Construction and validation of co-expression vector for rice alpha tubulin and microtubule associated protein respectively fused with fluorescent proteins

Construction of p35S::GFP-α-tubulin vector

Step 1 Extracting total RNA

Weigh about 0.1 g of fresh rice young inflorescence, and grind to powder in liquid nitrogen; the next operations follow the RNeasy^® Mini Handbook; mortar, pestle, tips, tubes, etc. are RNAase-free.

Step 2 Reverse transcription

A total of 1 μg total RNA is used for reverse transcription to obtain cDNA by SuperScript^®III First-Strand Synthesis System for RT-PCR; the operations follow the instructions of the Kit; temperature is controlled with a PCR amplifier.

Step 3 Amplification of GFP, α-tubulin ORF and NOS terminator sequences, and vector linearization

The plasmid pBI221-GFP, cDNA in Step 2, and plasmid pCambia1391 are used as templates to respectively amplify GFP, α-tubulin ORF and NOS terminator sequences; sequence-specific primers are listed in Table S1; high-fidelity polymerase KOD-Plus-Neo is used in the PCR, and reaction setup and thermocycling conditions for PCR are performed according to the protocol for KOD-Plus-Neo polymerase. pCambia1301 is digested with restricted enzyme BglII to linearization, and the reaction system and operations follow the product instructions.

Step 4 Electrophoresis and gel extraction of target fragments

•  Agarose powder is mixed with electrophoresis buffer (1×TAE buffer) to 1% concentration (w/v), and heated in a microwave oven until completely melted; DNA stain dye (SYBR^® Green I) is added to the gel (final concentration 0.5 μg/mL); cool the solution to 60 °C and pour into a casting tray containing a sample comb and allow to solidify at room temperature.

•  Gel is placed into the electrophoresis apparatus and immersed in 1×TAE buffer; DNA fragments in Step 3 mixed with loading buffer, and DNA marker are pipetted into the sample wells.

•  Electrophoresis is performed immediately at a voltage between 60–100 V; when adequacy has occurred, stop the electrophoresis.

•  Transfer the gel to a UV gel imager system for imaging, and excise under the TGreen blue light transmittance instrument; the excised gel containing DNA fragments should be as small as possible, and exposure time under the UV light should be kept as short as possible to minimize the damage to the DNA.

•  Use Monarch^® DNA Gel Extraction Kit to extract the DNA fragments from agarose gels; operations follow the protocol of kit.

•  Measure DNA fragment concentrations with the ultramicro spectrophotometer; wash the sample chamber three times with deionized H₂O before use. Immediately use extracted DNA fragments for seamless cloning or store them at −20 °C.

Step 5 Seamless cloning (Gibson assembly)

The extracted DNA fragments (including GFP, α-tubulin, NOS terminator, and linearized pCambia1301), deionized H₂O, and Assembly Master Mix are mixed in proportion according to the concentrations on ice; the mixture is incubated at 50 °C and transformed to DH5α competent E. coli; use kanamycin (final concentration 50 μg/mL) LB plate to select positive bacterial colonies; all the operations follow the NEBuilder^® HiFi DNA Assembly Master Mix protocol.

Step 6 E. coli colony PCR, sequencing, and plasmid purification

•  Prepare a PCR amplification reaction mixture; Taq DNA polymerase is used for colony PCR; PCR amplification reaction setup is conducted following the product instruction; amplification primers are listed in Table S1.

•  Stab a transformed colony with a sterile toothpick and swirl cells from the colony into the amplification reaction mixture in a superclean bench; gently mix each reaction.

•  Perform PCR and analyze the PCR products using standard 1% (w/v) agarose gel electrophoresis; thermocycling conditions is conducted following the product instructions.

•  Inoculate 0.5 mL of the kanamycin (final concentration 50 μg/mL) LB medium into a 2 mL sterile tube with an isolated colony that is confirmed by colony PCR and incubate at 37 °C at 200 rpm until the culture becomes turbid.

•  Transfer 0.2 mL inoculum into a new sterile tube to send to the company (Tsingke Biotech Co., Ltd., Beijing, China) for sequencing verification; add sterile 75% glycerin to the remaining inoculum to a final concentration of 15% and store at −80 °C.

•  Inoculate 4 mL of the kanamycin (final concentration 50 μg/mL) LB medium into a sterile vessel that allows some aeration (culture tubes on a roller drum) with a stored inoculum that had been verified by sequencing; incubate at 37 °C at 200 rpm about 12–16 h.

