Hairy root transformation using Agrobacterium rhizogenes as a tool for exploring cell type-specific gene expression and function using tomato as a model - PubMed (original) (raw)

. 2014 Oct;166(2):455-69.

doi: 10.1104/pp.114.239392. Epub 2014 May 27.

Kaisa Kajala 1, Germain Pauluzzi 1, Dongxue Wang 1, Mauricio A Reynoso 1, Kristina Zumstein 1, Jasmine Garcha 1, Sonja Winte 1, Helen Masson 1, Soichi Inagaki 1, Fernán Federici 1, Neelima Sinha 1, Roger B Deal 1, Julia Bailey-Serres 1, Siobhan M Brady 2

Affiliations

Hairy root transformation using Agrobacterium rhizogenes as a tool for exploring cell type-specific gene expression and function using tomato as a model

Mily Ron et al. Plant Physiol. 2014 Oct.

Abstract

Agrobacterium rhizogenes (or Rhizobium rhizogenes) is able to transform plant genomes and induce the production of hairy roots. We describe the use of A. rhizogenes in tomato (Solanum spp.) to rapidly assess gene expression and function. Gene expression of reporters is indistinguishable in plants transformed by Agrobacterium tumefaciens as compared with A. rhizogenes. A root cell type- and tissue-specific promoter resource has been generated for domesticated and wild tomato (Solanum lycopersicum and Solanum pennellii, respectively) using these approaches. Imaging of tomato roots using A. rhizogenes coupled with laser scanning confocal microscopy is facilitated by the use of a membrane-tagged protein fused to a red fluorescent protein marker present in binary vectors. Tomato-optimized isolation of nuclei tagged in specific cell types and translating ribosome affinity purification binary vectors were generated and used to monitor associated messenger RNA abundance or chromatin modification. Finally, transcriptional reporters, translational reporters, and clustered regularly interspaced short palindromic repeats-associated nuclease9 genome editing demonstrate that SHORT-ROOT and SCARECROW gene function is conserved between Arabidopsis (Arabidopsis thaliana) and tomato.

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Figures

Figure 1.

Figure 1.

Cortex layer numbers differ between adventitious roots and hairy roots. Cross sections show an S. lycopersicum cv M82 adventitious root (A) and a hairy root derived from transformation of S. lycopersicum cv M82 with A. rhizogenes (B). C1 to C4, Cortex layers 1 to 4; En, endodermis; Ep, epidermis; P, pericycle. Bars = 100 μm.

Figure 2.

Figure 2.

Promoter activity is recapitulated in _A. rhizogenes_-transformed hairy roots relative to _A. tumefaciens_-transformed primary roots of tomato. A and B, The SlSHR promoter drives expression in the vasculature in both an SlSHRpro-nlsGFP (G10-90pro-TagRFP-LTI6b)-transformed hairy root (A) and an _SlSHRpro-ERGFP_-transformed primary root (B). C and D, The SlSCR promoter drives expression in the QC and endodermis in both an SlSCRpro-nlsGFP (G10-90pro-TagRFP-LTI6b)_-_transformed hairy root (C) and an _SlSCRpro-ERGFP_-transformed primary root (D). In A to D, GFP fluorescence is green, TagRFP fluorescence is red, and the green component of autofluorescence is white. E, SlSHRpro-SHR-GFP protein fusion shows cytosolic subcellular localization of the SHR protein in the vasculature and movement of the protein outside of the vasculature, where it localizes to nuclei. En, Endodermis. This image was taken without linear unmixing, so green represents both GFP and autofluorescence. All images were taken with a 20× objective. Bars = 100 μm.

Figure 3.

Figure 3.

