, respectively. Plant growth conditions and transformation procedures were as described previously ( Liu et al., 2014 ). The tiny first or second true leaves in the seedlings at 10-day-old stage were dissected and used for live-cell imaging of trichomes at various developmental stages.

Additional files 10.7554/eLife.09351.027 Supplementary file 1. List of primer sequences used in this study. DOI: http://dx.doi.org/10.7554/eLife.09351.027