•  Pellet bacterial culture by centrifugation and purify plasmid by Monarch^® Plasmid Miniprep Kit; the operations follow the Kit protocol; plasmid DNA is eluted with nuclease-free water (pH 7–8.5) and stored at −20 °C.

Step 7 Plasmid transformation into Agrobacterium tumefaciens competent cells and colony PCR

The plasmid p35S::GFP-α-tubulin (Fig. 1B; Vector S5, GenBank accession: OR797711) purified in Step 6 is transformed into Agrobacterium tumefaciens EHA105 competent cells, following the chemically competent cells transformation protocol; use kanamycin (final concentration 50 μg/mL) and rifampicin (final concentration 20 μg/mL) LB plate to select positive bacterial colonies and incubate at 28 °C. After 2–3 d, carry out colony PCR following the Step 6 operations to further verify the positive colonies.

Step 8 Tobacco transient expression assay

•  Grow N. benthamiana to the age of 4 weeks; water the N. benthamiana plants 1 d (day) before the infiltration to make the infiltration easier.

• Separately  incubate Agrobacterium cultures p35S::GFP-α-tubulin, and P19 (Garabagi et al., 2012), a viral suppressor of gene silencing, overnight at 28 °C at 200 rpm in 3 mL LB medium with kanamycin (final concentration 50 μg/mL) and rifampicin (final concentration 20 μg/mL).

• Inoculate Agrobacterium from the LB medium into the fresh medium for enlarged cultivation; 1/100 dilution for overnight culture or 1/25 for about 8 h culture.

• Incubate at 28 °C at 200 rpm for about 8–12 h, so that the Agrobacterium cell is in the logarithmic growth phase; the best OD600 (optical density at 600 nm) value should be 0.5–0.8.

• Centrifuge Agrobacterium culture in 50 mL Corning tube for 15 min at 5,000×g at room temperature to pellet the cells.

• Prepare infiltration solution, and resuspend Agrobacterium pellet in 10 mL of infiltration solution to a desired OD600. Note: Mix different kinds of Agrobacteria, with the desired OD600 (usually 0.1–0.5) for each one; use a larger OD600 value for Agrobacteria expressing lower protein while using a smaller OD600 value for Agrobacteria expressing abundant protein in N. benthamiana.

• Let the inoculum stand and away from light for 2–3 h.

• Choose the healthiest looking plants and leaves; leaves 3–5 from top to bottom are usually chosen.

• Swirl the inoculum in the tube, and use a 1 mL blunt syringe to gently inject the inoculum into the underside of the leaf. Successful infiltration is often observed as a spreading ‘wetting’ area in the leaf.

• Place the infiltrated plants in the dark for 1 d, then grow them under 16 h day/8 h night at 22 °C.

• After 2–5 d of infiltration, cut the infiltrated leaf to an appropriate size, and immediately immerse it in MTSB buffer on a microscope slide, and carefully cover it with cover glass.

• Immediately transfer the microscope slide into the sample chamber of confocal microscopy to detect the fluorescence. Fluorescence is recorded sequentially after an excitation at 488 nm for the GFP and at 561 nm for the mCherry; the micrograph is projections of Z-stack confocal images (Drevensek et al., 2012).

Through above Steps 1–6, the GFP, α-tubulin ORF, and NOS terminator fragments have been fused and inserted into the pCambia1301 to construct p35S::GFP-α-tubulin vector, in which the α-tubulin gene is driven by CaMV 35S promoter, a strong promoter is inherent in the pCambia1301 itself and drive GUS gene expression before.

Furthermore, the p35S::GFP vector previously constructed by our laboratory to express GFP protein was used as a control. With the tobacco transient expression assay, the GFP protein distributed into the cytoplasm (Fig. 1C) but the GFP-α-tubulin protein localized to MTs (Fig. 1D), which suggested that the rice α-tubulin was able to localize to MTs.

By the tobacco transient expression assay (Step 8), it has been verified that α-tubulin was successfully expressed and localized to MTs (Fig. 1D). So the p35S::GFP-α-tubulin vector can be used for the subsequent procedures.