A cell type- and tissue-specific promoter toolbox. GFP expression patterns driven by a variety of promoters were tested in the hairy root transformation system by cloning them upstream of an nlsGFP-GUS fusion in a vector containing a ubiquitously expressing plasma membrane marker: G10-90pro-TagRFP-LTI6b (A–D, G–I, and K) or 35Spro-TagRFP-LTI6b (E and F). A and B, AtACT2pro drives near constitutive expression (under 63× and 10× magnification, respectively). The nlsGFP (green) shows nuclear localization, and TagRFP (red) shows plasma membrane localization. C, AtS18pro drives expression in the maturing xylem (10×). This image contains an overlay of GFP fluorescence from two different focal planes to show both phloem strands. D, SlRPL11Cpro expression is most pronounced in the meristematic zone (10×). E, AtPEPpro drives expression in the cortex throughout the root, including the elongation zone (20×). F and G, AtS32pro drives expression in the phloem, starting in the meristematic zone (F; 40×) all the way to mature root (G; 20x). H, SlCO2pro drives expression in cortex layers only in the meristematic zone (20×). I and J, AtWERpro drives expression in the lateral root cap (I; 20×) and all epidermal cells throughout the root (I and J; 20×). K, SlCYCD6pro drives expression in the QC and vascular initials (20×). L, SlWOX5pro drives expression in the QC and vascular cells in the proximal meristem (20×). M, SlCO2pro drives expression of AtRPL18CDS-GFP in the meristematic cortex of S. pennellii (20×). This image was taken without linear unmixing, so green represents both GFP and autofluorescence. N, AtACT2pro also drives near constitutive expression in L. hyssopifolium hairy roots (20×). Bars = 100 μm.

Figure 4.

Figure 4.

Application of INTACT methodology to tomato with the hairy root transformation system. A, _35Spro_-driven NTF protein expression in tomato hairy roots shows GFP localization (green) in the nuclear envelope and cytosol. Bar = 100 μm. B, Detection of biotinylated NTF protein in root extracts. Total protein was isolated from tomato roots that were either untransformed or transformed with 35Spro-NTF and ACT2pro-BirA transgenes. Transgenic Arabidopsis roots carrying the ADF8pro-NTF and ACT2pro-BirA transgenes were included as a positive control (+) for biotinylated NTF. Molecular mass markers are shown on the right. The expected size of NTF is 46 kD. C and D, Capture of biotinylated nuclei from transformed tomato roots carrying the 35Spro-NTF and ACT2pro-BirA transgenes with streptavidin-coated magnetic beads. Nuclei were stained with 4′,6-diamino-phenylindole (DAPI) to visualize DNA. C, View with mixed white light and DAPI-channel fluorescence illumination. Seven bead-bound nuclei are visible in the field shown, with a representative nucleus indicated by the yellow arrowhead. The nuclei are DAPI-bright patches surrounded by darker colored spherical magnetic beads. Bar = 50 μm. D, Magnification of the boxed region in C. Bar = 20 μm. E, Quantitative analysis of selected transcripts in INTACT-purified tomato nuclei. qPCR was used to assay the relative abundance of mRNAs. Data represent averages ±

sd

of two biological replicates of nuclei purification experiments. F, ChIP and qPCR to examine the abundance of H3K4me3 in INTACT-purified tomato nuclei. Relative enrichment of the modification in the promoter of the ACT2 and SHR genes as well as within the SHR gene body is shown relative to input. Data represent average ±

sd

enrichment from two biological replicate experiments. Solyc01g014230 was used as a reference for normalization.

Figure 5.

Figure 5.