Construction of p35S::GFP-α-tubulin-p35S::mCherry vector

Step 9 Amplification of 35S, mCherry, and NOS terminator sequences, and vector linearization

The 35S promoter is amplified by two rounds of PCR. The plasmid pBI221-H2B-mCherry is used as a template to amplify with the first primer pair; the PCR product is also used as a template to amplify with the second primer pair. Through the two rounds of PCR, we obtain a 35S promoter sequence with the restriction enzyme cutting sites XbaI, KpnI/Acc65I at its 3′ terminus. Using plasmid pBI221-H2B-mCherry as a template, the mCherry sequence is amplified with a primer pair, and the AscI restriction site is added to the 3′ terminus of the mCherry sequence. By adding restriction endonuclease sites XbaI, KpnI/Acc65I at the 5′ terminus and AscI at the 3′ terminus of mCherry, another gene, e.g., MAP could be fused with mCherry at its 5′ or 3′ terminus, facilitating the universality of vector’s application. Plasmid pBI221-H2B-mCherry is also used as a template to amplify the NOS terminator with a primer pair. All sequence specific primers are listed in Table S1. p35S::GFP-α-tubulin vector is digested with restricted enzyme KpnI-HF to linearization. All operations are the same as in Step 3.

Step 10 Electrophoresis and gel extraction of target fragments

The PCR products of 35S, mCherry and NOS terminator, and linearized p35S::GFP-α-tubulin vector are subjected to electrophoresis and gel extraction. All operations are the same as in Step 4.

Step 11 Seamless cloning (Gibson assembly).

The extracted DNA fragments 35S promoter, mCherry, and NOS terminator are fused and inserted into the p35S::GFP-α-tubulin vector at the KpnI site to construct p35S::GFP-α-tubulin-p35S::mCherry vector (Fig. 2A; Vector S6, GenBank accession: OR797712). All operations are the same as in Step 5.

Step 12 E. coli colony PCR, sequencing, and plasmid purification

Through the colony PCR, sequencing, and plasmid purification, we obtain the p35S::GFP-α-tubulin-p35S::mCherry vector. All operations are the same as in Step 6.

Step 13 Plasmid transformation into Agrobacterium tumefaciens competent cells and colony PCR.

Transform the p35S::GFP-α-tubulin-p35S::mCherry vector into Agrobacterium tumefaciens EHA105 competent cells and confirm it by colony PCR. All operations are the same as in Step 7.

Step 14 Tobacco transient expression assay.

Through this assay, GFP-α-tubulin and mCherry are transiently co-expressed in N. benthamiana leaves and detected by confocal microscopy. All operations are the same as in Step 8.

Based on the p35S::GFP-α-tubulin vector, we constructed a universal dual-transgene expression vector p35S::GFP-α-tubulin-p35S::mCherry (Fig. 2A) through above Steps 9–12, in which GFP-α-tubulin and mCherry genes are driven by 35S promoters, respectively, and could be simultaneously expressed. Through Steps 13–14, the vector p35S::GFP-α-tubulin-p35S::mCherry was transformed into N. benthamiana leaves, and GFP-α-tubulin and mCherry were co-expressed in leaf epidermal cells (Figs. 2B–2D). The availability of the p35S::GFP-α-tubulin-p35S::mCherry vector was validated by the observation that GFP-α-tubulin localized to MTs (Fig. 2B) and mCherry distributed into the cytoplasm (Fig. 2C).

Validation of p35S::GFP-α-tubulin-p35S::mCherry vector

To further verify the availability and universality of the p35S::GFP-α-tubulin-p35S::mCherry vector, several putative MAP genes in rice including GL7 (Sequence S1), OsKCBP (Sequence S2), and OsCLASP (Sequence S3) were inserted into the 3′ terminus of mCherry in the vector respectively, and OsMOR1 (Sequence S4) was inserted into the 5′ terminus of mCherry. All of the constructed dual-transgene vectors were transiently expressed in N. benthamiana leaves, respectively.

GL7, a major quantitative trait locus (QTL) for grain size diversity in rice, encodes a protein homologous to Arabidopsis thaliana LONGIFOLIA proteins (Wang et al., 2015). The GL7 protein has 19–22% amino acid sequence identity with the LONGIFOLIA1 and LONGIFOLIA2 proteins (Wang et al., 2015; Fig. S1). LONGIFOLIA2 is a MAP which may be involved in directing cell expansion by recruiting TON1 to cortical MT arrays (Drevensek et al., 2012; Lee et al., 2006). So the GL7 appears to be a MAP in rice, but the direct experimental evidence is lacking. Here, we used the p35S::GFP-α-tubulin-p35S::mCherry vector to construct the p35S::GFP-α-tubulin-p35S::mCherry-GL7 vector and transformed it into N. benthamiana leaves. The results suggested that mCherry-GL7 colocalized with GFP-α-tubulin and presented a punctate pattern along MTs (Fig. 3A), consistent with the subcellular localization of LONGIFOLIA2/TRM1 in N. benthamiana leaf epidermal cells (Drevensek et al., 2012). This result supplies the experimental evidence that GL7 might be a MAP.

Kinesin-like calmodulin-binding protein (KCBP), a calmodulin-binding kinesin of the Kinesin-14 sub-family (Richardson, Simmons & Reddy, 2006), occurs solely within the plant kingdom and acts as a hub that brings together MTs and actin filaments to modulate the cytoskeleton during trichome formation in Arabidopsis thaliana (Tian et al., 2015). The homologous protein of KCBP in rice has 70% amino acid sequence identity with KCBP (Fig. S2) but has not been studied. By the p35S::GFP-α-tubulin-p35S::mCherry vector, we constructed p35S::GFP-α-tubulin-p35S::mCherry-OsKCBP vector and transformed it into N. benthamiana leaves. The results showed that mCherry-OsKCBP colocalized with GFP-α-tubulin and somewhat decorated MTs as a punctate pattern in N. benthamiana leaf epidermal cells (Fig. 3B), indicating that the OsKCBP might be a MAP.

Cytoplasmic linker associated proteins (CLASPs) are considered as MT plus end-tracking proteins and well-studied MAPs found across plant, fungus, and animal systems. CLASPs have a broad range of functions in cell motility, cell expansion and proliferation, and localize at kinetochores, the mitotic spindle midzone, centrosomes, the Golgi network, and the cell cortex (Bratman & Chang, 2008; Efimov et al., 2007; Kumar et al., 2009; Mimori-Kiyosue et al., 2005; Ruan et al., 2018). The functions of CLASPs have been characterized for stabilizing overlapping MTs in mitotic spindles of fission yeast (Bratman & Chang, 2007), promoting MT bundling in metaphase spindle in fission yeast (Ebina, Ji & Sato, 2019), inducing MT pausing in Drosophila S2 cells (Sousa et al., 2007), stimulating MT rescues at leading cell edges (Mimori-Kiyosue et al., 2005), suppressing MT catastrophes (Aher et al., 2018; Al-Bassam et al., 2010), and promoting γ-tubulin-dependent MT nucleation at the Golgi (Efimov et al., 2007). CLASP localizes to MT lattice and induces MT bundles at transient high expression level in tobacco leaf epidermal cells, but enriches at growing MT plus ends at stable low expression in Arabidopsis thaliana leaf guard cells (Ambrose et al., 2007). CLASP is a very important MAP, but it has not been studied in rice. The OsCLASP has 63% amino acid sequence identity to AtCLASP (Fig. S3). In our study, the OsCLASP coding sequence was cloned into p35S::GFP-α-tubulin-p35S::mCherry vector and co-expressed with α-tubulin in N. benthamiana leaf epidermal cells. The results showed that mCherry-OsCLASP colocalized with GFP-α-tubulin to MT lattices and induced MT bundles (Fig. 3C), which is similar with AtCLASP location at transient high expression level in tobacco leaves. The OsCLASP always kept great fluorescence to MTs and merely distributed into the cytoplasm (Fig. 3C), suggesting its strong bonding ability to MTs. This implies that OsCLASP might be a MAP and have similar functions with its homologs in other species.

Microtubule organization 1 (MOR1) of Arabidopsis thaliana belongs to the MAP215 family of MAPs (Whittington et al., 2001). MOR1 regulates cortical MT organization and function, as well as the structure and function of MT arrays during mitosis and cytokinesis in Arabidopsis root (Chen et al., 2022; Kawamura et al., 2006). MAP215 proteins are essential MT polymerases that use multiple TOG domains to bind MT plus ends and catalyze fast MTs (Ayaz et al., 2014). These proteins may function as MT polymerases by adding new tubulin dimers to the plus ends, and localize to MT plus ends, MT organizing centers, kinetochores, and MT lattices. Here, we constructed the p35S::GFP-α-tubulin-p35S::OsMOR1-Cherry vector and transformed it into N. benthamiana leaves. The transient expression showed that OsMOR1-Cherry colocalized with GFP-α-tubulin to MTs, and presented a linear pattern along MTs, which suggested an association with cortical MTs (Thawani, Kadzik & Petry, 2018; Fig. 3D). In addition, the OsMOR1 has 62–68% amino acid sequence identity with the Arabidopsis MOR1 (Fig. S4). Thus, we speculate that OsMOR1 might be a MAP.

By the above tobacco transient expression assays, we validated the availability and universality of the dual-transgene expression vector (p35S::GFP-α-tubulin-p35S::mCherry), and provided experimental evidence that GL7, OsKCBP, OsCLASP, and OsMOR1 might be MAPs for the first time.