Application of TRAP methodology to tomato with the hairy root transformation system. A, _35Spro_-driven His6-FLAG epitope-tagged GFP-RPL18 (HF-GFP-RPL18) fusion protein in the tomato hairy root system shows GFP localization (green) in the nucleolus and cytosol. Bar = 100 μm. B, HF-GFP-RPL18 is efficiently immunopurified by TRAP. Equal fresh weight of tissue from wild-type roots (control) and HF-GFP-RPL18 hairy roots was extracted in a polysome-stabilizing buffer and processed to obtain a clarified cell supernatant (total), which was incubated with anti-FLAG agarose beads to obtain the TRAP fraction containing ribosomes and associated mRNA. Cell components not bound to the matrix remained in the unbound fraction. Molecular mass markers are shown on the left. The expected size of HF-GFP-RPL18 is 51 kD. C, Comparison of immunopurified proteins from untransformed roots or HF-GFP-RPL18-transformed hairy roots by TRAP and ribosomal proteins isolated by conventional ultracentrifugation (P-170). Proteins were visualized by silver staining. Equal proportions of TRAP samples were loaded in lanes 1 and 2. Molecular mass markers are shown on the left. Asterisks indicate bands corresponding to IgG from the affinity matrix. D, Ethidium bromide staining of RNA isolated from the total and TRAP fractions from wild-type (control) and HF-GFP-RPL18 roots. The 25S and 18S rRNA sizes are indicated on the left. E, Quantitative reverse transcription-PCR analysis of selected transcripts in the TRAP fractions. Relative transcript level in the TRAP versus total fraction was determined. Data represent average ±

sd

level of each transcript in two biological replicates. ACT2 was used as a reference for normalization. F, HF-GFP-RPL18 is incorporated into small to large polysomal complexes. HF-GFP-RPL18 hairy root ribosomal complexes were fractionated by ultracentrifugation through a 15% to 60% Suc density gradient, and the _A_254 was recorded. The positions of ribosomal subunits 40S and 60S, monosomes (80S), and polysomes are indicated. Fourteen fractions were analyzed by immunoblot with anti-FLAG and anti-RPS6 antisera. Two electrophoretic variants of RPS6 were detected by the antisera. Molecular mass markers are indicated on the left.

Figure 6.

Figure 6.

The CRISPR/Cas9 system introduces mutations in hairy root transformants and can be used to study gene function in roots. A, Schematic representation of SlSCRpro-mGFP5 and SlSHR gene structures, DNA sequences, and relative locations of the targets designed for use with CRISPR. The restriction site used to screen for mutations in each target is underlined. The target sequence is in red. SNPs between mGFP5 and eGFP are in blue. The PAM follows the consensus sequence NGG. B, Control hairy root (containing no CRISPR binary vector) induced from the SlSCRpro-mGFP5 transgenic line shows strong _ER_GFP expression in the QC and endodermis. C to E, sgRNAs designed to complement mGFP5 and SlSHR coding sequences were transformed in an SlSCRpro-mGFP5 transgenic background using hairy root transformation. An sgRNA designed to complement mGFP5 led to the reduction or elimination of SlSCRpro-mGFP5 expression (C). Hairy root transformation with an sgRNA designed against the endogenous SHR coding sequence induced short roots with stunted meristematic and elongation zones as well as the reduction or elimination of SlSCRpro-mGFP5 expression (D and E). White arrows point to root hairs and the start of the maturation zone 0.3 mm from the root tip. This domain is outside the field of view in the control (B). B to E were imaged with a 20× objective and constant settings of 488-nm excitation, 70% laser power, 1.87-Airy unit pinhole, and 583 gain. GFP expression is shown in green with both autofluorescence spectra, and residuals are shown in white. Bars = 100 μm. F, Genomic DNA was extracted from roots transformed with Cas9 and sgRNAs against SHR, mGFP5, and eGFP. DNA was predigested with _Ale_I and _Afl_II for SHR and mGFP5/eGFP, respectively, and genes were amplified using primers flanking the target sequence. Amplicons were digested again. Untransformed indicates SlSCRpro-mGFP5 (same as in B) but not transformed with Cas9/CRISPR. Black arrows indicate no digestion. G and H, Alignment of sequences with Cas9-induced mutations obtained from roots transformed with Cas9/sgRNA for mGFP5 (G) and SHR (H). The wild-type (WT) sequence is shown at the top. The sequence targeted by the synthetic sgRNA is shown in red, and mutations are shown in blue. The changes in length relative to the wild type are shown to the right.

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